山羊卵巢无腔卵泡的体外发育

在三维琼脂凝胶培养体系中 ,山羊无腔卵泡的生长模式类似于体内。在一定培养时间内能善为保持其完整的立体结构和形态。原始卵泡能在体外启动 ,并得到一定程度的生长。初级卵泡能发育为次级卵泡 ,次级卵泡发育为有腔卵泡。卵泡在体外发育过程中 ,表现出了明显的优势化。本试验首次初步揭示了山羊无腔卵泡的体外发育基本过程和规律。     

Immunohistochemical Localization of Androgen Receptor in Two Subpopulations of Porcine Granulosa Cells In Vitro

In the ovarian follicles of mammals, androgens are utilized as substrates for estrogen synthesis or can act via androgen receptor (AR). Within the ovary, granulosa cells generally display the strongest immunoreactivity for AR protein. Therefore, the aim of the present study was to analyse possible changes in the intensity of AR staining in cultured mural and antral granulosa cells isolated from follicles at various stages of development. Porcine ovaries were obtained at a local slaughterhouse and the follicles were classified as small, medium and large dependently on their diameter. Isolated granulosa cells were separated into two populations, mural and antral, and then the cells were cultured during 48 h. After this time the cells were fixed and immunostained to visualize the AR. Androgen receptor immunostaining was detected in both, antral and mural granulosa cell cultures isolated from follicles at all stages of the development. In the mural granulosa cultures the immunostaining was comparatively weaker than that in the antral granulosa cells originating from all types of follicles. Our results provide immunohistochemical evidence that antral granulosa layer is the main site of AR immunoexpression.     

猪circ0015292的鉴定及其在卵泡中的表达研究

试验旨在通过高通量测序及生物信息学分析circRNA在猪卵泡发育过程中的作用及其可能的机制,为进一步探索circRNA对猪卵泡发育的调控机理奠定生物学基础。采集梅山和杜洛克猪不同级别的卵泡及各组织样本,提取总RNA,采用RNA-Seq技术对卵泡中差异表达的circRNA进行筛选,同时验证并分析其在梅山猪和杜洛克猪卵泡中的表达情况,并用实时荧光定量PCR进行组织表达谱分析。结果显示:试验证实了circ0015292的存在,且其在肌肉、心脏、肝脏、脾脏、肺脏、肾脏、卵巢、输卵管、子宫、垂体及下丘脑中均有不同程度的表达,在心脏、脾脏、肺脏、卵巢中的表达量相对较高;circ0015292在梅山猪S卵泡中的表达量极显著高于杜洛克猪(P<0.01),在杜洛克猪M1、M2、L卵泡中的表达量均显著高于梅山猪(P<0.05);其潜在靶基因GADD45G、CCR2、IL20RB、CFL1、LAMB3与猪的卵泡发育相关。综合试验结果,circ0015292在不同组织、不同时期卵泡中均有不同水平的表达;部分靶标基因参与细胞周期及卵巢发育相关信号通路,提示其可能通过对靶基因的调控作用间接参与卵泡发育过程,这为猪繁殖机理的研究提供了新思路。     

鹌鹑卵泡发育过程中颗粒细胞黄体生成素受体mRNA的表达

用Ｎorthern杂交的方法研究了鹌鹑排卵泡内颗粒细胞黄体生成素受体（ＬＨＲ）mＲＮＡ的表达。在预计排卵前２０h和３h分别取出最大的３个卵泡（Ｆ１、Ｆ３卵泡）以及小黄卵泡（ＳＹＦ）,剥离颗粒层提取出总ＲＮＡ,并经变性凝胶电泳后将ＲＮＡ转移到滤膜上。杂交所用的探针是用特异的引物经反转录一多聚栈链式反应（ＲＴ－ＰＣＲ）扩增出编码ＬＨＲcＤＮＡ的细胞外区（ＥＣ）和跨膜区（ＴＭ）cＤＮＡ。结果表明,颗粒细胞ＬＨＲmＲ     

绵羊卵巢卵泡FoxO1表达模式的研究

本研究旨在探究Fox O1在绵羊卵巢卵泡的表达模式。屠宰场取卵巢获得卵泡,分为小卵泡(直径≤3 mm)、中卵泡(3 mm<直径<5 mm)、大卵泡(直径≥5 mm)3组。利用免疫组化技术对不同直径卵泡的FoxO1进行定位分析,通过机械分离法分离卵泡颗粒细胞,qRT-PCR和Western blotting技术检测FoxO1在小、中、大卵泡颗粒细胞的表达量。结果显示:在绵羊卵泡中,FoxO1表达于颗粒细胞层;中卵泡的FoxO1 mRNA表达量高于小卵泡和大卵泡(P<0.01),小卵泡FoxO1mRNA表达量高于大卵泡(P<0.01);FoxO1蛋白在大卵泡的表达量低于小卵泡和中卵泡(P<0.01),但小卵泡和中卵泡表达量差异不显著。综上表明,FoxO1在绵羊卵泡颗粒细胞层表达,且在大卵泡中的表达量极显著低于小卵泡和中卵泡。     

Effects of IGF‐1 on In Vitro Culture of Bovine Preantral Follicles are Dose‐Dependent

This study aimed at assessing the effect of different concentrations of the growth factor similar to insulin 1 (IGF‐1) in the development, survival and ultrastructure of the bovine preantral follicles cultured in situ. Fragments of bovine ovarian cortical tissue were cultured during 1 and 7 days in 1 ml of α‐MEM⁺, supplemented with different concentrations of human recombinant IGF‐1 (0, 30, 70 and 100 ng/ml), in an incubator at 37°C and 5% of CO₂ in 24‐well plates with total replacement of the medium every 2 days. Non‐cultured ovarian fragments (control) and ovarian fragments cultured during 1 and 7 days were processed for classic histology, mechanical isolation and electron transmission microscopy (ETM). Parameters such as normality, viability, activation, development, diameter and ultrastructure were evaluated. All statistical analyses were carried out using sas Version 9.2. The results showed that the percentage of follicles morphologically normal in the IGF‐1 30 ng/ml treatment was similar to the fresh control (p > 0.05) both on the day 1 and on the day 7 of in vitro culture. In the viability analysis, the cultured treatments maintained the percentage of viable follicles during the entire culture period (p > 0.05). After 7 days of culture, the IGF‐1 30 ng/ml treatment showed higher percentages of developing follicles (48.33%) than those of the fresh control (22.22%) and the cultured treatments (p < 0.05). Also, after 7 days of culture, IGF‐1 30 ng/ml presented a higher follicular diameter when compared to the control and other concentrations of IGF‐1 tested. Ultrastructurally, the non‐cultured control and IGF‐1 30 ng/ml, after 7 days of culture, showed conserved oocytes, nuclei and organelles. Hence, it is concluded that IGF‐1 30 ng/ml was the most efficient concentration for the development of bovine preantral follicles cultured in vitro.     

Influence of the Cumulus and Gonadotropins on the Metabolic Profile of Porcine Cumulus–Oocyte Complexes During In Vitro Maturation

The aim of this work was to examine the influence of the cumulus and gonadotropins on the metabolic profile of porcine cumulus oocyte complexes (COCs) during in vitro maturation. Immature COCs were assigned to morphological classes A₁ (with a dense cumulus), A₂ (with a translucent cumulus), B₁ (with the corona radiata), B₂ (with only some remaining cumulus cells) and matured with or without gonadotropins. Glycolysis and ammonia production were higher in the A class COCs; gonadotropins increased both, especially in the A₁ COCs (p < 0.05). The A class COCs had the highest initial protein contents and at the end of in vitro maturation. Furthermore, hormonal stimulation induced a similar increase in protein contents of both A classes (p < 0.05). The neutral lipid content and reactive oxygen species (ROS) levels were similar in the immature oocytes of the COCs of all classes. A reduction was seen in both these variables when maturation proceeded either in the presence or absence of gonadotropins. The cumulus type surrounding the oocyte is related to the metabolism of carbohydrates and amino acids by the COC during in vitro maturation under gonadotropic stimulation. Oocyte lipolytic activity and ROS production appear to be independent of the surrounding cumulus and the presence of gonadotropins.     

Expression of Endothelin‐1 and Endothelin Receptor A in Canine Ovarian Tumours

Ovarian tumours have a low incidence in bitch. Endothelin (ET‐1) and endothelin A receptor (ET‐A) are overexpressed in human ovarian cancer. Twenty canine ovarian tumours and five normal samples were first evaluated by western blotting and then immunohistochemically for ET‐1 and ET‐A expression. Seventeen out of twenty tumours were ET‐1 positive. Eight out of twenty tumours were ET‐A immunohistochemically positive. At molecular level both proteins were proven to be expressed in normal as well as in tumour samples. Our results show that ET‐1 and ET‐A are overexpressed in canine ovarian tumours, suggesting a potential role of these two molecules in canine ovarian carcinogenesis.     

Effect of Prenatal and Neonatal Anti‐Androgen Flutamide Treatment on Aquaporin 5 Expression in the Adult Porcine Ovary

The growth of ovarian follicles is accompanied by fluid‐filled antrum formation. Water movement within the follicular wall is predominantly transcellular via membranous water channels named aquaporins (AQPs). Androgens are important regulators of mammalian folliculogenesis, and their prenatal and/or neonatal deficiency affects female fertility in adulthood. Therefore, this study was performed to determine whether gestational or neonatal exposure to the anti‐androgen flutamide influences androgen‐dependent AQP5 expression in pre‐antral and large antral follicles of adult pigs. Flutamide was injected into pregnant gilts between days 80 and 88 of gestation and into female piglets between days 2 and 10 post‐natally. The ovaries were collected from flutamide‐treated and non‐treated (control) sexually mature pigs. In pre‐antral follicles, AQP5 mRNA and protein levels were both downregulated following maternal (p < 0.01 and p < 0.01, respectively) and neonatal (p < 0.01 and p < 0.01, respectively) flutamide exposure. Likewise, the expression of mRNA (p < 0.01 and p < 0.001, respectively) and protein (p < 0.05 and p < 0.01, respectively) for AQP5 were diminished in large antral follicles in both groups. Immunohistochemistry showed decreased intensity of AQP5 immunoreaction in pre‐antral (p < 0.01) and large antral (p < 0.001) follicles following flutamide treatment. Moreover, radioimmunological analysis revealed that changes observed in AQP5 expression corresponded with diminished follicular androgens production after both maternal (p < 0.05 and p < 0.05, respectively) and neonatal (p < 0.05 and p < 0.01, respectively) flutamide administration. Therefore, AQP5 appears to be a potential regulator of follicular fluid accumulation, under androgen control, and may be a key factor in antral follicle growth.     

一氧化氮对猪腔前卵泡生长发育的影响

旨在研究在培养液中添加不同浓度（0、0.001、0.01、0.1和1 mmol.L^-1）的一氧化氮供体硝普钠（Sodium Nitroprusside,SNP）对猪腔前卵泡体外生长发育的影响。结果显示,体外培养后,各处理组卵泡直径均增加,但未达显著水平（P〉0.05）;第6天1 mmol.L^-1SNP处理组卵泡存活率要显著低于1μmol.L^-1SNP（61.61%vs81.52%,P〈0.05）,与其它各组间差异不显著（P〉0.05）;卵泡成腔率在第4天以1μmol.L^-1SNP组最高,达到50%;第6天,1μmol.L^-1SNP组的卵泡成腔率显著高于0.1和1 mmol.L^-1SNP组（73.07%vs50%,47.62%,P〈0.05）,也高于对照组和0.01 mmol.L^-1SNP组,但差异不显著（P〉0.05）。培养结束（6 d）后,除1mmol.L^-1SNP组卵母细胞正常率显著低于1μmol.L^-1SNP组（71.21%vs 48.18%,P〈0.05）外,其余各组间差异不显著（P〉0.05）,其中0.001 mmol.L^-1SNP组卵母细胞正常率最高,为71.21%;0.001 mmol.L^-1SNP组COC回收率要显著高于其余各组（37.27%vs 22.88%、25.59%、20.74%和19.39%,P〈0.05）。结果表明,NO对猪腔前卵泡的存活、发育成腔及卵母细胞发育都有促进作用,但高浓度会产生毒性作用。     

Effects of Nitric Oxide on the Growth and Development of Porcine Preantral Follicles Cultured in Vitro

<正>In order to investigate the effects of nitric oxide(NO) on the growth and development of porcine preantral follicles,we treated the follicles with different concentrations of sodium nitroprusside(SNP,0, 0.001,0.01,0.1 and 1 mmol/L),a NO donor.The results showed that the follicle diameter increased during in vitro culture,but there were no significant differences between the treatments(P0.05);the survival rate in the 1 mmol/L SNP group was significantly lower than that in the 1μmol/L SNP group(61.61% vs 81.52%,P0.05),but no significant differences were found between other treatments(P0.05);the rate of antrum formation in the 1μmol/L SNP group peaked at 50%on day 4,and the rate in the 1μmol/L SNP group on day 6 was higher than that in the 0.01 mmol/L SNP group;in addition,the rate of antrum formation in the 1μmol/L SNP group was significantly higher than that in the 0.1 and 1 mmol/L SNP groups (Day 6:73.07%vs 50%,47.62%,P0.05).After 6 days of culture,the proportion of normal oocytes in the1 mmol/L SNP group was significantly lower than that in the 1μmol/L SNP group(71.21%vs 48.18%, P0.05),with no significant differences between other treatments(P0.05).The recovery rate of cumulus cells oocyte complexes(COCs) in the 1μmol/L SNP group was significantly higher than that in the controls and all other treatments(37.27%vs 22.88%,25.59%,20.74%and 19.39%,P0.05).The results indicate that during the in vitro culture of porcine preantral follicles,low concentration of NO released from SNP improves growth and development of oocytes and follicular antrum formation while high levels of NO are toxic to follicular survival.     

Effects of N‐acetyl‐cysteine and N‐acetyl‐cysteine‐amide Supplementation on In Vitro Matured Porcine Oocytes

This study was conducted to evaluate the effects of different concentrations of the antioxidant N‐acetyl‐cysteine (NAC) supplemented to the maturation medium on porcine embryo development. Concentrations of NAC and its synthetic derivative, NAC‐amide (NACA) were evaluated for effects on nuclear maturation, fertilization success and embryo development. Concentrations of NAC (0, 0.5, 1.0, 1.5, 2.0, 2.5 and 5.0 mm ) were supplemented to maturing oocytes, and embryo development was analysed at 48 and 144 h post‐fertilization. There were no differences among cleavage rates for any of the treatment groups. Blastocyst formation for 1.5 mm NAC (56.5 ± 9.2%) was higher (p < 0.05) than all other supplementations. There were no differences in nuclear maturation or fertilization or in cleavage rates when comparing 1.5 mm NAC and 1.5 mm NACA supplementation to the control. Blastocyst formation for 1.5 mm NAC (44.4 ± 4.7%) and 1.5 mm NACA (46.2 ± 3.4%) supplementation were higher (p < 0.05) than the control (32.1 ± 6.2%) oocytes. These results indicate that supplementing 1.5 mm of NAC or NACA to the oocyte maturation medium increased the percentage of viable embryos reaching the blastocyst stage of development.     

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.     

哺乳动物腔前卵泡体外培养及研究进展

哺乳动物卵巢内的原始卵泡库是雌性生殖资源的储存库,但大部分卵泡在腔前阶段已退化闭锁了,这无疑是对这一资源的巨大浪费。因此,越来越多的学者致力于腔前卵泡的开发和利用,建立了一系列的培养体系,尤其在小鼠上做的比较成功,已经得到了少量后代。文章主要就卵泡的发生发育过程,腔前卵泡的分离方法,培养基的选择,不同直径腔前卵泡培养体系的建立,影响腔前卵泡发育的因素及研究进展进行了阐述,为进一步完善腔前卵泡培养体系,揭示其发育机理提供参考。     

Effect of Follicle Size on In Vitro Maturation of Pre‐Pubertal Porcine Cumulus Oocyte Complexes

Very small follicles (<3.0 mm diameter) are over‐represented on the surface of ovaries of non‐cycling pigs, and the oocytes collected from these follicles generally have reduced developmental competence in vitro. This study examined the effect of follicle size on the nuclear maturation (n = 608), the potential of parthenogenetic activation (n = 243) and the cyclic AMP (cAMP) content of pre‐pubertal porcine oocytes (n = 480). In addition, the influence of follicle size on steroid hormone synthesis was analysed. Cumulus oocyte complexes (COCs) flushed from small (2.5–4.0 mm) or large (4.5–6.0 mm) ovarian follicles were cultured for 0, 28 and 46 h. After 46 h of IVM, a greater proportion of oocytes from 4.5‐ to 6.0‐mm follicles reach metaphase II (MII) compared with those from follicles with 2.5–4.0 mm of diameter (96.1 vs 77.0%, respectively; p < 0.001). Parthenogenetic activation of oocytes from large follicles produced higher developmental rates than oocytes from large follicles (p < 0.05). At 28 h, the IVM medium with oocytes from large follicles contained significantly more 17ß‐oestradiol (E₂) than the medium with oocytes from small follicles (5.55 vs 3.45 ng/ml, respectively; p < 0.05) and at 46 h, the medium with oocytes from small follicles contained significantly more progesterone (P₄) than the medium with oocytes from large follicles (276.7 vs 108.2 ng/ml, respectively, p < 0.05). Porcine oocytes from large follicles have higher nuclear and cytoplasmic maturation capacities, but the differences did not appear to be cAMP‐mediated. Our findings also suggest that COCs from small follicles undergo more intensive luteinization than COCs from large follicles. The results show that oocytes from follicles with a diameter greater than 4.0 mm are more suitable for in vitro studies.     

Antral Follicle Populations and Embryo Production – In Vitro and In Vivo – of Bos indicus–taurus Donors from Weaning to Yearling Ages

Interest in indicus–taurus cattle has been increasing, as these animals are likely to present the best characteristics of Zebu and European bovine breeds. The aim of this study was to compare the embryo production of indicus–taurus donors with high vs low antral follicle counts obtained by ovum pickup/in vitro production (OPU/IVP) and superovulation (SOV)/embryo collection. Braford females at weaning age (3/8 Nelore × 5/8 Hereford, n = 137, 9 ± 1 month old) were subjected to six serial ovarian ultrasonographs and were assigned to two groups according to the number of antral follicles ≥3 mm as follows: G‐High antral follicular count (AFC, n = 20, mean ≥40 follicles) and G‐Low AFC (n = 20, mean ≤10 follicles). When the females (n = 40) reached 24 months of age, they were subjected to both OPU/IVP and SOV/embryo collection. The average number of follicles remained highly stable throughout all of the ultrasound evaluations (range 0.90–0.92). The mean number of COCs recovered (36.90 ± 13.68 vs 5.80 ± 3.40) was higher (p < 0.05) for females with high AFC, resulting in higher (p < 0.05) numbers of total embryos among females with high vs low AFC (6.10 ± 4.51 vs 0.55 ± 0.83). The mean number of embryos per collection was also higher (p < 0.05) for G‐High vs G‐Low (6.95 ± 5.34 vs 1.9 ± 2.13). We conclude that a single ultrasound performed at pre‐pubertal ages to count antral follicles can be used as a predictor of embryo production following IVP and SOV/embryo collection in indicus–taurus females.     

Expression of Oestrogen Receptor α and Oestrogen Receptor β in the Uterus of the Pregnant Swine

The uterus is a well‐known target of endocrine, paracrine and autocrine acting molecules among which steroid hormones are of special importance. The objective of our work was to localize oestrogen receptors (ERα and ERβ) mRNA and protein in the pig uterus throughout pregnancy (10, 18, 32, 50, 71, 90 days post coitum) using RT‐PCR, Western‐blot and immunohistochemistry. The present study is the first one to demonstrate the presence of ERs protein in the porcine uterus not only at the beginning but also at mid‐ and late pregnancy. In the pregnant swine, ERα was immunolocalized in the luminal epithelium (LE) and glandular epithelium (GE) and the myometrium of the uterus with differences in the intensity of staining at different stages of pregnancy studied. The LE and GE of pregnant swine stained for ERβ regardless of the day of pregnancy examined, whereas only a few cells within the myometrium showed a weak immunoreactivity. Western blot analysis confirmed the presence of ERα and ERβ proteins on all investigated days of gestation. The expression of ERα and ERβ mRNA was detected by RT‐PCR in all examined samples corresponding to each of the consecutive stages of pregnancy. The obtained results show that ERα is more abundant in comparison to ERβ within the porcine pregnant uterus. The presence of ERα and ERβ in all compartments of the pig uterus during pregnancy may indicate direct action of oestrogens on proliferation and differentiation of these cells.