Sex-reversed Mice Research Articles

Live Birth in Sex-Reversed XY Mice Lacking the Nuclear Receptor Dax1

The nuclear hormone receptor Dax1 functions during development as a testes-determining gene. However, the phenotype of male mice lacking Dax1 is strain-dependent due to the background-specific abundance of male-determining Sry gene-transcripts. We hypothesised that inter-individual variation in Sry mRNA-abundance would result in a spectrum of phenotypes even within-strain. We found that while all XY C57BL/6J mice lacking Dax1 presented as phenotypic females, there was a marked inter-individual variability in measures of fertility. Indeed, we report rare occasions where sex-reversed mice had measures of fertility comparable to those in control females. On two occasions, these sex-reversed XY mice were able to give birth to live offspring following mating to stud-males. As such, this work documents within-strain variability in phenotypes of XY mice lacking Dax1, and reports for the first time a complete sex-reversal capable of achieving live birth in these mice.

Of sex and determination: marking 25 years of Randy, the sex-reversed mouse.

On Thursday 9 May 1991, the world awoke to front-page news of a breakthrough in biological research. From Washington to Wollongong, newspapers, radio and TV were abuzz with the story of a transgenic mouse in London called Randy. Why was this mouse so special? The mouse in question was a chromosomal female (XX) made male by the presence of a transgene containing the Y chromosome gene Sry This sex-reversal provided clear experimental proof that Sry was the elusive mammalian sex-determining gene. Twenty-five years on, we reflect on what this discovery meant for our understanding of how males and females arise and what remains to be understood.

Gonadal development.

The commitment of the embryonic gonad towards the male or female fate is a sequential and complex developmental process. The sex-independent growth and development of the adrenogonadal primordium into the bipotential gonadal ridge is committed to the formation of testis in the presence of the SRY gene on the Y chromosome. SRY upregulates the expression of SOX9 that sets into motion a cascade of complex genetic interactions for the formation of male internal and external genitalia whilst repressing the formation of female genitalia. The initiation and maintenance of somatic sex of the gonad as either male or female is achieved by suppression of the alternate fate. However, at least in mice, the primary sex-determining decision is not final but is maintained in adulthood by a mutually antagonistic double-repressive pathway. In the human, any imbalance between these two antagonistic genetic and physiological pathways results in inappropriate gonad differentiation and function leading to disorders of sex development (DSD). Genetic analysis of individuals presenting with DSD and sex-reversed mice has revealed a number of sexually dimorphic genes that are involved in the formation of mammalian gonads, which are discussed in this chapter. Despite an increase in the knowledge of genes involved in mammalian sex determination, the molecular mechanisms remain by and large undetermined. The use of novel 'omics' technologies for analyzing a large number of patients with DSD, and careful assessment of the resulting datasets may result in the identification of novel genetic factors in human sex determination and lead to the development of novel ex vivo cellular models.

The behavior of the X- and Y-chromosomes in the oocyte during meiotic prophase in the B6.YTIR sex-reversed mouse ovary

Sexual differentiation of the germ cells follows gonadal differentiation, which is determined by the presence or the absence of the Y-chromosome. Consequently, oogenesis and spermatogenesis take place in the germ cells with XX and XY sex chromosomal compositions respectively. It is unclear how sexual dimorphic regulation of meiosis is associated with the sex-chromosomal composition. In the present study, we examined the behavior of the sex chromosomes in the oocytes of the B6.Y(TIR) sex-reversed female mouse, in comparison with XO and XX females. As the sex chromosomes fail to pair in both XY and XO oocytes during meiotic prophase, we anticipated that the pairing failure may lead to excessive oocyte loss. However, the total number of germ cells, identified by immunolabeling of germ cell nuclear antigen 1 (GCNA1), did not differ between XY and XX ovaries or XO and XX ovaries up to the day of delivery. The progression of meiotic prophase, assessed by immunolabeling of synaptonemal complex components, was also similar between the two genotypes of ovaries. These observations suggest that the failure in sex-chromosome pairing is not sufficient to cause oocyte loss. On the other hand, labeling of phosphorylated histone gammaH2AX, known to be associated with asynapsis and transcriptional repression, was seen over the X-chromosome but not over the Y-chromosome in the majority of XY oocytes at the pachytene stage. For comparison, gammaH2AX labeling was seen only in the minority of XX oocytes at the same stage. We speculate that the transcriptional activity of sex chromosomes in the XY oocyte may be incompatible with ooplasmic maturation.

Onset and progress of meiotic prophase in the oocytes in the B6.YTIR sex-reversed mouse ovary.

When the Y chromosome of a Mus musculus domesticus male mouse (caught in Tirano, Italy) is placed on a C57BL/6J genetic background, approximately half of the XY (B6.YTIR) progeny develop into normal-appearing but infertile females. We have previously reported that the primary cause of infertility can be attributed to their oocytes. To identify the primary defect in the XY oocyte, we examined the onset and progress of meiotic prophase in the B6.YTIR fetal ovary. Using bromo-deoxyuridine incorporation and culture, we determined that the germ cells began to enter meiosis at the developmental ages and in numbers comparable to those in the control XX ovary. Furthermore, the meiotic prophase appeared to progress normally until the late zygotene stage. However, the oocytes that entered meiosis early in the XY ovary failed to complete the meiotic prophase. On the other hand, a considerable number of oocytes entered meiosis at late developmental stages and completed the meiotic prophase in the XY ovary. We propose that the timing of entry into meiosis and the XY chromosomal composition influence the survival of oocytes during meiotic prophase in the fetal ovary.

Smcy transgene does not rescue spermatogenesis in sex-reversed mice.

In mouse, the Sxr(b) deletion interval (delta Sxr(b)) maps to the small short arm of the Y chromosome and is known to contain gene(s) required for normal spermatogenesis; in particular, Spy, which is essential for the postnatal mitotic proliferation of spermatogonia. This deletion interval is approximately 1-2 Mb and contains eight known genes. In this paper we report the construction of YAC transgenic mice containing different regions of the delta Sxr(b) interval including Zfy1, Ube1y, Smcy, and Eif2s3. Two male and one female founder mice, transgenic for all four genes, were sterile. However, a fertile transgenic, carrying a full-length copy of the Smcy gene integrated into central Chr 12, was identified. Smcy is a highly conserved Y chromosome-located gene, encoding peptides corresponding to epitopes of the male-specific antigen, H-Y. The Smcy transgene was ubiquitously expressed in all organs and tissues tested in male and female carriers. Introduction of the transgene into an X Sxr(b)/O genetic background did not rescue the early arrest of spermatogenesis characteristic of these males. These data indicate that the presence of Smcy is not sufficient to restore spermatogenesis, making it a highly unlikely candidate for Spy.

Follicular development and atresia in the B6.Y(TIR) sex-reversed mouse ovary.

The B6.Y(TIR) mouse fails to develop normal testes despite transcription of Sry, the primary testis-determining gene on the Y chromosome. Consequently, B6.Y(TIR) fetuses with bilateral ovaries develop into apparently normal but infertile females. This infertility can be mainly attributed to oocyte incompatibility for postfertilization development. In addition, abnormality in preovulatory follicles and rapid loss of oocytes have been observed in XY ovaries. This study examined the effect of gonadotropins on follicular development and atresia in B6.Y(TIR) prepubertal females. The results show that untreated XY females had fewer late preantral follicles and their frequency of atresia was lower. No other difference was found when they were compared with XX females. After treatment with gonadotropins for 24 h, frequency of atresia decreased in both XX and XY ovaries. After 48 h, most preovulatory follicles in XY ovaries were nonatretic, but the oocytes often were denuded. Immunocytochemical staining for connexin 43 detected punctate foci along the oocyte plasma membrane. The density of these foci changed during follicular development, which was similar in XX and XY ovaries. In conclusion, follicular development and atresia under the control of gonadotropins is not influenced by defective oocytes until the preovulatory phase.

Sertoli Cell Differentiation and Y-Chromosome Activity: A Developmental Study of X-Linked Transgene Activity in Sex-Reversed X/XSxraMouse Embryos

The requirement of Y-chromosome activity for the differentiation of somatic cells and germ cells was studied in the fetal gonads of X/XSxramouse embryos where the activity of the Sxrafragment of the Y chromosome is influenced by the inactivation and reactivation of the X chromosome. In the interstitial somatic cells, random inactivation of the X and the XSxrachromosomes took place which was revealed by the mosaic expression of an X-linkedlacZtransgene. The Sertoli cells, however, displayed a preferentially active XSxrachromosome and the presence of Sxra-active Sertoli cells was associated with the morphogenesis of testicular tubules in the sex-reversed gonads. The activity of the Y-chromosome fragment is therefore necessary for the differentiation of the Sertoli cells which may direct the development of the testis. The expression pattern of the X-linked transgene in X/XSxragerm cells suggests that both the X and the XSxrachromosomes are active. This finding suggests that the presence of Sxrahas no impact on the reactivation of the X chromosome in the germ cells and that the X chromosome can be reactivated even though the germ cells are found in the testicular environment. Our results are consistent with the concept that the activity of genes on the XSxrafragment is essential for the differentiation of Sertoli cells and the morphogenesis of the testis, but not for premeiotic differentiation of germ cells in sex-reversed mice.

Ott, a mouse X-linked multigene family expressed specifically during meiosis.

The tissue expression patterns of 10 mouse testis cDNAs were analysed by RT-PCR to search for new mammalian meiotic genes. The homologue of the rat synaptonemal complex protein gene SCP1 is expressed in embryonic ovary, adult brain and testis. One novel gene is stringently testis specific and another is expressed exclusively in testis and embryonic ovary. The latter clone is not expressed in the testes of adult sex-reversed mice which lack germ cells, and therefore represents a meiosis-specific gene. It is part of a mouse multigene family, members of which are clustered and map genetically and physically to a single region of the X chromosome. We have named this family Ott (ovary testis transcribed). Steady-state levels of a 2.3 kb polyadenylated Ott mRNA are high throughout meiotic prophase in the testis when the X chromosome is generally transcriptionally inactive. A second transcript of 1 kb is also detectable from 4 weeks of age onwards. The two mRNAs have different 3' ends and contain different protein coding information. At least seven Ott genes are transcribed specifically during meiosis and are predicted to encode "pioneer' proteins with an unusual structure, containing tandem arrays of a degenerate eight amino acid repeat. This work could lead to the identification of a human Ott homologue, which is likely to be X-linked and would provide a candidate locus for some cases of male infertility.

An RBM homologue maps to the mouse Y chromosome and is expressed in germ cells.

We have isolated a murine homologue of the human Y-linked RBM genes (previously termed YRRM), a gene family implicated in spermatogenesis and which encodes proteins containing an RNA recognition motif. A number of very similar copies of this gene (called Rbm) are present in the mouse. These mouse homologues are also Y-encoded, mapping on the short arm of the chromosome, proximal to Sry. Expression is confined to the testis, specifically the germ line on the basis of lack of expression in the germ-line negative testes of adult sex-reversed mice. The timing of Rbm transcription is regulated, with fetal message levels reaching a peak at 15 d.p.c. Transcripts are clearly detectable by 4 days after birth and reach their highest level at 14 d.p.p. which is the time at which the Y chromosome condenses during meiotic prophase. These results suggest that Rbm is functionally involved in germline RNA metabolism.

Normal Onset, but Prolonged Expression, of Sry Gene in the B6.YDOM Sex-Reversed Mouse Gonad

When the Y chromosome of the Mus musculus domesticus mouse (Tirano, Italy) is placed onto the C57BL/6J (B6) background, the XY progeny (B6.YDOM) develop only ovaries or ovotestes in fetal life. In the present study, we examined transcription of genes known to be involved in gonadal sex differentiation in the B6.YDOM gonad. RNA was isolated from each gonad at 10 to 18 days of gestation and subjected to RT-PCR analysis. The results indicate that the onset of Sry, a putative testis-determining gene, transcription was normal, whereas its inactivation was delayed in all B6.YDOM gonads. Transcripts of Müllerian inhibiting substance, the earliest biochemical product of fetal Sertoli cells, appeared in the B6.YDOM ovotestis slightly later than in the control B6, XY gonad, whereas they were either absent or detected transiently in the B6.YDOM ovary. Transcription of 3β-hydroxysteroid dehydrogenase and 17α-hydroxylase, essential for testosterone synthesis, were equally delayed in the B6.YDOM ovotestis and completely absent in the B6.YDOM ovary. P450 aromatase (P450arom), which converts androgens to estrogens, was transcribed in both control XX and XY gonads at the onset of morphological sexual differentiation. The overall pattern of P450arom transcription was delayed by 1 day in both B6.YDOM ovotestes and ovaries. In conclusion, the testis-determining pathway in the B6.YDOM gonad appears to be impaired downstream of Sry transcription, resulting in persistent Sry transcripts and a delay in onset of other genes involved in testicular differentiation.

The influence of male-specific genes on female muscle fiber types: studies on the sex-reversed (Sxr) mouse.

Previous experiments on the effects of either male castration or injection of androgens have concluded that levels of androgens are responsible for different muscle fiber type proportions between the sexes. However, these conclusions are based on invasive techniques which may involve secondary factors. The sex-reversed (Sxr) mouse is genetically female (X/X) but phenotypically male due to the presence of part of the short-arm of the Y chromosome containing the testis determining gene (Tdy). Serum testosterone in this mouse is in the low normal range and therefore provides a model for investigating the possible control of muscle fiber types by male specific genetic factors. Ten males, ten females, and ten Sxr mice of approximately 60 days of age were used, together with ten males weight-matched to the females. The animals were killed and biceps brachii and soleus muscles removed and prepared for routine muscle histochemistry. Body and muscle weights were similar in the males and Sxr mice and significantly greater than in the females and the weight-matched males. Muscle fiber sizes in biceps brachii reflected the differences in muscle weights and there were no significant differences in fiber type proportions for this muscle. In the soleus muscle, the percentage of slow, oxidative (SO) fibers was higher in the female mice than in any other group. Furthermore, although the fast, oxidative, glycolytic (FOG) fibers were larger in the heavier animals, SO fibers were largest in the female mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Endocrine differentiation of the XY sex-reversed mouse ovary during postnatal development

When the mouse Y chromosome of Mus musculus domesticus is placed onto the C57BL/6J genetic background, half of the XY progeny develop bilateral ovaries and the female phenotype, but lack regular estrous cyclicity and lose embryos after fertilization. In the present study, we compared the endocrinological activity of XY ovaries with XX ovaries during postnatal development by measuring steroids in the incubation medium by radioimmunoassay. At 1 day postpartum (d.p.p.), production of progesterone and estradiol was significant while testosterone was undetectable in both ovaries. At 14 and 35 d.p.p., amounts of testosterone and estradiol produced by XY ovaries were half of those by XX ovaries. Production of progesterone by XY ovaries was slightly higher than XX ovaries at 14 d.p.p., but only half of that at 35 d.p.p. Addition of gonadotropins increased testosterone production by XX ovaries but not by XY ovaries at either 14 or 35 d.p.p. Progesterone production in XY ovaries at 35 d.p.p. was increased by gonadotropins to a much lesser extent than in XX ovaries. Gonadotropins increased estradiol production similarly in both ovaries at 35 d.p.p. Striking differences were found in the histochemical distribution of 3β-hydroxysteroid dehydrogenase between XY and XX ovaries at 14, but not at 35 d.p.p. In conclusion, the XY ovary develops abnormal endocrine features during the postnatal period, which likely lead to the fertility problems at puberty.

A mouse zinc finger gene which is transiently expressed during spermatogenesis.

Zinc finger proteins are polypeptides with sequence-specific, nucleic acid-binding properties. Substantial evidence has established them as a class of trans-acting molecules with regulatory roles in cellular growth and differentiation. We have screened an 11.5 day post coitum urogenital ridge cDNA library with an oligonucleotide encoding a sequence conserved between a variety of zinc finger proteins. By cDNA cloning and sequencing we show that a novel mouse gene, Zfp-35, encodes a protein with a block of 18 zinc finger domains and an N-terminal region rich in acidic residues. The 2.4 kb mRNA encoding this polypeptide is selectively expressed in adult testis, by comparison with other organs. We have analysed Zfp-35 expression in whole testes of sex-reversed mice, whole testes of prepuberal XY animals, germ cell fractions from XY adult testes and by in situ hybridization to sections from adult XY testes. Our studies show that a considerable increase in expression is restricted to spermatocytes at the pachytene stage of meiotic prophase. These experiments suggest that Zfp-35 may act to control gene activity during this particular stage of spermatogenesis.

Where next for mammalian male-specific (H-Y) antigen(s)?

The use of sexual differentiation as a paradigm in the understanding d development has a long and honourable history. In mammals, the main outlines of the process (where androgens pr~luced by the ~erentiated testis bring about many features of male development) are well understood at both the biochemical and genetic levels ~'~. Unfortunately, the crucial element needed to complete the model is missing;, there is no agreement as to how the male-determining gone(s) on the ~ Y chromosome initiate the whole process by inducing the indifferent embryonic gonad to differentiate as a testis, which can then secrete the androgens. More than ten years ago, Wachtel, Ohno and colleagues proposed that H-Y (male speci~c) antigen, whose expressionisposithelyregulated by the Y chromosome, has a role as the effector inthis primary sex determination ~. Recent work on the sex-reversed mouse (Sxv) and a new derivative of it (Sxr') *'s, reviewed recently by Good,low ~ and Simpson ? makes it seem unlikely, except on rather extreme ~ ~ hypo. theses ~, that the H-Y antigen as detected by cytoto~c T-cell a ~ y s or by skin transplantation -l',~s arty such primary role. Yet this may not be the end of the story; there may be more than one male-specitic antiged. H-Y antigens are defined operationally as any substances that are detected by immunological techniques on the cell surface of cells taken from males and are apparently absent from females of the same mammalian species. It is not necessarily the case that different classes of immunological techniques will always see either the same molecule or the same part of the same molecule. Although the immune-response genes controllingtherejectionofskingrafts are different from those regulating cytotoxic T cells im v~o ~, there has never been any evidence ~hat these two techniques detect separable male-specific antigens s. Thus, the antigen detected by cell-mediated assays may fairly be called the H-Y antigen. In contrast, because of cases where some animals (most notably the X0 mou~) had been typed as positive by serological techniques but were nngative for H-Y antigen, it was suggested in 1982 that serological assays Where next for mammalian male-specific (H-Y) antigen(s)?

Androgen levels and androgenization in sex-reversed (XXSxr pseudomale) mouse: absence of initial segment of epididymis is independent of androgens.

The XXSxr pseudomale ("sex-reversed") mouse has an apparently normal male phenotype. This is in accordance with Jost's Principle that, in an animal with androgen-producing testes, phenotype will be fully masculinized, irrespective of chromosomal sex. However, the epididymis of this mutant lacks the Initial Segment. Several alternative explanations were proposed. In the present study we examined the primary of these, viz, androgen insufficiency. We studied androgen levels in serum and tissues of the mutant, and we investigated whether injection of exogenous androgens could induce normal development. The XXSxr pseudomale is not deficient in androgens, nor do exogenous additional androgens induce development of the Initial Segment. It would appear that the abnormal genotype acts on the organ phenotype without intervention of androgens.

Correlation between sexual phenotype and X-chromosome inactivation pattern in the X*XY wood lemming.

Replication patterns of the X chromosomes were studied in X*XY wood lemmings with male and female phenotypes. The wild-type X was late replicating (ie, inactivated) in all cells of the X*XY female, whereas the mutated X* was late replicating in all cells of the X*XY male. These findings are compared with those obtained in sex-reversed (Sxr) mice.

Spermatogenesis and sperm-specific phosphoglycerate kinase B and lactate dehydrogenase C4 isoenzymes in sex-reversed mice.

The activities of phosphoglycerate kinase B and lactate dehydrogenase C4 were correlated with the state of spermatogenesis in testes of sex-reversed (Sxr) mice. Both isoenzymes were present in Sxr/+,XO testes, which contained late spermatids. LDH-C4 was detected in testes from 5 of 9 Sxr/+,XX mice, but PGK-B was present in testes of only 1 of the 5 mice. Pachytene spermatocytes were the predominant cell types in those Sxr/+,XX testes that contained spermatogenic cells. These observations confirm that PGK-B is restricted to spermatogenic cells and is not present in somatic testicular cells. Furthermore, they suggest that PGK-B synthesis may be initiated later than pachytene, when synthesis of the LDH-C polypeptide is known to commence.

X-chromosome activity in the germ cells of sex-reversed mouse embryos.

Germ cells were isolated from XX ovaries and XY and XX Sex-reversed (Sxr/+) testes of mouse embryos 14-16 days post coitum, and the activity of an X-chromosome-coded enzyme, hypoxanthine phosphoribosyl transferase (HPRT), relative to an autosomal one, adenine phosphoribosyl transferase was determined. The ratio of enzyme activities in XX Sxr/+ prospermatogonia was significantly higher than that in XY prospermatogonia, up to 2-fold, suggesting that the silent X chromosome is reactivated in XX male germ cells before birth, as it is in female germ cells. The ratio was several times higher still in XX oocytes than in XX prospermatogonia, confirming that the increase in HPRT activity reported in oocytes is only partly due to an X-chromosome dosage effect.

Meiosis in Sxr male mice. I. Does a Y-autosome rearrangement exist in sex-reversed (Sxr) mice?

Cytological evidence for the existence of a Y-autosomal rearrangement in meiotic cells of Sxr mice has been sought. At peachytene, in silver-stained light microscope spread preparations of XY, Sxr/+ and XO, Sxr/+ spermatocytes, evidence for pairing or association between a possible Y-bearing segment of a specific autosome and a sex chromosome could not, however, be found. Such sex chromosome-autosome associations as were seen were non-specific in nature, and occurred no more often in Sxr mice than in controls.