Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion.

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.

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.

Differential proteins associated with plasma membrane in X- and/or Y-chromosome bearing spermatozoa in indicus cattle.

The differential proteins associated with plasma membrane of spermatozoa are less known, identification of which shall help overcome limitations of currently used methods of sperm sexing, considered as a high priority for livestock sector of many countries. This study has reported plasma membrane proteomics of unsorted spermatozoa and differential expression of plasma membrane-associated proteins between X- and Y-chromosome bearing spermatozoa of indicus cattle (Bos indicus). Isolation of plasma membrane fraction using percoll gradient, relatively a rapid method, from bovine spermatozoa has been reported to enrich isolation of plasma membrane proteins. Significant enrichment for plasma membrane-associated proteins was observed in plasma membrane fraction (p < .05) as compared to the total cell lysate using LC-MS/MS. Furthermore, these experiments were conducted in flow cytometry sorted, sexed-semen samples. Thirteen proteins were identified as differentially abundant between X- and Y-sorted spermatozoa. Among these, two proteins were downregulated in Y-sorted spermatozoa compared to the X-sorted spermatozoa (p < .05), while four and seven proteins could be noted in X- and Y-sorted spermatozoa, respectively. Proteins that are presumed to support sperm capacitation and sperm migration velocity were found to be abundant in Y-sorted spermatozoa while those associated with structural molecule activity were identified as abundant in X-sorted spermatozoa in the present study. Our study provides better insight into the plasma membrane proteomics of spermatozoa of indicus cattle and furnishes data that might aid in design and development of alternate and open technology for sex-sorting of semen.

De novo assembly of the olive fruit fly (Bactrocera oleae) genome with linked-reads and long-read technologies minimizes gaps and provides exceptional Y chromosome assembly.

BACKGROUND: The olive fruit fly, Bactrocera oleae, is the most important pest in the olive fruit agribusiness industry. This is because female flies lay their eggs in the unripe fruits and upon hatching the larvae feed on the fruits thus destroying them. The lack of a high-quality genome and other genomic and transcriptomic data has hindered progress in understanding the fly's biology and proposing alternative control methods to pesticide use. RESULTS: Genomic DNA was sequenced from male and female Demokritos strain flies, maintained in the laboratory for over 45 years. We used short-, mate-pair-, and long-read sequencing technologies to generate a combined male-female genome assembly (GenBank accession GCA_001188975.2). Genomic DNA sequencing from male insects using 10x Genomics linked-reads technology followed by mate-pair and long-read scaffolding and gap-closing generated a highly contiguous 489 Mb genome with a scaffold N50 of 4.69 Mb and L50 of 30 scaffolds (GenBank accession GCA_001188975.4). RNA-seq data generated from 12 tissues and/or developmental stages allowed for genome annotation. Short reads from both males and females and the chromosome quotient method enabled identification of Y-chromosome scaffolds which were extensively validated by PCR. CONCLUSIONS: The high-quality genome generated represents a critical tool in olive fruit fly research. We provide an extensive RNA-seq data set, and genome annotation, critical towards gaining an insight into the biology of the olive fruit fly. In addition, elucidation of Y-chromosome sequences will advance our understanding of the Y-chromosome's organization, function and evolution and is poised to provide avenues for sterile insect technique approaches.

Dosage compensation of the sex chromosomes.

Differentiated sex chromosomes evolved because of suppressed recombination once sex became genetically controlled. In XX/XY and ZZ/ZW systems, the heterogametic sex became partially aneuploid after degeneration of the Y or W. Often, aneuploidy causes abnormal levels of gene expression throughout the entire genome. Dosage compensation mechanisms evolved to restore balanced expression of the genome. These mechanisms include upregulation of the heterogametic chromosome as well as repression in the homogametic sex. Remarkably, strategies for dosage compensation differ between species. In organisms where more is known about molecular mechanisms of dosage compensation, specific protein complexes containing noncoding RNAs are targeted to the X chromosome. In addition, the dosage-regulated chromosome often occupies a specific nuclear compartment. Some genes escape dosage compensation, potentially resulting in sex-specific differences in gene expression. This review focuses on dosage compensation in mammals, with comparisons to fruit flies, nematodes, and birds.

More sex chromosomes than autosomes in the Amazonian frog Leptodactylus pentadactylus.

Heteromorphic sex chromosomes are common in eukaryotes and largely ubiquitous in birds and mammals. The largest number of multiple sex chromosomes in vertebrates known today is found in the monotreme platypus (Ornithorhynchus anatinus, 2n = 52) which exhibits precisely 10 sex chromosomes. Interestingly, fish, amphibians, and reptiles have sex determination mechanisms that do or do not involve morphologically differentiated sex chromosomes. Relatively few amphibian species carry heteromorphic sex chromosomes, and when present, they are frequently represented by only one pair, either XX:XY or ZZ:ZW types. Here, in contrast, with several evidences, from classical and molecular cytogenetic analyses, we found 12 sex chromosomes in a Brazilian population of the smoky jungle frog, designated as Leptodactylus pentadactylus Laurenti, 1768 (Leptodactylinae), which has a karyotype with 2n = 22 chromosomes. Males exhibited an astonishing stable ring-shaped meiotic chain composed of six X and six Y chromosomes. The number of sex chromosomes is larger than the number of autosomes found, and these data represent the largest number of multiple sex chromosomes ever found among vertebrate species. Additionally, sequence and karyotype variation data suggest that this species may represent a complex of species, in which the chromosomal rearrangements may possibly have played an important role in the evolution process.

X inactivation in a mammal species with three sex chromosomes.

X inactivation is a fundamental mechanism in eutherian mammals to restore a balance of X-linked gene products between XY males and XX females. However, it has never been extensively studied in a eutherian species with a sex determination system that deviates from the ubiquitous XX/XY. In this study, we explore the X inactivation process in the African pygmy mouse Mus minutoides, that harbours a polygenic sex determination with three sex chromosomes: Y, X, and a feminizing mutant X, named X*; females can thus be XX, XX*, or X*Y, and all males are XY. Using immunofluorescence, we investigated histone modification patterns between the two X chromosome types. We found that the X and X* chromosomes are randomly inactivated in XX* females, while no histone modifications were detected in X*Y females. Furthermore, in M. minutoides, X and X* chromosomes are fused to different autosomes, and we were able to show that the X inactivation never spreads into the autosomal segments. Evaluation of X inactivation by immunofluorescence is an excellent quantitative procedure, but it is only applicable when there is a structural difference between the two chromosomes that allows them to be distinguished.

Alterations of sex determination pathways in the genital ridges of males with limited Y chromosome genes†.

We previously demonstrated that in the mouse only two Y chromosome genes are required for a male to produce an offspring with the help of assisted reproduction technologies (ART): testis determinant Sry and spermatogonial proliferation factor Eif2s3y. Subsequently, we have shown that the function of these genes can be replaced by transgenic overexpression of their homologs, autosomally encoded Sox9 and X-chromosome encoded Eif2s3x. Males with Y chromosome contribution limited to two (XEif2s3yOSry), one (XEif2s3yOSox9 and XOSry,Eif2s3x), and no genes (XOSox9,Eif2s3x) produced haploid germ cells and sired offspring after ART. However, despite successful assisted reproductive outcome, they had smaller testes and displayed abnormal development of the seminiferous epithelium and testicular interstitium. Here we explored whether these testicular defects originated from altered pro-testis and pro-ovary factor signaling in genital ridges at the time of sex determination. Timed pregnancies were generated to obtain transgenic XEif2s3yOSry, XEif2s3yOSox9, XOSry,Eif2s3x, XOSox9,Eif2s3x, and wild-type XX and XY fetuses at 12.5 days post coitum. Dissected genital ridges were assessed for their morphology and anatomy, and expression of pro-testis and pro-ovary transcripts. All transgenic males displayed incomplete masculinization of gonadal shape, impaired development of testicular cords and gonadal vasculature, and decreased expression of factors promoting male pathway. Fetal gonad masculinization was more effective when sex determination was driven by the Sry transgene, in the presence of Y chromosome genes, and to a lesser extent a double dosage of X genes. The study adds to the understanding of the role of Y chromosome genes and their homologs during sex determination.

Lysine demethylases KDM6A and UTY: The X and Y of histone demethylation.

Histone demethylases remove transcriptional repressive marks from histones in the nucleus. KDM6A (also known as UTX) is a lysine demethylase which acts on the trimethylated lysine at position 27 in histone 3. The KDM6A gene is located on the X chromosome but escapes X inactivation even though it is not located in the pseudoautosomal region. There is a homologue of KDM6A on the Y chromosome, known as UTY. UTY was thought to have lost its demethylase activity and to represent a non-functional remnant of the ancestral KDM6A gene. However, results with knockout mice suggest that the gene is expressed and the protein performs some function within the cell. Female mice with homozygous deletion of Kdm6a do not survive, but hemizygous males are viable, attributed to the presence of the Uty gene. KDM6A is mutated in the human condition Kabuki syndrome type 2 (OMIM 300867) and in many cases of cancer. The amino acid sequence of KDM6A has been conserved across animal phyla, although it is only found on the X chromosome in eutherian mammals. In this review, we reanalyse existing data from various sources (protein sequence comparison, evolutionary genetics, transcription factor binding and gene expression analysis) to determine the function, expression and evolution of KDM6A and UTY and show that UTY has a functional role similar to KDM6A in metabolism and development.

Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.

The essential role of the Y chromosome in male sex determination has largely overshadowed the possibility that it may exert other biologic roles. Here, we show that Y-chromosome lineage is a strong determinant of perivascular and renal T-cell infiltration in the stroke-prone spontaneously hypertensive rat, which, in turn, may influence vascular function and blood pressure (BP). We also show, for the first time to our knowledge, that augmented perivascular T-cell levels can directly instigate vascular dysfunction, and that the production of reactive oxygen species that stimulate cyclo-oxygenase underlies this. We thus provide strong evidence for the consideration of Y-chromosome lineage in the diagnosis and treatment of male hypertension, and point to the modulation of cardiovascular organ T-cell infiltration as a possible mechanism that underpins Y- chromosome regulation of BP.-Khan, S. I., Andrews, K. L., Jackson, K. L., Memon, B., Jefferis, A.-M., Lee, M. K. S., Diep, H., Wei, Z., Drummond, G. R., Head, G. A., Jennings, G. L., Murphy, A. J., Vinh, A., Sampson, A. K., Chin-Dusting, J. P. F. Y-chromosome lineage determines cardiovascular organ T-cell infiltration in the stroke-prone spontaneously hypertensive rat.

Impact of deleterious mutations, sexually antagonistic selection, and mode of recombination suppression on transitions between male and female heterogamety.

Deleterious mutations accumulating on non-recombining Y chromosomes can drive XY to XY turnovers, as they allow to replace the old mutation-loaded Y by a new mutation-free one. The same process is thought to prevent XY to ZW turnovers, because the latter requires fixation of the ancestral Y, assuming dominance of the emergent feminizing mutation. Using individual-based simulations, we explored whether and how an epistatically dominant W allele can spread in a young XY system that gradually accumulates deleterious mutations. We also investigated how sexually antagonistic (SA) polymorphism on the ancestral sex chromosomes and the mechanism controlling X-Y recombination suppression affect these transitions. In contrast with XY to XY turnovers, XY to ZW turnovers cannot be favored by Y chromosome mutation load. If the arrest of X-Y recombination depends on genotypic sex, transitions are strongly hindered by deleterious mutations, and totally suppressed by very small SA cost, because deleterious mutations and female-detrimental SA alleles would have to fix with the Y. If, however, the arrest of X-Y recombination depends on phenotypic sex, X and Y recombine in XY ZW females, allowing for the purge of Y-linked deleterious mutations and loss of the SA polymorphism, causing XY to ZW turnovers to occur at the same rate as in the absence of deleterious and sex-antagonistic mutations. We generalize our results to other types of turnovers (e.g., triggered by non-dominant sex-determining mutations) and discuss their empirical relevance.

Chromosome-specific nonrandom sister chromatid segregation during stem-cell division.

Adult stem cells undergo asymmetric cell division to self-renew and give rise to differentiated cells that comprise mature tissue. Sister chromatids may be distinguished and segregated nonrandomly in asymmetrically dividing stem cells, although the underlying mechanism and the purpose it may serve remain elusive. Here we develop the CO-FISH (chromosome orientation fluorescence in situ hybridization) technique with single-chromosome resolution and show that sister chromatids of X and Y chromosomes, but not autosomes, are segregated nonrandomly during asymmetric divisions of Drosophila male germline stem cells. This provides the first direct evidence, to our knowledge, that two sister chromatids containing identical genetic information can be distinguished and segregated nonrandomly during asymmetric stem-cell divisions. We further show that the centrosome, SUN-KASH nuclear envelope proteins and Dnmt2 (also known as Mt2) are required for nonrandom sister chromatid segregation. Our data indicate that the information on X and Y chromosomes that enables nonrandom segregation is primed during gametogenesis in the parents. Moreover, we show that sister chromatid segregation is randomized in germline stem cell overproliferation and dedifferentiated germline stem cells. We propose that nonrandom sister chromatid segregation may serve to transmit distinct information carried on two sister chromatids to the daughters of asymmetrically dividing stem cells.

Endogenous annexin A1 (AnxA1) modulates early-phase gestation and offspring sex-ratio skewing.

Annexin A1 (AnxA1) is a glucocorticoid-regulated anti-inflammatory protein secreted by phagocytes and other specialised cells. In the endocrine system, AnxA1 controls secretion of steroid hormones and it is abundantly expressed in the testis, ovaries, placenta and seminal fluid, yet its potential modulation of fertility has not been described. Here, we observed that AnxA1 knockout (KO) mice delivered a higher number of pups, with a higher percentage of female offsprings. This profile was not dependent on the male features, as sperm from KO male mice did not present functional alterations, and had an equal proportion of Y and X chromosomes, comparable to wild type (WT) male mice. Furthermore, mismatched matings of male WT mice with female KO yielded a higher percentage of female pups per litter, a phenomenon which was not observed when male KO mice mated with female WT animals. Indeed, AnxA1 KO female mice displayed several differences in parameters related to gestation including (i) an arrested estrous cycle at proestrus phase; (ii) increased sites of implantation; (iii) reduced pre- and post-implantation losses; (iv) exacerbated features of the inflammatory reaction in the uterine fluid during implantation phase; and (v) enhanced plasma progesterone in the beginning of pregnancy. In summary, herein we highlight that AnxA1 pathway as a novel determinant of fundamental non-redundant regulatory functions during early pregnancy.

Escape of X-linked miRNA genes from meiotic sex chromosome inactivation.

Past studies have indicated that transcription of all X-linked genes is repressed by meiotic sex chromosome inactivation (MSCI) during the meiotic phase of spermatogenesis in mammals. However, more recent studies have shown an increase in steady-state levels of certain X-linked miRNAs in pachytene spermatocytes, suggesting that either synthesis of these miRNAs increases or that degradation of these miRNAs decreases dramatically in these cells. To distinguish between these possibilities, we performed RNA-FISH to detect nascent transcripts from multiple miRNA genes in various spermatogenic cell types. Our results show definitively that Type I X-linked miRNA genes are subject to MSCI, as are all or most X-linked mRNA genes, whereas Type II and III X-linked miRNA genes escape MSCI by continuing ongoing, active transcription in primary spermatocytes. We corroborated these results by co-localization of RNA-FISH signals with both a corresponding DNA-FISH signal and an immunofluorescence signal for RNA polymerase II. We also found that X-linked miRNA genes that escape MSCI locate non-randomly to the periphery of the XY body, whereas genes that are subject to MSCI remain located within the XY body in pachytene spermatocytes, suggesting that the mechanism of escape of X-linked miRNA genes from MSCI involves their relocation to a position outside of the repressive chromatin domain associated with the XY body. The fact that Type II and III X-linked miRNA genes escape MSCI suggests an immediacy of function of the encoded miRNAs specifically required during the meiotic stages of spermatogenesis.

The Analysis of Pendolino (peo) Mutants Reveals Differences in the Fusigenic Potential among Drosophila Telomeres.

Drosophila telomeres are sequence-independent structures that are maintained by transposition to chromosome ends of three specialized retroelements (HeT-A, TART and TAHRE; collectively designated as HTT) rather than telomerase activity. Fly telomeres are protected by the terminin complex (HOAP-HipHop-Moi-Ver) that localizes and functions exclusively at telomeres and by non-terminin proteins that do not serve telomere-specific functions. Although all Drosophila telomeres terminate with HTT arrays and are capped by terminin, they differ in the type of subtelomeric chromatin; the Y, XR, and 4L HTT are juxtaposed to constitutive heterochromatin, while the XL, 2L, 2R, 3L and 3R HTT are linked to the TAS repetitive sequences; the 4R HTT is associated with a chromatin that has features common to both euchromatin and heterochromatin. Here we show that mutations in pendolino (peo) cause telomeric fusions (TFs). The analysis of several peo mutant combinations showed that these TFs preferentially involve the Y, XR and 4th chromosome telomeres, a TF pattern never observed in the other 10 telomere-capping mutants so far characterized. peo encodes a non-terminin protein homologous to the E2 variant ubiquitin-conjugating enzymes. The Peo protein directly interacts with the terminin components, but peo mutations do not affect telomeric localization of HOAP, Moi, Ver and HP1a, suggesting that the peo-dependent telomere fusion phenotype is not due to loss of terminin from chromosome ends. peo mutants are also defective in DNA replication and PCNA recruitment. However, our results suggest that general defects in DNA replication are unable to induce TFs in Drosophila cells. We thus hypothesize that DNA replication in Peo-depleted cells results in specific fusigenic lesions concentrated in heterochromatin-associated telomeres. Alternatively, it is possible that Peo plays a dual function being independently required for DNA replication and telomere capping.

The XY Body of the Cat (Felis catus): Structural Differentiations and Protein Immunolocalization.

The heteromorphic X and Y chromosomes behave in a special way in mammalian spermatocytes; they form the XY body and synapse only partially. The aim of this article was to study the origin and the role of the special differentiations in the XY pair of the domestic cat during pachytene by analyzing its fine structural characteristics and the immunolocalization of the main meiotic proteins SYCP3, SYCP1, SYCE3, SMC3, γ-H2AX, BRCA1, H3K27me3, and MLH1. The cat XY body shows particularly striking structures: an extreme degree of axial fibrillation in late pachynema and a special location of SYCP3-containing fibrils, bridging different regions of the main X axis, as well as one bridge at the inner end of the pairing region that colocalizes with the single mandatory MLH1 focus. There are sequential changes, first bullous expansions, then subdivision into fibrils, all involving axial thickening. The chromatin of the XY body presents the usual features of meiotic sex chromosome inactivation. An analysis of the XY body of many eutherians and metatherians suggests that axial thickenings are primitive features. The sequential changes in the mass and location of SYCP3-containing fibers vary among the clades because of specific processes of axial assembly/disassembly occurring in different species.

The Drosophila simulans Y chromosome interacts with the autosomes to influence male fitness.

The Y chromosome should degenerate because it cannot recombine. However, male-limited transmission increases selection efficiency for male-benefit alleles on the Y, and therefore, Y chromosomes should contribute significantly to variation in male fitness. This means that although the Drosophila Y chromosome is small and gene-poor, Y-linked genes are vital for male fertility in Drosophila melanogaster and the Y chromosome has large male fitness effects. It is unclear whether the same pattern is seen in the closely related Drosophila simulans. We backcrossed Y chromosomes from three geographic locations into five genetic backgrounds and found strong Y and genetic background effects on male fertility. There was a significant Y-background interaction, indicating substantial epistasis between the Y and autosomal genes affecting male fertility. This supports accumulating evidence that interactions between the Y chromosome and the autosomes are key determinants of male fitness.

Delayed administration of allogeneic cardiac stem cell therapy for acute myocardial infarction could ameliorate adverse remodeling: experimental study in swine.

BACKGROUND: The optimal timing of cardiac stem cells administration is still unclear. We assessed the safety of same-day and delayed (one week) delivery and the possible influence of the timing on the therapeutic outcomes of allogeneic porcine cardiac stem cells administration after acute myocardial infarction in a closed-chest ischemia-reperfusion model. METHODS: Female swine surviving 90 min occlusion of the mid left anterior descending coronary artery received an intracoronary injection of 25x10(6) porcine cardiac stem cells either two hours (n = 5, D0) or 7 days (n = 6, D7) after reperfusion. Controls received intracoronary injection of vehicle on day 7 (n = 6, CON). Safety was defined in terms of absence of major cardiac events, changes to the ECG during injection, post-administration coronary flow assessed using the TIMI scale and cardiac troponin I determination after the intervention. Cardiac Magnetic Resonance was performed for morphological and functional assessment prior to infarction, before injection (D7 and CON groups only), at one and 10 weeks. Samples were taken from the infarct and transition areas for pathological examination. RESULTS: No major adverse cardiac events were seen during injection in any group. Animals receiving the therapy on the same day of infarction (D0 group) showed mild transient ST changes during injection (n = 4) and, in one case, slightly compromised coronary flow (TIMI 2). Cardiac function parameters and infarct sizes were not significantly different between groups, with a trend towards higher ejection fraction in the treated groups. Ventricular volumes indexed to body surface area increased over time in control animals, and decreased by the end of the study in animals receiving the therapy, significantly so when comparing End Diastolic Volume between CON and D7 groups (CON: 121.70 ml/m(2) ± 26.09 ml/m(2), D7: 98.71 ml/m(2) ± 8.30 ml/m(2), p = 0.037). The treated groups showed less organization of the collagenous scar, and a significantly (p = 0.019) higher amount of larger, more mature vessels at the infarct border. CONCLUSIONS: The intracoronary injection of 25x10(6) allogeneic cardiac stem cells is generally safe, both early and 7 days after experimental infarction, and alleviates myocardial dysfunction, with a greater limitation of left ventricular remodeling when performed at one week.

Distribution of the sex chromosome during mouse spermatogenesis in testis tissue sections.

During mammalian spermatogenesis, spermatogenic cells undergo mitotic division and are subsequently divided into haploid spermatids by meiotic division, but the dynamics of sex chromosomes during spermatogenesis are unclear in vivo. To gain insight into the distribution of sex chromosomes in the testis, we examined the localization of sex chromosomes before and after meiosis in mouse testis sections. Here, we developed a method of fluorescence in situ hybridization (FISH) using specific probes for the X and Y chromosomes to obtain their positional information in histological testis sections. FISH analysis revealed the sex chromosomal position during spermatogenesis in each stage of seminiferous epithelia and in each spermatogenic cell. In the spermatogonia and leptotene spermatocytes, sex chromosomes were distantly positioned in the cell. In the zygotene and pachytene spermatocytes at prophase I, X and Y chromosomes had a random distribution. After meiosis, the X and Y spermatids were random in every seminiferous epithelium. We also detected aneuploidy of sex chromosomes in spermatogenic cells using our developed FISH analysis. Our results provide further insight into the distribution of sex chromosomes during spermatogenesis, which could help to elucidate a specific difference between X and Y spermatids and sex chromosome-specific behavior.

Identification and characterization of genes differentially expressed in X and Y sperm using suppression subtractive hybridization and cDNA microarray.

Differential expression of genes leads to variations in the phenotypes of X and Y sperm, although some differentially expressed gene products are shared through intercellular bridges. Genes differentially expressed in bovine X and Y sperm were identified by a combination of suppression subtractive hybridization (SSH), cDNA microarray, and sequence-homology analysis. Microarray data and Significance Analysis of Microarrays software were used to identify 31 differentially expressed genes, only four of which were previously identified. These genes are involved in fundamental life processes of mature sperm, and may be associated with the differences between X and Y sperm since 27 versus 4 were upregulated in X versus Y sperm, respectively. The levels of expression of seven genes-including the known genes UTY, DPH3, CYTB, and ISCU, and the unknown genes X + Y contig 41, X + Y contig 18, and Y + X contig 16-were validated by quantitative real-time PCR, and some genes were clearly differentially expressed by X and Y sperm, despite the presence of intercellular bridges among spermatids. These results provide a theoretical basis for research on gene expression during sperm development, as well as on sex control at the level of sperm.