Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function, and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic, but data on the use of other potential carbon substrates are lacking in humans. This prospective and blinded cohort study was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure then, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in an M + 2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M + 2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate in 278 CCs from 21 women ≤34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p = 0.0004, t-test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M + 2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: -6.02, CI: -1.74,-13.79, p = 0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility.Abbreviations: CCs: cumulus cells; COC: cumulus-oocyte complex; LC-MS: liquid chromatography-mass spectrometry; acetyl-CoA: acetyl-Coenzyme A; CoA: Coenzyme A.
In this study, the expression of the androgen receptor (AR) and estrogen receptor alpha (ERα) in testicular tissue of male patients with obstructive azoospermia (OA) and non-obstructive azoospermia (NOA) were evaluated by immunohistochemistry. NOA (n = 23) and OA (n = 21) groups were created according to clinical and laboratory archival records. Testicular sperm extraction tissue sections were evaluated according to Johnsen's tubular biopsy scoring (JTBS) method. ERα and AR immunostaining results were evaluated semiquantitatively. Serum follicle-stimulating hormone (FSH), luteinizing hormone (LH), testosterone, and estradiol were analyzed. Serum FSH and LH concentrations were greater, and testosterone concentrations were lower than the normal values in the NOA group, whereas the OA group revealed normal hormonal values. Serum estradiol concentrations in groups were in the normal range. JTBSs were significantly lower in the NOA group. Decreased AR expression and increased ERα expression were observed in the NOA group compared to the OA group. This suggests that ERα and AR are expressed in Sertoli cells, Leydig cells, and myoid cells and are required for normal testicular function. Decreased expression of the AR and increased expression of ERα in the testis may negatively affect spermatogenesis.Abbreviations: AR: androgen receptor; ER: estrogen receptor; ERα: estrogen receptor alpha; FSH: follicle-stimulating hormone; JTBS: Johnsen's tubular biopsy scoring; LH: luteinizing hormone; NOA: non-obstructive azoospermia; OA: obstructive azoospermia; TESE: testicular sperm extraction.
Ovine cysteine-rich secretory protein 1 (CRISP-1) is an acidic glycoprotein of epididymal origin under CRISP, antigen 5, pathogenesis-related protein 1 (CAP) super-family. The aim of the present study was the optimization of bacterial production and partial characterization of putative mature ovine CRISP-1 protein. The cDNA corresponding to T23 - C242 peptide fragment of ovine CRISP-1 protein was cloned into THE pET32b(+) expression vector using E. coli DH5α. Protein expression was carried out in E. coli BL21(DE3) by inducition with 1 mM IPTG at 37°C for 4 h. The recombinant protein was expressed as inclusion bodies and purified by Ni-NTA affinity chromatography using a pH gradient. Further purification of the protein was carried out by gel extraction following zinc sulfate negative staining. SDS-PAGE analysis of the purified recombinant CRISP-1 protein revealed a 43.8 kDa band. Bioactivity of the purified CRISP-1 protein was examined on sperm motility and capacitation. The recombinant ovine CRISP-1 protein at 5 µg/ml caused significant inhibition of sperm motility, and the activity was lost following heating the protein at 100°C for 5 min. The protein also demonstrated decapacitation activity, and at a concentration of 2 µg/ml, it caused a significant (P < 0.05) reduction in sperm capacitation. In conclusion, the thioredoxin-tagged ovine CRISP-1 protein was successfully produced in E. coli and purified in the soluble form by a combination of Ni-NTA affinity chromatography, gel purification, and dialysis. The recombinant protein exhibited both motility-inhibiting and decapacitating activities. Further study is needed to elucidate the mechanism of action and evaluate it's possible use in semen preservation.Abbreviations: CRISP-1: Cysteine-rich secretory protein-1; PCR: polymerase chain reaction; IPTG: isopropyl-β-D-thiogalactopyranoside; LB: Luria Bertani; SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis; EDTA: ethylene diamine tetraacetic acid; Ni-NTA: Nickel nitrilotriacetic acid.
This study examines the association of the -617 C > A polymorphism in the Nrf2 gene (rs6721961) with male infertility in a Turkish population and determines its functional role in spermatogenesis in correlation with the impact of different levels of DNA damage on the genotypes. A total of 100 infertile men and 100 healthy fertile men were included in the study. Nrf2 genotyping was performed with the PCR-based restriction fragment length gene polymorphism (RFLP-PCR) analysis. According to our results, the Nrf2 CC, CA, and AA genotype distribution frequencies were 58.6%, 38.4%, and 3% in the control group, respectively, and 38%, 48%, and 14% in the infertile men, respectively. The AA genotype was significantly higher in the patient group. In smokers, a significant difference was found in progressive motility values between the genotypes (p = 0.001). Also, sperm progressive motility and concentration decreased significantly in those smokers with the AA genotype; smokers carrying this genotype were also 5.75 times more likely to have oligoasthenozoospermia than those with CC (p < 0.05). There was a significant relationship between the number of cases with high sperm-DNA damage when comparing the frequency of Nrf2 AA genotype carriers with the CC genotype 16.3% vs. 6.9%, respectively (p < 0.001). These results suggest the importance of the Nrf2 gene C > A (rs 6,721,961) polymorphism in the etiology of sperm DNA damage as a risk factor for male infertility. Smokers carrying the AA genotype are more likely to impair seminal parameters through antioxidant mechanisms.Abbreviations: Polymerase chain reaction (PCR)-based restriction fragment length gene polymorphism (RFLP-PCR); reactive oxygen species (ROS); deoxyribonucleic acid (DNA); catalases (CATs); superoxide dismutase (SOD); glutathione peroxidase (GPX); glutathione-S-transferase (GST); Nuclear factor erythroid 2 (NF-E2)-related factor 2 (Nrf2); basic leucine zipper (bZIP); antioxidant response element (ARE); World Health Organization (WHO);normospermia(NS);asthenozoospermia(AS);oligozoospermia(OS);oligoasthenozoospermia (OAS); follicle stimulating hormone (FSH); ultraviolet (UV); low-melting-point agarose (LMA); normal-melting-point agarose (NMA); arbitrary units (AU); total comet score (TCS); A one-way analysis of variance (ANOVA); standard deviation (SD); N-acetyltransferase (NAT2); small non-coding RNAs (ncRNAs); microRNAs (miRNA).
Gestational diabetes mellitus (GDM) can cause a variety of adverse maternal and fetal complications. The purpose of this study was to screen and identify the urinary polypeptides related to the severity of GDM and to analyze the correlation between urinary peptide levels and neonatal metabolic indices. A total of 31 normal pregnant women (N group) and 74 patients with GDM (GDM group) were randomly selected between February 2018 and August 2019. Patients with GDM were divided into two groups according to their fasting plasma glucose (FPG) levels. The urine samples were enriched using weak cation-exchange magnetic beads (MB-WCX), and eight different urine polypeptides were screened and analyzed. The peptide spectra were obtained using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). The urinary peptide signatures of the two groups were compared using the BioExplorer software. The difference analysis of the eight urinary polypeptides between the normal pregnant (N) group and GDM group showed that two polypeptides with mass-to- charge ratios (m/z) of 2175.7 and 2318.8, respectively, were significantly different between the two groups (P < 0.01). The m/z 2175.7 polypeptide was analyzed by liquid chromatography-tandem mass spectrometry (LC-MS), and the corresponding name of the molecule was inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4). The changes in ITIH4 levels correlated with those in the neonatal metabolic indices. By establishing the Fisher discriminant function equation for the GDM group, the difference in sample distribution and mean value of the two groups could be observed directly.Abbreviations: GDM: gestational diabetes mellitus; FPG: fasting plasma glucose; MB-WCX: weak cation exchange magnetic beads; MALDI-TOF MS: matrix-assisted laser desorption ionization time-of-flight mass spectrometry; m/z: mass charge ratio; LC-MS: liquid chromatography-tandem mass spectrometry; glycosylated hemoglobin (HbA1c); PPG: postprandial plasma glucose; ITIH4: inter-alpha-trypsin inhibitor heavy chain H4; IR: insulin resistance; NFPG: neonatal fasting plasma glucose; NH: neonatal height; NW: neonatal weight; BMI: body mass index; RPL: recurrent pregnancy loss; OGTT: oral glucose tolerance test; ADA: American Diabetes Association; LIS: Laboratory Information System.