MicroRNA profiling reveals the role of miR-133b-3p in promoting apoptosis and inhibiting cell proliferation and testosterone synthesis in mouse TM3 cells.

Late-onset hypogonadism (LOH) is an age-related clinical and biological syndrome in which serum testosterone deficiency is an important characteristic and diagnostic indicator. In this study, we firstly analyzed the difference in the expression level of three miR-133 s (including miR-133a-3p, miR-133a-5p, and miR-133b-3p) in rat testis samples, blood samples from mice before and 1 wk after testis removal, and mouse TM3 cells. Secondly, the mimics and inhibitors corresponding to the three miR-133 s of mouse were transfected into TM3 cells separately to determine the correlation between the three miRNAs. Finally, using mouse TM3 cells to analyze the effect of miR-133b overexpression or inhibition on the proliferation and apoptosis of mouse testicular Leydig cells, the effect on genes related to testosterone synthesis, and the effect on the level of testosterone in the culture medium. We found that, compared with the testis tissue of newborn rats, miR-133a-5p was increased in adult rats, and miR-133a-3p and miR-133b-3p were decreased. In addition, 1 wk after the testis was removed, the expression levels of these three miRNAs in the blood of adult mice decreased. The correlation of the three miRNAs was summarized, and it was found that miR-133b-3p played an important role in it. In TM3 cells, overexpression of miR-133b-3p suppressed the proliferation and promotes apoptosis of cells, suppressed the expression level of most genes related to cell proliferation and testosterone synthesis, and the concentration of testosterone in the culture medium decreased while these phenomena can be reversed by the inhibition of miR-133b-3p expression. It was found that miR-133b-3p can regulate testosterone production in TM3 cells at least by targeting FSCN1. The above results suggest that miR-133b-3p plays an important role in regulating testosterone synthesis. These findings also provide new candidate diagnostic indicators for late-onset hypogonadism in men and provide new clues for the further study of pathogenesis.

LncRNA HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression.

LncRNA HOX antisense intergenic RNA (HOTAIR) can regulate cancer-related gene expression and promote stem cell and tumor cell proliferation via mechanisms including the competing endogenous RNA (ceRNA) mechanism. HOTAIR is abundantly expressed in the genital tubercle of E11.5, E12.5, and E13.5 embryos, whereas it became barely detectable at E13.5 and expressed again in adult mouse testis. However, the underlying function and mechanism of HOTAIR in spermatogenesis have not been elucidated. Interestingly, other researchers reported that the function of gene Nanos C2HC-Type Zinc Finger 2 (nanos2) includes the maintenance of both the primordial germ cells (PGCs) and germline stem cells, and Nanos2 protein and transcripts (NANOS2) were detected only in PGCs from day E11.5 and undifferentiated spermatogonia in spermatogenesis. We therefore investigated the relationship between HOTAIR and NANOS2 in maintaining spermatogonial stem cell population. We found that, compared to the adult mouse, the expression levels of HOTAIR and NANOS2 in embryo mouse were significantly higher and miR-761expression level was lower. In mouse GC-1 spermatogonia cells, overexpression of miRNA-761 significantly inhibited the expression of NANOS2 and HOTAIR, suppressed the proliferation, and promotes apoptosis of cells. Knock down and overexpression of HOTAIR indicated that HOTAIR expression was positively correlated with NANOS2 expression; overexpressed HOTAIR could promote proliferation and suppresses apoptosis of GC-1 cells. By a rescue experiment and dual luciferase reporter assay, miR-761 was identified as a direct target of HOTAIR, and NANOS2 was identified as the direct target of miR-761. The above results indicate that HOTAIR promotes proliferation and suppresses apoptosis of mouse spermatogonium GC-1 cells by sponging miR-761 to modulate NANOS2 expression. Our findings elucidate one of possible mechanisms and importance of HOTAIR in maintaining spermatogonial stem cell population, and provide new candidate genes and possible pathogenesis for male infertility.

The association of rs11457523 in HSP90AA1 with idiopathic male infertility in the Chinese population.

The association of single nucleotide polymorphisms (SNPs) in heat shock protein 90 (HSP90) genes with idiopathic male infertility remains unclear. In this study, the five selected SNPs in HSP90AA1 namely rs10133307, rs10873531, rs11547523, rs11621560 and rs7145597 were genotyped in 116 idiopathic infertile males and 185 ethnically matched fertile males using the Sequenom MassARRAY assay. The role of these SNPs in male infertility was then studied using multiple genetic models. We observed that genotype distribution (p = .028) and allelic frequency (p = .032) of rs11547523 were significantly different between the infertile and fertile groups. In particular, A genotype of rs11547523 was associated with an increased risk of infertility in the allele (OR = 2.508, p = .048), dominant (OR = 2.733, p = .030) and additive models (OR = 0.366, p = .031). However, there were no significant differences in semen parameters including seminal volume (p = .452), sperm concentration (p = .727), total sperm number (p = .588), motility (p = .282) and morphology (p = .975) between A and A/G genotypes of rs11547523. These results indicate that rs11547523 in HSP90AA1 may be associated with idiopathic male infertility in the Chinese population. The outcome of this study contributes to the development of the diagnosis of male infertility.

The progesterone-induced sperm acrosome reaction is a good option for the prediction of fertilization in vitro compared with other sperm parameters.

Progesterone (P4 ) is crucial for the physiological function of spermatozoa. In the study, we investigated the correlation between P4 -induced sperm acrosome reaction (AR) and parameters including sperm progressive motility, normal morphology and sperm DNA fragmentation (SDF), and compared the in vitro fertilization (IVF) predictive values of these indicators based on the multivariate regressions analysis and receiver operator characteristics (ROC) curve analyses. The results demonstrated a negative correlation between P4 -induced sperm AR and the SDF, with the correlation -9.05 (-17.25 to -0.84), p<0.05, n = 47). No relationship was found between the sperm progressive motility, normal morphology and the induced AR. The P4 -induced AR and SDF were both significantly correlated to the fertilization rate. ROC curve analyses indicated that P4 -induced AR was a better prognostic predictor for the fertilization rate compared with the SDF, with the areas under the curve 0.729 (0.580-0.849), p<0.01 and 0.637 (0.484-0.772), p=0.16 respectively. The cut-off value for P4 -induced AR to predict "50% fertilization rate" was 23.4% with sensitivity and specificity of 63.3% and 88.2% respectively. The overall results indicated that the assessment of P4 -induced AR seemed to be a more sensitive indicator for fertilization rate in vitro compared with other sperm parameters.

Effect of sperm DNA fragmentation on the clinical outcomes for in vitro fertilization and intracytoplasmic sperm injection in women with different ovarian reserves.

OBJECTIVE: To investigate effect of sperm DNA fragmentation (SDF) on clinical outcomes of assisted reproductive technology in women with normal ovarian reserve (NOR) versus reduced ovarian reserve (ROR). DESIGN: Retrospective clinical study. SETTING: University-affiliated tertiary teaching hospital. PATIENT(S): A total of 2,865 consecutive couples undergoing their first in vitro fertilization (IVF) or intracytoplasmic sperm injection (ICSI) cycle. INTERVENTION(S): SDF assessed using sperm chromatin dispersion in sperm samples 1-2 months before treatment. MAIN OUTCOME MEASURE(S): SDF, IVF, and ICSI outcomes. RESULT(S): The grouping criteria were [1] basal follicle stimulating hormone >10 IU/L, [2] antral follicle count <6, and [3] female age ≥38 years. Women fulfilling two of the three criteria were considered to have ROR, and those not meeting any criteria were considered to have NOR. The area under the receiver operating characteristic curve was 0.594 (0.539-0.648) for the ROR group and 0.510 (0.491-0.530) for the NOR group. A cutoff value for SDF to predict the clinical pregnancy rate (CPR) in the ROR group was 27.3%. When the SDF exceeded 27.3%, the live-birth and implantation rates in the ROR group were statistically significantly decreased, but the clinical pregnancy, live-birth, and implantation rates were not affected in the NOR group. The risk of early abortion increased significantly in the NOR group when the SDF exceeded 27.3%. CONCLUSION(S): Sperm DNA fragmentation has a greater impact on IVF and ICSI outcomes among women with ROR, so SDF testing may be of particular clinical significance for these couples.

Effect of sperm DNA fragmentation on clinical outcome of frozen-thawed embryo transfer and on blastocyst formation.

During the last decades, many studies have shown the possible influence of sperm DNA fragmentation on assisted reproductive technique outcomes. However, little is known about the impact of sperm DNA fragmentation on the clinical outcome of frozen-thawed embryo transfer (FET) from cycles of conventional in vitro fertilization (IVF) and intra-cytoplasmic sperm injection (ICSI). In the present study, the relationship between sperm DNA fragmentation (SDF) and FET clinical outcomes in IVF and ICSI cycles was analyzed. A total of 1082 FET cycles with cleavage stage embryos (C-FET) (855 from IVF and 227 from ICSI) and 653 frozen-thawed blastocyst transfer cycles (B-FET) (525 from IVF and 128 from ICSI) were included. There was no significant change in clinical pregnancy, biochemical pregnancy and miscarriage rates in the group with a SDF >30% compared with the group with a SDF ≤30% in IVF and ICSI cycles with C-FET or B-FET. Also, there was no significant impact on the FET clinic outcome in IVF and ICSI when different values of SDF (such as 10%, 20%, 25%, 35%, and 40%) were taken as proposed threshold levels. However, the blastulation rates were significantly higher in the SDF ≤30% group in ICSI cycle. Taken together, our data show that sperm DNA fragmentation measured by Sperm Chromatin Dispersion (SCD) test is not associated with clinical outcome of FET in IVF and ICSI. Nonetheless, SDF is related to the blastocyst formation in ICSI cycles.

[Diagnostic value of sperm DNA fragmentation for male infertility].

OBJECTIVE: To assess the diagnostic value of sperm DNA fragmentation (SDF) for male infertility. METHODS: Two hundred and ninety-nine males attending infertility clinic were classified into 157 primary infertile cases and 142 fertile controls. Semen analysis was performed as recommended by the World Health Organization (WHO). SDF was assessed by sperm chromatin dispersion (SCD) assay, and the results were expressed as DNA fragmentation index (DFI). RESULTS: The DFI was significantly higher in infertile males than that in fertile controls [(17.1± 9.3)% vs. (14.2± 9.0)%](P< 0.01). No significant difference was detected in the age of male and female partners, seminal volume, sperm count, motility and morphology between infertile males and fertile controls (P> 0.05). The area under the receiver operating characteristic curve (AUC) was 0.861 [95% confidence interval (CI)= 0.814-0.907] for 15.1% of SDF. The threshold level of 15.1% was derived as cut-off value to discriminate infertile men from fertile controls. By this threshold, specificity was 88.2% and sensitivity was 81.8%. The 299 men were divided into group A (n= 120) with DFI≥ 15.1% and group B (n= 179) with DFI< 15.1% based on the cut-off value. The percentage of infertile men in group A was significantly higher than that in group B (79.2% vs. 34.6%) (P< 0.01). The odds ratio (OR) for infertility in the two groups was 7.2 (95%CI= 4.2-12.3). CONCLUSION: Sperms with high-level of DNA fragmentation can impair male fertility. DFI can be used as a good diagnostic marker for male infertility.

[Variation of sperm DNA fragmentation index in male partners from infertile couples].

OBJECTIVE: To investigate variation of sperm DNA fragmentation index (DFI) in male partners of infertile couples. METHODS: A total of 539 males between April 2009 and April 2012 were analyzed. At least one repeated routine semen analysis and sperm DNA fragmentation test were performed for each sample by sperm chromatin dispersion (SCD) analysis following World Health Organization guidelines. Coefficient of variation (CV) for DFI was calculated. RESULTS: Respectively, 1, 2, 3 and 4 repeated SCD analyses were carried out on 473, 59, 6 and 1 semen samples. The median interval between the first and repeated SCD measurements was 3.0 (1.0-11.0) months. For the first tested samples, the between-sample coefficient of variation (CVB) for DFI was 71.2%. A significant difference has been found between DFI of the first measurement and DFI of repeated measurement in 0.5 to 3 months, 3 to 12 months and 12 to 34 months (P< 0.01). Compared with the first test, 26.3% of males were on both sides of the cut-off point of 18%. The median within-subject coefficient of variation (CVw) for DFI of 539 men was 26.0% (12.6%-42.8%). And the median CVw DFI was significantly lower compared with CVw of sperm count, concentration, progressive motility and normal morphology (P< 0.01). Significant correlations were found between the CVw DFI and sperm count, concentration and interval among the samples (P< 0.05). CONCLUSION: DFI of male partners for infertile couples is a parameter with substantial variation, repeated SCD measurements are therefore recommended.

The CFTR polymorphisms poly-T, TG-repeats and M470V in Chinese males with congenital bilateral absence of the vas deferens.

Congenital bilateral absence of the vas deferens (CBAVD) is a frequent cause of obstructive azoospermia, and mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene have also been frequently identified in patients with CBAVD. However, the distribution of the CFTR polymorphisms M470V, poly-T, TG-repeats and F508del mutation in the Chinese CBAVD population with presumed low cystic fibrosis (CF) frequency remains to be evaluated. Samples obtained from 109 Chinese infertile males with CBAVD and 104 normal controls were analyzed for the presence of CFTR (TG)m(T)n, M470V and F508del by PCR amplification followed by direct sequencing. Our study showed that the F508del mutation was not found in our patients. The 5T mutation was present with high frequency in Chinese CBAVD patients and IVS8-5T linked to either 12 or 13 TG repeats was highly prevalent among CBAVD patients (97.22% of 72 cases and 96.91% of 97 alleles with IVS8-5T). Moreover, a statistically significant relationship between TG12-5T-V470 haplotype and CBAVD was detected. This study indicated that the CFTR polymorphisms poly-T, TG-repeats and M470V might affect the process of CBAVD in the Chinese population.

Secretory phospholipase A2 group IID Is involved in progesterone-induced acrosomal exocytosis of human spermatozoa.

Phospholipase A2 (PLA(2)) plays a major role during acrosomal exocytosis (AE) in mammalian spermatozoa, but the identity of PLA(2) subtypes present in spermatozoa remains elusive. This study explored whether secretory PLA(2) Group IID (sPLA(2)-IID) isoform is present in human spermatozoa and whether it is involved in AE. Localization and expression of sPLA(2)-IID in human spermatozoa were explored by immunofluorescence staining and Western blot analysis. Occurrence of AE was evaluated by triple staining, and arachidonic acid (AA) levels were quantified by gas chromatography-mass spectrometry. Sperm motion parameters and hyperactivation were analyzed by computer-assisted sperm analysis. sPLA(2)-IID was localized in the postacrosomal region of the head and the midpiece of tail in human sperm. A 16-kd protein band was detected by Western blotting in sperm extracts. Progesterone-induced AE was significantly inhibited in a concentration-dependent manner using a sPLA(2)-IID neutralizing antibody. The increase in AA levels seen during progesterone-stimulated exocytosis was significantly abrogated by the antibody. The sPLA(2)-IID antibody significantly inhibited hyperactivation, sperm curvilinear velocity, and amplitude of lateral head displacement, but it did not affect the proportion of motile sperm. In conclusion, sPLA(2)-IID is present at the head and midpiece in the human sperm, and activation of such sPLA(2)-IID seems to be involved in AE. Therefore, sPLA(2)-IID isoform plays a functional role during the AE in human sperm.

[Sperm DNA damage and sperm-nucleoprotein transition correlate to acrosin activity and seminal parameters].

OBJECTIVE: To investigate the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters. METHODS: We collected 535 semen samples, assessed sperm DNA damage by sperm chromatin dispersion test, and analyzed the correlation of sperm DNA damage and sperm-nucleoprotein transition with acrosin activity and seminal parameters according to the WHO criteria. RESULTS: Statistically significant differences were observed in sperm DNA damage among sperm-nucleoprotein transition, acrosin activity, sperm concentration and the percentage of grade a + b sperm (P < 0.01). Sperm DNA damage was positively correlated with age, sperm-nucleoprotein transition, sperm concentration and the percentage of grade d sperm (P < 0.01 or P < 0.05), but negatively correlated with acrosin activity (P < 0.001). Stepwise linear regression analysis demonstrated that age, sperm concentration, the percentage of grade d sperm, sperm-nucleoprotein transition and acrosin activity were independent variables related to the DNA fragmentation index (DFI). The abnormality rates of sperm-nucleoprotein transition, acrosin activity, sperm concentration and graded a + b sperm were significantly higher in the sperm DNA damage group (DFI > or = 30%) than in the normal control (DFI < 30%) (P < 0.01). CONCLUSION: Sperm DNA damage is closely related with sperm-nucleoprotein transition, acrosin activity and seminal parameters, which may become another important independent parameter for the evaluation of sperm quality.

Activation of GABAA receptor/Cl- channel and capacitation in rat spermatozoa: HCO3- and Cl- are essential.

This study was designed to determine whether HCO(3)(-) and Cl(-) are required for the activation of the GABA(A) receptor/Cl(-) channel (GBRC) by GABA and the subsequent capacitation of rat sperm. Spermatozoa from adult Sprague Dawley rats were incubated in four different media: modified complete rat fertilization medium (mRFM), Cl(-)-deficient (Cl(-)-DF) mRFM, HCO(3)(-)-DF mRFM, and Cl(-)-DF HCO(3)(-)-DF mRFM, with or without GBRC agonists (GABA and progesterone) or GBRC antagonists (bicuculline and picrotoxin) for 0-6 h under capacitating conditions. Sperm capacitation and hyperactivation were assessed by chlortetracycline staining and computer-assisted sperm analysis, respectively. The results showed that GABA added to the mRFM accelerated capacitation and hyperactivation, followed by increase in the acrosome reaction, reaching maximum value after 5 h. Progesterone also accelerated sperm capacitation and hyperactivation. Bicuculline and picrotoxin, antagonists of GABA, blocked the effects of both GABA and progesterone acceleration of sperm capacitation and hyperactivation. Sperm capacitation required both Cl(-) and HCO(3)(-). These results indicate that activation of GBRC may contribute to sperm capacitation and hyperactivation, and that both HCO(3)(-) and Cl(-) are essential. This is the first report of a close relationship between HCO(3)(-)/Cl(-) transport and the activation of GBRC in rat sperm capacitation and hyperactivation.