The RNA binding protein DND1 is elevated in a subpopulation of pro-spermatogonia and targets chromatin modifiers and translational machinery during late gestation.

DND1 is essential to maintain germ cell identity. Loss of Dnd1 function results in germ cell differentiation to teratomas in some inbred strains of mice or to somatic fates in zebrafish. Using our knock-in mouse line in which a functional fusion protein between DND1 and GFP is expressed from the endogenous locus (Dnd1GFP), we distinguished two male germ cell (MGC) populations during late gestation cell cycle arrest (G0), consistent with recent reports of heterogeneity among MGCs. Most MGCs express lower levels of DND1-GFP (DND1-GFP-lo), but some MGCs express elevated levels of DND1-GFP (DND1-GFP-hi). A RNA-seq time course confirmed high Dnd1 transcript levels in DND1-GFP-hi cells along with 5-10-fold higher levels for multiple epigenetic regulators. Using antibodies against DND1-GFP for RNA immunoprecipitation (RIP)-sequencing, we identified multiple epigenetic and translational regulators that are binding targets of DND1 during G0 including DNA methyltransferases (Dnmts), histone deacetylases (Hdacs), Tudor domain proteins (Tdrds), actin dependent regulators (Smarcs), and a group of ribosomal and Golgi proteins. These data suggest that in DND1-GFP-hi cells, DND1 hosts coordinating mRNA regulons that consist of functionally related and localized groups of epigenetic enzymes and translational components.

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.

The RNA-binding protein DND1 acts sequentially as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers required for germ cell reprogramming.

The adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day (E)10.5. PGCs undergo rapid mitotic proliferation, then enter prolonged cell cycle arrest (G1/G0), during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein Dnd1 (Dnd1Ter/Ter ), many male germ cells (MGCs) fail to enter G1/G0 and instead form teratomas: tumors containing many embryonic cell types. To investigate the origin of these tumors, we sequenced the MGC transcriptome in Dnd1Ter/Ter mutants at E12.5, E13.5 and E14.5, immediately prior to teratoma formation, and correlated this information with DO-RIP-Seq-identified DND1 direct targets. Consistent with previous results, we found DND1 controls downregulation of many genes associated with pluripotency and active cell cycle, including mTor, Hippo and Bmp/Nodal signaling pathway elements. However, DND1 targets also include genes associated with male differentiation, including a large group of chromatin regulators activated in wild-type but not mutant MGCs during the E13.5 and E14.5 transition. Results suggest multiple DND1 functions and link DND1 to initiation of epigenetic modifications in MGCs.

A transgenic DND1GFP fusion allele reports in vivo expression and RNA-binding targets in undifferentiated mouse germ cells†.

In vertebrates, the RNA-binding protein (RBP) dead end 1 (DND1) is essential for primordial germ cell (PGC) survival and maintenance of cell identity. In multiple species, Dnd1 loss or mutation leads to severe PGC loss soon after specification or, in some species, germ cell transformation to somatic lineages. Our investigations into the role of DND1 in PGC specification and differentiation have been limited by the absence of an available antibody. To address this problem, we used CRISPR/Cas9 gene editing to establish a transgenic mouse line carrying a DND1GFP fusion allele. We present imaging analysis of DND1GFP expression showing that DND1GFP expression is heterogeneous among male germ cells (MGCs) and female germ cells (FGCs). DND1GFP was detected in MGCs throughout fetal life but lost from FGCs at meiotic entry. In postnatal and adult testes, DND1GFP expression correlated with classic markers for the premeiotic spermatogonial population. Utilizing the GFP tag for RNA immunoprecipitation (RIP) analysis in MGCs validated this transgenic as a tool for identifying in vivo transcript targets of DND1. The DND1GFP mouse line is a novel tool for isolation and analysis of embryonic and fetal germ cells, and the spermatogonial population of the postnatal and adult testis.

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.

Sry-Independent Overexpression of Sox9 Supports Spermatogenesis and Fertility in the Mouse.

The Y chromosome gene Sry is responsible for sex determination in mammals and initiates a cascade of events that direct differentiation of bipotential genital ridges toward male-specific fate. Sox9 is an autosomal gene and a primary downstream target of SRY. The activation of Sox9 in the absence of Sry is sufficient for initiation of male-specific sex determination. Sry-to-Sox9 replacement has mostly been studied in the context of sex determination during early embryogenesis. Here, we tested whether Sry-to-Sox9 replacement affects male fertility in adulthood. We examined males with the Y chromosome carrying a deletion removing the endogenous Sry, with testes determination driven either by the Sox9 (XY(Tdym1)Sox9) or the Sry (XY(Tdym1)Sry) transgenes as well as wild-type males (XY). XY(Tdym1)Sox9 males had reduced testes size, altered testes shape and vasculature, and increased incidence of defects in seminiferous epithelium underlying the coelomic blood vessel region when compared to XY(Tdym1)Sry and XY. There were no differences between XY(Tdym1)Sry and XY(Tdym1)Sox9 males in respect to sperm number, motility, morphology, and ability to fertilize oocytes in vitro, but for some parameters, transgenic males were impaired when compared to XY. In fecundity trials, XY(Tdym1)Sry, XY(Tdym1)Sox9, and XY males yielded similar average numbers of pups and litters. Overall, our findings support that males lacking the testis determinant Sry can be fertile and reinforce the notion that Sry does not play a role in mature gonads. Although transgenic Sox9 overexpression in the absence of Sry results in certain testicular abnormalities, it does not translate into fertility impairment.

Updates in Sertoli Cell-Mediated Signaling During Spermatogenesis and Advances in Restoring Sertoli Cell Function.

Since their initial description by Enrico Sertoli in 1865, Sertoli cells have continued to enchant testis biologists. Testis size and germ cell carrying capacity are intimately tied to Sertoli cell number and function. One critical Sertoli cell function is signaling from Sertoli cells to germ cells as part of regulation of the spermatogenic cycle. Sertoli cell signals can be endocrine or paracrine in nature. Here we review recent advances in understanding the interplay of Sertoli cell endocrine and paracrine signals that regulate germ cell state. Although these findings have long-term implications for treating male infertility, recent breakthroughs in Sertoli cell transplantation have more immediate implications. We summarize the surge of advances in Sertoli cell ablation and transplantation, both of which are wedded to a growing understanding of the unique Sertoli cell niche in the transitional zone of the testis.

Modeling development of genitourinary birth defects to understand disruption due to changes in gene dosage.

Genitourinary development is a delicately orchestrated process that begins in the embryo. Once complete, the genitourinary system is a collection of functionally disparate organs spread throughout the abdominal and pelvic regions. These distinct organs are interconnected through an elaborate duct system which aggregates the organs' products to a common exit point. The complicated nature of the genitourinary system makes it highly susceptible to developmental disruptions that produce anomalies. In fact, genitourinary anomalies are among the most common class of human birth defects. Aside from congenital anomalies of the kidney and urinary tract (CAKUT), for males, these birth defects can also occur in the penis (hypospadias) and testis (cryptorchism), which impact male fertility and male mental health. As genetic technology has advanced, it has become clear that a subset of cases of genitourinary birth defects are due to gene variation causing dosage changes in critical regulatory genes. Here we first review the parallels between human and mouse genitourinary development. We then demonstrate how translational research leverages mouse models of human gene variation cases to advance mechanistic understanding of causation in genitourinary birth defects. We close with a view to the future highlighting upcoming technologies that will provide a deeper understanding of gene variation affecting regulation of genitourinary development, which should ultimately advance treatment options for patients.

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.

Macroscopic demonstration of the male urogenital system with evidence of a direct inguinal hernia utilizing room temperature plastination.

The male urogenital system represents a morphologically complex region that arises from a common embryological origin. However, it is typically studied separately as the excretory system is dissected with the posterior wall of the abdomen while the reproductive features are exposed with the pelvis and perineum dissection. Additionally, the reproductive structures are typically dissected following pelvic and perineal hemisection obviating a comprehensive and holistic examination. Here, we performed a dissection of the complete male urogenital system utilizing a 70-year-old donor and room temperature silicon plastination. Identification of a direct inguinal hernia during the dissection facilitated a unique opportunity to incorporate a common abdominal wall defect into the plastination requiring a novel approach to retain patency of relevant structures. Results showed that the typical structures identified in medical gross anatomy were retained in addition to the hernia. Thus, the described approach and the resulting specimen provide valuable and versatile teaching tools for male urogenital anatomy.

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.