The expression of Y-linked Zfy2 in XY mouse oocytes leads to frequent meiosis 2 defects, a high incidence of subsequent early cleavage stage arrest and infertility.

Outbred XY(Sry-) female mice that lack Sry due to the 11 kb deletion Sry(dl1Rlb) have very limited fertility. However, five lines of outbred XY(d) females with Y chromosome deletions Y(Del(Y)1Ct)-Y(Del(Y)5Ct) that deplete the Rbmy gene cluster and repress Sry transcription were found to be of good fertility. Here we tested our expectation that the difference in fertility between XO, XY(d-1) and XY(Sry-) females would be reflected in different degrees of oocyte depletion, but this was not the case. Transgenic addition of Yp genes to XO females implicated Zfy2 as being responsible for the deleterious Y chromosomal effect on fertility. Zfy2 transcript levels were reduced in ovaries of XY(d-1) compared with XY(Sry-) females in keeping with their differing fertility. In seeking the biological basis of the impaired fertility we found that XY(Sry-), XY(d-1) and XO,Zfy2 females produce equivalent numbers of 2-cell embryos. However, in XY(Sry-) and XO,Zfy2 females the majority of embryos arrested with 2-4 cells and almost no blastocysts were produced; by contrast, XY(d-1) females produced substantially more blastocysts but fewer than XO controls. As previously documented for C57BL/6 inbred XY females, outbred XY(Sry-) and XO,Zfy2 females showed frequent failure of the second meiotic division, although this did not prevent the first cleavage. Oocyte transcriptome analysis revealed major transcriptional changes resulting from the Zfy2 transgene addition. We conclude that Zfy2-induced transcriptional changes in oocytes are sufficient to explain the more severe fertility impairment of XY as compared with XO females.

Clinical, cytogenetic, and molecular analysis with 46,XX male sex reversal syndrome: case reports.

PURPOSE: To investigate the clinical characteristics of different categories of sex-reversed 46,XX individuals and their relationships with chromosomal karyotype and the SRY gene. METHODS: Chromosome karyotyping for peripheral blood culture and multi-PCR and FISH were performed. RESULTS: Endocrinological data showed that their endocrine hormone levels were similar to that observed for Klinefelter syndrome, with higher FSH and LH levels and lower T levels. Chromosome karyotyping for peripheral blood culture revealed 46, XX complement for 11 males. Molecular studies showed that there were locus deletions at SY84, SY86, SY127, SY134, SY254 and SY255 in AZF on chromosome Y in 9 cases, with the SRY gene present at the terminus of the X chromosome short arm. In one case, besides 6 locus deletions in AZF, there was also SRY gene deletion. In another case, there were locus deletions only at SY254 and SY255, with SY84, SY86, SY127 SY134 loci and SRY present. CONCLUSIONS: The majority (10/11) of 46,XX males were SRY positive, with the SRY gene translocated into the terminus of the X chromosome short arm. These patients were caused mainly by an X/Y chromosomal inter-change during paternal meiosis, leading to the differentiation of primary gonads into testes. Only a single patient (1/11) was SRY-negative, in which there might be some unknown downstream genes involved in sex determination.

Hernia uteri inguinalis in a case of ovotesticular disorder of sexual differentiation.

An 18-year-old phenotypic male presented with an irreducible left inguinal mass, gynecomastia, and hypospadias. This mass on exploration was found to be a nonfunctional uterus with ipsilateral ovary and was excised. Further investigation confirmed the presence of a contralateral testis and a genotype of 46, XX. This confirmed the diagnosis of ovotesticular disorder of sexual differentiation (formerly true hermaphroditism) with obstructed hernia uteri inguinalis. The patient was raised as a male. Subcutaneous mastectomy for gynecomastia and neourethra construction with full thickness skin graft for hypospadias were performed. Hernia uteri inguinalis is rarely seen in this condition with only 2 cases being reported worldwide thus far, including our case.

Sry-negative XX true hermaphroditism in a roe deer.

A two-year-old roe deer was brought down in the course of a hunt in the north of Spain (Asturias). On physical examination the individual presented well-developed bared antlers, but surprisingly a female external genitalia. Several anatomical, histological and genetic analyses were performed in order to explain the observed phenotype. Necropsy evidenced ovary-like structures with follicles on the surface; histological analyses of testes evidenced positive immunolabel against testosterone in Leydig cells; genetic analyses showed that the sex of the individual was consistent with a female individual. PCR analysis failed to detect SRY sequences; no PIS deletion, which is responsible for XX sex-reversal in goats, was detected. On the basis of its presumptive normal female sexual karyotype (XX) and the presence of two functional abdominal bilateral testes and ovaries, the roe deer was finally diagnosed as possessing an XX hermaphroditism syndrome. However, as in many other cases, the specific reason for the occurrence of this case of hermaphroditism could not be determined.

[Effect of Sry silencing by siRNA on the expression of sex determining genes in mouse embryos].

In order to investigate Sry regulation network in the development of male embryo, we inhibited the Sry gene expression by RNAi and then examined expression of other sex-related genes. Six genes (Sox9, Wt1, Sf1, Dax1, Gata4 and Amh), which are suggested to be closely related to Sry regulation were studied. Two siRNA expression vectors pSilencer4.1/Sry217 and pSilencer4.1/Sry565 were constructed and injected into gestated mouse through tail vein at 9.5 day of conception (dpc). The inhibition efficiency of Sry and the expression of other six genes were examined in male embryos at 11.5 dpc by RT-PCR and Western-blot. Expressions of the other six genes were analyzed by fluorescence quantity PCR. The results indicated both the pSilencer4.1/Sry217 and pSilencer4.1/Sry565 could inhibit significantly increased after Sry silencing. In contrast, no significant changes were observed in the expression of Sf1, Amh, Gata4, Dax1 and Sox9 when silencing Sry by siRNA. Our results suggested that the Wt1 transcription was regulated by Sry, whereas the Sox9 expression is not directly regulated by Sry in the development of genital ridge.

Extended pedigree with multiple cases of XX sex reversal in the absence of SRY and of a mutation at the SOX9 locus.

It is well established that testicular differentiation of the human embryonic gonad depends on the action of the Y-chromosomal gene SRY. However, exceptional cases such as SRY-negative cases of 46,XX testicular disorder of sexual development (DSD), and of 46,XX ovotesticular DSD document that testicular tissue can develop in the absence of the SRY gene. These SRY-negative XX sex reversal cases are very rare and usually sporadic, but a few familial cases have been reported. We present a large, consanguineous family with nine affected individuals with phenotypes ranging from 46,XX testicular DSD to 46,XX ovotesticular DSD, with predominance of male characteristics. Absence of SRY in peripheral blood was documented by fluorescence in situ hybridization (FISH) and PCR analysis in all nine affected individuals, and by FISH analysis on gonadal sections with testicular tissue in four affected individuals. By quantitative PCR, a duplication of the SOX9 gene was excluded. In addition, as linkage analysis showed that the nine affected members of the family do not share a common SOX9 haplotype, any mutation at the SOX9 locus could be ruled out. Together, these findings implicate a mutation at a sex-determining locus other than SRY and SOX9 as the cause for the XX sex reversal trait in this family.