Testis-specific Expression Research Articles

Tissue distribution of cysteine string protein/DNAJC5 in C. elegans analysed by CRISPR/Cas9-mediated tagging of endogenous DNJ-14.

Cysteine string protein (CSP) is a member of the DnaJ/Hsp40 family of molecular chaperones. CSP is enriched in neurons, where it mainly localises to synaptic vesicles. Mutations in CSP-encoding genes in flies, worms, mice and humans result in neuronal dysfunction, neurodegeneration and reduced lifespan. Most attention has therefore focused on CSP's neuronal functions, although CSP is also expressed in non-neuronal cells. Here, we used genome editing to fluorescently tag the Caenorhabditis elegans CSP orthologue, dnj-14, to identify which tissues preferentially express CSP and hence may contribute to the observed mutant phenotypes. Replacement of dnj-14 with wrmScarlet caused a strong chemotaxis defect, as seen with other dnj-14 null mutants. In contrast, inserting the reporter in-frame to create a DNJ-14-wrmScarlet fusion protein had no effect on chemotaxis, indicating that C-terminal tagging does not impair DNJ-14 function. WrmScarlet fluorescence appeared most obvious in the intestine, head/pharynx, spermathecae and vulva/uterus in the reporter strains, suggesting that DNJ-14 is preferentially expressed in these tissues. Crossing the DNJ-14-wrmScarlet strain with GFP marker strains confirmed the intestinal and pharyngeal expression, but only a partial overlap with neuronal GFP was observed. DNJ-14-wrmScarlet fluorescence in the intestine was increased in response to starvation, which may be relevant to mammalian CSPα's role in microautophagy. DNJ-14's enrichment in worm reproductive tissues (spermathecae and vulva/uterus) parallels the testis-specific expression of CSPβ and CSPγ isoforms in mammals. Furthermore, CSPα messenger RNA is highly expressed in the human proximal digestive tract, suggesting that CSP may have a conserved, but overlooked, function within the gastrointestinal system.

Novel molecular aberrations involved in testicular germ cell tumor (TGCT) development and cisplatin resistance.

520 Background: Although highly curable, a proportion of testicular germ cell tumors (TGCT) develop resistance to cisplatin-based chemotherapy with subsequent disease relapses and possible patient death. The causes of disease progression and cisplatin (CDDP) resistance have not yet been well elucidated. Methods: We analyzed 56 samples of 15 patients with advanced and relapsing TGCT including primary tumors, metastases persisting after CDDP-containing chemotherapy, and circulating free tumor DNA (cfDNA) from the disease progression, with gDNA from blood leucocytes used as a background control. The quantity and quality of cfDNA was checked by capillary electrophoresis. All samples were sequenced by whole exome sequencing with SureSelect libraries on Illumina platform. Only mutations that were found in malignant samples or cfDNA with variant allele frequency (VAF) ≥ 5% and at least in 3 reads, not present in gDNA, with a minimum total read depth of 5 quality reads, excluding synonymous variants, were further analyzed. Results: Mutations of 6 genes with a significant role in carcinogenesis and/or testis development were identified in more than one patient: RBMX (in 4 pts), TPTE2 and ANKRD30A (in 3 pts), CDC27, PRAMEF8, and PRDM9 (in 2 pts). All the mutations were missense, with VAF 5 - 86%. They were all detectable in patients’ cfDNA, with increasing VAF during the tumor progression; the VAFs in cfDNA collected at advanced disease stages were even higher than those in primary tumors. PRDM9 mutations were found only in cfDNA from disease progression, but not in the primary testicular tumors. Of the identified genes, only CDC27 mutations have been previously described in TGCT. The genes newly associated with TGCT in this study are involved in genomic stability maintenance ( RBMX), spermiogenesis ( TPTE2), cell proliferation and apoptosis ( PRAMEF8), and several of them show testis-specific expression ( TPTE2, PRAMEF8, ANKRD30A, PRDM9). PRDM9 encodes a zinc-finger protein with histone methyltransferase activity, responsible for H3 methylation during meiosis. As epigenetic changes are supposed to play a crucial role in TGCT pathogenesis and resistance, and aberrant H3 methylation has been previously related to abnormal expression of OCT3/4 transcription factor promoting TGCT development, this gene is a strong candidate for TGCT progression and evolving CDDP resistance. Conclusions: Mutations of 5 novel genes have been identified in association with TGCT progression and cisplatin resistance. Their impact is strongly suggested by their functions in normal and malignant regulatory pathways and testis-specific expression patterns but has to be confirmed in experimental studies. The National Institute for Cancer Research project (Programme EXCELES, ID Project No. LX22NPO5102) - Funded by the European Union - Next Generation EU. Supported by grants MH CZ - DRO (00064190, 00064203).

MRNA-Seq of testis and liver tissues reveals a testis-specific gene and alternative splicing associated with hybrid male sterility in dzo.

Alternative splicing (AS) plays an important role in the co-transcription and post-transcriptional regulation of gene expression during mammalian spermatogenesis. The dzo is the male F1 offspring of an interspecific hybrid between a domestic bull (Bos taurus ♂) and a yak (Bos grunniens ♀) which exhibits male sterility. This study aimed to identify the testis-specific genes and AS associated with hybrid male sterility in dzo. The iDEP90 program and rMATS software were used to identify the differentially expressed genes (DEG) and differential alternative splicing genes (DSG) based on RNA-seq data from the liver (n = 9) and testis (n = 6) tissues of domestic cattle, yak, and dzo. Splicing factors (SF) were obtained from the AmiGO2 and the NCBI databases, and Pearson correlation analysis was performed on the differentially expressed SFs and DSGs. We focused on the testis-specific DEGs and DSGs between dzo and cattle and yak. Among the top 3,000 genes with the most significant variations between these 15 samples, a large number of genes showed testis-specific expression involved with spermatogenesis. Cluster analysis showed that the expression levels of these testis-specific genes were dysregulated during mitosis with a burst downregulation during the pachynema spermatocyte stage. The occurrence of AS events in the testis was about 2.5 fold greater than in the liver, with exon skipping being the major AS event (81.89% to 82.73%). A total of 74 DSGs were specifically expressed in the testis and were significantly enriched during meiosis I, synapsis, and in the piRNA biosynthesis pathways. Notably, STAG3 and DDX4 were of the exon skipping type, and DMC1 was a mutually exclusive exon. A total of 36 SFs were significantly different in dzo testis, compared with cattle and yak. DDX4, SUGP1, and EFTUD2 were potential SFs leading to abnormal AS of testis-specific genes in dzo. These results show that AS of testis-specific genes can affect synapsis and the piRNA biosynthetic processes in dzo, which may be important factors associated with hybrid male sterility in dzo.

An efficient and safe strategy for germ cell-specific automatic excision of foreign DNA in F1 hybrid transgenic silkworms.

The safety of transgenic technology is a major obstacle in the popularization and use of transgenic silkworms and their products. In sericulture, only the first filial generation (F1 ) hybrid eggs produced by cross-breeding Japanese and Chinese original strains are usually used for the large-scale breeding of silkworms, but this may result in uncontrolled transgene dispersal during the popularization and application of the F1 hybrid transgenic eggs. To address this issue, we developed a safe and efficient strategy using the GAL4/Upstream activating sequence (UAS) system, the FLP/flippase recognition target (FRT) system, and the gonad-specific expression gene promoters (RSHP1p and Nanosp) for the germ cell-specific automatic excision of foreign DNA in the F1 hybrid transgenic silkworms. We established 2 types of activator strains, R1p::GAL4-Gr and Nsp::GAL4-Gr, containing the testis-specific GAL4 gene expression cassettes driven by RSHP1p or Nanosp, respectively, and 1 type of effector strain, UAS::FLP-Rg, containing the UAS-linked FLP gene expression cassette. The FLP recombinase-mediated sperm-specific complete excision of FRT-flanked target DNA in the F1 double-transgenic silkworms resulting from the hybridization of R1p::GAL4-Gr and UAS::FLP-Rg was 100%, whereas the complete excision efficiency resulting from the hybridization of Nsp::GAL4-Gr and UAS::FLP-Rg ranged from 13.73% to 80.3%. Additionally, we identified a gene, sw11114, that is expressed in both testis and ovary of Bombyx mori, and can be used to establish novel gonad-specific expression systems in transgenic silkworms. This strategy has the potential to fundamentally solve the safety issue in the production of F1 transgenic silkworm eggs and provides an important reference for the safety of transgenic technology in other insect species.

Duplicate genes as sources for rapid adaptive evolution of sperm under environmental pollution in tree sparrow.

Environmental pollution can result in poor sperm quality either directly or indirectly in birds. However, adaptive and compensatory sperm morphology changes and motility improvements have rapidly evolved in tree sparrows (Passer montanus) inhabiting polluted areas over the past 65 years. To identify the genetic underpinnings of the rapidly evolving sperm phenotype, we carried out population genomics and transcriptomics on tree sparrow populations in the two differently polluted places. We identified a gene encoding the serine/threonine protein kinase PIM1, which may drive rapid phenotypic evolution of sperm. An unprecedented and remarkable expansion of the PIM gene family, caused by tandem and segmental duplication of PIM1, was subsequently observed in the tree sparrow genome. Most PIM1 duplicates showed a testis-specific expression pattern, suggesting that their functions are related to male reproduction. Furthermore, the elevated expression level of PIM1 was consistent with our earlier findings of longer and faster swimming sperm in polluted sites, indicating an important role for duplicated PIM1 in facilitating the rapid evolution of sperm. Our results suggest that duplicated PIM1 provides sources of genetic variation that may enable the rapid evolution of sperm under environmental heavy metal pollution. The findings of this study indicated that duplicated genes can be targets of selection and predominant sources for rapid adaptation to environmental change and shed light on sperm evolution under pollution stress.

Loss of Cep72 affects the morphology of spermatozoa in mice.

The centrosome regulates mammalian meiosis by affecting recombination, synapsis, chromosome segregation, and spermiogenesis. Cep72 is one of the critical components of the centrosome. However, the physiological role of Cep72 in spermatogenesis and fertility remains unclear. In this study, we identify Cep72 as a testis-specific expression protein. Although Cep72 knockout mice were viable and fertile, their sperms were morphologically abnormal with incomplete flagellum structures. Transcriptome analysis reveals significant differences in six genes (Gm49527, Hbb-bt, Hba-a2, Rps27a-ps2, Gm29647, and Gm8430), which were not previously associated with spermatogenesis. Overall, these results indicate that Cep72 participates in regulating sperm morphology and yet is dispensable for fertility in mice.

Genome-wide and expression-profiling analyses of the cytochrome P450 genes in Tenebrionidea.

Cytochrome P450 monooxygenases (CYPs) are present in almost all areas of the tree of life. As one of the largest and most diverse superfamilies of multifunctional enzymes, they play important roles in the metabolism of xenobiotics and biosynthesis of endogenous compounds, shaping the success of insects. In this study, the CYPome (an omics term for all the CYP genes in a genome) diversification was examined in the four Tenebrionidea species through genome-wide analysis. A total of 483 CYP genes were identified, of which 103, 157, 122, and 101 were respectively deciphered from the genomes of Tebebrio molitor, Asbolus verucosus, Hycleus cichorii and Hycleus phaleratus. These CYPs were classified into four major clans (mitochondrial, CYP2, CYP3, and CYP4), and clans CYP3 and CYP4 are most diverse. Phylogenetic analysis showed that most CYPs of these Tenebrionidea beetles from each clan had a very close 1:1 orthology to each other, suggesting that they originate closely and have evolutionally conserved function. Expression analysis at different developmental stages and in various tissues showed the life stage-, gut-, salivary gland-, fat body-, Malpighian tubule-, antennae-, ovary- and testis-specific expression patterns of T. molitor CYP genes, implying their various potential roles in development, detoxification, immune response, digestion, olfaction, and reproduction. Our studies provide a platform to understand the evolution of Tenebrionidea CYP gene superfamily, and a basis for further functional investigation of the T. molitor CYPs involved in various biological processes.

COX4-like, a Nuclear-Encoded Mitochondrial Gene Duplicate, Is Essential for Male Fertility in Drosophila melanogaster.

Recent studies on nuclear-encoded mitochondrial genes (N-mt genes) in Drosophila melanogaster have shown a unique pattern of expression for newly duplicated N-mt genes, with many duplicates having a testis-biased expression and playing an essential role in spermatogenesis. In this study, we investigated a newly duplicated N-mt gene—i.e., Cytochrome c oxidase 4-like (COX4L)—in order to understand its function and, consequently, the reason behind its retention in the D. melanogaster genome. The COX4L gene is a duplicate of the Cytochrome c oxidase 4 (COX4) gene of OXPHOS complex IV. While the parental COX4 gene has been found in all eukaryotes, including single-cell eukaryotes such as yeast, we show that COX4L is only present in the Brachycera suborder of Diptera; thus, both genes are present in all Drosophila species, but have significantly different patterns of expression: COX4 is highly expressed in all tissues, while COX4L has a testis-specific expression. To understand the function of this new gene, we first knocked down its expression in the D. melanogaster germline using two different RNAi lines driven by the bam-Gal4 driver; second, we created a knockout strain for this gene using CRISPR-Cas9 technology. Our results showed that knockdown and knockout lines of COX4L produce partial sterility and complete sterility in males, respectively, where a lack of sperm individualization was observed in both cases. Male infertility was prevented by driving COX4L-HA in the germline, but not when driving COX4-HA. In addition, ectopic expression of COX4L in the soma caused embryonic lethality, while overexpression in the germline led to a reduction in male fertility. COX4L-KO mitochondria show reduced membrane potential, providing a plausible explanation for the male sterility observed in these flies. This prominent loss-of-function phenotype, along with its testis-biased expression and its presence in the Drosophila sperm proteome, suggests that COX4L is a paralogous, specialized gene that is assembled in OXPHOS complex IV of male germline cells and/or sperm mitochondria.

Testis-specific expression pattern of dmrt1 and its putative regulatory region in the sea urchin (Mesocentrotus nudus).

Sea urchin (Mseocentrotus nudus) is an economically important mariculture species in several Asian countries. The growth rate and immunocompetence differ by sex in this species. However, the mechanisms of sex determination in M. nudus have remain unclear. In the present study, we focus on the dmrt1 gene of M. nudus (Mndmrt1) to investigate its dynamic expression pattern during different developmental stages. Real-time quantitative PCR (RT-qPCR) revealed that Mndmrt1 exhibits testis-specific expression and undetectable during the whole embryogenesis. With the development of ontogenetic, Mndmrt1 transcripts are first detected at 9months post-fertilization (mpf). In addition, both the transcripts and protein of Mndmrt1 gene were specifically expressed in spermatogonia and spermatocytes, indicating that it might be a male germ cells marker in sea urchin. Significantly, the 1441bp promoter sequence of Mndmrt1 gene was obtained by DNA walking, and one positive regulatory region at -1197/ -968 in the promoter, as well as one negative regulatory region at -1441/ -1198 have been identified by promoter activity analysis. Moreover, two regulatory regions contain multiple putative binding sites for transcription factors, including Sp1, Egr1, Sox5, CEBP, GATA and SRY. These findings suggest that Mndmrt1 may be related to testis differentiation and spermatogenesis in sea urchin and will provide an insight into understanding the regulatory mechanism of the dmrt1 gene.

Distinct roles of haspin in stem cell division and male gametogenesis

The kinase haspin phosphorylates histone H3 at threonine-3 (H3T3ph) during mitosis. H3T3ph provides a docking site for the Chromosomal Passenger Complex at the centromere, enabling correction of erratic microtubule-chromosome contacts. Although this mechanism is operational in all dividing cells, haspin-null mice do not exhibit developmental anomalies, apart from aberrant testis architecture. Investigating this problem, we show here that mouse embryonic stem cells that lack or overexpress haspin, albeit prone to chromosome misalignment during metaphase, can still divide, expand and differentiate. RNA sequencing reveals that haspin dosage affects severely the expression levels of several genes that are involved in male gametogenesis. Consistent with a role in testis-specific expression, H3T3ph is detected not only in mitotic spermatogonia and meiotic spermatocytes, but also in non-dividing cells, such as haploid spermatids. Similarly to somatic cells, the mark is erased in the end of meiotic divisions, but re-installed during spermatid maturation, subsequent to methylation of histone H3 at lysine-4 (H3K4me3) and arginine-8 (H3R8me2). These serial modifications are particularly enriched in chromatin domains containing histone H3 trimethylated at lysine-27 (H3K27me3), but devoid of histone H3 trimethylated at lysine-9 (H3K9me3). The unique spatio-temporal pattern of histone H3 modifications implicates haspin in the epigenetic control of spermiogenesis.

An actin-related protein that is most highly expressed in Drosophila testes is critical for embryonic development.

Most actin-related proteins (Arps) are highly conserved and carry out well-defined cellular functions in eukaryotes. However, many lineages like Drosophila and mammals encode divergent non-canonical Arps whose roles remain unknown. To elucidate the function of non-canonical Arps, we focus on Arp53D, which is highly expressed in testes and retained throughout Drosophila evolution. We show that Arp53D localizes to fusomes and actin cones, two germline-specific actin structures critical for sperm maturation, via a unique N-terminal tail. Surprisingly, we find that male fertility is not impaired upon Arp53D loss, yet population cage experiments reveal that Arp53D is required for optimal fitness in Drosophila melanogaster. To reconcile these findings, we focus on Arp53D function in ovaries and embryos where it is only weakly expressed. We find that under heat stress Arp53D-knockout (KO) females lay embryos with reduced nuclear integrity and lower viability; these defects are further exacerbated in Arp53D-KO embryos. Thus, despite its relatively recent evolution and primarily testis-specific expression, non-canonical Arp53D is required for optimal embryonic development in Drosophila.

H2A.B is a cancer/testis factor involved in the activation of ribosome biogenesis in Hodgkin lymphoma.

Testis-specific regulators of chromatin function are commonly ectopically expressed in human cancers, but their roles are poorly understood. Examination of 81 primary Hodgkin lymphoma (HL) samples showed that the ectopic expression of the eutherian testis-specific histone variant H2A.B is an inherent feature of HL. In experiments using two HL cell lines derived from different subtypes of HL, H2A.B knockdown inhibited cell proliferation. H2A.B was enriched in both nucleoli of these HL cell lines and primary HL samples. We found that H2A.B enhanced ribosomal DNA (rDNA) transcription, was enriched at the rDNA promoter and transcribed regions, and interacted with RNA Pol I. Depletion of H2A.B caused the loss of RNA Pol I from rDNA chromatin. Remarkably, H2A.B was also required for high levels of ribosomal protein gene expression being located at the transcriptional start site and within the gene body. H2A.B knockdown reduced gene body chromatin accessibility of active RNA Pol II genes concurrent with a decrease in transcription. Taken together, our data show that in HL H2A.B has acquired a new function, the ability to increase ribosome biogenesis.

The Dispensable Roles of X-Linked Ubl4a and Its Autosomal Counterpart Ubl4b in Spermatogenesis Represent a New Evolutionary Type of X-Derived Retrogenes.

X-derived retrogenes contribute to genetic diversity in evolution and are usually specifically expressed in testis and perform important functions during spermatogenesis. Ubl4b is an autosomal retrogene with testis-specific expression derived from Ubl4a, an X-linked housekeeping gene. In the current study, we performed phylogenetic analysis and revealed that Ubl4a and Ubl4b are subject to purifying selection and may have conserved functions in evolution. Ubl4b was knocked out in mice using CRISPR/Cas9 genome editing technology and interestingly, we found no alterations in reproductive parameters of Ubl4b–/– male mice. To get insights into whether Ubl4a could compensate the absence of Ubl4b in vivo, we further obtained Ubl4a–/Y; Ubl4b–/– mice that lack both Ubl4a and Ubl4b, and the double knockout (dKO) mice also displayed normal spermatogenesis, showing that Ubl4a and Ubl4b are both dispensable for spermatogenesis. Thus, through the in vivo study of UBL4A and UBL4B, we provided a direct evidence for the first time that some X chromosome-derived autosomal retrogenes can be unfunctional in spermatogenesis, which represents an additional evolutionary type of X-derived retrogenes.

Effect and in silico characterization of genetic variants associated with severe spermatogenic disorders in a large Iberian cohort.

Severe spermatogenic failure (SpF) represents the most extreme manifestation of male infertility, as it decreases drastically the semen quality leading to either severe oligospermia (SO, <5 million spermatozoa/mL semen) or non-obstructive azoospermia (NOA, complete lack of spermatozoa in the ejaculate without obstructive causes). The main objective of the present study is to analyze in the Iberian population the effect of 6 single-nucleotide polymorphisms (SNPs) previously associated with NOA in Han Chinese through genome-wide association studies (GWAS) and to establish their possible functional relevance in the development of specific SpF patterns. We genotyped 674 Iberian infertile men (including 480 NOA and 194 SO patients) and 1058 matched unaffected controls for the GWAS-associated variants PRMT6-rs12097821, PEX10-rs2477686, CDC42BPA-rs3000811, IL17A-rs13206743, ABLIM1-rs7099208, and SOX5-rs10842262. Their association with SpF, SO, NOA, and different NOA phenotypes was evaluated by logistic regression models, and their functional relevance was defined by comprehensive interrogation of public resources. ABLIM1-rs7099208 was associated with SpF under both additive (OR=0.86, p=0.036) and dominant models (OR=0.78, p=0.026). The CDC42BPA-rs3000811 minor allele frequency was significantly increased in the subgroup of NOA patients showing maturation arrest (MA) of germ cells compared to the remaining NOA cases under the recessive model (OR=4.45, p=0.044). The PEX10-rs2477686 SNP was associated with a negative testicular sperm extraction (TESE) outcome under the additive model (OR=1.32, p=0.034). The analysis of functional annotations suggested that these variants affect the testis-specific expression of nearby genes and that lincRNA may play a role in SpF. Our data support the association of three previously reported NOA risk variants in Asians (ABLIM1-rs7099208, CDC42BPA-rs3000811, and PEX10-rs2477686) with different manifestations of SpF in Iberians of European descent, likely by influencing gene expression and lincRNA deregulation.

Chromosome level reference of Atlantic halibut Hippoglossus hippoglossus provides insight into the evolution of sexual determination systems.

Changes in the genetic mechanisms that control sexual determination have occurred independently across the tree of life, and with exceptional frequency in teleost fishes. To investigate the genomic changes underlying the evolution of sexual determination, we sequenced a chromosome-level genome, multitissue transcriptomes, and reduced representation population data for the Atlantic halibut (Hippoglossus hippoglossus), which has an XY/XX sex determination mechanism and has recently diverged (0.9-3.8Ma) from the Pacific halibut (Hippoglossus stenolepis), which has a ZZ/ZW system. We used frequency and coverage-based population approaches to identify a putative sex-determining factor, GSDF. We characterized regions with elevated heterozygosity and linkage disequilibrium indicating suppression of recombination across a nascent sex chromosome. We detected testis-specific expression of GSDF, the sequence of which is highly conserved across flatfishes. Based on evidence from genome-wide association, coverage, linkage disequilibrium, testis and brain transcriptomes, and sequence conservation with other flatfishes, we propose a mechanism for the recent evolution of an XY sex-determination mechanism in Atlantic halibut. Changes to the ancestral sex-determining gene DMRT1 in regulating the downstream gene GSDF probably coincided with GSDF, or a proximal regulatory element of it, becoming the primary sex-determining factor. Our results suggest changes to a small number of elements can have drastic repercussions for the genomic substrate available to sex-specific evolutionary forces, providing insight into how certain elements repeatedly evolve to control sex across taxa. Our chromosome-level assembly, multitissue transcriptomes, and population genomic data provide a valuable resource and understanding of the evolution of sexual systems in fishes.

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.

Expression profile analysis of a new testis-specifically expressed gene C17ORF64 and its association with cell apoptosis in MCF-7 cells.

With the increasing incidence of male infertility, identification and investigation the functions of new genes related to spermatogenesis are effective avenues to elucidate the decline of testicular function. In this study, a new gene, C17ORF64 (chromosome 17 open reading frame 64), was identified from mouse testes and its potential function was studied.RT-PCR and qRT-PCR assay showed that C17ORF64 mRNA was expressed exclusively in mouse testes and up-regulated from the 3-week old to 6-month old testes during postpartum development, which is consistent with C17ORF64 protein expression profile by western blotting analysis. Immunohistochemical analysis revealed that C17ORF64 protein was mainly localized in the cytoplasm of spermatogonia and spermatocytes, which is verified by GFP- labeled C17ORF64 gene expressed in GC-1 cells. C17ORF64 overexpression not only promoted cell apoptosis in MCF-7 cells, but also significantly decreased cell viability via MTT assay. Flow cytometric assay showed that C17ORF64 overexpression could inhibit cell cycle progression by arresting G1/S transition. Western blot and qRT-PCR analysis revealed that C17ORF64 overexpression inhibited the expression of anti-apoptotic protein bcl-2 and increased the expressions of pro-apoptotic protein caspase-3, caspase-8, caspase-9, Bax, P21 and P53. Taken together, our results confirmed C17ORF64 testis-specific expression pattern and, for the first time, demonstrated that C17ORF64 could inhibit cell viability and accelerate apoptosis in MCF-7 cells through caspase-3 regulatory pathways.

Normal fertility in male mice lacking ADAM32 with testis-specific expression

The a disintegrin and metalloprotease (ADAM) family proteins comprise a group of membrane-anchored proteins. ADAM32 is expressed specifically in testis and is closely related phylogenetically to ADAM2 and ADAM3, which are known to be critical for fertilization in mice. To assess the biological role of ADAM32, we analyzed Adam32-mutant mice. We found that male mice lacking ADAM32 have normal fertility, testicular integrity, and sperm characteristics. ADAM32 was found to exist at lower levels than ADAM2 and ADAM3 in wild-type testis and sperm, respectively. The present study demonstrates that ADAM32 is dispensable for fertility and appears to be functionally unrelated to ADAM2 and ADAM3 in mice.

Gonadal Transcriptome Analysis of Sex-Related Genes in the Protandrous Yellowfin Seabream (Acanthopagrus latus).

Yellowfin seabream (Acanthopagrus latus), a protandrous hermaphroditic fish, is a good model for studying the mechanism of sex reversal. However, limited knowledge is known about the genetic information related to reproduction and sex differentiation in this species. Here, we performed de novo transcriptome sequencing analysis of the testis, ovotestis, and ovary to identify sex-related genes in yellowfin seabream. The results assembled 71,765 unigenes in which 16,126 and 17,560 unigenes were differentially expressed in the ovotestis and ovary compared to the testis, respectively. The most differentially expressed gene (DEG)-enriched Kyoto Encyclopedia of Genes and Genomes and GO pathways were closely associated with the synthesis of sex steroid hormones. Functional analyses identified 55 important sex-related DEGs, including 32 testis-biased DEGs (dmrt1, amh, and sox9, etc.), 20 ovary-biased DEGs (cyp19a, foxl2, and wnt4, etc.), and 3 ovotestis-biased DEGs (lhb, dmrt2, and foxh1). Furthermore, the testis-specific expression of dmrt1 and the brain-pituitary-ovary axis expression of foxl2 were characterized, suggesting that they might play important roles in sex differentiation in yellowfin seabream. Our present work provided an important molecular basis for elucidating the mechanisms underlying sexual transition and reproductional regulation in yellowfin seabream.

CRISPR/Cas9 in Male Factor Infertility

Male factor infertility is common and often multifactorial. A subset of these patients have underlying genetic etiologies. CRISPR/Cas9 is a simple and flexible gene editing tool with promising applications in this space. This review aims to summarize the advances that CRISPR/Cas9-based tools have brought to the field and propose future directions for study. CRISPR/Cas9 has been applied successfully to spermatogonial stem cells (SSCs) and via pronuclear injection of zygotes to generate animal models of male factor infertility. These approaches have led to the high-throughput validation of candidate male fertility genes obtained either through genome-wide associated studies or testis-specific gene expression studies. One group has applied this further to SSCs in the correction of genetic infertility due to a mutation in the Kit gene. Application of CRISPR/Cas9 to the investigation and treatment of male infertility holds promise in identifying novel genetic causes of NOA. Gene editing in germ cells to treat genetic infertility is technically feasible, but has not been used in humans due to significant ethical concerns. Stringent regulations are imperative to ensure safe translation of this technology into human populations.