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INI'RAGONADAL CONTROL OF TESTIS F UNCTION William W. Wright Introduction There is growing evidence that the establishment and maintenance of fertility in the male requires a series of precise interactions between the cells in the testis (1- 4). Specific interactions have been identified between the major somatic cell types in the testis, the Sertoli cells, the Leydig cells and the peritubular mycid cells. Cellular interactions are not restricted to the somatic cells of this organ as there is substantial morphological and physiological evidence for the interaction of germ cells with one another and with the Sertoli cells. However, we know much less about the interactions involving germ cells than we know about the interactions restricted to the somatic cell types. This review will examine cellular interactions in the testis with emphasis on interactions involving cell division and differentiation. In this review, we will first describe some of the basic events of the life history of a cell. We will discuss how growth factors and hormones can affect cellular replication and differentiation. Then we will list the growth factors and hormones which have been identified in the testis. Some of these factors will be shown to potentially have pronounced affects on the replication of Leydig and Sertoli cells. Our emphasis will then shift to an examination of the regulation of spermatogenesis. Little is known about the mechanisms which regulate spermatogenesis. However, we will see that the male gamete undergoes a precisely timed set of developmental changes and that this occurs in a highly organized tissue, the seminiferous epithelium. This appreciation will lead to the conclusion that the regulation of spermatogenesis is complex and requires a number of interlocking controlling mechanisms. The last part of this review will describe research ongoing in our laboratory which is aimed at determining how Sertoli cells regulate a specific step in germ cell development. Regulation of the cell cycle and cell differentiation. The fate of a cell is regulated, in part, by its position in the cell cycle. The cell cycle is divided into four parts: [1] the mitotic phase, when cells divide, [2] GO ~ period of time between mitosis and the next round of DNA replication, [3] S phase, when DNA is synthesized and [4] G2 the period between DNA replication and mitosis (5) (Fig. 1). Cells can be diverted from the cell cycle during the G1 phase via two processes. They can enter Go in which the cells stop dividing, but remain at their current state of differentiation. Alternatively at a specific point in G1 labeled GD on figure 1, they can enter a pathway which leads to cellular differentiation (6~. — 191 —

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Growth factors and hormones regulate the cell cycle at or near the G1 phase (Fig. 13. A cell enters the cell cycle from the Go state in response to specific growth factors called competence factors, two of which are platlet derived growth factor (PDGF) and fibroblast growth factor (FGF) (7, 83. Once the cell enters the cell cycle, somatomedin C and epidermal growth factor are often required for the cells to progress through the first half of G1 Somatomedin C is sufficient to carry the cells through the second half of G1(5,9). Once a cell has entered S phase, it usually will progress through S. G2 and mitosis without any requirements for external stimulation (5~. Upon completion of mitosis the cells can go through a second cell cycle or exit the cell cycle by entering Go state. For some cells, entry into Go is inhibited by interleukin 1 (101. Cells can also exit the cell cycle by following a pathway leading to cellular differentiation. This process has been shown to be stimulated by hormones, some paracrine factors, especially SmC, and may be inhibited by the same factors, the competence factors, which promote entry of cells into the cell cycle. A recently described example of a competence factor inhibiting the differentiation of a cell is the effect of platlet derived growth factor on progenitors of oligodendrocyte in the developing optic nerve. Nobel and colleagues (11,12) demonstrated that when oligodendrocyte progenitors were placed in culture in the absence of platlet derived growth factor (a competence factor), these cells quickly ceased cell division and differentiated into oligodendrocytes. In contrast, when oligodendrocyte progenitors were cultured with platlet derived growth factor, these cells continued to divide for the same period of time that was observed In viva. After that they differentiated into oligodendrocytes. The authors concluded that PDGF stimulated the progenitor cells to progress through the maximum possible number of cell cycles for these cells. In the testis, the proliferation and differentiation of spermatogonia, may be regulated in a similar manner (see below). Growth factors and hormones in the testis. Having seen which factors generally regulate the cell cycle, it is appropriate to ask whether factors with the ability to regulate cell proliferation and differentiation are made in the testis. Table 1 demonstrates that a large number of growth factors and hormones are synthesized in the testis. It is noteworthy that included in this list are somatomedin C, an EGF-like molecule, fibroblast growth factor and interleukin 1, all of which have demonstrated effects on the cell cycle. Indeed, it is possible that the sequential changes in the synthesis of these factors may be responsible for the maturational changes in Sertoli cell and Leydig cell number and function. - 192 -

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Potential Role of Growth Factors and How cones in Repl ication and Differentiation of Leydig and Sertoli Cells. The potential significance of a number of the growth factor" and hormones to establ ishment of the stature number of functional Leydig and Sertol i cells may be seen when we consider the time course of the replication of these cells during pubertal maturation of the rat testis (Fig 27. Sertoli cells are actively replicating in the 22 day old embryo ~ 38 ~ . Subsequent to this, the labeling index of Sertoli cells with 3H-thymidine decreases linearly, indicating either that the percent of the Sertoli cells in the cell cycle decreases, or that the duration of the cell cycle increases. What ever the case, by 20 days of age, DNA synthesis by Sertoli cells has ceased. The replication of Leydig cells appears to follow a completely different time sequence than is observed with Sertoli cells. Zirkin and Ewing (39), demonstrated that Leydig cells numbers per testis did not begin to increase until 2 to 3 days after birth and then increased to only 20% of their adult numbers by 21 days. Subsequent to this, however, there was a rapid rise in Leydig cell numbers, and thus, possibly the rate of replication of these cells. By 35 days of age, Leydig cell numbers had reached 83% of adult numbers. Thus, the replication of Sertoli cells and Leydig cells appear to exhibit opposite patterns, with numbers of Leydig cells increasing rapidly only after the end of Sertoli cell proliferation. An examination of the source and biological activities of some of the growth factors and hormones in the testis lead to the hypothesis that the replication of Leydig cells and Sertoli cells are related processes. A defense of this hypothesis follows. Fibroblast growth factor, which occurs in high concentration in the testis (32) stimulates Sertoli cell replication and stimulates the synthesis of FSH receptors (40~. The stimulation of FSH receptor numbers eight be important since FSH has been shown to be mitogenic to immature Sertoli cells in culture (41). Thus, we propose that the conditions in the late fetal testis may be optimal for stimulating Sertoli cell proliferation. Since Sertoli cells synthesize Somatomedin C ~13,14) , the increased numbers of Sertoli cells might be expected to lead to an increase in the intratesticular SmC concentration. This event would stimulate the synthesis of LH receptors on the Leydig cells (42) which should cause an increase in Leydig cell numbers since LH is a mitogenic to immature Leydig cells (43~. This increased numbers of Leydig cells would partially explain the increase in beta endorphin concentration during pubertal maturation of the testis (30~. Since Sertoli cells have receptors for beta endorphins (44) and as immunoneutralization of beta endorphin in the immature testis causes an increase in DNA synthesis by the Sertoli cells (31), it follows that the Leydig cells must suppress Sertoli cell proliferation. Thus, growth in Sertoli cell and D-ydig cells numbers during prepubertal maturation of the testis may be regulated by the effect of paracrine factors originating from both cell types. If this hypothesis is true, it must represent a very precise regulatory mechanism, for at the end of this process, the numbers of Sertoli cells and Leydig cells per testis are almost identical (45~.

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A General Review of Cell-Cell Interactions in the Seminiferous Epithelium. While the location of the Sertoli cells and Leydig cells constrains them to interact via secreted factors, all basic forms of cellular interaction are apparent in the seminiferous epithelium. These interactions are: secretion of paracrine factors (le. hormones and growth factors) which bind to receptors on a second cell, cell adhesion, stromal-epithelial interactions and gap junction communication. Table 1 lists the growth factors and hormones which have been identified in the testis and the source of these factors. There are only a few reports of growth factor receptors within the seminiferous epithelium. One of these reports is the observation of somatomedin C receptors on human spermatocytes and spermatids (461. It should be noted, though, that the presence of these receptors on rat germ cells has been both affirmed (47) and denied (48~. However, if germ cells do contain somatomedin C receptors and since somatomedin C has been shown to be both a progression factor in the cell cycle and to stimulate differentiated function in specific cells (please refer to figure 1), somatomedin C may play a biologically significant role in germ cell proliferation and development. Cell adhesion has been found to be a powerful regulator of cell survival and differentiation during organogenesis. In the development of the cerebellum, binding of neurons to one other via the use of nerve cell adhesion molecules, promotes the survival of these cells while cells which do not adhere to other neurons die (49~. The surviving neurons go on to form the functional cerebellum. Cell adhesion may also promote survival and differentiation of spermatogenic cells. A specific cell adhesion molecule has been identified on pachytene spermatocytes and shown to mediate adhesion of these cells to Sertoli cells in vitro (50~. There is also evidence for the presence of similar molecules on round on cell adhesion to Parvinen et al (52) in of the development of germ cells in vitro when those cells within intact segments of seminiferous tubules. Those reported that late meiotic cells would complete meiosis and , ~ bound to the Sertoli cells within the tubule. They also noted that this binding required the maintenance of the normal morphology of the Sertoli cells. spermatids (51~. The biological importance of spermatogenesis is suggested by experiments of their studies were cultured investigators early snermiocenesis when these cells remained The Sertoli cell is a highly polarized epithelial cells with numerous cytoplasmic process, which, in viva surround and make contact with the surrounding germ cells. The polarity of this cell is promoted by the basement membrane (53, 54~. This basement membrane has been shown to be synthesized in vitro by the Sertoli cell" and the peritubular myoid cells when these two cell types are in direct contact with one another (551. Finally, gap junctions have been described between Sertoli cells and shown to electrically couple these cells (56, 571. In general, gap junctions allow for the direct passage of ions and cyclic nucleotides between cells. This can allow for the direct stimulation of the function of one cell by another (58~. _ ~ 9 4 —

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Functional Interaction of Germ Cells and Sertoli Cells. The above review indicates that Sertoli cells have mechanisms which allow them to interact with surrounding germ cells. It is therefore pertinent to ask whether such interactions have an impact on the function of either the Sertoli or the germ cells. That Sertoli cells can stimulate germ cell function is apparent from data from Rivarola et al (59, see Fig. 3~. In this study, pools of germ cells (containing predominantly pachytene spermatocytes and round spermatids) were isolated from immature rats. These germ cells were cultured alone for one day and then transferred to one of four culture conditions: Condition ~ was the culture of germ cells alone without FSH, condition 2 was the culture of germ cells alone with ESH, condition 3 was the coculture of germ cells with Sertoli cells in the absence of PSH and condition 4 was the coculture of germ cells with Sertoli cells in the Presence of FSH. After an additional 24 hours of culture, or coculture, H-thymidine was added to some calls to measure the rate of DNA synthesis by the germ cells while H-uridine was added to the other cultures to measure the rat of RNA synthesis by the germ cells. After two hours of incubation with the radionucleotides, the germ cells were collected from the dishes and RNA and DNA synthesis determined. This analysis demonstrated that germ cells cocultured with Sertoli cells exhibited substantially more RNA and DNA synthesis than did germ cells cultured alone. Additionally, this experiment demonstrated that Sertoli cells cultured with FSH had a greater effect on RNA and DNA synthesis of germ cells than did Sertoli cells cultured without FSH. As Sertoli cells can influence germ cell function, so to, germ cells can modulate Sertoli cell function. Magueresse and Jegou (51) demonstrated that the addition of a pool of germ cells to cultured Sertoli cells (obtained from 45 day old rats) stimulated androgen binding protein secretion by the Sertoli cells (Fig. 4~. They also noted that both pachytene spermatocytes and round spermatids were stimulatory to this function of Sertoli cells. In contrast, they noted that germ cells suppressed the conversion of testosterone to estradiol by Sertoli cells. Thus, it was concluded that germ cells have different affects on different Sertoli cell functions. The process of spermatogenesis and the organization of the seminiferous epithelium. While it is established that germ cells and Sertoli cells can interact, we do not know how these interactions regulate the diverse cellular events required for spermatogenesis. Neither do we know how the high dearer Off Orson i 7.A~ i an Of The - ~1 1 within the aspects of testis cell biology are necessary before we can complex and precise must be the intragonadal regulation of spermatogenesis. Thus, it is pertinent to review the basic aspects of spermatogenesis and the organization of the spermatogenic cells in the seminiferous epithelium. ~ , ~ _, ~ , ~ _ _— · _ ~ _. ~ _ ~ . ~ ~ seminiferous epithelium is achieved. An appreciation of both testis cell biology are necessary before we can appreciate how There are three basic processes in spermatogene~is. Once the stem spermatogonia have differentiated into Al spermatogonia, these - 195 -

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spermatogonia undergo 6 mitotic cell cycles. In the rat, these cycles are completed every 42 hours and yield 64 preleptotene spermatocytes, assuming there is no death of cells during this process (60). These preleptotene spermatocytes then enter the meiotic cell cycle. At the end of 19.4 days, the meiotic cells complete the first meiotic cell cycle and then rapidly traverse the second meiotic cell cycle, giving rise to haploid, round spermatids (613. For the next 22 . 1 days. these haploid cells differentiate into immature testicular spermatozoa (61~. At the end of this time, they are released into the lumen of the seminiferous tubule. Spermatogenic cells are not distributed randomly throughout the seminiferous epithelium. Rather they occur in specific cellular associations, called the stages of the cycle. In the rat, there are 14 such stages (61~. Each stage contains specific types of spermatogonia, spermatocytes and spermatids at particular phases of development. All of the germ cells within a specific segment of tubule mature synchronously. For example, in the 3.3 days that are required for a tubule to progress from stage VI to stage VIII of the cycle, the B spermatogonia complete the last mitotic cell cycle and give rise to the preleptotene spermatocytes. Additionally, the pachytene spermatocytes found in the stage VI tubule significantly increase in volume. Also, during this time the round spermatids complete the synthesis of the constituents of the acrosome. Finally, the compacted spermatids move towards the tubular lumen and then are released from the epithelium. The cycle of the seminiferous epithelium is completed every 12.9 days in the rat (61~. The continuing slow proliferation of the stem spermatogonia and the differentiation of some of these cells into Type A1 spermatogonia insures the continuous production of sperm in the fertile testis. Strategy for identifying Sertol i cell products which regulate specific events during spermatocenesis. The analysis of the process of spermatogenesis and the stages of the cycle of the seminiferous epithelium "uggests that there must be some central organizer within this epithelium. For years, it has been assumed that this organizer was the Sertoli cell. However. it has not been known how the Sertoli cell exerted its control over specific events during spermatogenesis. The purpose of the research in our laboratory has been to identify, purify and characterize secretory products from Sertoli cells which may be involved in regulating specif ic steps in spermatogenesis. We assumed that Sertoli cells products which regulated specific steps in the development of spermatogenic cells would be secreted at a time when these factors were required by those germ cells. Based on this assumption, it followed that such factors would be secreted at specific stages of the cycle of the seminiferous epithelium. This hypothesis could be tested because Dr. Martti Parvinen had developed a technique for the isolation of seminiferous tubules at discrete stages of the cycle of the seminiferous epithelium (627. Therefore, in collaboration with Dr — 19o

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Parvinen, we set out to identify secretory products which were secreted in a stage dependent manner by Sertoli cells within intact seminiferous tubules (62~. To do this, t ~ ules at each stage of the cycle were cultured in the presence of S-methionine and the proteins in the medium analyzed by two dimensional gel electrophoresis. This analysis revealed that the proteins recovered from the medium changed dramatically with progression of the cycle of the seminiferous epithelium and suggested that there were profound changes in the synthesis of Sertoli cell secretory products with progression of the cycle (637. Of particular note was an extremely heterogeneous protein or family of proteins we called Cyclic protein 2 or CP-2 which had a mean pI of 5.5 and mean molecular size of 37,000. CP-2 was the predominant protein resolved at stages V1 and Vll. Subsequent to these stages, however, the amount of CP-2 detected by 2D gel analysis decreased dramatically and the protein was undetectable by stage Xll (Fig. 5~. Thus, Cyclic protein-2 fit the criteria we had set for a molecule which might regulate specific events in spermatogenesis, for its appearance changed dramatically with progression of the cycle of the seminiferous epithelium. Therefore, CP-2 deserved further analysis and we set about to isolate the protein and generate a specific antiserum to it. Isolation Cyclic Protein-2 and Generation of an Antiserum. A four step chromatographic procedure was developed for the isolation of CP.2 from rat seminiferous tubule fluid (64~. In order to obtain sufficient protein for the generation of an antiserum, 225 ml of this fluid were committed to the purification procedure. Analysis of the purified protein by SDS gel electrophoresis demonstrated that a single protein, CP-2 had been isolated from the pool of proteins present in seminiferous tubule fluid (65, Fig. 6~. The purified protein was then used to generate an antiserum in one rabbit. Using this antiserum, we demonstrated that CP-2 was ~ single, but heterogeneous glycoprotein and that the only cell within the seminiferous epithelium that synthesized CP-2 was the Sertoli cell. Stage-specific synthesis of CP-2. The first question we posed using the antiserum was, are the changes in the accumulation of CP-2 in medium surrounding cultured tubules the result of a change in the rate of synthesis of the protein? This question was important for it was just as likely that the rate of synthesis of CP-2 was constant but that the rate of degradation of the protein in the tubules varied in a stage-specific manner. To answer this question, we measured both the in vitro rates of synthesis and secretion of CP-2 by cultured seminiferous tubules at specific stages of the cycle (see table 2~. This analysis demonstrated that there were dramatic stage-specific changes in both the rates of synthesis and secretion of CP-2. However, it was apparent that the rate of synthesis, as measured, was in excess of the apparent rate of secretion (65~. One possible explanation for this result was that all of CP-2 was secreted into the lumen of the tubule and that export of the protein out the lumen and into the surrounding culture medium required a considerable period of time. - 197 -

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To address this possibility, we examined the rate of export of CP-2 out of cultured tubule. ~ ales at stage VI and vII of the cycle were cultured for one hour with S-methionine, the tubules removed from the radioactive amino acid and the amount of radiolabeled CP-2 in the medium and in the tubules measured at varying times thereafter (651. This analysis demonstrated that all CP-2 was secreted but that at least 17 hours were required for the export of CP-2 from the tubules. This observation could be explained in part by the fact that fluid flow through the tubules is very slow; the entire volume of the fluid in segment of tubule in replaced only in 9 hours (65, 66~. The conclusion that there is a very slow fluid flow through the tubules is important to an understanding of the physiology of paracrine factors within the testis. Once a molecule is secreted into the lumen of the tubule, it will not be flushed out the testis, but rather will have the opportunity to diffuse up and down the length of the tubule, and thus come in contact with cells in tubules at many different stages of the cycle. How then could such factors exert their effect only on germ cells at specific stages of development? First, it is possible that the factors, once secreted, are degraded by proteases. In this regard, preliminary results using Western blotting techniques demonstrate that there is considerable degradation of CP-2 in viva in the seminiferous tubule (data not shown). An alternative means by which paracrine factors may have specific effects on particular types of germ cells is the expression of the receptors for these factors on specific types of those cells. Finally, it is possible that many paracrine factors are secreted into microenvironments within the seminiferous tubule and that these factors never reach the lumen of the seminiferous tubule. Summary and Conclusions : ~ _ ~ _ ~ ~ This review has stressed the general mechanisms of cell-cell interactions and has examined how these mechanisms are manifest in the testis. We have seen that changes in the secretion or concentration of specific growth factors may regulate the growth of the Leydig cell and Sertoli cell population. We have seen that all forms of cell-cell communication are active in the seminiferous epithelium and have argued that the control of spermatogenesis must be a precise but complex process . Finally, we have reviewed work in this laboratory to identify Sertoli cell products which have a specific effect on germ cell development . A protein which we have discovered, isolated and to which an antiserum has been generated is Cyclic Progein-2. The synthesis and secretion of this protein has been shown to exhibit profound stage-specific changes. We have shown that it is retained within the tubule, in part because of the slow fluid through these structures. As in the case with many other testis products, the function of CP-2 in the regulation of spermatogenesis has not been documented. However, sequencing the purified protein and a c DNA for the mRNA which encodes this protein will allow us to determine its primary structure and thus search for sequence homology between CP-2 and proteins of known function. Use of the antiserum as an immunohistochemical reagent should begin to delineate potential cellular targets for this protein. - 198 -

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REFERENCES Parvinen, N., K.R. vihko and J. Toppari, 1986. Cell interactions during the seminiferous epithet ial cycle . Int . Rev . Cytol . 104: 115-151. 2. Russell, L.D., 1980. Sertoli-Germ Cell Interrelations: A review. Gamete Research 3:179-202. 3. de Kretser, D.M. , 1987. Local Regulation of Testicular Function. Int. Rev. Cytol. 109:89-112. 4. Saez , J. M., M. H. Perrard-Sapori , P. G. Chatelain , E . Tabone and M.A.Rivarola, 1987 . J. steroid Biochem. 27: 317-329 . Pledger, W.J., 1985. Regulation of Cell Proliferation: Serum Growth factors control an ordered series of G1 events . In C. M. Venez~ale, ed. Control of Cell Growth and Proliferation, Van Norstrand Reinhold Co., New York, pp. 108-131. Scott, R.E., J.J. Wille, Jr., M.L. Wier, B.J. Hoerl and P.B. Maercklein, 1985. Carcinogenesis and defects in the integrated control of cell proliferation and differentiation. In C.M. Veneziale, ed. Control of Cell Growth and Proliferation, Van Norstrand Reinhold Co., New York, pp.132-146. 7. Stiles, C.D., W.J. Pledger, R.W. Tucker, R.G. Martin and C.D. Scher, 1980. Regulation of the Balb/c-3T3 cell cycle-effects of growth factors..J. Supremolecular Structure 13:489-499. 8. Gospodarowicz, D. N. Ferrara, L. Schweigerer and G. Neufeld, 1987. Structural characterization and biological functions of fibroblast growth factor. Endocrine Rev. 8:95-114. 9. Russell, W.E., J.J. van Wyk, and W.J. Pledger, 1984. Inhibition of the mitogenic effects of plasma by a monoclonal antibody to somatomedin C. Proc. Natl. Acad. Sci. 81:2389-2392. 10. Singh, J.P., L.D. Adams and P.D. Bonin, 1988. Mode of fibroblast growth enhancememt by human interleukin-l. J. Cell Biology 106:813-819. 11. Nobel, M., K. Murray, P. Stroobant, M.D. Waterfield and P. Riddle, 1988. Platlet-derived growth factor promotes division and motility and inhibits premature differentiation of the oligodendrocyte/type 2-astrocyte progenitor cell. Nature 333:560-562. 12. Raff, M.C., L.E. Lillien, W.D. Richardson, J.~. Burne and M.D. Nobel, 1988. Platlet-derived growth factor from astrocytes drives the clock that times oligodendrocyte development in culture. Nature 333:562-565. — 199 —

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13. Chatelain, P.G., D. Naville and J.M. Saez, 1987. Somatomedin-C/Insulin-like growth factor I-like material secreted by porcine Sertoli cells in vitro: characterization and regulation. Biochem. Biophys Res. Commun. 146:1009-1017. 14. Smith, E.P., H.E. Svoboda, J.J. van Wyk, A.L. Kier~zenbaum and L.L. Tres, 1987. Partial characterization of a somatomedin-like peptide from the medium of cultured rat Sertoli cells. Endocrinology 120:186-193. 15. Holmes, S.D., G. Spotts and R.G. Smith, 1986. Rat Sertoli cells secrete a growth factor that blocks epidermal growth factor (EGF) binding to its receptor. J. Biol. Chem. 261:4076-4080. 16. Buch, J.P., D.J. Lamb, L.I. Lipshultz and R.G. Smith, 1988. Partial characterization of a unique growth factor secreted by h~man Sertoli cells. Fertility and Sterility 49: 658-66S. 17. Rivier, C., S. Cajander, J. Vaughan, A.J.W. Hsueh and W. Vale, 1988. Age-dependent changes in physiological action, content and immostaining of inhibin in male rats. Endocrinology 123: 120-126. 18. Meunier, H.C. Rivier, R.M. Evans and W. Vale, 1988. Gonadal and extragonadal expression inhibin alpha, beta A and beta B subunits in various tissues predicts diverse function. Proc. Natl. Acad. Sci. 85:247-251. 19. Feig, L.A., A.R. Bellve, N.H. Erickson and M. Klagsbrun, 1980. Sertoli cells contain a mitogenic polypeptide. Proc. Natl. Acad. Sci. 77:4774-4778. 20. Feig, L.A., M. Elagsbrun and A.R. Bellve, 1983. Mitogenic poly- peptide of the mammalian seminiferous epithelium: biochemical characterization and partial purification. J. Cell Biol. 97: 1435-1443. 21. Bellve, A.R., and L.A. Feig. 1984. Cell proliferation in the mammalian testis: biology of the seminiferous growth factor (SGF). Recent Prog. Hormone Res. 40:531-560. 22. Kahn, S.A., 0. Soder, V. Syed, K. Gustaffason, M. Lindh and E.M. Ritzen, 1987. The rat testis produces large amounts of an interleukin-l-like factor. Int. J. of Andrology 10: 495-S03. 23. Skinner, M. K. and I. B. Fritz, 1985. Testicular peritubular cells secrete a protein under androgen control that modulates Sertoli cell function. Proc. Natl. Acad. Sci. 82:114-118. 24. Skinner. M.~. and P.M. Fetterolf and C.T. Anthony, 1988. Purification of a paracrine factor, F.Mod-S, Produced by testicular peritubular cells that modulates Sertol i cell function . J . Biol . Chem. 263: 2884-2890. - 200

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25. Ayer-leLievre c., L. Olson, T. Ebendal, F. Hallbook and H. Persson, 1988. Nerve growth factor mRNA and protein in the testis and epididymis of mouse and rat. Proc. Natl. Acad. Sci. 85:2628-2632. 27. de Rocij, D.G., D. Lok and D. Weenk, 1985. Feedback regulation of the proliferation of the undifferentiated spermatogonia in the Chinese hamster by the differentiation "permatogonia. Cell. Tissue Finet. 18:71-81. 28. Clermont, Y. and A. Mauger, 1984. Existence of a spermatogonial chalone in adult irradiated testes. Cell Tissue Xinet. 12:81-99. 29. Rlinefelter, G.R. and L. L. Ewing, 1988 . Optimizing testosterone production by purified adult rat Leydig cells in vitro. In Vitro Cellular and Developmental Biology.24:545-549. 3Q. Bardin, C.W., C-L.C. Chen, P.L. Morris, I.Gerendai, C. Boitani, A.S. Liotta, A. Margioris and D.T. Krieger, 1987. Proopiomelanocortin-derived peptides in testis, ovary and tissues of reproduction. Rec. Prog. Hormone Res. 43:1-28. 31. Orth, J.M., 1986. FSH-induced Sertoli cell proliferation in the developing rat is modified by beta endorphin produced in the testis. Endocrinology 11 9 : 1 8 7 6 - 1 8 7 8 . 32. Ueno, N., A. Baird, F.Esch, N. Ling and R. Guillemin, 1987. Isolation and partial characterization of basic fibroblast growth factor from bovine testis. Mol. Cell. Endocr., 49:189-194. 33. Berry, S.A., and O.H. Pescovitz, 1988. Identification of a rat GERH-like substance and its messenger mRNA in rat testis. Endocrinology 123: 661-663. 34. Yoon, D.J., C. Sklar, and R. David, 1988. Presence of immuno- reactive corticotropin-releasing factor in rat testis. Endocrinology 122:757-761. 35. Pekary, A.E., T. Yameda, B. Sharp, S. Basin, R.S. Swerdloff, and J. Hershman, 1984. Somatostatin-14 and -28 in the male reproductive tract. Life Sci 34:939-945. 36. Morley, J.M. r N. Meyer, A.E. Pekery, S. Melmed, H.E. Carlson, J.E. Briggs and J.M. Hershman, 1980. A prolactin inhibitory factor with immunocharacteristics similar to thyrotropin releasing factor (~RH) is present in rat pituitary tumors (GH3 and W5), testicular tissue and a plant material, alfafa. Biochem. Biophys. Res. Commun. 96:47-53. 37. Bhasin, S., D. Herber, M. Peterson and R. Swerdloff, 1983. Partia1 ~solation and characterization of testicular Gn-RH like factors. Endocrinology 112: 1144-1146. - 201 -

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38. Orth, J.M., 1982. Proliferation of Sertoli cells in fetal and post natal rats: A quantitative autoradiographic study. Anat. Rec. 203:485-492. 39. Zirkin, B.R. and L.L. Ewing, 1987. Leydig cell differentiation during maturation of the rat testis: a stereological study of cell number and ultrastructure. Anatomical Record 219:157-163. 40. Jaillard, C., P.G. Chatelain and J.N Saez, 1987. In vitro regulation of pig Sertoli cell growth and function: }:ffects of 41. fibroblast growth factor and somatomedin C. Biol. Reprod. 37: 665-674 . Rich, R.A., C.W. Bardin, Gig. Gunsalus, and J.P. Mather, 1983. Age dependent secretion of androgen binding protein by cultured Sertoli cells. Endocrinology 113:2284-2293. 42. Perrard-Sapori, M-R., P.G. Chatelain, C. Jaillard and J.M. Saez, 1987.Characterization and regulation of somatomedin-C/insulin-like growth factor I(Sm-C/IGF-l) receptors on cultured pig Leydig cells. Eur. J. 8iochem. 165: 209-214 . 43. Orth, J.M., 1984. The role of FSH in controlling Sertoli cell proliferation in testes of fetal rats. Endocrinology 115:1248-1255. 44. Fabbri, A., C.H. Tsai Morris, S. Luna, F. Fraioli and M.L Dufau, 1985. Opiate receptors are present in the rat testis. Identification and localization in Sertoli cells. Endocrinology 117:2544-2546- 45. Wing, T.Y., anc A.K. Christensen, 1982. Morphometirc studies on rat seminiferous tubules. Amer. J. Anat. 165:13-25. Vannelli, B.G. , T. Barni, C. Orlando, A. Natali, M. Serio and G. C. Balboni , 1988 . Insulin-like growth facotor-1 (IGF-1) and IGF-1 receptor in human testis: An immuno2'istochemical study. Fertil ity and Sterility 49: 666-669 . 47. Tres, L.L., E.P. Smith, J.J. van Wyk and A.L. Rierazenbaum, 1986. Immunoreactive sites and accumulation of somatomedin-C in rat Sertoli-spermatogenic cell co-cultures. Exp. Cell Res. 162:33-50. 48. Oonk, R. B. and J.A. Grootegoed, 1988 . Insulin-like growth factor I (IGF-I) receptors on Sertoli cells from immature rats and age-dependent testicular binding of TGF-I and insulin. Mol. Cell Endocrinol. 55:33-43. 49. Rutishauser, A. B. Acheson, A.K. Hall, D.M. Mann and J. Sunshine, 1988. The neural cell adhesion molecule (NCAM) as a regulator of cell-cell interactions. Science 240:53-57. · 2u: -

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50. D'Agostino, and M. Stefanini, 1987. A rat spermatocyte surface protein is involved in adhesion of pachytene spermatocytes to Sertoli cells in vitro. Mol. Cell Biol. 7:1250-1255. 51. le Magueresse, B and B. Jegou, 1988. In vitro effects of germ cells on the secretory activity of Sertoli cells recovered from rats of different ages. Endocrinology 22:1672-1680. 52. Parvinen, M., W.W. Wright, D.M. Phillips, J.P. Mather, N.~. Musto and C.W. Bardin, 1983. Spermatogenesis in vitro: completion of meiosis and early spermiogenesis. Endocrinology 112:1150-1152. 53. Tung, P.S. and I.B. Fritz, 1984. Extracellular matrix promotes rat Sertoli cell histotypic expression in vitro. Viol. Reprod. 23:207-217. 54 . Hadley, M. A ., S . W. Byers , C . A . Suarez -Quian , H . K. Kleinman and M. Dym, 1985. Extracellular matrix regulates Sertoli cell differentiation, testicular cord formation, and germ cell development in vitro. J. Cell. Biology 101:1511-1512. 55. Tung, P.S. and I.B. Fritz, 1988. Morphogenetic restructuring and formation of basement membranes by Sertoli cells and testis peritubular cells in co-culture: Inhibition of the morphogenetic cascade by cyclic AMP derivatives and by blocking direct cell contact . Developmental Biology 120:139-153. 56. Eusebi, F. E. Ziparo, G. Fratamico, M.A. Russo and M. Stefanini, 1983. Intercellular communication in rat seminiferous tubules. Developmental Biology 100:249-255. 57. Gilula, N.B., D.W. Fawcett and A. Aoki, 1976. The Sertoli cell occluding j unctions and gap j unctions in mature and developing mammalian testis. Develop~nemtal Biology 50: 142-168 . 58. Lawrence, T. S ., W. H. Beers and N. B. Gilula , 1978 . Transmission of hormonal stimulation by cell-to-cell communication. Nature 272: 501-506. 59. Rivarola, M.A., P. Sanchez , and J.M. Saez , 1985 . Stimulation of ribonucleic acid and deoxyribonucleic acid synthesis in spermatogenic cells by their coculture with Sertoli cells. Endocrinology 117 :1796-1802 . 60. Huckins, C., 1971. The spermatogonial stem cell population in adult rats. Their morphology, proliferation and maturation. Anat. Rec. 169:533-558. 61. Clermont, Y., 1972. Kinetics of spermatogenesis in mammals: seminiferous epithelium cycle and spermatogonial renewal. Physiol Rev. 52:198.110. - 2~3

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62. Parvinen. M., and A. Runkonen, 1982. Endogenous steroids in rat seminiferous tubule. Comparison of different spe~matogenic stages isolated by transillumination-assi~ted microdissection. J. Androl. 3:211.220. 63.'Wright, W.W., M. Parvinen, N.A. Musto, G.L. Gunsalus, Did. Phillips, J.P. Mather and C.W. Bardin. 1983. Identification of stage.specific proteins synthesized by rat seminiferous tubules Biol. Reprod. 29:257.270. . 64. Wright, W.W. and M.~. Luzarraga, 1986. Isolation of cyclic protein-2 from rat seminiferous tubule fluid and Sertoli cell culture medium. Biol. Reprod. 35:761-772. 65. Wright, W.W., 1988. Germ Cell-Sertoli Cell Interactions: Analysis of the biosynthesis and secretion of cyclic protein-2 Developmental Biology, 130:45-56. , i - 204 .

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EGF (a) ~ `:Go ; In(erleukin I Figure 1. / \ M / ~)~SmC, EGF PDGF LO ,`~ Differentiation Hormo~s, `:J, Growth Factors \ ~ ~ S ~/(+) SmC Cell Cycle - me cell c~rcle ~d the role of.grawth factore ~d hormaDes ~ c~trolllog this cycle. Mitosis; GI-period between mitosis and DWA s~thesle; S-period of DNA synthesis G2-period between DNA synthesis Id mitosis. _ - . 3 ~ - = · 10 - - - 5 Figure 2. r — . 18 \ i 1 s~ ~ _ — ~ \ ~ ~ ~ A- ~ -30 '0 ED 80 40 80 60 ~ ~~ ~~) Replication of Sertoli cells and Leydig cells during prepUbert~l development of the rat testis. The replication of the Sertoli cells is expressed as the labeling index of these cells when they are exposed In Vivo to 3~-thym~dine. Leydig cell numbers are obtained from stereological analysis of different age testes. Data are from references 38 and 39. - 205 -

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Figure 3 P< 0~02 __ l o ._ - a o c ._ , 2 . O —. - - o o E c o ._ - ~ ~ 10 o o 0 0 ~ at: ._ o ~ O a: ~ · - . 5 E - - :E pFSH - p< 0.05 l g alone g.Sertoli 1 The effect of Sertoli cells and FSH on DNA and RNA synthesis by rat spermatogenic cells. RNA synthesis by the spermatogenic cells impressed as 3~-uridine incorporated by the germ cells during a two hour period. DNA `;ynthesi'; by the permatogenic cells is expressed as 3~-thymidine incorporated by the germ cells during a two hour period. Data are from reference S9. i ., ~ ~06 —

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Figure 4 Z 20 C) CR \ ~ 15 - o Q 10 m 5' Germ Cells: T The effect of rat ~permatogenic cells on ABP secretion by Sertoli cells. Data are expressed as the amount of ABP secreted in 24 hours per micro- gram of Sertoli cell DNA. Data are from reference 51. - 207 -

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::::: :~ ~~ ~~ ~ ~ :, ~~ . . ~~ :~:O ~ ... :::::: .. .~.~.~.~ ::::::: ~ ~ ~~ ~~ ::::::: ... : : : : :.: : : : : : ~ ~ ~ ~ ~~ ~ ~ ~ ~ i: : I: : : . a: .: - :::::::::::::: ::: ::::::::: ::::: :::::: .~ ~ ~~ Aft. ? i: :~ i:':::: :~:~::~2:: ::: :::::::::: :~ it, ~~ , :::: :::::::, $ ................. ..'.'....',', ,:::::',:,:: ::,:...'.. '...'''' . ~ ' ' ' '.,'.,'. ..... : ~,.'.',2 $ '' : .. ' ~''22'''.2.'.''.''.''.'. '.,., ' ' ,.~' '.' '.2 2.' ',:,:, '? ~ ~~ ~~ ~ ~~ In. ? ~ :~:~'~.~ ~~ ................. ............ - . —A: A:::: :: :: : : : ..... - a i' ~~: ~ .. ~~ ~ ~~ ::~ :. ~~ ~~ :.~.~: i: :~ ~~ ~ ~~ ~~ ):~ i:: ~ :: : ~~ ~~: ~:::~:~:~:~: Figure 5. Analysis by two dimensional gel electrophoresis of radiolabeled proteins released into culture medium from stage VI and XII tubules when these tubules are cultured in 35S-methionine for 17 hours. Cyclic Protein-2 is the major protein recovered from the Stage VI tubules. It has a mean pI of 5.5 and a mean molecular weight of 37,000. Note that CP-2 is not detected in the medium from stage XII tubules. Data are from reference 63. . ~76~ .~ ....... .O 6Ck . i. _. ~ ~ v`.. ~~ ~ ~~ .. ~::~0~ ~~ : :: _ :: .~ ~ ~ . . ~~ ~ ~~ ~ . ~~ ~ ~~ : ~~ _ A. a;. .... . ha_ ..... 29-~ ~ ~c' Figure 6. Purification of CP-2. CP-2 was purified fray seminiferous tubule fluid by a four step chromatographic procedure. This figure shows SDS-gel analysis of proteins in STF and in the purified preparation of CP-2. Proteins were detected by staining with silver. 208

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TABLE 1: GROWTH FACTORS. "D BORMONES IN' THE TESTIS Cell Source Factor Attributes Reference ~ . Sertoli cell Soo~atometin C Progreasion factor 13,14 EGF-like factor Progression factor 15,16 Inhibin Related to 'hormones' which Induce differentiation 17,18 Seminlferous-growth Unique to testis ~ r factor 19-21 Interleukin I Suppresses entry of~cells , ,,, into Go 22 . . . . . . . . Ferieubular myoid P-Mod S c6116 Somatomedin C Progression factor . . . .. .. . .. Stimulatory to Ser~coli cells 23, 24 14 Gene cells Nerve Growth Factor: : Unknown~n testis Chalone Suppresses replication of stem spermatogonla ; , .:. Leipzig Cells Testosterone Required for sper~aeogenesis POMC-related peptides Suppress replication of immature Sertoll cells - .25 27, 28 29 30,31 Unison Fibroblast growth ~ competence factor factor 32 GERB Bormone 33 Corticotropin- releasing horaone Rormone 34 Somatostatin- 1 4 and -28 Boneone 35 TAR Hormone 36 LIlRB lIor~one 37 - — 209 —

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TABLE 2: STAGE SPECIFIC SYNTHESIS attD SECRETION OF CP-2 (X+SEM) ,, STAGE: II HI Eta, b VIII XI Synthesis 3 1765 15005 14860 2670 119 ape x 10 /hr 596 1775 850 387 739 Secretion 624 4015 4143 702 19 ppm x 10~3/hr 444 670 372 131 22 i, ... .... 5 cm of seminiferous tubules were cultured for 1 hr (synthesis) or 16 h (secretion) and CP-2 was immunoprecipitated from tubular homogenates 6gynthesis) or media (secretion). Data are expressed as cpm of S-methionine synthesized or secreted per hour. . . - 210 -