Forskolin and the meiosis inducing substance synergistically initiate meiosis in fetal male germ cells

We have shown that Meiosis Inducing Substance (MIS) and forskolin synergistically and dose dependently induce meiosis in germ cells of cultured fetal mouse testes. We used a bioassay which consists of fetal mouse testes and ovaries cultured for 6 days. In this study MIS media are spent culture media from 24 hour cultures of minced adult mouse testes. In the bioassay one gonad of each fetus is cultured either in MIS medium, in control medium with forskolin, or in MIS medium with forskolin. The other gonad serves as the control and is cultured in control medium. After culture the gonads are fixed, squashed, and DNA-stained. In these preparations germ cells and somatic cells can be distinguished, and the number of germ cells in the different stages of meiosis is counted as is the number of somatic cells in mitosis. MIS activity is defined to be present in a medium when meiosis is induced in male germ cells during culture. We found that MIS media as well as forskolin induced meiosis in fetal male germ cells in a dose-dependent manner. In addition, MIS media and forskolin acted synergistically by inducing meiosis. Female germ cells seem to be unaffected by the various culture media. These findings indicate that receptors for stimuli of meiotic initiation may exist in germ cells or neighbouring somatic cells. In addition to induction of meiosis, MIS media and forskolin also dose dependently increase the number of male germ cells compared to controls. This increase is correlated with induction of advanced stages of meiosis: Male germ cells seem to survive better if they are triggered to enter meiosis. Neither MIS media nor forskolin affected the growth of somatic cells. We therefore propose that MIS media has a growth factor activity with a specific effect on meiotic initiation. © 1993 Wiley-Liss, Inc.     

Meiosis-inducing and meiosis-preventing effects of sex steroid hormones on hamster fetal ovaries in organ culture

Day 11 to day 15 p.c. female gonads were cultured for 6-8 days in chemically-defined media. In day 11 and day 12 p.c. ovaries grown in a non-hormonal medium, the germ cells were unable to enter meiosis; they were retained at a stage of oogonia or more frequently at a preleptotene stage. Ovaries of the same ages cultured in an estradiol-containing medium showed germ cells progressing through meiotic prophase in a way close to that in ovaries of equivalent age in vivo. That was the case of the germ cells in day 13 to day 15 p.c. ovaries maintained in a non-hormonal medium. In a testosterone-containing medium, the germ cells in day 13 and day 14 p.c. ovaries were prevented from entering meiosis; by contrast, those in day 15 p.c. ovaries underwent meiotic prophase normally. These results indicated that each of both hormones was able to exert its corresponding (meiosis-inducing or meiosis-preventing) effect before a definite critical time of ovarian development. The possibility is suggested that the germ cell differentiation in the female and male gonads in vivo would also depend on estrogens or androgens precociously synthesized in the gonads or supplied from other organs via the fetal blood.     

A study of meiosis in chimeric mouse fetal gonads

The influence of somatic environment on the onset and progression of meiosis in fetal germ cells was studied in chimeric gonads produced in vitro by dissociation-reaggregation experiments. Germ cells isolated from testes or ovaries of 11.5-13.5 days post coitum (dpc) CD-1 mouse embryos were loaded with the fluorescent supravital dye 5-6 carboxyfluorescein diacetate succinimyl ester (CFSE) and mixed with a cell suspension obtained by trypsin-EDTA treatment of gonads of various ages and of the same or opposite sex. Whereas 11.5 dpc donor germ cells appeared unable to survive in the chimeric gonads obtained, about 76% of the CFSE-labeled female germ cells obtained from 12.5 dpc donor embryos (premeiotic germ cells) found viable within host ovarian tissues showed a meiotic nucleus. In contrast, a smaller number (about 19%) were in meiosis in chimeric testes. None or very few of donor male germ cells entered meiosis in testes or ovarian host tissues. Aggregation of meiotic 13.5 dpc female germ cells with testis tissues from 13.5 to 14.5 dpc embryos resulted in inhibition of meiotic progression and pyknosis in most donor germ cells. These results support the existence of a meiosis-preventing substance or a factor causing oocyte degeneration in the fetal mouse testis, but not of a meiosis-inducing substance in the fetal ovary.     

Cyp26b1 Expression in Murine Sertoli Cells Is Required to Maintain Male Germ Cells in an Undifferentiated State during Embryogenesis

In mammals, germ cells within the developing gonad follow a sexually dimorphic pathway. Germ cells in the murine ovary enter meiotic prophase during embryogenesis, whereas germ cells in the embryonic testis arrest in G0 of mitotic cell cycle and do not enter meiosis until after birth. In mice, retinoic acid (RA) signaling has been implicated in controlling entry into meiosis in germ cells, as meiosis in male embryonic germ cells is blocked by the activity of a RA-catabolizing enzyme, CYP26B1. However, the mechanisms regulating mitotic arrest in male germ cells are not well understood. Cyp26b1 expression in the testes begins in somatic cells at embryonic day (E) 11.5, prior to mitotic arrest, and persists throughout fetal development. Here, we show that Sertoli cell-specific loss of CYP26B1 activity between E15.5 and E16.5, several days after germ cell sex determination, causes male germ cells to exit from G0, re-enter the mitotic cell cycle and initiate meiotic prophase. These results suggest that male germ cells retain the developmental potential to differentiate in meiosis until at least at E15.5. CYP26B1 in Sertoli cells acts as a masculinizing factor to arrest male germ cells in the G0 phase of the cell cycle and prevents them from entering meiosis, and thus is essential for the maintenance of the undifferentiated state of male germ cells during embryonic development.     

New testicular mechanisms involved in the prevention of fetal meiotic initiation in mice

In mammals, early fetal germ cells are unique in their ability to initiate the spermatogenesis or oogenesis programs dependent of their somatic environment. In mice, female germ cells enter into meiosis at 13.5 dpc whereas in the male, germ cells undergo mitotic arrest. Recent findings indicate that Cyp26b1, a RA-degrading enzyme, is a key factor preventing initiation of meiosis in the fetal testis. Here, we report evidence for additional testicular pathways involved in the prevention of fetal meiosis. Using a co-culture model in which an undifferentiated XX gonad is cultured with a fetal or neonatal testis, we demonstrated that the testis prevented the initiation of meiosis and induced male germ cell differentiation in the XX gonad. This testicular effect disappeared when male meiosis starts in the neonatal testis and was not directly due to Cyp26b1 expression. Moreover, neither RA nor ketoconazole, an inhibitor of Cyp26b1, completely prevented testicular inhibition of meiosis in co-cultured ovary. We found that secreted factor(s), with molecular weight greater than 10 kDa contained in conditioned media from cultured fetal testes, inhibited meiosis in the XX gonad. Lastly, although both Sertoli and interstitial cells inhibited meiosis in XX germ cells, only interstitial cells induced mitotic arrest in germ cell. In conclusion, our results demonstrate that male germ cell determination is supported by additional non-retinoid secreted factors inhibiting both meiosis and mitosis and produced by the testicular somatic cells during fetal and neonatal life.     

Retinoic acid prevents germ cell mitotic arrest in mouse fetal testes

During mouse fetal development, meiosis is initiated in female germ cells only, with male germ cells undergoing mitotic arrest. Retinoic acid (RA) is degraded by Cyp26b1 in the embryonic testis but not in the ovary where it initiates the mitosis/meiosis transition. However the role of RA status in fetal germ cell proliferation has not been elucidated. As expected, using organ cultures, we observed that addition of RA in 11.5 days post-conception (dpc) testes induced Stra8 expression and meiosis. Surprisingly, in 13.5 dpc testes although RA induced Stra8 expression it did not promote meiosis. On 11.5 and 13.5 dpc, RA prevented male germ cell mitotic arrest through PI3K signaling. Therefore 13.5 dpc testes appeared as an interesting model to investigate RA effects on germ cell proliferation/differentiation independently of RA effect on the meiosis induction. At this stage, RA delayed SSEA-1 extinction, p63γ expression and DNA hypermethylation which normally occur in male mitotic arrested germ cells. In vivo, in the fetal male gonad, germ cells cease their proliferation and loose SSEA-1 earlier than in female gonad and RA administration maintained male germ cell proliferation. Lastly, inhibition of endogenous Cyp26 activity in 13.5 dpc cultured testes also prevented male germ cell mitotic arrest. Our data demonstrate that the reduction of RA levels, which occurs specifically in the male fetal gonad and was known to block meiosis initiation, is also necessary to allow the establishment of the germ cell mitotic arrest and the correct further differentiation of the fetal germ cells along the male pathway.     

Autonomous regulation of sex-specific developmental programming in mouse fetal germ cells

In mice, unique events regulating epigenetic programming (e.g., genomic imprinting) and replication state (mitosis versus meiosis) occur during fetal germ cell development. To determine whether these processes are autonomously programmed in fetal germ cells or are dependent upon ongoing instructive interactions with surrounding gonadal somatic cells, we isolated male and female germ cells at 13.5 days postcoitum (dpc) and maintained them in culture for 6 days, either alone or in the presence of feeder cells or gonadal somatic cells. We examined allele-specific DNA methylation in the imprinted H19 and Snrpn genes, and we also determined whether these cells remained mitotic or entered meiosis. Our results show that isolated male germ cells are able to establish a characteristic "paternal" methylation pattern at imprinted genes in the absence of any support from somatic cells. On the other hand, cultured female germ cells maintain a hypomethylated status at these loci, characteristic of the normal "maternal" methylation pattern in endogenous female germ cells before birth. Further, the surviving female germ cells entered first meiotic prophase and reached the pachytene stage, whereas male germ cells entered mitotic arrest. These results indicate that mechanisms controlling both epigenetic programming and replication state are autonomously regulated in fetal germ cells that have been exposed to the genital ridge prior to 13.5 dpc.     

Retinoic acid derived from the fetal ovary initiates meiosis in mouse germ cells

Meiotic initiation of germ cells at 13.5 dpc (days post‐coitus) indicates female sex determination in mice. Recent studies reveal that mesonephroi‐derived retinoic acid (RA) is the key signal for induction of meiosis. However, whether the mesonephroi is dispensable for meiosis is unclear and the role of the ovary in this meiotic process remains to be clarified. This study provides data that RA derived from fetal ovaries is sufficient to induce germ cell meiosis in a fetal ovary culture system. When fetal ovaries were collected from 11.5 to 13.5 dpc fetuses, isolated and cultured in vitro, germ cells enter meiosis in the absence of mesonephroi. To exclude RA sourcing from mesonephroi, 11.5 dpc urogenital ridges (UGRs; mesonephroi and ovary complexes) were treated with diethylaminobenzaldehyde (DEAB) to block retinaldehyde dehydrogenase (RALDH) activity in the mesonephros and the ovary. Meiosis occurred when DEAB was withdrawn and the mesonephros was removed 2 days later. Furthermore, RALDH1, rather than RALDH2, serves as the major RA synthetase in UGRs from 12.5 to 15.5 dpc. DEAB treatment to the ovary alone was able to block germ cell meiotic entry. We also found that exogenously supplied RA dose‐dependently reduced germ cell numbers in ovaries by accelerating the entry into meiosis. These results suggest that ovary‐derived RA is responsible for meiosis initiation. J. Cell. Physiol. 228: 627–639, 2013. © 2012 Wiley Periodicals, Inc.     

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.     

Effect of the fetal testis on the number of germ cells in the fetal ovary of rats in vitro

When ovaries from 13.5-day-old fetuses are explained and cultured in vitro for 4 days in a synthetic medium, the number of germ cells increases 6 fold, on average. This increase is only approximately 2 fold if a pair of 16.5-day fetal testes is cultured together with the ovaries or if the ovaries are cultured in a medium in which testes have previously been grown for 4 days. The effect of the latter medium persists if it is dialysed against fresh medium, which suggests that the conditioned medium contains one or several substance(s) of molecular weight superior to the cut-off of the membrane. The testicular effect seems to be effective mainly during the final phase of intense multiplication of the germ cells.     

A test of the production line hypothesis of mammalian oogenesis

Summary Germ cells in female mammals become committed to meiosis and enter its prophase sequentially in fetal life and, according to the Production Line Hypothesis, the oocytes thus generated are released after puberty as mature ova in the same sequence as that of meiotic entry in fetu. This hypothesis in its original and complete form has a subordinate proposition that concerns chiasma (Xma) frequency; it postulates that Xma number would decrease with fetal age. Consequently, univalents would increase, leading to errors of chromosome disjunction at the first meiotic division (MI), and thus to maternal age-dependent numerical chromosome anomalies. By using an in vitro/in vivo approach, we radioactively labelled the DNA of germ cells at premeiotic synthesis as they sequentially entered meiosis, while the fetal ovaries were in culture. At the end of this in vitro phase, pachytene/diplotene (P/D) stages were studied to determine their labelled fraction. The ovaries were then transplanted to spayed females and, after the in vivo phase, mature ova were harvested and the proportion of labelled first and second meiotic metaphases (MI/MII) determined. By marking the germ cells with label while in vitro during periods equivalent to early and late gestation, and by comparing the observed proportions of labelled MI/MII with those of oocytes labelled at P/D, we concluded that, in the mouse, ova do not mature at random for release, but are formed according to a production line system in which the time of release after puberty is related to the time of entry into meiosis in fetu.     

In vitro culture of mouse primordial germ cells

Germ cells were isolated from mouse fetal gonads 11 1/2-16 1/2 days post coitum (dpc), and exposed to various methods of in vitro culture. From 13 1/2 dpc onwards, both male and female germ cells survived well at 37 degrees C for several days. During the culture period the proportion of female germ cells in meiosis increased and later stages of meiotic prophase were seen. The gonadal environment is therefore not essential for the progress of meiosis. Male germ cells in vitro did not enter meiosis. Germ cells isolated from gonads 11 1/2 or 12 1/2 dpc did not survive at 37 degrees C in any of the three culture systems used (Petri dishes, microtest plate wells, drops under oil); cell density, substrate and culture medium were varied, and several additives tested, but no improvement in viability was detected. Below 30 degrees C, on the other hand, 11 1/2 and 12 1/2 day germ cells survived in vitro for at least a week. They did not enter meiosis in culture, but continued to undergo mitotic proliferation.     

Vitamin A deficiency results in meiotic failure and accumulation of undifferentiated spermatogonia in prepubertal mouse testis

Vitamin A (retinol) is required for maintenance of adult mammalian spermatogenesis. In adult rodents, vitamin A withdrawal is followed by a loss of differentiated germ cells within the seminiferous epithelium and disrupted spermatogenesis that can be restored by vitamin A replacement. However, whether vitamin A plays a role in the differentiation and meiotic initiation of germ cells during the first round of mouse spermatogenesis is unknown. In the present study, we found that vitamin A depletion markedly decreased testicular expression of the all-trans retinoic acid-responsive gene, Stra8, and caused meiotic failure in prepubertal male mice lacking lecithin:retinol acyltransferase (Lrat), encoding for the major enzyme in liver responsible for the formation of retinyl esters. Rather than undergoing normal differentiation, germ cells accumulated in the testes of Lrat(-/-) mice maintained on a vitamin A-deficient diet. These results, together with our previous observations that germ cells fail to enter meiosis and remain undifferentiated in embryonic vitamin A-deficient ovaries, support the hypothesis that vitamin A regulates the initiation of meiosis I of both oogenesis and spermatogenesis in mammals.     

Histological and ultrastructural investigation of early gonad development and sex differentiation in Adriatic sturgeon (Acipenser naccarii,Acipenseriformes, Chondrostei)

Gonad development and sex differentiation from embryos to 594‐day‐old individuals were investigated in farmed Acipenser naccarii using light and transmission electron microscopy. The migrating primordial germ cells first appear along the dorsal wall of the body cavity in embryos 1.5 days before hatching. The gonadal ridge, containing a few primary primordial germ cells (PGC‐1) surrounded by enveloping cells, appears in 16‐day‐old larvae. At 60 days, the undifferentiated gonad is lamellar and PGC‐1 multiply, producing PGC‐2. In 105‐day‐old juveniles, a distinct germinal area with advanced PGC‐2 appears on the lateral side near the mesogonium and the first blood vessels are visible. At 180 days, putative ovaries with a notched gonadal epithelium and putative testes with a smooth one appear, together with adipose tissue on the distal side. In 210‐day‐old juveniles, active proliferation of germ cells begins in the putative ovaries, whereas putative testes still contain only a few germ cells. The onset of meiosis and reorganization of stromal tissue occurs in ovaries of 292‐day‐old individuals. Ovaries with developed lamellae enclosing early oocyte clusters and follicles with perinucleolar oocytes occur at 594 days. Meiotic stages are never found, even in anastomozing tubular testes of 594‐day‐old individuals. Steroid producing cells are detected in the undifferentiated gonad and in the differentiated ones of both sexes. Anatomical differentiation of the gonad precedes cytological differentiation and female differentiation largely precedes that of the male. Gonad development and differentiation are also associated with structural changes of connective tissue, viz. collagen‐rich areas are massive in developing testes and reduced in ovaries. J. Morphol., 2008. © 2008 Wiley‐Liss, Inc.     

Differentiation of mouse primordial germ cells into female or male germ cells.

Mouse primordial germ cells (PGCs) migrate from the base of the allantois to the genital ridge. They proliferate both during migration and after their arrival, until initiation of the sex-differentiation of fetal gonads. Then, PGCs enter into the prophase of the first meiotic division in the ovary to become oocytes, while those in the testis become mitotically arrested to become prospermatogonia. Growth regulation of mouse PGCs has been studied by culturing them on feeder cells. They show a limited period of proliferation in vitro and go into growth arrest, which is in good correlation with their developmental changes in vivo. However, in the presence of multiple growth signals, PGCs can restart rapid proliferation and transform into pluripotent embryonic germ (EG) cells. Observation of ectopic germ cells and studies of reaggregate cultures suggested that both male and female PGCs show cell-autonomous entry into meiosis and differentiation into oocytes if they were set apart from the male gonadal environments. Recently, we developed a two-dimensional dispersed culture system in which we can examine transition from the mitotic PGCs into the leptotene stage of the first meiotic division. Such entry into meiosis seems to be programmed in PGCs before reaching the genital ridges and unless it is inhibited by putative signals from the testicular somatic cells.     

Role of the pituitary in controlling oogenesis in the rabbit

The involvement of the pituitary and mesonephros in oogenesis was examined with the rabbit used as an experimental model. Fetal and neonatal ovaries were cultured for 8 days with and without their corresponding pituitaries or as half cranial or caudal portions. Progression of meiosis was followed histologically and autoradiographically by the uptake of tritiated thymidine. Pulse labeling for 24 h occurred on Days 0, 2, 4 and 7 of culture. No difference was noted in labeling or progression of meiosis in neonatal ovaries with or without the pituitary or in sectioned ovaries. By contrast, fetal ovaries with pituitary that were labeled on Day 4 had a labeling index (positive cells/1000 microns 2 of cortex) of 282.1 +/- 32.9 (mean +/- SEM) compared to 107.1 +/- 24.9 for ovaries without pituitary. Those labeled on Day 7 showed the same differences (263.7 +/- 46.0 vs. 160.2 +/- 20.8). Histologically, there was a threefold difference in percentage of germ cells with pituitary undergoing meiosis (21.2 +/- 2.9% compared to 7.1 +/- 2.8% without). These data suggest that the fetal pituitary has an important role in inducing meiotic prophase in the rabbit.