Regulation of Miwi-mediated mRNA stabilization by Ck137956/Tssa is essential for male fertility.

BACKGROUND: Sperm is formed through spermiogenesis, a highly complex process involving chromatin condensation that results in cessation of transcription. mRNAs required for spermiogenesis are transcribed at earlier stages and translated in a delayed fashion during spermatid formation. However, it remains unknown that how these repressed mRNAs are stabilized. RESULTS: Here we report a Miwi-interacting testis-specific and spermiogenic arrest protein, Ck137956, which we rename Tssa. Deletion of Tssa led to male sterility and absence of sperm formation. The spermiogenesis arrested at the round spermatid stage and numerous spermiogenic mRNAs were down-regulated in Tssa-/- mice. Deletion of Tssa disrupted the localization of Miwi to chromatoid body, a specialized assembly of cytoplasmic messenger ribonucleoproteins (mRNPs) foci present in germ cells. We found that Tssa interacted with Miwi in repressed mRNPs and stabilized Miwi-interacting spermiogenesis-essential mRNAs. CONCLUSIONS: Our findings indicate that Tssa is indispensable in male fertility and has critical roles in post-transcriptional regulations by interacting with Miwi during spermiogenesis.

Gonadal white adipose tissue is important for gametogenesis in mice through maintenance of local metabolic and immune niches.

Gonadal white adipose tissue (gWAT) can regulate gametogenesis via modulation of neuroendocrine signaling. However, the effect of gWAT on the local microenvironment of the gonad was largely unknown. Herein, we ruled out that gWAT had a neuroendocrine effect on gonad function through a unilateral lipectomy strategy, in which cutting off epididymal white adipose tissue could reduce seminiferous tubule thickness and decrease sperm counts only in the adjacent testis and epididymis of the affected gonad. Consistent with the results in males, in females, ovary mass was similarly decreased by lipectomy. We determined that the defects in spermatogenesis were mainly caused by augmented apoptosis and decreased proliferation of germ cells. Transcriptome analysis suggested that lipectomy could disrupt immune privilege and activate immune responses in both the testis and ovary on the side of the lipectomy. In addition, lipidomics analysis in the testis showed that the levels of lipid metabolites such as free carnitine were elevated, whereas the levels of glycerophospholipids such as phosphatidylcholines and phosphatidylethanolamines were decreased, which indicated that the metabolic niche was also altered. Finally, we show that supplementation of phosphatidylcholine and phosphatidylethanolamine could partially rescue the observed phenotype. Collectively, our findings suggest that gWAT is important for gonad function by not only affecting whole-body homeostasis but also via maintaining local metabolic and immune niches.

DNAJB7 is dispensable for male fertility in mice.

BACKGROUND: DNAJBs are highly conserved proteins that are involved in various biological processes. Although several DNAJBs are highly expressed in the testis, the function of DNAJB7 in spermatogenesis and male fertility remains unclear. METHODS: To identify the role of DNAJB7 in the male reproduction process, Dnajb7-deficient mice were generated by the CRISPR/Cas9-mediated genome editing system. Histological and immunofluorescence assays were performed to analyze the phenotype of the Dnajb7 mutants. RESULTS: DNAJB7 is specifically expressed in haploid germ cells. Dnajb7 knockout mice are fertile and do not have any detectable defects in Sertoli cells, spermatogonia, meiotic and postmeiotic cells, indicating that DNAJB7 is not essential for spermatogenesis. CONCLUSIONS: Our findings suggest that DNAJB7 is dispensable for male fertility in mice, which could prevent duplicative work by other groups.

Genetic characterization of a missense mutation in the X-linked TAF7L gene identified in an oligozoospermic man†.

Although hundreds of knockout mice show infertility as a major phenotype, the causative genic mutations of male infertility in humans remain rather limited. Here, we report the identification of a missense mutation (D136G) in the X-linked TAF7L gene as a potential cause of oligozoospermia in men. The human aspartate (D136) is evolutionally conserved across species, and its change to glycine (G) is predicted to be detrimental. Genetic complementation experiments in budding yeast demonstrate that the conserved aspartate or its analogous asparagine (N) residue in yeast TAF7 is essential for cell viability and thus its mutation to G is lethal. Although the corresponding D144G substitution in the mouse Taf7l gene does not affect male fertility, RNA-seq analyses reveal alterations in transcriptomic profiles in the Taf7l (D144G) mutant testes. These results support TAF7L mutation as a risk factor for oligozoospermia in humans.

Sox30 initiates transcription of haploid genes during late meiosis and spermiogenesis in mouse testes.

Transcription factors of the Sox protein family contain a DNA-binding HMG box and are key regulators of progenitor cell fate. Here, we report that expression of Sox30 is restricted to meiotic spermatocytes and postmeiotic haploids. Sox30 mutant males are sterile owing to spermiogenic arrest at the early round spermatid stage. Specifically, in the absence of Sox30, proacrosomic vesicles fail to form a single acrosomal organelle, and spermatids arrest at step 2-3. Although most Sox30 mutant spermatocytes progress through meiosis, accumulation of diplotene spermatocytes indicates a delayed or impaired transition from meiotic to postmeiotic stages. Transcriptome analysis of isolated stage-specific spermatogenic cells reveals that Sox30 controls a core postmeiotic gene expression program that initiates as early as the late meiotic cell stage. ChIP-seq analysis shows that Sox30 binds to specific DNA sequences in mouse testes, and its genomic occupancy correlates positively with expression of many postmeiotic genes including Tnp1, Hils1, Ccdc54 and Tsks These results define Sox30 as a crucial transcription factor that controls the transition from a late meiotic to a postmeiotic gene expression program and subsequent round spermatid development.

Sexually transmitted infections and semen quality from subfertile men with and without leukocytospermia.

BACKGROUND: The role of sexually transmitted infections (STIs) in semen parameters and male infertility is still a controversial area. Previous studies have found bacterial infection in a minority of infertile leukocytospermic males. This study aims to investigate the prevalence of STIs in semen from subfertile men with leukocytospermia (LCS) and without leukocytospermia (non-LCS) and their associations with sperm quality. METHODS: Semen samples were collected from 195 men who asked for a fertility evaluation. Infection with the above 6 pathogens was assessed in each sample. Sperm quality was compared in subfertile men with and without LCS. RESULTS: The LCS group had significantly decreased semen volume, sperm concentration, progressive motility, total motility and normal morphology. The infection rates of Ureaplasma urealyticum (Uuu), Ureaplasma parvum (Uup), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), herpes simplex virus-2 (HSV-2) and Neisseria gonorrhoeae (NG) were 8.7 %, 21.0 %, 8.2 %, 2.1 %, 3.6 %, 1.0 and 0 %, respectively. The STI detection rates of patients with LCS were higher than those of the non-LCS group (52.3 % vs. 39.3 %), although there was no statistically significant difference between the two groups (P = 0.07). All semen parameters were not significantly different between LCS with STIs and without STIs, except the semen volume in the MG-infected patients with LCS was significantly lower than that in the noninfected group. CONCLUSIONS: LCS was associated with a reduction in semen quality, but was not associated with STIs.

Biological and RNA regulatory function of MOV10 in mammalian germ cells.

BACKGROUND: RNA regulation by RNA-binding proteins (RBPs) involve extremely complicated mechanisms. MOV10 and MOV10L1 are two homologous RNA helicases implicated in distinct intracellular pathways. MOV10L1 participates specifically in Piwi-interacting RNA (piRNA) biogenesis and protects mouse male fertility. In contrast, the functional complexity of MOV10 remains incompletely understood, and its role in the mammalian germline is unknown. Here, we report a study of the biological and molecular functions of the RNA helicase MOV10 in mammalian male germ cells. RESULTS: MOV10 is a nucleocytoplasmic protein mainly expressed in spermatogonia. Knockdown and transplantation experiments show that MOV10 deficiency has a negative effect on spermatogonial progenitor cells (SPCs), limiting proliferation and in vivo repopulation capacity. This effect is concurrent with a global disturbance of RNA homeostasis and downregulation of factors critical for SPC proliferation and/or self-renewal. Unexpectedly, microRNA (miRNA) biogenesis is impaired due partially to decrease of miRNA primary transcript levels and/or retention of miRNA via splicing control. Genome-wide analysis of RNA targetome reveals that MOV10 binds preferentially to mRNAs with long 3'-UTR and also interacts with various non-coding RNA species including those in the nucleus. Intriguingly, nuclear MOV10 associates with an array of splicing factors, particularly with SRSF1, and its intronic binding sites tend to reside in proximity to splice sites. CONCLUSIONS: These data expand the landscape of MOV10 function and highlight a previously unidentified role initiated from the nucleus, suggesting that MOV10 is a versatile RBP involved in a broader RNA regulatory network.

Maternal circadian disruption before pregnancy impairs the ovarian function of female offspring in mice.

Circadian disturbance brought on by shift employment, nighttime light pollution, and other factors is quite prevalent in contemporary culture. However, the effect of maternal circadian disruption before pregnancy on the reproduction of offspring in mice requires further research. Herein, we exposed female ICR mice to constant light to establish a model of preconceptional circadian disruption and then checked the ovarian function of female offspring (named the CLE group below). Our results revealed obesity, abnormal lipid metabolism and earlier puberty onset in the CLE group. Additionally, impaired ovarian follicle development, oocyte quality and preimplantation embryo development were shown in the CLE group. Moreover, the expression levels of Gnrh1 in the hypothalamus and Cyp17a1, Bmper, Bdnf and Lyve1 in ovaries, as well as circadian clock genes, including Clock, Cry1, Nr1d2 and Per2, were significantly downregulated in the CLE group. Mechanistically, immune responses, including the interleukin-17 (IL-17) signalling pathway, cytokine-cytokine receptor interaction and the chemokine signalling pathway, were altered in the CLE group, which may be responsible for the damaged ovarian function.

Maternal and embryonic signals cause functional differentiation of luminal epithelial cells and receptivity establishment.

Embryo implantation requires temporospatial maternal-embryonic dialog. Using single-cell RNA sequencing for the uterus from 2.5 to 4.5 days post-coitum (DPC) and bulk sequencing for the corresponding embryos of 3.5 and 4.0 DPC pregnant mice, we found that estrogen-responsive luminal epithelial cells (EECs) functionally differentiated into adhesive epithelial cells (AECs) and supporting epithelial cells (SECs), promoted by progesterone. Along with maternal signals, embryonic Pdgfa and Efna3/4 signaling activated AECs and SECs, respectively, enhancing the attachment of embryos to the endometrium and furthering embryo development. This differentiation process was largely conserved between humans and mice. Notably, the developmental defects of SOX9-positive human endometrial epithelial cells (similar to mouse EEC) were related to thin endometrium, whereas functional defects of SEC-similar unciliated epithelial cells were related to recurrent implantation failure (RIF). Our findings provide insights into endometrial luminal epithelial cell development directed by maternal and embryonic signaling, which is crucial for endometrial receptivity.

Homeodomain protein HOMEZ is dispensable for male fertility in mice.

Background: Homeodomain (HD) proteins contain an evolutionarily conserved helix-turn-helix (HTH) DNA-binding motif and act as transcription factors to control gene expression. A previous study showed that the HD gene Homez is highly enriched in adult testes. However, the role of HOMEZ in spermatogenesis and male fertility remains unknown. Methods: Using CRISPR/Cas9 technology, Homez mutant mice were generated and performed histological, immunofluorescence, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), Western blot and mating assays to analyze the phenotype of Homez mutants. Results: Molecular phylogenetic analyses indicated that the HOMEZ is evolutionarily conserved among mammalian species. qRT-PCR and Western blot analyses showed that Homez is highly expressed in the testis, with a relatively increased expression trend during spermatogenesis. Homez mutant males were viable and showed no differences in body and testis weight compared to their wild-type. In addition, mating between Homez mutant males and wild-type females produced normal litter sizes. Moreover, histopathology detected complete spermatogenesis in the seminiferous tubules and mature spermatozoa in the epididymides from Homez knockout males. Furthermore, significantly increased transcription of three Zhx genes were found in Homez mutatnt testes compared with wild-type testes. Conclusions: Homez knockout mice are fertile and are not essential for germ cell development. These findings could prevent unnecessary duplicative work by other groups.

FHL1 mediates HOXA10 deacetylation via SIRT2 to enhance blastocyst-epithelial adhesion.

Recurrent implantation failure (RIF) is a rather thorny problem in the clinical practice of assisted reproductive technology. Due to the complex aetiology of RIF, its pathogenesis is far from fully understood, and there is no effective treatment available. Here, We explored the regulatory mechanism of the four half-domains of LIM domain 1 (FHL1), which is significantly downregulated in the endometrium of RIF patients, in blastocyst-epithelial adhesion. Indeed, FHL1 expression was dramatically increased in normal female mid-secretory endometrial epithelial cells and was abnormally reduced in RIF patients. Furthermore, FHL1 overexpression promoted blastocyst-epithelial adhesion, and interfering with FHL1 expression in the mouse uterus significantly inhibited embryo implantation. Mechanistically, FHL1 did not regulate HOXA10 mRNA expression but increased HOXA10 protein stability and activated HOXA10, thereby promoting its regulation of downstream gene expression and the ß3 integrin/FAK pathway. Meanwhile, FHL1 regulates HOXA10 function by increasing HOXA10 deacetylation through enhanced binding of HOXA10 and SIRT2. SIRT2-specific inhibitors can significantly inhibit this effect. In the endometrial epithelial cells of RIF patients, the correlation between FHL1 and HOXA10 and its downstream target genes has also been verified. Finally, our data indicated FHL1 is a regulatory molecule that promotes blastocyst-epithelial adhesion. Altogether, downstream dysfunction due to aberrant FHL1 expression is an important molecular basis for embryo implantation failure in patients with RIF and to provide new potential therapeutic targets.

Association of sexually transmitted infection with semen quality in men from couples with primary and secondary infertility.

This study aims to compare the prevalence of sexually transmitted infections (STIs) with semen quality in men from couples with primary and secondary infertility. Semen samples were collected from 133 men who requested fertility evaluation. Seminal tract infection with Ureaplasma spp. (UU), Mycoplasma hominis (MH), Mycoplasma genitalium (MG), Chlamydia trachomatis (CT), Neisseria gonorrhoeae (NG), and herpes simplex virus-2 (HSV-2) was assessed by PCR-based diagnostic assays. Among all patients, the prevalence of STIs was higher in men from couples with primary infertility than that in men from couples with secondary infertility (39.7% vs 21.7%, P = 0.03). The prevalence of UU was 28.8% and 13.3% in men from couples with primary and secondary infertility, respectively. Men from couples with primary infertility were more likely to be positive for UU than men from couples with secondary infertility (P = 0.04). Regarding the UU subtype, the prevalence of Ureaplasma urealyticum (Uuu) and Ureaplasma parvum (Uup; including Uup1, Uup3, Uup6, and Uup14) did not differ between the two groups. No associations between the prevalence rates of MH, MG, and CT were found in men from either infertility group. A lower sperm concentration was associated with STI pathogen positivity in men with primary infertility according to the crude model (P = 0.04). The crude and adjusted models showed that semen volume (both P = 0.03) and semen leukocyte count (both P = 0.02) were independently associated with secondary infertility. These findings suggest the importance of classifying the type of infertility during routine diagnosis of seminal tract infections.

Endometrial stromal PRMT5 plays a crucial role in decidualization by regulating NF-κB signaling in endometriosis.

Decidualization is a prerequisite for successful embryo implantation, in which elongated fibroblast-like endometrial stromal cells differentiate into more rounded decidual cells. Accumulating evidence has stressed the important role of the defective eutopic endometrium in infertility in endometriosis patients. However, the role of arginine methylation in the process of physiological decidualization and pathological decidualization defects is not clear. Here, we observed that the expression level of PRMT5, the main type II PRMT, was decreased in the endometrium of endometriosis patients, predominantly in stromal cells. Compared with the undecidualized state, PRMT5 was increased in the stromal cells of normal secretory endometrium in humans and in the decidua of normal pregnant mice or mice with artificially induced decidualization. The inhibition of PRMT5 resulted in a significant decrease in uterine weight and decidualization-related regulator expression, including FOXO1, HOXA10 and WNT4, in mice and IGFBP1 and prolactin levels in human endometrial stromal cells. Transcriptome analysis showed that decreased PRMT5 activity led to NF-κB signaling activation by inducing p65 translocation to the nucleus, which was also observed in endometriosis patients. Finally, overexpression of PRMT5 rescued the defective expression of IGFBP1 and prolactin in primary endometrial stromal cells from endometriosis patients. Our results indicate that promotion of PRMT5 may provide novel therapeutic strategies for the treatment of decidualization defects in infertile women, such as those with endometriosis.

TULP2, a New RNA-Binding Protein, Is Required for Mouse Spermatid Differentiation and Male Fertility.

Spermatogenesis requires a large number of proteins to be properly expressed at certain stages, during which post-transcriptional regulation plays an important role. RNA-binding proteins (RBPs) are key players in post-transcriptional regulation, but only a few RBPs have been recognized and preliminary explored their function in spermatogenesis at present. Here we identified a new RBP tubby-like protein 2 (TULP2) and found three potential deleterious missense mutations of Tulp2 gene in dyszoospermia patients. Therefore, we explored the function and mechanism of TULP2 in male reproduction. TULP2 was specifically expressed in the testis and localized to spermatids. Studies on Tulp2 knockout mice demonstrated that the loss of TULP2 led to male sterility; on the one hand, increases in elongated spermatid apoptosis and restricted spermatid release resulted in a decreased sperm count; on the other hand, the abnormal differentiation of spermatids induced defective sperm tail structures and reduced ATP contents, influencing sperm motility. Transcriptome sequencing of mouse testis revealed the potential target molecular network of TULP2, which played its role in spermatogenesis by regulating specific transcripts related to the cytoskeleton, apoptosis, RNA metabolism and biosynthesis, and energy metabolism. We also explored the potential regulator of TULP2 protein function by using immunoprecipitation and mass spectrometry analysis, indicating that TUPL2 might be recognized by CCT8 and correctly folded by the CCT complex to play a role in spermiogenesis. Our results demonstrated the important role of TULP2 in spermatid differentiation and male fertility, which could provide an effective target for the clinical diagnosis and treatment of patients with oligo-astheno-teratozoospermia, and enrich the biological theory of the role of RBPs in male reproduction.

Dnajb8, a target gene of SOX30, is dispensable for male fertility in mice.

BACKGROUND: The DNAJ family of molecular chaperones maintains protein homeostasis in mitotic and postmeiotic cells, especially germ cells. Recently, we found that the transcription factor SOX30 initiates transcription of Dnajb8 during late meiosis and spermiogenesis in mouse testes. METHODS: We used the CRISPR/Cas9 system to generate Dnajb8 mutant mice and analyze the phenotype of the Dnajb8 mutants. RESULTS: Although Dnajb8 is an evolutionarily conserved gene, it is not essential for spermatogenesis and male fertility. We provide this phenotypic information, which could prevent duplicative work by other groups.

Enhanced Crosslinking Immunoprecipitation (eCLIP) Method for Efficient Identification of Protein-bound RNA in Mouse Testis.

Spermatogenesis defines a highly ordered process of male germ cell differentiation in mammals. In testis, transcription and translation are uncoupled, underlining the importance of post-transcriptional regulation of gene expression orchestrated by RBPs. To elucidate mechanistic roles of an RBP, crosslinking immunoprecipitation (CLIP) methodology can be used to capture its endogenous direct RNA targets and define the actual interaction sites. The enhanced CLIP (eCLIP) is a newly-developed method that offers several advantages over the conventional CLIPs. However, the use of eCLIP has so far been limited to cell lines, calling for expanded applications. Here, we employed eCLIP to study MOV10 and MOV10L1, two known RNA-binding helicases, in mouse testis. As expected, we find that MOV10 predominantly binds to 3' untranslated regions (UTRs) of mRNA and MOV10L1 selectively binds to Piwi-interacting RNA (piRNA) precursor transcripts. Our eCLIP method allows fast determination of major RNA species bound by various RBPs via small-scale sequencing of subclones and thus availability of qualified libraries, as a warrant for proceeding with deep sequencing. This study establishes an applicable basis for eCLIP in mammalian testis.

In Vitro Biochemical Assays using Biotin Labels to Study Protein-Nucleic Acid Interactions.

Protein-nucleic acid interactions play important roles in biological processes such as transcription, recombination, and RNA metabolism. Experimental methods to study protein-nucleic acid interactions require the use of fluorescent tags, radioactive isotopes, or other labels to detect and analyze specific target molecules. Biotin, a non-radioactive nucleic acid label, is commonly used in electrophoretic mobility shift assays (EMSA) but has not been regularly employed to monitor protein activity during nucleic acid processes. This protocol illustrates the utility of biotin labeling during in vitro enzymatic reactions, demonstrating that this label works well with a range of different biochemical assays. Specifically, in alignment with previous findings using radioisotope 32P-labeled substrates, it is confirmed via biotin-labeled EMSA that MEIOB (a protein specifically involved in the meiotic recombination) is a DNA-binding protein, that MOV10 (an RNA helicase) resolves biotin-labeled RNA duplex structures, and that MEIOB cleaves biotin-labeled single-stranded DNA. This study demonstrates that biotin is capable of substituting 32P in various nucleic acid-related biochemical assays in vitro.

Rapamycin-mediated mTOR inhibition impairs silencing of sex chromosomes and the pachytene piRNA pathway in the mouse testis.

Mechanistic target of rapamycin (mTOR) controls cell growth and metabolism in response to environmental and metabolic signals. Rapamycin robustly extends the lifespan in mammals and has clinical relevance in organ transplantation and cancer therapy but side effects include male infertility. Here, we report that chronic rapamycin treatment causes spermatogenic arrest in adult male mice due to defects in sex body formation and meiotic sex chromosome inactivation (MSCI). Many sex chromosome-linked genes were up-regulated in isolated pachytene spermatocytes from rapamycin-treated mice. RNA-Seq analysis also identified mRNAs encoding the core piRNA pathway components were decreased. Furthermore, rapamycin treatment was associated with a drastic reduction in pachytene piRNA populations. The inhibitory effects of rapamycin on spermatogenesis were partially reversible, with restoration of testis mass and sperm motility within 2 months of treatment cessation. Collectively, we have defined an essential role of mTOR in MSCI and identified a novel function as a regulator of small RNA homeostasis in male germ cells.