Does Rbmy have a role in sperm development in mice?

The Y(d1) deletion in mice removes most of the multi-copy Rbmy gene cluster that is located adjacent to the centromere on the Y short arm (Yp). XY(d1) mice develop as females because Sry is inactivated, probably because it is now juxtaposed to centromeric heterochromatin. We have previously produced XY(d1)Sry transgenic males and found that they have a substantially increased frequency of abnormal sperm. Staining of testis sections with a polyclonal anti-RBMY antibody appeared to show a marked decrease of RBMY protein in the spermatids of XY(d1)Sry males compared to control males, which led us to suggest that this may be responsible for the increase in sperm anomalies. In the current study we sought to determine whether augmenting Rbmy expression specifically in the spermatids of XY(d1)Sry males would ameliorate the sperm defects. An expressing Rbmy transgene driven by the spermatid-specific mouse protamine 1 promotor (mP1Rbmy) was therefore introduced into XY(d1)Sry males. This failed to reduce the frequency of abnormal sperm. In the course of this study, a new RBMY antibody was generated that, in contrast to the original antibody, failed to detect RBMY in spermatid stages by immunostaining. The lack of RBMY was confirmed by western blotting of lysates from purified round spermatids and elongating spermatids. The implications of these results for the proposed role for RBMY in sperm development are discussed.

Evidence that the testis determination pathway interacts with a non-dosage compensated, X-linked gene.

In a number of mammals, including mouse and man, it has been shown that at equivalent gestational ages, males are developmentally more advanced than females, even before the gonads form. In mice, although some strains of Y chromosome exert a minor accelerating effect in pre-implantation development, it is a post-implantation effect of the difference in X chromosome constitution that is the major cause of the male/female developmental difference. Thus XX females are retarded in their development by about 1.5 h relative to X(M)O females or XY males; however, they are more advanced than X(P)O females by about 4 h. It has been suggested that this early developmental difference between XX and XY embryos may "weight the dice" in favour of ovarian and testicular development, respectively, although expression of Sry will normally overcome any such bias. Here we test this proposal by comparing the relative frequencies of female, hermaphrodite and male development in X(P)O, XX and X(M)O mice that carry an incompletely penetrant Sry transgene. The results show that testicular tissue develops more frequently in XX,Sry transgenics than in either of the two types of XO transgenics. Thus the incidence of testicular development is affected by X dosage rather than by the developmental hierarchy. This implies there is a non-dosage compensated gene (or genes) on the X chromosome, which interacts with the testis-determining pathway. Since the pseudoautosomal region (PAR) is known to escape X-inactivation, penetrance of the Sry transgene was also assessed in X(M)Y(*X) mice that have two doses of the PAR but have a single dose of all genes proximal to the distal X marker Amel. These mice showed similar levels of testicular development to X(M)O mice with the transgene; thus the non-dosage compensated X gene maps outside the PAR.

Mouse homologues of the human AZF candidate gene RBM are expressed in spermatogonia and spermatids, and map to a Y chromosome deletion interval associated with a high incidence of sperm abnormalities.

An RNA-binding motif (RBM) gene family has been identified on the human Y chromosome that maps to the same deletion interval as the 'azoospermia factor' (AZF). We have identified the homologous gene family (Rbm) on the mouse Y with a view to investigating the proposal that this gene family plays a role in spermatogenesis. At least 25 and probably >50 copies of Rbm are present on the mouse Y chromosome short arm located between Sry and the centromere. As in the human, a role in spermatogenesis is indicated by a germ cell-specific pattern of expression in the testis, but there are distinct differences in the pattern of expression between the two species. Mice carrying the deletion Yd1, that maps to the proximal Y short arm, are female due to a position effect resulting in non-expression of Sry ; sex-reversing such mice with an Sry transgene produces males with a high incidence of abnormal sperm, making this the third deletion interval on the mouse Y that affects some aspect of spermatogenesis. Most of the copies of Rbm map to this deletion interval, and the Yd1males have markedly reduced Rbm expression, suggesting that RBM deficiency may be responsible for, or contribute to, the abnormal sperm development. In man, deletion of the functional copies of RBM is associated with meiotic arrest rather than sperm anomalies; however, the different effects of deletion are consistent with the differences in expression between the two species.

Y353/B: a candidate multiple-copy spermiogenesis gene on the mouse Y chromosome.

There is evidence from Y Chromosome (Chr) deletion mapping that there is a gene on the long arm of the mouse Y Chr that is needed for the normal development of the sperm head. Since mice with partial Y long arm deletions show incomplete penetrance of the sperm head defect, whereas mice with no Y long arm show complete penetrance, it has been suggested that the 'spermiogenesis' gene may be present in multiple copies. A Y-specific genomic DNA sequence (Y353/B) has previously been described that is present in multiple copies on the long arm of the mouse Y and identifies testis-specific transcripts. We have suggested that Y353/B could be the proposed multiple copy 'spermiogenesis' gene. In support of this suggestion, we show here that mice with a partial Y long arm deletion associated with a 3.5-fold increase in the frequency of abnormal sperm heads have a marked reduction in genomic Y353/B copies and a corresponding reduction in Y353/B-related transcripts. Thus, the incompletely penetrant phenotype correlates with a reduction in Y353/B-related transcription. Furthermore, by in situ hybridization with a Y353/B riboprobe to testis sections, we show that the Y353/B-related transcripts are confined to the round spermatid stage of spermiogenesis, just prior to the shaping of the sperm head. The transcripts sediment with the fraction of cytoplasmic RNA in adult testis that is loaded on polysomes, suggesting that the transcripts are actively translated.