ZFY Genes Research Articles

MhGeneS: An Analytical Pipeline to Allow for Robust Microhaplotype Genotyping.

Microhaplotypes are small linked genomic regions comprising two or more single-nucleotide polymorphisms (SNPs) that are being applied in forensics and are emerging in wildlife monitoring studies and genomic epidemiology. Typically, targeted in non-coding regions, microhaplotypes in exonic regions can be designed with larger amplicons to capture functional non-synonymous sites and minimise insertion/deletion (indel) polymorphisms. Quality control is an important first step for high-confidence genotyping to counteract such false-positive variants. As genetic markers with higher polymorphism compared to biallelic SNPs, it is critical to ensure sequencing errors across the microhaplotype amplicon are filtered out to avoid introducing false-haplotypes. We developed the MhGeneS pipeline which works in tandem with Seq2Sat to help validate microhaplotype genotyping of the coding region of genes, with broader applicability to any microhaplotype profiling. We genotyped microhaplotype regions of the Zfx (≅ 160 bp) and Zfy (≅ 140 bp) genes, as well as an exon of the prion protein (Prnp) gene (≅ 370 bp) in caribou (Rangifer tarandus) using paired-end Illumina technology. As important quality metrics affecting microhaplotype calling, we identified the sequencing error rate profile related to the overlap or non-overlap of paired-end reads as well as the read depth as significant. In the case of Prnp, we achieved confident microhaplotype calling through MhGeneS by removing small sections of the 5' and 3' amplicons and using a minimum read depth of 20. Read depth and sequence trimming may be locus-specific, and validation of these parameters is recommended before the high-throughput profiling of samples.

Unusual sex chromosomal DSD in a domestic Shorthair cat with a 37,X/38,XY mosaic karyotype

BackgroundSex chromosome abnormalities associated with disorders of sexual development (DSD) are rarely described in cats, mainly due to the lack of chromossome studies that precisely reveal the condition. Genetic approaches are therefore required in order to detect sex chromossomes abnormalities as variations in the number and structure of chromosomes, or the presence of a second cell line as mosaicim or chimerism.Case presentationA male Shorthair cryptorchid cat was presented with clinical signs of anorexia, tenesmus and hyperthermia. Ultrasonography revealed a fluid-filled structure, with approximately 1 cm in diameter, adjacent to the descending colon. Computed tomography evidenced a tubular structure, ventral to the descending colon and caudal to the bladder, which extended cranially, through two branches. Histopathological evaluation confirmed the presence of two atrophic uterine horns and one hypoplastic testicle with epididymis at the end of one of the uterine horns. The end of the other uterine horn was attached to a structure composed by a mass of adipocytes. Cytogenetic analysis revealed a mosaic 37,X/38,XY karyotype. The two cell lines were found in 15% and 85% of the lymphocytes, respectively. Genetic analysis confirmed the presence of SRY and ZFY genes in blood and hair bulbs, and revealed a marked reduction in SRY expression in the testicle. Additionally, this case presented exceptionally rare features, such as a Leydig’ cell tumour and a chronic endometritis in both uterine horns.ConclusionsComplete imaging workup, cytogenetic analysis and SRY gene expression should be systematically realized, in order to properly classify disorders of sexual development (DSD) in cats.

Return of the forgotten hero: the role of Y chromosome-encoded Zfy in male reproduction.

The Y-linked zinc finger gene ZFY is conserved across eutherians and is known to be a critical fertility factor in some species. The initial studies of the mouse homologues, Zfy1 and Zfy2, were performed using mice with spontaneous Y chromosome mutations and Zfy transgenes. These studies revealed that Zfy is involved in multiple processes during spermatogenesis, including removal of germ cells with unpaired chromosomes and control of meiotic sex chromosome inactivation during meiosis I, facilitating the progress of meiosis II, promoting spermiogenesis, and improving assisted reproduction outcomes. Zfy was also identified as a key gene in Y chromosome evolution, protecting this chromosome from extinction by serving as the executioner responsible for meiosis surveillance. Studies with targeted Zfy knock-outs revealed that mice lacking both homologues have severe spermatogenic defects and are infertile. Based on protein structure and in vitro assays, Zfy is expected to drive spermatogenesis as a transcriptional regulator. The combined evidence documents that the presence of at least one Zfy homologue is required for male fertility and that Zfy2 plays a more prominent role. This knowledge reinforces the importance of these factors for mouse spermatogenesis and informs our understanding of the human ZFY variants, which are homologous to the mouse Zfy1 and Zfy2.

Evaluation of protein expression of microchimeric Y-chromosome in ovarian endometriosis

Introduction: Endometriosis is a benign chronic gynaecological disorder with the presence of endometrium-like tissue outside the uterus. It affects 10% of the reproductive age group of women. The symptoms include infertility 30 – 50%, and dysmenorrhea 40 – 60%. Thus, endometriosis is a global burden with a wide range of symptomatology and hence causes difficulty for women who suffer from the disease. But the pathogenesis of the disease is yet to be understood. The presence of fetal Y-chromosome microchimerism in endometriosis is first reported using the FISH (Fluorescence In-Situ Hybridization) technique. Later, with a similar technique, conflicting results have been reported. The detection probes used were Y-specific chromosomes Yp11.1 – q11.1-Alpha Satellite DNA and Yq12 Satellite III DNA. The genomic evidence of Y chromosome microchimerism is evaluated using microarray expression followed by RNA-seq and quantitative RT-PCR experiments of target inserts for selected targets for further validation. This study reported the presence of 21 non-coding, and 33 protein-coding genes present in the Male Specific region of Y-chromosome (MSY) in ovarian endometriosis. Genomic studies have been reported in the literature but protein expression from Y-chromosome genes in ovarian endometriosis has not been reported. So, we aimed to evaluate protein expressions of EIF1aY, NLGN4Y, and ZFY in the eutopic endometrium of ovarian endometriosis. Method: The 2 study groups are allotted; Group A (Control) and Group B (Endometriosis). The tissue samples collected were annotated, processed, and performed western blot for identification of EIF1aY, ZFY, and NLGN4Y gene expression. The blots were scanned and analyzed using Bio-Rad Pharos FX TM Plus Molecular Imager and NIH Image J software ( http://imagej.nih.gov/ij/ ) respectively. Result: There was a significant increase in the gene expression of EIF1aY in the endometriosis group when compared to the control group (p < 0.01). The Spearman correlation scores for NLGN4Y and ZFY are strongly associated (r = 0.617). Conclusion: The ectopic site promotes the niche and eutopic tissue has an error which enhances the histogenesis. It appears from the present study that proteins coded from Y-chromosome genes, namely EIF1aY, NLGN4Y and ZFY are involved in the pathogenesis of ovarian endometriosis, which could be a potential therapeutic target in future. To the best of our knowledge, this is the first report suggesting the involvement of Y-microchimerism at the protein level in endometriosis. Further studies with larger sample sizes and different stages and phenotypes of endometriosis may bring forth more conclusive knowledge in this regard that seems to promote the histogenesis of endometriosis at ectopic sites. Department of Physiology, AIIMS, New Delhi. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Genetic variation in Zfy final intron region on Y-chromosome of Canis aureus, Canis lupus and Vulpes vulpes and sex-determination for samples of three canid species

The proportion of females and males in the wild can be a result of past ecological impacts on mammalian populations, and it can offer valuable insights into the evolutionary history, demographics, and behavior of mammalian species. We aimed to determine sex of road-killed samples belonging to three canid species (Canis aureus, C. lupus and Vulpes vulpes from Türkiye, and V. vulpes from Iran), and to reveal whether there was genetic variation within and among three species by sequencing the Zfy final intron region on Y–chromosome. When sex-determination testing was performed on 109 canid samples using a specific primer pair for the Zfx and Zfy genes, two specific DNA bands were detected in males while one DNA band was detected in females. The ratio of males was relatively lower than females. This indicated that there were clearly differences between females and males in terms of the band patterns. We also sequenced partially the Zfy final intron region for V. vulpes, C. aureus and C. lupus; 539 bp, 546 bp and 562 bp, respectively. When analyzed together the partial Zfy sequences, there was no variation among the sequences of each canid species and no haplotype was shared among three species. No phylogeographic structure was observed for each species throughout the sampling areas due to the partial Zfy final intron region highly being conserved. This might be resulted from gene flow due to an extensive male-biased dispersal presumably have homogenized the genetic variation in the Y–chromosome throughout the distributional areas of the canid species. In phylogenetic tree, the Canis species were clustered together and the Vulpes species were grouped in basale position and this was compatible with previous studies.

ミトコンドリアDNA D-loop 領域およびY 染色体<I>ZFY</I> 遺伝子を用いた ラオス在来スイギュウ集団の遺伝的多様性

In Laos, water buffaloes are traditionally used as draft animals, but their role as movable property for wealth accumulation has increased in recent years. However, little is known about the genetic makeup of these water buffaloes. This study aimed to clarify the gene composition and genetic diversity of four native water buffalo populations in Laos using the mtDNA D-loop region as maternal information and the ZFY gene on the Y chromosome as paternal information. A total of 82 D-loop sequences from four populations were compared, and 18 haplotypes with 36 substitution sites were identified. From the neighbor-joining phylogenetic tree and the median-joining network, it was confirmed that the Laos native water buffaloes belong to the swamp buffalo, and two lineages were clarified. It was also found that the nucleotide sequence of the ZFY gene was identical to that of the swamp buffalo and that there were two haplotypes. Both the neutrality test and the analysis of molecular variance suggested that the population size of Houaphan and Attapeu had decreased in the past. These results indicate that the native water buffaloes of Laos are a genetically diverse population with at least two maternal and paternal ancestors each.

XX/XY Chimerism in Internal Genitalia of a Virilized Heifer.

Five DSD heifers underwent genetic analysis in the present study. We cytogenetically analyzed in vitro cultured leukocytes and searched for SRY, AMELX/AMELY and ZFX/ZFY genes in leukocytes and hair follicles, finding that four of the studied heifers were freemartins (XX/XY leukocyte chimerism). The fifth case had an underdeveloped vulva localized ventrally and cranially to the mammary gland, a normal female sex chromosome complement (60,XX) in the leukocytes, and a lack of Y-chromosome-derived genes in the leukocytes and hair follicles. Postmortem anatomical examination of this heifer revealed the presence of normal ovaries with follicles, uterus, and oviducts, but molecular detection of the SRY, ZFX, ZFY,AMELX, and AMELY genes in these organs indicated the presence of a cell line carrying the Y chromosome. Further analysis of twelve microsatellite markers revealed the presence of additional variants at six loci in DNA samples derived from the reproductive organs; XX/XY chimerism was thus suspected in these samples. On the basis of the detection of AMELY (Y-linked) versus AMELX (X-linked) and SOX9 (autosomal) versus AMELY genes by droplet digital PCR (ddPCR), the Y/X and Y/autosome ratios were evaluated; they indicated the presence of XX and XY cell lines in the reproductive tissues. Our study showed that XX/XY chimerism can be present in the internal reproductive organs of the virilized heifers with a normal female set of sex chromosomes (60,XX) and a lack of Y-chromosome-derived genes in the leukocytes. The etiology of this phenomenon remains unknown.

Geographical contrasts of Y-chromosomal haplogroups from wild and domestic goats reveal ancient migrations and recent introgressions.

By their paternal transmission, Y-chromosomal haplotypes are sensitive markers of population history and male-mediated introgression. Previous studies identified biallelic single-nucleotide variants in the SRY, ZFY and DDX3Y genes, which in domestic goats identified four major Y-chromosomal haplotypes, Y1A, Y1B, Y2A and Y2B, with a marked geographical partitioning. Here, we extracted goat Y-chromosomal variants from whole-genome sequences of 386 domestic goats (75 breeds) and seven wild goat species, which were generated by the VarGoats goat genome project. Phylogenetic analyses indicated domestic haplogroups corresponding to Y1B, Y2A and Y2B, respectively, whereas Y1A is split into Y1AA and Y1AB. All five haplogroups were detected in 26 ancient DNA samples from southeast Europe or Asia. Haplotypes from present-day bezoars are not shared with domestic goats and are attached to deep nodes of the trees and networks. Haplogroup distributions for 186 domestic breeds indicate ancient paternal population bottlenecks and expansions during migrations into northern Europe, eastern and southern Asia, and Africa south of the Sahara. In addition, sharing of haplogroups indicates male-mediated introgressions, most notably an early gene flow from Asian goats into Madagascar and the crossbreeding that in the 19th century resulted in the popular Boer and Anglo-Nubian breeds. More recent introgressions are those from European goats into the native Korean goat population and from Boer goat into Uganda, Kenya, Tanzania, Malawi and Zimbabwe. This study illustrates the power of the Y-chromosomal variants for reconstructing the history of domestic species with a wide geographical range.

Loss of mouse Y chromosome gene Zfy1 and Zfy2 leads to spermatogenesis impairment, sperm defects, and infertility.

Using mice with Y chromosome deficiencies and supplementing Zfy transgenes, we, and others, have previously shown that the loss of Y chromosome Zfy1 and Zfy2 genes is associated with infertility and spermiogenic defects and that the addition of Zfy transgenes rescues these defects. In these past studies, the absence of Zfy was linked to the loss of other Y chromosome genes, which might have contributed to spermiogenic phenotypes. Here, we used CRISPR/Cas9 to specifically remove open reading frame of Zfy1, Zfy2, or both Zfy1 and Zfy2, and generated Zfy knockout (KO) and double knockout (DKO) mice. Zfy1 KO and Zfy2 KO mice were both fertile, but the latter had decreased litters size and sperm number, and sperm headshape abnormalities. Zfy DKO males were infertile and displayed severe spermatogenesis defects. Postmeiotic arrest largely prevented production of sperm and the few sperm that were produced all displayed gross headshape abnormalities and structural defects within head and tail. Infertility of Zfy DKO mice could be overcome by injection of spermatids or sperm directly to oocytes, and the resulting male offspring had the same spermiogenic phenotype as their fathers. The study is the first describing detailed phenotypic characterization of mice with the complete Zfy gene loss. It provides evidence supporting that the presence of at least one Zfy homolog is essential for male fertility and development of normal sperm functional in unassisted fertilization. The data also show that while the loss of Zfy1 is benign, the loss of Zfy2 is mildly detrimental for spermatogenesis.

Phylogenetic relationships between different raccoon dog (Nyctereutes procyonoides) populations based on four nuclear and Y genes.

The raccoon dog (Nyctereutes procyonoides), endemic to East Asia, is classified as six subspecies according to their geographical distribution including a population introduced to Europe. Studies on phylogenetic relationship or population genetics in both native and introduced areas have been carried out recently. Lately, opinions that Japanese raccoon dogs should be classified as a different species were asserted based on several studies using karyotypes, morphometric characters, mtDNA, and microsatellites analysis. However, no data pertaining to the nuclear DNA (nDNA) or Y chromosome are available. To estimate the relationship among the species using different genes is necessary in understanding of the history of this species. Therefore, we investigated nDNA and Y chromosomes in our study to define relationships: (1) between continental raccoon dog populations, (2) between original and introduced groups, and (3) between continental and Japanese groups. The analysis of four nuclear (CHRNA1, VTN, TRSP, WT1) and ZFY genes indicated that there had been no genetic differentiation among the continental populations. However, significant differences were observed between continental and Japanese raccoon dogs in VTN and ZFY genes implying genetic differentiation has been going between them. To better understand the phylogenetic relationship among raccoon dog populations, further study will be necessary.

Non-invasive prenatal diagnosis of foetal gender through maternal circulation in first trimester of pregnancy

Owing to the lack of perfect accuracy and sufficient sample size in previously performed studies on cell-free foetal DNA (cffDNA) for detection of foetal gender through maternal plasma, this study aimed to investigate the efficiency of using two Y-chromosome specific probes in foetal sex determination during first trimester of pregnancy. Five millilitres of whole blood was drawn from 192 pregnant women (10–12 weeks) and was subjected to isolate cffDNA following separation of plasma. TaqMan Real-time PCR was performed on isolated cffDNA using primer pairs and probes specific for SRY, ZFY and β-globin genes. Co-amplification of ZFY and SRY genes was detected in 103 samples confirmed after the birth. Sensitivity and specificity of the test were calculated to be 100%. Further study on larger sample size is required to confirm the reproducibility of the present test in early and non-invasive determination of foetal sex.IMPACT STATEMENTWhat is already known on this subject? Foetal gender analysis through maternal plasma has been investigated in some cell-free foetal DNA (cffDNA) analysis. However, the detection rate and method of cffDNA analysis were different among various studies.What do the results of this study add? This study introduced a modified simple probe based real time analysis with perfect detection rate.What are the implications of these findings for clinical practice and/or further research? The proposed method can be used as diagnostic test in all laboratories around the world using real-time PCR to non-invasively determine the foetal gender in the initial weeks of pregnancy following confirmation in larger sample size.

STUDY ON INSPECTION OF THE PUTATIVE HYBRIDS BETWEEN TRACK-LACKING WILD MALE GAUR (BOS GAURUS) AND DOMESTIC FEMALE COW (BOS TAURUS) IN PHUOC BINH NATIONAL PARK, VIETNAM

Hybridization between wild and domestic bovine occurs widely due to the overlapping of the natural habitat of the wild and human farm animals. Due to the loss of habitat, the number of wild gaur (Bos gaurus) in Vietnam was in serious decline. Since 2009, a male wild gaur (Bos gaurus) has appeared and incorporated with female domestic cows (Bos taurus) in buffer zone Phuoc Binh National Park, on the border with Lam Dong and Ninh Thuan provinces. Then, several calves were born that carried some traits of wild gaur including physical characteristics and behaviors. These calves were supposed to be offspring between wild gaur and domestic cows. In previous study, the karyotypes of putative calves were identified with 2n = 58 and non-homologous chromosome 28 and 29 in these hybrids. In this study, we characterized the putative hybrids between track-lacking Bos gaurus and Bos taurus for breeding and preservation using Cytochrome b analysis, microsatellites and a novel marker growth hormone factor 1 (POU1F1). Cytochrome b analysis indicated the maternal lineage of the putative hybrids, with 100% nucleotide sequence identity. Microsatellite BM861 and sequence of ZFY gene region reveal Bos taurus chromosome Y origin was among the male putative hybrids. Importantly, the analysis of POU1F1 gene sequence on 1 chromosome showed efficacy in determining both Bos gaurus and Bos taurus lineage in the putative hybrids. The karyotyping results were confirmed by molecular analysis and our results provide a feasible way for detecting the putative hybrids between wild and domestic cattle in case of lacking the wild trace.

Sex control by Zfy siRNA in the dairy cattle

Zinc-finger Y is located in the short arm of the Y-chromosome and is a highly conserved gene that plays an important role in spermatogenesis. The objective of this study was to investigate the influence of silencing the Zfy gene during spermatogenesis on Y-sperm formation and offspring sex determination in Bos taurus cattle. Three recombinant expression vectors pLL3.7/a, pLL3.7/b and pLL3.7/c were evaluated and only pLL3.7/a effectively silenced the Zfy gene. The pLL3.7/a recombinant expression vector was injected into bull testes, using three injections. Semen was collected and preserved by extending and freezing. The frozen semen was subsequently used in artificial insemination of cows during a breeding season in accordance with the production plan on the farm where the experiment was conducted. Results showed that, after exposure to pLL3.7/a, sperm motility decreased (P < 0.01), but the sperm density was similar (p > 0.05) to the non-treated control semen. Injection of pLL3.7/a resulted in 72.0% female offspring, and was greater than the 49.4% female calves in the control (P ＜ 0.01), Results from this research suggests that the Zfy gene plays a role in the process of Y-sperm formation, and Zfy siRNA is a potential useful approach to control sex of offspring in cattle.

Distinct prophase arrest mechanisms in human male meiosis.

ABSTRACTTo prevent chromosomal aberrations being transmitted to the offspring, strict meiotic checkpoints are in place to remove aberrant spermatocytes. However, in about 1% of males these checkpoints cause complete meiotic arrest leading to azoospermia and subsequent infertility. Here, we unravel two clearly distinct meiotic arrest mechanisms that occur during prophase of human male meiosis. Type I arrested spermatocytes display severe asynapsis of the homologous chromosomes, disturbed XY-body formation and increased expression of the Y chromosome-encoded gene ZFY and seem to activate a DNA damage pathway leading to induction of p63, possibly causing spermatocyte apoptosis. Type II arrested spermatocytes display normal chromosome synapsis, normal XY-body morphology and meiotic crossover formation but have a lowered expression of several cell cycle regulating genes and fail to silence the X chromosome-encoded gene ZFX. Discovery and understanding of these meiotic arrest mechanisms increases our knowledge of how genomic stability is guarded during human germ cell development.

RNAi as a tool to control the sex ratio of mouse offspring by interrupting Zfx/Zfy genes in the testis.

The objective of this study was to explore a novel method to alter the sex-ratio balance of mouse offspring by silencing the paralogous genes Zfx/Zfy (Zinc finger X/Y-chromosomal transcription factor gene) during spermatogenesis. Four recombined vectors PRZ1, PRZ2, PRZ3, and PRZ4 (RNAi-Ready-pSIREN-RetroQ-ZsGreen) were constructed for interrupting the Zfx gene. Additionally, a recombined vector Psilencer/Zfy-shRNA was constructed for interrupting the Zfy gene. Male mice were randomly divided into 8 groups, with 20 animals per group. Five groups of mice were injected with PRZ1, PRZ2, PRZ3, PRZ4, and Psilencer/Zfy-shRNA vectors, respectively. The three control groups were injected with an equal volume of physiological saline, empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector, and empty Psilencer/Zfy-shRNA vector, respectively. All groups were injected every 7days for a total of four injections. Fourteen days after the fourth injection, 10 male mice from each group were mated individually with 10 females. Testicular tissue of 10 male mice in each group was collected, and the expression level of Zfx/Zfy mRNA was determined by qRT-PCR. Results showed that, compared with the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and the physiological saline group, expression of Zfx mRNA decreased significantly after injection of PRZ1 (p < 0.01), PRZ3 (p < 0.01), and PRZ4 (p < 0.01), and 78.75 ± 7.50% of the offspring were male in PRZ4 group, significantly higher than the offspring derived from the empty RNAi-Ready-pSIREN-RetroQ-ZsGreen vector and physiological saline group (p < 0.01). In the PRZ1 group, the expression of Zfx mRNA was also significantly lower (p < 0.01), but the male rate of offspring was not different (p > 0.05). Conversely, the expression of Zfy mRNA decreased significantly after injection of Psilencer/Zfy-shRNA (p < 0.01) and 31.00 ± 11.00% of the offspring were male, significantly lower than in the physiological saline group (p < 0.01). In conclusion, our findings show that RNAi-mediated disruption of Zfx/Zfy in mouse testis affected X/Y spermatogenesis. Additionally, results suggest that the paralogous genes Zfx/Zfy play an important role in the process of X and Y sperm development. The individual interference of Zfx/Zfy may predict the outcome of X and Y haploid sperms. Presented herein is an advanced method developed to control mouse X/Y spermatogenesis and sex ratio of offspring.

Complementary Critical Functions of Zfy1 and Zfy2 in Mouse Spermatogenesis and Reproduction.

The mammalian Y chromosome plays a critical role in spermatogenesis. However, the exact functions of each gene in the Y chromosome have not been completely elucidated, partly owing to difficulties in gene targeting analysis of the Y chromosome. Zfy was first proposed to be a sex determination factor, but its function in spermatogenesis has been recently elucidated. Nevertheless, Zfy gene targeting analysis has not been performed thus far. Here, we adopted the highly efficient CRISPR/Cas9 system to generate individual Zfy1 or Zfy2 knockout (KO) mice and Zfy1 and Zfy2 double knockout (Zfy1/2-DKO) mice. While individual Zfy1 or Zfy2-KO mice did not show any significant phenotypic alterations in fertility, Zfy1/2-DKO mice were infertile and displayed abnormal sperm morphology, fertilization failure, and early embryonic development failure. Mass spectrometric screening, followed by confirmation with western blot analysis, showed that PLCZ1, PLCD4, PRSS21, and HTT protein expression were significantly deceased in spermatozoa of Zfy1/2-DKO mice compared with those of wild-type mice. These results are consistent with the phenotypic changes seen in the double-mutant mice. Collectively, our strategy and findings revealed that Zfy1 and Zfy2 have redundant functions in spermatogenesis, facilitating a better understanding of fertilization failure and early embryonic development failure.

Y-chromosome variation in Indian native cattle breeds and crossbred population

The paternally inherited Y chromosome markers have been used widely in population genetic studies to trace paternal lineages, to understand differences in migration pattern and populations admixture in animals. In the absence of crossing over, Y-chromosomal markers in the non-recombining male-specific region (MSY) are mostly transmitted as a haplotype. Recent studies of five polymorphic sites on DDX3Y, UTY and ZFY genes of bull MSY assisted in the identification of three haplotypes (Y1, Y2 and Y3) in contemporary cattle. Here we report the screening of five SNPs (ZFY9- 120&gt; C/T; ZFY10- 655&gt; C/T; DDX3Y1- 425&gt;C/T; DDX3Y7 -123&gt;C/T and UTY19-423&gt;C/A) of bull MSY employing optimized and validated allele-specific PCR (AS-PCR) protocols that are useful in effective differentiation of bull/semen samples of Bos indicus and Bos taurus origin. Three haplotypes (Y1, Y2 and Y3) were identified in the present study by the screening of 181 bulls from 10 native cattle breeds and 50 HF crossbred. Y1 and Y2 haplotypes were restricted to HF crossbred with a frequency of 0.98 and 0.02, respectively. The high frequency of Y1 haplotype is possibly due to the occurrence of Y1 lineage predominantly in HF bulls. All the native cattle breeds were observed to have pure indicine haplotype (Y3). These cost effective AS-PCR protocols may be useful for reliable and accurate genotyping of Y-SNPs in diverse native cattle breeds, exotic and crossbred cattle populations.

Zfy genes are required for efficient meiotic sex chromosome inactivation (MSCI) in spermatocytes.

During spermatogenesis, germ cells that fail to synapse their chromosomes or fail to undergo meiotic sex chromosome inactivation (MSCI) are eliminated via apoptosis during mid-pachytene. Previous work showed that Y-linked genes Zfy1 and Zfy2 act as ‘executioners’ for this checkpoint, and that wrongful expression of either gene during pachytene triggers germ cell death. Here, we show that in mice, Zfy genes are also necessary for efficient MSCI and the sex chromosomes are not correctly silenced in Zfy-deficient spermatocytes. This unexpectedly reveals a triple role for Zfy at the mid-pachytene checkpoint in which Zfy genes first promote MSCI, then monitor its progress (since if MSCI is achieved, Zfy genes will be silenced), and finally execute cells with MSCI failure. This potentially constitutes a negative feedback loop governing this critical checkpoint mechanism.

Sex determination of three Neotropical canids by high resolution melting analysis

We describe a fast and reliable genetic method for sexing Neotropical canids by high resolution melting analysis of amplicons obtained by real time polymerase chain reaction. We used a primer set that amplifies a 195 bp fragment of the ZFX and ZFY genes to determine the sex of pampas fox (Pseudalopex gymnocercus), crab eating fox (Cerdocyon thous), maned wolf (Chrysocyon brachyurus), and domestic dog (Canis familiaris). This new method is useful, fast and reliable for sexing of noninvasively obtained samples with low DNA quality and quantity. This protocol could be easily modified to sex non-invasive samples of other canid species.

Y-chromosomal variation of local goat breeds of Turkey close to the domestication centre.

Genetic variations in chromosome Y are enabling researchers to identify paternal lineages, which are informative for introgressions and migrations. In this study, the male-specific region markers, sex-determining region-Y (SRY), amelogenin (AMELY) and zinc finger (ZFY) were analysed in seven Turkish native goat breeds, Angora, Kilis, Hair, Honamlı, Norduz, Gürcü and Abaza. A SNP in the ZFY gene defined a new haplotype Y2C. All domestic haplogroups originate from Capra aegagrus, while the finding of Y1A, Y1B, Y2A and Y2C in 32, 4, 126 and 2 Turkish domestic goats, respectively, appears to indicate a predomestic origin of the major haplotypes. The occurrence of four haplotypes in the Hair goat and, in contrast, a frequency of 96% of Y1A in the Kilis breed illustrate that Y-chromosomal variants have a more breed-dependent distribution than mitochondrial or autosomal DNA. This probably reflects male founder effects, but a role in adaptation cannot be excluded.