Changes of Connexin43 Expression in Non‐pregnant Porcine Myometrium Correlate with Progesterone Concentration During Oestrous Cycle

Connexin43 (Cx43) is a major protein of myometrial gap junctions. The number of Cx43 gap junctions increase dramatically with the onset of labour in association with development of synchronized uterine contractions. The formation of myometrial gap junctions follows an increase in the oestrogen to progesterone ratio indicating an important role of steroid hormones in regulating Cx43 expression at term. However, no relationship has been established between the expression of Cx43 in the non‐pregnant myometrium and concentration of steroid hormones during the oestrous cycle. Here, we used immunofluorescence and Western blotting to analyse the expression of Cx43 gap junctions in the myometrium of pre‐pubertal pigs (n = 7) and mature pigs at pre‐ovulatory (n = 7), luteal (n = 5) and late luteal (n = 3) stages of the oestrous cycle. The number of Cx43 gap junctions calculated per 1 mm² of the myometrial section was low in pre‐pubertal pigs and significantly higher (p < 0.022) in pre‐ovulatory animals. In relation to pre‐ovulatory animals the number of myometrial gap junctions was significantly lower (p < 0.019) at the luteal phase and correlated with significantly higher (p < 0.005) concentration of endogenous progesterone. Phosphorylated isoforms of Cx43 protein were expressed in the myometrium of pre‐pubertal pigs and mature animals at pre‐ovulatory and late luteal phases, while they were down regulated at the luteal stage. These results indicate that changes of Cx43 expression in the porcine myometrium during the oestrous cycle may be regulated by progesterone concentration and may contribute to the modulation of uterine motility.     

Quantification of Connexin43 Gap Junctions in Porcine Myometrium by Confocal Microscopy and Stereology

The aim of this study was to quantify the number and size of connexin43 (Cx43) gap junctions in the circular and longitudinal layers of myometrium of the non‐pregnant pig. We developed a novel approach to measure the mean surface area (), numerical density (N_v) and surface density (S_v) of gap junctions using confocal microscopy and stereological analysis. Immunolabelled Cx43 gap junctions were measured in the subendometrial and deep regions of the circular layer and in the longitudinal layer of the myometrium of pre‐pubertal pig and mature pig at pre‐ovulatory and secretory stages of the oestrous cycle. In the circular subendometrial region, all investigated stereological parameters of Cx43 gap junctions (, N_v and S_v) were significantly higher (p < 0.05) than those of the circular deep region and the longitudinal layer in all three groups of animals. These results indicate the large‐scale heterogeneity of the number and size of Cx43 gap junctions across the myometrium in non‐pregnant pig and emphasize the existence of functional diversity among myometrial cells.     

猪缝隙连接蛋白43(GJA1)结构和功能的预测与分析

为了探究猪缝隙连接蛋白43（GJA1）的结构和相关功能,本研究对16个哺乳动物的GJA1蛋白进行多重序列比对及系统发育分析,并通过相关的生物信息学数据库和软件从理化性质、蛋白修饰位点、跨膜区、亚细胞定位、二级结构、三级结构、多态位点、功能区域和互作蛋白等方面对猪GJA1蛋白的结构和功能进行系统分析。系统发育分析结果显示,GJA1蛋白在16种哺乳动物中变异未达到饱和,物种间遗传距离小且同源性高,其中猪与马的GJA1蛋白序列最为接近,同源性达93.3%,在进化树上的分类也证实了这一结果。蛋白结构分析结果显示,猪GJA1蛋白分子质量为43.08 ku,理论等电点为8.88,由20种氨基酸组成,不稳定指数为44.06,平均疏水系数为-0.240,脂溶指数为82.67,不分泌信号肽,有4个跨膜区、6个O-糖基化位点和38个磷酸化位点,最有可能位于细胞质膜和高尔基体内,其二级结构和三级结构主要由α-螺旋和无规则卷曲组成。蛋白功能分析结果显示,在猪GJA1蛋白的CDS序列中有2个超级家族结构功能区域和6个错义突变位点,且猪GJA1蛋白还与ZO-1和Cx36等蛋白及多种蛋白激酶存在相互作用。本研究通过生物信息学分析系统地揭示了猪GJA1蛋白的结构和功能,为后续进一步研究GJA1蛋白在猪体内发挥生物学功能的遗传调控机制提供一定的理论支持。     

Expression Pattern of CD34 at the Maternal–Foetal Interface During Pregnancy in Pigs

The pig exhibits a non‐invasive, epitheliochorial placentation. Adhesion molecules are indispensable for successful implantation and establishment of placentation. CD34 is an adhesion molecule belonging to the immunoglobulin superfamily (IgSF). To take the first step to investigate the role of CD34 in placentation, we examined the expression pattern of CD34 at the maternal–foetal interface in Yorkshire gilts on days 15, 26, 50 or 95 and in Meishan gilts on days 26, 50 or 95 of pregnancy (n = 3 gilts/breed/day of pregnancy) by immunohistochemical technique. The CD34‐positive signals were detected in uterine luminal epithelium and trophectoderm in Yorkshire pigs; the staining for CD34 was located in trophectoderm but barely detectable at the uterine luminal epithelium on day 15 of pregnancy. Then, the expression of CD34 increased dramatically in both the uterine luminal epithelium and trophectoderm by day 26, and weak staining intensity was observed at the maternal–foetal interface on days 50 and 95 of pregnancy. The expression pattern of CD34 in Meishan pigs is similar to that in Yorkshire pigs except that only a few positive signals were observed at the luminal epithelium on day 26 of pregnancy. These results suggest that CD34 may be involved in mediating the cell‐to‐cell adhesion between trophectoderm and the luminal epithelial cells during early pregnancy in pigs.     

Expression of Adiponectin and its Receptors in the Porcine Hypothalamus During the Oestrous Cycle

Adiponectin is a hormonal link between obesity and reproduction, and its actions are mediated by two types of receptors: adiponectin receptor 1 (AdipoR1) and adiponectin receptor 2 (AdipoR2). This study compares the expression levels of adiponectin and adiponectin receptor mRNAs and proteins in selected areas of the porcine hypothalamus responsible for GnRH production and secretion: the mediobasal hypothalamus (MBH), pre‐optic area (POA) and stalk median eminence (SME). The tissue samples were harvested on days 2–3, 10–12, 14–16 and 17–19 of the oestrous cycle. Adiponectin mRNA expression in MBH was significantly lower on days 14–16, whereas in SME, the most pronounced gene expression was found on days 2–3 of the cycle (p < 0.05). Adiponectin protein in MBH was most abundant on days 17–19 and in POA on days 2–3 (p < 0.05). Adiponectin protein expression in SME was at similar level throughout the most of the cycle with a statistically significant drop (p < 0.05) on days 14–16. AdipoR1 gene expression in POA was potentiated on days 2–3 and 10–12 of the oestrous cycle (p < 0.05). In SME, the highest AdipoR1 mRNA expression was noted on days 2–3 (p < 0.05). The concentrations of the AdipoR1 protein in POA were similar throughout the luteal phase (days 2–14 of the cycle), and they decreased on days 17–19 (p < 0.05). In SME, AdipoR1 protein expression peak occurred on days 2–3 (p < 0.05). The expression patterns of the AdipoR2 gene in MBH, POA and SME revealed the highest mRNA levels on days 2–3 of the cycle (p < 0.05). The highest content of AdipoR2 protein in MBH was reported on days 2–3 (p < 0.05), while in POA on days 17–19 and in SME on days 10–12 and 14–16 (p < 0.05). This study demonstrated that adiponectin and adiponectin receptor mRNAs and proteins are present in the porcine hypothalamus and that their expression levels are determined by the pig's endocrine status related to the oestrous cycle.     

Expression of Genes Associated with Apoptosis in the Porcine Corpus Luteum During the Oestrous Cycle

The corpus luteum (CL) of the pig lacks luteolytic sensitivity (LS) to prostaglandin (PG) F‐2α until after day 12 of the oestrous cycle, but the mechanisms underlying this phenomenon are poorly understood. As luteolysis involves apoptosis, we hypothesized that critical apoptotic proteins may be deficient in CLs that lack LS. The specific aim of these studies was to examine mRNA expression and protein levels of apoptosis genes/proteins (BAX/Bax, BCLX/Bcl‐x, CASP3/Caspase‐3, CASP8/Caspase‐8, NFΚB1/NFκB, TP53/p53) in porcine CLs collected at different stages of the oestrous cycle. CLs were collected surgically, mRNA and protein extracted, and expression/levels analyzed by semi‐quantitative (SQ) PCR and Western blots, respectively. At the mRNA expression level, only BAX (maximal on day 4) and TP53 (maximal on day 7) showed significant variations during the oestrous cycle. At the protein level, only Bcl‐x and Caspase‐3 showed significant changes during the cycle; Bcl‐x decreased on day 13 and Caspase‐3 increased on day 13. It is concluded that apoptosis‐associated proteins (i.e. Bcl‐x and Caspase 3) may play a critical role in luteolytic sensitivity in the pig.     

Altered Expression of 3β‐HSD,CYP17 and 17β‐HSD in the Foetal Porcine Gonads in Response to Anti‐androgen Flutamide Exposure

We investigated whether the limited access to androgens during late prenatal period alters expression of steroidogenic enzymes involved in androgen production: 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3β‐HSD), cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17) and 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) or type 3 (17β‐HSD3) in the foetal porcine gonads. Pregnant gilts were injected with anti‐androgen flutamide (for seven days, 50 mg/day/kg bw) or corn oil (control) starting at 83 (GD90) or 101 (GD108) gestational day. To assess 3β‐HSD, CYP17 and 17β‐HSD1 or 17β‐HSD3 expression, real‐time PCR and immunohistochemistry were performed. In testes from flutamide‐treated foetuses, increased 3β‐HSD and CYP17 mRNA expression was observed in the GD90 group, while decreased 3β‐HSD and 17β‐HSD3 mRNA expression and increased CYP17 mRNA expression were found in the GD108 group. CYP17 and 17β‐HSD3 were localized in Leydig cells. Following flutamide administration, the intensity of CYP17 immunostaining was higher in both treated groups, while 17β‐HSD3 intensity was lower in the GD108 group. In ovaries from flutamide‐treated foetuses in the GD90 group, mRNA level for 3β‐HSD was elevated, but it was diminished for CYP17 and 17β‐HSD1. In the GD108 group, flutamide treatment led to lower mRNA level for 3β‐HSD but higher for CYP17. 3β‐HSD was found in granulosa cells, while CYP17 was localized within egg nests and oocytes of forming follicles. Following flutamide treatment, the intensity of 3β‐HSD and CYP17 immunostaining was higher in the GD90 and GD108 groups, respectively. Immunohistochemical staining for 3β‐HSD was restricted to the ovary. Concluding, diminished androgen action in the porcine foetal gonads during late gestation induces changes in steroidogenic enzymes expression, which may led to changes in gonadal function. However, it seems that androgens exert diverse biological effects depending on the gestational period.     

Melatonin Supplementation During In Vitro Maturation and Development Supports the Development of Porcine Embryos

Melatonin has been reported to improve the in vitro development of embryos in some species. This study was conducted to investigate the effect of melatonin supplementation during in vitro maturation (IVM) and development culture on the development and quality of porcine embryos. In the first experiment, when the in vitro fertilized embryos were cultured with different concentrations of melatonin (0, 10, 25 and 50 ng/ml) for 8 days, the blastocyst formation rate of embryos cultured with 25 ng/ml melatonin (10.7%) was significantly increased (p < 0.05) compared to the control embryos cultured without melatonin (4.2%). The proportion of DNA‐fragmented nuclei in blastocysts derived from embryos cultured with 50 ng/ml melatonin was significantly lower (p < 0.05) than that of embryos cultured without melatonin (2.1% vs 7.2%). In the second experiment, when oocytes were cultured in the maturation medium supplemented with different concentrations of melatonin (0, 10, 25 and 50 ng/ml), fertilized and then cultured with 25 ng/ml melatonin for 8 days, there were no significant differences in the rates of cleavage and blastocyst formation among the groups. However, the proportions (2.7–5.4%) of DNA‐fragmented nuclei in blastocysts derived from oocytes matured with melatonin were significantly decreased (p < 0.05) compared to those (8.9%) from oocytes matured without melatonin, irrespective of the concentration of melatonin. Our results suggest that supplementation of the culture media with melatonin (25 ng/ml) during IVM and development has beneficial effects on the developmental competence and quality of porcine embryos.     

Expression of Perilipin 2 (PLIN2) in Porcine Oocytes During Maturation

Perilipins have been reported to limit the interaction of lipases with neutral lipids within the droplets, thereby regulating neutral lipid accumulation and utilization. This study aimed to identify the location and expression of PLIN1 and PLIN2 in porcine oocytes during maturation. Quantitative real‐time polymerase chain reaction (qRT‐PCR), immunostaining and Western blot methods were used to characterize the expression and distribution patterns of PLIN1 and PLIN2 in porcine oocytes. The results showed that PLIN1 was not detectable in porcine oocytes. PLIN2 and BODIPY 493/503‐detected neutral lipid droplets appeared identical distribution patterns and extensive colocalization in both GV and MII porcine oocytes. PLIN2 protein expression was higher in GV oocytes than that in MII oocytes (p < 0.05), although PLIN2 mRNA expression was similar in both groups. These findings suggested that PLIN2 was a major lipid droplet‐associated protein in porcine oocytes.     

缝隙连接蛋白43对小鼠子宫基质细胞蜕膜化的影响

缝隙连接蛋白43(connexin 43,Cx43)是缝隙连接蛋白家族成员之一,广泛存在于哺乳动物器官和组织中,参与细胞增殖、分化、凋亡等生理过程。本试验参照Gen Bank中已发表的Cx43基因序列,设计特异性引物,进行PCR扩增。为了提高构建过表达重组质粒的效率,PCR产物先与p GEM-T载体进行连接,经双酶切后与pc DNA3.1载体融合得到过表达重组质粒,经双酶切和测序鉴定后将其转染到体外分离培养的小鼠子宫基质细胞中,利用实时荧光定量PCR(qRT-RCR)方法检测Cx43 mRNA表达变化及Cx43过表达对蜕膜化标志性分子表达的影响。结果表明,与空载体转染组相比,重组质粒pc DNA3.1-Cx43转染子宫基质细胞后可使小鼠子宫基质细胞Cx43 mRNA的表达量显著升高,同时Cx43过表达可使子宫基质细胞蜕膜化标志性分子Prl8a2和Prl3c1 mRNA的表达量均显著升高,表明Cx43可促进小鼠子宫基质细胞发生蜕膜化。     

一氧化氮合酶在猪早期胚胎发育中表达的初步研究

本研究运用分子生物学手段探究了一氧化氮(NO)在猪早期胚胎发育过程中的表达情况及其相关的规律。应用RT-PCR方法将猪各发育阶段的早期胚胎的内皮型一氧化氮合酶(eNOS)和诱导型一氧化氮合酶(iNOS）进行检测,然后对产物进行半定量分析。结果表明,iNOS在猪早期胚胎发育的整个过程中都有表达,其相对表达量随着胚胎发育进程呈现上升的趋势,在桑葚/囊胚阶段达到最高;eNOS仅仅是在2-细胞期和4-细胞期有表达,其相对表达量在2～4-细胞期间的差异不明显;在猪2～4-细胞胚胎发育过程中,iNOS的相对表达量高于eNOS的相对表达量。结果表明,在猪早期胚胎发育中NO的产量主要由iNOS调节。     

Sialomucin Complex (Muc4) Expression in Porcine Endometrium During the Oestrous Cycle and Early Pregnancy

The non‐invasive type of implantation in the pig is characterized by the maintenance of a thick glycocalyx coating on the uterine epithelial surface microvilli. Present study investigated the alteration in the sialomucin complex (Muc4) expression during the oestrous cycle and early pregnancy in the pig. Endometrial tissue samples were immunostained with the primary antibody to the Muc4 transmembrane subunit ASGP‐2. Muc4 immunostaining increased in the surface and glandular epithelia between days 5 and 10 of oestrous cycle. Immunostaining continued to increase on day 12 with the greatest intensity of uterine Muc4 immunostaining detected on day 15 of the oestrous cycle and early pregnancy. Endometrial Muc4 expression in cyclic gilts decreased dramatically during early proestrous but continued to remain abundant in the surface and glandular epithelium of pregnant gilts during the period of conceptus attachment to the uterine surface.     

Growth Hormone Receptors in the Porcine Testis During Prepuberty

Supplementation of exogenous growth hormone (GH) during prepuberty advances onset of spermatogenesis in boars, but the mechanism of action is unknown. The present study is an investigation of the presence and characteristics of testicular growth hormone receptors (GHR). A total of 36 boars were castrated, three boars every 10 days, between the ages of 10 and 120 days. Testicular membrane preparations of 10, 20, 30, 50, 70, 100 and 120‐day‐old boars were used to determine ¹²⁵I‐bGH binding and Scatchard analysis. Liver from a 60‐kg barrow was used for comparison. Specific ¹²⁵I‐bGH binding to testicular membrane preparations occurred in all age groups with the exception of 20‐day‐old boars at levels of 30–40% of liver binding. At 30 days of age the unlabelled bGH at 1.1 ng/tube achieved half maximal inhibition (ID₅₀). Results of Scatchard analysis indicated a single class of binding sites. Binding affinity was 2.89 × 10⁹m with a binding capacity of 12 fmole/mg membrane protein. The results from this study suggest that GH may act directly on the cells of the prepubertal boar testis.     

Changes in the Expression Patterns of the Genes Involved in the Segregation and Function of Inner Cell Mass and Trophectoderm Lineages During Porcine Preimplantation Development

In mouse embryos, segregation of the inner cell mass (ICM) and trophectoderm (TE) lineages is regulated by genes, such as OCT-4, CDX2 and TEAD4. However, the molecular mechanisms that regulate the segregation of the ICM and TE lineages in porcine embryos remain unknown. To obtain insights regarding the segregation of the ICM and TE lineages in porcine embryos, we examined the mRNA expression patterns of candidate genes, OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc, in blastocyst and elongated stage embryos. In blastocyst embryos, the expression levels of OCT-4, FGF4 and FGFR1-IIIc were significantly higher in the ICM than in the TE, while the CDX2, TEAD4 and GATA3 levels did not differ between the ICM and TE. The expression ratio of CDX2 to OCT-4 (CDX2/OCT-4) also did not differ between the ICM and TE at the blastocyst stage. In elongated embryos, OCT-4, NANOG, FGF4 and FGFR1-IIIc were abundantly expressed in the embryo disc (ED; ICM lineage), but their expression levels were very low in the TE. In contrast, the CDX2, TEAD4 and GATA3 levels were significantly higher in the TE than in the ED. In addition, the CDX2/OCT-4 ratio was markedly higher in the TE than in the ED. We demonstrated that differences in the expression levels of OCT-4, CDX2, TEAD4, GATA3, NANOG, FGF4, FGFR1-IIIc and FGFR2-IIIc genes between ICM and TE lineages cells become more clear during development from porcine blastocyst to elongated embryos, which indicates the possibility that in porcine embryos, functions of ICM and TE lineage cells depend on these gene expressions proceed as transition from blastocyst to elongated stage.     

Human Foetal Sacral Length Measurement for the Assessment of Foetal Growth and Development by Ultrasonography and Dissection

This study investigated the value of prenatal ultrasonographic measurements of sacrum as a predictor of in utero development and gestational age (GA), and to make a comparison between the data obtained by ultrasonography and dissection. A prospective cross-sectional study of ultrasonography was conducted in 186 pregnant women with uneventful single pregnancies. GA ranged from 14 to 40 weeks, and the relationships of sacrum length (SL) with GA, femur length (FL), biparietal diameter (BPD), head circumference (HC) and abdominal circumference (AC) were evaluated. In addition, the sacral bones of 101 spontaneously aborted foetuses aged from 13 to 39 weeks were dissected and measured. The relationship of the results with GA was investigated. There was a linear relationship between the ultrasonographic measurements of the SL and the GA (R2 = 0.93, P < 0.001), the FL (R2 = 0.93, P < 0.001), the BPD (R2 = 0.93, P < 0.001), the HC (R2 = 0.92, P < 0.001) and the AC (R2 = 0.90, P < 0.001). The rate of increase of SL was significantly higher before 28 weeks of gestation than in later pregnancy, with a correlation coefficient between SL and GA of 0.96 and 0.88, respectively. The SL measurements obtained by dissection were similar to those of ultrasonography. There was a linear relationship between the SL and the GA (R2 = 0.96, P < 0.001) and the correlation coefficients between SL and GA were 0.97 before 28 weeks of gestation, and 0.94 in later weeks. In conclusion, this study demonstrates a high correlation between the SL, GA and other standard measurements of foetal growth, and suggests that SL measurement is a valuable predictor of GA in the foetuses with normal growth.     

显微注射针对猪ICSI胚胎的发育和EGFP表达效率的影响

本实验以猪体外成熟卵子以及冷冻解冻后失活的精子为材料,以无BSA且成分明确的TL-HEPES溶液为操作液,探讨显微注射过程中分别使用钝口针和磨口针进行注射,对猪卵子的激活、ICSI胚胎的发育及EGFP表达效率的影响。结果表明:成熟后的猪卵子在不进行精子注射和电激活的情况下,使用钝口针空注射后,卵裂率显著高于磨口针(P<0.05)。分别使用钝口针和磨口针对成熟后的猪卵子进行精子注射,不论进行或不进行电激活,使用钝口针注射,卵子的卵裂率、囊胚率和胚胎EGFP的表达效率均显著高于磨口针(P<0.05)。本实验研究发现,使用钝口针进行注射有利于猪ICSI卵子的激活,胚胎的发育以及胚胎中EGFP表达效率。     

Inflammatory Cytokine Gene Expression in Blood During the Development of Oligofructose-Induced Laminitis in Horses

Systemic inflammation is a risk factor for laminitis in horses and precedes the onset of lameness in experimental models. We therefore hypothesized that whole-blood inflammatory cytokine expression would increase during the development of laminitis in a carbohydrate overload model. Blood samples were obtained from 14 horses undergoing laminitis induction with 10 g/kg oligofructose as part of another study. Samples were collected at 0, 8, 12, 16, 20, and 24 hours, and lameness evaluations were performed every 4 hours. Expression levels of interleukin-1β (IL-1β), IL-6, IL-8, IL-10, and tumor necrosis factor-α were measured in whole blood by using real-time PCR. IL-1β, IL-8, and IL-10 expression increased above baseline from 8 to 24 hours (P < .001), and IL-6 expression increased at 16 and 20 hours (P = .005). Expression of tumor necrosis factor-α did not change over time. All horses developed clinical laminitis between 12 and 24 hours. Increased mean IL-1β, IL-8, and IL-10 expression detected at 8 hours therefore preceded the onset of lameness. We conclude that peripheral leukocyte cytokine expression increases as systemic inflammation develops in an alimentary carbohydrate overload model of laminitis, and this precedes detection of lameness. Results support current recommendations to control the systemic inflammatory response in order to lower the risk of laminitis in horses.