[Long non-coding RNA in spermatogenesis regulation and male infertility: Progress in research].

Long non-coding RNA (lncRNA) is an RNA molecule transcribed by RNA polymerase II, longer than 200 nt, and not translated into proteins. During gonadal development and spermatogenesis, lncRNAs are involved in epigenetic mechanisms, including DNA methylation, chromatin remodeling, and histone tail modification, which play important regulatory roles at the transcriptional or post-transcriptional level. Epigenomics including lncRNA is considered to be the second dimension of DNA sequence that can be adapted to environmental factors to specifically regulate gene expressions in some cells. Based on the functional action mechanism of lncRNAs, we reviewed the advances in the studies of lncRNAs in the direction of spermatogenesis and male infertility and analyzed the potential of lncRNAs as a biomarker of male infertility. The potential application of lncRNA in the treatment of male infertility diseases can be further explored based on the lncRNA target, RNA interference, competitive binding closed target and structural disruption of lncRNAs.

[Regulation of the ubiquitin-proteasome system in spermiogenesis].

The ubiquitin-proteasome system (UPS) is an adenosine triphosphate (ATP)-dependent enzymatic machinery that targets substrate proteins for degradation by the 26S proteasome by tagging them with an isopeptide chain composed of covalently linked molecules of ubiquitin, a small chaperone protein. UPS is the main pathway of protein degradation in eukaryotic cells, and plays an important role in spermatogenesis. The dysfunction of various ubiquitin systems results in impaired sperm development with abnormal morphology and function, which is highly associated with male infertility. This review focuses on the roles of UPS in histone-to-protamine exchange, acrosome formation, sperm mitochondrial degradation and regulation of sperm function and quality.

Pharmacological characterization of JWX-A0108 as a novel type I positive allosteric modulator of α7 nAChR that can reverse acoustic gating deficits in a mouse prepulse inhibition model.

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a ligand-gated Ca2+-permeable homopentameric ion channel implicated in cognition and neuropsychiatric disorders. Pharmacological enhancement of α7 nAChR function has been suggested for improvement of cognitive deficits. In the present study, we characterized a thiazolyl heterocyclic derivative, 6-(2-chloro-6-methylphenyl)-2-((3-fluoro-4-methylphenyl)amino)thiazolo[4,5-d]pyrimidin-7(6H)-one (JWX-A0108), as a novel type I α7 nAChR positive allosteric modulator (PAM), and evaluated its ability to reverse auditory gating and spatial working memory deficits in mice. In Xenopus oocytes expressing human nAChR channels, application of JWX-A0108 selectively enhanced α7 nAChR-mediated inward current in the presence of the agonist ACh (EC50 value = 4.35 ± 0.12 µM). In hippocampal slices, co-application of ACh and JWX-A0108 (10 µM for each) markedly increased both the frequency and amplitude of spontaneous inhibitory postsynaptic currents (sIPSCs) recorded in pyramidal neurons, but JWX-A0108 did not affect GABA-induced current in oocytes expressing human GABAA receptor α1ß3γ2 and α5ß3γ2 subtypes. In mice with MK-801-induced deficits in auditory gating, administration of JWX-A0108 (1, 3, and 10 mg/kg, i.p.) dose-dependently attenuates MK-801-induced auditory gating deficits in five prepulse intensities (72, 76, 80, 84, and 88 dB). Furthermore, administration of JWX-A0108 (0.03, 0.1, or 0.3 mg/kg, i.p.) significantly reversed MK-801-induced impaired spatial working memory in mice. Our results demonstrate that JWX-A0108 is a novel type I PAM of α7 nAChR, which may be beneficial for improvement of cognitive deficits commonly found in neuropsychiatric disorders such as schizophrenia and Alzheimer's disease.

Antipruritic Effect of Natural Coumarin Osthole through Selective Inhibition of Thermosensitive TRPV3 Channel in the Skin.

Coumarin osthole is a dominant bioactive ingredient of the natural Cnidium monnieri plant commonly used for traditional Chinese herbal medicines for therapies and treatments including antipruritus and antidermatitis. However, the molecular mechanism underlying the action of osthole remains unclear. In this study, we report that osthole exerts an antipruritic effect through selective inhibition of Ca2+-permeable and thermosensitive transient receptor potential vanilloid 3 (TRPV3) cation channels that are primarily expressed in the keratinocytes of the skin. Coumarin osthole was identified as an inhibitor of TRPV3 channels transiently expressed in HEK293 cells in a calcium fluorescent assay. Inhibition of the TRPV3 current by osthole and its selectivity were further confirmed by whole-cell patch clamp recordings of TRPV3-expressing HEK293 cells and mouse primary cultured keratinocytes. Behavioral evaluation demonstrated that inhibition of TRPV3 by osthole or silencing by knockout of the TRPV3 gene significantly reduced the scratching induced by either acetone-ether-water or histamine in localized rostral neck skin in mice. Taken together, our findings provide a molecular basis for use of natural coumarin osthole from the C. monnieri plant in antipruritic or skin care therapy, thus establishing a significant role of the TRPV3 channel in chronic itch signaling or acute histamine-dependent itch sensation.

[L-carnitine improves sperm acrosin activity in male infertility patients].

OBJECTIVE: To evaluate the effect of L-carnitine (LC) on low sperm acrosin activity in infertile man. METHODS: A total of 240 male infertility patients with low sperm acrosin activity were randomly assigned to an LC group (n = 180) and a control group (n = 60) to be treated with LC (1g, tid) and vitamin E (VE) capsules (100 mg, tid) respectively, both for 3 months. Based on the results of routine semen analysis, the patients in the experimental group were further divided into oligozoospermia, asthenozoospermia and normozoospermia subgroups. Semen parameters and sperm acrosin activity were examined before and after treatment. RESULTS: Totally, 220 of the patients completed the treatment and follow-up, 163 in the LC medication and 57 in the VE control group. Compared with the baseline, the percentage of progressively motile sperm (PMS) was significantly increased in the LC group after 3 months of treatment (ï¼»32.58 ± 1.13ï¼½% vs ï¼»36.35 ± 1.26ï¼½%, P < 0.05), and so was sperm acrosin activity (ï¼»37.05±0.66ï¼½ vs ï¼»58.61±1.93ï¼½ µIU/106 sperm, P < 0.01). Sperm concentration, PMS and sperm acrosin activity were also improved in the VE control group after treatment, but with no statistically significant difference (P > 0.05). In comparison with pretreatment, remarkable increases were observed after LC medication in sperm concentration in the oligozoospermia subgroup (ï¼»11.27 ± 0.73ï¼½ vs ï¼»21.82 ± 4.21ï¼½ ×106/ml, P < 0.01) and PMS in the asthenozoospermia patients (ï¼»20.61 ± 0.85ï¼½% vs ï¼»29.81 ± 1.88ï¼½%, P < 0.01). And sperm acrosin activity was even higher after treatment in the asthenozoospermia than in the oligozoospermia and normozoospermia subgroups (ï¼»60.85 ± 3.04ï¼½ vs ï¼»56.32 ± 2.86ï¼½ and ï¼»57.09 ± 6.31ï¼½ µIU/106 sperm, P < 0.05). CONCLUSIONS: L-carnitine can effectively elevate sperm acrosin activity in male infertility patients, particularly in those with asthenozoospermia.

Exposure to Brefeldin A promotes initiation of meiosis in murine female germ cells.

In mammals, ontogenesis starts from a fusion of spermatozoon and oocyte, which are produced by reductive nuclear division of a diploid germ cell in a specialised but complex biological process known as meiosis. However, little is known about the mechanism of meiotic initiation in germ cells, although many factors may be responsible for meiosis both in male and female gonads. In this study, 11.5 days post coitum (dpc) female fetal mouse genital ridges were cultured in vitro with exposure to Brefeldin A (BFA) for 6h, and the changes in meiosis were detected. Synaptonemal-complex analysis implied that BFA played a positive role in meiosis initiation and this hypothesis was confirmed by quantitative PCR of meiosis-specific genes: stimulated by retinoic acid gene 8 (Stra8) and deleted in a zoospermia-like (DAZL). At the same time, mRNA expression of retinoic acid synthetase (Raldh2) and retinoic acid (RA) receptors increased in female gonads with in vitro exposure to BFA. Transplanting genital ridges treated with BFA into the kidney capsule of immunodeficient mice demonstrated that the development capacity of female germ cells was normal, while formation of primordial follicles was seen to be a result of accelerated meiosis after exposure to BFA. In conclusion, the study indicated that BFA stimulated meiosis initiation partly by RA signalling and then promoted the development of follicles.

Exposure to diethylhexyl phthalate (DEHP) results in a heritable modification of imprint genes DNA methylation in mouse oocytes.

Diethylhexyl phthalate (DEHP) is an estrogen-like compound widely used as a plasticizer in commercial products and is present in medical devices, and common household items. It is considered an endocrine disruptor since studies on experimental animals clearly show that exposure to DEHP can alter epigenetics of germ cells. This study was designed to assess the effects of DEHP on DNA methylation of imprinting genes in germ cells from fetal and adult mouse. Pregnant mice were treated with DEHP at doses of 0 and 40 µg DEHP/kg body weight/day from 0.5 to 18.5 day post coitum. The data revealed DEHP exposure significantly reduced the percentage of methylated CpG sites in Igf2r and Peg3 differentially methylated regions (DMRs) in primordial germ cells from female and male fetal mouse, particularly, in the oocytes of 21 dpp mice (F1), which were produced by the pregnant micetreated with DEHP. More surprisingly, the modification of the DNA methylation of imprinted genes in F1 mouse oocytes was heritable to F2 offspring which exhibit lower percentages of methylated CpG sites in imprinted genes DMRs. In conclusion, DEHP exposure can affect the DNA methylation of imprinting genes not only in fetal mouse germ cells and growing oocytes, but also in offspring's oocytes.

Cutaneous applied nano-ZnO reduce the ability of hair follicle stem cells to differentiate.

The ability of metal oxide nanoparticles to penetrate the skin has aroused a great deal of interest during the past decade due to concerns over the safety of topically applied sunscreens that contain physical UV-resistant metal particles, such as nano-Zinc oxide (nZnO). Previous studies demonstrate that metal oxide nanoparticles accumulate in skin furrows and hair follicles following topical application while little is known about the consequence of these nanoparticles on skin homeostasis. The current investigation tested the effects of nZnO (0.5 mg/day mouse) on hair follicle physiology. Topical application of Vaseline containing nZnO, bulk ZnO (bZnO), or ionized Zn to newborn mice vibrissa pad over a period of 7 consecutive days revealed that nZnO accumulated within hair follicles, and this induced the apoptosis of hair follicle stem cells (HFSCs). In vitro studies also indicated that nZnO exposure caused obvious DNA damage and induced apoptosis in HFSCs. Furthermore, it was found that nZnO exposure perturbed genes associated with HFSC apoptosis, cell communication, and differentiation. HFSCs transplantation assay demonstrated that the potential of HFSCs to differentiate was reduced. This investigation indicates a potential risk of topically applied ZnO nanoparticles on skin homeostasis.

Differentiation of early germ cells from human skin-derived stem cells without exogenous gene integration.

Infertility has long been a difficult issue for many couples. The successful differentiation of germ cells and live progeny from pluripotent stem cells brings new hope to the couples suffering with infertility. Here we successfully isolated human fetus skin-derived stem cells (hfSDSCs) from fetus skin tissue and demonstrated that hfSDSCs can be differentiated into early human germ cell-like cells (hGCLCs). These cells express human germ cell markers DAZL and VASA. Moreover, these pluripotent stem cell-derived hGCLCs are free of exogenous gene integration. When hfSDSCs were differentiated in porcine follicle fluid (PFF) conditioned media, which has been shown to promote the differentiation of mouse and porcine SDSCs into oocyte-like cells (OLCs), we observed some vesicular structures formed from hfSDSCs. Moreover, when hfSDSCs were cultured with specific conditioned media, we observed punctate and elongated SCP3 staining foci, indicating the initiation of meiosis. Ploidy analysis and fluorescent in situ hybridization (FISH) analysis indicated that a small percentage of putative 1N populations formed from hfSDSCs when compared with positive controls. In conclusion, our data here, for the first time, demonstrated that hfSDSCs possess the differentiation potential into germ lines, and they may differentiate both male and female hGCLCs in vitro under appropriate conditions.

[Expression of GFP gene in mouse early embryo produced by sperm-mediated gene transfer].

The efficiency of the exogenous DNA transfecting mouse sperm was studied by the DIG end labeled and immunohistochemistry technology. The results suggested that: the efficiency of transfecting positive rate of individual mouse sperm was distinct difference (P < 0.01), and the average rate was 13%. The acrosomal reaction was evaluated using the technology of Coomassie brilliant blue stained, and the appropriate in vitro fertilization (IVF) medium TYH was elected. Mouse sperms were transferred with GFP gene in vitro, and the mature oocytes were fertilized using IVF, and then the zygotes were cultured in vitro. The embryos were observed using the fluorescence microscopy, and the transgenic rate was 4.7%. The results suggested that sperm mediated gene transfer (SMGT) was an effective and feasible method.