Search

To explore whether resveratrol (Res) pretreatment could exert a protective effect on cyclophosphamide (Cy) induced ovarian toxicity in a rat model.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.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 (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.

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 100 μmol/L H2O2 for 24 h. The LGCs in the Res group were treated with 50 μmol/L Res for 2 h, followed by H2O2. The LGCs in the Sirt1 blockage group were treated with 2.5 μmol/L EX527 + 50 μmol/L Res for 2 h, 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.

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 pro- duced 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.

To explore the correlation of males'age with sperm apoptosis, sperm DNA integrity and other seminal parameters.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), andor = 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.There were no statistically significant differences among the35 yr, 35 -39 yr andor = 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) (P0.05). The percentage of progressively motile sperm was significantly lower in theor = 40 yr ([39.00 +/- 8.35 %) than in the35 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 theor = 40 yr than in the35 yr group ([11.11 +/- 8.26]% vs [16.43 +/- 8.75 ]%, P0.01). The percentage of apoptotic sperm was markedly higher in theor = 40 yr than in the35 yr group ([11.82 +/- 5.77]% vs [7.04 +/- 3.50]%, P0.01), while the sperm DNA integrity significantly reduced in theor = 40 yr group ([75.52 +/- 10.60]%) as compared with the35 yr ([86.55 +/- 5.60])% and 35 -39 yr group ( [81.39 +/- 8.94]%) (P0.01). The males' age was correlated positively with the rate of sperm apoptosis (P0.01), and negatively with sperm DNA integrity and the percentage of progressively motile sperm (P0.01).The advance in males' age increases sperm apoptosis and reduces sperm progressive motility, normal morphology and DNA integrity.

To study the influence of male age on the outcome of conventional IVF-ET.Based on male age, 170 couples undergoing conventional IVF-ET were divided into three groups:35 yr (n = 60), 35 -39 yr (n = 77) andor = 40 yr (n = 33). We observed the rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy and abortion in different groups.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]%) (P0.05). The men of theor = 40 yr group showed a significantly lower percentage of grade a + b sperm ([40.97 +/- 11.91]%) than those of the35 and 35 - 39 yr groups ([48.47 +/- 11.78]% and [46.84 +/- 13.51]%) (P0.05), and morphologically normal sperm ([11.76 +/- 5.97]%) than those of the35 yr group ([15.25 +/- 6.94]% (P0.05). The rates of fertilization, cleavage, good quality embryo, implantation, clinical pregnancy were 81.52%, 82.61%, 52.33%, 18.06% and 33.33% in theor = 40 yr group, with no significant differences from those of the35 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%) (P0.05), while the abortion rate was markedly increased in theor = 40 yr group as compared with the35 yr group (36.36% vs 8.33%, P0.05).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.

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.

To clone a novel gene expressed specifically in human embryonic stem cell and to analyze its characteristics.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.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).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.