妊娠山羊黄体组织中ghrelin的表达

运用组织学和免疫组化方法对不同妊娠时期山羊黄体的组织学结构及ghrelin的分布规律进行了研究,结果表明:黄体外覆结缔组织性被膜,实质主要由粒性(大)黄体细胞和膜性(小)黄体细胞组成;在各时期均存在大黄体细胞的退化现象,退化黄体细胞数量随周期进程逐渐增多,结构变化表现为:细胞形态多变,胞质呈强嗜酸性,胞核固缩或碎裂.ghrelin阳性反应主要位于被膜和黄体细胞,各阶段被膜上均存在ghrelin表达,主要为纤维及血管的着色;黄体内ghrelin主要定位于大黄体细胞,另外部分小细胞也有表达,从分布范围和染色强度看,呈现出少-多再渐少的变化趋势;结合各周龄山羊黄体组织中ghrelin阳性产物相对表达量的分析,表明在妊娠早中期,黄体组织内有较高的ghrelin表达,而在妊娠后期表达较低.不同妊娠时期山羊黄体组织中ghrelin的这种周期性表达模式与黄体的功能相适应,提示ghrelin的表达与黄体的功能密切相关.     

IFN-γ在胚泡植入期大鼠卵巢的分布

为研究IFN-γ对妊娠的免疫调控机制,本文采用免疫组化SP法,对大鼠胚泡植入期卵巢组织内IFN-γ的分布进行了研究。结果表明,IFN-γ在不同发育阶段卵泡的颗粒细胞中均有分布,原始卵泡、初级卵泡、次级卵泡和成熟卵泡的卵母细胞呈中等阳性反应,在原始卵泡、初级卵泡及次级卵泡的卵泡内膜细胞呈IFN-γ强阳性反应,成熟卵泡内膜细胞则呈弱阳性反应;在卵巢组织基质细胞、血管内皮细胞、粒性黄体细胞、生殖上皮也出现强阳性反应,卵泡液呈阳性着色。结果表明,妊娠有机体生理剂量的IFN-γ通过影响卵巢功能参与了胚泡的植入,与妊娠的建立和维持密切相关。     

瘦素及其长型瘦素受体在雌性小鼠体内的表达

采用免疫组织化学SP染色法对长型瘦素受体在雌性小鼠生殖周期不同阶段的下丘脑、卵巢、子宫中细胞定位和分布进行了研究,并用ELISA方法对小鼠血清中的瘦素浓度进行了检测。结果显示,下丘脑神经元胞质中有棕褐色阳性颗粒,且阳性细胞数量随妊娠日龄增加逐渐增加,妊娠期与间情期阳性细胞数差异显著(P0.05);在卵巢中,卵母细胞胞质中有长型瘦素受体表达,随卵泡的发育,卵泡细胞中免疫反应阳性细胞数量逐渐增加;在胚泡附植期,瘦素受体在子宫腺和子宫内膜上皮细胞中大量表达。妊娠期血清瘦素浓度均高于间情期,且瘦素浓度从间情期到妊娠4日龄,呈上升趋势,妊娠5日龄稍下降,后随妊娠日龄逐渐增加。结果表明,瘦素及其长型受体能够促进卵泡发育,有利于小鼠胚泡的附植。     

ERα免疫反应产物在山羊卵巢中的分布

为了研究不同生殖周期山羊卵巢中雌激素受体α（estrogen receptor α，ERα）的表达，本试验应用高灵敏度的免疫组织化学SP法检测了ERα免疫反应产物在不同生殖周期山羊卵巢中的分布。结果显示，ERα免疫反应产物主要存在于成熟卵泡的颗粒细胞、发情期和妊娠期生长卵泡的部分颗粒细胞、原始卵泡卵母细胞胞质、粒黄体细胞胞质及膜黄体细胞胞核、卵泡膜细胞和类固醇细胞胞质；间情期生长卵泡的颗粒细胞中的ERα免疫反应产物较少，主要存在于细胞膜上；卵母细胞中ERα免疫反应产物在初级卵泡阶段含量较少，从次级卵泡开始其含量很丰富。以上结果提示，ERα以激活不同信号途径的方式参与了对卵泡生长、排卵及妊娠维持的调控，这为研究雌激素及雌激素受体α在卵巢中的作用及其生殖调控机理提供了形态学资料。     

血管活性肠肽及其基因在小鼠卵巢中的定位

为探明血管活性肠肽（VIP）及其基因在青春期、妊娠期、哺乳期和泌乳后期小鼠卵巢中分布的变化规律,分别采用免疫组织化学PV-9000两步法和原位杂交法确定VIP及其mRNA在卵巢中的定位。结果显示：VIP及其基因主要定位在是黄体细胞和卵泡上,通过单因素方差分析表明在小鼠卵巢内,泌乳后期表达量相对其他3期差异极显著（P〈0.01）。VIP及其基因在不同生理时期内小鼠卵巢的表达量不同,即随着日龄的增加表达量呈上升趋势,且泌乳后期表达量最大。     

溶血磷脂酸受体1-3在牦牛不同时期卵巢中表达定位

为探究正常生理下条件下溶血磷脂酸受体(LPAR)1-3在牦牛不同时期卵巢(卵泡期、黄体期、妊娠期)表达及生物学作用,利用RT-qPCR检测LPAR1-3基因在不同时期卵巢上的表达,同时利用蛋白质免疫印迹、免疫组织化学(IHC)等方法对LPAR1-3基因和蛋白的表达进行分析和定位。结果显示,LPAR1-3在牦牛不同时期卵巢上均有表达,其中LPAR1-3的表达量在妊娠期显著高于卵泡期和黄体期(P0.05),卵泡期表达量最低;在卵泡期、黄体期、妊娠期LPAR3的表达量均显著高于LPAR1和LPAR2(P0.05),LPAR2的表达量最低;IHC结果显示,LPAR1-3在卵巢生殖上皮、颗粒细胞、卵泡液、卵泡膜和黄体细胞均有表达。研究结果提示LPAR1-3可能在卵泡生长、发育、排卵以及维持妊娠等一系列生殖过程中发挥重要的作用,其中LPAR3可能在这一过程中起主要作用,该结果有助于对牦牛繁殖机能的进一步研究,可为高原哺乳动物生殖生理的研究提供理论依据。     

Caspase-9在肉用绵羊主要生殖器官中的分布研究

通过试验研究凋亡因子Caspase-9在肉用绵羊主要生殖器官中的表达,并探讨其生理意义.利用免疫组织化学的方法对Caspase-9在肉用绵羊主要生殖器官中的表达进行研究.研究结果显示,Caspase-9只在细胞质中表达,其在卵巢中主要分布于原始卵泡、初级卵泡及颗粒性黄体细胞,而卵泡膜性黄体细胞中未见表达;黄体期,主要表达于子宫的浅层腺体腺上皮细胞、子宫内膜上皮细胞、子宫颈黏膜固有层的淋巴小结、输卵管黏膜上皮分泌细胞和纤毛细胞、峡部的浆液性腺的腺上皮细胞;卵泡期,子宫和输卵管各个部位阳性反应不明显.在正常生理情况下,Caspase-9参与了肉用绵羊主要生殖器官周期性变化的调控和子宫黏膜免疫,对生殖器官功能的稳定发挥起着重要作用.     

AR与VIPR1蛋白在不同表型鸡冠的免疫组织化学与比较组织学研究

为了研究下丘脑-垂体-性腺(HPG)轴相关基因雄激素受体(AR)与血管活性肠肽受体1(VIPR1)对鸡冠发育的影响,试验采用免疫组织化学和比较组织学方法对17羽不同冠型的16周龄宁都黄公鸡的鸡冠组织中AR与VIPR1蛋白的表达规律进行了研究。结果表明:AR与VIPR1蛋白在不同冠型中均有免疫组织化学阳性表达,宁都黄公鸡细齿冠AR与VIPR1蛋白免疫组织化学阳性细胞比率显著低于其他冠型(P<0.05),倒冠AR与VIPR1蛋白的免疫组织化学阳性细胞比率均显著高于其他冠型(P<0.05),并且在鸡冠窦状血管网内皮细胞及生发层细胞中具有较高免疫组织化学阳性反应。鸡冠中间层由于胶原纤维网较为松散,其中散乱分布的成纤维细胞具有AR和VIPR1蛋白的免疫组织化学阳性反应;几种冠型外周层均有血管分布,并具有AR与VIPR1蛋白的免疫组织化学阳性反应,相对于其他冠型细齿冠生发层中血管分布较少。说明AR与VIPR1蛋白的表达水平对鸡冠血管的诱导及扩张具有一定的影响,同时可能对外周层细胞具有迁移趋化作用,从而间接影响了鸡冠生发层的发展趋势,形成不同冠型。     

INHβB、FSHR和LHR在牦牛不同生殖生理阶段卵巢组织中的表达及差异性分析

为了从受体角度研究抑制素(INH)、促卵泡素(FSH)和促黄体素(LH)对牦牛不同生殖生理阶段卵巢发挥的功能,采用实时荧光定量PCR(RT-qPCR)、蛋白印迹技术(Western blot)和免疫组织化学技术(IHC)检测抑制素受体(INHβB)、促卵泡素受体(FSHR)和促黄体素受体(LHR)在牦牛发情期、间情期和妊娠期卵巢组织中的表达和定位。结果显示,INHβB在妊娠期牦牛卵巢组织中的表达量显著高于发情期和间情期(P<0.05),而FSHR在发情期的表达量显著高于间情期和妊娠期(P<0.05),LHR则在间情期的表达量显著高于发情期和妊娠期(P<0.05);这3个受体主要分布在卵泡颗粒细胞、卵丘细胞、黄体细胞和卵泡膜上。结果表明,INHβB、FSHR和LHR在牦牛发情期、间情期、妊娠期卵巢组织中均有表达,但其表达量存在一定差异,提示INH、FSH和LH在牦牛不同阶段对卵泡生长发育、卵泡成熟、排卵及黄体的形成发挥着不同的作用,该结果为进一步研究牦牛生殖活动中INH与FSH、LH的互作机制提供参考。     

受体山羊卵巢有无卵泡对移植妊娠率的影响

为了提高受体山羊移植妊娠率,试验在生产条件下对111只波尔山羊供体实施超数排卵处理,子宫生产胚胎,然后对977只奶山羊受体分两组实施胚胎移植,一组卵巢上只有黄体,另一组卵巢上除有黄体外,还有卵泡(卵泡较小,一般小于黄体),并对移植妊娠率进行比较分析。结果表明:有卵泡受体移植后妊娠率为52.65%;无卵泡受体移植后妊娠率为60.13%,差异显著(P<0.05),说明选择卵巢上无卵泡的受体进行移植妊娠率较高。卵巢上有卵泡的受体妊娠率为52.65%,在生产上也属于理想效果。     

促性腺激素受体在雌性水牛生殖器官的表达定位研究

为研究促性腺激素受体(FSHR、LHR)在广西雌性水牛生殖器官中的分布情况,运用免疫组化SABC法对处于不同发情周期(卵泡期、黄体期)成年水牛的卵巢、子宫、输卵管中FSHR、LHR分别进行染色定位。结果表明,FSHR/LHR阳性细胞在卵巢主要见于卵巢内膜细胞及卵泡颗粒细胞;子宫主要见于子宫内膜上皮细胞和腺体细胞;输卵管主要见于柱状上皮纤毛细胞。其中,随着发情周期不同,FSHR、LHR的表达量也有所差异,卵巢中卵泡期FSHR、LHR的表达量均高于黄体期;子宫中FSHR的表达量卵泡期高于黄体期,LHR的表达量黄体期高于卵泡期;而输卵管并没有显著差异。     

The expression of thrombopoietin and its receptor during different physiological stages in the bovine ovary

Thrombopoietin (TPO) is known to be involved in megakaryocytopoiesis, but its role in the control of ovarian function is unknown in cattle. The aims of this study were to demonstrate the expression of TPO and its receptor (c-MPL) in detail in bovine corpus luteum (CL) obtained from different stages of the oestrous cycle and during pregnancy--and to demonstrate that TPO/c-MPL system is expressed clearly in bovine follicles. Real-time RT-PCR (qPCR) and ELISA were applied to investigate mRNA expression of examined factors and TPO protein, respectively. In this investigation, increases in the concentrations of TPO protein and the mRNA expression of TPO and c-MPL were noticed during both early luteal stage and late luteal stage of the oestrous cycle. Furthermore, the expression of TPO/c-MPL system does not show any significant regulation in the CL throughout pregnancy. Highest co-expression of TPO/c-MPL system in both theca interna (TI) and granulosa cells (GC) in small follicles (<10 mm in diameter) was observed in this study that may suggest the possible role of TPO/c-MPL system in proliferation of TI and GC cells. To conclude, the results demonstrate the possible involvement of locally produced TPO/c-MPL system as a 'physiological filter' in bovine ovary where they may promote cell selection by inducing proliferation of viable cells and scavenging non-viable cells and thereby may play an important role in modulation of ovarian function.     

Expression of estrogen receptor alpha (ERalpha) and estrogen receptor beta (ERbeta) in the ovarian follicles and corpora lutea of pregnant swine

The objective of the study was to demonstrate the presence of estrogen receptor alpha (ERalpha) and beta (ERbeta) protein and corresponding mRNA in porcine ovarian follicles and corpora lutea obtained on day 10, 18, 32, 50, 71 and 90 post coitum (p.c.) using immunohistochemistry, Western blot, and RT-PCR analysis. Immunohistochemistry showed that ERalpha protein was located in the granulosa cells of ovarian follicles and the strongest immunoreaction was observed on days 32 and 50 p.c. The ERbeta protein was found mainly in theca cells of follicles as well as in luteal cells. The most intense immunoreaction was observed on day 18 p.c. within theca cells, while in the corpus luteum (CL) the intensity of ERbeta staining gradually increased and remained elevated at mid and late pregnancy. In CL by day 50 p.c. immunoreaction for ERbeta was present only in small luteal cells, but starting from day 71 to 90 p.c. it was observed in both small and large luteal cells. Western blot analysis was performed and validated data obtained from immunohistochemistry. RT-PCR results indicated that ERalpha mRNA was expressed only in ovarian follicles of the pregnant swine, while that of ERbeta in both follicles and CL. The results suggest an autocrine/paracrine role of estrogens acting via both ERalpha and ERbeta in the regulation of the ovarian function during pregnancy and for the process of successful reproduction.     

Immunohistochemical Localization of Luteinizing Hormone Receptor in the Cyclic Gilt Ovary

Luteinizing hormone receptor (LHR) is a specific membrane receptor on the granulosa and theca cells that bind to luteinizing hormone (LH), resulting in androgen and progesterone production. Hence, the regulation of LHR expression is necessary for follicle maturation, ovulation and corpus luteum formation. We examined the immunolocalization of LHR in cyclic gilt ovaries. The ovaries were obtained from 21 gilts aged 326.0 ± 38.7 days and weighing 154.6 ± 15.7 kg. The ovarian tissues were incubated with rabbit anti‐LHR polyclonal antibody. The follicles were categorized as primordial, primary, preantral and antral follicles. Ovarian phase was categorized as either follicular or luteal phases. The immunolocalization of LHR was clearly expressed in primary, preantral and antral follicles. LHR immunostaining was detected in the cytoplasm of granulosa, theca interna and luteal cells. LHR immunostaining was evaluated using imaging software. LHR immunostaining in the theca interna cells in antral follicles was almost twice as intense as that in preantral follicles (65.4% versus 38.3%, P < 0.01). LHR immunostaining was higher in the follicular phase than in the luteal phase (58.6% versus 45.2%, P < 0.05). In conclusion, the expression of LHR in the theca interna cells of antral follicles in the follicular phase was higher than in the luteal phase. The expression of LHR in all types of the follicles indicates that LHR may impact follicular development from the primary follicle stage onwards.     

Immunolocalization of steroidogenic enzymes in the corpus luteum and placenta of the Japanese Shiba goat

In this study, we performed immunohistochemistry of cholesterol side-chain cleavage cytochrome P450 (P450scc), 3beta-hydroxysteroid dehydrogenase (3betaHSD), cytochrome 17alpha-hydroxylase P450 (P450c17), and cytochrome P450 aromatase (P450arom) in the corpus luteum and placenta of Shiba goats. The aim was to clarify the steroidogenic capability of the corpus luteum and placenta of Shiba goats. Ovaries containing corpora lutea were obtained from four adult Shiba goats during the luteal phase (day10; n=2) and pregnancy (90 and 120 days of gestation). Placenta was obtained from one Shiba goat on day 120 of gestation. The sections of the ovaries and placentae were immunostained using the avidin-biotin-peroxidase complex method (ABC) with polyclonal antibodies generated against steroidogenic enzymes of mammalian origin. All luteal cells expressed P450scc, 3betaHSD, P450c17 and P450arom. The distribution of P450scc, 3betaHSD, P450c17 and P450arom were not different during the luteal phase and pregnancy. P450arom showed a weak positive staining in late pregnancy (120 days). In addition, immunoreactions for P450c17 and P450arom were observed in syncytiotrophoblast of the placenta of one Shiba goat. These results indicate that, in Shiba goats, corpus luteum is not only an important source of progesterone but also has the ability to synthesize androgen and estrogen during the luteal phase and pregnancy. Also the placenta has the ability to synthesize androgen and estrogen in late pregnancy.     

雌性牦牛主要生殖器官中DBI的表达与定位分析

为加深对DBI基因功能的探究,揭示其在牦牛生殖生理过程中的作用.本试验采集健康母牦牛(3～6岁)繁殖周期不同阶段(卵泡期、黄体期和妊娠期)的卵巢、输卵管和子宫组织,共分为9组,每组设置3个生物学重复.采用实时荧光定量PCR (qRT-PCR)和蛋白免疫印迹技术(Western blotting,WB)检测牦牛DBI在繁...     

The ovarian insulin and insulin-like growth factor system with an emphasis on domestic animals

Insulin and insulin-like growth factors (IGFs) have direct effects on cultured ovarian cells. These effects include stimulation of granulosa cell mitogenesis, granulosa and luteal cell progesterone production, and thecal cell androgen production and appear similar among species. However, species differences exist with regard to insulin and IGF-I effects on granulosa cell estradiol production. In addition to endocrine effects of insulin and IGFs, IGFs are produced by granulosa, thecal, and luteal cells, allowing for an intraovarian autocrine and paracrine system. Granulosa, thecal, and luteal cells contain receptors for insulin and IGFs, and these receptors appear to mediate the effects of insulin and IGFs. Adding to the complexity of the regulatory role of IGFs is the presence of IGF-binding proteins (IGFBPs) within the ovary. These IGFBPs are produced by granulosa, thecal, and luteal cells, and their production is hormonally regulated. Evidence for a coherent mechanism by which insulin, IGFs, and IGFBPs interact and regulate ovarian function in vivo has yet to be found.     

Expression Pattern of Markers in the Canine Ovarian Cycle

Although hormonal changes during different phases of the oestrous cycle of bitches are well-described, knowledge about the luteal phase and anoestrus is incomplete. Furthermore, which paracrine and autocrine critical factors that differentiate between follicles destined for atresia and those that continue to develop are unknown. In this study, ovarian tissue was collected from 39 healthy bitches that were subject to ovariectomy or ovariohysterectomy for surgical neutering or medical purposes such as unwanted pregnancy. Bitches were allocated to different groups depending on the stage of the oestrous cycle. Serum progesterone, LH, FSH and 17β-estradiol (E₂) -levels were determined and immunhistochemistry was performed for a variety of receptor antigens; Ki-67, vimentin, pan cytokeratin antibody, p53 and oestrogen receptor (ER) α antigens. Marked differences were found in progesterone concentration between pregnant and non-pregnant animals. Oestrogen concentration was significantly lower in pro-oestrus and ovulation than during the luteal phase. Although progesterone could be detected in cytoplasm of ovarian cells at each stage, its presence was restricted to follicular cells during anoestrus. A strong presence of AE1/AE3, vimentin and p53 was found in each oestrous stage, in contrast with Ki67. The localization of ERα appeared to vary during the oestrous cycle, a phenomenon that suggests a switch between target cells of oestrogen; while as a proliferation marker, the mild reaction of p53 during parturition suggests an apoptotic process at this stage of the cycle.