MEIOSIS-INDUCING AND MEIOSIS-PREVENTING SUBSTANCES IN HUMAN MALE REPRODUCTIVE ORGANS*

Transcription

1 FERTILITY AND STERILITY Copyright < 1981 The American Fertility Society Vol. 35, No.2, February 1981 Printed in U.S A. MEIOSIS-INDUCING AND MEIOSIS-PREVENTING SUBSTANCES IN HUMAN MALE REPRODUCTIVE ORGANS* J0RGEN GRINSTED, M.D.t ANNE GRETE BYSKOV, PH.D., DR.SCI. The Finsen Laboratory, The Finsen Institute, 2100 Copenhagen 0, DK-Denmark The initiation of meiosis is controlled by two substances, a meiosis-inducing substance (MIS) and a meiosis-preventing substance (MPS). These have been shown to be present in reproductive organs of both sexes of different mammals. In this investigation MIS and MPS were also shown to be present in man. MIS was found in fetal and adult epididymides and testes. MPS was obtained from the testes of one fetus, which was delivered by laparotomy. MPS could not be found in the testes of another fetus, which was aborted by prostaglandin F2o. and oxytocin. No or only weak MPS activity was detectable in the adult testis. This report indicates that continued spermatogenesis might be influenced by the MIS:MPS ratio. MPS was shown not to be species-specific. MIS and MPS activity have been evaluated by qualitative scoring of the different germ cell stages present in fetal gonads after culture in used media containing MIS or MPS. Fertil Steril35:199, 1981 The initiation of the meiotic division of the germ cells in mice/' 2 hamsters,3, 4 and bulls 5 seems to be regulated by two substances opposing each other. In both sexes the onset of meiosis is triggered by a meiosis-inducing substance (MIS).1-8 In the female this occurs early in life, but in the male meiosis is not induced until puberty. The male germ cells, which are enclosed in seminiferous cords during fetal and infant life, seem to be prevented from entering meiosis by a meiosis-preventing substance (MPS).I, 2, 5 MIS has been shown to be secreted by parts of the mesonephric-derived organs, in the female the rete ovarii and in the male the rete testis and the epididymis.i-8 MPS is secreted by the testis, as shown in culture experiments. 1, 2 Several authors have suggested that the male somatic cells are Received July 21, 1980; revised and accepted October 28, *Supported by Grants and from the Danish Medical Research Council, by EURATOM Conttact BIO-DK, and by the Nordic Insulin Foundation. treprint requests: Jliirgen Grinsted, M.D., The Finsen Laboratory, The Finsen Institute, Strandboulevarden 49, 2100 Copenhagen 0, DK-Denmark. 199 involved in blocking the initiation of meiosis In this investigation we studied the existence of MIS and MPS in two parts of the reproductive system, epididymides and testes of fetal and adult human males. MATERIALS AND METHODS Human material was obtained from induced abortions and from orchidectomy of an adult human male. Abortions. The abortions were carried out for social reasons in accordance with Danish law. The fetal material in this study was derived from two fetuses of 16 weeks' (fetus a) and 19 weeks' (fetus b) gestation. The abortion offetus a was performed by a sectio parva and that offetus b by instillation of prostaglandin F2o< (Dinoprost; Upjohn, Kalamazoo, Mich.) into the amniotic fluid and by simultaneous intravenous stimulation with oxytocin (Syntocinon, Sandoz A.G., Basel, Switzerland). The testes and epididymides were isolated from the fetuses under sterile conditions and prepared for culture by cleaving the tissues into small pieces. Parts of these tissues were fixed in Bouin's solution and processed for histologic examination.

2 200 GRINSTED AND BYSKOV February 1981 Orchidectomy. The orchidectomy was performed on a 44-year-old man who had persistent testicular pain of unknown origin. All attempts to diagnose a pathologic process were negative. The epididymis was prepared for culture and histology as described above. Before culture, the testis was roughly separated into two fractions by pressing the tissue through a sieve (Cellector, 1.91-mm pore size; Bellco Glass Inc., Vineland, N. J.). The tissue above the sieve contained numerous testicular tubules (upper fraction), whereas only few tubules were present in the fraction that passed through the sieve (lower fraction). Microscopic evaluation of both the epididymis and the testis appeared to be normal. Preparation of Used Medium. Fetal and adult tissues were cultured at 35 0 C in Falcon flasks (0.02 gm of tissue/ml) in Alpha Medium (Flow Laboratories Ltd., Irvine, Scotland) with added penicillin (50 IU/ml), streptomycin (50 f.l.g/ml), glutamine (2 mm), adenosine (0.8 mg/ml), cytidine (1.02 mg/ml), guanosine (0.85 mg/ml), uridine (0.73 mg/ml), and thymidine (0.24 mg/ml). After 2 days in culture, pieces of the tissues were fixed in Bouin's solution and processed for histologic examination. Thereafter the medium and the remainder of the tissues were centrifuged at 200 x g for 10 minutes. The supernatant medium, i.e., the "used medium" was kept frozen at C until used for the test (for further details see reference 5). The Test. Fetal mouse gonads of both sexes were cultured in the used medium. The behavior of the germ cells indicated activity of MIS or MPS. Ten per cent fetal calf serum (Flow Laboratories) was added to the used media after thawing. A small amount (0.5 ml) of this mixture served for 7 days as the growth medium for undifferentiated fetal mouse gonads obtained from fetuses 11 to 12 days postcoitum. The day of the vaginal plug was considered day 1 postcoitum. The gonads were identified and, when possible, isolated from all adjacent tissue, including the mesonephros, the Wolffian ducts, and the Mullerian ducts. One gonad from each fetus was cultured in the used medium. The other served as a control and was cultured in freshly prepared Alpha Medium containing 10% fetal calf serum, i.e., "normal medium." Since the age of the fetuses was critical for sex differentiation and induction of meiosis, the developmental stage of each litter was judged according to size and form of the hind limb. 13 Evaluation. The sex of the mouse gonads could not be identified on the day when the cultures were set up (i.e., undifferentiated gonads). The sex was determined later by histologic examination of the gonad cultured in the normal medium. The gonad was judged to be a testis by the presence of seminiferous cords and germ cells that had not entered meiosis. If seminiferous cords were absent and all of the germ cells had entered meiosis, the gonad was judged to be an ovary. A testis cultured in a medium with MIS contained more meiotic germ cells than did the contralateral testis cultured in normal medium. An ovary cultured in a medium with MPS contained fewer meiotic germ cells than did its control. 1, 2, 5 TABLE 1. MIS:MPS Activity of the Used Media a Mean score Used medium No. offetu8es Sex MIS MPS Control Test Epididymis Fetus a 3 Male Female 4 4 Fetus b 2 Male Female Testis Fetus a 1 Male Female Fetus b 3 Male Female 5 4 (+) Adult epididymis 11 Male Female Adult testis Upper fraction 3 Male Female (+ ) Lower fraction 3 Male Female "The mean core of the germ cell stages is given. The resultant activity of MIS and MPS in the used media is indicated by + or -. The ( + ) indicates a weak activity. Upper and lower fractions refer to a separation of the testicular tissue (see text).

3 Vol. 35, No.2 REGULATION OF MEIOSIS IN MAN 201 cell stages. The germ cells were counted in every 5th section of 12 pairs of ovaries and testes. If possible, two pairs of gonads cultured in the same used medium were counted. The effect on the distribution ofthe germ cell stages was analyzed by a contingency table analysis (x 2 test); the stages were classified into four groups: nonmeiotic cells, preleptotene, leptotene, and zygotene together with more advanced stages. The effect of the total germ cell number was analyzed by a p~ired ranksum test (Wilcoxon's). A significance level of 5% was chosen. RESULTS FIG. 1. A, Control ovary; all of the germ cells are in meiosis (zygotene and pachytene, score 5). B, Higher magnification of A. C, Contralateral ovary grown in used medium obtained from fetal testis containing MPS. No germ cells are seen (score 0). The arrow points to a pyknotic cell that may be a germ cell. D, Higher magnification of C. The clear cells seen in C are shown to be somatic cells (original magnifications: A, x 610; B, x 2500; C, x 610; D, x 2500). To evaluate the activity of the used media, the stages of the germ cells of ea~h mouse gonad were scored in the following way: 0, less than 10% of the cells were in meiosis (Le., preleptotene, leptotene, zygotene, and later stages); 1, between 10% and 50% of the germ cells had entered meiosis (leptotene and zygotene); 2, between 50% and 90% of the germ cells were in leptotene or later stages of meiosis; 3, more than 90% of the germ cells had entered meiosis, most were in leptotene and some in later stages of meiosis; 4, all germ cells had entered meiosis-half were in leptotene, the others had reached zygotene or later stages of meiosis; 5, all germ cells had entered meiosis and most were in zygotene or in later stages-only a few leptotene stages were seen. The precision ofthis qualitative scoring system was judged by differential counting of the germ Fifty-eight mouse fetuses were included in the experiment. The mean scores of the germ cell stages of the cultured test and control gonads are given in Table 1. From this score, MIS or MPS activity of the tested media was indicated by a plus or a minus. The used media obtained from all epididymides and testes contained MIS activity (Table 1). MPS activity was clearly expressed only in the ovaries grown in the used medium obtained from the testes of fetus a (Fig. 1, A to D). Counting the germ cells did not change the judgment of the used media with respect to the content of MIS (Tables 2 and 3). MPS was shown to be present in the lower fraction of the adult testes only by the differential count. The correlation between the two different evaluations of the germ cell stages indicated that the qualitative scoring was sufficient for evaluating the content of MIS and MPS in the media. The numbers of the germ cells had an insignificant tendency to increase in both sexes when the gonads were grown in used medium from epididymal tissue and seemed to decrease when grown in medium obtained from testicular tissue (all P values 0.05 to 0.15). In all testes in which meiosis was induced, the testicular cords were absent or their formation was impaired (Fig. 2, A to D). The cords of the control testes were well-defined. Histologically, the cultured human tissues appeared to be healthy with cells in mitosis, and necrotic areas were found only in the central parts of the largest pieces. The histologic features were similar to those of tissues from fetus a and fetus b. The content of testicular tubuli was higher in the upper testicular fraction than in the lower fraction. DISCUSSION In previous experiments, fetal tissues of both sexes and pubertal epididymides and testes of

4 202 GRINSTED AND BYSKOV February 1981 Used medium TABLE 2. Differential Counts of the Germ Cell Stages in the Female in Relation to the Qualitative Score of MIS:MPS Activity" Experiment Germ cell stage Ovogonia Preleptotene Leptotene Zygotene Total Epididymis Fetus a, + MIS, X2 = Control , P = 0.09 Test Control Fetus b, + MIS, X2 = Test Control , P = 0.00 Test Testis Fetus a, + MIS + MPS, Control X2 = , P = 0.00 Test Control Test Fetus b, + MIS ( + ) MPS, Control X2 = , Test P = 0.00 Control Test Adult epididymis, + MIS, Control X2 = 5.00, Test P = 0.41 Control Test Adult testis Upper fraction, + MIS ( + ) MPS, Control X2 = 4.24, P = 0.11 Test Lower fraction, + MIS, Control X2 = , Test P = 0.00 Control Test "The resultant MIS and MPS activities are given together with the source ofthe used media. If only one pair of gonads was counted, the others could not be counted quantitatively. TABLE 3. Differential Counts of the Germ Cell Stages in the Male in Relation to the Qualitative Score of MIS:MPS Activitya Used medium Experiment, Germ cell stage Prespermato Preleptotene Leptotene Zygotene Total gonia Epididymis Fetus a, + MIS, Control X2 = , P = 0.00 Test Control Fetus b, + MIS, Test Control X2 = 87.50, P = 0.00 Test Testis Fetus a, + MIS + MPS, Control X2 = ,P = 0.00 Test Fetus b, + MIS ( + ) MPS, Control X2 = , Test P = 0.00 Control Adult epididymis, + MIS, Test Control X2 = , Test P = 0.00 Control Test Adult testis Upper fraction, + MIS ( + ) MPS, Control X2 = , Test P = 0.00 Control Lower fraction, + MIS Test Control X2 = , Test P = 0.00 Control Test "The resultant MIS and MPS activities are given together with the source of the used media. If only one pair of gonads was counted, the others could not be counted quantitatively. Score Score

5 Vol. 35, No.2 REGULATION OF MEIOSIS IN MAN FIG. 2. A, Control testis with cords and germ cells in interphase (score 0). B, Higher magnification of A. C, Contralateral testis grown in used medium obtained from human adult epididymis containing MIS. All of the germ cells are in meiosis (zygotene); no cords are seen (score 5). D, Higher magnification ofc(originalmagnifications:a, x610;b, x2500;c, x610;d, x 2500). different species have been evaluated for content of MIS and MPS. It has been shown that the fetal ovary, fetal and pubertal epididymides, and the pubertal testis possess MIS activity. MPS is found only in the fetal testis and is absent or low at puberty. In addition, MIS has been shown not to be species-specific. 1-8 In this experiment, MIS was found in fetal and adult epididymides as well as in fetal and adult testes. MIS activity has not been found previously in fetal testis, but only in the epididymides of all ages examined and in the pubertal testis. Wartenberg 14 distinguished between two types of Sertoli cells, a meiosis-preventing cell (MP cell) and a meiosis-inducing cell (MI cell). These two cell types might be responsible for the intratesticular secretion of MIS and MPS. The MP cell may derive from the superficial celomic epithelium. The MI cells were suggested to originate in 203 the central gonadal blastema under the influence of the mesonephros. Whether this testicular blastema itself is derived from mesonephros remains to be clarified. In the ovary the gonadal blastema is shown to develop from the mesonephros Byskov6 showed that fetal male mouse germ cells situated outside the cords but close to the rete testis and epididymis enter meiosis. This finding suggests that a high concentration of MIS is present in the fetal testis close to the epididymis and rete testis in vivo, although earlier in vitro examinations of the fetal mouse testis indicated absence of MIS. I, 2 In the hamster the rete ovarii has been shown to possess alternate activity of MIS and MPS: on the 5th day postpartum the rete ovarii is MIS-active, but on the 14th day postpartum the rete ovarii possesses MPS activity.4 MPS activity in this investigation was clearly present only in the testis of the 16-week-old fetus (fetus a) which was aborted without medication except for the anesthesia given to the mother. In the 19-week-old fetus (fetus b), as in the adult testis, no or only very weak MPS activity was found. Grinsted et al. 5 suggested that the reduced MPS activity at puberty might determine the onset of spermatogenesis. Kula et al. 18 proposed that a reduction in the number of Sertoli cell precursors initiates meiosis in the testis. These hypotheses are in accordance with previous findings suggesting that the somatic cells are involved in blocking the initiation ofmeiosis9-12 and that low or absent MPS activity is found in pubertal testes. 5 As MIS activity but no MPS activity was found in the adult human testis, it is likely that the MIS:MPS ratio might influence continued spermatogenesis throughout the fertile period in man. Fetus b was only 3 weeks older than fetus a. This small age difference might not account for the lack of MPS activity in this fetus aborted with drugs. The drugs, most likely the prostaglandin, which induced labor could have interfered with the gonadal function of the fetus. Either prostaglandin itself and/or the extended time between instillation of the drug and abortion of the fetus could have interfered with MPS production, secretion, metabolism, or binding, without influencing MIS activity. The qualitative judgment ofthe germ cell stages shown by the differential counts to be adequate and to afford a quick and reliable evaluation ofthe content of MIS and MPS in the culture medium. Whether the preleptotene stage is included in the meiotic prophase offemales and males is discussed

6 204 GRINSTED AND BYSKOV February 1981 elsewhere. 19 The germ cell number was not significantly altered after growth in used medium from testis or epididymis, whether or not MIS or MJ;>S was present. However, fewer germ cells tended to be present in the gonads grown in testis medium and more in gonads cultured in epididymis medium. de Rooij20 has recently shown that testicular extracts decrease the production of spermatogonia in mice in vivo. This action of testicular extracts is probably localized in the spermatogonial compartment. The germ cell numbers were markedly but not significantly lowered in the fetal gonads grown in the medium obtained from the upper fraction of the adult testicular tissue as compared with controls. The germ cell number of gonads cultured in the lower fraction was comparable to that of the controls. This finding is in accordance with de Rooij's observations.20 This report indicates that MPS is not speciesspecific and supports the finding of speciesunspecificity of MIS.5 We are now studying the biochemical compositions of these two substances. Acknowledgments. Professor J. Philip, Department ofgynecology and Obstetrics, and Professor Fl. Lund, Department of Urology, The University Hospital of Copenhagen, are gratefully acknowledged for providing the human tissues. We wish to thank the technicians, Mrs. Jette Lise Hansen, Mrs. Lene Ahrenst, Miss Ros Sapawi, Mrs. Annelise Mohr, Mrs. Inga Husum, and Mr. John Post, for skillful technical assistance, and Dr. Ib Christensen for statistical evaluation. REFERENCES 1. Byskov AG, Saxen L: Induction of meiosis in fetal mouse testis in vitro. Dev BioI 52:193, Byskov AG: Regulation of initiation of meiosis in fetal gonads. Int J Androl [Suppl] 2:29, Wai-sum 0, BakerTG: Initiation and control of meiosis in hamster gonads in vitro. J Reprod Fertil 48:399, Fajer AB, Schneider JA, McCall D, Ances!G, Polakis SE: The induction of meiosis by ovaries of newborn hamsters and its relation to the action of the extra ovarian structures in the mesovarium (rete ovarii). Ann BioI Anim Biochim Biophys 19(4B):1273, 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 Fertil56:653, Byskov AG: The meiosis inducing interaction between germ cells and rete cells in the fetal mouse gonads. Ann BioI Anim Biochim Biophys 18(2B):327, Byskov AG: Does the rete ovarii act as a trigger for the onset of meiosis? Nature (Lond) 252:397, Byskov AG: Regulation of meiosis in mammals. Ann BioI Anim Biochim Biophys 19(4B):1251, Tarkowski AK: Are genetic factors controlling sexual dif ferentiation of somatic and germinal tissues of a mammalian gonad stable or labile? In Environmental Influences on Genetic Expression: Biological and Behavioral Aspects of Sexual Differentiation, Edited by N Kretchmar, DN Walcher. New York, Fogarthy International Center Proceedings No.2, 1969, p Jost A: Hormonal factors in the sex differentiation of the mammalian foetus. Philos Trans R Soc Lond [BioI Sci] 259:119, Jost A, Magre S, Cressent M, Perlman S: Sertoli cells and early testicular differentiation. In Male Fertility and Sterility, Edited by RE Mancini, L Martini. New York, Academic Press, 1974, p McLaren A, Chandley AS, Kofman-Alfaro S: A study of meiotic germ cells in the gonads offoetal mouse chimaeras. J Embryol Exp MorphoI27:515, Byskov AG, Grinsted J: Feminizing induction by mesonephros on cultured differentiating mouse gonads and ducts. Science. In press, Wartenberg H: Human testicular development and the role of the mesonephros in the origin of a dual Sertoli cell system. Andrologica 10(1):1, Byskov AG: The role of the rete ovarii in meiosis and follicle formation in the cat, mink and ferret. J Reprod Fertil 45:201, Zamboni L, BezardJ, Mauleon P: The role ofthe mesonephros in the development of the fetal sheep ovary. Ann BioI Anim Biochim Biophys 19(4B):1153, Upadhyay S, Luciani JM, Zamboni L: The role of the mesonephros in the development of indifferent gonads and ovaries of the mouse. Ann BioI Anim Biochim Biophys 19(4B):1179, Kula K, RomerTE, Wlodarczyk WP: Somatic and germinal cells' interrelationship in the rourse of seminiferous tubule maturation in man. Arch Androl 4:9, Byskov AG, Grinsted J: Production of germ cells and regulations of meiosis. In Bioregulators of Reproduction, P & S Biomedical Sciences Symposia, Edited by G Jagiello, HJ Vogel. New York, Academic Press. In press, de Rooij DG: Effect of testicular extracts on proliferation of spermatogonia in the mouse. Virchows Arch [Zellpathol] 33:67,1980