Upregulated lncRNA Gm2044 inhibits male germ cell development by acting as miR-202 host gene

Long non-coding RNAs (lncRNAs) have been found to participate in the regulation of human spermatogenic cell development. However, little is known about the abnormal expression of lncRNAs associated with spermatogenic failure and their molecular mechanisms. Using lncRNA microarray of testicular tissue for male infertility and bioinformatics methods, we identified the relatively conserved lncRNA Gm2044 which may play important roles in non-obstructive azoospermia. The UCSC Genome Browser showed that lncRNA Gm2044 is the miR-202 host gene. This study revealed that lncRNA Gm2044 and miR-202 were significantly increased in non-obstructive azoospermia of spermatogonial arrest. The mRNA and protein levels of Rbfox2, a known direct target gene of miR-202, were regulated by lncRNA Gm2044. Furthermore, the miR-202-Rbfox2 signalling pathway was shown to mediate the suppressive effects of lncRNA Gm2044 on the proliferation of human testicular embryonic carcinoma cells. Understanding of the molecular signalling pathways for lncRNA-regulated spermatogenesis will provide new clues into the pathogenesis and treatment of patients with male infertility.     

LncRNA Gm2044 promotes 17β‐estradiol synthesis in mpGCs by acting as miR‐138‐5p sponge

Long noncoding RNAs (lncRNAs) have been demonstrated to play vital roles in mammalian reproduction. Our previous research revealed that lncRNA Gm2044 is highly expressed in mouse spermatocytes and regulates male germ cell function. The gene annotation database BioGPS shows that Gm2044 is not only highly expressed in testicular tissue but also in ovarian tissue, which suggests that Gm2044 may be involved in female reproductive development. In this study, we confirmed that lncRNA Gm2044 promotes 17β‐estradiol synthesis in mouse pre‐antral follicular granulosa cells (mpGCs). Furthermore, bioinformatics methods, western blot, and the luciferase assay proved that Gm2044 functions as a miR‐138‐5p sponge to inhibit the direct target of miR‐138‐5p, Nr5a1, which enhances 17β‐estradiol synthesis through cyp19a1 activation. Taken together, our results provide an insight into the mechanistic roles of lncRNA Gm2044 for 17β‐estradiol synthesis by acting as competing‐endogenous RNAs to modulate the function of mpGCs. Studying the potential lncRNAs, which regulate estradiol release, will be beneficial for the diagnosis and treatment of steroid hormone‐related disease.     

Melatonin enhances spermatogonia activity through promoting KIAA1429-mediated m6A deposition to activate the PI3K/AKT signaling

Melatonin is a key neuroendocrine hormone that promotes spermatogenesis and sperm motility, but the underlying mechanisms remains poorly understood. In this study, we aimed to investigate the possible roles of m⁶A (N⁶--methyl-adenosine) in mediating melatonin-regulated spermatogonia activity alterations. In this study, mouse-derived GC-1 spermatogonia (spg) cell line was used as the in vitro cellular model. The viability, proliferation rates and apoptosis of spermatogonia were detected via CCK-8, Edu staining and flow cytometry respectively. Total m⁶A level was quantitated by dot blot, while mRNA and proteins contents in spermatogonia were measured by qRT-PCR and western blot respectively. Differentially expressed mRNAs were characterized by deep RNA sequencing method. Results showed that melatonin significantly promoted viability and proliferation rate while inhibited apoptosis in the GC-1 spg cells. The total m⁶A levels in GC-1 spg cells were also greatly increased by melatonin treatment, accompanied by remarkable expressional elevation of the m⁶A writer KIAA1429. Moreover, the regulation of GC-1 spg cell viability, proliferation and apoptosis by melatonin were greatly abrogated by KIAA1429 silencing but effectively strengthened by KIAA1429 overexpression. In addition, KIAA1429 overexpression regulates multiple biological process and signaling pathways in spermatogonia such as the PI3K/AKT signaling. The PI3K inhibitor LY294002 effectively mitigated the regulation of spermatogonia activity by KIAA1429 overexpression under melatonin treatment. Taken together, melatonin promotes spermatogonia activity via enhancing KIAA1429 expression and m⁶A RNA methylation to activate the downstream PI3K/AKT signaling pathway.     

GC-1 mRHBDD1 knockdown spermatogonia cells lose their spermatogenic capacity in mouse seminiferous tubules

Background  

Apoptosis is important for regulating spermatogenesis. The protein mRHBDD1 (mouse homolog of human RHBDD1)/rRHBDD1 (rat homolog of human RHBDD1) is highly expressed in the testis and is involved in apoptosis of spermatogonia. GC-1, a spermatogonia cell line, has the capacity to differentiate into spermatids within the seminiferous tubules. We constructed mRHBDD1 knockdown GC-1 cells and evaluated their capacity to differentiate into spermatids in mouse seminiferous tubules.     

d-aspartate and N-methyl-d-aspartate promote proliferative activity in mouse spermatocyte GC-2 cells

D-Aspartate (D-Asp) and its methylated form N-methyl-d-aspartate (NMDA) promote spermatogenesis by stimulating the biosynthesis of sex steroid hormones. d-Asp also induces spermatogonia proliferation directly by activating the ERK/Aurora B pathway. In the present study, a mouse spermatocyte-derived cell line (GC-2) which represents a stage between preleptotene spermatocyte and round spermatids was exposed to 200 μM d-Asp or 50 μM NMDA for 30 min, 2 h, and 4 h to explore the influence of these amino acids on cell proliferation and mitochondrial activities occurring during this process. By Western blotting analyses, the expressions of AMPAR (GluA1-GluA2/3 subunits), cell proliferation as well as mitochondria functionality markers were determined at different incubation times. The results revealed that d-Asp or NMDA stimulate proliferation and meiosis in the GC-2 cells via the AMPAR/ERK/Akt pathway, which led to increased levels of the PCNA, p-H3, and SYCP3 proteins. The effects of d-Asp and NMDA on the mitochondrial functionality of the GC-2 cells strongly suggested an active role of these amino acids in germ cell maturation. In both d-Asp- and NMDA-treated GC-2 cells mitochondrial biogenesis as well as mitochondrial fusion are increased while mitochondria fission is inhibited. Finally, the findings showed that NMDA significantly increased the expressions of the CII, CIII, CIV, and CV complexes of oxidative phosphorylation system (OXPHOS), whereas d-Asp induced a significant increase in the expressions only of the CIV and CV complexes. The present study provides novel insights into the mechanisms underlying the role of d-Asp and NMDA in promoting spermatogenesis.     

H19 regulates the proliferation of bovine male germline stem cells via IGF-1 signaling pathway

Self-renewal and differentiation of male germline stem cells (mGSCs) provide the basic function for continual spermatogenesis. Studies of in vitro culture of germline stem cells are important and meaningful for basic biological research and practical application. Growth factors, such as GDNF, bFGF, CSF1, and EGF, could maintain the self-renewal of mGSCs. Insulin-like growth factor 1 (IGF-1), an important growth factor, and its pathway have been reported to maintain the survival of several types of stem cells and play important roles in male reproduction. However, the mechanism through which the IGF-1 pathway acts to regulate the self-renewal of mGSCs remains unclear. We analyzed the effect of IGF-1 on the proliferation and apoptosis of bovine mGSCs. We evaluated the expression profile of long noncoding RNA (LncRNA) H19 in bovine and mouse tissues. Moreover, we investigated whether LncRNA H19 could regulate the IGF-1 pathway. Results showed that IGF-1 could activate the phosphorylation of AKT and ERK signaling pathways, and the IGF-1 pathway played an important role in regulating the proliferation and apoptosis of bovine mGSCs. The proliferation rate of mGSCs decreased, whereas the apoptosis rate of mGSCs increased when the IGF-1 receptor (IGF-1R) was blocked using the IGF-1R-specific inhibitor (picropodophyllin). LncRNA H19 could regulate the IGF-1 signaling pathway and, consequently, the proliferation and apoptosis of mGSCs. The number of cells in the seminiferous tubule decreased when H19 was interfered by injecting a virus-containing supernatant. Hence, LncRNA H19 participated in the regulation of the proliferation and apoptosis of mGSCs via the IGF-1 signaling pathway.     

The Sumo proteome of proliferating and neuronal-differentiating cells reveals Utf1 among key Sumo targets involved in neurogenesis

Post-translational modification by covalent attachment of the Small ubiquitin-like modifier (Sumo) polypeptide regulates a multitude of processes in vertebrates. Despite demonstrated roles of Sumo in the development and function of the nervous system, the identification of key factors displaying a sumoylation-dependent activity during neurogenesis remains elusive. Through a SILAC (stable isotope labeling by/with amino acids in cell culture)-based proteomic approach, we have identified the Sumo proteome of the model cell line P19 under proliferation and neuronal differentiation conditions. More than 300 proteins were identified as putative Sumo targets differentially associated with one or the other condition. A group of proteins of interest were validated and investigated in functional studies. Among these, Utf1 was revealed as a new Sumo target. Gain-of-function experiments demonstrated marked differences between the effects on neurogenesis of overexpressing wild-type and sumoylation mutant versions of the selected proteins. While sumoylation of Prox1, Sall4a, Trim24, and Utf1 was associated with a positive effect on neurogenesis in P19 cells, sumoylation of Kctd15 was associated with a negative effect. Prox1, Sall4a, and Kctd15 were further analyzed in the vertebrate neural tube of living embryos, with similar results. Finally, a detailed analysis of Utf1 showed the sumoylation dependence of Utf1 function in controlling the expression of bivalent genes. Interestingly, this effect seems to rely on two mechanisms: sumoylation modulates binding of Utf1 to the chromatin and mediates recruitment of the messenger RNA-decapping enzyme Dcp1a through a conserved SIM (Sumo-interacting motif). Altogether, our results indicate that the combined sumoylation status of key proteins determines the proper progress of neurogenesis.Subject terms: Enzyme mechanisms, Cell signalling, Chromatin, Sumoylation     

The cloning of cyclin B3 and its gene expression during hormonally induced spermatogenesis in the teleost, Anguilla japonica

We cloned cyclin B1, B2, and B3 cDNAs from the eel testis. Northern blot analysis indicated that these cyclin B mRNAs were expressed and increased from day 3 onward after the hormonal induction of spermatogenesis, and that cyclin B3 was most dominantly expressed during spermatogenesis. In situ hybridization showed that cyclin B1 and B2 were present from the spermatogonium stage to the spermatocyte stage. On the other hand, cyclin B3 mRNA was present only in spermatogonia. Although mouse cyclin B3 is expressed specifically in the early meiotic prophase, these results indicate that eel cyclin B3 expression is limited during spermatogenesis to spermatogonia, but is not present in spermatocytes. These facts together suggest that eel cyclin B3 is specifically involved in spermatogonial proliferation (mitosis), but not in meiosis.     

The inflammatory mediators TNFα and nitric oxide arrest spermatogonia GC-1 cell cycle

During an inflammatory process of the testis, the network of somatic, immune, and germ cell interactions is altered leading to organ dysfunction. In testicular biopsies of infertile men, spermatogenesis impairment is associated with reduced spermatogonia proliferation, increased number of immune cells, and content of pro-inflammatory cytokines. TNFα-TNFR and nitric oxide (NO)-NO synthase systems are up-regulated in models of testicular damage and in human testis with maturation arrest. The purpose of this study was to test the hypothesis that TNFα-TNFR system and NO alter the function of spermatogonia in the inflamed testis. We studied the effect of TNFα and NO on GC-1 spermatogonia cell cycle progression and death by flow cytometry. GC-1 cells expressed TNFR1 and TNFR2 (immunofluorescence). TNFα (10 and 50 ng/ml) and DETA-Nonoate (0.5 and 2 mM), a NO releaser, increased the percentage of cells in S-phase of the cell cycle and reduced the percentage in G1, inducing also cell apoptosis. TNFα effect was not mediated by oxidative stress unlike NO, since the presence of N-acetyl-l-cysteine (2.5 and 5.0 mM) prevented NO induced cell cycle arrest and death. GC-1 spermatogonia overpass NO induced cell cycle arrest but no TNFα, since after removal of NO, spermatogonia progressed through the cell cycle. We propose TNFα and NO might contribute to impairment of spermatogenesis by preventing adequate functioning of the spermatogonia population. Our results showed that TNFα and NO impaired spermatogonia cell cycle, inducing GC-1 arrest in the S phase.     

Identification of a novel testis-specific gene mtLR1, which is expressed at specific stages of mouse spermatogenesis

A novel testis-specific gene termed mtLR1 was identified by digital differential display. Sequence analyses revealed that mtLR1 protein contains an amino terminus leucine-rich repeat domain and shows 33% similarities to PIDD which functions in p53-mediated apoptosis. Northern blot analysis showed that mtLR1 mRNA was specifically expressed in adult mouse testis, and RT-PCR results also showed that mtLR1 was exclusively expressed in adult testis and not in spermatogonial cells. The expression of mtLR1 mRNA was developmentally upregulated in the testes during sexual maturation and was, conversely, downregulated by experimental cryptorchidism in vivo. We also showed that the expression of mtLR1 mRNA was relatively highly sensitive to heat stress in vitro. The green fluorescent protein produced by pEGFP-C3/mtLR1 was only detected in the cytoplasm of spermatogonia cell line GC-1 after 24 h posttransfection. Immunohistochemical analysis revealed that the protein is most abundant in the cytoplasm of spermatocytes and round spermatids within seminiferous tubules of the adult testis. The time-dependent expression pattern of mtLR1 in postnatal mouse testes suggested that mtLR1 gene might be involved in the regulation of spermatogenesis and sperm maturation.     

Lrh1 can help reprogram sexual cell fate and is required for Sertoli cell development and spermatogenesis in the mouse testis

The mammalian nuclear hormone receptors LRH1 (NR5A2) and SF1 (NR5A1) are close paralogs that can bind the same DNA motif and play crucial roles in gonadal development and function. Lrh1 is essential for follicle development in the ovary and has been proposed to regulate steroidogenesis in the testis. Lrh1 expression in the testis is highly elevated by loss of the sex regulator Dmrt1, which triggers male-to-female transdifferentiation of Sertoli cells. While Sf1 has a well-defined and crucial role in testis development, no function for Lrh1 in the male gonad has been reported. Here we use conditional genetics to examine Lrh1 requirements both in gonadal cell fate reprogramming and in normal development of the three major cell lineages of the mouse testis. We find that loss of Lrh1 suppresses sexual transdifferentiation, confirming that Lrh1 can act as a key driver in reprogramming sexual cell fate. In otherwise wild-type testes, we find that Lrh1 is dispensable in Leydig cells but is required in Sertoli cells for their proliferation, for seminiferous tubule morphogenesis, for maintenance of the blood-testis barrier, for feedback regulation of androgen production, and for support of spermatogenesis. Expression profiling identified misexpressed genes likely underlying most aspects of the Sertoli cell phenotype. In the germ line we found that Lrh1 is required for maintenance of functional spermatogonia, and hence mutants progressively lose spermatogenesis. Reduced expression of the RNA binding factor Nxf2 likely contributes to the SSC defect. Unexpectedly, however, over time the Lrh1 mutant germ line recovered abundant spermatogenesis and fertility. This finding indicates that severe germ line depletion triggers a response allowing mutant spermatogonia to recover the ability to undergo complete spermatogenesis. Our results demonstrate that Lrh1, like Sf1, is an essential regulator of testis development and function but has a very distinct repertoire of functions.     

Downregulation of lncRNA HCP5 has inhibitory effects on gastric cancer cells by regulating DDX21 expression

LncRNA HCP5 has been confirmed to play crucial roles in many types of cancers. However, the role of lncRNA HCP5 in regulating the occurrence and development of gastric cancer (GC) remains unknown. In the current study, we aimed to investigate the precise effects of lncRNA HCP5 on cell proliferation, migration and invasion and molecular mechanisms in gastric cancer. Using RT-qPCR analysis, we found that lncRNA HCP5 was differentially expressed in GC cell lines. CCK-8, wound healing and transwell assay indicated that the proliferation, migration and invasion of gastric cancer cells were inhibited by downregulation of lncRNA HCP5 and lncRNA HCP5 overexpression exhibited the opposite effects in gastric cancer cells. Mechanistically, RNA binding protein immunoprecipitation and dual luciferase reporter assay confirmed the interaction between lncRNA HCP5 and DDX21. The effects of lncRNA HCP5 overexpression the proliferation, migration and invasion of GC cells were partly rescued by DDX21 silencing. Taken together, downregulation of lncRNA HCP5 exerted inhibitory effects on GC cell proliferation, migration and invasion through modulation of DDX21 expression, demonstrating the function of lncRNA HCP5 and DDX21 in GC progression.     

Removal of serum factors by charcoal treatment promotes adipogenesis via a MAPK-dependent pathway

Human TSPY is a candidate oncogene and is supposed to function as a proliferation factor during spermatogenesis. It is the only mammalian protein-coding gene known to be organized as a tandem repeat gene family. It is expressed at highest level in spermatogonia and to a lower amount in primary spermatocytes. To characterize the human TSPY promoter we used the luciferase reporter system in a mouse spermatogonia derived cell line (GC-1spg) and in a GC-4spc cell line, that harbour prophase spermatocytes of the preleptotene and early pachytene stage. We isolated a 1303 bp fragment of the 5′-flanking region of exon 1 that shows significant promoter activity in GC-1spg and reduced activity in GC-4spc cells. In order to gain further insight into the organization of the TSPY-promoter, stepwise truncations of the putative promoter sequence were performed. The resulting fragments were cloned into the pGL3-vector and analysed for reporter gene activity in the murine germ cell lines GC-1spg and GC-4spc, leading to the characterization of a core promoter (−159 to −1), an enhancing region (−673 to −364) and a silencing region (−1262 to −669). Database research for cis-active elements yielded two putative SOX-like binding sites in the enhancing region and reporter gene activity was drastically reduced when three nucleotides of the AACAAT SOX core sequence were mutated. Our findings strongly suggest that testis-specific expression of human TSPY is mediated by Sox proteins. (Mol Cell Biochem 276: 159–167, 2005)     

Molecular cloning of a novel mouse testis-specific spermatogenic cell apoptosis inhibitor gene mTSARG7 as a candidate oncogene

A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and.ll introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and AU041707 protein shared 83.9% identity in 402 amino acid residues. Expression of the mTSARG7 gene was restricted to the mouse testis. The results of the in situ hybridization analysis revealed that the mTSARG7 mRNA was expressed in mouse spermatogonia and spermatocytes. Subcellular localization studies showed that the EGFPtagged mTSARG7 protein was localized in the cytoplasm of GC-1 spg cells. The mTSARG7 mRNA expression was initiated in the mouse testis in the second week after birth, and the expression level increased steadily with spermatogenesis and sexual maturation of the mouse. The results of the heat stress experiment showed that the mTSARG7 mRNA expression gradually decreased as the heating duration increased. The pcDNA3.1 Hygro(-)/mTSARG7 plasmid was constructed and introduced into GC- 1 spg cells by liposome transfection. The mTSARG7 can accelerate GC-1 spg cells, causing them to traverse the S-phase and enter the G2-phase, compared with the control group where this did not occur as there was no transfection of mTSARG7. In conclusion, our results suggest that this gene may play an important role in spermatogenesis and the development of cryptorchid testes, and is a testis-specific apoptosis candidate oncogene.     

LncRNA ABHD11‐AS1 promotes the development of endometrial carcinoma by targeting cyclin D1

To investigate the expression, role and mechanism of action of long non‐coding RNA (lncRNA) ABHD11‐AS1 in endometrial carcinoma. The expression of lncRNA ABHD11‐AS1 was quantified by qRT‐PCR in human endometrial carcinoma (n = 89) and normal endometrial tissues (n = 27). LncRNA ABHD11‐AS1 was stably overexpressed or knocked‐down in endometrial carcinoma cell lines to examine the cellular phenotype and expression of related molecules. Compared to normal endometrial tissue, lncRNA ABHD11‐AS1 was significantly overexpressed in endometrial carcinoma. Overexpression of lncRNA ABHD11‐AS1 promoted the proliferation, G1‐S progression, invasion and migration of endometrial cancer cells; inhibited apoptosis; up‐regulated cyclin D1, CDK1, CDK2, CDK4, Bcl‐xl and VEGFA; and down‐regulated p16, while ABHD11‐AS1 down‐regulation has the opposite effect. RNA pull down demonstrated that lncRNA ABHD11‐AS1 binds directly to cyclin D1. Knockdown of cyclin D1 can reverse the effect of ABHD11‐AS1. Overexpression of lncRNA ABHD11‐AS1 increased the tumorigenicity and up‐regulated cyclin D1 in an in vivo model of endometrial cancer in nude mice. LncRNA ABHD11‐AS1 functions as an oncogene to promote cell proliferation and invasion in endometrial carcinoma by positively targeting cyclin D1.