Sex Reversal Factor Research Articles

Only three Y chromosome genes are enough for obtaining sperm functional in assisted fertilization and yielding live offspring in the mouse

Recently, we have shown that live mice can be obtained from males with the Y chromosome contribution limited to only two genes, the testis determinant factor Sry and the spermatogonial proliferation factor Eif2s3y when round spermatid injection (ROSI) is used. ROSI efficiency, measured as proportion of offspring from embryos transferred was <10%. When ROSI was attempted with males, in which Sry was substituted for sex reversal factor Sxrb encoding Zfy2/1, Sry, H2al2y and Rbmy genes, the efficiency increased 2-fold.

The Y-Encoded Gene Zfy2 Acts to Remove Cells with Unpaired Chromosomes at the First Meiotic Metaphase in Male Mice

SummaryDuring male but not female mammalian meiosis, there is efficient apoptotic elimination of cells with unpaired (univalent) chromosomes at the first meiotic metaphase (MI) [1]. Apoptotic elimination of MI spermatocytes is seen in response to the univalent X chromosome of XSxraO male mice [2], in which the X chromosome carries Sxra [3, 4], the Y-chromosome-derived sex-reversal factor that includes the testis determinant Sry. Sxrb is an Sxra-derived variant in which a deletion has removed six Y short-arm genes and created a Zfy2/Zfy1 fusion gene spanning the deletion breakpoint [4, 5]. XSxrbO males have spermatogonial arrest that can be overcome by the re-addition of Eif2s3y from the deletion as a transgene; however, XSxrbOEif2s3y transgenic males do not show the expected elimination of MI spermatocytes in response to the univalent [6]. Here we show that these XSxrbOEif2s3y males have an impaired apoptotic response with completion of the first meiotic division, but there is no second meiotic division. We then show that Zfy2 (but not the closely related Zfy1) is sufficient to reinstate the apoptotic response to the X univalent. These findings provide further insight into the basis for the much lower transmission of chromosomal errors originating at the first meiotic division in men than in women [7].

Live Offspring from Mice Lacking the Y Chromosome Long Arm Gene Complement1

The mouse Y chromosome long arm (Yq) comprises approximately 70 Mb of repetitive, male-specific DNA together with a short (0.7-Mb) pseudoautosomal region (PAR). The repetitive non-PAR region (NPYq) encodes genes whose deficiency leads to subfertility and infertility, resulting from impaired spermiogenesis. In XSxr(a)Y*(X) mice, the only Y-specific material is provided by the Y chromosome short arm-derived sex reversal factor Sxr(a), which is attached to the X chromosome PAR; these males (NPYq- males) produce sperm with severely malformed heads and are infertile. In the present study, we investigated sperm function in these mice in the context of intracytoplasmic sperm injection (ICSI). Of 261 oocytes injected, 103 reached the 2-cell stage, and 46 developed to liveborn offspring. Using Xist RT-PCR genotyping as well as gamete and somatic cell karyotyping, all six predicted genotypes were identified among ICSI-derived progeny. The sex chromosome constitution of NPYq- males does not allow production of offspring with the same genotype, but one of the expected offspring genotypes is XY*(X)Sxr(a) (NPYq-(2)), which has the same Y gene complement as NPYq-. Analysis of NPYq-(2) males revealed they had normal-sized testes with ongoing spermatogenesis. Like NPYq- males, these males were infertile, and their sperm had malformed heads that nevertheless fertilized eggs via ICSI. In vitro fertilization (IVF), however, was unsuccessful. Overall, we demonstrated that a lack of NPYq-encoded genes does not interfere with the ability of sperm to fertilize oocytes via ICSI but does prevent fertilization via IVF. Thus, NPYq-encoded gene functions are not required after the sperm have entered the oocyte. The present work also led to development of a new mouse model lacking NPYq gene complement that will facilitate future studies of Y-encoded gene function.

Fertility in mice requires X-Y pairing and a Y-chromosomal “Spermiogenesis” gene mapping to the long arm

There is accumulating evidence that the mammalian Y chromosome, in addition to its testis-determining function, may have other male limited functions, particularly in spermatogenesis. We have previously shown that the short arm of the mouse Y carries information needed for spermatogonial proliferation. This information, together with the testis-determining gene Sry, is contained within the Y-derived sex reversal factor Sxr a. XO males carrying a copy of Sxr a attached to the X chromosome are nevertheless sterile owing to an almost complete arrest during the meiotic metaphase stages. Here we show that this meiotic block can be overcome by providing a meiotic pairing partner (with no Y-specific DNA) for the XSxr a chromosome. However, this does not restore fertility because the sperm produced all have abnormal heads. It is concluded that the Y-specific region of the mouse Y chromosome long arm includes information essential for the normal development of the sperm head.

Illegitimate pairing of the X and Y chromosomes in Sxr mice

X/Y male mice carrying the sex reversal factor, Sxr, on their Y chromosomes typically produce 4 classes of progeny (recombinant X/X Sxr male male and X/Y non-Sxr male male, and non-recombinant X/X female female and X/Y Sxr male male) in equal frequencies, these deriving from obligatory crossing over between the chromatids of the X and Y during meiosis. Here we show that X/Y males that, exceptionally, carry Sxr on their X chromosome, rather than their Y, produce fewer recombinants than expected. Cytological studies confirmed that X-Y univalence is frequent (58%) at diakinesis as in X/Y Sxr males, but among those cells with X-Y bivalents only 38% showed normal X-Y pseudo-autosomal pairing. The majority of such cells (62%) instead showed an illegitimate pairing between the short arms of the Y and the Sxr region located at the distal end of the X, and this can be understood in terms of the known homology between the testis-determining region of the Y short arm and that of the Sxr region. This pairing was sufficiently tenacious to suggest that crossing over took place between the 2 regions, and misalignment and unequal exchange were suggested by indications of bivalent asymmetry. Metaphase II cells deriving from meiosis I divisions in which the normal X-Y exchange had not occurred were also found. The cytological data are therefore consistent with the breeding results and suggest that normal pseudo-autosomal pairing and crossing over is not a prerequisite for functional germ cell formation.(ABSTRACT TRUNCATED AT 250 WORDS)

Sex chromosome pairing patterns in male mice of novel Sxr genotypes.

The influence of the sex-reversal factor (Sxr) on X and Y chromosome pairing was examined by comparing males with "novel" and "standard" Sxr genotypes. The novel Sxr males were exceptional in carrying Sxr on their X rather than their Y chromosome, or homozygously on both their X and Y chromosomes, or on a Y chromosome of different origin to that on which the factor arose. Regardless of its chromosomal location, Sxr was found to elevate the frequency of X-Y separation. Univalent X and Y chromosomes were observed to undergo self-association in a variable proportion of spermatocytes of all Sxr-carrying males. There was a suggestion that chromosomal location of the factor could influence the frequency of univalent self-association. Our observations do not support the published hypothesis of Y self-pairing as the cause of the elevated rate of X-Y separation at pachytene in Sxr-carrying males. Rather, they suggest that heterozygosity due to the presence of Sxr in the XY pairing region may be sufficient to disrupt pairing and cause univalence, or alternatively, that Sxr is an inefficient promoter of X-Y pairing initiation.

Intersex mice composed of androgen insensitive Tfm and wild-type cells analysed by 3H dihydrotestosterone autoradiography.

The X-linked testicular feminization mutation (Tfm) in the mouse is characterized by an androgen receptor defect. Due to random X-chromosome inactivation, XTfm/X+ heterozygotes are mosaics with respect to Tfm. They are composed of androgen receptor deficient XTfm cells and normal X+ wild-type cells. If Tfm heterozygotes are converted to XX males by the sex reversal factor (Sxr) the mosaicism is expressed. Therefore in sex reversed Tfm heterozygotes (XTfm/X+-Sxr) intersexual sex organs develop. In five intersexes with small male accessory glands and hypospadia and one heavily feminized intersex with vagina and caudally dislocated deferent ducts the mosaic is visualized by 3H-DHT-autoradiography. In the epididymis differentiated wild-type cells show nuclear labeling, whereas undifferentiated Tfm cells are unlabeled. Unlabeled Tfm cells are also encountered in the vesicular glands of the heavily feminized animal, demonstrating that Tfm cells can participate in the formation of male sex glands. The urethral glands of the mosaic animals are composed of unlabeled Tfm lobules exhibiting the female phenotype of the glands, and of labeled wild-type lobules exhibiting the male phenotype. Formation of a vagina and deviation of the deferent ducts is correlated with lack of androgen binding sites in the connective tissue.

Androgen receptor-deficient Tfm cells in the mosaic epididymis of sex-reversed mice heterozygous for Tfm: an autoradiographic study with [3H]-dihydrotestosterone and [3H]-estradiol.

Testicular feminization (Tfm) in the mouse is characterized by androgen insensitivity of the target cells. We describe the presence of androgen-insensitive Tfm cells in the epididymis of mosaic mice produced by converting female carriers of the Tfm mutation (XTfm/X+) to males via the sex reversal factor (Sxr). The mosaic arises by random X-inactivation. In the epididymal duct, flat undifferentiated Tfm cells are interspersed between high columnar wild-type cells. By thaw-mount autoradiography we show that after injection of [3H]dihydrotestosterone, radioactivity is concentrated in the nuclei of high columnar wild-type cells, whereas the nuclei of the low cuboid Tfm cells remain free of nuclear radioactivity. After injection of [3H]estradiol, both Tfm and wild-type cells show nuclear labeling. Our observations demonstrate that Tfm cells in the mosaic epididymis selectively lack nuclear dihydrotestosterone-binding sites, whereas estradiol-binding sites are intact.

Meiosis in Sxr male mice: II. Further absence of cytological evidence for a Y-autosome rearrangement in sex-reversed (Sxr) mice.

If the sex-reversal factor (Sxr) in mice is represented by a segment of the Y chromosome translocated on to an autosome, this segment might be expected to show some evidence of attempted pairing with the normal Y chromosome at the first meiotic division. Our failure to find such evidence in a light microscope study of the silver-stained pachytene spermatocytes from Sxr/4, XY males further supports existing cytological information that the factor is more likely to be represented by an autosomal dominant gene mutation.

Meiotic studies in mice carrying the sex reversal (Sxr) factor.

A sex reversal factor (Sxr) that causes mice having apparently normal X chromosomes to become phenotypically male is transmitted in an autosomal pattern. The origin of the Sxr factor is still unknown. It seems most likely that it has originated from an autosomal gene mutation or is the result of a translocation of part of the Y chromosome to one of the autosomes. Chromosomes from four XY and six XO mice carrying this sex reversal factor were examined in the diakinesis stage of meiosis. The following unusual observations were noted: (1) in XY males carrying the Sxr factor, the X and Y chromosomes were separated more often than in controls. (2) The Y chromosome tends to be closer to an autosome when the X and Y are separate than when the X and Y are attached. (3) A chromosome fragment was present in 4/226 cells from two XO males and a single cell from an XY, Sxr carrier. Although there is no direct evidence, these observations seem to favor the possibility that the Sxr factor involves a chromosomal rearrangement rather than a single gene mutation.

Sex-chromosome aberrations in wood lemmings (&lt;i&gt;Myopus schisticolo&lt;/i&gt;&lt;i&gt;r&lt;/i&gt;)

The wood lemming displays certain peculiar features: (1) The sex ratio shows a prevalence of females (FRANK, 1966; KALELA and OKSALA, 1966), and some females produce only female offspring (KALELA and OKSALA, 1966). (2) In a considerable proportion (in the present material, slightly less than half) of the females, an XY chromosome complement is found in the somatic tissues, but the Y is absent in the germ line of those studied (Fredga et al., 1976). Therefore, (3) a mechanism of double nondisjunction in early fetal life of XY females has to be postulated, which replaces the Y in the germ line by duplication of the X. It is assumed (4) that the X of XY females bears a sex-reversal factor that affects the male determining action of the Y (Fredga et al., 1977). There is (5) a strong presumption that in most cases the XY females are those that produce daughters only, but (6) a few exceptions may occur (FRANK, unpublished observations), suggesting that the regulation according to assumption 3 (perhaps also to 4) is incomplete in XY females. In the present report, four females are described with a 31,XO karyotype, two females with 33,XYY or 32,XY/33,XYY, respectively, two males with a 33,XXY, and one male with a 32,XX/33,XXY karyotype, as observed in a consecutive series of 502 wood lemmings. The incidence of sex-chromosome anomalies in liveborn and adult animals was 2.3%; the overall incidence, including embryos, was 1.79%. Neither the somatic XO constitution nor the existence of an extra Y in females precludes fertility. However, the XXY condition in the male results in sterility. There is certain evidence that an instability of the proposed mechanism for double mitotic nondisjunction of the sex chromosomes in oogonia accounts for the high rate of sex-chromosome aberrations in wood lemmings, at least when the mother is XY.