Regulation of the Onset of M eiosis in the D eveloping Testis*

Transcription

1 ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 26, No. 5 Copyright 1996, Institute for Clinical Science, Inc. Regulation of the Onset of M eiosis in the D eveloping Testis* BERNARD GONDOS, M.D.,t ANNE GRETE BYSKOV, Dr.Sci.t and JETTE LISE HANSEN, B.Sc.t tsansum Medical Research Foundation, Santa Barbara, CA and tlaboratory of Reproductive Biology, Juliane Marie Center fo r Women, Children and Reproduction, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark ABSTRACT Meiotic division begins the process of spermatogenic maturation leading to sperm formation. In contrast to the ovary, in which meiosis is initiated early in development, onset of meiosis in the testis is delayed until the time of puberty. An assay procedure was utilized to evaluate factors responsible for the activation and prevention of meiosis in the developing rabbit testis. Testicular specimens from postnatal rabbits at different ages were used to determine if meiosis-activating substance (MAS) activity and meiosispreventing substance (MPS) activity could be demonstrated prior to the onset of spermatogenesis. An in vitro system in which undifferentiated gonads from 11.5 day old mouse fetuses are cultured in test and control media was employed. The findings indicate that MAS activity is associated with the onset of spermatogenesis and is also present shortly after birth. Activity of MPS is present throughout much of the prespermatogenic period, with a decline to reach low levels at the onset of spermatogenesis. well established for various mammalian species, but it is still uncertain how this process is regulated.1,2 Both the activation of meiosis at the appropriate time and the prevention of meiosis prior to this time require clarification. An assay procedure to assess the activation and prevention of meiosis was developed to evaluate the mechanisms involved. Utilization of this assay has indicated the presence of a m eiosisactivating substance (MAS) and a meio /96/ $00.90 Institute for Clinical Science, Inc. Introduction A key event in the onset of spermatogenesis at the time of puberty is the initiation of m eiosis. M eiotic division begins the process of spermatogenic maturation leading to sperm formation. The timing of meiotic onset has been * Address reprint requests to: Bernard Gondos, M.D., Sansum Medical Research Foundation, 2219 Bath Street, Santa Barbara, CA 93105

2 4 2 2 GONDOS, BYSKOV, AND HANSEN sis-preventing substance (MPS) in the ovary of the mouse,3 hamster,4 human,5 and cow,6 and in the m ouse,7,8 rat,9 bull,10 and human11 testis. Recently, two closely related sterols which are able to induce resumption of oocyte meiosis in the mouse were isolated from bull testis and human preovulatory follicular fluid and characterized.12 Studies in the testis have been difficult to evaluate, in part because the species investigated have short prespermatogenic periods. The rabbit was chosen for the present study because its relatively prolonged period of meiotic arrest prior to the onset of spermatogenesis enables detailed investigation of the regulation of meiotic initiation. Meiosis in the rabbit testis begins at 7 to 8 weeks postnatally.13 Prior to this time there is only limited mitotic activity. It has previously been shown by us that mitotic activity of testicular germ cells is high during late fetal development and declines sharply in the first few days after birth.14 Mitotic activity subsequently remains low until the time that spermatogenesis begins. Interestingly, preleptotene figures are present during a transitory period in the neonatal testis, but there is no further progression into m eiosis until sperm atogenesis is initiated. The present study was undertaken to determine if MAS and MPS activity could be demonstrated in the prepubertal rabbit testis. The findings indicate that both types of activity are present and can be correlated with germ cell activity prior to and during the onset of spermatogenesis. Materials and Methods The study was performed using testicular tissue from postnatal Ssc:Copenhagen white rabbits at 3 days, 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 weeks of age and adults. Specimens were minced and placed in Eagle s m odified Minimal Essential Medium (MEM), supplemented with streptomycin (50 xg/ml), penicillin (50 IU/ml), glutam ine (2mM) and heatinactivated 10 percent fetal calf serum, using 1 g of testicular tissue/100 ml medium. After 24 hours in culture, the spent medium (test medium) was centrifuged, and the supernatant stored at 20 C until use. The control medium was MEM supplem ented as previously described. To test for MAS and MPS activity, modification of a previously described in vitro system was used.15 In this test, sexually undifferentiated gonads from 11.5 day old mouse fetuses are dissected out together with adjacent mesonephric tissue. One gonad of each fetus was cultured in the test medium, and the contralateral gonad was cultured in the control medium. After 6 days in culture at 35 C in 95 percent air, and 5 percent C 0 2 in a closed water-saturated chamber, squash preparations were made of the gonadal tissue using a previously described method.16 Sex of the fetuses was determined by Barr body analysis of the amnion correlated with the appearance of the control gonads in the squash preparations, with the presence of meiosis indicating an ovary and the absence of meiosis indicating a testis. Evidence of MAS activity in the test media was indicated by the presence of meiotic cells in fetal testes. Evidence of MPS activity in the test media was indicated by the presence in fetal ovaries of fewer meiotic germ cells and germ cells in less advanced stages of meiosis than the control ovaries. Active oogenesis is normally present in the fetal mouse ovary during the period under study. Analysis of the squash preparations was performed in a blind fashion by one of the authors (B.G.). For each specimen, the presence or absence of meiotic cells was determined and, if present, the relative numbers of cells in leptotene, zygotene, pachytene, and diplotene were

3 REGULATION OF THE ONSET OF MEIOSIS IN THE DEVELOPING TESTIS scored. For evaluation of MAS activity, gonadal specimens from male fetuses were scored as follows: 0, no meiotic activity; +, presence of leptotene, occasional zygotene figures; + +, numerous leptotene, zygotene, some pachytene figures. For evaluation of MPS activity, gonadal specimens from female fetuses were scored as follows: 0, presence of leptotene, zygotene, pachytene and some d ip lo te n e fig u res; +, le p to te n e, zygotene, occasional pachytene, no diplotene figures; + +, leptotene, occasio n al zy g o ten e, no p ach yten e or diplotene figures. Results were determined for each specimen tested and the findings arranged according to the ages of the animals. Evaluation of the effects of the test media for each age group was analyzed by the chi-square method. TABLE I Meiosis-activating Substance Activity in Postnatal Rabbit Testis at Different Ages Meiotic Activity in Fetal Mouse Testis Rabbits No. of Test Control Age No. Fetuses Medium Medium 3 days wk * wk wk wk wk wk wk b 0 Adult b 0 Superscripts indicate significant difference between test and control: ap < 0.05; bp < Results The findings indicated that MAS and MPS activity could be demonstrated in the postnatal rabbit testis. The MAS activity was present in 1 week old animals and after 8 weeks of age (table I). The MAS activity was not seen at 3 days, but there were only two male mouse fetuses available for study at this age. No effect was seen using spent media from the 2nd to the 8th week. The MPS activity was present beginning at 2 weeks, with a peak at 5 weeks, followed by a decline to reach a low level at 7 to 8 weeks (table II). The activity at 5 weeks was remarkable in that most of the test ovaries showed almost complete inhibition of meiosis. Only leptotene figures were seen in 5 of the 7 specimens, com pared w ith ad van ced m eio tic prophase in the corresponding controls. Slight activity lacking statistical significance was present at 7 to 10 weeks and in adult animals. There was no effect during the first week after birth. Discussion Differentiation of male and female germ cells in the mammalian gonads following their early proliferation involves different patterns in the timing of meiosis. In the ovary, oocytes begin meiosis during fetal development, then become arrested, resuming meiosis at the time of ovulation. In the testis, there is a prolonged period of premeiotic arrest which lasts until the initiation of spermatogenesis at the time of puberty. Previous studies have suggested that a dual regulatory system involving a meiosis-inducing substance (MIS) and a meiosis-preventing substance (MPS) may be responsible for the regulation of meiosis in the ovary and testis.1,2 The former is now d esign ated as MAS (m eiosisactivating substance) to avoid confusion with the Mullerian inhibiting substance, also known as anti-mullerian hormone. The findings of the present study indicate that MAS activity in the postnatal testis is associated with the onset of sper-

4 424 GONDOS, BYSKOV, AND HANSEN TABLE II Meiosis-preventing Substance Activity in Postnatal Rabbit Testis at Different Ages Meiotic Activity in Fetal Mouse Ovarv Rabbits No. of Test Control Age No. Fetuses Medium Medium 3 days wk wk 2 4 +a wk a wk 3 7 0b ++ 6 wk wk 2 6 +a wk Adult O o +I Superscripts indicate significant difference between test and control: p < 0.05; bp < 0.01; cp < matogenesis and is also present at 1 week of age. The MPS activity is present throughout much of the prespermatogenic period with a peak effect at 5 weeks. The association of MAS activity with spermatogenesis is similar to observations in several other mammalian species.2 In the rabbit, the rise of MAS activity occurs simultaneously with the beginning of sperm atogenesis. The activity at 1 week correlates with the presence of preleptotene figures in the neonatal rabbit testis.14 Comparable time-related studies have not been performed in other species. The transient early rise of MAS in the rabbit is followed by a rise in MPS activity, which may be responsible for the failure of germ cells to progress beyond the preleptotene stage. Conversely, the onset of spermatogenesis is associated with a fall in MPS followed by a rise in MAS activity. The high level of MPS activity in the prespermatogenic rabbit testis is comparable to that seen in the fetal mouse7 and human fetal testis.11 In the rabbit, the effect was most evident between 3 and 6 weeks. It is interesting in this regard that there is active incorporation of tritiated thymidine by testicular germ cells at 3 weeks,17 which is not associated with increased mitotic or meiotic activity. Production of MPS at this time might play a role in preventing premature onset of sperm atogenesis. The low le v e l of mitotic activity throughout the prespermatogenic period suggests that MPS may not only interfere with meiosis but could also have a general effect on mitotic activity of germ cells. The precise role of MAS and MPS in regulating entry into meiosis in the rabbit testis remains to be clarified. Another factor likely to be involved is testosterone, which undergoes a gradual rise at 5 weeks of age and then a sharper rise at 8 to 9 weeks.18 Studies in rodents suggest that follicle stimulating hormone (FSH) is required for the initiation and support of spermatogenesis, most likely in association with testosterone.19 Clinical evidence from cases of precocious puberty indicates that testosterone can initiate premature onset of meiosis. Other factors which may be involved include retinoids,20 growth factors,21 and Sertoli cellgerm cell membrane interactions.22 The relative importance of MAS/MPS, hormones, and other factors remains to be investigated. Whatever the mechanisms involved, it may be that MPS activity in the prepubertal testis is important in allowing time for the proper conditions to develop, e.g., Sertoli cell maturation,23 before spermatogenesis begins. The findings of the present study suggest that the MPS:MAS ratio may be more important than either alone. While the MAS level began to rise only after spermatogenesis was initiated, it was the sharp drop in MPS from its previously high levels that preceded and possibly led to the onset of meiosis. Further studies will be needed to confirm this. Another potentially significant aspect of the high level of MPS found in the postnatal rabbit testis relates to its availa

5 REGULATION OF THE ONSET OF MEIOSIS IN THE DEVELOPING TESTIS bility for other studies. The recent isolation and synthesis of several closely related C29 sterols which induce resumption of meiosis in mouse oocytes indicate that this family of sterols is crucial to meiotic regulation.12 Comparable investigations on the basis for meiosis-preventing activity are needed. The current observations could be useful in this regard. Acknowledgment Thanks are extended to Inga Husum for technical assistance. References 1. Byskov AG. Regulation of meiosis in mammals. Ann Biol Anim Biochem Biophys 1979;19: Byskov AG. Differentiation of the mammalian embryonic gonad. Physiol Rev 1986;66: Byskov AG, Saxen L. Induction of meiosis in fetal mouse testis in vitro. Dev Biol 1976;52: O WS, Baker TG. Initiation and control of meiosis in hamster gonads in vitro. J Reprod Fertil 1976;48: W estergaard L, Byskov AG, Van Look PFA, Angell R, Aitken J, Swanton IA, et al. Meiosisinducing substances in human preovulatory follicular fluid related to time of follicle aspiration and to the potential of the oocyte to fertilize and cleave in vitro. Fertil Steril 1985;44: Westergaard L, Callesen H, Hyttel P, Greve T, Byskov AG. Meiosis inducing substance (MIS) in bovine preovulatory follicles. Zuchthyg 1985;20: Byskov AG. The meiosis inducing interaction between germ cells and rete cells in the fetal mouse gonad. Ann Biol Anim Biochem Biophys 1978;18: Dolci S, De Felici M. A study of meiosis in chim eric mouse fetal gonads. Developm ent 1990;109: Parvinen M, Byskov AG, Yding Andersen C, Grinsted J. Is the spermatogenic cycle regulated by MIS and MPS? Ann NY Acad Sci 1982; 383: Grinsted J, Byskov AG, Andreasen MP. Induction of meiosis in fetal mouse testis in vitro by rete testis tissue from pubertal mice and bulls. J Reprod Fertil 1979;56: Grinsted J, Byskov AG. Meiosis inducing and meiosis preventing substances in human male reproductive organs. Fertil Steril 1981;35: Byskov AG, Yding Andersen C, Nordholm L, Th0gersen H, Guoliang X, Wassmann O, et al. Chem ical structure of sterols that activate oocyte meiosis. Nature 1995;374: Gondos B, Renston RH, Conner LA. Ultrastructure of germ cells and Sertoli cells in the postnatal rabbit testis. Am J Anat 1973;136: Gondos B, Byskov AG. Germ cell kinetics in the neonatal rabbit testis. Cell Tiss Res 1981;215: Westergaard L, Byskov AG, Yding Andersen C, Grinsted J, McNatty KP. Is resumption of meiosis in the human preovulatory oocyte triggered by a meiosis-inducing substance (MIS) in the follicular fluid? Fertil Steril 1984;41: Randolph M, Gondos B. Evaluation of premeiotic activity in the developing rabbit testis using a modified squash technique. Gamete Res 1982;6: Sun EL, Gondos B. Proliferative activity in the rabbit testis during postnatal development. In: Byskov AG, Peters H, editors. Development and function of reproductive organs. Amsterdam: Excerpta Medica, 1981: Chubb C, Ewing L, Irby D, Desjardins C. Testicular maturation in the rabbit: Secretion of testosterone, dihydrotestosterone, 5a-androstan-3a,17fS-diol and 5a-androstan-3 3,170-dio] by perfused rabbit testes-epididym ides and spermatogenesis. Biol Reprod 1978;18: Fritz I. Sites of action of androgens and follicle stimulating hormone on cells of the seminiferous tubule. In: Litwack G, editor. Biochemical actions of hormones. New York: Academic Press, 1978: Eskild W, Hansson V. Vitamin A functions in the reproductive organs. In: Blomhoff R, editor. Vitamin A in health and disease. New York: Marcel Dekker, Inc, 1994: Skinner MK. Secretion of growth factors and other regulatory factors. In: Russell LD, Griswold MD, editors. The Sertoli cell. Clearwater FL: Cache River Press, 1993: Orth JM, Jester WF. NCAM mediates adhesion between gonocytes and Sertoli cells in cocultures from testes of neonatal rats. J Androl 1995; 16: Griswold MD. Interactions between germ cells and Sertoli cells in the testis. Biol Reprod 1995; 52:211-6.