牛卵泡颗粒细胞无血清培养的形态学观察

McCoy’s5a为基础培养液添加硒、转铁蛋白、雄烯二酮、谷氨酰胺 ,对牛卵巢上大 (直径 >8mm)、中 ( 4mm <直径 <8mm)、小 (直径 <4mm)非闭锁卵泡的颗粒细胞进行无血清培养。McCoy’s5a无血清培养中的颗粒细胞形态与分化特点类似于体内情况 ,说明颗粒细胞在该培养条件下 ,其功能可能更接近体内的生理状况     

Notch2在牛黄体化颗粒细胞中的调控作用

试验旨在探究Notch2在牛黄体化颗粒细胞中的调控机制.将免疫荧光鉴定采集到的原代颗粒细胞,通过慢病毒过表达技术感染牛黄体化颗粒细胞,最后应用qRT-PCR技术检测周期基因、抗氧化基因和孕酮合成相关基因的表达.结果显示:牛黄体化颗粒细胞过表达Notch2胞内功能片段NICD2后,周期基因CyclinD1、CyclinD...     

Association Between Leptin, LH and its Receptor and Luteinization and Progesterone Accumulation (P4) in Bovine Granulosa Cell In Vitro

A full understanding of the cellular events that occur during in vitro luteinization of bovine granulosa cells, stimulated by LH and by leptin, is a complex goal that has not been completely achieved. The aim of this work was to study the effects of leptin, LH and leptin + LH on progesterone accumulation (P4) and on the expression of LH receptors (LHR) in bovine granulosa cells in culture. The results confirm that this in vitro model is representative of functional and morphological luteinization/differentiation. The pattern of expression of LHR with time of incubation was an important marker of in vitro luteinization, with 50–90% of cells expressing LHR by 96 h in culture. Cytoplasmic lipidic droplets were highly abundant in granulosa cells, suggesting a sufficient source of precursors for steroid hormone synthesis: P4 accumulation ranged between 40 and 550 ng/ml. In addition, a positive correlation ( r  = 0.58, p < 0.05) between the expression of LHR and accumulation of P4 throughout the time of incubation was observed. The expression of LHR was inhibited by LH and leptin + LH treatment. In conclusion, we found an inverse modulation between the expression of LHR and the concentration of LH, and the expression of LHR could be regulated by P4 produced by the luteinized granulosa cells. These findings are contributing to elucidate further the panoply of interactions during the differentiation of granulosa cells into luteal cells in vitro .     

瘦素和FSH对绵羊卵泡颗粒细胞孕激素分泌的影响

试验旨在探明瘦素和卵泡刺激素(FSH)对绵羊卵泡颗粒细胞孕激素分泌的影响并建立瘦素和FSH之间的相互作用.从屠宰场收集健康母羊的卵巢,机械分离卵泡,收集卵泡颗粒细胞.在细胞培养液中加入不同浓度的FSH(0 U/mL,2.5 U/mL,5 U/mL,7.5 U/mL,10 U/mL)培养24 h和48 h,通过MTT检测...     

褪黑素对体外培养绵羊卵泡颗粒细胞分泌雌二醇和孕酮的影响

采集绵羊卵泡颗粒细胞进行体外培养,根据试验设计加入褪黑素(MLT)和HCG,分别于不同培养时间收集细胞培养液,用放射免疫测定法(RIA)测定细胞培养液中雌二醇(17β-E2)和孕酮(P4)含量。结果表明:卵泡颗粒细胞在无血清培养体系中可体外培养,并能够分泌相应17β-E2;用MLT或MLT与HCG共同处理卵泡颗粒细胞,能促进细胞分泌17β-E2,并呈时间和剂量的依赖性,但对P4的分泌没有影响。     

Expression of adiponectin and its receptors (AdipoR1 and AdipoR2) in chicken ovary: potential role in ovarian steroidogenesis

Adiponectin and its receptors (AdipoR1 and AdipoR2) mRNAs are expressed in various chicken tissues including ovary. However, the cellular expression and the role of adiponectin system have never been investigated in chicken ovary. Here, we have shown that the level of adiponectin mRNA is about 10- to 30-fold higher (p < 0.001) in theca cells than in granulosa cells from each hierarchical yellow follicle studied (F4–F1). In contrast, the level of AdipoR1 mRNA expression was about two-fold lower in theca cells than in granulosa cells (p < 0.05) whereas those of AdipoR2 was similar in both ovarian cells. Whereas expression of adiponectin mRNA increased with follicular differentiation in theca cells, it decreased in granulosa cells. In contrast, mRNA expression of AdipoR1 and AdipoR2 in both theca and granulosa cells remained stable during yellow follicle development. To determine whether adiponectin is involved in the ovarian steroidogenesis, LH (100 ng/ml)-, FSH (100 ng/ml)- and IGF-1 (100 ng/ml)-induced progesterone production was measured in absence or presence of human recombinant adiponectin (10 μg/ml) for 36 h in cultured granulosa cells from F1, F2 and mixed F3 and F4 follicles. In absence of LH, FSH and IGF-1, adiponectin treatment had no effects on progesterone production whatever vitollegenic follicle studied. However, it increased by about two-fold IGF-1-induced progesterone secretion in F2 and F3/4 follicles whereas it halved progesterone production in response to gonadotropins (LH and FSH) in F3/4 follicles. Thus, in chicken, adiponectin, mainly expressed in theca cells, could exert paracrine or autocrine effect on the ovarian steroidogenesis.     

牛卵泡颗粒细胞中生长相关基因的表达研究

随着卵巢卵泡的生长,卵泡内雌激素分泌量逐渐上升,雌激素通过其受体(Estrogen Receptors,Esrs)对卵泡的调节作用也在发生改变。该研究通过手术法获取牛卵巢上不同直径的卵泡颗粒细胞,对颗粒细胞中雌激素受体及相关基因表达进行研究。结果表明:Esr1和Esr2都能在牛卵泡颗粒细胞中表达,且随着卵泡的生长而显著下降(P0.05);Fshr的表达随着卵泡的生长而显著上升(P0.05),但在中、大型卵泡颗粒细胞中差异不显著(P0.05);Lhr在大型卵泡颗粒细胞中的表达量极显著高于小、中型卵泡(P0.01);随着卵泡的生长,Cox2在颗粒细胞中的表达显著上升(P0.05),而Hsd17b1的表达则显著下降(P0.05)。综上所述,随着卵泡生长,卵泡颗粒细胞对Esr1、Esr2作用的依赖性逐渐减弱;大卵泡在排卵前Lhr表达量急剧上升,同时诱导Cox2表达量显著上升而Hsd17b1表达量显著下降,表明大卵泡已启动排卵反应。     

A Retrospective Analysis of 2,253 Cases Submitted for Endocrine Diagnosis of Possible Granulosa Cell Tumors in Mares

Endocrine diagnoses of granulosa cell tumors (GCTs) in the mare are frequently based upon determination of serum concentrations of inhibin, testosterone, and progesterone (GCT panel). In the present study, we examined results from 2,253 samples submitted for determination of GCT panel. In an additional subset of samples (n = 75), diagnosis of GCT was confirmed based upon surgery or necropsy. The objective of the current study was to examine the agreement between serum inhibin and testosterone concentrations in mares with serum progesterone concentrations less than or ≥1 ng/mL. Across all samples, elevations in serum inhibin or testosterone were noted in 15.2% or 17.3% of samples, respectively. For samples with progesterone <1 ng/mL, more (P = .001) samples had elevated inhibin than elevated testosterone, whereas for samples with progesterone ≥1 ng/mL, more (P < .0001) samples had elevated testosterone than elevated inhibin. For samples with progesterone <1 ng/mL, the agreement between elevated inhibin and testosterone was greater than that for samples with progesterone ≥1 ng/mL. In a subset of 53 samples with progesterone ≥1 ng/mL, 28 samples had endocrine evidence of a granulosa cell tumors based upon elevations of serum anti-Müllerian hormone, and 4 of these samples had endocrine evidence of pregnancy based upon estrone sulfate concentrations. For samples from mares with confirmed GCTs, 85% and 55% had elevated inhibin or testosterone, respectively. Based upon endocrine diagnosis, GCTs occur in mares with elevated progesterone and/or estrone sulfate, albeit at a low rate.     

Granulosa cell tumour in two speyed bitches

Granulosa cell tumours are uncommon ovarian tumours in the bitch and are rare in speyed bitches. This case report describes two cases of granulosa cell tumour in bitches that were speyed at less than 1-year-of-age. Both animals presented with persistent vulval bleeding. Although the majority of granulosa cell tumours are large enough to be palpated by the time of presentation, both tumours were relatively small. Granulosa cell tumour is a possible complication of incomplete ovarian excision at the time of ovariohysterectomy. In cases of granulosa cell tumour in previously speyed bitches, with no evidence of metastases, tumour resection should be curative. Ovaries should be double-checked at the initial ovariohysterectomy to ensure all normal ovarian tissue has been excised.     

牛卵泡闭锁与颗粒细胞凋亡基因表达研究

颗粒细胞凋亡与卵泡闭锁是颗粒细胞功能基因及凋亡相关基因的表达结果,通过手术法获取三种不同直径的牛卵泡(小型卵泡2 mm;中型卵泡2~6 mm;大型卵泡6mm),分离卵泡颗粒细胞并提取总RNA,以研究不同直径卵泡中颗粒细胞相关基因的表达。结果表明,牛卵巢上小型卵泡颗粒细胞中Fshr的表达显著低于中型和大型卵泡(P0.05),而中型和大型两者之间差异不显著(P0.05);中型卵泡和小型卵泡颗粒细胞中CYP19的相对表达显著低于大型卵泡(P0.05);中型卵泡和小型卵泡颗粒细胞中促细胞凋亡基因BAX的表达显著低于大型卵泡(P0.05),而在中型和大型卵泡颗粒细胞中抑制细胞凋亡基因BCL2的表达显著低于小型卵泡(P0.05),且随着卵泡的增大而下调;Casapase 8与Caspase3的表达模式相同,其表达量均随着卵泡直径的增加显著上升(P0.05)。随着卵泡生长与直径增加,卵泡内部分颗粒细胞虽然在继续增殖,部分颗粒细胞已经开始凋亡,卵泡逐渐进入闭锁阶段。     

小鼠卵巢颗粒细胞的优化培养

为了找到小鼠卵巢颗粒细胞更加合适的体外生长环境,本研究分别向分离的小鼠卵巢颗粒细胞中加入无血清RMPI1640、含10%血清RMPI1640、无血清DMEM（高糖）、含10%血清DMEM（高糖）及含15%血清DMEM（高糖）的培养液对小鼠卵巢颗粒细胞进行体外培养。试验经观察结果显示,DMEM（高糖）总体培养效果好于RMPI1640,且含15%血清的DMEM（高糖）是较理想的培养用液。     

孕酮对体外培养的牛子宫内膜细胞上孕酮受体表达的影响

本试验分别用50、100、200和400 ng/mL浓度的孕酮处理体外培养的牛子宫内膜细胞1、3、6、12、24 h,然后收集细胞通过半定量RT-PCR分析孕酮对子宫内膜细胞孕酮受体（PR）表达的影响。结果表明：在细胞层次水平上,孕酮能够抑制子宫内膜体外培养细胞上孕酮受体的表达。     

颗粒细胞对卵泡发育的影响

卵泡发育是一个复杂的生理过程,通过间隙连接,颗粒细胞在卵母细胞的生长发育过程中起营养作用,并促进卵母细胞的成熟;颗粒细胞和膜细胞的相互作用是卵泡发育和维持正常功能的重要条件。作为卵泡发育的标志,颗粒细胞的生长分化是原始卵泡启动和生长的关键,并通过受体介导途径调控生长期卵泡的发育及卵泡闭锁,从而在卵泡发育过程中起重要的调控作用。     

Low Molecular Mass Factors from Follicular Fluid Inhibit Steroidogenesis in Bovine Granulosa Cells

Gonadotropins are required for follicular growth and differentiation, but increasing amounts of evidence indicate that intrafollicular factors modulate their effects at granulosa cell level. In order to study the effect of factors present in bovine follicular fluid, a partial purification of low molecular mass factors from fluids collected from small (< 5 mm), medium (5–8 mm) and large (> 8 mm) follicles was performed and the biological activity of these peptides on steroidogenesis of granulosa cells from small and large follicles was examined. The purification was carried out by filtration through membranes in 25 and 10 kDa molecular weight cutoffs. After filtration, samples were analysed by polyacrylamide gel electrophoresis and protein concentration was measured by spectrophotometric analysis. Granulosa cells from small and large follicles were cultured in serum‐free DMEM/Ham's F12 (1 : 1) plus transferrin (5 mg/l) and selenium (5 µg/l) for 2 days. At the end of the culture period, media were renewed and follicular extracts from two different preparations (< 25 and < 10 kDa) were added at the concentrations of 1–10–100–1000 ng/ml. After 24 h the media were collected and stored until estradiol 17β (E2) and progesterone (P4) determination by validated radio‐immuno‐assays. Basal P4 production was 18.3 ± 1.4 (mean ± SEM)and 9.8 ± 1.8 ng/24 h per 3 × 10⁴ cells from small and large follicles, respectively. Both < 10 and < 25 kDa extracts reduced P4 production by cells from both the types of follicles (p < 0.05). Basal E2 release was 671.8 ± 21.4 and 5500 ± 800 pg/24 h per 3 × 10⁴ cells from small and large follicles, respectively. Both extracts reduced E2 production by either cells from small and large follicles (p < 0.05). No differences were observed in the inhibition of steroidogenesis by purifications obtained from large, medium or small follicles. Results of this study indicate that factors present in bovine follicular fluid can reduce steroidogenesis in granulosa cells in vitro.     

促卵泡激素和胰岛素对牛卵泡颗粒细胞长期培养的影响

以McCoy’s5a为基础培养液添加硒、转铁蛋白、雄烯二酮、谷氨酰胺，对牛卵巢上大（直径〉8mm）、中（4mm〈直径〈8mm）、小（直径〈4mm）非闭锁卵泡的颗粒细胞进行无血清培养，研究添加不同浓度的促卵泡激素（FSH）和胰岛素对颗粒细胞增生、分化和激素分泌的影响。结果，FSH能诱导颗粒细胞增生及提高其雌二醇合成能力，颗粒细胞雌二醇的合成能力与生理浓度的FSH存在剂量依赖关系。胰岛素对大、中、小卵泡颗粒细胞增生和雌激素分泌有促进作用。     

FSH和胰岛素对牛卵泡颗粒细胞长期培养的影响

McCoy’s5a为基础培养液添加硒、转铁蛋白、雄烯二酮、谷氨酰胺 ,对牛卵巢上大 (直径 >8mm)、中 (4mm <直径 <8mm)、小 (直径 <4mm)非闭锁卵泡的颗粒细胞进行无血清培养 ,研究添加不同浓度的促卵泡激素和胰岛素对颗粒细胞增生、分化和激素分泌的影响。结果 :FSH可以诱导颗粒细胞增生及雌二醇合成能力 ,颗粒细胞的雌二醇合成能力与生理浓度的FSH呈剂量依赖方式。胰岛素对大、中、小卵泡颗粒细胞增生和雌二醇分泌有促进作用。     

牛卵泡颗粒细胞无血清培养的形态学观察

以McCoy＇s 5a为基础培养液添加硒、转铁蛋白、雄烯二酮、谷氨酰胺，对牛卵巢上大（直径＞8mm）、中（4mm＜直径＜8mm）、小（直径＜4mm）非闭销卵泡的颗粒细胞进行无血清培养。结果表明，McCoy＇s 5a无血清培养中的颗粒细胞形态与分化特点类似于体内情况，说明颗粒细胞在该培养条件下，其功能可能更接近体内的生理状况。