The gonads of 53 foetuses (16♀, 37♂) derived from fusion of embryos at about the 8-cell stage were examined. Ovotestes were found in two of the male foetuses. When gonads were sectioned days post coitum, germ cells in meiotic prophase were seen in 6/6 female and 4/14 male foetuses. When air-dried preparations were made from gonads days post coitum, germ cells in meiotic prophase were seen in 7/7 females and 4/13 males. At days post coitum, 2/2 female but only 2/10 male foetuses showed any meiotic cells, and these were degenerating.

[3H]Thymidine injected into the mother days post coitum resulted in labelling of the meiotic germ cells, but the sex vesicle characteristic of meiotic cells in normal males was never seen. The timing of DNA synthesis and meiosis was identical to that seen in the germ cells in foetal ovaries.

We conclude that the meiotic cells seen in the testes of foetal chimaeras are probably XX in constitution, and rarely survive beyond the 19th day of gestation.

The occurrence and timing of meiosis in the germ cells of the foetal ovary of the mouse were described by Brambell (1927), and later analysed in more detail by Borum (1961), Peters, Levy & Crone (1962), and Crone, Levy & Peters (1965). The pre-meiotic DNA synthesis takes place 13-15 days post coitum, and the germ cells enter meiosis from 13 days onwards. At 16-18 days post coitum, many oocytes are seen in zygotene and pachytene, and at birth (19-20 days post coitum) the ovary contains about equal numbers of oocytes in pachytene and in early diplotene. Many degenerating pachytene oocytes are seen before and shortly after birth (Borum, 1961).

In contrast, germ cells in the male mouse do not enter meiosis until after birth: the foetal testis therefore contains large numbers of spermatogonia, many of which are undergoing mitosis, but no meiotic cells are seen.

An exceptional situation occurs in experimental chimaeras, formed by fusion of cleaving mouse embryos. Given an equal sex ratio, half on average of such fusions will be between male and female embryos. On the basis of the low proportion of hermaphrodites and the predominantly male sex ratio in his material, Tarkowski (1961, 1963) postulated that most XX/XY chimaeras develop as fertile males, a suggestion which was supported by evidence obtained later by Mystkowska & Tarkowski (1968). An examination of primary spermatocytes in adult male XX/XY chimaeras revealed no germ cells of XX chromosome constitution (Mystkowska & Tarkowski, 1968). No oocytes were found in the testes of 12 male chimaeras examined at 8–12 days of postnatal life, but some oocytes as well as spermatogonia were seen in a single 5-day-old animal (Mystkowska & Tarkowski, 1968, 1970). Prenatally (15–16 days post coitum), five out of 11 male chimaeras contained germ cells in meiotic prophase, as well as gonocytes (Mystkowska & Tarkowski, 1970). Three out of these five animals were examined cytologically and all proved to be XX/XY chimaeras.

It is tempting to assume that the meiotic germ cells seen in the testes of foetal XX/XY chimaeras are of XX constitution, behaving autonomously in the sense that they enter meiosis at the same chronological time as they would in the foetal ovary. However, another possibility exists. The somatic tissue in the testis of a sex chromosome chimaera consists of patches of XX and patches of X Y constitution. It is possible that any germ cell located in the neighbourhood of predominantly XX tissue will enter meiosis before birth, in which case the meiotic germ cells observed in the testes of foetal chimaeras will be indifferently XX and XY.

The aim of the present experiments was: (a) to confirm the observations of Mystkowska & Tarkowski (1970) on the occurrence of meiotic germ cells in the testes of foetal chimaeras; (b) to collect further information on the fate of such germ cells; and (c) to make use of the very striking late-labelling characteristic of the mouse Y chromosome (contained in the sex vesicle) in the DNA synthesis preceding the first meiotic division (Kofman-Alfaro & Chandley, 1970), in order to determine the chromosome constitution of the meiotic germ cells.

The mice were from the randomly bred Q strain. Fusion, culture and transfer of embryos were as described by Bowman & McLaren (1970). The first series of foetuses were those derived by Bowman & McLaren (1970) from fused embryos. The recipient foster-mothers were killed days post coitum, the foetuses were fixed in formol saline, and their gonads later dissected out and serially sectioned at 8 µm, stained with haematoxylin and eosin and examined for the occurrence of meiotic germ cells. Although the quality of fixation was poor, meiotic cells were in general easy to recognize. In the second series, the foster-mothers received an intraperitoneal injection of 0·25 mCi [3H]thymidine (Radiochemical Centre, Amersham; specific activity 22·8 Ci/mM) days post coitum, and were killed 2-5 days later. Some of the foetal gonads from these mothers were fixed in Bouin and processed as before for histological examination, but most were placed in a 1 % solution of sodium citrate for 30 min, fixed in 3:1 methanol:glacial acetic acid for at least 15 min and transferred to 45% acetic acid for 1–2 min prior to spreading on slides pre-heated at 60 °C. Air-dried preparations were stained with carbol fuchsin and filmed for autoradiography using Ilford L.4 liquid emulsion with an exposure period of 3 weeks at 4 °C.

The first series consisted of 20 foetuses, days post coitum, derived from embryo fusion. From the position and external appearance of the gonads, the foetuses were classified as 6 females and 14 males. Histological examination revealed that one of the males possessed an ovotestis very similar to that described by Mystkowska & Tarkowski (1970). The distal end of the gonad consisted of clearly differentiated sex cords, but the proximal end was ovarian in appearance, with no sex cords and the majority of the germ cells in meiotic prophase (Figs. 1,2).

FIGURES 1-3

8µm sections of foetal gonads, 1612 days post coitum, stained with haematoxylin and eosin. Fig. 1. Ovotestis from male foetus derived from embryo fusion, showing sex cords in distal region (T) and ovarian tissue proximally (O). × 110 Fig. 2. Higher magnification of area outlined in Fig. 1, to show germ cells in meiotic prophase (pachytene) (arrowed), × 1100 Fig. 3. Testis from phenotypically normal male foetus derived from embryo fusion, showing germ cell in meiotic prophase (pachytene) (arrowed), × 1100

FIGURES 1-3

8µm sections of foetal gonads, 1612 days post coitum, stained with haematoxylin and eosin. Fig. 1. Ovotestis from male foetus derived from embryo fusion, showing sex cords in distal region (T) and ovarian tissue proximally (O). × 110 Fig. 2. Higher magnification of area outlined in Fig. 1, to show germ cells in meiotic prophase (pachytene) (arrowed), × 1100 Fig. 3. Testis from phenotypically normal male foetus derived from embryo fusion, showing germ cell in meiotic prophase (pachytene) (arrowed), × 1100

FIGURES 4-8

Air-dried preparations from the testes of foetuses derived from embryo fusion. No sex vesicles visible. Fig. 4. Group of six germ cells in the zygotene and early pachytene stages of meiotic prophase, 1512 days post coitum. Fig. 5. Two labelled pachytenes, 1512 days post coitum. Fig. 6. Labelled meiotic cell thought to be in the diplotene stage, 1712 days post coitum. Fig. 7. Degenerate meiotic cells, 1812 days post coitum. Fig. 8 a. Germ cell in the pachytene stage of the first meiotic prophase, from an air-dried preparation of the testis of a normal male mouse 13 days old, to show the prominent sex vesicle (arrowed) containing the X and Y chromosomes. Fig. 8b. Germ cells in the pachytene stage of meiotic prophase from an adult male mouse showing the characteristic late-labelling pattern of the sex vesicle and centric heterochromatin. (From Kofman-Alfaro & Chandley, 1970.)

FIGURES 4-8

Air-dried preparations from the testes of foetuses derived from embryo fusion. No sex vesicles visible. Fig. 4. Group of six germ cells in the zygotene and early pachytene stages of meiotic prophase, 1512 days post coitum. Fig. 5. Two labelled pachytenes, 1512 days post coitum. Fig. 6. Labelled meiotic cell thought to be in the diplotene stage, 1712 days post coitum. Fig. 7. Degenerate meiotic cells, 1812 days post coitum. Fig. 8 a. Germ cell in the pachytene stage of the first meiotic prophase, from an air-dried preparation of the testis of a normal male mouse 13 days old, to show the prominent sex vesicle (arrowed) containing the X and Y chromosomes. Fig. 8b. Germ cells in the pachytene stage of meiotic prophase from an adult male mouse showing the characteristic late-labelling pattern of the sex vesicle and centric heterochromatin. (From Kofman-Alfaro & Chandley, 1970.)

Histological examination (Table 1) showed germ cells in meiotic prophase not only in the ovotestis, but in 6/23 of the otherwise normal testes examined (Fig. 3). The total, 7/24 (or 4 out of 14 males examined), does not differ significantly from the proportion (5/11) reported by Mystkowska & Tarkowski (1970).

Table 1.

The occurrence of germ cells in meiotic prophase in the gonads of 14 male and 6 female foetuses derived from pre-implantation fusion (sectioned material)

The occurrence of germ cells in meiotic prophase in the gonads of 14 male and 6 female foetuses derived from pre-implantation fusion (sectioned material)
The occurrence of germ cells in meiotic prophase in the gonads of 14 male and 6 female foetuses derived from pre-implantation fusion (sectioned material)

In the second series, 16/33 foster-mothers became pregnant, and 33/104 fused embryos transferred to them survived to the time of autopsy. Ten were females and 23 males, giving a sex ratio very similar to that found in the first series. For the two series combined, the sex ratio deviated significantly from a 1:1 ratio (P < 0·01) but not from the 3:1 ratio expected if all XY chimaeras had developed as males. In this series also, one of the males proved on histological examination to possess an ovotestis.

Air-dried preparations were made from the ovaries of nine female foetuses derived from fused embryos and two control female foetuses derived from transfer of non-fused embryos, days post coitum. At days the germ cells were typically in leptotene and zygotene, at and days most were in zygotene and pachytene and at days post coitum zygotene, pachytene and some diplotene stages were seen. The ovaries of female foetuses derived from transferred embryos (whether fused or single) resembled those of controls of the same developmental stage.

Air-dried preparations were also made from one or both gonads of the 23 male foetuses. The results are given in Table 2. Combining the data from , and days post coitum, four out of thirteen males contained germ cells in meiotic prophase, closely resembling the meiotic cells seen in female foetuses of the same developmental age (Figs. 4-6). By contrast, at days post coitum, no normal germ cells in meiotic prophase were seen, though small numbers of cells believed to be degenerate pachytene stages were found in two foetuses out of ten (Fig. 7).

Table 2.

The occurrence of germ cells in meiotic prophase in the testes of foetuses derived from fused embryos, at different stages of pregnancy (air-dried material)

The occurrence of germ cells in meiotic prophase in the testes of foetuses derived from fused embryos, at different stages of pregnancy (air-dried material)
The occurrence of germ cells in meiotic prophase in the testes of foetuses derived from fused embryos, at different stages of pregnancy (air-dried material)

When meiotic cells were found in one testis of a foetus, they were invariably present in the other testis also. No similar cells were seen in air-dried preparations from the testes of control male foetuses days post coitum.

A total of 164 meiotic cells were examined after autoradiography, of which 94 were from the male with an ovotestis. None of the meiotic cells contained a sex vesicle, nor did any show a pattern of incorporation of [3H]thymidine characteristic of XY cells.

The results of the present study amply confirm Mystkowska & Tarkowski’ s (1970) finding of meiotic germ cells in the testes of foetal chimaeras. In the earlier study and in the two series reported in the present paper, the proportion of day male foetuses possessing such cells was 5/11, 4/14 and 4/13 respectively, in good agreement with each other. Cells in meiosis have never been seen in the testes of male foetuses not derived from embryo fusion, and the results of Mystkowska & Tarkowski (1970) suggest that they occur only in the testes of sex chromosome chimaeras (XX/XY). We may therefore inquire what proportion of male foetuses derived from embryo fusion may be expected to be sex chromosome chimaeras.

Not all foetuses derived from embryo fusion are chimaeric, since one of the two component cell populations may fail for a number of reasons to become incorporated in the developing foetus (for discussion, see McLaren & Bowman, 1969). In the strain combination described by McLaren & Bowman (1969), for example, 28 mice have been raised to an age when they could be classified for chimaerism in respect of a number of characters involving different tissues, and six (21 %) failed to show chimaerism in any tissue (A. McLaren, unpublished observations). If a similar frequency be assumed in the present study, 69·75% of foetuses derived from embryo fusion will be expected to be of either XY or XX/X Y constitution (Table 3). Since 69-8 % of our foetuses (37/53) were male, it is reasonable to conclude that, in the present study, all XX/XY chimaeras developed as males. Mystkowska & Tarkowski (1970), on the other hand, found a more nearly equal sex ratio (57% males) and detected two XX/XY foetuses which were developing as normal females.

Table 3.

Expected numbers of embryos of different chromosome constitutions on the assumption of 21 % non-chimaerism

Expected numbers of embryos of different chromosome constitutions on the assumption of 21 % non-chimaerism
Expected numbers of embryos of different chromosome constitutions on the assumption of 21 % non-chimaerism

From Table 3 it will be seen that the best estimate that can be derived for the expected proportion of XX/XY chimaeras among our male foetuses is 57% (39-50/69-75). During the period days post coitum, the proportion of male foetuses showing meiotic germ cells in our material is only 30% (8/27), significantly lower than the expected 57% (P < 0·01), indicating either that some of the meiotic cells eluded detection, or that not all XX/XY chimaeras have meiotic cells in the testis. Probably both explanations are true: if meiotic cells were present in very low frequency they could well have been missed, particularly in the sectioned material, while Mystkowska & Tarkowski (1970) identified one XX/XY chimaera without any meiotic cells in the testis.

At days post coitum, the few remaining meiotic cells appeared very degenerate. Borum (1961) reports large numbers of germ cells degenerating at the pachytene stage in squash preparations of foetal ovaries days post coitum. We therefore wondered whether the degeneration of meiotic cells in chimaera testes days post coitum might represent part of a normal process of atresia, which would also affect meiotic cells in the foetal ovary. However, the air-dried preparations of ovaries at this stage of development showed no cells comparable to the degenerating meiotic cells seen in the chimaera testes. It would seem that the environment of the foetal testis is hostile to the development of meiotic germ cells beyond the pachytene stage, and that few are likely to survive further than the nineteenth day of gestation. The presence of oocytes in the testis of a 5-day-old male chimaera (Mystkowska & Tarkowski, 1968) may have been an exceptional occurrence. In mules, where meiosis also fails to be completed, it is again at the pachytene stage that the germ cells degenerate (Short, 1972).

Are the germ cells in meiotic prophase which we have observed in the testes of foetal chimaeras XX in constitution, or do they include both XX and XY germ cells which have been induced to enter meiosis under the influence of XX somatic cells? The second alternative raises the further question of the ultimate fate of those XX germ cells in an XX/XY chimaera which do not enter meiosis prenatally, since no XX germ cells are seen at the primary spermatocyte stage.

The fact that we were unable to detect a sex vesicle in any of the meiotic germ cells in the foetal testes strongly supports the first alternative, since the first male germ cells to enter meiosis after birth in the mouse show, by the late zygotene stage, a large sex vesicle (Fig. 8a). Also, the pattern of incorporation of [3H]thymidine into XY meiotic cells is very characteristic in the mouse, with parts of the X and Y chromosomes giving rise to late-labelling regions (Fig. 8 b) (Kofman-Alfaro & Chandley, 1970). This labelling pattern was never seen in the meiotic cells of the chimaera testes. Another piece of circumstantial evidence suggesting that the germ cells in meiotic prophase are indeed XXcells is that the meiotic cells in the chimaeric testes label on precisely the same day and develop at the same rate as do female germ cells in the foetal ovaries of normal and chimaeric mice. Spontaneous sex reversal, observed in the goat, pig and mouse (for references, see Short, 1972), can also lead to XX germ cells developing in a testis, and it is perhaps relevant that in each instance the germ cells have been found to degenerate before birth.

However, if it is the germ cell’ s own genetic constitution which alone determines the timing of its entry into meiosis, so that all XX germ cells in sex chromosome chimaeras enter meiosis prenatally, it is hard to see why the meiotic germ cells should constitute such a small minority of the total germ cell population of the testis. Primordial germ cells presumably reach the gonadal ridge in equal numbers regardless of their sex, and if subsequent development is autonomous, one might expect 50 % of the germ cells in a sex chromosome chimaera to enter meiosis prenatally. Yet Mystkowska & Tarkowski’s (1970) estimates of the proportion of meiotic germ cells in phenotypically normal chimaeric testes range from 0·08 to 16·3 %, averaging only just over 5%. Because of this finding, and because of the large differences in the frequency of occurrence of meiotic cells in the two testes of a single foetus which they sometimes observed, these authors incline to the view that it is the genetic constitution of the somatic tissue of the testis rather than of the germ cell itself which determines entry of the germ cell into meiosis. Their ‘environmental’ and our ‘genetic’ model could both be satisfied, and all existing data accommodated, on the assumption that the meiotic germ cells that are seen in the foetal testis are in fact XX cells, but that such cells only enter meiotic prophase under the influence of neighbouring XX somatic tissue.

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