Expression of mitochondrial transcription factor A in granulosa cells: implications for oocyte maturation and in vitro fertilization outcomes.

OBJECTIVE: In vitro fertilization-embryo transfer (IVF-ET) is a widely used treatment for infertility, with oocyte maturation and quality having a significant impact on oocyte fertilization, embryo development, and fetal growth. Mitochondrial transcription factor A (TFAM) is essential for maintaining the mitochondrial oxidative respiratory chain and supplying energy for oocyte development, fertilization, and embryonic development. In this study, we aimed to examine TFAM expression in women undergoing IVF-ET and assess its impact on the IVF outcomes. METHODS: We recruited 85 women who underwent IVF-ET treatment for infertility. On the date of egg collection, granulosa cells were extracted from the clear follicular fluid of the first mature egg using ultrasound-guided needle aspiration. The collected granulosa cells served three purposes: (1) detecting TFAM gene expression in granulosa cells via immunocytochemistry, (2) determining TFAM mRNA expression using reverse transcription-PCR (RT-PCR), and (3) measuring TFAM protein expression through western blotting. RESULT: Based on the results, we found that TFAM was localized and expressed in the cytoplasm of granulosa cells, whereas no expression was detected in the nucleus. Granulosa cells exhibited a linear correlation between TFAM mRNA and TFAM protein expression. The study participants were divided into three groups using the ternary method based on relative TFAM mRNA expression thresholds of 33% and 76%: the low-expression group (n = 30), the moderate-expression group (n = 27), and the high-expression group (n = 28). When compared to the other two groups, the moderate expression group exhibited a significantly higher egg utilization rate, 2 pronucleus rate, fertilization rate, and clinical pregnancy rate (P < 0.05). CONCLUSION: TFAM was detected in the cytoplasm of human ovarian granulosa cells. Women with moderate TFAM expression demonstrate enhanced outcomes in IVF.

Resveratrol improves ovarian function in aged rat by inhibiting oxidative stress and activating the Sirt1.

Oxidative stress is a leading driver of ovarian aging. Silent mating-type information regulation 2 homolog-1 (Sirt1) plays an role in ovarian function. Resveratrol has numerous effects, including anti-oxidant and Sirt1 activator. The aim of the study was to investigate the effect of resveratrol on aging-induced ovarian change in rats. The female Sprague Dawley rats were randomly divided into three groups: young control (Con), Aged+Res (20 mg/kg/day resveratrol for 45 days), and Aged. Anti-Müllerian hormone (AMH) was detected by ELISA assay. Malondialdehyde (MDA), glutathione peroxidase (GSH-Px) and superoxide dismutase (SOD) were detected by conventional method. The ovarian structure and follicles were observed by hematoxylin staining, the caspase-3 and Sirt1 were detected by immunohistochemistry and Western blotting. The AMH in the Aged+Res group was elevated, compared to that in Aged group (p < 0.05). The MDA was decreased and GSH-Px and SOD were increased in the Aged+Res group (p < 0.05). The primordial and primary follicles were increased in the Aged+Res group (p < 0.05). The Sirt1 was increased and caspase-3 was decreased in the Aged+Res group (p < 0.05). These results indicate that resveratrol can delay ovarian aging, probably by reducing oxidative damage and increasing Sirt1.

Growth hormone improved oxidative stress in follicle fluid by influencing Nrf2/Keap1 expression in women of advanced age undergoing IVF.

OBJECTIVES: To investigate whether growth hormone (GH) can improve oxidative stress (OS) by affecting) /nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap1) in women of advanced age undergoing in vitro fertilization (IVF). METHODS: This retrospective study enrolled 141 patients, including 65 aged C patients (patients not treated with GH) and 76 aged GH patients (patients treated with GH). The outcomes included IVF-ET results, OS markers in follicle fluid (FF) and Nrf2 and Keap1 mRNA and protein expressions in granulosa cells (GCs). RESULTS: The results showed that GH improved the available blastocyst (p=.047) and implantation rate (p=.043) in women of advanced age undergoing IVF. The malondialdehyde (MDA) content of FF was significantly higher in the aged-C group than in the aged-GH group (p=.013). The antioxidant enzyme activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-px) and catalase (CAT) were significantly lower in the aged-C group than in the aged-GH group (p= .000, p= .049, p= .012 respectively). Nrf2 mRNA and protein expression was significantly higher and Keap1 mRNA and protein expression was lower in the aged-GH group than in the aged-C group (p= .000, p= .000 respectively). CONCLUSIONS: The study showed that GH improved embryo quality and implantation rate and alleviated OS in FF, which may be related to Nrf2/Keap1.

Resveratrol protects human luteinised granulosa cells against hydrogen peroxide-induced oxidative injury through the Sirt1.

Granulosa cells (GCs) control follicular development and are important for female reproduction. Resveratrol (Res) was considered as an antioxidant and Sirt1 inducer. Hydrogen peroxide (H2O2) is the classical reagent to study oxidative stress. The study was conducted to investigate the role of Res against H2O2 in human luteinised granulosa cells (LGCs). The LGCs in the H2O2 group were treated with 100µmol/L H2O2 for 24h. The LGCs in the Res group were treated with 50µmol/L Res for 2h, followed by H2O2. The LGCs in the Sirt1 blockage group were treated with 2.5µmol/L EX527+50µmol/L Res for 2h, followed by H2O2. Results showed that Res significantly increased LGCs viability in H2O2-induced LGCs. The apoptotic rate and ROS in the H2O2 group was higher and the antioxidant enzyme activity was lower compared with other groups. Following the Res, the apoptotic rate and ROS level were reduced and the antioxidant enzyme activity were increased. In the Res blockage group, no significant alterations in the cell apoptosis, ROS and antioxidant enzyme activity were observed compared with the H2O2 group. The Res group had a Caspase-3 downregulation and Sirt1 upregulation compared with the other groups. In conclusion, Res had a protective effect against the H2O2-induced LGCs, and the mechanism may be associated with Sirt1.

The protective effects of resveratrol pretreatment in cyclophosphamide-induced rat ovarian injury: an vivo study.

OBJECTIVES: To explore whether resveratrol (Res) pretreatment could exert a protective effect on cyclophosphamide (Cy) induced ovarian toxicity in a rat model. METHODS: Twenty-four female 7-week old Sprague-Dawley rats were randomly divided into four groups: Con, administered with vehicle solutions; Cy, treated with Cy; Res + Cy, treated with Cy + Res combined; Res, treated with Res. After 21 d of treatments, the rats were euthanized and blood samples were collected to evaluate the levels of anti-Müllerian hormone (AMH). The Ovaries were processed for immunohistochemical and western blotting. RESULTS: Cy-treat caused the decrease of body weights and ovarian weight. AMH was lower in Cy group, whereas AMH levels were similar among other groups. Histomorphology showed a large number of primordial follicles were activated in Cy groups, whereas the primordial follicles were inhibited in the Res and Res + Cy groups. The expressions of Sirt1, Foxo3a were up-regulated and p53, Caspase-3, and Bax were down-regulated in Res + Cy and Res groups (p < .05). CONCLUSIONS: Res can prevent the primordial follicle activation and decrease apoptosis induced by Cy. Res may be an effective protection for ovarian function during chemotherapy, which means a new nonsurgical application for protection of ovarian reserve.

The protective effects of pretreatment with resveratrol in cyclophosphamide-induced rat ovarian granulosa cell injury: In vitro study.

Cyclophosphamide (Cy), a chemotherapeutic agent, is widely used to treat tumoursand is also associated with premature ovarian insufficiency. 4-Hydroperoxycyclophosphamide (4-HC), an active metabolite of Cy, was used for in vitro experiments. Granulosa cells (GCs) are crucial for maintaining follicle development and are also used in reproductive toxicity research in vitro. Resveratrol (Res), a polyphenolic compound, exhibits multiple effects in cells and animal models. To date, whether Res pretreatment has a protective effect on GCs induced by Cy remains unclear. This was an in vitro study, and primary cultures of rat GCs were used. Rat GCs were treated with 4-HC alone, Res + 4-HC or Res + 4-HC + EX527, and GCs survival rates, oxidative stress levels, apoptosis rates and related Sirt1 pathway proteins were evaluated. We demonstrated that 4-HC caused GC damage by increasing oxidative stress, autophagy and apoptosis. Res pretreatment improved 4-HC-induced GC damage by increasing Sirt1 expression, reducing oxidative stress levels and decreasing Beclin1, LC3B, Bax and Caspase-3 levels. Importantly, the addition of EX527, which is a selective inhibitor of Sirt1, reversed the protective effect of Res pretreatment, indicating that Sirt1 may be an important mediator of the protective effect of Res. Taken together, we demonstrated that Res may be a potential drug to improve fertility preservation for patients undergoing chemotherapy.

[Effects of males' age on sperm apoptosis and DNA integrity].

OBJECTIVE: To explore the correlation of males'age with sperm apoptosis, sperm DNA integrity and other seminal parameters. METHODS: We collected 104 semen samples and divided them into three groups according to the males' age: <35 yr (n = 43), 35 -39 yr (n = 31), and > or = 40 yr (n = 30). Based on the WHO Laboratory Manual (4th ed), we detected the seminal parameters, calculated the percentage of apoptotic sperm by flow cytometry (FCM), determined sperm DNA integrity by Acridine orange staining, and compared the results among the three groups. RESULTS: There were no statistically significant differences among the < 35 yr, 35 -39 yr and > or = 40 yr groups in semen volume ([2.87 +/- 0.89] ml vs [2.98 +/- 1.09] ml vs [2.65 +/- 0.95] ml), sperm concentration ([60.40 +/- 25.43] x 10(6)/ml vs [69.74 +/- 28.33] x 10(6)/ml vs [55.97 +/- 27.22] x 10(6)/ml) (P>0.05). The percentage of progressively motile sperm was significantly lower in the > or = 40 yr ([39.00 +/- 8.35 %) than in the <35 and 35 -39 yr groups ([48.73 +/- 9.89]% and [45.65 +/- 10.55]%) (P<.0.1), and so was the percentage of morphologically normal sperm in the > or = 40 yr than in the < 35 yr group ([11.11 +/- 8.26]% vs [16.43 +/- 8.75 ]%, P<0.01). The percentage of apoptotic sperm was markedly higher in the > or = 40 yr than in the <35 yr group ([11.82 +/- 5.77]% vs [7.04 +/- 3.50]%, P<0.01), while the sperm DNA integrity significantly reduced in the > or = 40 yr group ([75.52 +/- 10.60]%) as compared with the <35 yr ([86.55 +/- 5.60])% and 35 -39 yr group ( [81.39 +/- 8.94]%) (P<0.01). The males' age was correlated positively with the rate of sperm apoptosis (P<0.01), and negatively with sperm DNA integrity and the percentage of progressively motile sperm (P<0.01). CONCLUSION: The advance in males' age increases sperm apoptosis and reduces sperm progressive motility, normal morphology and DNA integrity.

[Influence of male age on the outcome of conventional IVF-ET].

OBJECTIVE: To study the influence of male age on the outcome of conventional IVF-ET. METHODS: Based on male age, 170 couples undergoing conventional IVF-ET were divided into three groups: <35 yr (n = 60), 35 -39 yr (n = 77) and > or = 40 yr (n = 33). We observed the rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy and abortion in different groups. RESULTS: There were no significant differences among the three groups in semen volume ([3.10 +/- 1.22] ml vs [2.84 +/- 1.05] ml vs [2.80 +/- 0.79] ml), sperm concentration ([54.23 +/- 26.07] x 10(6)/ml vs [60.27 +/- 24.80] x 10(6)/ml vs [60.21 +/- 27.42] x 10(6)/ml) and sperm viability ([53.93 +/- 13.25]% vs [56.10 +/- 16.58]% vs [51.82 +/- 15.45]%) (P>0.05). The men of the > or = 40 yr group showed a significantly lower percentage of grade a + b sperm ([40.97 +/- 11.91]%) than those of the <35 and 35 - 39 yr groups ([48.47 +/- 11.78]% and [46.84 +/- 13.51]%) (P<0.05), and morphologically normal sperm ([11.76 +/- 5.97]%) than those of the <35 yr group ([15.25 +/- 6.94]% (P<0.05). The rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy were 81.52%, 82.61%, 52.33%, 18.06% and 33.33% in the > or = 40 yr group, with no significant differences from those of the <35 yr group (83.18%, 82.68%, 56.99%, 22.40% and 40.00%) and the 35 - 39 yr group (78.78%, 80.66%, 55.01%, 21.74% and 38.96%) (P>0.05), while the abortion rate was markedly increased in the > or = 40 yr group as compared with the <35 yr group (36.36% vs 8.33%, P>0.05). CONCLUSION: Increasing male age is related with decreasing percentages of progressively motile sperm and morphologically normal sperm, but not obviously with the rates of fertilization, good quality embryo, implantation, pregnancy and abortion.

Molecular cloning of a novel rat gene Tsarg1, a member of the DnaJ/HSP40 protein family.

Beginning with a mouse gene mTSARG3, which was related to apoptosis of spermatogenic cells, bioinformatics was applied and a predicted novel rat gene full-length cDNA sequence was attained. Gene-specific primers were designed for PCR in rat testis cDNA library. A new gene Tsarg1 (GenBank Accession No. AY380804) was cloned, which is related to apoptosis in rat spermatogenic cells. The gene whose full cDNA length is 1176 bp containing 8 exons and 7 introns is located in rat chromosome 1q32-1q33, which encoded a protein containing 316 amino acid residues and being a new member of HSP40 protein family since the sequence contains the highly conserved J domain, which is present in all DnaJ-like proteins and is supported to have a critical role in DnaJ-DnaK protein-protein interactions. The results of RT-PCR and Northern blot analysis showed that Tsarg1 was specifically expressed in rat testis, which probably inhibits rat testis spermatogenic cell apoptosis.

Molecular cloning of MSRG-11 gene related to apoptosis of mouse spermatogenic cells.

Beginning with a new contig of the expressed sequence tags (Mm.63892) obtained by comparing testis libraries with other tissue and cell line libraries using the digital differential display program, we cloned a new gene which is related to the apoptosis of mouse spermatogenic cells using the Genscan program and polymerase chain reaction (PCR) technology. The sequence data have been submitted to the GenBank database under accession number AY747687. The full cDNA length is 1074 bp, and the gene with 7 exons and 6 introns is located in mouse chromosome 1 H5. The protein is recognized as a new member of calmodulin (CaM) binding protein family because the sequence contains three short calmodulin-binding motifs containing conserved Ile and Gln residues (IQ motif) and is considered to play a critical role in interactions of IQ motif-containing proteins with CaM proteins. The putative protein encoded by this gene has 192 amino acid residues with a theoretical molecular mass of 23.7 kDa and a calculated isoelectric point of 9.71. The sequence shares no significant homology with any known protein in databases. RT-PCR and Northern blot analyses revealed that 1.3 kb MSRG-11 transcript was strongly expressed in adult mouse testis but weakly expressed in the spleen and thymus. The MSRG-11 gene was expressed at various levels, faintly at two weeks postpartum and strongly from three weeks postpartum in adult testes. The green fluorescence produced by pEGFP-C2/MSRG-11 was detected in the cytoplasm of COS7 cells 24 h post-transfection. The pcDNA3.1(?-)/MSRG-11 plasmid was constructed and introduced into COS7 cells using Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, USA). MSRG-11 can accelerate COS7 cell apoptosis, which suggests that this gene may play an important role in the development of mouse testes and is a candidate gene of testis-specific apoptosis. Based on these observations, it was considered that we cloned a new gene which probably accelerates spermatogenetic cell apoptosis in mouse.

[Molecular cloning and characterization analysis of HPESCRG1, a novel gene expressed specifically in human embryonic stem cell].

OBJECTIVE: To clone a novel gene expressed specifically in human embryonic stem cell and to analyze its characteristics. METHODS: Based on an expression sequence tags(EST) CF948547 which expressed specifically in human embryonic stem cell, the full-length cDNA sequence of a novel gene was cloned by using bioinformatic and molecular biological technique. Its expression profile was analyzed by reverse transcription-polymerase chain reaction(RT-PCR), and subcellular location was determined by enhanced green fluorescent protein (EGFP) eukaryotic expression system. RESULTS: A novel gene HPESCRG1(homo sapiens pluripotent embryonic stem cell-related gene) was cloned successfully. Its GenBank accession number was AY283672. Its cDNA length was 1395 bp. It comprised 9 exons and 8 introns, and its opening reading frame was 250-1146 bp. Its chromosomal mapping was located in 3q13.13, and the putative protein contained 297 amino acids. The theoretical molecular weight of the putative protein was 33 784 and the isoelectric point was 9.35. The protein primary structure of this gene contained a SAP motif and it was subcellularly located in nuclei. Expression analysis showed that this gene was expressed specifically in human ES cells, but not expressed in the adult human tissues, the multiple tissues of embryo aborted in over 5 months' pregnancy, the differentiated cells of HESC-1, and the human mesenchymal stem cells (hMSCs) and human embryonic fibrocytes (hEFCs). CONCLUSION: HPESCRG1 was found to be a novel gene expressed specifically in human ES cell, which might be related to self-renewal of human ES cell and maintaining its undifferentiated state.