Effects of adding luteinizing hormone to a medium containing follicle stimulating hormone on progesterone-induced differentiation of cumulus cells during meiotic resumption of porcine oocytes

In the present study, we investigated the effects of adding luteinizing hormone (LH) to a medium containing follicle stimulating hormone (FSH) on the shift in expression of progesterone receptor (PR) isoforms (PR‐A and PR‐B) and the roles in function of cumulus cells of cumulus‐oocyte complexes (COC). The level of PR‐B mRNA in cumulus cells was up‐regulated by FSH during the first 16‐h cultivation but the level was significantly decreased at 20 h. The decrease of PR‐B mRNA was accelerated when COC were cultured with FSH and LH. Still, a high level of total PR mRNA was maintained in cumulus cells cultured with or without the addition of LH up to 20 h, suggesting that the expression of PR isoforms was shifted from PR‐B to PR‐A in cumulus cells. The reduction of PR‐B was also induced by addition of progesterone to FSH‐containing medium. The addition of LH or progesterone to FSH‐containing medium stimulated cumulus expansion of COC as compared with that of COC cultured with FSH. In the expanded COC, ADAMTS‐1 which is expressed in granulosa cells and cumulus cells in rodent follicles through LH‐induced progesterone‐ and PR‐dependent pathway, was more accumulated within the COC matrix. These results suggest that the addition of LH or progesterone to FSH‐containing medium is required for the differentiation of cumulus cells, such as cumulus expansion, mediated by the shift from PR‐B to PR‐A in them.     

Polo‐like kinase 1 inhibition results in misaligned chromosomes and aberrant spindles in porcine oocytes during the first meiotic division

Polo‐like kinase 1 (Plk1), a type of serine/threonine protein kinase, has been implicated in various functions in the regulation of mitotic processes. However, these kinase's roles in meiotic division are not fully understood, particularly in the meiotic maturation of porcine oocytes. In this study, the expression and spatiotemporal localization of Plk1 were initially assessed in the meiotic process of pig oocytes by utilizing Western blotting with immunofluorescent staining combined with confocal microscopy imaging technique. The results showed that Plk1 was expressed and exhibited a dynamic subcellular localization throughout the meiotic process. After germinal vesicle breakdown (GVBD), Plk1 was detected prominently around the condensed chromosomes and subsequently exhibited a similar subcellular localization to α‐tubulin throughout subsequent meiotic phases, with particular enrichment being observed near spindle poles at MI and MII. Inhibition of Plk1 via a highly selective inhibitor, GSK461364, led to the failure of first polar body extrusion in porcine oocytes, with the majority of the treated oocytes being arrested in GVBD. Further subcellular structure examination results indicated that Plk1 inhibition caused the great majority of oocytes with spindle abnormalities and chromosome misalignment during the first meiotic division. The results of this study illustrate that Plk1 is critical for the first meiotic division in porcine oocytes through its influence on spindle organization and chromosome alignment, which further affects the ensuing meiotic cell cycle progression.     

Influence of co‐culture with denuded oocytes during in vitro maturation on fertilization and developmental competence of cumulus‐enclosed porcine oocytes in a defined system

Co‐culture of cumulus‐oocyte complexes (COCs) with denuded oocytes (DOs) during in vitro maturation (IVM) was reported to improve the developmental competence of oocytes via oocyte‐secreted factors in cattle. The aim of the present study was to investigate if addition of DOs during IVM can improve in vitro fertilization (IVF) and in vitro culture (IVC) results for oocytes in a defined in vitro production system in pigs. The maturation medium was porcine oocyte medium supplemented with gonadotropins, dbcAMP and β‐mercaptoethanol. Cumulus‐oocyte complexes were matured without DOs or with DOs in different ratios (9 COC, 9 COC+16 DO and 9 COC+36 DO). Consequently; oocytes were subjected to IVF as intact COCs or after denudation to examine if DO addition during IVM would affect cumulus or oocyte properties. After fertilization, penetration and normal fertilization rates of zygotes were not different between all tested groups irrespective of denudation before IVF. When zygotes were cultured for 6 days, no difference could be observed between all treatment groups in cleavage rate, blastocyst rate and cell number per blastocyst. In conclusion, irrespective of the ratio, co‐culture with DOs during IVM did not improve fertilization parameters and embryo development of cumulus‐enclosed porcine oocytes in a defined system.     

The involvement of N‐glycosylation of zona glycoproteins during meiotic maturation in sperm–zona pellucida interactions of porcine denuded oocytes

The present study was conducted to delineate whether N‐glycosylation of zona pellucida (ZP) glycoproteins occurred during meiotic maturation and whether this N‐glycosylation played a role in sperm–ZP interactions of porcine cumulus denuded oocytes (DOs). After mechanical removal of cumulus cells from cumulus oocyte complexes (COCs), DOs were cultured for 44 h in in vitro maturation (IVM) culture. The experiments were carried out to determine the effects of tunicamycin, a specific N‐glycosylation inhibitor, for various intervals during IVM on sperm–ZP interactions in porcine DOs. The results determined that DOs could induce meiotic maturation, although the maturation rate of DOs was earlier than that of COCs. In addition, N‐glycosylation of ZP glycoproteins occurred during meiotic maturation and was crucial in sperm–ZP interactions, was responsible for sperm penetration, sperm binding to ZP and induction of acrosome reaction in ZP‐bound sperm. However, the inhibition of N‐glycosylation by tunicamycin during IVM did not influence ZP hardness and male pronuclear formation, indicating that this N‐glycosylation was involved in the initial stage of fertilization. We conclude that 24–44 h of N‐glycosylation of ZP glycoproteins during meiotic maturation was crucial in sperm penetration and sperm binding to ZP and the induction of acrosome reaction in sperm bound to ZP of porcine DOs.     

Changes in the amount of proteins, glycogen and lipids in porcine oocytes during in vitro meiotic maturation

Changes in the cytoplasmic inclusions during meiotic maturation were histochemically examined in cultured porcine oocytes. The oocytes contained a small amount of protein and glycogen granules throughout the maturation culture, as well as Sudanophilic lipids composed of small, medium and large droplets. Soon after collection, the amount of Sudanophilic lipid droplets of small and medium size was small and there were 167 ± 11.2 large droplets. After being cultured for 22 h, the number of large lipid droplets decreased remarkably, while the number of small and medium ones increased. There were no differences in the number of Sudanophilic lipid droplets of different sizes between ovulated oocytes and the oocytes cultured for 44 h. The oocytes always contained a large amount of neutral fats and lipoids, but not cholesterols. In the oocytes cultured for 22 h with olomoucine, both the resumption of nuclear maturation and the decrease in the size of the Sudanophilic lipid droplets were inhibited. From the present findings, it appears that the change in the size of the Sudanophilic lipid droplets in the cytoplasm of porcine oocytes is closely related to nuclear maturation.     

猪蛋白酪氨酸磷酸酶-1B基因第7内含子多态性与猪脂肪沉积性状的关联性

本研究旨在分析蛋白酪氨酸磷酸酶-1B(PTP-1B)基因部分SNP与猪脂肪沉积性状的关联性。选取国外瘦肉型猪种大约克夏16头、长白猪21头和中国脂肪型地方猪种梅山猪22头以及"大白×梅山"F2代资源家系213头为研究材料,利用PCR-Eco88I-RFLP方法检测PTP-1B基因第7内含子序列存在G/A突变的多态性,并进行方差分析。结果表明,大白和长白猪种为单一的A等位基因,而梅山猪种以杂合子GA基因型为主,对213头"大白×梅山"F2代资源家系进行性状关联分析,该位点与猪板油质量、内脂率、胸腰椎间背膘厚等脂肪沉积性状显著相关(P<0.05),且杂合子GA基因型个体的板油质量和内酯率都最低(GA基因型个体相似文献   

Cyclin D1 immunohistochemical expression and somatic mutations in canine oral melanoma

Canine oral melanoma (COM) is the most frequent tumour with oral localization in dogs. Copy number gains and amplifications of CCND1, a gene coding for Cyclin D1, are the most frequent chromosomal aberrations described in human non‐UV induced melanomas. Twenty‐eight cases of COM were retrieved from paraffin‐blocks archives. A total of 4 μm thick sections were immunostained with an antibody against human Cyclin D1 and Ki‐67. Cyclin D1 and Ki‐67 expressions were scored through two counting methods. DNA was extracted from 20 μm thick sections of formalin‐fixed paraffin‐embedded blocks. Pathological and surrounding healthy tissue was extracted independently. Cyclin D1 immunolabelling was detected in 69% (18/26) while Ki‐67 was present in 88.5% (23/26) of cases. Statistical analysis revealed correlation between two counting methods for Cyclin D1 (r = 0.54; P = .004) and Ki‐67 (r = 0.56; P = .003). The correlation found between Ki‐67 and Cyclin D1 indexes in 16/26 cases labelled by both antibodies (r = 0.7947; P = .0002) suggests a possible use of Cyclin D1 index as prognostic marker. Polymerase chain reaction analysis on CCND1 coding sequence revealed the presence of nine somatic mutations in seven samples producing synonymous, missense and stop codons. Since none of the single‐nucleotide polymorphisms was found to be recurrent, it is suggested that overexpression of Cyclin D1 may be the consequence of alterations of CCND1 upstream regions or other genetic aberrations not detectable with the methodology used in this study. Future studies are needed to verify the potential use of Cyclin D1 index as prognostic indicator and to highlight the molecular events responsible for Cyclin D1 overexpression in COMs.     

Insights into the NAD+ biosynthesis pathways involved during meiotic maturation and spindle formation in porcine oocytes

Treatments that elevate NAD⁺ levels have been found to improve oocyte quality in mice, cattle, and pigs, suggesting that NAD⁺ is vital during oocyte maturation. This study aimed to examine the influence of different NAD⁺ biosynthetic pathways on oocyte quality by inhibiting key enzymes. Porcine oocytes from small antral follicles were matured for 44 h in a defined maturation system supplemented with 2-hydroxynicotinic acid [2-HNA, nicotinic acid phosphoribosyltransferase (NAPRT) inhibitor], FK866 [nicotinamide phosphoribosyltransferase (NAMPT) inhibitor], or gallotannin [nicotinamide mononucleotide adenylyltransferase (NMNAT) inhibitor] and their respective NAD⁺ pathway modulators (nicotinic acid, nicotinamide, and nicotinamide mononucleotide, respectively). Cumulus expansion was assessed after 22 h of maturation. At 44 h, maturation rates were determined and mature oocytes were fixed and stained to assess spindle formation. Each enzyme inhibitor reduced oocyte maturation rate and adversely affected spindle formation, indicating that NAD⁺ is required for meiotic spindle assembly. Furthermore, NAMPT and NMNAT inhibition reduced cumulus expansion, whereas NAPRT inhibition affected chromosomal segregation. Treating oocytes with gallotannin and nicotinamide mononucleotide together showed improvements in spindle width, while treating oocytes with 2-HNA and nicotinic acid combined showed an improvement in both spindle length and width. These results indicate that the salvage pathway plays a vital role in promoting oocyte meiotic progression, while the Preiss-Handler pathway is essential for spindle assembly.     

Low expression of mitofusin 1 is associated with mitochondrial dysfunction and apoptosis in porcine somatic cell nuclear transfer embryos

Mitochondria are necessary for the transition from oocyte to embryo and for early embryonic development. Mitofusin 1 is the main mediator of mitochondrial fusion and homeostasis. We investigated Mitofusin 1 expression levels in porcine somatic cell nuclear transfer (SCNT) embryos. The rate of blastocyst formation in SCNT embryos was reduced significantly compared with that of parthenogenetic activation embryos. SCNT embryos showed significantly decreased Mitofusin 1 expression and mitochondrial membrane potential, while exhibiting increased reactive oxygen species and apoptosis. Mitochondrial functional changes were observed in the SCNT embryos and may be correlated with low levels of Mitofusin 1 to negatively affect development.     

Phosphorylation of histone H3 on Ser10 by auto-phosphorylated PAK1 is not essential for chromatin condensation and meiotic progression in porcine oocytes

Background

The p21-activated kinase 1 (PAK1) is essential for mitosis and plays an important role in the regulation of microtubule assembly during oocyte meiotic maturation in mice; however, little is known about its role in porcine oocytes.

Result

Total p21-activated kinase 1 (PAK1) and phosphorylated PAK1 at Thr423 (PAK1^Thr423) were consistently expressed in porcine oocytes from the germinal vesicle (GV) to the second metaphase (MII) stages, but phosphorylation of histone H3 at Ser10 (H3^Ser10) was only expressed after the GV stage. Immunofluorescence analysis revealed that PAK1^Thr423 and H3^Ser10 colocalized on chromosomes after the GV stage. Blocking of endogenous PAK1^Thr423 by injecting a specific antibody decreased the phosphorylation level of H3^Ser10; however, it had no impact on chromatin condensation, meiotic progression, cleavage rate of blastomeres or the rate of blastocyst formation.

Conclusion

Phosphorylation of PAK1^Thr423 is a spontaneous activation process and the activated PAK1^Thr423 can promote the phosphorylation of H3^Ser10; however, this pathway is not required for meiotic maturation of porcine oocytes or early embryonic development.     

Effects of resveratrol on in vitro maturation of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer

As a natural plant‐derived antitoxin, resveratrol possesses several pharmacological activities. This study aimed to evaluate the effects of resveratrol addition on nuclear maturation, oocyte quality during in vitro maturation (IVM) of porcine oocytes and subsequent early embryonic development following somatic cell nuclear transfer (SCNT). Our experiments showed that the treatment of porcine oocytes with 5 µM resveratrol during IVM resulted in the highest rate of the first polar body extrusion. Treatment of oocytes with resveratrol had no influence on cytoskeletal dynamics, whereas it significantly increased glucose uptake ability compared to the control oocytes. Oocytes matured with 5 μM resveratrol displayed significantly lower intracellular reactive oxygen species (ROS) levels and higher relative mRNA expression levels of the genes encoding such antioxidant enzymes as catalase (CAT) and superoxide dismutase 1 (SOD1). In addition, resveratrol also prevented onset and progression of programmed cell death in porcine oocytes, which was confirmed by significant upregulation of the anti‐apoptotic B‐cell lymphoma 2 (BCL‐2) gene and significant downregulation of the pro‐apoptotic BCL2‐associated X (BAX) gene. Furthermore, the blastocyst rates and the blastocyst cell numbers in cloned embryos derived from the oocytes that had matured in the presence of 5 μM resveratrol were significantly increased. In conclusion, supplementation of IVM medium with 5 μM resveratrol improves the quality of porcine oocytes by protecting them from oxidative damage and apoptosis, which leads to the production of meiotically matured oocytes exhibiting enhanced developmental potential following SCNT.     

17种植物性饲料原料中伏马毒素(B1+B2)、赭曲霉毒素A、T-2毒素、黄曲霉毒素B1污染状况调查检测分析

为了解掌握伏马毒素（B₁+B₂）、赭曲霉毒素A、T-2毒素、黄曲霉毒素B₁在植物性饲料原料中的污染状况,指导饲料生产企业和养殖企业开展霉菌毒素防控,降低霉菌毒素对饲料产品质量及畜禽养殖危害,减少经济损失,2019年对17种62份植物性饲料原料进行采集,采用液相色谱—串联质谱法、免疫亲和柱净化—高效液相色谱法检测,依据《饲料卫生标准》（GB 13078—2017）判定分析。结果表明：伏马毒素（B₁+B₂）、赭曲霉毒素A、黄曲霉毒素B₁在17种植物性饲料原料中的污染状况差别明显,伏马毒素（B₁+B₂）、赭曲霉毒素A、黄曲霉毒素B₁平均检出率分别为37.09%、8.06%、29.03%,最大检测值分别为15.96 mg/kg、26.60 μg/kg、351.00 μg/kg。从检测结果得出,4种霉菌毒素在17种植物性饲料原料中的污染较重,整体污染率达48.40%,玉米皮、喷浆玉米皮、花生粕3种植物性饲料原料中黄曲霉毒素B₁超标,污染率与超标率不一定呈正比,表明霉菌毒素在植物性饲料原料中污染普遍,对饲料产品及养殖安全造成严重影响。针对该问题,提出控制植物性饲料原料质量建议,为今后控制饲料原料中霉菌毒素含量提供参考。     

神经介素B及其受体NMBR参与抗A型流感病毒H1N1亚型感染的信号通路

NMB/NMBR通过调节A型流感病毒（IAV/H1N1/PR8）感染诱导的细胞因子表达而参与抗IAV的先天性免疫反应。为探究其发挥抗IAV/H1N1感染的信号通路,本文用PR8和WSN毒株分别感染MLE-12细胞和小鼠,用NF-κB抑制剂BAY11-7028单独或联合NMB处理MLE-12细胞,小鼠后腿肌内注射NMB和NMBRA,采用RT-PCR和qRT-PCR分析NMB、NMBR、IL-6、IFN-α和NP基因表达变化,采用Western blot分析NMB、NMBR、P65/p-P65、IκBα和NP蛋白表达的变化。结果显示,BAY11-7028可促使PR8和WSN感染的MLE-12细胞中NMB、NMBR、IL-6和IFN-α基因表达水平均下降和NP基因表达水平上升,并降低NMB、NMBR和p-P65蛋白表达水平和提升IκBα和NP蛋白表达水平。然而,NMB联合BAY 11-7028诱导PR8或WSN感染后的细胞中IL-6和NP表达出现极显著下降和IFN-α显著上升。此外,NMB抑制PR8和WSN感染的小鼠肺组织内p-P65和NP蛋白表达水平和促进IκBα蛋白表达水平;NMBRA联合NMB抵消NMB对PR8或WSN感染后的这些蛋白表达水平的调节作用。综上表明,NMB/NMBR通过调节PR8和WSN感染的MLE-12细胞和小鼠体内的NF-κB信号通路上P65蛋白磷酸化和IκBα的表达,进而影响下游细胞因子IL-6和IFN-α基因的表达,从而发挥抗IAV/H1N1感染的先天性免疫应答反应。     

猪日本乙型脑炎病毒NS1基因重组腺病毒的构建及表达

应用RT-PCR方法扩增出猪日本乙型脑炎病毒（JEV）的NS1基因,通过Sal Ⅰ＋EcoR Ⅰ双酶切后把该基因插入到经过同样双酶切的穿梭质粒pDC315中.重组穿梭载体经过双酶切和PCR鉴定后进行测序,序列测定正确.将获得的重组穿梭质粒与腺病毒骨架质粒共转染293细胞后获得重组腺病毒.PCR鉴定及间接ELISA检测的结果证明所构建的重组腺病毒成功地表达了JEV的NS1.     

PS48 promotes in vitro maturation and developmental competence of porcine oocytes through activating PI3K/Akt signalling pathway

Oocyte maturation plays a vitally important role in porcine reproduction. Regrettably, the quality of oocytes matured in vitro is weaker than that of in vivo matured oocytes. We collected and cultivated porcine cumulus oocyte complexes (COCs) in vitro with phosphoinositide-dependent kinase 1 (PDK1) activator 5-(4-chloro-phenyl)-3-phenyl-pent-2-enoic acid (PS48), whose concentrations were 0, 2, 5, 10 and 20 µM to investigate whether the phosphatidylinositol-3-kinase/protein kinase B (PI3K/Akt) signalling pathway would impact the oocyte quality. The results showed that 10 µM PS48 increased the oocyte proportion of metaphase II (MII) stage and improved the expansion of cumulus cells (CCs). What's more, the activation of PI3K/Akt signalling pathway could regulate the expression of maturation-related genes and proteins. The results of quantitative real-time PCR showed that 10 µM PS48 increased the mRNA and protein levels of Akt and regulated maturation-related genes, including cyclin B1, MOS, BMP15, GDF9, CDC2, mTOR, BAX, BCL2 and caspase-3. The results of Western blot indicated that 10µM PS48 increased the protein abundance of Akt, phosphorylation of Akt Thr308 (p-Akt^Thr308) and cyclin B1, but decreased the protein abundance of pro-apoptotic BAX. These results suggested that adding 10 µM PS48 to mature culture medium could promote the maturation of porcine oocytes, potentially through activating the PI3K/Akt signalling pathway.     

抑制UCHL1对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响

试验旨在研究抑制泛素羧基末端水解酶-1（UCHL1）对猪卵母细胞体外成熟、透明带泛素化及多精入卵的影响。利用DAPI染色、Hoechst染色及SDS-PAGE等方法检测猪卵母细胞体外成熟率、透明带泛素化水平及多精入卵等情况。结果表明,添加不同浓度UCHL1抑制剂（10、20、25、30 μmol/L,二甲基亚砜（DMSO）及对照组）体外培养猪卵母细胞46 h后,对照组成熟率为86.22%,而30 μmol/L UCHL1抑制剂组的成熟率为15.30%,且各处理组间成熟率差异显著（P<0.05）;经Western blotting检测,各处理组的透明带在约61、80、106 ku处均发生不同程度的泛素标记,通过灰度值分析差异显著（P<0.05）;进行体外受精试验后,发现对照组透明带精子黏附数最多,精子入卵数较少,添加30 μmol/L UCHL1抑制剂组的透明带精子黏附数最少,几乎没有精子入卵。结果显示,UCHL1抑制剂对猪卵母细胞的体外成熟有一定影响,随着UCHL1抑制剂浓度的增加,透明带发生泛素化的程度逐渐降低,且UCHL1可调节透明带精子黏附及多精入卵。     

Effects of UCHL1 Inhibition on Porcine Oocyte Matuation in vitro,Zona Pellucida Ubiquitination and Polyspermy

This study was aimed to examine the effects of UCHL1 inhibition on porcine oocyte maturation in vitro, zona pellucida (ZP) ubiquitination and polyspermy. DAPI staining, Hoechst staining and SDS-PAGE methods were used to detect the matuation rate of porcine oocytes in vitro, the level of ubiquitination of zona pellucida (ZP) and polyspermy. The results showed that after different concentrations UCHL1 inhibitor (10, 20, 25 and 30 μmol/L, DMSO and control group) were added into maturation medium for culturing 46 h in vitro, the mature rate of control group was 86.22%, while the maturation rate of 30 μmol/L group was 15.30%, and the maturation rate of every treatment group had significant difference (P<0.05). Western blotting result showed that every group generated ubiquitin markers of ZP were about 61, 80 and 106 ku in different degree. According to the analysis of gray value, the result had significant difference (P<0.05). Conducting fertilization in vitro, the number of sperm adhered on oocyte ZP in control group was the most, the number of sperm running into oocyte was fewer, the number of sperm adhered on oocyte ZP with 30 μmol/L UCHL1 inhibitor was the fewest, and there was almost no sperm running into the oocyte. The results showed that UCHL1 inhibitor had an impact on maturation of porcine oocytes in vitro. With the higher concentration of UCHL1, the lower degree of ZP protein ubiquitinated, UCHL1 could regulate sperm attachment and polyspermy.     

Expression of TGFBR1, TGFBR2, TGFBR3, ACVR1B and ACVR2B is altered in ovaries of cows with cystic ovarian disease

The objective of this study was to examine the expression of transforming growth factor beta receptor (TGFBR)1, TGFBR2, TGFBR3, activin receptor (ACVR)1B and ACVR2B in ovaries of cows with cystic ovarian disease (COD). The expression of the selected receptors was determined by immunohistochemistry in sections of ovaries from cows with ACTH‐induced and spontaneous COD. Expression of TGFBR1 and TGFBR3 was higher in granulosa cells of cysts from cows with spontaneous COD than in tertiary follicles from the control group. Additionally, TGFBR3 expression was higher in granulosa cells of cysts from cows with ACTH‐induced COD than in those from the control group and lower in theca cells of spontaneous and ACTH‐induced cysts than in tertiary control follicles. There were no changes in the expression of TGFBR2. ACVR1B expression was higher in granulosa cells of tertiary follicles of cows with spontaneous COD than in the control group, whereas ACVR2B expression was higher in cysts of the spontaneous COD group than in tertiary follicles from the control group. The alterations here detected, together with the altered expression of the ligands previously reported, indicate alterations in the response of the ligands in the target cells, modifying their actions at cellular level.