Metabonomics analysis of Zi goose follicular granulosa cells using ENO1 gene expression interference

The Zi goose is native to North-east China and is noted for its high egg production. Alpha enolase (ENO1) is a glycolytic enzyme which functions as a plasminogen receptor in follicular granulosa cells (FGCs), with several studies showing that FGCs can support follicular development. By transfecting the ENO1 interfering plasmid (shRNA) into FGCs, ENO1 expression in these cells was downregulated, suggesting the successful knock-down of ENO1 in these cells. In this knock-down model, we detected 13 metabolites from FGCs using LC/MS. When compared with the non-coding shRNA (NC) group, the lower level metabolites were (R)-(+)-citronellic acid, altretamine, 3-hydroxycaproic acid, heptadecanoic acid, cholecalciferol vitamin D3, indole, benzoic acid, capric acid, caffeic acid, azelaic acid, 3,4-dihydroxyhydrocinnamic acid and cholic acid, while oleic acid was detected at high levels. To further examine the results of metabolomics, six key metabolites were verified by gas chromatography-mass spectrometry (GC-MS). We found that vitamin D3, indole, benzoic acid, capric acid and cholic acid were significantly downregulated in the shRNA group, while oleic acid was significantly upregulated. This observation was consistent with the metabolomics data. Through these studies, we found that decreased ENO1 levels altered certain metabolite levels in FGCs.     

The impact of bisphenol S on bovine granulosa and theca cells

Bisphenol S (BPS) is an endocrine‐disrupting chemical with multiple potential mechanisms of action, including as an oestrogen receptor agonist. BPS is increasingly used in plastics and thermal receipts as a substitute for bisphenol A, which has been phased out due to concerns about human health implications. The ability of BPS to alter female reproductive function in mammals has not been widely studied, despite the importance of normal hormone signalling for female reproduction. The aim of this study was to investigate how BPS (in a wide range of doses, including very low doses) affects granulosa cell and theca cell steroid hormone production and cell viability in the bovine. Granulosa cell oestradiol production was stimulated when cells were exposed to 100 μM BPS under basal conditions, but there was no effect of BPS when cells were stimulated with follicle‐stimulating hormone (FSH). Additionally, there was no effect of BPS on granulosa cell progesterone production or cell viability under basal or FSH‐stimulated conditions. BPS did not affect theca cell androstenedione or progesterone production, or theca cell viability under basal or luteinizing hormone‐stimulated conditions. This study suggests for the first time that BPS may alter oestradiol production by bovine granulosa cells, albeit at a concentration that is unlikely to be physiologically relevant. Further studies are needed to determine the effects of BPS on the bovine oocyte and on other functions of follicular cells.     

卵泡大小和颗粒细胞对山羊卵泡卵母细胞体外成熟和体外受精的影响

用切割法采集卵泡液,收集卵丘一卵母细胞复合体（Cumulus oocytes comlexs,COCs）和自然裸卵,将部分COCs去除卵丘细胞获得机械裸卵,COCs放入体外成熟培养液中培养为成熟卵母细胞,加入获能的精子液,进行体外受精。结果表明：卵母细胞的体外成熟率和卵裂率与卵泡直径密切相关,大卵泡（80．95％,P〈0．01）和中等卵泡（75．50％,P〈0．05）的卵母细胞成熟率高于小卵泡（50．27％）;犬卯泡（53．53％）和中等卵泡（47．13％）的卵裂率显著高于小卵泡的32．26％（P〈0．05）。COCs、机械裸卵和自然裸卵的体外成熟率分别为75．0％、54．2％和10．5％,差异极显著（P〈0．01）,卵裂率分别为53．8％、10．8％和0％,差异极显著（P〈0．01）。对照组和1×10^5、1×10^6个／mL颗粒细胞组卵母细胞体外成熟率分别为68．6％、69．6％和67．8％,无显著差异（P〉0．05）,但均显著高于1×10^7个／mL（51．5％,P〈0．05）和1×10^10个／mL（35．5％,P〈0．05）颗粒细胞组,但各组间的体外受精率无显著差异（P〉0．05）。结果提示,大卵泡和中卵泡的卵母细胞的体外成熟率和卵裂率显著高于小卵泡,体外成熟培养液中添加高浓度的颗粒细胞能显著抑制卵母细胞的体外成熟。     

miRNA调控动物卵泡发育研究进展

雌性动物的繁殖潜力基于卵泡的发育,该过程受到复杂的基因网络调控。microRNA （miRNA）是一种短的、非编码RNA,在转录后调控基因表达。在过去的十年中,对动物卵泡中非编码RNA的深入研究揭示了miRNA在卵泡发育过程中的关键作用。作者简述了雌性动物卵泡发育过程,总结了miRNA调控卵泡发育的作用机制,包括原始卵泡的激活、生长卵泡的发育选择、卵泡颗粒细胞和卵泡膜细胞的生长调控等,分析了卵泡液外囊泡携带miRNA的功能。但当前卵泡miRNA的研究多集中在细胞敲低模型研究,敲除模型研究较少,因此增加细胞及活体敲除模型的研究有助于更好地了解miRNA在卵泡发育中的功能。本综述可为深入解析雌性动物繁殖性状调控的分子机制提供参考。     

水牛健康与闭锁优势卵泡中颗粒细胞凋亡的生物学特征

采用组织学苏木精伊红(HE)染色与光镜检查、荧光显微镜检查和DNA电泳技术,检查了水牛健康优势卵泡(HDF)和闭锁优势卵泡(ADF)中颗粒细胞(GCs)凋亡的生物学特征。此外还应用孕酮处理早期的ADF,应用流式细胞术(FCM)方法检查该激素诱发GCs凋亡的情况。HE染色和荧光显微镜检查结果显示,HDF和ADF都含有凋亡的GCs,只是ADF中凋亡的GCs较多。凋亡GCs的HE染色表现细胞体积缩小,染色浓黑,荧光原位检测发现核浓缩。DNA电泳分析表明,在HDF中未发现GCs的DNA降解,而在ADF中因闭锁程度不同而表现不同程度的DNA降解,出现典型的"拖尾"带,但均未出现具有细胞凋亡特征的DNA"Ladder"。FCM检查结果见到早期的ADF存在着DNA的降解,孕酮处理使DNA降解碎片显著增加。上述结果提示,在水牛的HDF中,GCs的凋亡很少,在ADF中GCs的凋亡较多。水牛ADF中GCs的死亡,少量是以凋亡方式发生的,而坏死是主要方式。     

scRNA-seq of ovarian follicle granulosa cells from different fertility goats reveals distinct expression patterns

The new technology of high-throughput single-cell RNA sequencing (10 × scRNA-seq) was developed recently with many advantages. However, it was not commonly used in farm animal research. There are few reports for the gene expression of goat ovarian follicle granulosa cells (GCs) during different developmental stages. In the current investigation, the gene expression of follicle GCs at different stages from two populations of Ji'ning grey goats: high litter size (HL; ≥3/L; 2 L) and low litter size (LL; ≤2 /L; 2 L) were analysed by scRNA-seq. Many GC marker genes were identified, and the pseudo-time showed that GCs developed during the time course which reflected the follicular development and differentiation trajectory. Moreover, the gene expression difference between the two populations HL versus LL was very clear at different developmental stages. Many marker genes differentially expressed at different developmental stages. ASIP and ASPN were found to be highly expressed in the early stage of GCs, INHA, INHBA, MFGE8 and HSD17B1 were identified to be highly expressed in the growing stage of GCs, while IGFBP2, IGFBP5 and CYP11A1 were found to be highly expressed in late stage. These marker genes could be used as reference genes of goat follicle GC development. This investigation for the first time discovered the gene expression patterns in goat follicle GCs in high- or low-fertility populations (based on litter size) by scRNA-seq which may be useful for uncovering the oocyte development potential.     

东北地区农业系统发展的阶段性特征

农业作为国民经济的基础,具有较强的内在有机性和外部关联性。宏观地考察农业系统的演变历史及发展规律,对于客观判断农业结构变动是否合理以及功能衍变是否良性,具有重要意义。东北地区农业系统的发展经历了4个阶段(渔猎系统同采集系统的分化阶段,农牧系统同渔猎系统的分化阶段,农牧渔猎长期共存互补阶段以及农耕系统同畜牧系统的分化阶段)。在每个发展阶段,农业系统的结构形式和功能表现均有所不同,其发展趋势是系统结构的多元化和功能的稳态化。     

Histological and immunohistochemical evaluation of granulosa cells during different stages of folliculogenesis in bovine ovaries

Bovine granulosa cells (GC) vary in their morphological aspect during different stages of folliculogenesis. In this study, 10 morphologically normal bovine ovaries were collected to study the structural aspects of different stages of GC using intermediate filament protein antibodies including cytokeratin AE1/AE3 (AE1/AE3), vimentin, nectin‐4 and desmin. Hormonal immunolocalization was assessed using the immunomarkers anti‐Müllerian hormone (AMH) and inhibin alpha. In addition, tumour markers and proliferation markers using c‐erbB‐2 oncoprotein and proliferating cell nuclear antigen, respectively, were investigated. The immunolabelling of AE1/AE3 in GC was strongest in the early follicle stage and gradually decreased when reaching the Graafian follicle stage. Its immunolabelling increased again as the stage progressed from stage I to stage III. The immunolabelling of inhibin alpha was inversely proportional to that of AE1/AE3 in the developing ovarian follicles as their immunolabelling is opposite to each other during folliculogenesis. AMH was immunopositive in almost all GC stages in different intensities and percentages, except for some negative staining in the atretic IV follicles. The atretic IV follicle is a unique type of atretic follicle that shows Call‐Exner body formation, which was mainly found in older cows in this study. The distinct patterns of immunoreactivity for various types of immunomarkers in the different GC stages will play an important role in diagnostic assistance of various follicle conditions, including cystic ovaries and GC tumours.     

共培养系统的体细胞类型和状态对猪胚胎早期发育的影响

经体外成熟、受精和培养获得猪胚胎 ,采用体细胞共培养研究了体细胞类型和状态对胚胎早期发育的影响。取体外成熟的不同直径卵泡 (>5 m m,2～ 5 mm)卵母细胞的卵丘团 (颗粒细胞团 ) ,培养铺层后与猪受精卵共培养 ,组间受精卵的卵裂率和发育能力无显著差异 ;根据卵巢的状况对猪输卵管上皮细胞 (POECs)的状态进行分组 ,受精卵和卵巢表面布满卵泡的 POECs共培养 ,卵裂率显著低于卵巢表面有黄体和 /或红体的 POECs共培养组 (P<0 .0 5 ) ,虽然各组间 3～ 4-细胞的发育率无显著差异 ,但卵巢表面有红体和卵泡的 POECs共培养组的 >4-细胞的发育率显著高于卵巢表面布满卵泡的 POECs共培养组 (P<0 .0 5 ) ;受精卵在共培养系统和非共培养系统中的卵裂率无显著差异 ,受精卵非共培养系统中 3～ 4-细胞的发育能力显著低于颗粒细胞共培养组 (P<0 .0 5 ) ,极显著低于 POECs共培养组 (P<0 .0 1) ,无能力突破 4-细胞继续发育 ,与颗粒细胞单层、POECs单层共培养的受精卵 >4-细胞的发育率分别为 2 4.0 %、5 3.8% ,差异显著 (P<0 .0 5 )。结果表明 ,共培养系统对胚胎体外发育的作用 ,一方面与体细胞类型有关 ,另一方面也受输卵管上皮细胞状态的影响     

能量对家畜卵泡发育的影响及调控

家畜卵泡发育是被众多因素精确调控的生理过程。日粮能量可能通过不同的机制影响卵泡发育。胰岛素、瘦素和IGF-Ⅰ可能作为代谢信号而介导能量对卵泡发育的影响,即通过下丘脑-垂体-卵巢轴调节促性腺激素释放或直接作用于卵巢。各种代谢信号之间的相互作用也是其介导作用发挥的一个重要方面。     

VEGF对绵羊卵泡颗粒细胞FSHR、E2R表达的影响

血管内皮生长因子(VEGF)可以促进颗粒细胞的增殖,但是否影响同样分布于颗粒细胞上的FSHR、E2R表达效果尚属未知.因此,通过Western blotting和荧光定量PCR分别对添加0、5、10、15、20、25 μg/L VEGF卵泡颗粒细胞中FSHR、E2R表达量及FSHR mRNA、E2R mRNA表达量进行检测.Western blotting蛋白检测结果表明,空白对照组和各个不同质量浓度VEGF处理组的颗粒细胞上均有FSHR和E2R蛋白表达,FSHR相对分子质量为75 000,E2R相对分子质量为56 000；荧光定量PCR检测结果表明,添加VEGF的各个处理组中FSHR mR-NA、E2R mRNA表达量均显著高于对照组(P＜0.05),各处理组间的FSHR mRNA之间差异不显著(P＞0.05),而VEGF添加量20、25 μg/L组的E2R mRNA表达量显著高于其他3个处理组(P＜0.05),并且这2个组间则差异不显著(P＞0.05),其余3个处理组间亦差异不显著(P＞0.05).     

Ovarian follicle development and genital tract characteristics in different birthweight gilts at 150 days of age

In the last decades, selection for improved prolificacy has resulted in higher litter sizes and has thereby increased the proportion of low birthweight (LW) piglets. It is well documented that LW piglets have lower growth performance, muscle accretion and poor carcass quality. However, little is known about the relations of birthweight with subsequent reproductive performance in gilts. This study investigated the effects of birthweight on reproductive tract and ovarian follicle development in 150‐day‐old gilts. Twenty eight female pigs of different birthweight ranges (high‐HW: 1.8–2.2 kg; low‐LW: 0.8–1.2 kg) from higher parity commercial sows were reared until 150 days of age, and their body weights were recorded at weaning, end of nursery and end of the grower‐finisher phase. The animals were killed and their reproductive tracts collected for biometrical and histomorphometrical analysis. LW gilts showed significantly lower body weights and growth rates during all phases of production compared to their HW counterparts (p < .01). Most biometrical measurements of the reproductive tract were similar between the experimental groups, except vaginal length and the gonadossomatic index (relative ovarian weight), which were affected by birthweight class (p < .05). LW females also showed fewer medium size (3–5 mm; p < .01) ovarian follicles, pre‐antral follicles (p < .07) and more atretic follicles per ovarian cortex area (p < .05). Therefore, besides the effects on post‐natal growth performance, birthweight affects vaginal length and the follicular dynamics process, which may impair the reproductive performance of replacement gilts.     

诱导荷斯坦犊牛卵泡发育及体外受精研究

从日龄、培养条件和供体个体差异3个方面对4~9周龄犊牛JIVET(juvenile in vitro embryo transfer)技术的影响进行了初步研究。试验结果如下:不同日龄的10头母犊激素诱导后,30~40 d母犊平均获卵数为(31.8±9.75)枚,46~63 d的母犊平均获卵数为(21.8±11.67)枚,二者差异不显著(P>0.05);犊牛胚胎在3气(5%CO2,5%O2,90%N2)条件下和与颗粒细胞共培养条件(5%CO2,95%空气)下卵裂率分别为48.5%和54.1%,二者差异不显著(P>0.05),但是桑葚胚率差异显著(0%,15.2%,P<0.05);供体间个体差异很大,70%(7/10)的个体对激素诱导反应良好。     

miR-1307在猪卵巢颗粒细胞周期中的作用

为了解猪miR-1307序列和结构特征,阐明其在猪卵巢颗粒细胞周期中的作用,利用生物信息学方法分析猪miR-1307的序列特征、染色体定位和潜在的生物学功能;在体外培养的猪卵巢颗粒细胞中转染miR-1307模拟物(mimics)和抑制剂(inhibitor),利用流式细胞术检测细胞周期。结果显示:猪miR-1307前体序列长度为80 bp,其核苷酸序列(包括成熟序列和种子序列)和基因组定位等与哺乳动物其他物种高度一致;功能富集分析发现miR-1307靶基因富集在rRNA分解代谢进程和蛋白酪氨酸磷酸酶活性等多个重要的生物学进程和分子功能中;在猪卵巢颗粒细胞中,过表达miR-1307可使G0/G1期细胞比例增加,S期细胞比例显著降低(P<0.05);抑制miR-1307可使G0/G1期细胞比例降低,S期细胞比例显著增加(P<0.05)。结果表明:miR-1307是猪卵巢颗粒细胞周期的重要调节因子。     

输卵管和颗粒细胞单层对牛体外受精胚胎发育的影响

以屠宰场牛卵巢为试验材料,研究输卵管细胞单层(OCM)和颗粒细胞单层(GCM)对牛卵母细胞体外成熟(IVM)、体外受精(IVF)和体外培养(IVC)后胚胎发育能力的影响。(1)从卵泡抽取卵丘卵母细胞复合体(COCs),并根据卵母细胞外面卵丘细胞的层数将其分为3类:1级(≥4层);2级(2～3层);3级(0～1层)。作分别在IVM和IVC培养液中添加GCM(1×106个/mL)与不添加的对比试验。结果显示:添加GCM对1级卵母细胞的卵裂率、6～8细胞发育率和囊胚率无明显影响(P>0.05);但添加GCM的2级、3级卵母细胞,受精后的卵裂率、6～8细胞发育率和囊胚率分别高于未添加组(P<0.05)。(2)所有卵母细胞(包括COCs和裸卵)被随机分为3个组,在其IVM和IVC培养液中分别添加OCM、GCM或不添加体细胞(对照组)。结果显示:OCM和GCM组的卵裂率、6～8细胞发育率和囊胚率均高于对照组(P<0.05),而两试验组之间差异不显著。     

Analysis of PPP1R11 expression in granulosa cells during developmental follicles of yak and its effects on cell function

The aims of this study were to analyse the protein phosphatase 1 regulatory subunit 11 (PPP1R11) expression and cellular localization in yak follicles and investigate its effects on cell proliferation, apoptosis and oestrogen secretion in granulosa cells (GCs). Ten healthy and non-pregnant female yaks (4-year-old) were used as experimental animals. The mRNA relative expression level of PPP1R11 in GCs from small (<3.0 mm), medium (3.0–5.9 mm) and large (6.0–9.0 mm) follicles was detected by RT-qPCR, and the cellular localization of PPP1R11 protein was detected by immunohistochemistry staining (IHC). After isolation, culture and identification of yak GCs in vitro, si-PPP1R11 and si-NC (negative control) were transfected into GCs. RT-qPCR and immunofluorescence staining were used to evaluate the interference efficiency, and ELISA was performed to detect oestrogen concentration. Then, EdU staining and TUNEL staining were conducted to analyse cell proliferation and apoptosis. In addition, the oestrogen synthesis, proliferation- and apoptosis-related genes were detected by RT-qPCR after knockdown PPP1R11. The results showed that PPP1R11 is mainly located in ovarian GCs, and the expression levels of PPP1R11 in GCs from large follicles were significantly higher than that from medium and small follicles. Transfection of si-PPP1R11 into GCs could significantly inhibit the expression of PPP1R11. Interestingly, the oestrogen secretion ability and the expression level of oestrogen pathway-related genes (STAR, CYP11A1, CYP19A1 and HSD17B1) were also significantly downregulated. Moreover, the proportion of positive cells was decreased, and cellular proliferation-related genes (PCNA, CCNB1 and CDC25A) were significantly downregulated after knockdown PPP1R11. However, the proportion of apoptotic cells was increased, and apoptosis-related genes (BAX, CASP3 and P53) were significantly upregulated. Taken together, this study was the first revealed the expression and cellular localization of PPP1R11 in yak follicles. Interference PPP1R11 could reduce oestrogen secretion, inhibit proliferation and promote apoptosis in GCs, which provided a basis for further studies on the regulatory mechanism of PPP1R11 in follicle development.     

不同生长时期沙打旺不同部位及其植株的化感作用研究

研究了营养生长期、花果期、成熟期沙打旺(3年生)不同部位及其植株的化感作用.结果显示,1)不同生长时期沙打旺各个部位(如根、茎、叶)及其植株的水提液(1:60 g/mL,下同)均对受体萝卜的幼苗生长显示化感活性.表明沙打旺释放化感物质可能有多种途径(如地上部淋溶、根系分泌等).2)各个生长时期沙打旺的不同部位及其植株的水提液通常对受体植物的幼苗生长显示化感抑制作用,而且以叶的抑制作用最为显著.表明叶片是整个生长周期内集中表现沙打旺化感作用的特定部位.3)沙打旺的单一部位(地下部除外)及其完整植株的水提液在花果期的化感作用较成熟期强烈,而成熟期的化感作用强度又较营养生长期为高.表明花果期可能是沙打旺表达化感作用的关键时期.本研究对农业生产实践中沙打旺草地的管理与应用具有积极意义.     

AQP8 participates in oestrogen-mediated buffalo follicular development by regulating apoptosis of granulosa cells

Aquaporins (AQPs), a family of small membrane-spanning proteins, are involved in fluid transport, cell signalling and reproduction. Regulating AQP8 expression influences apoptosis of granulosa cells (GCs), ovarian folliculogenesis, oogenesis and early embryonic development in mice, but its role has never been investigated in other species. The aim of the present study was to characterize the AQP8 function in buffalo follicular development. The expression pattern of AQP8 in buffalo follicle was analysed by immunohistochemistry method. 17β-Estradiol (E2) or oestrogen receptor antagonist ICI182780 was used to treat GCs cultured in vitro, and the expression of AQP8 was detected using qRT-PCR. Its roles in apoptosis of buffalo GCs were investigated by shRNA technology. AQP8 was found to be expressed higher in secondary follicles (p < .05), and its mRNA level in GCs was upregulated by E2 via receptor-mediated mechanism in a dose-dependent manner. A 732-bp buffalo AQP8 coding region was obtained, which was highly conserved at the amino acid level among different species. AQP8-shRNA2 had more effective inhibition on target gene than AQP8-shRNA1 (66.49% vs. 58.31%) (p < .05). Knockdown of AQP8 induced GCs arrested at G2/M stage and occurred apoptosis. Compared with the control group, higher Caspase9 expression were observed in AQP8-shRNA2 lentivirus infected GCs (p < .05), while Bcl-2 and Bax expression levels had no obvious change (p > .05). Altogether, the above results indicate that AQP8 is involved in oestrogen-mediated regulation of buffalo follicular development by regulating cell cycle progression and apoptosis of GCs.