The porcine Gpr3 gene: molecular cloning, characterization and expression level in tissues and cumulus–oocyte complexes during in vitro maturation

G protein-coupled receptor 3 (Gpr3) is a member of G protein-coupled receptor rhodopsin family, which is present throughout the follicle within the ovary and functions as a critical factor for the maintenance of meiotic prophase arrest in oocytes by a Gs protein-mediated pathway. In the current paper, attempts were made to clone and characterize a gene encoding Gpr3 from pigs and investigate its expression pattern in tissues and the whole cumulus-oocyte complexes (COCs) in vitro maturation (IVM). Rapid amplification of cDNA ends and RT-PCR gave rise to the full sequence of Gpr3 gene with its length being 2101?bp nucleotides, including an open reading frame of 993?bp, encoding a 331 amino acid polypeptide with the molecular weight of 35.2?kDa. Homology search and sequence multi-alignment demonstrated that the putative porcine Gpr3 protein sequence shared a high identity with other animal Gpr3 orthologs, including several highly conservative motifs and amino acids. Real-time PCR analysis showed that the Gpr3 gene was expressed in tissues of cerebrum, cerebellum, hypothalamus, pituitary, ovary, oviduct, uterus, heart, liver, spleen, lung, kidney, muscle, fat, testis, thymus and granulosa cell, oocyte and COCs at different expression levels. The expression levels of this gene in oocyte, uterus, liver, fat, pituitary and brain were higher than that in other tissues. Interestingly, the mRNA and protein levels of Gpr3 in the whole COCs were down-regulated, and its mRNA expression levels were significantly and negatively correlated with the degrees of cumulus expansion (r?=?-0.937, P?相似文献   

Gene expression profiles of cumulus cell oocyte complexes during ovulation reveal cumulus cells express neuronal and immune-related genes: does this expand their role in the ovulation process?

Ovulation is a complex process initiated by the preovulatory LH surge, characterized by cumulus oocyte complex (COC) expansion and completed by the release of a mature oocyte. Although many ovarian genes that impact ovulation have been identified, we hypothesized that genes selectively expressed in COCs would be overlooked by approaches using whole ovary or granulosa cell samples. RNA isolated from COCs collected from preovulatory follicles of equine chorionic gonadotropin (CG) primed mice and at selected times after human CG treatment was subjected to microarray analyses and results confirmed by RT-PCR analyses, Western blotting, and immunofluorescent studies. A remarkable number of genes were up-regulated in COCs including Areg, Ereg, and Btc. Several genes selectively expressed in cumulus cells compared with granulosa cells were related to neuronal (Mbp, Tnc, Nts) or immune (Alcam, Pdcd1, Cd34, Cd52, and Cxcr4) cell function. In addition to Sfrp2, other members of the Wnt/Fzd family (Sfrp4, Fdz1 and Fdz2) were expressed in COCs. Thus, there is a cumulus cell-specific, terminal differentiation process. Furthermore, immunofluorescent analyses documented that cumulus cells are highly mitotic for 4-8 h after human CG and then cease dividing in association with reduced levels of Ccnd2 mRNA. Other down-regulated genes included: Cyp19a1, Fshr, Inhb, and the oocyte factors Zp1-3 and Gja4. In summary, the vast number of matrix, neuronal, and especially immune cell-related genes identified by the gene- profiling data of COCs constitutes strong and novel evidence that cumulus cells possess a repertoire of immune functions that could be far greater than simply mediating an inflammatory-like response.     

Effect of temporary meiosis block during prematuration of bovine cumulus–oocyte complexes on pregnancy rates in a commercial setting for in vitro embryo production

Ovum pick up (OPU) associated with in vitro production (IVP) of embryos has been shown as an important tool in cattle breeding to increase the number of descendants from animals of high genetic value. In herds maintained distant from the laboratory, collecting cumulus–oocyte complexes (COCs) and transporting them to the laboratory may take several hours and decrease COCs viability, representing a challenge for commercial settings. In this study, a prematuration culture to induce temporary meiosis block was evaluated in a commercial scale IVP setting as a strategy to transport bovine OPU-derived COCs from Nelore and Brangus donors. Effects on embryo yield and pregnancy rates were assessed. Viable COCs from each donor were destined to one of the experimental groups (control, blocks 1 and 2). Control group COCs were placed in cryotubes with 1 mL TCM199–HEPES. In block groups (1 and 2), COCs were placed in cryotubes with 300 μL TCM 199 + 12 μM butyrolactone I (block medium). All groups were gassed and kept in a thermos bottle for 4 hours at 36 °C. Next, COCs in the control group were transferred to IVM medium and block 1 group to block medium, and cultured for 22 hours and 15 hours, respectively, at 38.5 °C and 5% CO₂ in air. Block 2 COCs were kept in the cryotubes and in the thermos bottle for another 15 hours at 36 °C to simulate long-term transport conditions. After meiosis block in prematuration culture, blocks 1 and 2 COCs were matured in vitro for 22 hours as for the control group. After IVM, COCs in all groups were submitted to IVF and IVC, and blastocyst rates were evaluated on day 7. Embryos were transferred and pregnancy rates evaluated at 60 days of gestation. The mean total number of COCs retrieved by OPU did not differ between Nelore and Brangus donors (16.8 and 17.2, respectively, P > 0.05), but Nelore donors produced more viable COCs than Brangus (10.1 and 7.6, respectively, P < 0.05) and more embryos/cow (3.8 and 2.7, respectively, P < 0.05). Blastocyst rates were similar for control (40.2% and 36.7%), block 1 (37.3% and 34.5%), and block 2 groups (34.7% and 33.6%) for Nelore and Brangus cattle, respectively (P > 0.05). Pregnancy rates did not differ regardless of breed or treatment (36.7%, P > 0.05). In conclusion, temporary meiosis block during prematuration culture did not affect embryo development or pregnancy rates; therefore, this strategy may be used to transport bovine COCs in a commercial IVP setting.     

Effects of RXRα gene silencing on the porcine adipocyte differentiation in vitro

The elucidation of the molecular mechanism of porcine adipocyte differentiation is important not only to domestic animal industry, but also to medical science because of the similarities in the trait of body lipid deposition between human and pigs. In this study, we investigated the roles of RXRα in porcine (Sus scrofa) adipocyte differentiation by using RNAi method and primary cultured porcine adipocytes. With the transfection of RXRα-siRNA, the gene and protein expressions of RXRα were successfully inhibited in differentiating porcine adipocytes. By the RXRα gene silencing, the adipocyte differentiation, as characterized by lipid accumulation and GPDH activity, was suppressed. Furthermore, in association with the decrease in RXRα gene expression level, PPARγ and C/EBPα genes expression level also decreased, suggesting that RXRα is involved in their expression. Thus, our study clearly demonstrated the pivotal roles of RXRα in porcine adipocyte differentiation.     

Follicle stimulating hormone and anti-Müllerian hormone per oocyte in predicting in vitro fertilization pregnancy in high responders: a cohort study

Background

Follicle stimulating hormone (FSH) and Anti-Müllerian hormone (AMH) are utilized to differentiate between good and poor response to controlled ovarian hyperstimulation. Their respective roles in defining functional ovarian reserve remain, however, to be elucidated. To better understand those we investigated AMH and FSH per oocyte retrieved (AMHo and FSHo).

Methodology/Principal Findings

Three-hundred and ninety-six women, undergoing first in vitro fertilization cycles, were retrospectively evaluated. Women with oocyte yields >75^th percentile for their age group were identified as high responders. In a series of logistic regression analyses, AMHo and FSHo levels were then evaluated as predictive factors for pregnancy potential in high responders. Patients presented with a mean age of 38.0±5.0 years, mean baseline FSH of 11.8±8.7 mIU/mL and mean AMH of 1.6±2.1 ng/mL. Those 88 women, who qualified as high responders, showed mean FSH of 9.7±6.5 mIU/mL, AMH of 3.1±3.1 ng/mL and oocyte yields of 15.8±7.1. Baseline FSH and AMH did not predict pregnancy in high responders. However, a statistically significant association between FSHo and pregnancy was observed in high responders, both after univariate regression (p = 0.02) and when adjusted for age, percentage of usable embryos, and number of embryos transferred (p = 0.03). Rate of useable embryos also significantly affected pregnancy outcome independently of FSHo (p = 0.01). AMHo was also associated with clinical pregnancy chances in high responders (p = 0.03) and remained significant when adjusted for usable embryos and number of embryos transferred (p = 0.04).

Conclusions

AMHo and FSHo are predictive of pregnancy potential in high responders, but likely reflect different responsibilities in recruitment and maturation of growing follicle cohorts.     

Mapping, expression and regulation of the TRα gene in porcine adipose tissue

Thyroid hormone receptors (TR) are members of the nuclear receptor superfamily. There are at least two TR isoforms, TRα and TRβ. The TRα isoform plays a critical role in mediating the action of thyroid hormone in adipose tissue. We mapped the porcine TRα gene to chromosome 12 p11-p13, by using the ImpRH panel. We examined tissue-localization of TRα and determined expression patterns of TRα in porcine adipose tissue with quantitative real-time PCR. TRα was expressed in all tissues, including heart, liver, spleen, stomach, pancreas, brain, small intestine, skeletal muscle, and subcutaneous adipose tissue. In the adipose tissue, the expression of TRα decreased postnatally. Compared to Yorkshire pigs, Jinhua pigs had significantly lower expression levels of TRα gene in the subcutaneous fat tissue. The expression levels of β2-AR, HSL and ATGL were also significantly lower in Jinhua pigs than in Yorkshire pigs. However, no significant differences in PPARγ and SREBP-1C expression levels were found between Jinhua and Yorkshire pigs. Incubation of porcine adipose tissue explants with high doses of isoproterenol (100 and 1000 nM) significantly increased the expression levels of TRα. We conclude that there is considerable evidence that TRα plays an important role in fat deposition in porcine adipose tissue.     

Cumulus cells during in vitro fertilization and oocyte vitrification in sheep: Remove,maintain or add?

The objective was to evaluate the developmental competence of immature and matured ovine oocytes after removing, maintaining or adding cumulus cells (CC) associated to vitrification by Cryotop method. Three experiments were performed involving 3,144 oocytes. In Experiment 1, CC were removed from immature, matured or fertilized oocytes subjected to in vitro embryo production. In Experiment 2, oocytes were vitrified either in MI or MII stage with or without CC, while a control group with CC remained unvitrified. In Experiment 3, oocytes partially denuded from CC were vitrified either in MI or MII stage, and a co-culture of fresh CC was added or not soon after warming to complete in vitro maturation (IVM) and in vitro fertilization (IVF), or IVF, respectively, while a control group remained unvitrified. In Experiment 1, the cleavage rate, development rate on Day 6 and blastocyst rate on Day 8 were improved when CC were maintained until the end of IVF (P < 0.05). In Experiment 2, vitrification of oocytes with enclosed CC showed a tendency to increase cleavage (P = 0.06) and improved blastocyst rate (P < 0.05). In Experiment 3, adding CC as co-culture after vitrification-warming tended to improve cleavage rate (P = 0.06) and increased hatching rate (P < 0.05). Regarding oocyte stage, vitrification of in vitro matured oocytes resulted in greater developmental competence than immature stages (P < 0.05). In conclusion, CC seems to have a relevant role for in vitro embryo development in either fresh or vitrified oocytes.     

Changes in intracellular location of DNA polymerase-α during oocyte maturation of the toad,bufo bufo japonicus

Intracellular location of DNA polymerase-α during oocyte maturation of the toad was studied. Quantitative and qualitative changes in the activity of DNA polymerase-α were not observed during the maturational process. Nearly all activity was found in isolated germinal vesicles from full grown oocytes and in enucleated mature oocytes. The cytoplasmic DNA polymerase-α of mature oocytes was recovered at buoyant densities equivalent to microsome by isopycnic centrifugation. These findings indicate that DNA polymerase-α in the germinal vesicle is released into the cytoplasm and binds to the endoplasmic reticulum when the germinal vesicle breaks down.     

Down-regulation of thyroid hormone receptor β1 gene expression in gastric cancer involves promoter methylation

Hypermethylation has been shown in the promoter region of the thyroid hormone receptor β1 (TRβ1) gene in several human tumors. However, its role in gastric cancer formation is still unclear. In the study, we analyzed mRNA expression of TRβ1 gene using real-time quantitative PCR (qPCR). A quantitative methylation-specific PCR (Q-MSP) assay was used to determine the methylation status of the TRβ1 gene promoter region in 46 pair-matched gastric neoplastic and adjacent non-neoplastic tissues. The results showed that TRβ1 mRNA expression was significantly reduced in gastric cancer specimens. The methylation of promoter of TRβ1 gene in gastric cancer tissues was significantly higher than in adjacent normal tissues. Promoter hypermethylation of the TRβ1 gene correlated with tumor infiltration, lymph node metastasis, and distant metastasis, but it was not associated with other clinicopathological characteristics. We treated gastric cancer cell lines MKN-45, MKN-28, SGC-7901, NCI-N87, and SNU-1 with 5-Aza-2-deoxycytidine (5-Aza-dC). The results showed the expression of TRβ1 mRNA was increased in MKN-45, MKN-28, SGC-7901, but not increased in NCI-N87 and SNU-1. These results suggest that the TRβ1 gene plays important roles in the development of gastric cancer partially through epigenetic mechanisms.     

Oocyte regulation of anti-Müllerian hormone expression in granulosa cells during ovarian follicle development in mice

In the ovarian follicle, anti-Müllerian hormone (Amh) mRNA is expressed in granulosa cells from primary to preovulatory stages but becomes restricted to cumulus cells following antrum formation. Anti-Müllerian hormone regulates follicle development by attenuating the effects of follicle stimulating hormone on follicle growth and inhibiting primordial follicle recruitment. To examine the role of the oocyte in regulating granulosa cell Amh expression in the mouse, isolated oocytes and granulosa cells were co-cultured and Amh mRNA levels were analysed by real-time RT-PCR. Expression in freshly isolated granulosa cells increased with preantral follicle development but was low in the cumulus and virtually absent in the mural granulosa cells of preovulatory follicles. When preantral granulosa cells were co-cultured with oocytes from early preantral, late preantral or preovulatory follicles, and when oocytes from preovulatory follicles were co-cultured with cumulus granulosa cells, Amh expression was increased at least 2-fold compared with granulosa cells cultured alone. With oocytes from preantral but not preovulatory follicles, this was a short-range effect only observed with granulosa cells in close apposition to oocytes. We conclude that stage-specific oocyte regulation of Amh expression may play a role in intra- and inter-follicular coordination of follicle development.