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.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ injury including testicular inflammation, reduced testosterone, and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells, however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury could be initiated by direct virus infection or exposure to systemic inflammatory mediators or viral antigens. We characterized SARS-CoV-2 infection in different human testicular 2D and 3D culture systems including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not productively infect any testicular cell type. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma decreased cell viability and resulted in the death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 Envelope protein caused inflammatory response and cytopathic effects dependent on TLR2, while Spike 1 or Nucleocapsid proteins did not. A similar trend was observed in the K18-hACE2 transgenic mice which demonstrated a disrupted tissue architecture with no evidence of virus replication in the testis that correlated with peak lung inflammation. Virus antigens including Spike 1 and Envelope proteins were also detected in the serum during the acute stage of the disease. Collectively, these data strongly suggest that testicular injury associated with SARS-CoV-2 infection is likely an indirect effect of exposure to systemic inflammation and/or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Genetic variation in the Y chromosome and sex-biased DNA methylation in somatic cells in the mouse.

Several lines of evidence suggest that the presence of the Y chromosome influences DNA methylation of autosomal loci. To better understand the impact of the Y chromosome on autosomal DNA methylation patterns and its contribution to sex bias in methylation, we identified Y chromosome dependent differentially methylated regions (yDMRs) using whole-genome bisulfite sequencing methylation data from livers of mice with different combinations of sex-chromosome complement and gonadal sex. Nearly 90% of the autosomal yDMRs mapped to transposable elements (TEs) and most of them had lower methylation in XY compared to XX or XO mice. Follow-up analyses of four reporter autosomal yDMRs showed that Y-dependent methylation levels were consistent across most somatic tissues but varied in strains with different origins of the Y chromosome, suggesting that genetic variation in the Y chromosome influenced methylation levels of autosomal regions. Mice lacking the q-arm of the Y chromosome (B6.NPYq-2) as well as mice with a loss-of-function mutation in Kdm5d showed no differences in methylation levels compared to wild type mice. In conclusion, the Y-linked modifier of TE methylation is likely to reside on the short arm of Y chromosome and further studies are required to identify this gene.

In vitro evidence against productive SARS-CoV-2 infection of human testicular cells: Bystander effects of infection mediate testicular injury.

The hallmark of severe COVID-19 involves systemic cytokine storm and multi-organ failure including testicular injury and germ cell depletion. The ACE2 receptor is also expressed in the resident testicular cells however, SARS-CoV-2 infection and mechanisms of testicular injury are not fully understood. The testicular injury can likely result either from direct virus infection of resident cells or by exposure to systemic inflammatory mediators or virus antigens. We here characterized SARS-CoV-2 infection in different human testicular 2D and 3D models including primary Sertoli cells, Leydig cells, mixed seminiferous tubule cells (STC), and 3D human testicular organoids (HTO). Data shows that SARS-CoV-2 does not establish a productive infection in any testicular cell types. However, exposure of STC and HTO to inflammatory supernatant from infected airway epithelial cells and COVID-19 plasma depicted a significant decrease in cell viability and death of undifferentiated spermatogonia. Further, exposure to only SARS-CoV-2 envelope protein, but not Spike or nucleocapsid proteins led to cytopathic effects on testicular cells that was dependent on the TLR2 receptor. A similar trend was observed in the K18h-ACE2 mouse model which revealed gross pathology in the absence of virus replication in the testis. Collectively, data strongly indicates that the testicular injury is not due to direct infection of SARS-CoV-2 but more likely an indirect effect of exposure to systemic inflammation or SARS-CoV-2 antigens. Data also provide novel insights into the mechanism of testicular injury and could explain the clinical manifestation of testicular symptoms associated with severe COVID-19.

Genetically-biased fertilization in APOBEC1 complementation factor (A1cf) mutant mice.

Meiosis, recombination, and gametogenesis normally ensure that gametes combine randomly. But in exceptional cases, fertilization depends on the genetics of gametes from both females and males. A key question is whether their non-random union results from factors intrinsic to oocytes and sperm, or from their interactions with conditions in the reproductive tracts. To address this question, we used in vitro fertilization (IVF) with a mutant and wild-type allele of the A1cf (APOBEC1 complementation factor) gene in mice that are otherwise genetically identical. We observed strong distortion in favor of mutant heterozygotes showing that bias depends on the genetics of oocyte and sperm, and that any environmental input is modest. To search for the potential mechanism of the 'biased fertilization', we analyzed the existing transcriptome data and demonstrated that localization of A1cf transcripts and its candidate mRNA targets is restricted to the spermatids in which they originate, and that these transcripts are enriched for functions related to meiosis, fertilization, RNA stability, translation, and mitochondria. We propose that failure to sequester mRNA targets in A1cf mutant heterozygotes leads to functional differences among spermatids, thereby providing an opportunity for selection among haploid gametes. The study adds to the understanding of the gamete interaction at fertilization. Discovery that bias is evident with IVF provides a new venue for future explorations of preference among genetically distinct gametes at fertilization for A1cf and other genes that display significant departure of Mendelian inheritance.

Two acquired mouse Y chromosome-linked genes, Prssly and Teyorf1, are dispensable for male fertility‡.

Prssly (Protease, serine-like, Chr Y) and Teyorf1 (Testis expressed, chromosome Y open reading frame 1) are two acquired single-copy genes located on the distal tip of the non-pairing short arm of the mouse Y chromosome adjacent to telomeric sequence. Both genes lack X chromosome-linked homologues and are expressed in testicular germ cells. We first performed analysis of Prssly and Teyorf1 genomic sequences and demonstrated that previously reported Prssly sequence is erroneous and the true Prssly sequence is longer and encodes a larger protein than previously estimated. We also confirmed that both genes encode pseudogenes that are not expressed in testes. Next, using CRISPR/Cas9 genome targeting, we generated Prssly and Teyorf1 knockout (KO) mice and characterized their phenotype. To create Prssly KO mice, we targeted the conserved exon 5 encoding a trypsin domain typical for serine proteases. The targeting was successful and resulted in a frame shift mutation that introduced a premature stop codon, with the Prssly KO males retaining only residual transcript expression in testes. The Teyorf1 targeting removed the entire open reading frame of the gene, which resulted in no transcript expression in KO males. Both Prssly KO and Teyorf1 KO males were fertile and had normal testis size and normal sperm number, motility, and morphology. Our findings show that Prssly and Teyorf1 transcripts with potential to encode proteins are dispensable for male fertility.

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.

Pregnancy environment, and not preconception, leads to fetal growth restriction and congenital abnormalities associated with diabetes.

Maternal diabetes can lead to pregnancy complications and impaired fetal development. The goal of this study was to use a mouse model of reciprocal embryo transfer to distinguish between the preconception and gestational effects of diabetes. To induce diabetes female mice were injected with a single high dose of streptozotocin and 3 weeks thereafter used as oocyte donors for in vitro fertilization (IVF) and as recipients for embryo transfer. Following IVF embryos were cultured to the blastocyst stage in vitro or transferred to diabetic and non-diabetic recipients. Diabetic and non-diabetic females did not differ in regard to the number of oocytes obtained after ovarian stimulation, oocytes ability to become fertilized, and embryo development in vitro. However, diabetic females displayed impaired responsiveness to superovulation. Reciprocal embryo transfer resulted in similar incidence of live fetuses and abortions, and no changes in placental size. However, fetuses carried by diabetic recipients were smaller compared to those carried by non-diabetic recipients, regardless hyperglycemia status of oocyte donors. Congenital abnormalities were observed only among the fetuses carried by diabetic recipients. The findings support that the diabetic status during pregnancy, and not the preconception effect of diabetes on oogenesis, leads to fetal growth restriction and congenital deformities.

Battle of the Sex Chromosomes: Competition between X and Y Chromosome-Encoded Proteins for Partner Interaction and Chromatin Occupancy Drives Multicopy Gene Expression and Evolution in Muroid Rodents.

Transmission distorters (TDs) are genetic elements that favor their own transmission to the detriments of others. Slx/Slxl1 (Sycp3-like-X-linked and Slx-like1) and Sly (Sycp3-like-Y-linked) are TDs, which have been coamplified on the X and Y chromosomes of Mus species. They are involved in an intragenomic conflict in which each favors its own transmission, resulting in sex ratio distortion of the progeny when Slx/Slxl1 versus Sly copy number is unbalanced. They are specifically expressed in male postmeiotic gametes (spermatids) and have opposite effects on gene expression: Sly knockdown leads to the upregulation of hundreds of spermatid-expressed genes, whereas Slx/Slxl1-deficiency downregulates them. When both Slx/Slxl1 and Sly are knocked down, sex ratio distortion and gene deregulation are corrected. Slx/Slxl1 and Sly are, therefore, in competition but the molecular mechanism remains unknown. By comparing their chromatin-binding profiles and protein partners, we show that SLX/SLXL1 and SLY proteins compete for interaction with H3K4me3-reader SSTY1 (Spermiogenesis-specific-transcript-on-the-Y1) at the promoter of thousands of genes to drive their expression, and that the opposite effect they have on gene expression is mediated by different abilities to recruit SMRT/N-Cor transcriptional complex. Their target genes are predominantly spermatid-specific multicopy genes encoded by the sex chromosomes and the autosomal Speer/Takusan. Many of them have coamplified with not only Slx/Slxl1/Sly but also Ssty during muroid rodent evolution. Overall, we identify Ssty as a key element of the X versus Y intragenomic conflict, which may have influenced gene content and hybrid sterility beyond Mus lineage since Ssty amplification on the Y predated that of Slx/Slxl1/Sly.

Rescue of Sly Expression Is Not Sufficient to Rescue Spermiogenic Phenotype of Mice with Deletions of Y Chromosome Long Arm.

Mice with deletions of the Y-specific (non-PAR) region of the mouse Y chromosome long arm (NPYq) have sperm defects and fertility problems that increase proportionally to deletion size. Mice with abrogated function of NPYq-encoded gene Sly (sh367 Sly-KD) display a phenotype similar to that of NPYq deletion mutants but less severe. The milder phenotype can be due to insufficient Sly knockdown, involvement of another NPYq gene, or both. To address this question and to further elucidate the role of Sly in the infertile phenotype of mice with NPYq deletions, we developed an anti-SLY antibody specifically recognizing SLY1 and SLY2 protein isoforms and used it to characterize SLY expression in NPYq- and Sly-deficient mice. We also carried out transgene rescue by adding Sly1/2 transgenes to mice with NPYq deletions. We demonstrated that SLY1/2 expression in mutant mice decreased proportionally to deletion size, with ~12% of SLY1/2 retained in shSLY sh367 testes. The addition of Sly1/2 transgenes to mice with NPYq deletions rescued SLY1/2 expression but did not ameliorate fertility and testicular/spermiogenic defects. Together, the data suggest that Sly deficiency is not the sole underlying cause of the infertile phenotype of mice with NPYq deletions and imply the involvement of another NPYq gene.

Juvenile Shank3b deficient mice present with behavioral phenotype relevant to autism spectrum disorder.

Autism spectrum disorder (ASD) is a pervasive, multifactorial neurodevelopmental disorder diagnosed according to deficits in three behavioral domains: communication, social interaction, and stereotyped/repetitive behaviors. Mutations in Shank genes account for ∼1% of clinical ASD cases with Shank3 being the most common gene variant. In addition to maintaining synapses and facilitating dendritic maturation, Shank genes encode master scaffolding proteins that build core complexes in the postsynaptic densities of glutamatergic synapses. Male mice with a deletion of the PDZ domain of Shank3 (Shank3B KO) were previously shown to display ASD-like behavioral phenotypes with reported self-injurious repetitive grooming and aberrant social interactions. Our goal was to extend these previous findings and use a comprehensive battery of highly detailed ASD-relevant behavioral assays including an assessment of mouse ultrasonic communication carried out on key developmental days and male and female Shank3B KO mice. We demonstrate that ASD-related behaviors, atypical reciprocal social interaction and indiscriminate repetitive grooming, are apparent in juvenile stages of development of Shank3B KO mice. Our findings underscore the importance of utilizing Shank mutant models to understand the impact of this gene in ASD etiology, whichmay enable future studies focusing on etiological gene-environment interactions in ASD.

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.

Intracytoplasmic Sperm Injection in Mice.

Intracytoplasmic sperm injection (ICSI) is the injection of a single spermatozoon directly into the cytoplasm of an oocyte using an injection pipette. Spermatozoa that are used for ICSI do not need to be motile or alive. Indeed, for mouse ICSI, the sperm tail is typically broken off before injection into the oocyte, resulting essentially in a nuclear transfer. The protocol describes creation of the injection pipettes, preparation of oocytes and spermatozoa, and steps of the ICSI procedure. The technique can be used to create both hybrid and inbred mice, as well as transgenic animals.

Testicular abnormalities in mice with Y chromosome deficiencies.

We recently investigated mice with Y chromosome gene contribution limited to two, one, or no Y chromosome genes in respect to their ability to produce haploid round spermatids and live offspring following round spermatid injection. Here we explored the normalcy of germ cells and Sertoli cells within seminiferous tubules, and the interstitial tissue of the testis in these mice. We performed quantitative analysis of spermatogenesis and interstitial tissue on Periodic acid-Schiff and hematoxylin-stained mouse testis sections. The seminiferous epithelium of mice with limited Y gene contribution contained various cellular abnormalities, the total number of which was higher than in the males with an intact Y chromosome. The distribution of specific abnormality types varied among tested genotypes. The males with limited Y genes also had an increased population of testicular macrophages and internal vasculature structures. The data indicate that Y chromosome gene deficiencies in mice are associated with cellular abnormalities of the seminiferous epithelium and some changes within the testicular interstitium.

Two genes substitute for the mouse Y chromosome for spermatogenesis and reproduction.

The mammalian Y chromosome is considered a symbol of maleness, as it encodes a gene driving male sex determination, Sry, as well as a battery of other genes important for male reproduction. We previously demonstrated in the mouse that successful assisted reproduction can be achieved when the Y gene contribution is limited to only two genes, Sry and spermatogonial proliferation factor Eif2s3y. Here, we replaced Sry by transgenic activation of its downstream target Sox9, and Eif2s3y, by transgenic overexpression of its X chromosome-encoded homolog Eif2s3x. The resulting males with no Y chromosome genes produced haploid male gametes and sired offspring after assisted reproduction. Our findings support the existence of functional redundancy between the Y chromosome genes and their homologs encoded on other chromosomes.