The effect of M‐phase stage‐dependent kinase inhibitors on inositol 1,4,5‐trisphosphate receptor 1 (IP3R1) expression and localization in pig oocytes

At fertilization, inositol 1,4,5‐trisphosphate receptor type 1 (IP₃R1) has a crucial role in Ca²⁺ release in mammals. Expression levels, localization and phosphorylation of IP₃R1 are important for its function, but it still remains unclear which molecule(s) regulates IP₃R1 behavior in pig oocytes. We examined whether there was a difference in localization of IP₃R1 after in vitro or in vivo maturation of pig oocytes. In mouse oocytes, large clusters of IP₃R1 were formed in the cortex of the oocyte except in a ring‐shaped band of cortex adjacent to the spindle. However, no such clusters of IP₃R1 were observed in pig oocytes and there was no difference in its localization between in vitro and in vivo matured oocytes. We next tried to clarify which factor(s) regulates IP₃R1 localization, phosphorylation and expression using M‐phase stage‐dependent kinase inhibitors. Our results show that treatments with roscovitine (p34^cdc2 kinase inhibitor) or U0126 (mitogen‐activated protein kinase inhibitor) did not affect IP₃R1 expression or localization in pig oocytes, although the latter strongly inhibited phosphorylation. However, treatment with BI‐2536, an inhibitor of polo‐like kinase 1 (Plk1), dramatically decreased the expression level of IP₃R1 in pig oocytes in a dose‐dependent manner. From these results, it is suggested that Plk1 is involved in the regulation of IP₃R1 expression in pig oocytes.     

Phosphorylation of inositol 1,4,5-triphosphate receptor 1 during in vitro maturation of porcine oocytes

During fertilization in mammalian species, a sperm-induced intracellular Ca²⁺ signal ([Ca²⁺]_i) mediates both exit of meiosis and oocyte activation. Recently, we demonstrated in mouse oocytes that the phosphorylation levels of inositol 1,4,5 trisphosphate receptor type1 (IP₃R1), the channel responsible for Ca²⁺ release and oscillations during fertilization, changed during maturation and fertilization. Therefore, we examined the expression and phosphorylation of IP₃R1 during in vitro maturation of pig oocytes. Here, our present study shows that expression of IP₃R1 protein did not change during maturation, although the phosphorylation status of the receptor, specifically at an MPM-2 epitope, did. We found that while at the beginning of maturation IP₃R1 lacked MPM-2 immunoreactivity, it became MPM-2 reactive by 24 h and reached maximal reactivity by 36 h. Interestingly, the acquisition of MPM-2 reactivity coincided with the activation of p34^cdc2 kinase and mitogen-activated protein kinase (MAPK), which are involved in meiotic progression. Following completion of maturation, inactivation of MAPK by U0126 did not affect IP₃R1 phosphorylation, although inactivation of p34^cdc2 kinase by roscovitine dramatically reduced IP₃R1 phosphorylation. Neither inhibitor affected total expression of IP₃R1. Altogether, our results show that IP₃R1 undergoes dynamic phosphorylation during maturation and this might underlie the generation of oscillations at fertilization.     

Molecular mechanism of fertilization in the pig

At fertilization, the sperm triggers resumption from the arrest, extrusion of the second polar body and pronuclear formation, the events of which are collectively acknowledged as ‘oocyte activation’. In all species up to date, oocyte activation requires a fertilization‐associated increase in the intracellular concentration of calcium. Especially in mammals, the signal of intracellular calcium rise at fertilization consists of periodical rises, which are also referred to as calcium oscillations. Our recent results suggest that these calcium oscillations have an important role in not only oocyte activation but also development of mammals. Pigs are animals of great agricultural value and ones in which assisted reproductive techniques, including somatic cell nuclear transfer, to produce gene‐modified pigs. Although reconstructed embryos require artificial activation stimuli which mimic fertilization‐associated increase of intracellular calcium in the oocytes, it has been known that the developmental ability of the oocytes after artificial activation is low and the regimen seems to be required for improvement. Recently we focused on two molecules, phospholipase C zeta and inositol 1,4,5‐triphosphate receptor which have important roles in regulation of calcium oscillations during fertilization in mammals, including pigs. In this review, we will discuss the present status and future perspective of molecular mechanisms during fertilization in pigs.     

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L‐LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L‐LPS. Here, we stimulated pig fibroblasts by L‐LPS and then quantitatively measured gene and protein expression levels of two toll‐like receptors (TLRs), TLR2 and TLR4, by real‐time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up‐regulated within 24 h after L‐LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL‐6) and IL‐8 gene expressions were markedly up‐regulated by L‐LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L‐LPS stimulation, thereby inducing expression of cytokines.     

Sucrose assists selection of high‐quality oocytes in pigs

We investigated whether high‐quality in vitro matured (IVM) oocytes can be distinguished from poor ones based on the morphological changes after treatment with hyperosmotic medium containing 0.2 mol/L sucrose in pigs. We hypothesize that IVM oocytes maintaining round shape have higher quality than mis‐shapened oocytes following dehydration. Oocyte quality was verified by determining embryonic developmental competence using in vitro fertilization, nuclear transfer and parthenogenetic activation. In all cases, the round oocytes had greater (p < .05) developmental competence than that of mis‐shapened oocytes in terms of blastocyst rate and total cell number in blastocysts obtained after 6 days of in vitro culture. We also confirm that round aged oocytes are higher in quality than mis‐shapened aged oocytes. In an attempt to find out why high‐quality oocytes maintain a round shape whereas poorer oocytes become mis‐shapened following sucrose treatment, we examined the arrangement of actin microfilaments and microtubules. Abnormal organization of these cytoskeletal components was higher (p < .05) in mis‐shapened oocytes compared to round oocytes after 52 hr of IVM. In conclusion, sucrose treatment helps selection of high‐quality oocytes, including aged oocytes, in pigs. Abnormal cytoskeleton arrangements partly explain for low developmental competence of mis‐shapened oocytes.     

Roles of the zona pellucida and functional exposure of the sperm‐egg fusion factor ‘IZUMO’ during in vitro fertilization in pigs

The zona pellucida (ZP) is considered to play important roles in the prevention of polyspermy in mammalian oocytes. However, in pigs we have shown that the presence of the ZP accelerates sperm penetration into the ooplasm during in vitro fertilization (IVF). In the present study, we investigated the effects of the ZP on sperm binding, acrosomal status, and functional exposure of IZUMO, a critical factor involved in sperm‐egg fusion, during IVF in pigs. We evaluated the numbers and acrosomal statuses of sperm binding to the ZP and oolemma, and being present in the ZP and perivitelline space (PVS) using ZP‐intact and ZP‐free oocytes. More sperm bound to the ZP than to the oolemma. The average number of sperm present in the PVS was 0.44?0.51 per oocyte, and all sperm had lost their acrosomes. The proportion of sperm that were immunopositive for anti‐IZUMO antibody was significantly higher after they were passing or had passed through the ZP. Furthermore, addition of anti‐IZUMO antibody to the fertilization medium significantly inhibited the penetration of sperm into ZP‐free oocytes. These results suggest that, in pigs, the ZP induces the acrosome reaction, which is associated with the functional exposure of IZUMO, resulting in completion of fertilization.     

Comparison of opioid receptor binding in horse, guinea pig, and rat cerebral cortex and cerebellum

OBJECTIVE: To compare the density and binding characteristics of opioid receptor subtypes in horse, rat, and guinea pig cerebral cortex and cerebellum. STUDY DESIGN: Prospective receptor binding study. ANIMALS: Whole brains were obtained from four neurologically normal adult horses during necropsy. Rat and guinea pig brains were obtained commercially. METHODS: The cerebellum and cerebral cortex were dissected from each brain, and tissue homogenates prepared. A radioligand binding technique with the highly selective ligands [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE was used to identify the mu- (mu), kappa- (kappa) and delta- (delta) opioid receptors, respectively. Competitive binding assays were performed with these ligands and varying concentrations of one of multiple unlabeled ligands. RESULTS: While there were marked species differences in relative densities of opioid receptors, all radioligands interacted with their binding sites with high, nanomolar affinity in both the cerebral cortex and cerebellum. In the horse cerebral cortex, the percentages of total opioid binding sites for the mu-, kappa- and delta-receptors were 71%, 14% and 15%, respectively. In the rat and guinea pig cerebral cortex, the corresponding values were 56% mu-, 4% kappa- and 40% delta-receptors, and 25% mu-, 37% kappa- and 38% delta-receptors, respectively. In horse and guinea pig cerebellum, the binding was 37% mu-, 59% kappa- and 4% delta-receptors, and 15% mu-, 76% kappa- and 10% delta-receptors, respectively. For competitive analysis, all competitors of the mu-, kappa- and delta-receptors completely displaced [(3)H]-DAMGO, [(3)H]-U69593, and [(3)H]-DPDPE and had inhibitory constants in the nanomolar range. CONCLUSION AND CLINICAL RELEVANCE: Horses used in this study had a greater density of mu-receptors in the cerebral cortex compared with rats and guinea pigs but without further characterization of the functional role of these receptors it is impossible to determine the clinical significance of these data.     

Effect of L‐carnitine on maturation,cryo‐tolerance and embryo developmental competence of bovine oocytes

In this study, the effects of the addition of L‐carnitine in in vitro maturation (IVM) medium for bovine oocytes on their nuclear maturation and cryopreservation were investigated; they were matured in IVM medium supplemented with 0.0, 0.3, 0.6 and 1.2 mg/mL of L‐carnitine (control, 0.3, 0.6 and 1.2 groups, respectively) and some of them were vitrified by Cryotop. Moreover, the effects of L‐carnitine during in vitro fertilization (IVF) and in vitro culture (IVC) on the developmental potential and quality of IVF embryos were also examined. A significantly higher maturation rate of oocytes was obtained for 0.3 and 0.6 mg/mL groups compared with the control (P < 0.05). The blastocyst formation rate in the 0.6 group was significantly improved, whereas the rate in the 1.2 group was significantly decreased when compared with the control group (P < 0.05). No significant difference was found in embryo development between the control and the L‐carnitine group after oocyte vitrification. Supplementation of IVF and IVC media with L‐carnitine had no effect on development to the blastocyst stage of IVM oocytes treated with 0.6 mg/mL L‐carnitine. In conclusion, the supplementation of L‐carnitine during IVM of bovine oocytes improved their nuclear maturation and subsequent embryo development after IVF, but when they were vitrified the improving effects were neutralized.     

Nucleotide sequence polymorphisms of beta1‐, beta2‐, and beta3‐adrenergic receptor genes on Jinhua,Meishan, Duroc and Landrace pigs

The full amino acid coding sequences of adrenergic receptor genes beta1, beta2, and beta3 (ADRB1, ADRB2, and ADRB3)were determined for Jinhua, Meishan, Duroc and Landrace pigs. Non‐synonymous substitution of Arg458Pro was found in the porcine ADRB1 gene, resulting in a 469 amino acid sequence. Continuous substitutions of Asn29Asp and Glu30Gln were found in the porcine ADRB2 gene, resulting in a 418 amino acid sequence. Additionally, a Lys30 polymorphism of the ADRB2 gene was found in the Jinhua pigs. There were three non‐synonymous substitutions of Asn24Thr, Arg264Gln and Asn398Asp on the porcine ADRB3 gene. A thymine insertion in the ADRB3 gene, resulting in a protein with two fewer amino acids, was found in the Jinhua and Meishan pigs. To assess the effect of ADRB polymorphisms on porcine subcutaneous fat layer thickness, we calculated the genetic frequency of the variants in fatty and lean groups, each consisting of 24 pigs that were crossbreds of Duroc and Jinhua pigs. The effect of the ADRB3 gene polymorphism was not evaluated, because there was insufficient variation on the ADRB3 gene in the examined groups. Although Fisher's exact test showed no significant difference in the frequency of ADRB1 and ADBR2 variants between the two groups, the Arg458 variant of ADRB1 was higher (P = 0.11) in the lean group, and pigs in that group had a thinner fat layer than did those with the Pro458 variant. These results imply a possibility of ADRB1 polymorphism as a minor factor in porcine fat layer thickness. The Asp29 variant of ADRB2 was higher in the lean group (P = 0.11), and the Glu30 variant was higher in the fatty group (P = 0.15), but the Asp29 variant was found only in the Chinese pigs. Thus, the effect of ADRB2 polymorphisms was not clear in this study.     

Expression analysis of an α‐1, 3‐galactosyltransferase,an enzyme that creates xenotransplantation‐related α–Gal epitope,in pig preimplantation embryos

α‐1,3‐Galactosyltransferase (α‐GalT), an enzyme creating Galα1‐3Gal (α‐Gal) epitope on the cell surface in some mammalian species such as pigs, is known to be a key factor that causes hyperacute rejection upon transplantation from pigs to humans. To establish the RNA interference‐based suppression of endogenous α‐GalT messenger RNA (mRNA) synthesis in porcine preimplantation embryos, we determined the suitable embryonic stage at which stage such approach is possible by using the semi‐quantitative RT‐PCR (qRT‐PCR) and the cytochemical method using a fluorescence‐labeled Bandeiraea simplicifolia Isolectin B₄ (BS‐I‐B₄). Staining with BS‐I‐B₄ demonstrated that α‐Gal epitope expression was first recognized at the 8‐cell stage, and increased up to the hatched blastocyst stage. Single embryo‐based qRT‐PCR also confirmed this pattern. These results indicate that creation of α‐Gal epitope is proceeded by de novo synthesis of α‐GalT mRNA in porcine preimplantation embryos with peaking at the blastocyst stage.     

猪GnRH受体基因多态性分析及相关基因组织表达谱的构建

促性腺激素释放激素(GnRH)受体基因与猪的繁殖性状密切相关,本试验对不同猪种的GnRH受体基因的编码区进行了SSCP分析,未发现多态性。在GnRH受体基因5′侧翼序列上设计引物扩增了该基因大约2000 bp的侧翼序列,通过比对设计引物,对其中的一段含有GAGABox的插入突变序列和一段由于点突变而形成转录调控蛋白结合序列IRF-1的区域运用SSCP的方法进行了多态性分析。结果表明在插入突变处,基因频率在不同的猪种间存在着很大的差异性,其中民猪与其他猪种的差异性最大。IRF突变只存在于民猪中,并且只存在AA型和AB型。构建了3头大白猪母猪GnRH基因、GnRH受体基因、卵泡抑素(FST)基因、促卵泡素(FSH)基因、FSH受体基因、促黄体素(LH)基因、抑制素(inhibin)β(a)基因、inhibinβ(b)基因的组织表达谱,根据表达谱可以推测在下丘脑-脑垂体-性腺轴外不存在这些基因表达产物的相互作用系统。     

A substitution within erythropoietin receptor gene D1 domain associated with litter size in Beijing Black pig,Sus scrofa

Studies of uterine capacity and litter size in swine have suggested that erythropoietin receptor (EPOR) plays an important role in fetal survival through maturation of red blood cells. In this study, we screened the porcine EPOR gene for mutations and identified three single nucleotide polymorphisms (SNPs): two missense mutations and one synonymous mutation. We then genotyped 272 Beijing Black sows, Sus scrofa, and compared this data with litter sizes from a total of 1523 parities among the sows. The G allele of the nonsynonymous SNP, EPOR c.434A>G, was associated with greater litter size at both first parity (P < 0.05) and at later parities (P < 0.01). This SNP causes His92Arg adjacent to the fourth conserved cysteine residue in the mature protein and is in the D1 domain of the protein. Additionally, we determined the allele frequencies for this SNP among six Chinese indigenous pig breeds (Bamei, Erhualian, Laiwu Black, Mashen, Meishan and Min) and three Western commercial pig breeds (Duroc, Landrace and Large White). The c.434G allele was significantly more common among the more prolific Chinese breeds than the Western breeds, implying that EPOR c.434A>G could be a useful genetic marker to improve litter size in swine.     

L‐carnitine improves hydrogen peroxide‐induced impairment of nuclear maturation in porcine oocytes

We investigated the effect of oxidative stress induced by hydrogen peroxide (H₂O₂) on lipid peroxide (LPO) level and nuclear maturation in porcine oocytes cultured with or without cumulus cells. After 22 h of pre‐culture, oocytes with attached cumulus cells (COC group) or denuded oocytes (DO group) were cultured with H₂O₂, and intra‐oocyte H₂O₂ and LPO levels were quantitatively analyzed using immunofluorescence. This is the first report evaluating LPO levels in porcine oocytes. After H₂O₂ supplementation, the DO group showed severe accumulation of H₂O₂ and LPO in the oocytes. Similarly, while inhibition of progression of nuclear maturation was observed in both groups, the effect was more severe in the DO group. These results demonstrate that cumulus cells reduce the accumulation of H₂O₂ stress in oocytes. Furthermore, we attempted to reduce the oxidative stress by H₂O₂ with L‐carnitine, a H₂O₂ scavenger. L‐carnitine decreased H₂O₂ and LPO levels in the oocytes in both groups, and improvement in the progression of impaired nuclear maturation was observed. These effects were different by the presence of cumulus cells. Our results provide that L‐carnitine is useful for alleviating H₂O₂‐induced oxidative stress by reducing LPO levels and improving the progression of nuclear maturation.     

Activin‐A receptor expression patterns in prepubertal goat oocytes and derived embryos

This study examines the presence of activin IIA and IIB receptors (ActR‐IIA and ActR‐IIB) by Western blotting and immunocytochemistry in immature and IVM‐oocytes, 2 to 8‐cells embryos and blastocysts from prepubertal goats. Western blotting revealed that activin receptors are synthesized during oocyte maturation and embryo development. In the immunocytochemistry experiments, no immunostaining for either receptor was detected in oocytes while both receptors were immunolabelled in all the cells of cleaved embryos. In blastocysts, while ActR‐IIA expression appeared evenly distributed in the two cell lineages, inner cell mass and trophectoderm, the ActR‐IIB immunosignal was restricted mainly to the inner cell mass. Our findings reveal the presence of activin type II receptors (ActR‐IIA and ActR‐IIB) in in vitro matured prepubertal goat oocytes and blastocyst‐stage embryos. The expression of these receptors could be a key factor in understanding differences between competent and incompetent oocytes.     

The localization and function of p38α mitogen‐activated protein kinase in rat oocytes

P38α mitogen‐activated protein kinase (MAPK), which is a member of the canonical MAPK family, is activated in response to various extracellular stresses and plays a role in multiple cellular processes. In this study, we investigated the expression, subcellular localization and functional roles of p38α MAPK during the meiotic maturation of rat oocytes. We found that p38α MAPK phosphorylation (p‐p38α MAPK, indicative of p38α MAPK activation) was low at the germinal vesicle (GV) stage, increased 3 hr after germinal vesicle breakdown (GVBD) and maintained its maximum at metaphase I (MI) or metaphase II (MII). The p‐p38α MAPK mainly accumulated in the GV and had no obvious expression in the nucleus. From GVBD to MII, p‐p38α MAPK was distributed in the cytoplasm around either the chromosomes or the spindle. We used SB203580, an inhibitor of p38α MAPK, to investigate the possible functional role of p38α MAPK during rat oocyte meiotic maturation. Treatment of GV stage oocytes with 20 μM SB203580 blocked p‐p38α MAPK activity, and the spindles appeared abnormal. Additionally, the rate of GVBD after 3 hr of culture with 20 μM SB203580 (58.8%) was significantly inhibited compared with the control (82.5%, p < .05), and the polar body extrusion rate after 12 hr of culture with SB203580 was also significantly decreased compared with the control (40.1% vs 73.3%, p < .05). Taken together, these data indicate that p38α MAPK may play a vital role in rat oocyte meiotic maturation.     

In vitro maturation using αMEM with reduced NaCl enhances maturation and developmental competence of pig oocytes after somatic cell nuclear transfer

BackgroundCompared to medium containing 108 mM sodium chloride (NaCl), in vitro maturation (IVM) using a simple medium with reduced (61.6 mM) NaCl increases the cytoplasmic maturation and embryonic development of pig oocytes.ObjectivesThis study determines the effect of a complex medium containing reduced NaCl on the IVM and embryonic development of pig oocytes.MethodsPig oocytes were matured in Minimum Essential Medium Eagle-alpha modification (αMEM) supplemented with 61.6 (61αMEM) or 108 (108αMEM) mM NaCl, and containing polyvinyl alcohol (PVA) (αMEMP) or pig follicular fluid (PFF) (αMEMF). Medium-199 (M199) served as the control for conventional IVM. Cumulus cell expansion, nuclear maturation, intra-oocyte glutathione (GSH) contents, size of perivitelline space (PVS), and embryonic development after parthenogenesis (PA) and somatic cell nuclear transfer (SCNT) were evaluated after IVM.ResultsRegardless of PVA or PFF supplementation, oocytes matured in 61αMEM showed increased intra-oocyte GSH contents and width of PVS (p < 0.05), as well as increased blastocyst formation (p < 0.05) after PA and SCNT, as compared to oocytes matured in 108αMEMP and M199. Under conditions of PFF-enriched αMEM, SCNT oocytes matured in 61αMEMF showed higher blastocyst formation (p < 0.05), compared to maturation in 108αMEMF and M199, whereas PA cultured oocytes showed no significant difference.ConclusionsIVM in αMEM supplemented with reduced NaCl (61.6 mM) enhances the embryonic developmental competence subsequent to PA and SCNT, which attributes toward improved oocyte maturation.     

Molecular expression and identification of caprine estrogen receptor gene 1 for fertility status in bucks

Estrogen and its receptors are essential for sexual development and reproduction. Oestrogen receptor alpha (ERα) is a nuclear receptor activated by the hormone oestrogen. In male, ERα is encoded by the gene ESR1 (oestrogen receptor1) responsible for better fertility. ESR1 is involved in the reabsorption of luminal fluid during the transit of spermatozoa from the testis to the head of the epididymis which is important for their survival and maturation during epididymal storage. The absence of ESR1 leads to reduced epididymal sperm content, reduced sperm motility and fertilizing ability. The present study was undertaken to investigate the expression and presence of ESR1 gene in fertile and low-fertile male goat breeds. We identified ESR1 gene through various molecular tools. Genotyping was carried out by high resonance melting analysis using Roche Light Cycler 480(LC-480) system and found three different genotypes. Genotypic frequency-AA (blue-0.67), BB(Red-0.2), AB(Green-0.08) with allele frequency A(0.71 and B (0.29). The predominance of this gene in head of epididymis in fertile bucks was confirmed by SDS-PAGE, Western blotting and immunohistochemistry. From the results, we corroborated that the present study provides a useful and effective way to predict male fertility in goat breeds, which in turn increases the percentage of fertility in flock leading to more number of offspring in a kidding season.