Medically Assisted Conception: An Agenda for Research (1989)

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C~ER 2 AILING IN 1= l~AC,lICE: AND SCIENCE BASE OF MEDICALLY ASSISTED WONTON this *lapser is }mused on papers pi at a workshop on Basic Science Formations of MA ly Assisted Corruption sponsor by the Institute of Medicine (IOM) and the Lard on Agriculture of me National Research Council. It was held A - USL 21-23, 1988 at the An sold and M Beckman Center ~ Irvine, California. For the workshop, the organizing committee developed a program that explored the recent advances ~ reproductive and develop mental biology that apply to medically misted conception. this chapter represents a report of science topics selected by the committee. It is therefore not a comprehensive review of recent advances in reproductive research. The workshop also initiated an interchange of ideas among those involved in patient-related clinical practice, animal IVFET, and those working in basic research as it applies to humans and other animals. Thus, this chapter indicates research areas that promise improvements in the practice of rVFET. First are some developments in human and animal IVFET; subsequently, the processes and recent advances relating to gametogenes~s, fertilization, preimplantation development, and implantation are disruccPl. This chapter summarizes each talk given at the workshop. The full papers contributed by each author are found in Apperdix A. Developments in Human In Vitro Fertilization E1] Eastern Virginia Medical School, one of the premier centers for the clinical practice of in vitro fertilization, has a pregnant rate of 18.3 percent, bash on the Or of arcs used to stimulate the ovaries with either follicle stimulating hormone (ESH) or human n~a,~1 gonadotropin art, and a pregnancy late of 37.6 percent he on the ~ Or of patients. Both patient age and the cause of female infertility have an effect on the oubccme of in vitro fertilization. For women infertile because of tubal ligations or endometriosis, the rate of viable pregnancies obtained by [VFET at Eastern Virginia is 16.4 percent. Although the pregnancy rate did not vary according to whether infertility was due to tnhun ligation or endometrios~s, age has an effect. After the age of 40, it Ones Ore c3iffiadt to stimulate the ovaries to price mature Is. Because Rupees often seek in vitro fertilization to mrercc~ne mane infertility, the evaluation of semen is important. Arrantly, this evaluation is descriptive and relatively incise. Factors sup as number of ~ m, Epenn motility, arm ger ~ 1 shape of sperm ~ ntsfe.g. head Shape and tad) shape) are important variables in achieving pregnancy. For example, if 14 percent of the sperm are "normal," and there are 50~000 In per tic oentimeter of ejaculate, in - 25 -

vitro fertilization prig: arming pr~ies in 30 Perot of all attests. However, eve if there are 100,000 On per cubic centimeter, but only 4 pendant are Normal the Firs pro rate dog; greatly. Abnormal sunk often fail to fertilize an egg became of their inability to penetrate the e~'s protective omrerir~, the zeta pellucida, therefore workers have tried a t~niq~e ~ as "zone driller' Owlish holes are prig in the zone pellucida to permit direct an: of sin to the he plasm membrane. Bus far, this t~niq~e has not been successful. Archer technique, hen as zone ~?littir~, has }>Ben ~ at leery University, Atlanta. E'y this t~niq~e, the zone pellucida is Split hani~al ly. E~na~ies have been reports by use of this method. Basic science questions new to be at In order to Event better marks for normal art a~r~1 seem art to iT~prave the perforate of sham in IVIES. ~y~preservation (freezing) of in vitro fertilized ~ryos1 is another new ark promising technique that will be discuss in greater detail later In this Copter. If embryos are frozen for later use, the stage of Heir develc~xt at Be time they are placed In the uterus can be mashed with the stage of the uterine wall Arid, increasing the likelihood of a su~=cful pro. Before cryo preservation bare an option, Libby - C had either to be plan ~ He up or A. In order to avoid the ethical dilemma of Eat to do with excess embryos, Ore than the optimal ~ er of embryos were safeties transfer Eta the uterus. By allowing the preservation of embryos for later use, He technique of cryopreservation rues not only the Lance of multiple pregnancies, but also the Or of tog a ~n's ovaries night be subjects to hormonal stimulation to produce opiates for additional ads of Inter. He use of cry~preservation has item In ideal rate of pregnancy at the Eastern Virginia clinic. Neverthel—a, Eric science relearn is need to ~ the nary parameters for sulfur cry~preservation ark the possible deleterious effects of freezing on the embryo. In cases of ovarian failure, failure of in vitro fertilization, poor quality of eggs, genetic abnormality, or inam-~=ible c varies, the only option available to women wanting to bear a child is to use eggs freon a donor to perform in vitro fertilization with the husband's sperm. m e donated eggs most often come from IVFET patients who have received hormonal stimulation and produced more eggs than necessary for their own use. Scmetimes, however, women who are to undergo a tubal ligation agree to ovarian stimulation before the surgery so that eggs can be harvested simultaneously. mese eggs are then donated for in vitro fertilization and transfer. Donors are phenogypically matched with recipients and are screened for psychological previews and infectious diseases. Patients who 1 me t£=m embryo in this cog often refers to In vitro fertilized eggs that developed to twos to Farrell staged embryos. Alff~a~h there are Tree technical ly precise terms for variants early stages of de~rel~rent, the more precise term`; are used here only when the distinctions are important to the Carets under discussion. — 26 —

conceive through egg donation may need supplemental hormone therapy to replace the estrogen and progesterone normally released from the ovaries. In such a patient, however, the placenta produces detectable estrogen by week 6 of the pregnancy and progesterone by week 7. Fertile ized eggs from oocyte donation, if transferred to the uterus by day 17-19 of the recipient's cycle, resulted in a 36 percent pregnancy rate and 80 percent of these pregnancies going to term. One benefit of oocyte donation! in addition to overcoming the problems mentioned above, is that a Lime period of optimum implantation can be achieved such that the stage of the embryo and endometrium are synchrcrized. This technique has been found to Increase the success rate of IVFET. (food halite ryes are critically instant to the sumacs of IVFET. Experience at many clinics shows that a number of factors determine the quality of an embryo. Some important factors include the way ovaries are stimulated with hormone supplements and the synchrony between the age of the embryo and the endometrial stage. Mbre research is needed to understand fully these factors and to establish unambiguous and unbiased criteria with which to distinguish poor quality and good quality embryos. RP-~C~ fertilization is more likely with mature oocytes than immature ones, research to learn how to recruit a more synchronous population of follicles is important. Such follicles produce a more sync hrono~c population of of capable of responding to the maturational stimuli. Failing the recruitment of mature oocybes, research is needed to develop reliable methods of maturing oocybes in vitro. All of these areas, if improved by I knowledge of the cell biology of early reproductive events, should greatly increase the ability to identify couples with high prnh~hility of sucks== and may increase the Cc rat== of IVFET in human clinical practice. Developments ~ Assisted Conception in Food-Pro*ucing Animals [2] There are critical differences between the gnats of -misted conception in animals and human beings. In human beings, the gall ~ to increase the reproductive ability of these with impaired fertility or, perhaps in the future, to avoid the transmitted of genetic disorders. In contrast, the goal of -existed conception in food-producing animals often is to increase the yield of milk or meat. Because the ethical barriers are lower and the financial stakes higher, a number of advanced technologies are available for use ~ animals that are not available in the clinical practice of human [VFET. Techniques in commercial or research lace include artificial Insemination, superovulation, embryo transfer, freezing of embryo, sexing of embryos, multiplication of embryos by bisection and cloning, ~ vitro fertilization, and the modification of embryos by gene transfer. All western European dairy cows and 60 percent of U.S. dairy cows are impregnated by artificial insemination (AI). Al allows bulls, affected for their franc mission of bilk producing genes, to achieve up to 500 inseminations frml, One ejaculate. Thirty Yeats of AI has helped to double the milk production of each cow.

Arx~ t~niqwe ,~ widely in humans as well ~~ in other animals is a~latic~n, which irT`rolves ovarian stimulation by he administration of hormone suppler;. In bath anions and horn, Elation is uppr~ictable ark often ~sful. there has been son Us when came am given priming Be; of follicle sti~ating hormone (=H), ark Den highly purified forms of FSH am use, hat the reasons for these effects are ~lea'. In Tuition, studies of cattle and other specie; have fat that the cxx~; pried by Formulation safeties exhibit abnormal c~aracteristic:s. Beat urx]~tar~ of hormonal cycles arx] the effects of hormxes on the cell biology of merit oat; whim likely have dint abdication to the solution of scare of these p~lelmse the process of embryo transfer In cattle starts with Ovulation of a ax', ark subs~uerrt mating to a desirable Hal. He resultant erribryos are c:ollec~ Surgically and transfers to anther cow whose estrous cycle has ~ syr~zed with that of ache donor occur. Although the sat rate with embryo transfer Is r ~ table (a ~ ut 60 Entrant of transfers result in pregnancy), the technique has not produced great ~ in ~ Ik or meat production. The production of many ide ~ ira copies of embryos from cattle with highly desirable traits may improve this situation. For this reason, embryo freezing and embryo multiplication procedures are receiving a great de~1 of attention in the commercial breeding of cattle. Embryo freezing, in concert with embryo transfer, has been generally successful in terms of pregnancy rater. Freezing embryos allows for the s borage of rare breeds and preservation of a cattle surplus. However, embryo multiplication has greater potential for the production of large n ~ s of highly desirable cattle. TWO methods of embryo multiplication are used--embryo bisection and nuclear transfer. Embryo bisection is performed at a very early embryonic stage and yields at least two ~11 masses which are genetics ly identical . me bishop embryos can Aches be transferred for norm pro to a recipient oar. mere is a species specific limit on hcm many bisections of a given cell ~ can be done without compromising viability; with cattle, the maxim yield is four embryos free one. In n~ucl-=r transfer, a blasters (embryonic cell), or its nucleus, fray a valuable embryo is placed into an ~ e from which the nucleus has been removed. The transferred embryo nucleus promotes development of a multicellular mass that can be used to mate a number of copies or cranes. There ~ a great deal of interest in this method both in academic and commercial research. Since these techniques allow more precise selection of desirable traits, both have the pabential to effect rapid changes ~ the prevalence of ads with those traits. While the techniques divest above are frequently and s~ccfully used, other methods of assist repression are also being investigated. One of the newer approaches is embryo sexing. Sting of embryos; is of particular i~portarx~ to the dairy icy singe only female offspring are needed] for mink production. Sexing is done by three ethic. The - 28

first is kary~ping of embryos by bisecting then and using one half for ~r~genetic analysis. the thief did of this method are Damage that often oomurs to the ~~ and ache variable reliability of ~ kary~pic identification of sex. The sad ~~ drays antibcxlies to Specific antigens oar the ~hryo6 to identify the male embryos. He attract of this i~ologic=1 metha] is greater than that of Typing and may bed Vilely use, did on ache availability of antisera. Finally, ~ hybridization Is have bed adapt fan Ear biology to sex embryos }fly labeling specific parts of the male Y Dad Stains by embryo bict?sy. Were are Capella reasons to develop are fully IVFET pry in food-pr~ucing animals. Such techniques pride a large number of Embryos for transfer, ray, and embryo multiplication. However, as in human WFEr~ ~ success of in vitro fertilization and embryo transfer In - He cattle industry suffers from a lack of basic krxowI~ge of the ~~] biology arm biochemistry of early gamete maturation ad fertilization. ng the arms in Ail s lapin is he maturation. As in humans, Iv~r in cattle is Are fur if it begins with mature rather than ilr~nature dates;. me biochemistry of sperm capacitation arm entry into the eggs also reds to be better dud. A nod of cats have been identified that ad nectary for these presses, but precise identification of the role each *him?] play ~ fertilization is for further progress. Finally, being able to maintain the grad of an embryo in Suture for a longer period than is now possible weld be helpful. Such an ability w ~ d ~ se the ~ anc ~ of a favorable match between the embryos c and endometrial stager. Gene transfer, if developed further, also shows potential for enhancing the productivity of cattle. With the development of techniques by which genes can be microinjected into embryos, the ability to alter the phenogypic characteristics of food-pro~ucing animals has become a possibility. In an early castration of transgenic technology, world; injected Ruse eggs with the gene for human growth hormone. me rating mad grew to ad Rice the size of nonnal Moe. such teleology cold be used in cattle to alter the genes for skeletal muscle in order to prepuce higher quality meat prefects, or to alter genes in such a way that biologically active syntax wed be secreted ~ milk. mere are many areas in which the results of basic Beard Ward further the practice of a ~ isbed conception and embryo transfer in both human beings and over animals. these irxlude ornate maturation, Sperm morphology, the biochemistry of fertilization, cryopreservation, ovarian stimulation, and molecular genetics. In addition, topics such as membrane biochemistry, hormonal control of testicular and ovarian function, gene expression In early development, and the cell biology of implantation are identified as areas of exploration that would make major contributions to the sue of human and animal [VFET. — 29 —

His ~ Awes Otis of G~tog~;~ AL ~zvirulation t3] Comparative rearm con ovarian starvation of non-h~nran primal; and dc~restic animals such as cattle, horn;, shop, arm pigs raises Sting issues. Scam work highlights He diffen~es between Be normal estrous cycles of animals sum as cattle, arm the cycles of human arm r~on-hmnan primate. Yet there are promo that are On Hang species In the station of follia~lar develc~ent arm the in~ian of mature oceans. Ovarian stipulation arm ~wulation are exag~s interverltions into a highly regulate ~ysiologi~1 press. He rang physiology is not always racily manipulated. Develc ping strategic to cir ~ event the clifferenoes between nor ~ arx] in ~ ovulation Is a major goal of research proposed for this area. Non-Human Primates Non-human primates exhibit a menstrual cycle that closely approximate that of humans. In monkeys, the hormonal events of the cycle include a geometric increase in serum estradiol, mldcycle surges of luteinizing hormone (LH), follicle stimulating hormone (FSH), and a later increase in serum progesterone after EH and [~H decrease In concentration. These hormone levels are precisely synchronized in the normal cycle and lead to the development of one follicle, the dominant follicle, from which cvulation occurs. One of the purposes of superovulation is to encourage more than one follicle to develop fully, thereby producing multiple oocytes for in vitro fertilization. In monkeys, one of four chemicals is administered in combination with human chorionic gonadotropin to stimulate ovulation. these are human mencQa~sa~ gonadotropin, human FSH, pregnant mare's serum gonadotropin, or porcine FSH. However, monkeys sometimes produce antibodies that block the activity of hormones obtained flown cipher species activity, and clarion stimulation fails. Incus, them is a need to develop stores of monkey hormones deco be used for ovarian starvation in this species. the rinse to the a~ninistrution of hordes ~ stim~a~ce oration ~ also not uniform in non-in ~ an primates. In g ~ ral, estradiol levels are higher than in a natural cycle, the EH surge is lacking, EH concentration remains high over a long period, FSH is low, and progesterone remains high in concentration. However, in some subjects the ~ is a premature, spontanecus EH surge that is nck synchronized with fo1licular maturation; this surge StCp6 the follicles and oocytes fern maturing. Even if such an EH surge does not occur, there ~ a wide range of responses to gonadotropin administration. It is impossible to predict the response of any individual animal. me EH surge and the hetercgenous responses, however, can often be prevented by additional administration of gonadotropin rehearing hormone. Another difficulty often observed with superovulation protocols is an overstimulation of prolact~n secretion. These Repartees from the normal hormonal levels raise important questions relating to the efficacy and urn effects of ovarian stimulation. - 30 -

Apiece the problems of ovarian sti~ati~ ~ nca~-h~nan primate, reasonable so ret m; have Ben achieved. Nevertheless, ~ are additional barriers to progress ~ this field. ~ Sony of In primate is soberly limit - . In ~;tioa~, the animals often c~ be Are Can once for ovarian stimulation because of Me iTr~ogica~ r~ to human, I, and porcine ~a~ins usual in the stagnation I. Finally, As must be zippered fmn ~ }fly either I~t~y or Party, surgical Unit; that are reprice by ethical cor~siderations arm formal legal oc~ns~aints ~ a l~ Her of repetitions per animal. Although these prowls of avaiiabilit~r, ~se, arm Be lack of no~h~nan gona~r~ins are more difficult to avers than t~ebiologi=~1 pr~bl~ofaverridi~normal~ysiology, Inns questions a}x~t ~ e basic biological pa ~ sees Robin Ha ~ . Resolution of the problems a-=c^~iated with the use of non-human primates in this type of research could be facilitated by an increased ability to augment natural hormone and gonadotropin release, by improved resolution of ultrasound imaging to identify mature oocytes and guide their collection by non-surgimal means, and by the development of cell lines that cculd produce larger quantities of non-human primate gQns~ctro pies. Finally, it is important to note that many of the same questions reman unanswered regarding human ovarian stimulation and that particular non-human primate species serve as the best mcde] for human reprc~uctive physiology. Domestic Animals Extensive ~~.c~ of artificial insemination in cattle has ~ ease d the genetic contribution of desirable huts to the overall supply of cattle. However, there is little likelihood that the Hirable characteristics of fen ales will be further is tar augmenting the male genetic contributions. Ovulation, on the ocher harm, Dined with embryo freezing arx] embryo transfer, has the potential to irx:rease directly the germ pool of desirable female traits by increasing the ember of offspring fray a Valerie cow. There are a nor of different; beaten non-human prima`; and clarestic anneals ~ the ways ovarian stimulation is a ~ Cliched. Sare of these differs arise fmn differences in the reprc~uctive cycles of the two grcup6. In dairy and beef cattle, stimulation is accomplished with the administration of either pregnant mare's serum gonadakro pin (PMSG) or porcine FSH. m e latter is preferred to FUG because FUG has a long half-life and often results in asynchronous ovulations. Porcine FSH is nct without problems either since it is contami~ by variably high levels of luteinizing hormone. Dean dhorio~c gonac~c~rop~n, routinely beck ~ human primate, is not USA ~ the Facial practice of ovarian stimulation of cattle. ~ oont~1 the variability arm lack of prExlictabilit~r in the estrous cycle; of came, current propels often include an injection of a Specific type of pr=;taglarxlin (PGF-2 alpha). this treatment caress the reg~sicn of the corers lutes, thereby artificially restarting the cycle. The sum rate of Cation ~ came has been variable. this is caused by a moper of factors including seasonal variability, breed differences, dose and timing of gonadotropin administration, and history - 31 -

of previous formulations. Sew diff~s contribute ~ ctiffi~ Mortaring st~ies performed on different bows and, at tom, in Paring sties In we same breeds, Ott ~ with varied nines or in animals of varying age. It has been fat that, even if all the factors forcible are bled, ~ Swains a great Anal of friability In the success rate of ~r~ulati~. A number of stra~gi~c ~ Ore the gun rate are Firstly being tried. One is an attest to purify porcine FSH preparations to exclude the contaminating IlI ark, further, to detennire the FSH to IlI ratios that are bib for su~cctul Formulation. Ark strategy Is to try to ruralize R4SG with antibodies so that the complications bat arise fen ~ lord half-life of EME;G can be prevent - . Rf~ the lack of normal acordination of _ ~ a ~ endocrine egrets is a Cain charred in ~r~vLLLatlonl bravest lgators are also tryir~ to define He sources of ~py art develop ways; of normalizing a Ordinal series of biolc~ical events. Superovulation In c3c~ic animus other than cable also has had mixed results. ~ goats, the technique has been tr~c~usly ~ccful. Superovulation of sheep has also been largely su~mcful, art a few irritative strategies have been allied in this species. Sheep are seasonal brewers and attends have been made, with initial =~==, to induce ovulation in this species during tins Pen they are normally arts. In order to bypass the use of exogenous gonadotr~ins, workers; have er~ployec] antibodies to inhibin In s;h~?. In is a Stan made in an ovarian fold icle that suppresses FSH secretion. me blocking of ir~ibin with antibodies raises ~1 levels, thereby increasing the rntura1 stimulus for follicular develc~rent. Antibodies against steroids have also been tried in ~h-~-r to achieve the same end. In horses, Ovulation has proven to be difficult, and to require no~tar~ard protocols. me period of esteems or "heat" in mares is '~`c'~_1 1 or 1~ :~ bran.; at = ~1~1, .~ - ~~ .~_~_. . In addition, there are preps p ysiologim~l ~ nines In mare to prevent the develc ~ nt of grins. mese two aspects of e ~ reproductive physiology greatly complicate the sur~=c~ful application of ovarian striation techniques. Also of critical importance is the fact that PMSG, even in high doses, does not adequately stimulate the development of follicles in mares. Unlike other animals, where PMSG acts as both FSH and EH receptors, In mares PMSG has only AH activity. Some success has been achieved in mares with administration of porcine FSH, especially if combined with human chorionic gonadotropin. In general, however, progress in so superovulation in horses lags well behind that in other domestic animals, ewnent for the pig. me my limited of all dial abdications of s~run~lation in animals has been In the pig. Although possible, the technique does net confer maw of ache advantages that it holds for other species. One ran is that pigs deliver liters as q~ to one offspring at a tin - . therefore, the need to Pease He offspring foxy one individual, while helpful in certain cir~ta~es, Rustic animals. Never~c, Ovulation arx] embryo transfer may — 32 — __ , _ . is net as cc~maellina in Dices as in other

be able to lower the frequency of din by ~ sing the number of offspring f`=u di=~'=-free or disease-resistant populations. Mbre frequent reasons for the use of embryo ~ fer in pigs are to obtain sew-free embryos f ~ u infected pigs and to 1ntroduoe new genetic material into specific pathcgen-free herds. Thus, further research into the normal reproductive physiology of pigs will be useful. Tb improve rVFET in domestic animals as weJ1 as in human and nonrhuman primates, more needs to be known about the normal regulatory events of ovulation, including the physiology and biochemistry of the development of a dominant follicle. Improved ultrasonography he= potential for increasing the I of superovulation in all species. As with parleys, developing ways to bypass the use of exogenous arc like MEG and porcine FSH weld be of enough ,~ in domestic animals. One way of doing this may }'e by blodki~ ir~ibin as has been done in sleep. In order to imps w e the mY~~=c= rate of assisted c ~ oaption In animals, many of these rv~Y~mch areas must, nevertheless' be pursued in each species separately with realization of the inter-species differences. It is not always possible to predict when the results obtained from one species can contribute knowledge applicable to other species. Biology and Maturation [43 This section examines three topics relating to the biology and maturation of oocybes: 1) The sway of membrane biochemistry, which has relevance to the complex membrane interactions that occur at fertili- zation; 2) the study of molecules that are important in maturation events, Is also important to understanding the possible reasons for failure in IV BET; arx] 3) the biological are physical pr ~ erties of eggs are embryos that are affected by preservation techniques such as freezing, known as cryqpreservation. Membrane Biochemistry The membranes of all cells of the body share certa ~ standard structural characteristics. The basis of a ~~l membrane is wiled a bilayer, which is a two-layered collection of phospholipid molecules (lipids containing phosphorus). Phospholipid yes are polar ~ that each has one end that is stable in water (hydrcphilic), and one end that repels waver (hydrcph~bic). In a bilayer, then, the lipid molecules line up so that the by ~ Cubic ends meet each other on the inside of the bilayer. The hydrophilic ends are thus oriented on the outside of the bilayer such that one group faces the intracellular space are the ether far the extracellu~ar spar. Sub an arrangement Mars sense when ore oc~nsider:; that bath the Neoplasm of a cell arrt the extracellular span are ~1 largely of water. It is, in fact, a quite natural rinse of lipids to form bilayers in an aqueous vironr~nt; such a process Berlin soap bubble arm oil droplet formations. 2 Embryos can be rinsed free of pathogens or tread with enzyme to destroy pathogens Eking embryos the safest method of Ovid germ plamn win t pathogens around the world. — 33 —

Steins are part of Me membrane also. One proteins E - n Off thickress of Me m3ubrare and act as ~hilic Carts for ions or participate ~ Alar signal tr~tion Aim. Other proteins are partly erred in the lipid bilayer arm partly exposed to the extrar~llular Laos. Atta~ to sew proteins are Cog, often br~i~, chains of carbohydrate lies Cat also etch into the excra~llular space. Finally, large e~ra~1lular proteins often associate ~= or stick to the Nat Al parts of the carbohydrate molecules. Its, the ~11 membrane, ever In its scarab fond, is a complex structure made up of differ An; of molecules array In regions that each exhibit - ^ - - ~_ ' _~ _- - If: ~~ ~~ ~_: =~ ~ : ~~: ~~ ~¢ ~~ ~1 ~1 ~ 9 ~;1^ 1 ,pL~ ~1~. 110: i~—~1= AL—~ "~D Vet "~`w`~ `~ - ~ · ire since He variants Leaves are fertile within ache bilayer. these interactions orderly 03 Rally immense range of biological r~ of c - 1 is including ~1 1~1 ir~eractioa=, r~r~ia~ signal premising, arm t~brane transport of nutrients or t=XinD- Reproductive events In which Crane biochemistry is of particular i~ortarx~e include sperm capacitation arm fusion of serum arm egg membranes for fertilization. Relic assays of membrane biochemistry are Nat often sixties using membranes fmn owls such as red blood Ells or Bran manufacture in the laboratory, called likes. Such Rare are simpler Man the Umbra fmn eggs or sperm ark, therefore, easier to sky. Hirer, data Caine freon t:h~ Gel Ares nest be excrapola~ ~ Us arm sperm with Goat caution. Lillian Locke Maturation . . . In a nor foil icle there is Fornication between the As and me ~r~g reverse cells, called gra~osa ~1 IS, through specialized junctions (gap junctions) threw which smal l moleall.es Franc fold ore roll to ark. Developing A; undergo a type of ~11 division in which only half the normal nor of ~nc; are retained. This pr~c~;s is kncr~7n as Kiosks and he a Enter of stages. Until just before maturation, however, the growing oomph; remain In a sea te of susp ~ ed or arrests meiosis. It is th ~ ht that the granulosa cells help to ma Stain this arrest of meiosis by producing substances that enter the oocyte through the gap junctions. It ~ also possible that substances that induce final maturation of the oocybes are prod urea in the surrcundinq cells and are transmitted to the oocytes in the same manner. Since the ability to mature oocybes An vitro would be of enormous value In IVIES, the u~rsta~q of the s~s that either maintain or abolish meiotic arrest Is particularly desirable. It Is important to note that many of the studies satirized In this section were done with mouse 00~5 and these data were cc~panad to those Stained from other Spinals incl~i~ra~its and rats. Ir~vestigators have also studied frog oocytes, but the mouse is the my an animal meek. Evenh,~1ly, of Carrie, ~xr~= critical - Stints will have to be replicated using oocybes frill human or nonrhuman primates. Several substances are thought to maintain meiatic arrest. One, cyclic adenosine monophosphate (cyclic AMP), is a molecule present ubiquitously In the body. Another, hypaxanthine, is common in the body - 34 -

are Mulcts to a gasp of Yes 1~ as Enshrines. Guarx:6ine, art Enshrine, is also a potent inhibitor of Meiosis arxl the purine, Antoine, he been stun to aunt flee activity of hy~hine. E,y Dominic He effects of sane of Due Stan an eat Our, a hypes he arisen whip suggests that hy~chire are aver prim; may impose ache Intel of c~yclic~, which then ir~ibits~ios~s. Fire Drub, f~ ~ - n Be ampules of the cyclic AN pathway ithibitir~ meiosis, may eluc:idate ways In which i; can be maintain In Civic abut In vitro. Bile knee rqa~i~ the ithibiti~ of oboe maturation Is important, kr~ri~ hcr~v to macro i; in ~,l~re would have many applications that are describe later. ~ viva, Ye ma~rati~ ~ prig by ~ surge of 111 i~t-1y prior to Ablation. she Beanie of action of Ill Is uncial, however. Cue might assure ~at, if Chic AL mains Idiotic arctic, Awe IN sat cause; a Lease In Chic AL In ~ cue or ~ ~rr~ir~ nurse delis. It he been On ~ viva that administration of It in combination with human ~a~-~1 gonaclotrnpin rout cyclic ~ in the ovate, but not in the s~rm~di~ curse cells. His effect is puzzling since the gap jurx~tions between the ox; are the ocher Plus still appear to be functional, so cyclic AT dhalld Ego through these junctions Oily. An alternative hypothesis is that Ill acts icily by causing scale maburation-inducing Stag to be pr~x3u~ in the ~rr~ir~ Bus that duress the cyclic AD in the ~e. Since an Crease in ir~cra~lular calcium has been shun to be Naiad In sanatic cell division, it ~ bible that a ma~tion-ir~ucing ~ curate in the Cues by caroling an Case in ~tra~lular Cain. me sear for these suntan art their anti of action Is a major area for fug rehear=. she stay of o ~ ce ma Oration has led to etch ~ ]y sun ~ =ful methods for maturing mouse oocytes in vitro. Such work has shown that the in vitro culture of oocytes and their surrounding ~P1IS can be accomplished in ways that allow subsequent fertilization, implantation, and delivery of normal young to ~ Jr. Sur--==fu] in vitro fertilization of oocyte_ from other species, however, depends upon continued basic research on oocyte nutrition and met~holism and on the factors or substances that control differentiation and maturation. m e potential applications of an increased ability to mature oocybes in vitro are numerous. As disr~=c-d earlier, such an ability would be of enormous value in the production of agria naturally important animals. m ese techniques also have important applications in attempts to preserve endangered species. For example, it had been demonstrator that oocybes removed from mice up to six hours after Beth can be matured in vitro (Lazarus effect). Yet another application can be found in basic science research into genetic dic.~c c through the ,~== of transgonic ~ ce. Such mice are pro*uoe1 by introducing known gene fragments into the gonome of a mouse. These gene fragments have been inserted by m~crv~njection into eggs. Alternatively, the fragments, incorporated into a ha ~1~ virus, have been Injected into neonatal female mice. In this way, such fragments are incorporated into the DNA of developing oocytes in these females. It - 35 -

weld be untruly helpful to be able to transfer gene fragments by vi=31 infection of al;, which card In be mature in vitro for Ant fertilizations Finally, He ability to mat human As may have sane application for human IVF=, since mature Ares freon donors ~d Triage the Ned for hormonal stimulation therapy are permit sy~ran~zation of the c~vel~tal state of the embryo with Vie state of the u~rine lining. He degree to whip ata certain A ex~ri~s winch Use cx0:es can be allied to hum is not Ken. Before, for such applications to be realized in human IVES, it is pliably Rosary that basic resort be done using ooc~ from human ovaries roved for justified clinical reasons armor Ares from Nan primates. C~yapr~rvation of Tortes are Embryos Cryobiology ~ the stably of how livir~ tissues can be frozen and stored for later revive. ]~ are a Per of use for cryapreservati~ of Ax; arm embryos in Rich with IVES arm other directive Theologies. C:ry~preservatioa, of human arm came Mann has been a repine are] su~c~ful prepare for many year;. It is thought ~at, properly stored, frozen embryos can be preserved for many ci~c3~ before thawing and transfer to the uterine cavity. To date, embryo cryapr~ervation has been su~ful in many mammalian Emeries' incI=lir~ mouse, sheep, on, human, ark] baboon. Rare Cant animus have al so been preserved for later sty. The Use Embryo Bard at the Ja~son laboratory in Dine contain many Temples of cryq?resenred mutant Use embryos. Such mutants are valuable for Rio into genetic art cipher cli~c. Cry~preservation allay easy transportation of frozen embryos by re~arubers; To Ore frozen mice embryos freon one lab to archer, arx] for ache national arm international transportation of Abbe embryos. If proved safe for use In clinical practice, routine cry~prm;ervation of eggs or embryos ~d route ~tantia1ly the number of ti~; a An w ~ d have to ur~ergo hor ~ ne stim ~ ation, because the eggs harbored ~ could be frozen for later fertilization or fertilized in vitro and the extra embryos stored for future transfer. m ese stored embryos could be used if the first pregnancy failed or used at a later time so that an infertile couple could have more than one child freon one stimulation/IVFET procedure. Bus, such preservation techniques could also minimize the waste of extra eggs arm extra embryos prod by Formulation and other rvF~T pressures. Al~cha~h the specific me~ch~.c of cryc~reservation vary, the Uric sups involved are the As. ~ first Is chemical protection of the tissue against damage Frau frying. He tissue ~ sometimes Holed In sequential Saks to a subzero temperature before being rapidly cooled to -19 6° C for lo ~ term storage. Ibe pr ~ ures used for warming arxl cooling are variable. After rewarming, the chemical protective solutions are relived by rinsing. Protection from damage by freezing is critical to the survival of an embryo becalms, without protection, the formation of ice crystals causes tissue damage that can not be repaired. — 36 —

~ycprat~t:ive solutions, whim are made f~ Admirals sum as glyc~1 or dimethyl sulfo~de, furx~ion In a manner similar deco antifreeze In a car. They replace the water In the Wry. Because of the importance of this step in cryc~preservation, basic ~ Is being Acted to find aF~?r~riate cryaprc*ective solutions. ~ awn specific pa ~ ies of the solW ions are important determinants of the survival rate of eggs and embryos. For example, some chemicals, such as sucrose, cannot enter Calls and, therefore, do not protect from freezing damage in the some way, or with the same efficiency, as other molecules that can enter cells. Glycerol, an the other hand, can permeate a ~11, partially replace the water, and impart the cryqprokection necked" by. the osmotic characteristics of particular solutions are important also. In the Cal=- of glycerol, if one exposes a cell ~ a solution of water and glycerol, glycerol Eves into He cell by osmcs=, a process by which miles in a high concentration will more across a semi-permeable barrier, such as a ~11 membrane, into a region of leer con ~ Oration of that molecule. Li ~ rise, water TrOves out of ~ e cell by osmosis into the surrounding fluid. Eve ~ ly, the concentration of glycerol will equilibrate and be the Cam_ on both the inside and outside of the cell. Since glycerol moves into the cell scare slowly than water moves out of the cell, the cryqprotection procedure must be carefully controlled so that the cell ekes nck 1OCP water so quickly as to shrink and die. It ~ pecially important to control He movement of Cleaves across He cell membrar~e when thawing ova arx3 embryos. After thawing, it Is important ~ remove the glycerol friars ache owls and to return He ~n~cra~llular contents to normal. ~ever, a cell probed with glycerol, if su ~ enly exposed to a normal saline solution, will take up a great deal of water very quickly. This influx of water causes the cell to swell ark the nine may Ret. Although this pniblen On be avoid by exposing the cells to solutions of decreasing concentration of glycerol in a stepwise fashion, this is not always easy to accomplish. Research has shown that adding the impermeable solute, sucrc se, to the saline solution results in a Ore even excavate of glycerol and water across the Crane and Aviate the rapid influx of water into the cell. This Cheep method has proven to be of particular ~~-~ In the application of these techniques under field oonditions such as In work with endangered Dies. The method used to freeze and thaw the Ova ark embryos is also i:pportar~. Until recently, the tissue was first Holed to a few ~ rees below zero so that ice formation could be controlled. The tissue was then cooled to some intermediate subzero temperature before being rapidly cooled to the storage temperature of -196° C. research with mouse ova showed that the cooling procedure ~~.=A~ could affect the survival rate important ways. For example, it was demonstrated that, if ova 1ncP a considerable amount of their original volume during cooling, the survival rate is greater. Cells that retain meet of their original volume usually do not survive. This is probably beck. a ~e11 is primarily composed of water from which the damaging ice crystals fork. The method undo to - 37 -

freeze ~ tissue also det~mi~s the Ant thawing pry. Hawing is performed so in reverse order to the freezing steps. New freezing phenols have been desrel~1 that simplify the multiple step math - a. For Ale, nrx~se embryos have Ben fully frozen in one step after incubation in glycerc,1 ark shone. this Shod ~~-~ the glycerol to cryopr~t the tissue arx] the sure to dehydrate it before freezing, eliminating the need to slowly cool the tissue before freezing. Variations of this method have also been so. C,ne navel methal is called vitrification, which means freezing without crystal formaticn. Saltine, this can be Amish }fly untruly rapic] freezing. ver,in a variation of this a ~ roach, embryos are exEx~;ed to a solution of chemicals that form a glassy solid substance when cooled. No foe crystals form in the embryos exposed to this solution so freezing can be rapid and done in one step. These new freezing procedures are still being developed, but they have the potential to increase the su~--n~c~ rate of ova and embryo freezing. Improvements in cryobiology would have significant impact. Improved preservation and storage of embryos frmn valuable cattle and other agriculturally important ads Bald greatly Case the yield fmn s~valation and IVES pressures. the preservation of exchange red species and rare genetic bre - = of laboratory animals would be em. Finally, su~c~fu1 storage of human embryos that result fmn IVES techniques can Crease the chances of an infertile couple to have a baby. Intratesticular arx] Intraavarian Paracrine control to Development of made and f ~ le gametes a ~ recover varied periods of time ~ special body compartments, the ovaries and the testes. In this section, the biochemical control mechanisms that affect gamete development will be discussed. In contrast to hormonal control mechanisms in which bioactive sobetances are released mto the bloodstream and act at distant sides, paracrine control nechanisrs involve chemical interactions between neighboring cells. For example, one cell can release an active substance into the extracellular space, the substance then diffuses around other cells in the region and affects the activity of these other cells ~ some way. mese effects most often are mediated by cell surface receptors for the substance released. Intragonad~1 Control of testis Function m e testes are composed of a number of different ~P11 types, including germ cells in their various developmental stages (spermatogo~ a, spermatocytes, spermatids, and mature sperm) each of which displays unique characteristic. Other ~11 types include Sertoli cells, which are nurse cells for the developing spermatogonia a ~ spermatocytes, and Leydig rolls, which relics the hormone testosterone, required for sperm maturation. Many bioactive hormones and peptides have also been found ~ the testis and research has begun to determine the cell sources of these chemicals and their function in spermatogenesis. Some of the mulches found in the testis have been shown to affect cell cycles and ~11 differentiation. For example, somatc~redin C (also rolled insul~-like growth favor, IGF) ark epidermal — 38 —

grouch factor Lima cells ~ begm IMP. syrups for mimic cell division. Fibmblast growth factor st;nulat~ iesoent oelis to r~mr ache ~~l Cycle in order deco Implicate arm divide. Various hormone and grc~ factors act as stipulators to ~1l differentiation, pus causing the cells to exit ~a~ly fmn the cycle of ohr~ replication and mimetic division. Culls am inhibit fmn exiting the ~11 cycle by i~rleukin-l, which is a Allele me by cells of ache inane system. me funnies of ~ of the horn; arm gram factors in the Avis are net 1~. For a fear, however, certain furx~tions are beginning to be I;. In me rat, a Awn animal ~ for irwestigations of sperm develc~nt, Somali awls start to divide arm proliferate art me 22rx] day of embryonic life, am complete this proliferation by 20 days after birth. Leydig culls, in trash, do nab start ~ divide mail 2 or 3 days after birth arm ax~cinue ~ proliferate Aid 35 days after birth. By the erg of these pr ~ ses, al ~ It equivalent ~ ers of Sertoli and Leydig cells are found in the testis, despite very different developmental timetables. A hypothesis has been proposed that these Cools may regulate each other by cc~plicated interactions during development. Fibroblast growth factor stimulates replication of Serboli cells by causing an increase in the number of receptors for follicle stimulating hormone (FSH). As this occurs, the numbers of Sertoli cells increase and reload- somatome~in C in greater and greater amounts. Somat ~ C increases the number of receptors for luteinizing hormone (LH), which cats ~ the Leydig c-1 Is to divide and Increase in number. The increasing numbers of Leydig cells leads to an increase in beta-endorphin re1Pacp~ by the Ley~;g coals. A negative f"Pah~ck occurs such that beta-endorphin decreases the proliferation of Sertoli cells. Such paracrine interactions are probably not unusual and may occur between other types of calls in the testis. In addition, non-paracrine mechanisms of communication probably occur, including cell to cell adhesion and communication through gap junctions between reals. Very little is known regarding the mechanists of interaction between developing sperm calls and the Or nurse cells, the Sertoli cells. Cell adhesion molecules have been found on spermatocytes that' some suggest, prcmcte continued meiosis of the spermatocytes bound to the Sertoli calls. Experiments done In Sure have shown that sperm cells synthesize more DNA and RNA when they are cultured with Sertoli calls (especially if F5H is in the Couture medium), than if the sperm m-1 Is are cultured alone, or alone with FSH. Such data suggest that the Sertoli ceils are important to sperm ~11 develc~ment. Investigators have looked for proteins that can be related to specific stages of sperm cell development. The stages of sperm oe11 development are quite complex, but can be generally grouped into four main stages. The primordial germ calls are called spermatogonia, and undergo so totic divisions before Aim s~rmat~;. Mbi=;is begins in sperms. Further develc~nt forms sper~tids, which are immature germ cells in With meiosis is a~t=. ppermatogon~a, spermata~;, — 39 —

at early ~natids are ~ close a~ositi~ to Ser~li ce1 Is in me long tubular stn~tur~ of the testis where sperm are formal, m=1 leaf me rmnife~s files. me seninif~s files coon into fir ~r~ which in are transports. As s~rmatids mature and diff~tiate into early sin owls, hey are release freon Me Ser~li oils ink the seninfer~s file. An wing feature of the s~nifer~s tubule ~ scram species is it, in any given sent of the tile, the gerTn ~1 Is form a similar pain of stages. file this array art for sane kirk of cellular ~1 of develc~nent~1 stage, it also provides a definable tissue state with whim to merriment. Scientists have isolated Specific s ~ nts of seminif ~ s tubules and extracted the proteins contained in them. MAps of these proteins have shown that the different stages are associated with different proteins. By isolating some of them- proteins and producing mLnoclona1 antibodies to them, it has also been possible to determine which cells contain the protein of interest. One such protein has been found in Sertoli cells, ark its synthesis ark section have been chain to be stage~ific. mese experiments have also fat that such proteins diffuse item the ex~cellular spaces of the seminiferous tubes, but because of the slaw flaw ~~ within the tubes, remain In span= near developing firm ceils for quite a long time before being d ~ red ~ by prokeo)ytic enzy ~ . The ~ fore, there seems to be strong evidence that Sertoli cell pro ducts greatly influence the development of sperm cells. Continued research into the molecular events of male gamete development would fill some of the large gaps in knowledge about the normal development of sperm cells and would further the search for causes and cures of male infertility. Paracrine Control of the Ovary Although it is generally agreed that p~racrine control is important ~ the ~ve1~nt of follicles In the Ovary, Ash of the evidence Is ci~tial. mis stab of affairs derives fin the nature of au~rine/paracrine radiation. It has proved difficult to manipulate experinentally system In which the ~11 of origin of an coved signal is the same as or adjacent to the Urged ~11. Fatly Worriments to ~ the i~ortarx~e of intrac~varian * - nit control involved roving the pituitaries of rats. Withy Me pituitary hormones (especially Ill and FSH), whi*1 normally promote follia~lar development, investigators cold then look at the effects of a her of ~ ta ~ on Ovarian follicles. S ~ e ~ rim nts fa ~ that ~ t ~ , which ~ synthesized and released f ~ n cells in the c very, was needed for follicular development and that other steroids proud inhibitory effects. Further, it was demonstrated that estrogen could act in concert with or synergistically with AH and FSH to differentiate folli~lar reals. Later experiments used granulosa reels Of pig follicles ~ Ire to dhc~ various effects of outran. Following a brief irihibitory effect, edgers stigmas prod sy~h~is in the grarn~lc~ cells in a maser that was ti~cnt ark blo~cable by phannacologi~a~ — 40 —

ir~ibi~cors to oxygen. Incite ~ ~h~ that outran action, ~ ex~ after Me generation, of cyclic AM (~ earlier section ooze maturation for our actions of cyclic AMP). lure are sevem1 *emits folios of estrogen that are made by enzyme codification of a heroic struck. Cue type, ca~olestrugen, is fat in high cor~tratio~ in the walls of large ovarian follicles in the pig alxl probably equal, }fly paracrine banish, the effects of PE;H ~ the follicle. This corset arose few =~ expiring; Wick fag ~t ca~oles~gen with FSH spaces pr~one Synthesis arm prounion of cyclic Am. Hover, ar~ci~r~ do not block this effect. mus, Wile experiments have ~ at It plays an important role ~ the Petrol of folli=~1ar c~evelc~:, its Once Bole in the nears state regains ~ be determined. Estrogen ~ only one of the ~a~ over investigation in tree; of parac:rine regulation in the ovary. Tissue bare experts have shown a number of pepkides and growth factors to cause proliferation of granulosa cells. For some of Chew- factors, it is known that they have effects on follicular cells and that they are present in the ovary In viva. However, whether or not the factors are ne-=cc~ry for follicular develc~rent-and normal ovarian physiology is not } ~ . the nest r ~ ear has been done on insul~n-like growth factors (IGFs). IGFs are synthesized by many tissues and have been found to be a powerful local mediator of growth and differentiation. They seem to stimulate all kinds of effects in granulosa cells including increasing enzyme levels, ~11 numbers, prote~n-synthesis, glucose utilization, and various secretions. Insulin itself and the IGFs seem ~~r,tial for FSH induction of rectors for It, for generation of cyclic Am, art for FSH stimulation of s~ic3s. IGFs also infract with other growth factors In the Calvary to grease cell fir. Since my of Muse findings have bum freon in vim studies, it is i ~ ortant 0 denons~crate the presence of IGFs in follicles in viva. In fact, IGFs have been shown to be present in follicular fluid in in viva concentrations similar to those that produce effects in culture. Furthermore, the concentration of IGFs increases during follicular growth and under the influence of FSH, AH, and growth hormone. This is important because any substance active in follicular development and local regulation in the ovary would be expected to change in concentration in respo ~ e to pituitary hormones. A ~ her go ~ h factor fat in ~ awry is a type of ~;form~ growth factor called TGF-alpha, which is a molec`~1e related to epidermal grouch factor. TGF-alpha increases the rate of cell division of ovarian Ills ~ vitro and has a negative effect on the secretion of estrogen from granulosa calls. It also seems to decrease the effect of FSH on AH r ~ rs, although this effect varies. TGF-alpha has been found significant ooncentrations in follicular fluid and is in highest concentration in small follicles. Rb=-d an these findings, it has been proposed that TGF-alpha promotes cell replication in the follicles and iris differentiation. me other type of IGF, IGF-beta, is a molecule very similar to ir~ib~n arm MUllerian-iT~ibiting Stag, both of which — 41 —

have effects In the ovary. In general, sixties have sagged Mat TGF - em aunts the Scions of FSH to increase estrogen suction, 1H Adaptor activity, arm pr~terme synthesis. Ire effects seem to be dot on both the filtration of -beta and FSH ark An to vary acmes species. Fibrublast grub factor Is archer factor being investigate for its Fusible role In Alsatian of ovarian fur~tioa~. This grub factor Is also present In mark tin= and has been In to be particularly patent ~ stim,Qatir~ Me forTnati~ of new blood v--c~ls. An vitro, fibrobl~t gush factor has effects ~ oelis of the oodles lu~n and it stigmata gra ~ he cells to divide. In addition, the factor see as to support ache growth of blood vents ~ the corpus luteum. Its effects an synthesis of estrogen and progesterone are probably inhibitory. There are many other peptides that have been found in the ovary, but research on these substances is sparse. Many of them have not even been purified enough to allow analysis of their structure and examination of the Or effects in . . . vitro or In VlVO. Tnis cx~nplex area of research promises to provide useful knowledge abaft regulation In the Ovary ark owed have far-reaching ir~plicati~s for both IVFEr arrt nag reproductive biology. In order to demonstrate that a particular sitar has effects In the ovary, the substance nut be shown to be secret by ovarian ~1 Is into the local extract lular Ace and to produce Ranges In the activity of neighboring cells. Sum m~ninnml criteria have been rut for sane of the factors discuss above; Aver, elucidation of ache Units involve Is critical. Especially ignorant for fixture reseal Is to determine the biochemical r~ulati~ of ~11 replication ark cliff ~ ntiation in the ovary. Also, it is ran Vassal to det ~ how some of these factors are produced by the cells. For example, a ~ there large precursor molecules produced that are modified inside the cells for specific needs? The interaction of these factors with known hormones is also important and may be of special application evaluating the effects of superovulation. Strategies to block the activity of these factors could help~to determine if any of them are absolutely rYyy~=c~ry to normal ovarian function as this is entirely unXnewn. Finally, it is of utmost importance to relate the findings from in vitro studies to what is actually occurring in the natural state. AE~;sment of Gametes t63 There are many reasons why [VFET techniques in human clinical practice do not result ~ ongoing pregnancies one hundred percent of the time. Certainly, some of the failure rate is attributable to such things as the varied skill levels of the practitioners, the physiological problems of the infertile e male or female, or the difficulty m synchronizing the stage of the embryo with the stage of the uterine wail. Nevertheless, a significant contribution to the failure of [VFET derives Of-. poorly understood physiological responses of oocytes and early — 42 —

While the analys ~ of oocytes and eggs is important, it is equally nearby to analyze the processes that cause fertilized eggs to develop to varying stages before finally failing to develop. It has been estimated that only 10 to 15 percent of all fertilized eggs develop past a certain stage, and these failures occur at similar frequencies despite differences In ovarian stimulation or in vitro couture oonditi ~ . mus, failures are probably due to proms==== within the eggs or embryos themselves. In contrast to the analysis of oocytes, analysis of embryos is an extremely difficult endeavor. Between fertilization and implantation, a myriad of cellular and molecular changes take place In an embryo that have to be coordinated precisely and synchrcnized both spatially and temporally. Research, then' must fable upon individual stages beginning at the earliest stage of fealty ization. It has been found that 5 to 10 percent of human eggs that appear to be unfertilized actually do contain one or more sperm in the region just outside the egg membrane when viewed microscopically. At higher magnifications with electron m~crcsccQy, such oocytes exhibit an absence of binding between the sperm and the egg. Usually the membranes of the sperm form protrusions, Alec] m~villi, which seem ~ be important for span binding to an egg. IBM associated with these Ply unfertilized eggs lack this structural feature. Although the reasons for this failure in sper~/egg birdie are unarm, a Per of possible explanations are likely. For example, ~11 surface ~l~1 es, which faction as cell recognition markers, <~1d be agent. Farther forcible cause is an abnonr~ organization of specific proteins, cable filaments, that form the structural fork ~ for m~cravilli. His possibility is attractive Ruse eggs have been fat that lack crovilli except in small reprice regions of their nines. Since the abnormality has been caverned in mature, pr~vulatory o~tEs, it is not likely to be the result of culture conditions. Further, the lack of mercy li is also typical of oocytes from women with a history of failed fertilization by in vitro methods. It may well be necessary to analyze immature but fully grown oocytes from such women to determine if stimulation protocols affect the frequency of this problem. Other research to determine the molecular changes in the egg membrane necessary for fertilization would also be extremely helpful. me ~ ~ other oor~itions Piano with ~ rly failure at fertilization. In one condition, the DNA of the sperm fails to de con dense. Decondensation Is a new-= Chary prior condition to the combining of the male and female chucks. While it is possible that there is abnormal packaging of the sperm I, it is more likely that there is a problem in the egg. Animal studies have suggested that chemicals in the egg cytoplasm are responsible for causing the sperm DNA to Recondense. Cl-~rly, mare needs to be known about the process. Another' critical stage in fertilization that can go awry is the migration, juxtaposition, and fusion of the male and female pronuclei, each of which contains the respective chrorcscnal contribution to the - 44

resulting embryo. El~:tran Topic arialysis has An that failure here is Fiats with failure of the membranes between He two prmn~clei to dissolve. Before, the two pmn~clei cannot fuse. Very preliminary data Ding fluo~t IX stains have sin that, in these cases, ma; replication is incomplete or a ~ :. It is possible chat IN replication is required for membrane d;-=cnlution. research into this problem could proceed in two ways. First, analysis of fertilized eggs that have arrested development at this stage could be done to further knowledge about the mechanisms of such failure. Second, the mouse could provide an animal model with which to study this problem. It has already keen demarcated In bemuse that c~0oi~a~s, Mice specifically inhibit ~ replication, do not affect the formation of pronuclei, them m~g~tion, or their juxtaposition. Sum chemicals do, however, prevent the evolution of mE0rar~s between the pm~lei. -these results also For when ~ replication Is prevented by ether rearm, so probably are ret simply a side effect of Ache inhibiting chemicals. While this discussion has centered upon very early reps ~ ive failures, there rema ~ many other conditions that cam e the demise of early embryos. For example, human development is sometimes arrested when the embryo reaches the four-ce11 stage. This may be due to a failure in the expression of embryonic genes that results in a lack of proteins required for further development. At the 12-16 cell stage, some normally fertilized eggs develop multiple nuclei. Finally, during the first Static division of the zygote to form two cools, a chrome sore pair sometimes does not separate or disjoin. a his results in different numbers- of chromosomes in different r=11 lines of a developing embryo. These are called '~x~saics". Research into the calls-= of these problems can be of benefit to the clinical practice of IVFEr as well ~= to the understanding of human reproduction ~ general. While animal models, like the mouse, have great usefulness in this research, the Or of developmentally arrested zygotic and embryos produced by IVFET is also Cry. Such zygotes and embryos no longer have Me p~enti~ to d.evelcp into a fame but may, revere rennin information critical to a better undersbar~i~ of retractive suedes and failure. Fertilization t73 Fertilization er~passes spend and ED maturation as well as the Alex cascade of events that ours ante Me send Cares; Me egg. Although sane aspects of Mete maturation have been previously divest, thou that City infI~e fertilization rehire fury Basis. the first hurdle for an Be Is ~ admire the ability to continue T - iotas. 0~; fmn yam mice (~ than 15 days old) have bee On to be incapable of serum ~ meiosis if put into tissue couture. ~ fore, it seems that there is a grow~h-related biological process that allows for meiotic competence. Fo1 lowing the breakdown of the germinal vesicle, the chromosome pairs in the oocybe separate with emission of the first polar body and arrest at metaphase II; the polar body frequently degenerates. Resumption of meiosis is initiated by fertilization of the egg. - 45 -

Before ovulation ~ I; are staid within follicles and, as was previously dine - , it ~ tlx~t that the folli~,lar Ills ir~ibit the ode fmn matured. Many experi~;s with frog ~ muse axles have been Cone to elucidate the debar ~anis;ms that r~ula~ce maturation. Cyclic PI has been strongly implicate in maintains meiatic arrest, presumably by activating an enzyme railed protein kinase A. Eden kite A prudes Me steal ~ificatiar~ of ather proteins by adroit a plus grasp; this prowess is Leon as protein horylation. Often, proteins are active or inactive biologically, d~di~ upon Whether they are ph~rylat~ or not. In a sense then, modification of proteins }fly enzymes is one way in which calls turn m:~le~lar signals on arxt off. she proteins that participate in this mscade of reactions associated which oocyte maturation ad not krx~n, but are a subject of great r~ irk. In amition, it mast be ~ha~;ized that, in addition to cyclic Ad, ether type; of ~l~les present in the follicle are likely to participate in ~inb~ of biotic arrest. A molecule that Is likely to be involved ~ ode maturation has been isolate fen frog ~ and Is collect mutation pmr~kir~ factor (MIFF). MPF, when injected into immature mantes of many species, cause; c~ oondensation ~ bra of the nuclear membrane. Its biochemical pretties are i~tir~ since it behaves like a protein kinase ark is only activated when it has been ~ihosFhorylated. is fen Woe have a similar Stare mat has not been fully isolated. There have bed innovative experiments performs to measure this MPF-like activity. In or eriment. mature muse ooc~ wed fuse with _ _ , ~ _ _ , _ , ~_~ __ ~~ ~ ~~~ ~¢ - ~~ Are ~rl~_1 =~ _~10~ ;~ 0~ JIlEllature Orles. rile ~~ ~1 ~~ ~~ ~y~l^~'l~ 1W~ ~ ~1 ~~ ~ ~ _ ~ ~ · ~ ~ ~ ~ ~ ~ breakdown of the nuclear Crane of the denature cell. Fury ` - eriments using similar strategies- have s;hawn that the ~F-like fac~r's biological activity changes adroit to the develc~rent=1 stage of He Axle. It has also been s~sb~ that Ayes that do not Rely Biotic c~e~ may contam a Satan which blot; ~ activity of MBF. Greater ur~=s~r~ir~ of oracle Inaction and the Leaves that are directly involved in this pro; might result in improved ability to mature cedes frmn humans are Other animus in vitro. dead that tests this hypothesis might also further in vitro ornate maturation. Spenn recants di~y feel the testis carrot fertilize ems; like ems, these haploid Owls must also be mature after formation if fertilization of an aver ~ Deco Our. -this maturation takes place during sperm transit through the ~pididymis, a process that Sally tales abaft l - 14 days. Arid this time Arty Alar Charles ~ the spent, particularly on the surface of the sperm cell, Our. Ike Tunis which bring abaft these surface alterations have not yet been elucidated. Similarly, it is not ~ which of these ~ificati~s are related to the acquisition of fertilizer function by sperm. Identification of the releases use by sperm for garde inaction ~ By before the relevant minis can be armchair - . Cue way that has been ~ to examine sperm at different stages of maturation ~ the epidi~yrnis is to stain the cells with antibodies that Agonize particular sperm antigens. - — 46 -

In one Rae=, an antibody recognizes a sperm protein in the anterior region of the sperm head. The protein recognized by the antibody, termed Mat, is altered subtly during sperm maturation; prior to the proke~n~s alteration' sperm cannot fertilize eggs, whereas after its modification, fertilization can be achieved readily. It is likely that this change in the My protein represents only one example of many molecular changes in the sperm during epidi~ymal transit. Continued exam mation of individual mulches, like MM2, may reveal the mechanists by which the epidi~ymis ca As sperm maturation. This information, in turn, could be used productively in two different ways: to promote fertility by learning how to mature sperm in Vl=O, or to promote contraceptive c3esrelc~ent by preventing Sperm maturation E~pecifica~ly. After ejaculation, sperm mast urn a so, anal final, maturation phase. This press, termed c?pacitatic~n, Coors rascally In the female reproductive tract, but can be prowls experimentally for many sties by ~ ubation In vitro. Line may ration In the epidic~mis, capaci~cation Is associated with a large number of sperm alterations, the functions of which are largely unknown. But at least two changes in the sperm's surface membrane are now recognized for Ps=P~tially all mammalian sperm as a function of capacitation: changes in lipid composition and the loss of many surface-associated components. Capacitation is a prerequisite for fertilization, and these alterations apparently permit sperm to penetrate through the physical barriers that surround the egg at the time of fertilization. Cleanly, the ability to control this process would racist fertility regulation considerably. In the ideas circumstance, sperm and egg meet in the fallopian tube and fertilization takes place. However, fertilization is an exceedingly oumplex process in which the sequence of events is of the utmost importance. At the time of fertilization, the egg is surrounded by two kits of barriers. Enveloping the egg di~y is a jelly-like capering, nailed the zone pellucid, which Is surged itself by a cellular layer called the callus. the zone plays a major role during fertilization and Teeny of its Ear features have beg defined. gore are three important proteins in the muse zone ZP1, ZP2, arm ZP3. Eadh preen has spar stains attached to it and is, thus, a glycopr~e~n. me str~h~ of the ex~a~lular zone p-1lucida Is ~ of polymer; (~s pry by the Combination of two like Flexes) of ZP2 arm ZP3 for into log filaments whiff are ;s-lir~ed }fly ZP1. ZP2 arm ZP3 bath function as relxptor~; for Ann. ZP3 also induces the a ~ she n ~ brand of the ~ n to break c ~ a. ~ h ZP2 and ZP3 are chemically modified following fertilization and play important roles ~ keeping extra sperm from penetrating the egg. The genes coding for mouse ZP2 and ZP3 have been isolated. Each is a single-copy gene in the mouse genome and each is highly conserved among mammalian species. The zone genes are expressed only in oocytes where their expression is developmentally restricted to the growth phase of - 47 -

o~;is bat Irk; just prior ~ Cation. me determination of the stn~ and Me nucleic acid fix of ~ ZP2 and ZP3 Tier Is has 1~ to the prediction of We amino acid se~x of the ZP2 ark ZP3 proteins. mire the cic~1 gas, the zeta proteins can rx~ be expressed by ti~ Ol1~ cells. by ~difyir~ the clone zone ger—;, scientists will be able to produce nutant zone proteins for further anal; of how the zone proteins interact with me art as well as ~ biological function of irxlividua~ zone proteins. Antibodies have been pro Wick biro either to ZP2 or to ZP3. After Ejection into female In, ffi~e antibodies localize to We zonae pellucidae ~r~ir~ grw/i~ ooc~;. We preserve of these antibodies cache= highly effective, Icing term, but ultimately reversible Fraction. Rat sb~i~ have Stray scat ionization of fence with a syn~cheticZP] peptide (b~ on the ZPS INPUTS irxtuces the formation of ci~atir~ a~i-ZP3 antibodies that biro to eggs awl effectively preclude fertilization. Because the zone germ; are highly conserved, the development of this con~craa~ive stra~y using h~1ogous (same spriest peptize as a vaccine may have wide spread abdication. this method of conjuration is Hirable bemuse it ~d prE`re~ fertilization, not implantation, arx] ~1d avoid the dangers ark side effects of the hormones a~inist~ in birth control pills. Neverthel-==, the reversibility of ache strategy remains to be rigorously d~nrent~. me surface changes that Sperm Ego during capacitation permit them to navigate thrc,~h the cumulus ~1 is. A secretory organelle that is essentially an enzyme bomb located at the apex of the spend head must not be disrupt - , however. He Eon will use this boo' called the acre, to digest a path thresh the zone. If the across brea~-cic~wn p~turely, In stick to the cumulus Ills and cannot reach the zone surface. The ~rm's recognition site for the zone is located on its surface Irene arx] birds to the ZP3 pecan of the zone. me ZP3 lealle there triggers Sean to release its ac~a1 enzymes. Particular ~nents within the an bird to ZP2 art He a~1 enzymes, together with the Utility of the Sean cell, alleger ache sperm ~ penetrate the zone ark reach the ~ membrane. His process is remarkably similar access Fustian species. It is likely that factors few. the ED itself are not involved in fertilization up to this powt singe these erects Amour even if the zone Stains no egg, a so-called zone ghost. Lit the antibodies against zone proteins discussed abjure, antibodies di~J against specific send proteins can also inhibit fertilization. me sperm protein M42, described earlier, that is altered Purim epidi~mal maturation is one of sev~ carxtidate In proteins for this purpose. Sperm irrupted Ninth an anti+42 antibody carat fertilize eggs In vitro since the sperm are blocked four lo`;ir~ their ace ~ s. In a more natural experimental situation, injection of female Moe with the same anti-M42 antibody decreases pregnancy levels markedly. Strong evidence suggests that some forms of infertility in women may be caused by the presence of antis pens antibodies. Perhaps the continued examination — 48 —

of this experimental systemr-the induction of inferno ity with specific anti-sperm antikodies--will assist in explaining the mechanisms underlying such naturally occurring types of infertile ity. Once the sperm has penetrated through the zone, it Is with the egg surface rapidly. Membrane fusion between the two gamete= is initiated at a specific Anion, near the m~1e of the sperm head, and proceeds arc und the nail so that the entire sperm is incorporated into the egg. Once the sperm has fused with the egg, small packages curie] oortin=1 granules, which certain enzymes that modify the zone pellucida, move to the egg membrane, fuse with it, and release their contents into the space between the egg and the zone pellucids. The released enzymes modify be ZP2 protein so that abreacts In can no beer hire to ZP2. edification of zn also oars that affect; seem birding and the irrluction of an ac~;ane reaction. In brief, it seers; as though nature has devised bat Cyst to ensure that anly ore In ~ the egg. Very little is ~ n ~ i ~ tube precise biochemical events that e plan during the cortical grate reaction. In vitro experiments have shown that activated prctem kinase C inhibits fertilization by modifying ZP2 in the same way as occurs in the natural state. Protein kinase C also modifies ZP3 but it only prevents ZP3 frump inducing a complete acro some reaction. Sperm binding to ZP3 is unaffected. Other experiments have implicated other enzymes and co-factors (inositol phosphate, G proteins, and p ~ olipase C), all of which participate in cascades of biochemical reactions. Much research neons to be done before any of the actual biochemical events can be known. For example, it is not known what chemicals are red by the grannies. The precise changes in the zone proteins are not clear. Since some of these chemical cascades prc~uce changes in calcium storage and release, the role of calcium is not clear. knowledge of these events could contribute greatly to the ability to fertilize eggs in vitro. Finally, while the block to polyspermy is taking place, changes occur in the egg that allow the none and female prDnuciei to combine. When the sperm first penetrates the egg, its chromosomes are oon~ensed, held together by chromat~n-~=nriated proteins called prctamines. For the chromosomes of the male to Nadine with the female, the ohrorcEcmes must Recondense. It is thought that a chemical change in certain molecules of the prctamines is re~===ary for the chromat~n to Recondense. Such a chemical change can be induced by glutathione, which has been found in high concentration in mature eggs. Furthermore, depending upon the develcpmental state of the oocyte, glutathione can calms decondGnsation of oocyte chromat~n when applied in vitro. It is possible that the substance that cab- sperm chrcmat~n Recondensation is contained in the germinal vesicle, since sperm injected into cocytes before germinal vesicle breakdown retain their condensed chromatic. The substance cculd be a co-factor required for another reaction, could be an activator of some char cleave, aid be solely r~;ible for the effect, or card actually act as an inhibitor of a mile whose action is to prevent - 49 -

mnat~n Cation. E~r~ information AL He pros may Shea stogies; with whit IVES ~~d ~ I. To Prize, Be ~ of esrenb; for gamete in~acticn In Arms leading ~ fer~cilization is: 5. 1. me capacitate An penetrates between the Stylus Ells. 2. me a~ntact sperm binds ~ ZP3 in ~ zoo pellucid. 3. ZP3 primers the To of me acrosane (the Crane reaction), causing ~1~ of digestive enzymes face me Sam head. He abreact Germ birds to ZP2 arm perxatrat - ; throb zone matrix. me sperm arm egg fuse with the event formation of two pronuclei arm restoration of ache diploid state. 6. At the time of sperm fusion with the egg, a reaction is initiated that prevents any other sports foxy entering (the block to polygamy) . Etrei~plantation De~relc~nt [8] In this section, a variety of events that oozier beaten fertilization an] implantation are I. Thea- include the special metabolic ~ i ~ eats of early e ~ryo6 arx] the first escpr ~ lion of embryonic genes. Finally, the results of experiments involving micromanipulation of animal embryos and embryo splitting will be described. The short description that follows reviews the ma m events of preimplantation develcpment in a general way. It is also offered as an explanation of terms that will be used throughout this section. the major S - 6 in preimplantation ~velc~nt occur In the fallopian tub;. He fertilized egg, or zygote, urgers Atomic c~11 divisions called cleavage. me first cleavage results ~ the formation of two cells, each of which vivid in the set cleavage to form fair cells, and so on ~ 8~1 arm 16 cell stages until there are Cat 50 or 60 cells, at which time blastlllation occurs. During He cleavage divisions, the cells are ~1 led blast arx] all the blasts are ~ulated her the zone pellucida. Blast~lation begins abaft the fourth day after fertilization, just as He developing embryo reaches the uterus. At blash~lation, cavities form in the call new arm the calls s ~ agate into two regions, an inner r=11 Marc, from which the fen '= and some extra embryo ~ c membranes will form, and an outer oel1 layer, called the tr~phecLoderm. Coincident with these ~ s, the zone pellucida degenerates. m e blastocyst, as the embryo is called at this time, can be considered to be a polarized structure in that the inner cell mass is attached to the try layer at one side of the blastocyst. If At — 50 —

thz~ ~ bate, be blasts ~d lock like a ring of amps winch a clump of other amps sunk to one part of the inside of bat rims This polarization is inportar~ for inpla~atian later singe be pole of be blast~t ~tainir~ the inner cell n - .=c will first taxi be Minim of the ~ in pr~paratia', for i - lantatio', in sane Species (in ate, the c~;ite is true). bucolic Su~crat~ arm Pathways Realism Basses the pa by thigh cells extract fmn ~ r ~vir~nnent are synthesize the redry building blob; to form large suedes. Ibus, ~~bolimn ~ an exactingly ccaplex network of biochemical reactions bat can be viewed as ~n~;ect:ing sets of interactions. the sets overlap at key points that radiate dish set of reactions will be Are or 1~ active, ~i~ con the reds of the ~11 at be time. the radiators at he key points are enzyme proteins. In nod cycles of bioc~E'mira] reactions, cone enzyme's activity will be more informant than any other 's. Such an enzyme is called a ra~lim;ting enzyme artful is often positior.3d at the by pouts of Overlap been different pathways of metabolism. ~ orb cleper~ent In oxen, glucose is used as a heroic fuel for metabolism. There are three tire system that subserve glucose metabolism glyoolysis, the citric acid cycle, arm be dilative ;piratory chain. In glycolysis, glucose is chemically changed in a series of enzyns~=lyzed reactions to pyruvate. In the process, maladies of aderK:6ine ~criph~a~ce (ATP) are pro. ~ is be energy scx~ for vast no of biochemical reactions. The citric acid cycle Bad pynlvate, ford by glyoolysis, as a substrate for adder -dries of reactions to price carbon ctiox~cle ark maleness (N~ and FISH) that are r~i~i for a particular type of chemical reaction also ilrportarlt in biological synod;. Other pricks of ache citric acid cycle are E~r,; for the syr~es~s of Other m:,lecules. For ensemble, certain amino acids, which are the building blob; for proteins, are ford fmn the citric acid cycle. Finally, the NOSH ark FAR fmn the citric acid Cycle are \?~ ~ be Fictive ~im0~ ~~ ~ defer Liar open and form Critical flies of ATP. Gl~,cx~ is also r.~C=~ n ~ other system Cat synopsize important biological ct3emi~al s. Huh of these Sys—as i~c with be glycolytic pathway. The first reaction of glycolysis is the conversion of glum End glu~6-E~ate (~6-P) by the enzyme, hexokinas~. Ibe - 6-P produced can be divert" Len wary to ~ter the pent~p~o~ate path or to be Ural to Synthesize gly~en. In the pent - pate path, - 6-P is Averts to art kirk of spar by series of Practice l, in the praise, molecules of NADER are formal. NADER fashions as a hyc3r~ and Arch Air in bi~ethic — 51 —

reactions. In gly~gen synthesis, me G 6-P is Inverted to glen, which Is the way cells stem glee for later use. In Owls net ATP, for example, glycogen can be brown dam to form G 6-P, w}~ic~ can then contirnle ~ me glycol~rtic pathway. Eventually, thrum ~ citric acid cycle arm ox~dative respiratory chain, every - 6-P Delete 1~-c to the pr~uc:ti~ of 37 carp Alleles. [fir many years, glum metabolism was not st~ied in eggs am early embryos, because it was generally ~ mat Results of sties frc0 other cells ~d also ably to eggs. With the advent of tissue ire, it became al—reYc that eggs am early Abyss firm ~t~ nutrients in their Allege media, whim ~ to violate~isass~ ion. Specifically, iLwas~n that, for2~cell Norse mayor, glum alone ~1d not pie survival in culture. Rather, lactate was critical for these ~rbryos. lactate is form fmn pyruvate men there is little In in He tissue. E~ised muscles, for example, pace a great ~1 of lactate If pyruvate. lactate can also be converted to pyr~vate, whim may then be lace in the citric acid Cycle. mus, the finding of a lactate r~uir~t of these erdbryo6 s~3gest~1 that certain pa ~ ays of meta}'olimn were restricts, especially the glycolytic pathway. It was later found that maturing oocytes and embryos up to the 8-cell stage needed pyruvate and other citric acid cycle precursors to survive. While the exact requirements have been shown to differ from one animal species to another, it is inberesting that pyravate is often used in culture media for human and monkey eggs and early embryos despite the lack of experimental data to justify its Ace. The nutrient studies described above ~ not the only evidence that metabolism is restricted in oocytes and early embryos. Morphological studies have demonstrated that the ~ntrar=1lular organelles in which the oxidative respiratory chain takes place are not the same in early Ills - and embryos of 8 or more Owl is. These organelles are called ~ tochon~ria and are small membrane ~ packages of enzymes and ckher molecules. Inside each m~tochondrion is a complex labyrinth of membranes called cristae. It has been known for a long time that the arrangement of the cristae differ among ~1 types deserting on the function of the cell and the metabolic s bate of the cell. Studies of eggs and early embryos showed a pattern of cristae that include a concentric and transverse arrangement. However, The concentric cristae disappeared by the 8-cel1 Wage in mast specific. This change in structure correlated well with an observed Increase ~ oxygen oonsumpkion by these 8-cel1 embryos. Other studies have explored which substrate were taken up byes. These studies also showed that Leggy and early embryos took up pyruvate rather than glucose until they reached tbe 8-cel1 stage. Tb assess whether the rolls actually used the pyruvate, studies were done ~ measure the carbon dioxide produced by metabolism of pyruvate compared to gillrr=P. This is done by labeling the pyruvate or glucose with radioactive tags which, if the pyruvate is metabolized to carbon dioxide, results in the formation of radioactively tagged car ban dioxide. As might be expected, most carbon dioxide was prn~uKeA flue br3akdbbn of pyruvate in carry (1 to 2 Owl) embryos. At 8 ~Is, the carbon dioxide was mostly from glucose metabolize. - 52 -

Bile He its of he shies An clear, it is important to Be same forcible ~nfa~ factors ~ these exert. Ire inportar~ of Use Is ~at, singe mx~ of he work has been ~ with aells In Allure, ~ differences In he and of ashen available in culture art if available in the natural state may have afford ache results. IN anklet of Owen available can, by itself, cause metabol; - n to be switch fed oh pathway to air. It is also important to determine if the enzymes risible for glycolysis are present, arm if glade- is being us" in a pathway other than glycolysis. llarials sties live ~ the metabolic par.- at play }fly using the strategy of starvation and refeeding of cells with the avatar of ir~t~est. It was fad that, if As starves embryos of glad at any stage, the level of - 6-P Leash drastically. Ivan refiring with glad, ~ G~6-P levels Crease In all stages also. -this sets that trarY;port of glues:" into cells and Be fires enzymatic reaction by hexokinase to fonn—UP are bath node adds different Cages of elrbryomc devel~t. in Artist, the levels of fn~ 1,6, biplane (an initiate mal~tle ford In glycolysis) do not change with starvation of glucose at any embryonic stage. E~ver, Be levels of this ~nt~iate increase Potentially with refining of glad in late embryos, but net early ones. Such data provide strong evidence that, like all Ills, the rat~limiting enzyme for glycolys~s (E~o~ofructokinase) is the one that catalyses Be formation of fructose 1,6, biph - Hate. Mbreover, the results su west that this enzyme berates at a very low level In early embryos and does not reach its normal activity level until after the 8-cel1 stage. A similar starvatior/refeeding experiment has suggested that the presence of pyruvate inhibits the formation of fructose 1,6, biphosphate from glucose and that the addition of glare=- resulted in no increase in the metabolizes of the citric acid cycle, whereas pyruvate did result In an Increase In these metabolites at all stages of embryonic development. There is much that should be known regarding the control of glycolys~s in oocytes and early embryos. It is important to continue to investigate the rate-l~miting enzymes at the intersection points between different metabolic pathways. Since the energy charge of a cell determunes to a great extent the pathway chosen, it is also necessary to consider changes in the total energy charge of oocytes as they proceed through ~ turation and embryonic development. The energy charge can be expressed as the amount of AIP molecules available. Often, it Is measured by determining the ratio of ATP to adenosine di pcsphate, ADP, which is a lower energy state form. It is well known that if the energy charge of a cell is high, the pentose-phosphate and glycogen synth es" pathways ad favored. finis Is because more energy in the form of UP Alleles is rat needed. If there are not enough AMP Ales, the eddy level is To and glycolys;~s, citric acid Ale, and Fictive Aspiratory Rains are ache favored roes. It Is Ken that' In the ~e, zygotes and early embryos have a high energy charge that begins to fall as development pus. Such a firming Is consist with the evidence that these early embryos exhibit a restricted metabolism in the glycolytic and citric acid — 53 —

pathways. It is important to know the bucolic its of c~; and Ply "T~06 Bait? these its are critical to fur maid of thee ~1 Is in all sure. Germ Expression in Early E3rbry~ic Derelc~: The determination of Then embryonic ger - ; begin to be express for Be synods of new proteins Is important In ~ creation of genetic d;~ in embryos;. Of the aerial Cation, two dint are at risk ~ , ~ ~ ~ _,= __ _ , _ _ ~ _ _ _ _ _ to prod Lichen with genetically ~~ Ad. For sure me risk is as high as one *large ~ two. With the clevelc~nt of IVY denies, it may ~ Edible to fertilize ~ fed high risk couples in vitro to ally for early diagnc,6es of genetic (l _. In theory, ~ cell from a n.,lti~lled embryo could be removed, analyzed for ~;~ and, if fad to be afford. a decision ~1d be made no to · . . . . . · . . · — Implant Bat embryo. AS an alternative, nakedly fertilized eggs <Ella be revved fin the uterus by flushing, bicg?sied, ark then return via ~ bryo transfer. For such diagnostic pr ~ es to be realized, he ~ Her, certain prerequisites are necessary. First, there must be a realistic chance of maintaining a pregnancy after embryo transfer. F~bryo biopsy may adversely affect the change of a su~=cful transfer. Second, the embryo should not be damaged by biopsy. In cattle, embryo biopsy is a relatively common technique and has been done with embryos at various stages. The optimum stage for biopsy is, nevertheless, important to determine. For example, techniques using 8-cel1 embryos involve removing the zone pellucida and removing one cell. An advantage of Vichy at this stage is that the embryo can be transferred to the mother when it is well synchronized with the state of the uterine call. There are scene disadvantages at this stage, however. These include the fact that there Is only one ~11 with which to work, ad the fact that embryos ladki~ zonae can stick together during venture or transfer, and can d~—i0D a Chic or Bra Eat, at Ad, ~ Risible ad, at worst, is fatal to the embryo. In m Cation to these problems, r ~ r ~ has suggested that zone-free embryos do not exhibit normal cleavage and often exhibit cells that vary greatly in size. In later embryonic stages, it is possible to nick the zone and slice off the 5 to 10 calls that herniate cut the nick. q his approach will likely result in a lower implantation rate, beca ~=- the optimum time for implantation would be lost. However, the embryos cculd be frozen for later implantation. Clearly, much more needs to be known about these changes and their effects. The third prerequisite for preimplantation diagnoses is to have reliable probes with which to identify the presence of genetic dic~-=rc. Ideally these probes Fecund make the identification at the level of the genome or DNA, as is Forcible with new technologies, such as Polymerase Chain Reaction techniques. In situ hybridization is a technique whereby a strand of DNA is made that matches with the strand of DAN one is looking for. Tb ~~= in sibs hybridization, however, the sequence of the gene On

question mUL=t be known. Some of the sequences for human genetic dicpacpc are known, especially the sex-linked i... The problem, however, i_ that many in situ probes are linked to the Y sex chrarosome. Thi_ causes an unacceptable rate of false negative readings. A procedure with greater potential is to look at the defective gene products/ like hemoglobins as an indication of genetic blood d*co~=-c, or like hypaxanthine phosphoribosyl tran_fer~e tHPRI) as an indication of Lesch-Nyhan Hi.=P~=P. The last prerequisite is absolutely critical, and that i that the probes most a.==-== the activity of embryonic genes nek maternal genes. Studies ~ the mouse have suggested that the -embryonic gencme turns an between the 2- and 4-cel1 stage. Before this stage, intracellular proteins are determined by RNA which was stored in the oocybe. Investigators at the Medical Research Council Unit of Mammalian Deveiapment in Landon have developed a m~croassay for the presence of HART On embryos that, applied in mousse embryos, has begun to answer some of the questions about the time of appearance of embryonic gene activity. HpRr is an enzyme that is lacking in children born with the sex-linked Lesch-Nyhan dilate. The hallmark of the Use is self-mut~ ation and such children also exhibit strange motor behavior, are often mentally retarded, and usually die by the age of 10. Using the Acre essay for HPRr, workers have found that the enzyme increases in concentration in 8-cel1 mouse embryos suggesting that the embryonic genes have become active. So, it seems fairly clear that, in the mouse, gene activity begins at this point. However, before pre~mplantaticn diagnoses can be achieved in humans, one must know the timing of human embryonic gene activity. In Great Britain, research on human embryos is allowed under the con L`vl of the Voluntary Licensing Authority. In the Cambridge University clinic, all patients are asked to donate their excess eggs and embryos for research Is. Of 300 patients, only two refuse permission for the research, an] thick two were set 1 afforded all the clinical services available. I Investigators at this clinic have thus con ~ EN a number of stNdies on human embryos. In a series of experiments, radioactive methionine was added to the couture media of cultured embryos at various stages of development. Methionine is taken up by cells and incorporated into newly synthesized proteins and so is a qualitative measure of protein synthesis. Researchers found new proteins that contained the radioactively tagged methionine were synthesized for the first time between at the 4- and 8-cel1 stage. In another experiment, when chemicals, which blocked transcription of messenger RN\, were added to the culture medium, they had no effect on unfertilized oocytes or early embryos and development pro seedy normally. However the blockade of RNA, or protein synthesist from the 4-cel1 stage caused the embryos to stop developing. While the results suggested that embryonic gene activity! which directs synthesis of new proteins, is important at the 4- to 8-cel1 transition period, the data are complicated by the fact that embryos have a tendency in vitro to stop development spontaneously at this stage. m ere is, however, further support for the idea that the embryonic genes are active at this stage. Since earlier experiments had looked at the - 55 -

pattern of proteins present in early awl late Embryos and faced that the pattern chased quip clearly, the protein patterns were Irk with and without Me blockade of I_—r I. -these results ~trsted that, Men ~ synthesis was blocked, Me ct3a~ in protein patterT1 characteristic of Me 8~11 stage and beyond did rat War, but Me pants maim in the early state. Becky tile Objective of such remark was to develop p~i~plantation diagrx~tic techniques, Me pr~e of HI was also assayed Frau hen embryos. Unlike Me fifties ~ ~ mouse, there was a large variation in EM levels an human embryos am the massive rise ~ HEM at Me 8 cell stage Characteristic of the at of gene activity in Me Muse was not son in the human. lee Masons for these diffe~xs ark for di~ies between these data arx] that of others are not clear. It Weld be that EM is synopsized by the human Embryo later than in the noose embryo, or that the Hear Frau the maternal gee - ; ~ being broken dawn as rapidly as new HI is pry so that no net impose can bee seen. It is also possible that there are simply ur ~ rained cliff ~ ; in storage, culture conditions, or cipher variables. Whatever the reasons, the application of HART assays to preimplantation diagnosis of Lesch-Nyhan Syndrome in the human has been disappointing. The situation does point out, however, the dangers of extrapolating flown animal studies and undersoores the need to conduct some research with human embryos that, one ~ ise, would be wasted. Regulative Pobential of Micromanipulated Embryos Many of the classic studies on embryonic development involved manipulation of certain parts of animal embryos and subsequent analysis of the effects of that manipulation on find development. Such manipulations included red of certain Is (limb buds, for exile, to study regenerative capacity), transplantation or exchange of parts (as in Elisha ~ ing uE—r Spinal cord regions with lower Spinal chord to see if nerve outgrowth would follow the limb buds in the normal way), and grafting of parts of one embryo to another. Many new technologies allow m~cro-manipulation of embryos at the level of Calls and, sometimes, even individual molecules. It is clear that the developmental potential of cells gradually narrows as develcpment proceeds and that embryos have remarkable capabilities to readjust to disturbance. Yet little is known about what governs the point at which readjustment can no longer occur be the pot Partial of a given embryonic region Hal been irreversibly determir.cd. Further, it is expected that that point of determination will vary according to the specific region in question and according to the species involved. It he long been thought that prior to blastulatian an embryo is totipotent, meaning that no ~11 of the developing embryo is committed to any particular develop mental fate. In other wards, the minis are - 56 -

undifferentiated and uncommitted. However, it is becoming evident that embryos of different species, even though exhibiting the same morphological state and the same number of ~Is, may differ ~ the degree of determination and potential of each nail. Such findings call into question the long-held assumptions of totipokentiality. since an embryonic ~1 1 is totipatent only if it can Revels into an entire organism, e~eri~nts con embryo Splitting can answer question r~i~ ye real potential of embryonic calls at different sees are in different secrecies. If a 2~l Encage embryo of a 1~ - ratory muse Is bisect, develc~tpr~normally. Unto the stage, individual blasts can be dish from an embryo and aggregated Inch art embryo fully. Ever, such 'related blast Erg: develop or organize into viable fetes- an there En. he findings In the rat are s;~nilar. In contrast, refit blast isolate Emu 4- and 8~11 stage embryos have been shown to be totipotent. Other experi~s have Emil the effects of bisecting Fuse embryos exactly in half. File 65 Event of half embryos sunrive f`~ the 2~l stage, anly about 45 pi survive fern later stages. me Exam at later so may not Sean that all the cells are toting; rather, it may mean that each half embryo contain the r~ui~ ~ and type of ~11 to Deplete develc~nent. Information cn Panic Lies Is derived by fen ex`~rin~s with sheep and cattle embryos. In sheep, isolated blasts meres have been shown to develop normally from embryos up to the 8-cel1 stage. However, for unknown reasons, only some blastomeres are capable of such development. One case was reported in which an 8-cel1 embryo was divided into four equal parts. Transfer of these split embryos into ewes resulted in the birth of fair lands. Sheep embryos, which have been halved, seen to survive well fen the 2 ael1 to blast stage. :Cn cattle, similar sum= has been achieved with Eatery arx] halved embryos fog. the 8 oe11 stage to the blast~yab stage. ~ to the ~se, Lip art cattle exhibit blas~ation at a later cleavage stage. Therefore, the n ~ of cells ~ her at blasb~ation for Sheep arx] cattle. It is possible that a higher total oe11 number may allow for greater flexibility embryonic adjustment to manipulations such as splitting. Beyond questions of immediate survival of micromanipulated embryos, there are other questions that have been addressed by investigators. Some studies have assessed the size of embryc6 after splitting and the birth weight of resulting offspring. While me study in mice found no differences in these mEasuremcots between control and halved embryos, others have reporter differences at various times of development. An examination was made of blasbocyst formation following 2-cel1 stage embryos that had been bisected. One half of the embryo was discarded and He other was cultured, returned to a 5~ize~ fee, or placed In an immature oviduct. Iho~;e placed into culture critic e~;:bited Ayes - 57 -

<3evelc~rent. A hider Its of the half marry=; <3evelc~ nonnally men returr~ to a yr~ized fame or an in ature adrift. Sum fistic indicate that playgirl the split Embryos in vitro sashay Rises their c~velc~nt. However, it is possible that Be User of ted half efforts wee ~r~tilnat~ in Me in viAro ~itic~ns, since it Is Ire cliffiadt to recover arm identify Grated Oryx; fmm the USA; or Writs. Be sties f~ ~ Hat viability was oorrelat~ with cell er sum that the lacer the ~11 number achieved in the first few days after bisection, He lam He can es of sur~rival. Many of He half embryos; fails after implantation, irxlicating that ~ bisection did not rat in a failure to implant. One of He most clear suggestion Frau these sties is that ~ ~11 rnmixr at blas~atic~n owlish Augur'; at a particular time, irk of the cell Amber) ~ critical art It; blastlllation results in an embryo with a 1~ Beer of inns ~11 mass Ails. A reduction in the inner ~11 mass may then cause the Denise of scan half embryos. Is idea was tats directly by ark gram of skies that use diffe~ial stains to carfare the rumba of Ells in the Bar cell mass ~ me Gal namer of ~ is. Embryos with a lax ~1 1 Beer at blas~lation hac] a lacer inner ~11 mass ratio and exhibited a lower viability than embryos winch a higher cell Trim at blas~lation. these experiments further she that maintaining the half Oryx ~ viva, even in a nor'?regnant uterus, Gas the formation of the inner cell ~c=, resulting in a higher inner ~11 nsss ratio arx] higher viability than that achiever] in vitro. In a Sony =~ the lantern effects of Kayo splitting, genetically identical embryos Ore either bisected arc] immediately plasm it foster ~thers-or were left intact art plan into foster ok. He half erdiaryos were generally To C~cful am those born had a higher rate of neonatal mortality than did the controls. However, sane of ache neonatal Mortality ought have been I the half embryos were born fern smaller liters than He controls. small litters usually result in longer potencies, which are also associated with hither neonate] Gail rate=. ~i~n of pi~er~ypic c~cteristics of the negates born revealed no significant differs= between half am] whole By n measure such as the are at eve opening or Row curves. . . . . . . . _ _ thus, by birth, sex type of regulation has taken place. the stoics taken together argue strongly for ~ Lists of crisis points for half embryos ir2cludir~blastulationarx1 implantation. F=ther stay of the Gil of regulation in mi~nanipulated embryos net Ply can ~ to arc basic Repledge of Rive biology, but can also elucidate important rats for fur splitting of embryos in different species. :[nplantati<~n to] ~plantation, one of the Nat poorly urxierstood pros in r~pmductive biology, involves a Alex interaction bets the Kayo arm the Ells of the uterus. ~ are also significant Ties differences in the pro of implantation that can be generally divided 58

into Irk types. Cane type of i—la~atian fat in rabid s ear led fusion; Was the type fat in rows is calls display:. Plantation in hens ark Mar primary is rolled intrusive and is the inportarTt to ~ for the pa of this report. we germs Mel for implantation in the hen begins with the altar of the blas~st to Me Dial wall. At this point, He alter layer of trim of the rye proliferate arx] differentiate into two types of Blast Ills. He Ills clcee to He Frye Bose ~tr~lasts and the cools ~ dint Fact with He ~530rium few with each ather to be He ~yr~tiotr~l~t. A ~ytium is a large, n~ltirn~cleated, mass of ~rtaplasm for }fly the fusion of arty strafe ~to~h~blastic cells into me. He syn~,tiatz~l~t acts to Be a path into the e~anetrial tissue ~at, ~ turn, allows the embryo to burro it He wall of He uterus. Scan Ells of the e~anetriurn are ir~ucc~ to Kilt] up large stores of nutrient Leaves, which are Ten released into He excracellular span (close ~ the erdbryo3 as the=- Ells degenerate. ~ charges in erx~anetrial cells can be visualized with a ~ of Deal Cliques ark are called the decided ~action. Eventually, the try plastic cells penetrate cheaply Oh into the uterine wall to Apace maternal blew veals. He iT~ceractions between the tr—cblast and these bloat vows results ache formation of the vascular supply of the placenta. The sty of implantation involves ache sears for and i~icification of factors conning uterine r~ivity are maternal reposition of pi. Me of them factors seem to be pried arx] relend by the His of the blas~cyst prior ~ implantation. me Bard of the biology of implantation has far-reac~i~ implications for human and nonhuman reproduction. Both in the natural reproductive process and in the practice of medically assisted caption, there is a huge gap between the Oar of sue awfully fertilized Ed ark the Faber of offspring bon,. A large part of this gap can be absented for by the 1~C of embryos at or AL the point of implantation. For Ample, in human IVY practice, 60 to 70 pennant of the eggs are fertilized, but only twenty to thirty percent of the embryos placed in the uterus result ~ an ongoing pregnancy. moreover, it has been estimate that, in couple without fertility problems and not practicing birth control, a prearm ogre; ~ e art of three Trust Holes in Nigh a fert;1izec] egg is present. In *seep art cattle, twenty to forty pen sent of the fertilized eggs do not survive and, in the pig, the Len rate is thirty to forty percent. early embryonic 1~c~ is common across species. While some early embryonic lace can be explained by factors such as heat stress, nutritional deficiencies, and genetic abnormalities, it has been proposed that much of this loss results fray three other possible candy-. First, the uterine environment is probably only narrowly Dive to implantation by an embryo. Seed, embryos may fail to signal Heir preserve and, consequently, fail to induce the nor hormonal and uterine changes nary to maintain pro. third, embryos may be rejected as a foreign body by the Therms imnn~ne sylvan. — 59 —

ye idea that the uterus ally implantation ply user praise c~xlitions aft certainly underlies the primly dice els~e this chapter relating to By between the "cynic stage are the uterine stage. Iac:ik of Prey has bee Rated to be a problem in all species family. It has bee sin that if praise By cannot be achieved, it is Gully better to transfer embryos that are Are ~ than the uterine Nit. Tnis makes sense if ane residers that, foll~ir~ ovulation, the corpus lumen ~ the ovary begins to pry pr~ter~e, fillip acts to prepare the Us for pr~.If the corms lutes degenerates, the dreg in pmg~tem~ calicmc the uterirx3 lit ~ be ~ retire In ~tr~ticxl. A hormone sated by the therm, c~horionic g~adc~tr~in, prevent; the duration of the corpus luteum and, thus, maintains the pr~e suction. It is reasonable then that an embryo that Is trarmferr~ to a uterus Are Alvarez than the embryo may not prcx3~e High *,orionic Strain to be able ~ rescue the corms lumen few Gratis. there have been att~cs~to control the state of the uterine lining through the a~ninistration- of Us hormones. Attends to Vance the uterine errcriron~nt of pigs by a~ninistration of prone have been unsu~=cful. However, became pig embryos sate estrogen which also affect; the uterine lining, emus estrogen was tried. lhis Sarah worked, but only during a narrow time wire are, if given too early, was actually toxic to the embryos. Other rearm suggests that it Is too simplistic to assume a~inist~tion of one type of hormone Hula be sufficient to control ache uterine enrima~nt. Analysis of proteins synthesized are secreted by embryos indicat - : that there are a Or of different chemicals made by ethos that can affect the ~ erus or ache corpus lubeum. It is nevertheless probable that some of the embryo-produ~c~ proteins cause change_ ~ the secretions of uterine calls that am nenP=c~ry anchor supportive of an implanting embryo. A number of prokeins-are also secreted by endometrial malls, and much research has focused upon isolation of these proteins, analysis of their function_, and ~ pping the changing patterns of protein synthesis associated with implantation. One experimental strategy has keen to late] implantation sites in mouse uterus with a ~ye, pontamine blue. this ~ye, when injected into the veins of-a pregnant mouse, cards implantation site= to be colored blue without staining the rest of the endometrium. After this labeling, investigators can then remove the uterus and maintain explants of it in culture. Using precursors for protein synthesis that have been radioactively labeled, investigators con label the new proteins synthesized and compare the patron of proteins fran ~lantati~ and non-iT~plan~tion site. -this strategy he chain that me rate of protein accumulation in implantation site; is up to forty pent greater than aver site:. =dh of the grease is accamted for by Is ~ proteins destined to be secreted Frau erx~trial ~1 is. F~ work Ire the pattern of protein synthesis freon natural i~?lantaticn sit~- to those that were ~ animally Rued to look like implantation sites. Although there was an inazease in the synthesis of some proteins by the mechanical method, some proteins were not Increased. Such a finding - 60 -

art for there being embryo preteen synods In the anetri~ at implantation silo. As will be discos" In a later part of this car, it is on that "oryx release Main factor arm prcteins bat directly affect the cells of the er~anetrium. At this point, beer, it is -fur to cxx~;i~3er saw of the proteins fat ~ be reload by erxia~trial calls. E~c~netrial pried 15 has ban imaged In home. This pried only present in the secretory phase arxl first trier of pro. me pro of this protein has bum ~ to charge ~ on the hormonal state, but so far no Fannie has ban ascribe ~ this preteen. Erxic~trial panted 14 has been fad In human and Muse er~aretria. this protein is identical In Strom to an I0F, whim ~ present in as sites of ~ body. en rapids, a pried Select utemglob~n has been shaven to be irrluc~ by pr~e and present In only prearm or In F~u~c~pr~y. Ihis preteen has a dried of fur~ic~ns incl~i~ an a~i-infla~natory action. Factors isolate freon the mouse include ppidermal girth factor arm a type of colony stimulating factor, both of which increase in rinse to progesterone. It is surmised that these, arxt probably Her, gram factors may furs tion to amtro1 the proliferation of the placenta. Studies of ~lametrial proteins ~ the pig are of special interest since this .~ cics does not e Exhibit the intrusive type of implantation. In fact, the maternal and embryonic blood supplies never come clod to each other in the pig as they do in humans and other primate=. Such a situation sets up potential problems in bringing nutrients to the developing embryo. Nutrients must be red from the endometrium and diffuse to the embryo. Proteins have been found in the pig that seem to help in this respect. For example, uteroferr~n is a p Stein that carries iron to the embryo. Another type of p Stein, which increases in response to progesterone, transports water insoluble molecules to the embryo. Thee are Called retinol-binding proteins. Two other proteins plasmin and tryps~n inhibitors protect the uterine cells Frau destruction by embryonic enzymes, and lysozyme protects against infection. In summary, many Classic of proteins are produced and secreted by endometrial cells in response to estrogen, progesterone, or embryo-pro~uced factors. While some of these p~V~ein-c serve nutritive functions in species in which the embryo either invade_ the endometrium late or not at all, other functions of these proteins are not clear. It Is probable that early embryonic In== is, in some cases, due to abnormal eXprpccian of uterine prcteinC. A poor quality environment for the embryo could result from either excessively low levels of necessary proteins or excessively high levels of proteins, which card be tactic to the embryo. TPCC meal has been ~ to eking the incite or ani~5 of ~nologi~1 rejection of embryos. The uterus Is no isola~ fmn ache inform Dyson, Medially In species In whim the maternal and fetal blood are hardly strati. An embryo is like a trar~lant~ organ, whi~h~stbe transplar~t~1to a site that is p~W~ - 61 -

fmn ~ cells or an ingressive c3r~ ~st be given. In sane species, an i~plantir~ embryo CZ`~,,~-- what locilcs very ~ like an allergic inflammatory reactic~n ~ the Atrium, Cat is, dilatation of blood vowels, proliferation of blood capillaries, aTx] fluic] ablation. ~ addition, it Is McCann that ~~l surface antigen; Able of eliciting an ire reaction on the part of the mother are eventually present on embryos. me, ~ ~ why act embryos are scat rejoin ideological ly are contesting arm important areas of rag. It has been fag Hat embryos release Stage especially interferon. that. _ ~ _, _ _ _a~ _ _ _ . . . . . . . . . . 1n our tissues, act to suppress or mxhllate Me Gil act. therefore, it is possible Mat the embryo acts to control locally the in Sense of the Her by secretiall of inflating A. Aver, Bra rearm Is ~ before sub a Knin can be established. lie Obey of iT plantation in human beings is particularly difficult because there is no in vitro model available. Such a barrier has special importance becalms= of the wide species differences that exist in regard to implantation. Findings from other species cannot be assumed to be true for human beings. However, it is possible to ~ some aspects of human implantation by using in vitro methods recently developed. -One of these methods involves the isolation of cytotrophoblast cells. It has keen found that cytotrophoblast owls placed into culture under certain conditions will proliferate and differentiate into syncytioLrophoblast. Such studies have shown conclusively that sync ytiotrophoblast is derived from cytotroph~blasts. In a m~1bNre dish, this differentiation takes place in two steps. First, the troph~blast cells aggregate. Then they fuse to Bone a syncytimn. It is likely that this pro Is mediate by -adhesion Alleles or Cams, which have important roles in ~1 infractions during cievel~nent of the rearms system arm ather tissues. me CAMs are Prague by the tr~ablast cells because blockade of pr~ceirE(ynt: s prevents aberration of the cells. It Is also known that the process depends on Lain, since it d~ not aver in Are An catkin Lain. In contrast to the aggravation step, the Panic risible for fusion of the tr~ciblast are entirely Am. me Infractions of cage tr~oblastic cells with variants compor ~ s of ex ~aoellular matr ~ have also been s ~ ied. Excra~1lul matrix is simply the Intercellular space and its oc~Qanent molecules, which are usually synthesized by the surrounding cells and secreted into the extra~1lular space. m e exact composition of the matrix varies from tissue to tissue, but it is important in the uterus since interactions between it and the blastocyst occur during implantation. For trqphoblast aggregation and membrane fusion to occur, there must be serum proteins in the culture media an4/or the dishes mast be coated with extra~lular matrix proteins. It is Thought that the serum is required bec a11=r it contains matrix proteins, (e.g., fibronect~n). If these proteins are added, the serum is no longer requited. Collagen, fibronectin, and laminin are structural proteins that are plentiful in many extra~-llular spaces. — 62 -

The importance of extra~-llular matrix was underscored by additional experiments that and endometrial explants to cc-culture the trcphoblast ~Is. This method~is more like the natural situation, singe the traphoblast differentiates into sync ytiakraphablast, as before, but now interacts with endometrial tin- m. These experiments showed that trophoblast from first trimesber-and term placentas bind to epith£1ial a-us of secretory en~ometrium. m e Otis also bind to cut surface= of the explants' areas where extra~-llular matrix was e ~ . After 24 to 48 hours, a zone of tissue recauais deve~qped~in the endonekrial explants where the sync ytiotrophoblast ~=cn~iated ~ th the.tic=~e. Mbreover, - . trophoblastic celis.-bind to and invade nests-of Burke endcoetrial gland cells. The trophoblastic cells dislodge the en~ometrial cools and penetrate beneath them in a pro-.-== resembling intrusive implantation. . These experiments suggest that the extra~1lular matrix always permits attachment and differentiation. of the sync ytictrophoblast. That fact has important implications in d;~-~^ce stabs= where the epithelium is eroded and, especially, when the lining of the fallopian tubes is eroded. Such static may cause implantation to occur ~ either 1es~= desirable sines or totally undesirable sites such as the fallopian turf=. The mechanisms for trcphoblast invasion of the endometrium are nak known. There are a variety of pr~teases (enzymes which break down proteins) that have been implicated, such as plasm mogen activator. In some strains of mice, blas*ocysts are less invasive of the endometrium apparently because they produce 1-== plasminogen activator. Cultured human trqphdblasts produce plasminogen activator (urokinase). Urokinase may degrade fibronectin and activate other enzymes (e.g., collagenase). It is possible that the action of prc teases like urokinase could be controlled by the presence of ~11 surface receptors for the enzyme, which localize its actions, and by specific plasminogen activator inhibitors,two of which are known to be generated by trc~phoblastic ~Is. This control would be important since the invasion of the endometrium must have an endpoint since embryos do not burrow all the way through the endometrial wall. It is expected that many of the biochemical interactions between the traphoblast and the endometrium Could occur through paracrine Mechanisms. Substances released by the syncytiotccphoblast and cytotrophablact could reach a high load concentration in areas of the en~ometrium. Sobstanocs known to have powerful paracrine effects in other tiC=~Fc may play a role in implantation including protein and steroid hormones. Substances red from bath the trophabl~=t and the endometrial opals, could account for the inductive and interdependent changes in both these tissues. Further research, however, Is clearly necessary to answer these questions. On the This of the above studies, investigators have proposed a working model for implantation in the human. The first step of this model holds that the troph~blast binds to specific endometrial ~1 l-adhesion molecules. After binding, the trcphabla-=t penetrates the endometrium and - 63 -

attach to the extracellular matrix by Manic involvir~ pa ark controlled bar specific ir~ibitor';. Tnis Mel, then, form an Mate starting mint for further I. Kr~l~ge of the bi~istry is particularly deficient and <xx0d be fi~r~ bir ~ a~licatior~ of technologies such as ~nocl~al antibodies arm IN pro; for particular ~ll-adhesioaa lie;. Identification of the Alum 1 ar ~i~ of placenta formation may cleterm~ne We factors; that rebate the prnteas~. Sum fit Cold be of great tt~ in ~star~ die- states like toxemia ~ which abnor~1 i~nrasi~ of trc~l~tic Owls is ~ to be a cam-. Finally, irnrestigators seo a real need for the institution of a national or ~ternaticmal r~isbry of IVEEr program to track the incidence of abr~rmal implantation and correlate thee data with the types of Ovarian stimulation petrols used and hormone lament therapy given. In this way, the discs of implantation caused by variants amps of the procures of IVAN amid be~separated fit - u those which occur naturally. — 64 —

E21INO:ESS PI ~ tactic ~ Dr. Zev me. 2. Spray of tale given by Dr. Neal First; also ~ paper by this author In fix A. 3. Spray of talk giver by Or. Robert F. William and Dr. Jerome Fortune; also see pat by these authors c A. 4. Seminary of talk given by Or. Ray H~rst~, lDr. John E\:3?ig, and Jar. Stanley Leibo. Drs. }~c~st~t's art E=ig's pal are included- In fix A. For Optional information on crycibiolc~y, see S.P. Deibo, Physiology: Elation of Aeolian Embryos. In Genetic E~gin~ring of Animus, J.W. Evans arc] A. Hollacnder (Felt.), Plen~ma fess, New York, 1985. Smnnary of talks given by Dr. William W. Wright and fir. James M. H~r~; also see pat bar these authors In fix A. 6. Gunnery of talk ~ Dr. Jonathan Van Blerkc~n; see paper by this author in Admix A. fink sully of talks by Dr. Patricia N. thing, Dr. Richard Schultz, art Dr. torrid Dean; see Mix A for papers contributed them authors. S ~ Pries of tats by Dr. John D. Biggers, Dr. Peter Braude, and Dr. Virginia Papaioannou; summary of talk by Dr. Harry M. Weitlauf, which was given ~ the fertilization section of the workshop, is combined ~ this chapter with talks included under implantation. Sac papers by Drs. Biggers and Papaioannou In Appendix A. For additional information, of Braude, P.R., Bolton, V.N., and Mbore, S., Human gene expression first coats between the fair- arxI eightball stages of pre implantation development. Nature 332:459-461, 1988; and Saute, P.R., Bolton, V.N., ark J*~n, M.H., the ~~-~ of human pry in infertility 1~. In Embryo Plead: Yes or No, Ciba Faction Sty of, G. BcxJk arX] M. Orator (FAR.) Tavis~c fess, Unit Kinsman, Ed. 63-82, 1986. 9. Chirp series of Calf given by Dr. Harry M. Wbitlauf, Dr. Jerome Strauss, arxl lDr. R. Michael Roberts. Sac pairs by Or. P0erts ark 1~5. ~tifaris, Strap-, jars} Klein In fix A. For additional detail ~ Weitlauf, H.M. and S~a~rt~n, M., Cafes In seated uterine proteins Fiats with embryo implantation in the ~se. Jaarnal of P=r~x~ti~ and Fertility. 84: 539-549, 1988. — 65 —