Testis-specific Gene Research Articles

Combined Use of Whole Exome Sequencing and CRISPR/Cas9 to Study the Etiology of Non-Obstructive Azoospermia: Demonstration of the Dispensable Role of the Testis-Specific Genes C1orf185 and CCT6B.

The genetic landscape of male infertility is highly complex. It is estimated that at least 4000 genes are involved in human spermatogenesis, but only few have so far been extensively studied. In this study, we investigated by whole exome sequencing two cases of idiopathic non-obstructive azoospermia (NOA) due to severe hypospermatogenesis. After variant filtering and prioritizing, we retained for each patient a homozygous loss-of-function (LoF) variant in a testis-specific gene, C1orf185 (c.250C>T; p.Gln84Ter) and CCT6B (c.615-2A>G), respectively. Both variants are rare according to the gnomAD database and absent from our local control cohort (n = 445). To verify the implication of these candidate genes in NOA, we used the CRISPR/Cas9 system to invalidate the mouse orthologs 4930522H14Rik and Cct6b and produced two knockout (KO) mouse lines. Sperm and testis parameters of homozygous KO adult male mice were analyzed and compared with those of wild-type animals. We showed that homozygous KO males were fertile and displayed normal sperm parameters and a functional spermatogenesis. Overall, these results demonstrate that not all genes highly and specifically expressed in the testes are essential for spermatogenesis, and in particular, we conclude that bi-allelic variants of C1orf185 and CCT6B are most likely not to be involved in NOA and male fertility.

Rapid Cis-Trans Coevolution Driven by a Novel Gene Retroposed from a Eukaryotic Conserved CCR4-NOT Component in Drosophila.

Young, or newly evolved, genes arise ubiquitously across the tree of life, and they can rapidly acquire novel functions that influence a diverse array of biological processes. Previous work identified a young regulatory duplicate gene in Drosophila, Zeus that unexpectedly diverged rapidly from its parent, Caf40, an extremely conserved component in the CCR4–NOT machinery in post-transcriptional and post-translational regulation of eukaryotic cells, and took on roles in the male reproductive system. This neofunctionalization was accompanied by differential binding of the Zeus protein to loci throughout the Drosophila melanogaster genome. However, the way in which new DNA-binding proteins acquire and coevolve with their targets in the genome is not understood. Here, by comparing Zeus ChIP-Seq data from D. melanogaster and D. simulans to the ancestral Caf40 binding events from D. yakuba, a species that diverged before the duplication event, we found a dynamic pattern in which Zeus binding rapidly coevolved with a previously unknown DNA motif, which we term Caf40 and Zeus-Associated Motif (CAZAM), under the influence of positive selection. Interestingly, while both copies of Zeus acquired targets at male-biased and testis-specific genes, D. melanogaster and D. simulans proteins have specialized binding on different chromosomes, a pattern echoed in the evolution of the associated motif. Using CRISPR-Cas9-mediated gene knockout of Zeus and RNA-Seq, we found that Zeus regulated the expression of 661 differentially expressed genes (DEGs). Our results suggest that the evolution of young regulatory genes can be coupled to substantial rewiring of the transcriptional networks into which they integrate, even over short evolutionary timescales. Our results thus uncover dynamic genome-wide evolutionary processes associated with new genes.

Paternal factors in recurrent pregnancy loss: an insight through analysis of non-synonymous single-nucleotide polymorphism in human testis-specific chaperone HSPA2 gene.

Heat shock protein A2 (HSPA2) is a testis-specific molecular chaperone of the 70 kDa heat shock protein (HSP70) family and reported to play a key role in spermatogenesis as well as in the remodelling of the sperm surface during capacitation. It is established that mice lacking HSPA2 gene are infertile and spermatozoa that fail to interact with the zona pellucida of the oocyte consistently lack HSPA2 protein expression. However, its role in post fertilization events is not fully understood. Owing to the importance of HSPA2 in male reproduction, the present study is undertaken to reveal the association between genetic mutation and phenotypic variation in recurrent pregnancy loss (RPL) patients through an in silico prediction analysis. In this study, we used different computational tools and servers such as SIFT, PolyPhen2, PROVEAN, nsSNPAnalyzer, and SNPs & GO to analyse the functional consequences of the nsSNPs in human HSPA2 gene. The most damaging amino acid variants generated were subjected to I-Mutant 2.0 and ConSurf. Post-translational modifications such as phosphorylation mediated by these deleterious nsSNPs were analysed using NetPhos 2.0, and gene-gene interaction study was conducted using GeneMANIA. Finally, in-depth studies of the nsSNPs were studied through Project HOPE. The findings of the study revealed 18 nsSNPs to be deleterious using a combinatorial bioinformatic approach. Further functional analysis suggests that screening of nsSNP variants of HSPA2 that tend to be conserved and has potential to undergo phosphorylation at critical positions (rs764410231, rs200951589, rs756852956) may be useful for predicting outcome in altered reproductive outcome. The physicochemical alterations and its impact on the structural and functional conformity were determined by Project HOPE. Gene-gene interaction depicts its close association with antioxidant enzyme(SOD1) strongly supporting an inefficient oxidative scavenging regulatory mechanism in the spermatozoa of RPL patients as reported earlier. The present study has thus identified high-risk deleterious nsSNPs of HSPA2 gene and would be beneficial in the diagnosis and prognosis of the paternal effects in RPL patients.

New Genes in the Drosophila Y Chromosome: Lessons from D. willistoni.

Y chromosomes play important roles in sex determination and male fertility. In several groups (e.g., mammals) there is strong evidence that they evolved through gene loss from a common X-Y ancestor, but in Drosophila the acquisition of new genes plays a major role. This conclusion came mostly from studies in two species. Here we report the identification of the 22 Y-linked genes in D. willistoni. They all fit the previously observed pattern of autosomal or X-linked testis-specific genes that duplicated to the Y. The ratio of gene gains to gene losses is ~25 in D. willistoni, confirming the prominent role of gene gains in the evolution of Drosophila Y chromosomes. We also found four large segmental duplications (ranging from 62 kb to 303 kb) from autosomal regions to the Y, containing ~58 genes. All but four of these duplicated genes became pseudogenes in the Y or disappeared. In the GK20609 gene the Y-linked copy remained functional, whereas its original autosomal copy degenerated, demonstrating how autosomal genes are transferred to the Y chromosome. Since the segmental duplication that carried GK20609 contained six other testis-specific genes, it seems that chance plays a significant role in the acquisition of new genes by the Drosophila Y chromosome.

Original Research: Expression Profile of CAPZA3 And TR-KIT Genes in Men with Globozoospermia & Asthenoteratospermia that Undergo ICSI Protocol

Objective(s): Sperm-mediated oocyte activation depends upon suitable expression and assembly of sperm-borne oocyte-activating factors (SOAFs) during spermiogenesis. Several factors have been considered as candidates for oocyte activation in recent years. Globozoospermia is a severe sperm morphology disorder that is a rare type of teratozoospermia with an incidence of 0.1% among infertile individuals. testis-specific genes including CAPZA3 [capping protein (actin filament) muscle Z-line, alpha, which is considered as a nominee for sperm associated oocyte activating factors, an actin-capping protein, controlling actin polymerization during spermiogenesis. They contain a common bidirectional promoter. Another gene TR-KIT (a truncated form of the KIT receptor) which is a major sperm-associated oocyte-activating factor. The objective of this study was to investigate the expression profile of CAPZA3 and TR-KIT mRNA, in men with total globozoospermia, Asthenoteratospermia, and fertile individuals.
 Materials and Methods: Semen samples were collected from three groups including 25 fertile men, 20 Asthenoteratospermia and 12 Globozoospermia that undergo intra-cytoplasmic sperm injection (ICSI), Expression of CAPZA3 and TR-KIT were assessed by Real time PCR.
 Results: Individuals with Globozoospermia have presented significantly lower expression of CAPZA3 and TR-KIT mRNA when compared with fertile men. Asthenoteratospermia (male factor) showed significantly lower expression of CAPZA3 mRNA, whereas non-significantly of TR-KIT mRNA. Levels of CAPZA3 and TR-KIT mRNA in the spermatozoa of fertile men were significantly higher than the corresponding values of the globozoospermic and Asthenoteratospermia subjects.
 Conclusion: Analysis mRNA of CAPZA3 gene maybe assist the researcher to identify individuals with a lack of ability to induce oocyte activation and make them a candidate for artificial oocyte activation and help researcher to identify genetic defects associated with failed fertilization. whereas, mRNA of TR-KIT gene appears inability to induce oocyte activation

Microtubule Organizing Centers Contain Testis-Specific γ-TuRC Proteins in Spermatids of Drosophila.

Microtubule nucleation in eukaryotes is primarily promoted by γ-tubulin and the evolutionary conserved protein complex, γ-Tubulin Ring Complex (γ-TuRC). γ-TuRC is part of the centrosome and basal body, which are the best-known microtubule-organizing centers. Centrosomes undergo intensive and dynamic changes during spermatogenesis, as they turn into basal bodies, a prerequisite for axoneme formation during spermatogenesis. Here we describe the existence of a novel, tissue-specific γ-TuRC in Drosophila. We characterize three genes encoding testis-specific components of γ-TuRC (t-γ-TuRC) and find that presence of t-γ-TuRC is essential to male fertility. We show the diverse subcellular distribution of the t-γ-TuRC proteins during post-meiotic development, at first at the centriole adjunct and then also on the anterior tip of the nucleus, and finally, they appear in the tail region, close to the mitochondria. We also prove the physical interactions between the t-γ-TuRC members, γ-tubulin and Mozart1. Our results further indicate heterogeneity in γ-TuRC composition during spermatogenesis and suggest that the different post-meiotic microtubule organizing centers are orchestrated by testis-specific gene products, including t-γ-TuRC.

A new hemizygous missense mutation, c.454T＞C (p.S152P), in AKAP4 gene is associated with asthenozoospermia.

Asthenozoospermia (ASZ) is a condition characterized by reduced forward motility of spermatozoa affecting approximately 19% of infertile men. A kinase anchor protein 4 (AKAP4) is an X-linked testis-specific gene and plays a major role in sperm motility and flagella formation. However, few studies have reported its association with ASZ. Here, we sequenced for exonic mutations of human AKAP4 gene by high-fidelity PCR/Sanger sequencing in peripheral blood samples from 150 ASZ patients and 150 fertile men. We reported the identification of three novel hemizygous mutations unique to four ASZ patients, including one patient carrying missense mutation c.454T>C (p.S152P), two patient carrying synonymous mutation c.1173T>C (p.H391H), and one patient carrying synonymous mutation c.2007 A>G (p.R669R). The p.S152P mutation was located in a precursor pro-polypeptide domain of AKAP4 protein, which was predicted to be damaging by SIFT and PolyPhen-2 and could cause the protein accumulation in the cytoplasm of COS-7 cells. The mature protein of AKAP4 was absent in spermatozoa of ASZ patient harboring AKAP4 p.S152P mutation. Further in vitro cellular assays showed that reactive oxygen species (ROS), malondialdehyde (MDA), myeloperoxidase (MPO) levels, and apoptotic cells were increased in GC2-spd cells by AKAP4 p.S152P mutant protein, whereas superoxide dismutase (SOD) level was decreased. AKAP4 p.H391H and p.R669R mutant proteins were coimmunoprecipitated with ribonuclease T2 (RNASET2) protein in GC2-spd cells, whereas no interaction between the AKAP4 p.S152P mutant protein and RNASET2 protein was observed. In addition, AKAP4 p.S152P mutant protein could decrease the activity of PKA/PI3K signaling. Overall, our study identifies a novel AKAP4 p.S152P mutation is associated with ASZ probably through affecting oxidative stress and cell apoptosis by regulating the interaction with RNASET2 and the activity of the PKA/PI3K signaling pathway.

Characterization of the Impact of Density Gradient Centrifugation on the Profile of the Pig Sperm Transcriptome by RNA-Seq.

RNA-Seq data from human semen suggests that the study of the sperm transcriptome requires the previous elimination from the ejaculates of somatic cells carrying a larger load of RNA. Semen purification is also carried to study the sperm transcriptome in other species including swine and it is often done by density gradient centrifugation to obtain viable spermatozoa from fresh ejaculates or artificial insemination doses, thereby limiting the throughput and remoteness of the samples that can be processed in one study. The aim of this work was to evaluate the impact of purification with density gradient centrifugation by BoviPureTM on porcine sperm. Four boar ejaculates were purified with BoviPureTM and their transcriptome sequenced by RNA-Seq was compared with the RNA-Seq profiles of their paired non-purified sample. Seven thousand five hundred and nineteen protein coding genes were identified. Correlation, cluster, and principal component analysis indicated high—although not complete—similarity between the purified and the paired non-purified ejaculates. 372 genes displayed differentially abundant RNA levels between treatments. Most of these genes had lower abundances after purification and were mostly related to translation, transcription and metabolic processes. We detected a significant change in the proportion of genes of epididymal origin within the differentially abundant genes (1.3%) when compared with the catalog of unaltered genes (0.2%). In contrast, the proportion of testis-specific genes was higher in the group of unaltered genes (4%) when compared to the list of differentially abundant genes (0%). No proportion differences were identified for prostate, white blood, lymph node, tonsil, duodenum, skeletal muscle, liver, and mammary gland. Altogether, these results suggest that the purification impacts on the RNA levels of a small number of genes which are most likely caused by the removal of epididymal epithelial cells but also premature germinal cells, immature or abnormal spermatozoa or seminal exosomes with a distinct load of RNAs.

TSGA10 as a Potential Key Factor in the Process of Spermatid Differentiation/Maturation: Deciphering Its Association with Autophagy Pathway.

Testis-specific gene antigen 10 (TSGA10) plays an important role in spermatogenesis. However, the exact TSGA10 role and its relationship with the autophagy pathway in the process of spermatids differentiation/maturation is still not clear. Therefore, the present study evaluates the role of TSGA10 gene in the spermatid differentiation/maturation through its effect on autophagy and explores possible underlying pathway(s). Sperm samples from patients with teratospermia were collected. The mRNA and protein level of TSGA10 in these samples were assessed by real-time PCR and western blotting. Using the ingenuity pathway analysis (IPA) software, the gene network and interactions of TSGA10 involved in sperm maturation and autophagy were investigated. Based on these analyses, the expression levels of identified genes in patient's samples and healthy controls were further evaluated. Moreover, using flow cytometry analysis, the levels of reactive oxygen species (ROS( production in teratospermic sperm samples were evaluated. The results showed that the expression levels of TSGA10 mRNA and protein decreased significantly in the teratospermic patients compared to controls (P < 0.05). Moreover, a significant reduction in the expression of the important genes involved in sperm maturation and autophagy was observed (P < 0.05). Also, the levels of ROS production in teratospermic sperm samples were shown to be significantly higher compared to those in normal sperms (P < 0.05). Our findings provide new evidence that simultaneous decrease in TSGA10 and autophagy beside the increased level of ROS production in sperm cells might be associated with the abnormalities in the spermatids differentiation/maturation and the formation of sperms with abnormal morphology.

Comparative distributions of RSBN1 and methylated histone H4 Lysine 20 in the mouse spermatogenesis.

During spermatogenesis, nuclear architecture of male germ cells is dynamically changed and epigenetic modifications, in particular methylation of histones, highly contribute to its regulation as well as differentiation of male germ cells. Although several methyltransferases and demethylases for histone H3 are involved in the regulation of spermatogenesis, roles of either histone H4 lysine 20 (H4K20) methyltransferases or H4K20 demethylases during spermatogenesis still remain to be elucidated. Recently, RSBN1 which is a testis-specific gene expressed in round spermatids was identified as a demethylase for dimethyl H4K20. In this study, therefore, we confirm the demethylase function of RSBN1 and compare distributions between RSBN1 and methylated H4K20 in the seminiferous tubules. Unlike previous report, expression analyses for RSBN1 reveal that RSBN1 is not a testis-specific gene and is expressed not only in round spermatids but also in elongated spermatids. In addition, RSBN1 can demethylate not only dimethyl H4K20 but also trimethyl H4K20 and could convert both dimethyl H4K20 and trimethyl H4K20 into monomethyl H4K20. When distribution pattern of RSBN1 in the seminiferous tubule is compared to that of methylated H4K20, both dimethyl H4K20 and trimethyl H4K20 but not monomethyl H4K20 are disappeared from RSBN1 positive germ cells, suggesting that testis-specific distribution patterns of methylated H4K20 might be constructed by RSBN1. Thus, novel expression and function of RSBN1 could be useful to comprehend epigenetic regulation during spermatogenesis.

Inactivation of testis-specific gene C4orf46 is dispensable for spermatogenesis and fertility in mouse.

Several genes have been reported to be involved in spermatogenesis but their functional importance in male fertility is yet needed to be elucidated. Therefore, in current research, we focused to explore the in vivo role of evolutionary conserved and testis-specifically expressed, C4orf46, gene in male mouse fertility and spermatogenesis. The expression profile of C4orf46 is specific to testes and expressed in testes from 7 days of postpartum to onward.Thus, we generated the C4orf46 knockout mice by utilizing CRISPR/Cas9 genome editing technology and examined gene function in spermatogenesis and fertility. Surprisingly, C4orf46 knockout mice were completely fertile, displayed normal testes morphology, however, higher sperm contents were observed in knockout mice compared to wild type (WT) littermates. Subsequently, intact testis histology and architecture of seminiferous tubules were observed in C4orf46 knockout and WT mice. Similarly, sperm morphology and swimming velocity of C4orf46 knockout mice were comparable with the WT littermates. Furthermore, all type of germ cells ranging from spermatogonia to mature spermatozoa were observed in the testes and epididymis sections of C4orf46 knockout mice suggesting that disruption of C4orf46 did not impact spermatogenesis. Moreover, meiotic prophase I progression was normal, and each type of cell population was comparable between knockout and WT mice. Overall, finding from this research indicates that C4orf46 is not an essential gene for fertility in mice. This study will help researchers to avoid the repetition and duplication of efforts, and to explore the genes that are indispensable for spermatogenesis and male fertility.

Relation of gilthead seabream (Sparus aurata) seasonal reproductive activity to hematology, serum biochemistry, histopathology, and Brdt gene expression.

This study aimed to find the relation of Sparus aurata (gilthead seabream) reproductive activities to some blood parameters as complete blood count, liver enzymes, some hormones related to reproduction process and microscopic findings of gonads, as well as expression of Bromodomain testis-specific gene. Eighty-eight sexually mature seabream were collected and investigated through the four seasons. Red blood cells were higher in autumn and spring. Hemoglobin was high in summer, MCV highest values ​​were seen in winter and summer, while MCHC was highest in summer. The values ​​of white blood cells increased significantly in spring, summer, and autumn compared with winter. The highest value of lymphocytes was recorded in spring and autumn. Eosinophil was recorded the highest value in the spring. The highest value of segmented neutrophils was recorded in summer. The highest value of band neutrophil was recorded in summer and winter. Alanine aminotransferase and aspartate aminotransferase showed high values in the winter. Luteinizing hormone (LH) was higher in females, males, and hermaphrodites during winter. Follicle-stimulating hormone (FSH) was higher in females during spring. The highest value of estradiol 17-β and progesterone was recorded in summer. The highest value of total testosterone was recorded in spring. Microscopically, ovaries were immature and inactive during spring and summer but well developed in autumn and winter. During spring and summer, testes were immature and began spermatogenesis process but well developed with the appearance of spermatids and spermatozoa during autumn and winter. The expression of Brdt was higher in testes than ovary. Brdt recorded high expression in autumn and spring than in summer and winter.

Gonadoblastoma in Turner Syndrome: A Surprise in a Streak

Turner syndrome (TS) is a difference in sex development (DSD) affecting 25-50 per 100,000 females with many cardiac, endocrine, neurocognitive, genitourinary, and reproductive implications.1 There is a spectrum of TS depending on degree of chromosomal mosaicism, with particular interest given to those with Y chromosome material present (∼5.5% of TS), which may increase the risk of gonadal malignancy.2 In particular, the expression of testis-specific protein-Y gene is implicated in development of gonadoblastoma (Gb), a premalignant condition found in some patients with DSD.

SEPTIN12 c.474 G &gt; A polymorphism as a risk factor in teratozoospermic patients

Teratozoospermia is a condition related to poor morphologically normal sperm count below the lower reference limit, which could hinder natural conception. Single nucleotide polymorphisms (SNPs) in the genes involved in sperm production and testicular function are proved to be risk factors, resulting in decreased sperm parameters and defects in sperm morphology. c.474 G > A polymorphism in the SEPTIN12 gene which is one of the testis-specific genes creates a novel splice variant and the resulting truncated protein was previously found to be more prevalent in infertile men. We aimed to investigate the association of SEPTIN12 c.474 G > A polymorphism with male infertility in teratozoospermia patients. Forty-eight teratozoospermic patients, diagnosed according to Kruger's criteria and 164 fertile controls who fathered at least 1 child within 3years without assisted reproductive technologies were included into our prospective randomized controlled study. PCR-RFLP method was used for genotyping. Although no statistical difference was found between teratozoospermic patients and fertile controls in terms of genotype distributions, significance was identified between the genotypes of all and non-smoking teratozoopermic patients in terms of neck defects. SEPTIN12 c.474 G > A polymorphism was shown to be associated with sperm neck defects in teratozoospermic patients using the dominant statistical model. Smoking was identified as a risk factor for the sperm morphology defects in teratozoospermic A allele carriers.

Mitochondrial genotype alters the impact of rapamycin on the transcriptional response to nutrients in Drosophila

BackgroundIn addition to their well characterized role in cellular energy production, new evidence has revealed the involvement of mitochondria in diverse signaling pathways that regulate a broad array of cellular functions. The mitochondrial genome (mtDNA) encodes essential components of the oxidative phosphorylation (OXPHOS) pathway whose expression must be coordinated with the components transcribed from the nuclear genome. Mitochondrial dysfunction is associated with disorders including cancer and neurodegenerative diseases, yet the role of the complex interactions between the mitochondrial and nuclear genomes are poorly understood.ResultsUsing a Drosophila model in which alternative mtDNAs are present on a common nuclear background, we studied the effects of this altered mitonuclear communication on the transcriptomic response to altered nutrient status. Adult flies with the ‘native’ and ‘disrupted’ genotypes were re-fed following brief starvation, with or without exposure to rapamycin, the cognate inhibitor of the nutrient-sensing target of rapamycin (TOR). RNAseq showed that alternative mtDNA genotypes affect the temporal transcriptional response to nutrients in a rapamycin-dependent manner. Pathways most greatly affected were OXPHOS, protein metabolism and fatty acid metabolism. A distinct set of testis-specific genes was also differentially regulated in the experiment.ConclusionsMany of the differentially expressed genes between alternative mitonuclear genotypes have no direct interaction with mtDNA gene products, suggesting that the mtDNA genotype contributes to retrograde signaling from mitochondria to the nucleus. The interaction of mitochondrial genotype (mtDNA) with rapamycin treatment identifies new links between mitochondria and the nutrient-sensing mTORC1 (mechanistic target of rapamycin complex 1) signaling pathway.

PDE11A gene polymorphism in testicular cancer: sperm parameters and hormonal profile

PurposeTesticular germ cell tumours (TGCTs) is the most common malignancy among young adult males. The etiology is multifactorial and both environmental and genetic factors play an important role in the origin and development of TGCT. Genetic susceptibility may result from the interaction of multiple common and low-penetrance genetic variants and one of the main candidate genes is PDE11A. Many PDE11A polymorphisms were found responsible for a reduced PDE activity in TGCT patients, who often also display impaired hormone and sperm profile. The aim of this study was to investigate testicular function and PDE11A sequence in testicular cancer cases.MethodsSemen analysis was performed in 116 patients with unilateral and bilateral sporadic TGCTs and in 120 cancer-free controls. We also investigated hormone profile and PDE11A polymorphisms using peripheral blood samples.ResultsOur data revealed that TGCT patients showed lower testosterone levels, higher gonadotropins levels and worse semen quality than controls, although the mean and the medians of sperm parameters are within the reference limits. PDE11A sequencing detected ten polymorphisms not yet associated with TGCTs before. Among these, G223A in homozygosity and A288G in heterozygosity were significantly associated with a lower risk of testicular tumour and they displayed a positive correlation with total sperm number.ConclusionsOur findings highlight the key role of PDE11A in testis and suggest the presence of an underlying complex and fine molecular mechanism which controls testis-specific gene expression and susceptibility to testicular cancer.

The mRNA expression profile of the goat prion protein testis-specific (PRNT) gene and its associations with litter size

The goat PRNT gene was initially identified as a testis-specific gene with a role in spermatogenesis. In this study, we used quantitative real-time PCR (qPCR) to first determine the mRNA expression profile of this gene in different goat tissues. Surprisingly, we found that PRNT was expressed not only in the testis but also in nine other tissues in goats. Moreover, PRNT was weakly expressed in the testis, while its expression was strongest in the ovary. These results, combined with those of other studies, led us to hypothesize that the goat PRNT gene has a role in both male and female reproduction. We further used direct DNA sequencing to detect potential SNPs within this gene in Shaanbei whit cashmere (SBWC) rams and ewes, and identified three SNPs within the PRNT gene, namely, c.-58C > T, c.71A > G (p.Alanine24Valine), and c.102C > T (synonymous). In rams, c.-58C > T and c.102C > T were strongly linked with each other (D′ = 1.000, r2 = 0.504), whereas no significant association (P > 0.05) was found between the three SNPs and semen quality, which was consistent with the low expression of the PRNT gene in the testis. Interestingly, in ewes (n = 502), c.-58C > T and c.71A > G were also strongly linked with each other (D′ = 0.973, r2 = 0.537). Additionally, the c.71A > G locus, especially the AA genotype, had a significant influence on litter size (P = 0.006), consistent with the high PRNT expression in the ovary. Combined, the results of the expression profiling and analysis of the association between the SNPs and reproductive traits showed that two strongly linked nucleotide sequence variants within PRNT were significantly associated with goat litter size. These findings provide potential DNA markers for use in the marker-assisted selection (MAS) of goats with high-fertility traits.

The Germ Cell-Specific Markers ZPBP2 and PGK2 in Testicular Biopsies Can Predict the Presence as well as the Quality of Sperm in Non-obstructive Azoospermia Patients.

To assess the role of three testis-specific genes including ZPBP2, PGK2, and ACRV1 in the prediction of sperm retrieval result and quality of retrieved sperm by microdissection testicular sperm extraction (micro-TESE) in non-obstructive azoospermia (NOA) patients. This was a case-control study including 57 testicular samples of NOA patients including 32 patients with successful sperm retrieval (NOA+) and 25 patients with failed sperm retrieval (NOA-), and 9 samples of men with normal spermatogenesis in the testes as the positive control (OA). We investigated the expression of candidate genes by RT-qPCR and germ cell population patterns by DNA flow cytometry in testicular biopsy samples. The association between PGK2 expressions with the quality of retrieved spermatozoa was also evaluated. The RT-qPCR data revealed a significantly higher expression of ZPBP2 and PGK2 in the NOA+ in comparison to NOA- group (P = 0.002, and P = 0.002, respectively). Flow cytometry results revealed that the haploid cell percentage was significantly higher in NOA+ vs. NOA- group (P = 0.0001). In samples with a higher percentage of haploid cells, expression levels of ZPBP2 and PGK2 were higher (P = 0.001). The PGK2 expression was significantly associated with retrieved sperm quality (P = 0.01). Our results contribute to the search for the biomarkers for predicting the presence of testicular sperm and would be useful to avoid unnecessary multiple micro-TESE. Overall, the expression pattern of the ZPBP2 and PGK2 may be useful in predicting sperm recovery success and quality of retrieved sperm in NOA patients.

Spata19 Inactivation as a Cause of Oligospermia

Background: Spermatogenesis associated 19 (Spata19) was introduced as a testis-specific gene that was probably involved in spermatogenesis cell apoptosis. Therefore, this study aimed to investigate the effect of Spata19 inactivation on sperm count. Materials and Methods: We generated global Spata19 knockout mice by CRISPR/Cas9 nickase technology. Disability was validated in three levels of DNA, RNA, and protein using PCR, RT-PCR, and immunohistochemistry. Histological studies were performed for testis. Sperm characteristics were also assessed with CASA software. Results: Spata19 knockout mice had a 43 nucleotides deletion in exon 4 of this gene. The presence and absence of Spata19 were confirmed in normal and knockout mice, respectively. The presence of Spata19 in normal NMRI mice was detected in the brain, heart, and thymus by semi-nested RT-PCR and in Leydig cells by immunohistochemistry. Histological studies revealed a decrease in sperm count in knockout mice. Also, CASA parameters were significantly reduced (P&lt;0.05). Conclusion: These data indicate that Spata19 inactivation is a cause of oligospermia, and its role could be beyond an adhesive molecule.

Stellate Genes and the piRNA Pathway in Speciation and Reproductive Isolation of Drosophila melanogaster.

One of the main conditions of the species splitting from a common precursor lineage is the prevention of a gene flow between diverging populations. The study of Drosophila interspecific hybrids allows to reconstruct the speciation mechanisms and to identify hybrid incompatibility factors that maintain post-zygotic reproductive isolation between closely related species. The regulation, evolution, and maintenance of the testis-specific Ste-Su(Ste) genetic system in Drosophila melanogaster is the subject of investigation worldwide. X-linked tandem testis-specific Stellate genes encode proteins homologous to the regulatory β-subunit of protein kinase CK2, but they are permanently repressed in wild-type flies by the piRNA pathway via piRNAs originating from the homologous Y-linked Su(Ste) locus. Derepression of Stellate genes caused by Su(Ste) piRNA biogenesis disruption leads to the accumulation of crystalline aggregates in spermatocytes, meiotic defects and male sterility. In this review we summarize current data about the origin, organization, evolution of the Ste-Su(Ste) system, and piRNA-dependent regulation of Stellate expression. The Ste-Su(Ste) system is fixed only in the D. melanogaster genome. According to our hypothesis, the acquisition of the Ste-Su(Ste) system by a part of the ancient fly population appears to be the causative factor of hybrid sterility in crosses of female flies with males that do not carry Y-linked Su(Ste) repeats. To support this scenario, we have directly demonstrated Stellate derepression and the corresponding meiotic disorders in the testes of interspecies hybrids between D. melanogaster and D. mauritiana. This finding embraces our hypothesis about the contribution of the Ste-Su(Ste) system and the piRNA pathway to the emergence of reproductive isolation of D. melanogaster lineage from initial species.