H-Y antigen negative XOSxrb mice, like their H-Y positive XOSxra counterparts, have testes; but, in contrast to XOSxra males, XOSxrb testes almost totally lack meiotic and postmeiotic stages of spermatogenesis. The quantitative analysis of the testes of XOSxrb males and their XY±Sxrb sibs, described in. the present study, identified two distinct steps in this spermatogenic failure. First, there was a reduction in mitotic activity among T2 prospermatogonia, so that approximately half the normal number of T2 prospermatogonia were produced. Second, there was a dramatic decrease in the number of A3 and A4 spermatogonia and no Intermediate or B spermatogonia. These reductions were also largely due to decreased mitotic activity, there being a shortage of A1 and A2 spermatogonial divisions and no divisions among A3 or A4 spermatogonia. Mitotic activity among the T2 prospermatogonia and the undifferentiated A spermatogonia was normal. This means that the spermatogonial stem cells, which are a subset of the undifferentiated A spermatogonia, are unaffected in XOSxrb mice. Sxrb is now known to have arisen by deletion of DNA from Sxra. It is clear from the present findings that a gene (or genes) present in the deleted DNA plays a major role in the survival and proliferation of the differentiating A spermatogonia.

Sex-reversed (Sxr) is a factor that causes an inherited form of sex-reversal, such that XX and XO mice carrying Sxr develop as phenotypic males (Cattanach et al. 1971). In 1982 evidence was obtained that Sxr was in fact an extra copy of the testis-determining region of the mouse Y chromosome which had become located distal to the pairing and exchange region of the Y, so that it regularly crossed over onto the X chromosome during male meiosis (Singh and Jones, 1982; Evans et al. 1982; Burgoyne, 1982; Eicher, 1982; Hansmann, 1982).

In addition to testis-determining information, the original Sxr (now termed Sxra - McLaren et al. 1988) included information required for H-Y antigen expression (Bennett et al. 1977). In 1984 McLaren et al. discovered a variant of Sxra (originally designated Sxr′, but now Sxrb) that retained the testis-determining information, but which had lost the Y-chromosomal gene required for transplantation H-Y antigen expression (Simpson et al. 1981, 1986). This finding, recently confirmed by the separation of TDF from H-Y loci in humans (Simpson et al. 1987), negated the hypothesis of Wachtel et al. (1975) that H-Y antigen was the primary testis determinant (at least in so far as the transplantation H-Y antigen is concerned).

XOSxra males differ genetically from normal males not only in that they lack most of the Y chromosome, but also in having two X chromosomes. The presence of two X chromosomes is incompatible with male germ cell survival beyond the perinatal period (reviewed by McLaren, 1983) so that in order to investigate the effects of the Y-chromosomal deficiencies associated with Sxra and Sxrb, it is necessary to produce Sxr males with single X chromosomes.

XOSxrb mice were first described by Cattanach et al. (1971) and although all stages of spermatogenesis are represented in their testes, the later stages are severely depleted so that the testes are small and the mice are sterile. The majority of the spermatids are in fact diploid and the few sperm produced, whether haploid or diploid, are abnormal (Levy and Burgoyne, 1986a).

XOSxrb mice have a more severe spermatogenic impairment with only a few germ cells reaching early meiotic prophase (Burgoyne et al. 1986). The XO germ cells in an XO/XY/XYY mosaic male described by Levy and Burgoyne (1986b) suffered a similar fate despite a normal XY Sertoli cell environment. These findings led Burgoyne et al. (1986) to suggest that Sxra carries a spermatogenesis gene (Spy) that is lacking in Sxrb, and that Spy is expressed cell-autonomously in the germ line. Recent studies have shown that the Sxrb variant arose by deletion of DNA from Sxra (Bishop et al. 1988; Mardon et al. 1989).

The purpose of the present study was to define the spermatogenic block in XOSxrb mice by a quantitative analysis of germ cells in the two weeks following birth (when the block first becomes apparent) and from this deduce the function of Spy. During the course of the experiment, the finding of a significant body weight difference between XOSxrb and XOSxra mice supported a hypothesis, under separate study, that a growth and development gene (dubbed Gdy) may also be deleted.

Mice

XYSxrb males were mated with females heterozygous for the inversion In(X)lH. In(X)/X females produce some nullo-X eggs following crossing-over within the inversion (Evans and Phillips, 1975), and approximately 1 in 19 of the progeny from this cross have the XOSxrb genotype. The In(X)/X females were checked for vaginal plugs each morning, and coitus was presumed to have taken place at the midpoint of the previous dark cycle. Ages were calculated from conception, rather than birth, because it is known that the duration of pregnancy is affected by litter size. The majority of litters were born about days post coitum (dpc), so in what follows this is equated with the day of birth. 157 litters were bred of which 59 included XOSxrb males. Litters were processed from dpc (day of birth) through dpc (11 days post partum), dpc (13dpp) and dpc (40dpp).

A similar breeding cross was set up to produce XOSxr3 males as controls for a possible XO effect. Data from 35 litters are included in this study. The litters were processed at , through and dpc.

Body weights were recorded at autopsy. Following exclusion of ‘runts’ (Burgoyne et al. 19836), litters were evaluated provided at least one XOSxr and one XY±Sxr male was present. Since a qualitative analysis suggests that XY and XYSxr testes are not significantly different during the pre-meiotic stages (results not shown) XY and XYSxr males were not separately identified. 52 Sxrb and 35 Sxra litters finally provided data.

Karyotyping

Mitotic spreads were prepared either by dissociating liver fragments ( and dpc) or by flushing out bone marrow cells ( dpc onwards) in 0.04% colcemid in Hepes-buffered Eagle’s minimal essential medium, and incubating at 32°C for 60 min (liver) or 15 min (bone marrow). Cells were then treated with 0.56 % KC1 for 20 min followed by five changes of 3:1 methanol: glacial acetic acid. The cells were then air-dried on slides and stained for 15 min in 2% Giemsa in pH 6.8 buffer. XOSxr males were identified by scoring at least 5 consecutive spreads with 39 chromosomes and no evidence of a Y chromosome. XY±Sxr males were identified by 40 chromosomes, with a Y recognised by size and the presence of splayed short arms (Ford, 1966).

Histology

Both testes from each male were weighed using a Cahn electrobalance, and were then retained in Bouin’s fixative awaiting the results of karyotyping. Testes from XOSxr and XY±Sxr littermates were dehydrated and cleared according to standard procedures, embedded in paraffin wax, serially sectioned at 3 μm and stained with haematoxylin and eosin.

Quantitative analysis

This analysis was carried out ‘blind’ with respect to genotype of the mice from which the sections were taken. The sampling was one tubule cross-section from every 20th section, or every 10th section for smaller testes, such that between 25 and 35 tubule cross-sections were analysed per testis. The procedure for selecting tubules for analysis was as follows: (1) A 0.25 mm square grid (R-4 grid, Graticules Ltd, Tonbridge, Kent) was ‘stuck’ to the bottom of the microscope slide with a film of water and a Chalkley grid (G52, Graticules Ltd) was inserted in the eyepiece. (2) When a section was selected, the square grid was focused under low power with a ×10 objective and a square chosen at random. The central cross of the Chalkley grid was centered over the square and the section was brought back into focus. (3) The tubule cross-section adjacent to the central cross was analysed under oil immersion, provided it could be encompassed within the field of view.

This selection procedure ensures that all regions of the gonad have an equal chance of being sampled. Once a tubule was selected, all cells within the tubule cross-section were classified as to cell type except dead or dying cells which could not be classified. Sertoli cells were scored as being in interphase or division. Gonia were scored as being in interphase or division, and were also classified as to stage (i.e. T1 prospermatogonia, T2 prospermatogonia, undifferentiated A spermatogonia, differentiating A1 or A2 spermatogonia, A3 or A4 spermatogonia, Intermediate or B spermatogonia) using the criteria described by Clermont and Perey (1957), Oakberg (1971), Hilscher et al. (1974), Hilscher and Hilscher (1976), Bellve et al. (1977), Huckins and Oakberg (1978) and Kluin and de Rooij (1981). It was often difficult to assign divisions to specific spermatogonial stages and in these cases they were classified according to the adjacent interphase stages in the same tubule. A category existed for cells that could not be classified. This group formed less than 0.5% of germ cells scored and have been omitted from the analysis. It should be pointed out that these cell counts are crude counts, uncorrected for cell sizes and thickness of the sections.

The body weight data for the Sxra and Sxrb litters are given in Table 1. The best estimates for the body weights of the four genotypes (XOSxra, XY±Sxra, XOSxrb, XY±Sxrb) at the various ages studied are provided by the means of litter means. In order to compare the two genotypes in each cross, mean weighted differences between these genotypes and the significance of these differences have been calculated from ‘within litters’ as described by Burgoyne et al. (19836). From these mean weighted differences it is clear that XOSxrb mice are underweight when compared with XY±Sxrb mice. Despite the limited number of mice at each age, the difference is significant for 5/13 age groups, and pooling across age groups (the mean weighted differences are similar throughout the age range studied) gives an overall estimated weight deficit of −0.359±0.059g (P<0.005). XOSxra mice are not significantly underweight when compared with XY±Sxra mice (pooled mean weighted difference= −0.044 ± 0.069g).

Table 1.

Mean body weights for (A) XOSxrb and (B) XOSxra± Sxra mice and the estimated difference between them for the period

1912
-
3312
dpc

Mean body weights for (A) XOSxrb and (B) XOSxra± Sxra mice and the estimated difference between them for the period 1912-3312 dpc
Mean body weights for (A) XOSxrb and (B) XOSxra± Sxra mice and the estimated difference between them for the period 1912-3312 dpc

The testis weight data for the Sxra and Sxrb litters are given in Table 2. XOSxra testes (Table 2B) are not underweight when compared with XY±Sxra litter mates, but XOSxrb testes (Table 2A) are significantly underweight for 9/13 of the ages studied. Since XOSxrb mice are underweight, this testis weight deficit could simply be a reflection of the overall reduction in body weight. The XOSxrb testis weights were therefore corrected by dividing by individual body weight and multiplying by the mean XY±Sxrb body weight for the relevant litters. The mean weighted XOSxrb-XY±Sxrb differences for these corrected testis weights are plotted in Fig. 1. XOSxrb testes are significantly underweight by dpc and the weight deficit rapidly increases thereafter.

Table 2.

Mean testis weights for (A) XOSxrb and XY±Sxrb, and (B) XOSxra±Sxra mice and the estimated difference between them for the period inline-formula>

1912
3312
dpc

Mean testis weights for (A) XOSxrb and XY±Sxrb, and (B) XOSxra±Sxra mice and the estimated difference between them for the period inline-formula>1912 – 3312 dpc
Mean testis weights for (A) XOSxrb and XY±Sxrb, and (B) XOSxra±Sxra mice and the estimated difference between them for the period inline-formula>1912 – 3312 dpc
Fig. 1.

Mean weighted differences in testis weights (corrected for body weights) for XOSxrb and XY±Sxrh mice for the period inline-formula>

1912
3212
dpc. Where error bars are shown the differences are significant (t-test, 1-tailed).

Fig. 1.

Mean weighted differences in testis weights (corrected for body weights) for XOSxrb and XY±Sxrh mice for the period inline-formula>

1912
3212
dpc. Where error bars are shown the differences are significant (t-test, 1-tailed).

The reason for the reduced testis weight in XOSxrb mice is apparent in Fig. 2, which gives the mean number of germ cells and Sertoli cells per tubule cross-section in XOSxrb and XY±Sxrb mice, throughout the period studied. As expected, there is a marked increase in the number of germ cells per tubule cross-section in XY±Sxrb mice, but by contrast there is no increase in XOSxrb mice. There is no deficiency of Sertoli cells in XOSxrb mice. Indeed the mitotic index for Sertoli cells during the period days was found to be very similar in XOSxrb mice (0.85%) and XY±Sxrb mice (0.90%). The mitotic index for Sertoli cells drops to less than 0.3% after in both genotypes. Clearly, the testis weight deficiency in XOSxrb mice is due to germinal failure.

Fig. 2.

Mean number of Sertoli cells (SC) and germ cells (GC) per tubule cross-section in XOSxrb and XY±Sxrb mice for the period

1912
3212
dpc. The numbers in parentheses are the numbers of litters scored at each age. Asterisks indicate XOSxrb points which are significantly different from controls (t-test, 2-tailed). The significantly higher numbers of Sertoli cells in XOSxrb tubules at
2912
and
3212
dpc is a scoring artifact: at these ages some large tubule cross-sections from the controls had to be excluded because they would not fit in the field of view, resulting in an underestimate of the numbers of Sertoli cells and germ cells for controls at these ages.

Fig. 2.

Mean number of Sertoli cells (SC) and germ cells (GC) per tubule cross-section in XOSxrb and XY±Sxrb mice for the period

1912
3212
dpc. The numbers in parentheses are the numbers of litters scored at each age. Asterisks indicate XOSxrb points which are significantly different from controls (t-test, 2-tailed). The significantly higher numbers of Sertoli cells in XOSxrb tubules at
2912
and
3212
dpc is a scoring artifact: at these ages some large tubule cross-sections from the controls had to be excluded because they would not fit in the field of view, resulting in an underestimate of the numbers of Sertoli cells and germ cells for controls at these ages.

In view of the normal numbers of Sertoli cells in XOSxrb mice, in the more detailed analysis of the germ cell deficiency that follows, germ cell numbers are expressed per 100 Sertoli cells, rather than per tubule cross-section.

In Fig. 3, germ cell numbers are plotted against age for the various classes of germ cells identified in the scoring procedure. The numbers of Ti prospermatogonia are indistinguishable in XOSxrb and XY±Sxrb mice. However, XOSxrb mice clearly have fewer T2 prospermatogonia than the controls and pooling over the period dpc reveals that XOSxrb have only 39% of the control value. By contrast, XOSxr3 mice have 91 % of the control value. Since T2 prospermatogonia are assumed to be the progenitors of the undifferentiated A spermatogonia, a deficit of undifferentiated A spermatogonia is expected in XOSxrb mice, and is indeed observed (XOSxrb is 54% of XY±Sxrb). Similarly, there is the expected deficit of differentiating A3 /A2 spermatogonia (XOSxrb is 42% of XY±Sxrb). The number of A3/A4 spermatogonia, however, is reduced much more than expected (XOSxrb is 7 % of XY±Sxrb) and there are no Intermediate or B spermatogonia.

Fig. 3.

Number of germ cells per 100 Sertoli cells for each germ cell stage during the period inline-formula>

1912
3212
dpc. The asterisk denotes occasional XOSxrb zygotene or pachytene cells.

Fig. 3.

Number of germ cells per 100 Sertoli cells for each germ cell stage during the period inline-formula>

1912
3212
dpc. The asterisk denotes occasional XOSxrb zygotene or pachytene cells.

This pattern of germ cell deficiency in XOSxrb mice is largely accounted for by observations on mitotic index (Fig. 4). That is to say, there is a shortage of dividing Ti prospermatogonia, accounting for the drop in the number of T2 prospermatogonia; a reduced frequency of divisions among A1/A2 spermatogonia accounting for the much more severe shortage of A3/A4 spermatogonia; and no dividing A3/A4 spermatogonia accounting for the absence of In/B spermatogonia.

Fig. 4.

Histogram showing the mitotic index according to germ cell stage of XOSxrb and XY±Sxrb mice.

Fig. 4.

Histogram showing the mitotic index according to germ cell stage of XOSxrb and XY±Sxrb mice.

During the scoring procedure the gonia with the morphological characteristics of Aj and A2 spermatogonia were pooled, although it is assumed that they are distinct generations of spermatogonia, as in the adult. When the mitotic index of the A1 /A2 spermatogonia is plotted against age (Fig. 5), there is no marked shortage of divisions in XOSxrb mice until dpc, raising the possibility that it is the A2 rather than the A3 spermatogonia that are affected.

Fig. 5.

Mitotic index of A1/A2 spermatogonia in XOSxrb and XY±Sxrb mice during the period inline-formula>

1912
3212
dpc.

Fig. 5.

Mitotic index of A1/A2 spermatogonia in XOSxrb and XY±Sxrb mice during the period inline-formula>

1912
3212
dpc.

If A1 /A2 spermatogonia rarely divide to give A3 or A4, but the undifferentiated A spermatogonia continue to divide, one might expect a ‘piling up’ of A3/A2 stages. This is not observed, implying that the cells that fail to divide are degenerating. This is supported by observations on the germ cell degeneration index (Fig. 6), which has been calculated on the assumption that all the dying cells observed were germ cells. The degeneration index is very low in XOSxrb and XY±Sxrb mice. Nevertheless, from 261 days onwards XOSxrb mice clearly have more degenerating cells than controls, which is consistent with the increased degeneration of A1 /A2 spermatogonia. It is tempting to suggest that the increased degeneration index in XOSxrb mice at days is similarly due to the death of Tj prospermatogonia that failed to divide.

Fig. 6.

The germ cell degeneration index for the period

1912
3212
dpc was calculated on the assumption that all dying cells were germ cells. The two points marked with an asterisk are artifically high, in that only one of the males at each of these points showed an elevated degeneration index.

Fig. 6.

The germ cell degeneration index for the period

1912
3212
dpc was calculated on the assumption that all dying cells were germ cells. The two points marked with an asterisk are artifically high, in that only one of the males at each of these points showed an elevated degeneration index.

Although no Intermediate or B spermatogonia were scored during the quantification, very rare patches of these spermatogonia, and also of early meiotic stages, can be found in dpc and adult (dpc) XOSxrb testes. They occur without the normal hierarchy of stages, and in small patches, as if an occasional A3/A4 spermatogonium divides and the products proceed via the usual stages up to early pachytene.

The present results show that XOSxrb testes have normal numbers of germ cells at birth, but become severely deficient in germ cells in the ensuing two weeks. During the same period the numbers of Sertoli cells remain normal. These findings are consistent with the view of Burgoyne et al. (1986) and Levy and Burgoyne (1986b) that the spermatogenic failure in XOSxrb mice is due to the loss of a gene (Spy) that acts cell autonomously in the germ line.

The quantitative analysis of the germ cell deficiency in XOSxrb mice revealed a reduction in mitotic activity among T1 prospermatogonia, which resulted in a shortage of T2 prospermatogonia, and consequently a reduced pool of undifferentiated A spermatogonia. However, mitotic activity among the undifferentiated A spermatogonia, which include the spermatogonial stem cells, was found to be normal. It was during the early differentiating spermatogonial stages that the spermatogenic block occurred, with mitotic failure leading to an almost complete absence of Intermediate and B spermatogonia and subsequent meiotic stages.

XO female mice are developmentally retarded in early pregnancy (Burgoyne et al. 1983b) and are significantly underweight postnatally (Burgoyne et al. 1983a). It was anticipated that XOSxrb mice would also be underweight from birth, and this proved to be the case. Unexpectedly, however, the XOSxra mice originally included as controls for this ‘XO effect’ showed little, if any, postnatal weight deficit. Coincidentally, the genetic basis for the early developmental advantage of XY over XX embryos (Tsunoda et al. 1985; Seller and Perkins-Cole, 1987) was being investigated in this laboratory, concurrently with the present study of XOSxrb mice, and the findings may provide an explanation for this difference in postnatal weight between XOSxrb and XOSxra mice. Briefly, it was shown that the Y chromosome carries a factor that accelerates the early growth and development of XY embryos, and it appears that this factor (Gdy) may be present in Sxr3 (P. S. Burgoyne, S. Kalmus, E. P. Evans, K. Holland and M. J. Sutcliffe, unpublished) but deleted from Sxrb (P. S. Burgoyne and C. E. Bishop, unpublished). Thus it may be that the ‘XO effect’ is ameliorated by the presence of Gdy in XOSxra but not XOSxrb mice.

The deletion of Y-chromosomal material involved in the generation of Sxrb has thus removed genetic information required for H-Y antigen expression (McLaren et al. 1984), for spermatogenesis (Burgoyne et al. 1986) and for an early acceleration of growth and development (P. S. Burgoyne et al. unpublished). Burgoyne et al. (1986) pointed out that the spermatogenesis gene (Spy) and the gene controlling H-Y expression (Hya) might be one and the same, and this possibility still holds. Similarly, Gdy may not be a separate gene from Hya and/or Spy. At the molecular level, it has been shown that Zfy-2, one of the Y-chromosomal copies of a gene encoding a zinc finger protein, present along with Zfy-1 in Sxra, has been deleted from Sxrb (Roberts et al. 1988; Mardon et al. 1989; Nagamine et al. 1989a). Because it is strongly expressed in testes, probably in germ cells (Mardon and Page, 1989; Nagamine et al. 19896), it is an obvious candidate for Spy.

As to the function of the ‘spermatogenesis gene’ Spy, we have clearly shown that the spermatogenic failure seen in XOSxrb mice is due to a failure of proliferation during the differentiating A spermatogonial stages, and so by definition Spy is important for the survival/ proliferation of these spermatogonial stages. Whether the deficiency of T! or prospermatogonial divisions in XOSxrb mice is also a consequence of the deletion of Spy, or whether it is due to the deletion of a gene separate from Spy, remains to be determined.

M.J.S. is very grateful to Drs W. and B. Hilscher for training in the recognition of the germ cell stages. M.J.S. is a recipient of an MRC Studentship.

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