Yi-Feng Ge

Gonadotropin-releasing hormone positively regulates steroidogenesis via extracellular signal-regulated kinase in rat Leydig cells

Gonadotropin-releasing hormone (GnRH) is secreted from neurons within the hypothalamus and is necessary for reproductive function in all vertebrates. GnRH is also found in organs outside of the brain and plays an important role in Leydig cell steroidogenesis in the testis. However, the signalling pathways mediating this function remain largely unknown. In this study, we investigated whether components of the mitogen-activated protein kinase (MAPK) pathways are involved in GnRH agonist (GnRHa)-induced testis steroidogenesis in rat Leydig cells. Primary cultures of rat Leydig cells were established. The expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) and the production of testosterone in response to GnRHa were examined at different doses and for different durations by RT-PCR, Western blot analysis and radioimmunoassay (RIA). The effects of GnRHa on ERK1/2, JNK and p38 kinase activation were also investigated in the presence or absence of the MAPK inhibitor PD-98059 by Western blot analysis. GnRHa induced testosterone production and upregulated 3β-HSD expression at both the mRNA and protein levels; it also activated ERK1/2, but not JNK and p38 kinase. Although the maximum effects of GnRHa were observed at a concentration of 100 nmnol L⁻¹ after 24 h, activation of ERK1/2 by GnRHa reached peak at 5 min and it returned to the basal level within 60 min. PD-98059 completely blocked the activation of ERK1/2, the upregulation of 3β-HSD and testosterone production. Our data show that GnRH positively regulates steroidogenesis via ERK signalling in rat Leydig cells. ERK1/2 activation by GnRH may be responsible for the induction of 3β-HSD gene expression and enzyme production, which may ultimately modulate steroidogenesis in rat Leydig cells.

A girl with distinctive features of borderline high blood pressure, short stature, characteristic brachydactyly, and 11.47 Mb deletion in 12p11.21–12p12.2 by oligonucleotide array CGH

Interstitial deletions of the short arm of chromosome 12 are rare. Since Tenconi et al. [1975] described a male baby with a de novo interstitial deletion of 12p, a total of 10 additional patients have been reported [Orye and Craen, 1975; Magenis et al., 1981; Boilly-Dartigalongue et al., 1985; Fryns et al., 1990; Nagai et al., 1995; Gl€aser et al., 2003; Stumm et al., 2007]. Interstitial deletion of 12p could be divided into four groups according to the region of deletion: (1) deletions including the p1! p11; (2) deletions extending more distally (p13! p11); (3) deletions in band 12p13, and (4) deletions encompassing the terminal of 12p [Gl€aser et al., 2003]. Common phenotypic findings constituting the interstitial deletions of 12p include mental retardation, developmental delay, craniofacial anomalies, microcephaly, brachydactyly, and clinodactyly. Only one patient with borderline high blood pressure, short stature, brachydactyly, and a deletion in 12p11.21! 12.2 region was reported by Nagai et al. [1995]. We encountered a 13-year-old girl with moderate mental retardation, short stature, and characteristic brachydactyly. She was born at term of gestation with normal birth weight of 3,300 g and short stature of 46 cm ( 2.4 SD). The girl spoke at the age of 2 years and walked without assistance at the age of 3 years. Clinical observation showed a small slender girl with height 126 cm ( 4.4 SD), weight 25 kg ( 2.66 SD), borderline high blood pressure (120/80 mmHg), round face, strabimus and chaotic tooth arrangement (Fig. 1). The fifth toe overlapped the fourth one and both of them showed brachydactyly. Serum hormone levels of GH, TSH, T3, and T4 were normal for her age. Cerebral CT scan and MRI were normal. Roentgenograms of hands and feet disclosed the bilateral brachydactyly, with shortened metacarpals of digits 3–5, middle phalanges of digit 5, and metatarsals of digits 4 and 5 (Fig. 2). Cone-shaped epiphyses were not evident. No obvious skeletal abnormalities were found in vertebrae, pelvis, and other tube bones. Now she is enrolled in a special school for children with mental retardation. She can count numbers to 10. Chromosome analysis of G-banding at 400 band showed a possible deletion of 12p11.2 in all metaphases analyzed (Fig. 3A). Family history was negative. Chromosome analysis of both parents showed normal karyotypes, indicating a de novo deletion. This research was approved by Ethics Committee of the hospital, and the written informed consent was obtained from the patient and the parents. Multicolor fluorescent in situ hybridization (M-FISH) analysis using the Spectra Vysion WCP Probe (Vysis, Downers Grove, IL) was performed on metaphases. A reciprocal translocation was excluded. To refine the extent of the deletion on the molecular cytogenetic level, analysis of using a genomic-wide high-density oligo array (OaCGH44K) with 44,000 probes covering more than 30,000 mapped genes was carried out according to Agilent manufacturer’s procedures and statistical algorithms [Fan et al., 2007; www.agilent. com.chem/gocgh]. An 11.47 Mb interstitial deletion at genomic position 20, 724, 852 bp! 32, 201, 544 bp in

Follicle-stimulating hormone autoantibody is involved in idiopathic spermatogenic dysfunction

AIM To detect the anti-follicle-stimulating hormone (FSH) antibody in idiopathic infertile patients and fertile subjects in order to determine the role of this antibody in patients with spermatogenic dysfunction. METHODS The anti-FSH antibody in serum was detected by an enzyme-linked immunosorbent assay (ELISA). The functional and structural integrity of the sperm membrane was evaluated with hypo-osmotic swelling (HOS) test and the ultrastructure of the spermatozoa was investigated by transmission electron microscopy (TEM). RESULTS The extent of positive FSH antibody in the patients with oligozoospermia and/or asthenozoospermia was significantly higher than that in the fertile subjects and infertile patients with normal sperm concentration and motility, but it was significantly lower than that in the patients with azoospermia. The extent of anti-FSH antibody in the patients with azoospermia was significantly greater than that in patients with oligospermia and/or asthenospermia, infertile people with normal sperm density and motility and fertile people. The hypo-osmotic swelling test showed that the percentage of HOS-positive spermatozoa (swollen) was 45.1 mu 3.5% in the FSH antibody-positive group and 59.1% micro 6.2% in the FSH antibody-negative control group. The percentage of functional membrane damage to spermatozoa was significantly higher in the anti-FSH antibody-positive group than in the control group. TEM showed that the outer acrosomal membrane was located far from the nucleus, and detachment of the acrosome was found in the FSH autoantibody-positive group. CONCLUSION These data suggest that the presence of anti-FSH antibody is strongly correlated with the sperm quantity and quality in idiopathic male infertility. Anti-FSH antibody may be an important factor causing spermatogenic dysfunction and infertility.

Annexin A5 regulates Leydig cell testosterone production via ERK1/2 pathway.

This study was to investigate the effect of annexin A5 on testosterone secretion from primary rat Leydig cells and the underlying mechanisms. Isolated rat Leydig cells were treated with annexin A5. Testosterone production was detected by chemiluminescence assay. The protein and mRNA of Steroidogenic acute regulatory (StAR), P450scc, 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-hydroxysteroid dehydrogenase (17β-HSD), and 17α-hydroxylase were examined by Western blotting and semi-quantitative RT-PCR, respectively. Annexin A5 significantly stimulated testosterone secretion from rat Leydig cells in dose- and time-dependent manners and increased mRNA and protein expression of StAR, P450scc, 3β-HSD, and 17β-HSD but not 17α-hydroxylase. Annexin A5 knockdown by siRNA significantly decreased the level of testosterone and protein expression of P450scc, 3β-HSD, and 17β-HSD. The significant activation of ERK1/2 signaling was observed at 5, 10, and 30 min after annexin A5 treatment. After the pretreatment of Leydig cells with ERK inhibitor PD98059 (50 μmol l−1 ) for 20 min, the effects of annexin A5 on promoting testosterone secretion and increasing the expression of P450scc, 3β-HSD, and 17β-HSD were completely abrogated (P < 0.05). Thus, ERK1/2 signaling is involved in the roles of annexin A5 in mediating testosterone production and the expression of P450scc, 3β-HSD, and 17β-HSD in Leydig cells.

The effect of induced anti-follicle-stimulating hormone autoantibody on serum hormone level and apoptosis in rat testis.

AIMS Anti-follicle-stimulating hormone (FSH) autoantibody was found to highly correlate with oligospermia and asthenospermia, but the actual effect of FSH autoantibody on spermatogenesis is still unknown. MAIN METHODS In this study, 21-day rats were immunized seven times with FSH peptides linked with Keyhole Limpet Hemocyanin (KLH) (experimental group) or KLH (control group) every 2 weeks. Luteinizing hormone (LH) and inhibin B level in the immunized rat sera were measured by enzyme-linked immunosorbent assay (ELISA). Apoptosis of spermatogenic cells in the testis was detected by in situ end labeling method (TUNEL), and the mRNAs of Bax, Bcl-2 and Caspase-3 in testis were detected by fluorescent Quantitative PCR. KEY FINDINGS Compared with the control, serum inhibin B level was significantly decreased at all time points (34.49%, 23.20%, and 37.00%) (p<0.05). There was no difference in the serum LH level between experimental and control groups. FSH peptide immunization increased the apoptosis of spermatogenic cells in the testis that was associated with an imbalance of Bax and Bcl-2 expression and upregulation of Caspase-3. SIGNIFICANCE These results suggest that FSH autoantibody could cause the reduction of inhibin B, thereby inducing hypospermatogenesis via augment of spermatogenic cell apoptosis.

Relation of size of seminal vesicles on ultrasound to premature ejaculation.

Myriad biological factors have been proposed to explain premature ejaculation (PE). However, data correlating PE with seminal vesicles (SVs) are sparse. The study aimed to evaluate the relationship between the size of SV and PE. The cross-sectional study included 44 outpatients with PE and 44 volunteers without PE, and the size of SV was compared. Self-estimated intravaginal ejaculatory latency time, the Premature Ejaculation Diagnostic Tool (PEDT), the International Index of Erectile Function-15, and the National Institutes of Health-Chronic Prostatitis Symptom Index were used for assessment of symptoms. Compared to the control group, the PE group had significantly higher mean anterior-posterior diameter (APD) of SV (P < 0.001). The optimal mean APD of SV cutoff level was 9.25 mm for PE. In the PE group, PEDT was also higher with a mean APD of SV ≥9.25 mm compared with mean APD of SV <9.25 mm. PEDT was significantly correlated with the mean APD of SV (r = 0.326, P = 0.031). The seminal plasma proteins were compared between six PE and six matched control cases by mass spectrometry and it was shown that 102 proteins were at least 1.5-fold up- or down-regulated. Among them, GGT1, LAMC1, and APP were significantly higher in the PE group. These results indicated that men with a larger mean APD of SV might have a higher PEDT score. Transrectal ultrasound of SV should be considered in the evaluation of patients with premature ejaculation. SV might be a potential target for the treatment of patients with PE and ultrasound change in SV.

Meiotic pairing error in an infertile male bearing reciprocal deletion of chromosome 13.

A series of complex processes takes place during the first meiotic division, including pairing, synapsis, recombination, and segregation of homologous chromosomes. When any of these processes is altered, cellular checkpoints arrest the progression of meiosis and induce cell loss, causing a severe reduction in fertility, or even sterility. In this study, we report on a 29-year-old, healthy, but severe oligozoospermic male with a supernumerary, ring-neocentric 13q12.3 → 13q22 chromosome and a reciprocal deletion, which interfere with the meiotic pairing of chromosomes 13, causing spermatogenesis failure.

Analysis of human sperm DNA fragmentation index (DFI) related factors: a report of 1010 subfertile men in China

BackgroundMany factors may lead to sperm DNA damage. However, it is little known that the correlations of sperm DNA damage with obesity-associated markers, and reproductive hormones and lipids levels in serum and seminal plasma.MethodsIn our prospective study, a total of 1 010 subfertile men, aged from 18 to 50 years old, were enrolled from August 2012 through June 2015. Their obesity-associated markers, semen parameters, sperm acrosomal enzyme activity, seminal plasma biochemical markers, and reproductive hormones and lipids levels in serum and seminal plasma were detected. Sperm DNA fragmentation index (DFI) was determined by sperm chromatin structure assay. The correlations between DFI and each of the above-mentioned variables were analyzed.ResultsSpearman correlation analysis showed that sperm DFI was positively related to age and abstinence time (P<0.001). Sperm DFI was also positively related to semen volume and percent of abnormal sperm head (P<0.001), while negatively related to sperm concentration, progressive motility (PR), sperm motility, total normal-progressively motile sperm count (TNPMS), percent of normal sperm morphology (NSM), percent of intact acrosome and acrosomal enzyme activity (P<0.001). Sperm DFI was positively related to seminal plasma zinc level (P<0.001) but unrelated to seminal plasma total α-glucotase, γ-glutamyl transpeptidase (GGT) and fructose levels. There was no any correlation between sperm DFI and obesity-associated markers such as body mass index (BMI), waist-to-hip ratio (WHR), waist circumference (WC) and waist-to-height ratio (WHtR), and serum lipids levels, but there was positive correlation between sperm DFI and seminal plasma triglyceride (TG) and total cholesterol (TC) levels (P<0.001). Sperm DFI was positively related to serum luteinizing hormone (LH) and follicle stimulating hormone (FSH) levels and seminal plasma FSH and estradiol (E2) levels (P<0.001), but unrelated to serum and seminal plasma testosterone (T) levels. The multivariate regression analysis for the variables which were significantly correlated with sperm DFI in Spearman correlation analysis showed that age, semen volume, sperm concentration, progressive motility, TNPMS and intact acrosome were independently correlated with sperm DFI.ConclusionsThere are many potential factors associated with sperm DFI, including age, abstinence time, spermatogenesis and maturation, seminal plasma lipids and reproductive hormones levels. However, the potential effects of seminal plasma lipids and reproductive hormones on sperm DNA damage need still to be demonstrated by the studies with scientific design and a large size of samples.

AB185. Semen pH effects sperm motility and capacitation by influencing Na/K-ATPase activity and Ca concentration in spermoplasm

Background The pH of semen is one of the important elements for maintaining spermatic normal functions. Variation of semen pH could lead to male infertility, but the mechanism of which is still unknown. Methods The healthy human spermatozoa cultured in sperm nutrition solution with pH 5.2, 6.2, 7.2, 8.2, 9.2 and 10.2 respectively were analyzed for sperm motility, vitality, hypoosmotic swelling rate, Na/K-ATPase activity and the intracellular Caconcentration. Results The results showed that sperm motility, vitality, hypoosmotic swelling rate were all on the peak in pH 7.2 sperm nutrition solution, and decreased in different degree in pH 5.2, 6.2, 8.2, 9.2 and 10.2. Furthermore, the hypoosmotic swelling rate had a positive correlation with the sperm motility and viability. Sperm penetration meter test was displayed that in compared with pH 7.2, the sperm ascending altitude in sperm nutrition solution with pH 5.2, 6.2, 8.2, 9.2 and 10.2 were significantly declined. In addition, sperm Na/K-ATPase activity in sperm nutrition solution with different pHs was detected and the enzyme activity were significantly lower in pH 5.2, 6.2, 8.2, 9.2 and 10.2 medium compared to pH 7.2. Using flow cytometry and laser confocal scanning microscope to contrast the intracellular Ca concentration of sperm cultured in pH 5.2, 6.2, 7.2, 8.2, 9.2 and 10.2 sperm capacitation solution. Taking pH 7.2 group as a control, the mean fluorescence intensity of sperm in pH 5.2, 6.2 and 10.2 medium were decreased significantly, while pH 8.2 and 9.2 group had no differences with control. Conclusions All the results suggested that hydrogen-ion concentration outside spermatozoa could affect sperm motility and capacitation by influencing sperm Na/K-ATPase activity and spermoplasm Ca concentration revealing one of the mechanisms of male infertility.

Erratum to “A G560S mutation in α1 (I) collagen causes familial osteogenesis imperfecta type IV” [Clinica Chimica Acta 409 (2009) 145–146]

The Publisher regrets that the following error has occurred in the above article. Subsequently, a heterozygous substitution of 7872 g. G>A (AF017178.2) in exon 25 of COL1A1 was found by DNA sequencing in all affected individuals and was further confirmed by DHPLC analysis. However, to our knowledge, the mutation reported here had been proposed only by Mackay et al. [5] who reported a sporadic case in a six-year-old child without detailed clinical description.