Premature ovarian insufficiency in the XO female mouse on the C57BL/6J genetic background.

In humans, all but 1% of monosomy 45.X embryos die in utero and those who reach term suffer from congenital abnormalities and infertility termed Turner's syndrome (TS). By contrast, XO female mice on various genetic backgrounds show much milder physical defects and normal fertility, diminishing their value as an animal model for studying the infertility of TS patients. In this article, we report that XO mice on the C57BL/6J (B6) genetic background showed early oocyte loss, infertility or subfertility and high embryonic lethality, suggesting that the effect of monosomy X in the female germline may be shared between mice and humans. First, we generated XO mice on either a mixed N2(C3H.B6) or B6 genetic background and compared the number of oocytes in neonatal ovaries; N2.XO females retained 45% of the number of oocytes in N2.XX females, whereas B6.XO females retained only 15% of that in B6.XX females. Second, while N2.XO females were as fertile as N2.XX females, both the frequency of delivery and the total number of pups delivered by B6.XO females were significantly lower than those by B6.XX females. Third, after mating with B6 males, both N2.XO and B6.XO females rarely produced XO pups carrying paternal X chromosomes, although a larger percentage of embryos was found to be XO before implantation. Furthermore, B6.XO females delivered 20% XO pups among female progeny after mating with C3H males. We conclude that the impact of monosomy X on female mouse fertility depends on the genetic background.

The presence of X- and Y-chromosomes in oocytes leads to impairment in the progression of the second meiotic division.

The oocytes of B6.Y(TIR) sex-reversed female mice can be fertilized but the resultant embryos die at early cleavage stages. In the present study, we examined chromosome segregation at meiotic divisions in the oocytes of XY female mice, compared to those of XX littermates. The timing and frequency of oocyte maturation in culture were comparable between the oocytes from both types of females. At the first meiotic division, the X- and Y-chromosomes segregated independently and were retained in oocytes at equal frequencies. However, more oocytes retained the correct number of chromosomes than anticipated from random segregation. The oocytes that had reached MII-stage were activated by fertilization or incubation with SrCl(2). As expected, the majority of oocytes from XX females completed the second meiotic division and reached the 2-cell stage in 24 h. By contrast, more than half of oocytes from XY females initially remained at the MII-stage while the rest precociously entered interphase after SrCl(2) activation; very few oocytes were seen at the second anaphase or telophase and they often showed impairment of sister-chromatid separation. Eventually the majority of oocytes entered interphase and formed pronuclei, but very few reached the 2-cell stage. Similar results were obtained after fertilization. We conclude that the XY chromosomal composition in oocyte leads to impairment in the progression of the second meiotic division.

Protein transduction as a strategy for evaluating important factors in mammalian sex determination and differentiation.

Protein transduction is a powerful tool to deliver biologically active protein into mammalian cells and whole animals. Transduced proteins are folded properly and can mediate their respective functions in their hosts. To examine the feasibility of applying this strategy to study the molecular events of gonadogenesis, we have studied the kinetics of protein transduction and stability of transduced protein in in vitro mouse gonad culture systems using two reporter proteins, TAT-beta-gal and beta-gal fusion proteins with and without the TAT protein transduction domain (PTD) respectively. Our results indicate that the TAT-PTD was critical and essential for protein transduction to cultured fetal gonads. The TAT-beta-gal reporter entered the cells of the gonads and mesonephros efficiently for both sexes at E11.5 to E15.5 stages examined. The delivered protein persisted in the gonads for an extended period after an initial one-hour transduction. The distribution of the reporter was relatively even in gonads and mesonephros at E11.5 stage for both sexes and at later stages in female. The transduced protein was distributed heterogeneously in male gonads after seminiferous tubule differentiation in which the amount of reporter protein was higher outside than inside the tubules. Nevertheless, we surmise that such protein delivery technique should be useful in studies designed to evaluate the sex determining or differentiating functions of various new protein factors identified by advanced differential screening strategies.

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary.

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.

Low levels of Sry transcripts cannot be the sole cause of B6-Y(TIR) sex reversal.

Sry, a single-copy gene on the Y-chromosome, triggers the fetal gonad to begin testis differentiation in mammals. On the other hand, mutation or absence of Sry results in ovary differentiation and the female phenotype. However, cases of XY sex reversal in the presence of wild-type Sry exist in mice and man. One such example is the B6-Y(TIR) mouse, whose autosomes and X-chromosome are from the C57BL/6J mouse (an inbred strain of Mus musculus molossinus), whereas the Y-chromosome is from a Mus musculus domesticus mouse originating in Tirano, Italy. The B6-Y(TIR) mouse never develops normal testes and instead develops ovaries or ovotestes in fetal life. It has been suggested that low levels of Sry transcription may account for the aberrant testis differentiation in the B6-Y(TIR) mouse. In this study, however, we observed relatively low levels of Sry transcripts not only in B6-Y(TIR) but also in B6 mice, which develop normal testes. We conclude that low dosage of Sry transcripts cannot be the sole cause of sex reversal in the B6-Y(TIR) gonad.

Follicular development and atresia in the B6.Y(TIR) sex-reversed mouse ovary.

The B6.Y(TIR) mouse fails to develop normal testes despite transcription of Sry, the primary testis-determining gene on the Y chromosome. Consequently, B6.Y(TIR) fetuses with bilateral ovaries develop into apparently normal but infertile females. This infertility can be mainly attributed to oocyte incompatibility for postfertilization development. In addition, abnormality in preovulatory follicles and rapid loss of oocytes have been observed in XY ovaries. This study examined the effect of gonadotropins on follicular development and atresia in B6.Y(TIR) prepubertal females. The results show that untreated XY females had fewer late preantral follicles and their frequency of atresia was lower. No other difference was found when they were compared with XX females. After treatment with gonadotropins for 24 h, frequency of atresia decreased in both XX and XY ovaries. After 48 h, most preovulatory follicles in XY ovaries were nonatretic, but the oocytes often were denuded. Immunocytochemical staining for connexin 43 detected punctate foci along the oocyte plasma membrane. The density of these foci changed during follicular development, which was similar in XX and XY ovaries. In conclusion, follicular development and atresia under the control of gonadotropins is not influenced by defective oocytes until the preovulatory phase.

Both nuclear and cytoplasmic components are defective in oocytes of the B6.Y(TIR) sex-reversed female mouse.

In the mammalian gonadal primordium, activation of the Sry gene on the Y chromosome initiates a cascade of genetic events leading to testicular organization whereas its absence results in ovarian differentiation. An exception occurs when the Y chromosome of Mus musculus domesticus from Tirano, Italy (Y(TIR)), is placed on the C57BL/6J (B6) genetic background. The B6.Y(TIR) progeny develop only ovaries or ovotestes despite Sry transcription in fetal life. Consequently, the XY offspring with bilateral ovaries develop into apparently normal females, but their eggs fail to develop after fertilization. Our previous studies have shown that the primary cause of infertility can be attributed to oocytes rather than their surrounding somatic cells in the XY ovary. This study attempted to identify the defects in oocytes from the B6.Y(TIR) female mouse. We examined the developmental potential of embryos from XY and XX females after exchanging their nuclear components by microsurgery following in vitro maturation and fertilization. The results suggest that both nuclear and cytoplasmic components are defective in oocytes from XY females. In the XY fetal ovary, most germ cells entered meiosis and their autosomes appeared to synapse normally while the X and Y chromosomes remained unpaired during meiotic prophase. This lack of X-Y pairing probably caused aneuploidy in some secondary oocytes following in vitro maturation. However, normal numbers of chromosomes in the rest of the secondary oocytes indicate that aneuploidy alone can not explain the nuclear defect in oocytes.

Sex-specific exons control DNA methyltransferase in mammalian germ cells.

The spermatozoon and oocyte genomes bear sex-specific methylation patterns that are established during gametogenesis and are required for the allele-specific expression of imprinted genes in somatic tissues. The mRNA for Dnmt1, the predominant maintenance and de novo DNA (cytosine-5)-methyl transferase in mammals, is present at high levels in postmitotic murine germ cells but undergoes alternative splicing of sex-specific 5' exons, which controls the production and localization of enzyme during specific stages of gametogenesis. An oocyte-specific 5' exon is associated with the production of very large amounts of active Dnmt1 protein, which is truncated at the N terminus and sequestered in the cytoplasm during the later stages of oocyte growth, while a spermatocyte-specific 5' exon interferes with translation and prevents production of Dnmt1 during the prolonged crossing-over stage of male meiosis. During the course of postnatal oogenesis, Dnmt1 is present at high levels in nuclei only in growing dictyate oocytes, a stage during which gynogenetic developmental potential is lost and biparental developmental potential is gained.

Live-borns from XX but not XY oocytes in the chimeric mouse ovary composed of B6.Y(TIR) and XX cells.

When the Y chromosome of some Mus musculus domesticus subspecies is placed onto a C57BL/6J mouse background, the XY (B6.Y(TIR)) progeny develop only ovaries or ovotestes during fetal life. The XY sex-reversed female is infertile mainly because of death of embryos during preimplantation development. In the present study, we constructed female mouse chimera composed of B6.Y(TIR) and XX BALB/c cells to determine whether developmental incompetence of XY oocytes can be attributed to defects in the oocytes themselves or in the surrounding XY somatic cells. Distribution of XY cells in chimeric ovaries was examined by in situ hybridization. Of nine XX <--> XY chimeric females born, eight were composed of B6.Y(TIR) and XX BALB/c cells with a wide range of XY contribution (16-95%), whereas one had 12% XY components of the BALB/c strain. All these females produced progeny exclusively derived from XX oocytes. By comparison, most XX <--> XX chimeric females produced progeny derived from oocytes of either strain. Two XY <--> XY males also produced progeny of both strains. In conclusion, the XY chromosomal composition in the oocyte appears to be responsible for programming its incompetence for postfertilization development. On the other hand, the presence of XY somatic cells in the chimeric ovary allows development of fertile XX oocytes.

Interactions between the oocyte and cumulus cells in the ovary of the B6.Y(TIR) sex-reversed female mouse.

The XY (B6.Y(TIR)) sex-reversed female mouse is infertile, primarily because of the early death of its embryos. We have previously determined that the XY oocyte itself, not the surrounding somatic cells, is responsible for its failure in postfertilization development. In the present study, we assessed the ability of the XY oocyte to regulate granulosa cell differentiation and functions. Oocyte-cumulus complexes (OCC) were isolated from antral follicles and were cultured in the presence of FSH and testosterone. Microsurgical removal of oocytes prevented cumulus cell expansion and suppressed estradiol production while it promoted progesterone production. Coculture with denuded oocytes from either XX or XY ovaries restored cumulus expansion and the endocrine profile observed in intact OCC. Morphology of oocytes and OCC in the preantral and antral follicles in situ as well as after isolation was compared for XX and XY ovaries. The average area of XY oocytes was smaller by 20% only at the preantral stage, whereas the zona pellucida layer was thinner by 20% at all stages. Furthermore, the XY oocyte was found to be attached to fewer cumulus cells (60% of XX control) in antral follicles and isolated OCC. In conclusion, the XY oocyte develops the normal ability of regulating granulosa cell differentiation despite its inferiority with respect to some morphometric parameters when compared to the XX oocyte.

Competence of oocytes from the B6.YDOM sex-reversed female mouse for maturation, fertilization, and embryonic development in vitro.

When the Y chromosome of a Mus musculus domesticus mouse strain is placed onto the C57BL/6J (B6) inbred genetic background, the XY (B6.YDOM) progeny develop ovaries or ovotestes, but not normal testes, during fetal life. At puberty, while some of the hermaphroditic males become fertile, none of the XY sex-reversed females produce litters. We have previously demonstrated that the eggs ovulated from the B6.YDOM ovary undergo fertilization efficiently, but cannot develop beyond the 2-cell stage either in vivo or in vitro. In the present study, we collected oocytes directly from the XY ovary, and examined their maturation, fertilization, and embryonic development in vitro. The results show that the juvenile XY ovary yielded far more fertilizable oocytes by direct collection and in vitro maturation than through in vivo ovulation, but the majority of fertilized eggs failed to reach the blastocyst stage. Hence, developmental incompetence of oocytes in the XY ovary appears to be programmed during oocyte differentiation or growth. Nonetheless, in vitro matured oocytes showed a higher potential of embryonic development than the ovulated eggs, suggesting that fertility of the XY female may be impaired by multiple factors. We hypothesized that poor responsiveness of the XY ovary to gonadotropins, as we have previously demonstrated in testosterone production, may impair follicular development or proper recruitment of oocytes for ovulation. In the present study, we compared 125I-hCG binding in XX and XY ovaries, but did not find a significant difference. Hence, LH activity appears to be impaired after receptor binding in the XY ovary. On the other hand, the pattern of 125I-hCG binding indicated that the majority of antral follicles in the XY ovary failed to undergo normal preovulatory phases, which may explain the lower developmental capacity of eggs after ovulation.

Normal onset, but prolonged expression, of Sry gene in the B6.YDOM sex-reversed mouse gonad.

When the Y chromosome of the Mus musculus domesticus mouse (Tirano, Italy) is placed onto the C57BL/6J (B6) background, the XY progeny (B6.YDOM) develop only ovaries or ovotestes in fetal life. In the present study, we examined transcription of genes known to be involved in gonadal sex differentiation in the B6.YDOM gonad. RNA was isolated from each gonad at 10 to 18 days of gestation and subjected to RT-PCR analysis. The results indicate that the onset of Sry, a putative testis-determining gene, transcription was normal, whereas its inactivation was delayed in all B6.YDOM gonads. Transcripts of Müllerian inhibiting substance, the earliest biochemical product of fetal Sertoli cells, appeared in the B6.YDOM ovotestis slightly later than in the control B6,XY gonad, whereas they were either absent or detected transiently in the B6.YDOM ovary. Transcription of 3 beta-hydroxysteroid dehydrogenase and 17 alpha-hydroxylase, essential for testosterone synthesis, were equally delayed in the B6.YDOM ovotestis and completely absent in the B6.YDOM ovary. P450 aromatase (P450arom), which converts androgens to estrogens, was transcribed in both control XX and XY gonads at the onset of morphological sexual differentiation. The overall pattern of P450arom transcription was delayed by 1 day in both B6.YDOM ovotestes and ovaries. In conclusion, the testis-determining pathway in the B6.YDOM gonad appears to be impaired downstream of Sry transcription, resulting in persistent Sry transcripts and a delay in onset of other genes involved in testicular differentiation.

Developmental arrest of fertilized eggs from the B6.YDOM sex-reversed female mouse.

When the Y chromosome of a Mus musculus domesticus mouse strain is placed onto the C57BL/6J (B6) inbred background, the XY progeny develop ovaries or ovotestes but never normal testes during fetal life. While some of the hermaphroditic males become fertile, none of the XY females produces litters. Here, we examined the fertility and development of oocytes derived from the XY female mouse. With or without preceding injection of gonadotropins, female mice were mated with normal B6 males, and their embryos were recovered at various developmental stages. In vitro fertilization was performed with the eggs recovered from the oviduct after treatment with gonadotropins. Development of embryos was examined by both light and electron microscopy. The results indicate that the oocytes released from the B6.YDOM ovary were efficiently fertilized and often initiated the first cell cleavage, but all embryos died during early preimplantation periods. Even when oocytes were fertilized in vitro, minimizing their exposure to the XY oviduct/uterus environment, most embryos died at the 1- or 2-cell stage. A few exceptional embryos reached the 4- or 8-cell stage, but abnormalities were evident in both nuclear and cytoplasmic structures of all embryos. After cleavage, neighbouring blastomeres were only loosely associated, and microvilli were abundant at the intercellular interfaces. We postulate that oocytes of the B6.YDOM female mouse become defective during XY ovarian differentiation, and, hence, fail to proceed through normal embryonic development.

Müllerian inhibiting substance production associated with loss of oocytes and testicular differentiation in the transplanted mouse XX gonadal primordium.

The mouse XX gonadal primordium develops seminiferous-like tubules after transplantation into the renal subcapsular site of the adult male or female mouse. We examined the ontogeny of Sertoli cell differentiation in XX gonadal grafts by immunocytochemical staining and organ culture bioassay for Müllerian Inhibiting Substance (MIS). During normal in situ development of the XY gonad, MIS staining was first detected in fetal Sertoli cells at 12 days of gestation (d.g.) and remained intense until 4 days postpartum (d.pp.), after which it gradually diminished with progressive testicular development. In the normal in situ XX gonad, MIS was detected in granulosa cells of growing follicles at 7 d.pp. and thereafter. When the XX gonad at 12 d.g. was grafted beneath the renal capsule, a few testicular cords composed of MIS-positive cells appeared on Day 7 post-transplantation (equivalent to 19 d.g.), much earlier than the normal appearance of MIS production in the intact XX ovary. The ovarian region containing germ cells at the meiotic prophase was unstained for MIS in the same sections. The incidence of XX gonadal grafts containing MIS-positive testicular cords and the number of such cords per gonadal graft steadily increased from Day 7 to Day 14 post-transplantation. Germ cells were absent or scarce inside the MIS-positive testicular cords. The MIS bioactivity in both control gonads and gonadal grafts coincided with the immunocytochemical staining for MIS. These results support the hypothesis that XX cells differentiate into Sertoli cells as a consequence of oocyte loss in the gonadal graft.

Endocrine differentiation of the XY sex-reversed mouse ovary during postnatal development.

When the mouse Y chromosome of Mus musculus domesticus is placed onto the C57BL/6J genetic background, half of the XY progeny develop bilateral ovaries and the female phenotype, but lack regular estrous cyclicity and lose embryos after fertilization. In the present study, we compared the endocrinological activity of XY ovaries with XX ovaries during postnatal development by measuring steroids in the incubation medium by radioimmunoassay. At 1 day postpartum (d.p.p.), production of progesterone and estradiol was significant while testosterone was undetectable in both ovaries. At 14 and 35 d.p.p., amounts of testosterone and estradiol produced by XY ovaries were half of those by XX ovaries. Production of progesterone by XY ovaries was slightly higher than XX ovaries at 14 d.p.p., but only half of that at 35 d.p.p. Addition of gonadotropins increased testosterone production by XX ovaries but not by XY ovaries at either 14 or 35 d.p.p. Progesterone production in XY ovaries at 35 d.p.p. was increased by gonadotropins to a much lesser extent than in XX ovaries. Gonadotropins increased estradiol production similarly in both ovaries at 35 d.p.p. Striking differences were found in the histochemical distribution of 3 beta-hydroxysteroid dehydrogenase between XY and XX ovaries at 14, but not at 35 d.p.p. In conclusion, the XY ovary develops abnormal endocrine features during the postnatal period, which likely lead to the fertility problems at puberty.

The effect of diabetes on sexual behavior and reproductive tract function in male rats.

The effect of streptozotocin induced diabetes and sabeluzole (SBZ) on sexual function was evaluated in male rats. SBZ is a benzothiazole derivative with antihypoxic and antiischaemic activities. Rats were rendered diabetic by intraperitoneal injection of streptozotocin, 60 mg./kg. body weight, and either left untreated or treated with 1.0 mg./kg. of SBZ. Two groups of control rats treated with or without SBZ were also evaluated. Seven weeks after the induction of diabetes, all rats were studied in vivo for mating behavior. Animals were sacrificed one week later, and detrusor strip response in vitro was evaluated. The reproductive organ weight, sperm content and motility as well as in vitro testosterone secretion and serum levels of LH and testosterone were determined. Diabetes induced significant reduction in mating behavior. The diabetic rats that received SBZ showed a significant improvement in mating behavior. The percentage of animals that exhibited ejaculation was 0% in the diabetic group compared to 70% in the controls and 38% in diabetic plus SBZ group. The strips of the detrusor muscle of the diabetic group showed a marked hypersensitivity to bethanechol HCL. In the diabetic plus SBZ group, the strips of the detrusor muscle showed a response similar to that of the control. The diabetic rats had significantly diminished reproductive organ weight, testicular sperm content, epididymal sperm content and sperm motility relative to the control. In addition, marked decrease in the serum level of testosterone and in vitro testosterone secretion was observed in diabetic rats. In the diabetic plus SBZ group, the reproductive organ weight, sperm content and motility as well as serum testosterone and in vitro testosterone secretion showed an improvement compared to diabetic rats. In summary, our data suggest that sex behavior and reproductive tract functions are markedly affected by streptozotocin induced diabetes. Sabeluzole treatment could be beneficial in reducing the deleterious effect of diabetes on sexual functions.

True hermaphrodites: an experimental model in the mouse.

When the Y chromosome from the Mus musculus domesticus mouse strain is placed onto the C57BL/6J mouse background ovarian and testicular components develop in half of the XY progeny (B6.YDOM), providing an excellent model of true hermaphroditism. We examined the correlation between gonadal sex and development of the internal genital tract in the B6.YDOM mouse at puberty. Of 55 mice examined 20 had bilateral testes, 33 were true hermaphrodites and 2 had mixed gonadal dysgenesis. In all mice with bilateral testes male accessory sexual organs developed bilaterally. In the true hermaphrodites testes were found on either side but preferentially on the left side. When a male feature was present on the ipsilateral side of the ovary, the seminal vesicle was more frequently found than prostate or vas. Testicular mass was significantly different between the true hermaphrodites with and those without bilateral seminal vesicles. Similar difference was found in those with and without bilateral prostates. The serum testosterone level was not significantly different between these groups. We conclude that the testicular mass is a better discriminant than serum testosterone for the presence or absence of seminal vesicles and prostates at puberty.

Testicular differentiation in mammals under normal and experimental conditions.

Gonadal differentiation begins with the establishment of a sexually undifferentiated gonad, in which gonadal cords are formed by condensation of somatic cells and deposition of basal laminar components around the cluster of epithelial-like cells. The first event of sexual differentiation is the invasion of mesenchymal and endothelial cells into the genital ridge in the XY gonad. As a consequence of this event, the gonadal cords become conspicuous, recognized as seminiferous cords (or testis cords). Cytological differentiation of Sertoli cells follows these stromal changes. In the XX gonad, by contrast, the invasion of the mesenchyme is absent and gonadal cords remain associated with the surface epithelium. In the B6.YDOM XY ovotestis, seminiferous cords and ovarian gonadal cords are often enveloped by common basal laminae, confirming that both structures share the embryonic origin. It has been recently reported that seminiferous-like cords are formed after loss of oocytes in the rat XX ovary cultured in the presence of Müllerian inhibiting substance or after long-term culture in the basic medium alone. These results are comparable with our observation on the persistent gonadal cords in the ovary of busulphan-treated rats or W/WV mutant mice, in which oogonia are absent or scarce. Ultrastructural evidence for Sertoli cell differentiation from XX cells has been presented, so far, only in the fetal mouse ovary that has been grafted beneath the kidney capsule of adult male mice. Possible mechanism of gonadal sex determination is discussed based on these morphological studies.

Delay of testicular differentiation in the B6.YDOM ovotestis demonstrated by immunocytochemical staining for müllerian inhibiting substance.

It has been found that when the Y chromosome from Mus musculus domesticus (YDOM) is placed onto the C57BL/6J (B6) mouse background, the XY progeny (B6.YDOM) develop ovaries or ovotestes but not normal testes during fetal life. We examined the ontogeny of the abnormal testicular differentiation in the B6.YDOM ovotestis by immunocytochemical staining for Müllerian inhibiting substance (MIS). We found that the B6.YDOM ovotestis initiated testicular differentiation later in development than did the control B6 testis. When the YDOM was transferred onto the SJL J mouse background by crossing B6.YDOM males with SJL/J females, all XY progeny developed normal testes. The onset of testicular differentiation was at the same developmental stage as in the B6 male fetus. These results suggest that the delay of testicular differentiation is not due to the effect of the YDOM chromosome itself, but due to improper interaction of the testis-determining gene on the YDOM chromosome with autosomal genes of B6. In addition, we found a close correlation between the arrest of germ cells at the prespermatogonia stage and MIS production of adjacent somatic cells in the B6.YDOM ovotestis. This result may support the hypothesis that MIS is involved in the regulation of germ cell differentiation.