Regulation of meiosis in the foetal mouse gonad

In the mouse, as in other vertebrates, all fertile germ cells must undergo meiosis which consists of two cell divisions each composed of four phases (prophase, metaphase, anaphase and telophase). The first division is relatively complicated with an extended prophase divided into four stages (leptotene, zygotene, pachytene and diplotene). Although meiosis is fundamentally the same in both sexes, there are three significant differences between meiosis in the male and in the female :

1. Meiosis in the male does not commence until puberty, whereas in the female meiosis begins during foetal life or just after birth.

2. Meiosis in the male is an uninterrupted process which occurs throughout life after puberty. In the female, meiosis is arrested at diplotene and is not resumed until shortly before ovulation. Meiosis is arrested in the female for a second time at metaphase II and is not completed unless the oocyte has been fertilized.

3. Cytoplasmic divisions during meiosis in the male are equal, whereas in the female such divisions are unequal.

What mechanisms underly these differences in the meiotic pathways of the two sexes ? Analysis of in vitro experiments on meiotic progression in mammals has suggested a number of factors which may regulate meiosis in both males and females. Byskov (1974) suggested that a factor derived from the foetal rete ovarii (the Meiotic Inducing Substance, MIS) may be involved in initiating meiosis in the female while, according to Ohno & Smith (1964), contact with follicular (i.e. granulosa) cells may be responsible for arresting oocytes at diplotene. Since Byskov (1975,1978 a) has suggested that the rete ovarii contains precursors of the definitive granulosa cells, the possibility exists that meiotic control may lie within one cell type arising relatively early in gonadal development. In the male, the delay in the initiation of meiosis might involve some interaction between the germ cells and the Sertoli cells which are in close proximity within the confines of the testicular cords. It has been suggested that the Sertoli cells may secrete a substance which inhibits meiosis in the male until puberty (Byskov & Saxen, 1976; Byskov, 1978 b). Indeed, the interplay of such a substance (the Meiotic Preventing Substance, MPS) with the postulated inducing substance MIS in the presence of other components such as the endocrine hormones may contribute to the mechanism underlying the inhibition and onset of meiosis (Byskov, 1978b). However, at this stage the presence of inducing and preventing substances in both sexes has not been demonstrated conclusively. The present study was designed to test the effects of co-culturing testes and ovaries from foetal mice of differing ages on both male germ cells (in which meiosis has not been initiated) and on female germ cells (after or at the time of the initiation of meiosis but before arrest at diplotene).

Pregnant mice of the C57BL strain were killed by exposure to ether vapour on the appropriate day after coitus. The day when a vaginal plug was found was recorded as day 1 post coitum (day 1 p.c.). The foetuses were placed in Dulbecco’s phosphate-buffered saline (PBS) and the gonads (including their rete systems) were removed. The foetuses were sexed using morphological criteria or following examination of chromosome preparations (Evans, Burtenshaw & Ford, 1972).

Organ cultures consisted of two types (Fig. 1):

• Rafts: these were either blocks of 2% agar (Baker & Neal, 1973) or squares of Millipore filter (0 · 22 μm pore size) supported on expanded stainless-steel grids. In raft cultures, the gonads were placed on top of the agar or filter support in Petri dishes and the medium level was adjusted to just approach the junction between the gonads and their support.

• Vertical cultures: the gonads were placed on either side of a square of Millipore filter held vertically in a specially designed stainless steel grid (Fig. 1 ; Robb & Evans, 1981). Enough medium was added to the Petri dish containing the culture system to reach the bottom of the filter and pass up to the gonads by capillary action.

The medium used for all cultures was Eagle’s Minimal Essential Medjum with Earle’s Salts (Flow Labs.) at pH 7 · 3, supplemented with 20% newborn calf serum, 2 mM glutamine, 50 i.u. ml⁻¹ penicillin and 50 μg. ml⁻¹ streptomycin sulphate. Cultures were maintained at 37 °C in a humidified atmosphere of 95 % air and 5 % CO₂ in the presence or absence of additional pressure (5 p.s.i.) as indicated in Results. The medium was changed every second day and after the appropriate length of time the gonads and their supporting surfaces were removed from the Petri dishes, fixed in aqueous Bouin’s solution and prepared for histological examination using routine techniques.

The percentages of cells in different stages of meiosis in non-cultured control ovaries were determined for each age and the results are presented in Table 1. These results are in general agreement with those of Borum (1961) and confirm slight differences in the rate of progression of germ cells through meiosis in different strains of mice.

Variations in testicular cord structure in non-cultured control male gonads are shown in Table 2. It can be seen that there is an increase in cord diameter and in the number of Sertoli cells lining each testicular cord with increasing gestational age.

Table 3 summarizes the effects of culture on the structure of testicular cords in males and on meiotic progression in females. Culture has considerable effects on gonadal structure and development, significantly reducing the testicular cord diameter and the number of lining Sertoli cells in males and increasing the percentage of pyknotic oocytes in females.

From counts of the number of germ cells in diplotene and the volume of the ovaries in cultured and non-cultured (control) females, we have determined the effects of culture on germ cell progression through meiosis (Table 4). Culture of day 14 p.c. and day 15 p.c. ovaries results in about a 50 % decrease in oocyte density.

Table 5 illustrates the inhibitory effect of day 14 p.c. testes on the progression through meiosis of day 14 p.c. oocytes. In day 14 p.c. mixed-sex co-cultures, 26 · 4 ± 0 · 5 % of all germ cells remained as oogonia while the rest were pyknotic.

The variations in testicular cord structure in co-cultures prepared using the Millipore raft technique are shown in Table 6. The diameter of testicular cords and the number of Sertoli cells per cord cross-section are not significantly different when similar and mixed-sex co-cultures prepared from day 14 p.c. are compared. However, similar data from cultures prepared from day 15 p.c. could not be compared because testicular cord structure degenerated in day 15 p.c. mixed-sex co-cultures. Likewise, in all but one of the mixed-sex cocultures of different ages the cord structure also degenerated (Table 6). In the single case where the day 14 p.c. testicular cords did not break down it was apparent that the germ cells of the day 15 p.c. female did not progress into leptotene (result not shown).

Results from cultures prepared using the vertical culture system at 5 p.s.i. or agarose rafts were essentially similar to those obtained using the Millipore raft technique (summarized in Table 7). Again it was apparent that day 14 p.c. male gonads inhibited meiosis in day 14 p.c. oocytes. Cultures prepared from day 15p.c. testes also inhibited meiosis in the day 14 p.c. female providing male germ cells were present and providing the testicular cords were more or less intact. Day 14 p.c. testes affect day 15 p.c. oocytes in a similar manner, However, the results from day 15 p.c. mixed-sex co-cultures are somewhat more equivocal (Table 7). Thus, in agreement with earlier results, in six cultures meiosis in the ovary proceeded in the absence of both intact testicular cords and germ cells in the male portion of the co-culture. On the other hand, in nine cultures meiosis was able to proceed even in the presence of intact testicular cords and germ cells in the male.

Examination of testes in single and mixed-sex co-cultures provided no evidence for precocious meiosis in the germ cells of the foetal male gonad.

Reliable culture conditions are obviously a prerequisite for examining the events following the co-culture of mixed-sex gonads since apparent inhibitory effects may be due to the relatively greater sensitivity of the gonads of one sex to in vitro conditions. We find that during culture spreading of the gonads tends to occur over the supporting substrate although in cultured single organs the capsules remain intact. This spreading leads to the eventual fusion of the gonads in all co-cultures if the gonads are initially placed within a few millimetres of each other. As well as changes in the external structure, there are also changes in the internal structure at the cellular level. Thus, in the male, the testicular cords fail to reach their potential diameter during culture and the number of lining Sertoli cells is significantly decreased (Table 3). Comparison of the data in Tables 2 and 3 shows that in cultured testes (unlike control testes) there is no significant growth in the diameter of testicular cords and nor is there any increase in Sertoli cell number from the age at which the cultures were established. In the ovary, culture leads to an increase in pyknosis which is reflected in a smaller percentage of cells in pachytene (Table 3). In real terms, however, there is a 50 % loss in germ cells reaching diplotene when cultured ovaries are compared to uncultured ovaries of equivalent age (Table 4). These variations reflect the need to seek improved conditions for the culture of foetal gonads. However, we believe the present experimental regimes to be more than adequate to justify our conclusions following the co-culture of gonads for a number of reasons. First, although fewer oocytes progress through prophase I, enough germ cells reach meiosis in control cultures to allow us to assess the effects of experimental regimes designed to modulate germ cell development. Secondly, meiotic inhibition in the female partner of mixed-sex co-cultures is seen when the male gonad is healthy with intact testicular cords encompassing germ cells but has not been seen when the structure of the male portion is degenerating (Table 7). Thirdly, inhibition of meiosis is not due to the effects of culturing more than one gonad on the same substrate since control experiments show no specific effects on meiosis following co-culture of similar sex gonads (Tables 5 – 7).

From our studies of mixed-sex co-cultures we conclude that day 14 p.c. testes inhibit meiosis in day 14 p.c. ovaries when the gonads are cultured to the equivalent of day 1 p.p., i.e. 7 days (Tables 5, 7). It is unlikely that the high level of pyknotic oocytes (Table 5) reflects a feature of the culture system per se since other gonad combinations show that cultures can be maintained for this length of time without significant cell death. Meiotic inhibition requires intact testicular cords and the presence of germ cells in the male and is able to act across a vertical Millipore filter barrier (100 μ m thick, 0 · 22 μ m pore size) when appropriate gonads are placed on either side (Table 7). Histological sections of Millipore filters from vertical cultures failed to reveal the presence of direct physical contact across the filter, but the presence of a soluble inhibitory factor has not been proven because of the possibility of cellular microprojections across Millipore filters (Saxen et al. 1970).

In day 15 p.c. co-cultures and mixed-age co-cultures the results are less straightforward. The absence of significant numbers of male germ cells and the disruption of testicular cords again correlates consistently with progression through meiosis in the co-cultured female (Table 7). In two out of three combinations the presence of intact cords and germ cells in the male correlates with meiotic inhibition in the female. The set of results (day 15 p.c. female/day 15 p.c male) at variance with the others may reflect difficulties in assessing true foetal age (which will vary firstly with the exact time of fertilization of oocytes and secondly, with the time of experimentation, i.e. a variation in the order of 6-9 h) and could suggest that progression through the early stages of meiosis may be irrevocably committed in the female around day 15 p.c. The concept of a critical time beyond which meiosis in female germ cells cannot be inhibited by the testes gains circumstantial support from other studies (Ożdżenśki et al. 1976) but further experiments with more accurate assessment of foetal age will be required to fully evaluate this possibility.

We found no evidence in any of our day 14 and day 15 p.c. cultures to support the possibility that mixed-sex co-culture may lead to the precocious stimulation of meiosis in spermatogonia. Similarly, Ożdżenśki et al. (1976) failed to detect the initiation of meiosis in spermatogonia from the mouse when foetal testes were co-cultured in vivo with mouse ovaries. O and Baker (1976, 1978), however, have suggested that isolated spermatogonia which are not enclosed by the testicular cords may enter meiosis precociously. Byskov (1978b) has also suggested that extra-tubular germ cells may enter meiosis. It would seem from these conflicting lines of evidence that the early induction of meiosis in foetal spermatogonia may be a relatively rare event and that the role of the postulated meiotic-inducing substances in the precocious initiation of meiosis in the male requires further study.

We conclude, in agreement with Byskov (1974), that cellular activities associated with foetal testes may inhibit meiosis in foetal ovaries. This inhibitory effect may be mediated by a soluble meiosis-preventing substance and our results suggest a possible means of regulating the meiotic pathway in mammals.