Reproduction

Carroll, John

doi: 10.1530/rep-07-0521pmid: N/A

Livera, Gabriel; Petre-Lazar, Béatrice; Guerquin, Marie-Justine; Trautmann, Emilie; Coffigny, Hervé; Habert, René

doi: 10.1530/REP-07-0054pmid: 18159078

Female fertility in mammals is determined by the pool of primordial follicles and low doses of radiation induce a major lossof primordial follicles in the ovary. We investigated the expression of p53 and its homologues, p63 and p73, in the normaland irradiated neonatal ovary. p63 was the only member of the p53 family detected in oocyte nucleus. No p63 transcripts orprotein were detected in the early foetal ovary. p63 production began in late pachytene-stage oocytes and peaked in diploteneoocytes in mice and humans. The production of p63 was correlated with meiotic DNA double-strand break repair. Only transactivation(TA) isoforms were present in the ovary, with TAp63α by far the most abundant in terms of mRNA and protein levels. Completep63 null mutation did not affect normal ovary development. Irradiation rapidly triggered p63 phosphorylation. p63 null mutationprevented the cleavage of caspases-9 and -3 and the follicle loss induced by ionising radiation. Thus, our results evidencethat irradiation-induced depletion of the primordial follicle pool results from the activation of p63 in quiescent oocytes.

Livera, Gabriel; Petre-Lazar, Béatrice; Guerquin, Marie-Justine; Trautmann, Emilie; Coffigny, Hervé; Habert, René

doi: 10.1530/rep-07-0054pmid: 18159078

Female fertility in mammals is determined by the pool of primordial follicles and low doses of radiation induce a major loss of primordial follicles in the ovary. We investigated the expression of p53 and its homologues, p63 and p73, in the normal and irradiated neonatal ovary. p63 was the only member of the p53 family detected in oocyte nucleus. No p63 transcripts or protein were detected in the early foetal ovary. p63 production began in late pachytene-stage oocytes and peaked in diplotene oocytes in mice and humans. The production of p63 was correlated with meiotic DNA double-strand break repair. Only transactivation (TA) isoforms were present in the ovary, with TAp63α by far the most abundant in terms of mRNA and protein levels. Complete p63 null mutation did not affect normal ovary development. Irradiation rapidly triggered p63 phosphorylation. p63 null mutation prevented the cleavage of caspases-9 and -3 and the follicle loss induced by ionising radiation. Thus, our results evidence that irradiation-induced depletion of the primordial follicle pool results from the activation of p63 in quiescent oocytes.

Du, Y; Pribenszky, C S; Molnár, M; Zhang, X; Yang, H; Kuwayama, M; Pedersen, A M; Villemoes, K; Bolund, L; Vajta, G

doi: 10.1530/REP-07-0362pmid: 18159079

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcinein vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHPtreatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure)for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressureparameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4±2.4%) or 130 (13.1±3.2%) min recovery, when comparedwith the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperatureat HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performedat 37 °C or 25 °C. Oocytes pressurized at 37 °C had a significantly higher blastocyst (14.1±1.4%) rate than those treatedat 25 °C (5.3±1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrifiedpig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence atdifferent developmental stages in different species including humans.

Du, Y; Pribenszky, C S; Molnár, M; Zhang, X; Yang, H; Kuwayama, M; Pedersen, A M; Villemoes, K; Bolund, L; Vajta, G

doi: 10.1530/rep-07-0362pmid: 18159079

The purpose of the present study was to improve cryotolerance using high hydrostatic pressure (HHP) pretreatment of porcine in vitro matured (IVM) oocytes, to facilitate their further developmental competence after parthenogenetic activation. A total of 1668 porcine IVM oocytes were used in our present study. The pressure tolerance and optimal duration of recovery after HHP treatment were determined. Oocytes were treated with either 20 or 40 MPa (200 and 400 times greater than atmospheric pressure) for 60 min, with an interval of 10, 70, and 130 min between pressure treatment and subsequent vitrification under each pressure parameter. Oocytes from all vitrification groups had much lower developmental competence than fresh oocytes (P<0.01) measured as cleavage and blastocyst rates. However, significantly higher blastocyst rates (P<0.01) were obtained in the groups of 20 MPa pressure, with either 70 (11.4±2.4%) or 130 (13.1±3.2%) min recovery, when compared with the vitrification control group without HHP treatment where no blastocysts were obtained. The influence of temperature at HHP treatment on further embryo development was also investigated. Treatments of 20 MPa with 70 min recovery were performed at 37 °C or 25 °C. Oocytes pressurized at 37 °C had a significantly higher blastocyst (14.1±1.4%) rate than those treated at 25 °C (5.3±1.1%; P<0.01). Our results demonstrate that HHP pretreatment could considerably improve the developmental competence of vitrified pig in vitro matured (IVM) oocytes. The HHP pretreatment will be tested as a means to improve survival and developmental competence at different developmental stages in different species including humans.

Coy, Pilar ; Grullon, Luis; Canovas, Sebastian; Romar, Raquel; Matas, Carmen; Aviles, Manuel

doi: 10.1530/REP-07-0280pmid: 18159080

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion(ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence isavailable that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It wasthought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytesmatured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effectson ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP(P<0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF.In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZP or blockingof SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayedand produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization.When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed thatthe observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian speciesand can be imitated by the use of amine-reactive cross-linkers during IVF.

Coy, Pilar ; Grullon, Luis; Canovas, Sebastian; Romar, Raquel; Matas, Carmen; Aviles, Manuel

doi: 10.1530/rep-07-0280pmid: 18159080

One of the proposed mechanisms of polyspermy block is an increased resistance of the zona pellucida (ZP) to proteolytic digestion (ZP hardening) as a consequence of cortical granule exocytosis that occurs soon after fertilization. However, evidence is available that the zonae pellucidae of freshly ovulated pig and cow oocytes harden considerably before fertilization. It was thought that such pre-fertilization ZP hardening could be involved in the control of polyspermy, and its lack in the oocytes matured in vitro could be one of the reasons for the extremely high incidence of polyspermy in pig in vitro fertilization (IVF). To test this hypothesis, two different types of cross-linking reagents were employed and their effects on ZP hardening and IVF efficiency were examined. The sulfhydryl-reactive cross-linkers produced a slight hardening of ZP (P<0.001) of treated oocytes compared with control oocytes, and totally inhibited sperm penetration into pig oocytes after IVF. In the cow, sperm penetration into eggs was reduced to 10%. It is proposed that formation of disulfide bonds in ZP or blocking of SH groups in the oocyte plasma membrane proteins prevents sperm penetration. An amine-reactive cross-linker was then assayed and produced strong ZP hardening, increasing the incidence of monospermy in both pig and cow oocytes after fertilization. When the cross-linker concentration was optimized, a 45% improvement for pig IVF efficiency was reached. It is proposed that the observed physiological ZP hardening is a mechanism to control polyspermy, differentially affecting various mammalian species and can be imitated by the use of amine-reactive cross-linkers during IVF.

Thurston, Alexandra; Taylor, Jane; Gardner, John; Sinclair, Kevin D; Young, Lorraine E

doi: 10.1530/REP-07-0211pmid: 18159081

The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resultingin loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such asBeckwith–Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamicsof establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR wasused to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels betweenbiparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprintingof ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse andhuman, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of theH19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation ofthese imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined,monoallelic expression does not occur until after the blastocyst stage.

Thurston, Alexandra; Taylor, Jane; Gardner, John; Sinclair, Kevin D; Young, Lorraine E

doi: 10.1530/rep-07-0211pmid: 18159081

The preimplantation embryos of a range of mammals can be susceptible to disruptions in genomic imprinting mechanisms, resulting in loss of imprinting. Such disruptions can have developmental consequences involving foetal and placental growth such as Beckwith–Wiedemann syndrome in humans and large offspring syndrome in sheep. Our objective was to investigate the dynamics of establishing monoallelic expression of individual sheep imprinted genes post-fertilisation. Semi-quantitative RT-PCR was used to amplify cDNA from the sheep blastocyst, day 21 foetus and day 21 chorioallantois, to compare expression levels between biparental and parthenogenetic embryos in order to indicate allelic expression status. In common with other mammals, IGF2, PEG1 and PEG3 were paternally expressed in the day 21 conceptus, while H19, IGF2R, GRB10 and p57KIP were maternally expressed. Interestingly, GNAS was maternally expressed in the foetus, but paternally expressed in the chorioallantois at day 21. Overall, the imprinting of ovine GRB10 and IGF2R was comparable with mouse but not with human. Contrary to the trophoblast-restricted maternal expression in both mouse and human, SASH2 (sheep homologue of Mash2/HASH2) was expressed in the ovine foetus and was biallelically expressed in the chorioallantois. Differential methylation of the H19 CTCF III upstream region and IGF2R DMR2 in the chorioallantois revealed predominantly paternal and maternal methylation respectively, indicating conservation of these imprinting regulatory regions. In blastocysts, IGF2R, GRB10 and SASH2 were expressed biallelically, while the other genes were not detected. Thus, for the majority of ovine imprinted genes examined, monoallelic expression does not occur until after the blastocyst stage.

Ding, Tianbing; Song, Haengseok; Wang, Xiaohong; Khatua, Atanu; Paria, Bibhash C

doi: 10.1530/rep-07-0013pmid: 18159082

Blastocyst implantation occurs in the progesterone-primed uterus of hamsters, but not in mice where the progesterone-primed uterus requires estrogen influence. Leukemia inhibitory factor (Lif), an estrogen-regulated gene in mice, is an absolutely needed cytokine for uterine receptivity and implantation in this species. This study aimed to evaluate the importance of Lif ligand-receptor signaling during uterine receptivity and implantation in hamsters. We investigated whether or not the uterine expression patterns of Lif and its receptors, Lif-r and gp130, during the periimplantation period of pregnancy and its hormonal regulation in the ovariectomized hamster correlate with some of the vital phases of uterine changes during early pregnancy. Uterine Lif, Lif-r, and gp130 mRNA expressions were examined by Northern and in situ hybridization. During the uterine preparatory phase for implantation, Lif, Lif-r, and gp130 were expressed either in the gland, luminal epithelium or both. As the implantation process began, Lif expression was minimal, but Lif-r and gp130 extended to the decidual areas. This decidual expression of Lif-r and gp130 was not dependent on the presence of the embryo since these genes were expressed in the suture-induced deciduomata. We also observed that, while the uterine Lif was induced by estrogen, Lif-r and gp130 were induced by progesterone in ovariectomized hamsters. Additionally, we show that a Lif antibody when instilled intraluminally on day 3 of pregnancy reduced the number of implantation sites. Taken together, these data suggest that Lif signaling is important for uterine receptivity and implantation in hamsters.