不同营养水平对湖羊黄体期血液理化指标及卵泡发育的影响

【目的】研究不同营养水平对湖羊黄体期血液理化指标及卵泡发育的影响。【方法】选择28只经产母羊,于发情周期第6天分别按0.5倍体重维持需要量（R组）,1倍体重维持需要量（C组）和1.5倍体重维持需要量（S组）饲喂6 d,第12天每组屠宰6头;剩下的湖羊用于观察发情;分别于发情周期第7、8、10和12天采血。【结果】随着营养水平的提高,≥3.5 mm的卵泡数量显著增加（P＜0.05）、2.5-3.5 mm的卵泡数量显著降低（P＜0.05）,平均发情周期缩短（P＜0.05）,血液尿素、胆固醇和游离脂肪酸含量显著下降（P＜0.05）,甘油三酯含量显著提高（P＜0.05）;S组乳酸脱氢酶活性显著高于C组（P＜0.05）;血液尿酸、血氨、高密度脂蛋白、低密度脂蛋白浓度和谷草转氨酶和谷丙转氨酶活性差异不显著（P＞0.05）,但尿酸、血氨、低密度脂蛋白含量和谷草转氨酶活性具有时间效应（P＜0.05）。【结论】绵羊黄体期不同生理阶段具有不同的营养需求和代谢特点,黄体期限饲抑制卵泡发育与蛋白质和脂类合成降低、分解增强有关。     

Localization of thrombospondin‐1 and its receptor CD36 in the ovary of the ostrich (Struthio camelus)

Angiogenesis, the formation of new blood vessels from pre‐existing vasculature, plays a decisive role for the rapid growth of avian follicles. Compared to mammals, few data on the angiogenesis in the avian ovary are available. However, whereas several pro‐angiogenic factors in the avian ovary have been recently studied in detail, little information is available on the localization of anti‐angiogenic factors. The aim of this study was to determine the localization and possible function of the anti‐angiogenic factor thrombospondin‐1 (TSP‐1) and its receptor CD36 in the ovary of the ostrich using immunohistochemistry and to correlate the results with ultrastructural data. Whereas the oocytes and granulosa cells of all follicular stages were negative for TSP‐1, myofibroblasts of the theca externa and smooth muscle cells of blood vessels showed distinct reactions. A distinctly different staining pattern was observed for CD36. The oocytes were CD36 negative. No immunostaining for CD36 could be observed neither in the granulosa cells nor in the adjacent theca interna of vitellogenic follicles. In the theca externa, blood vessels protruding towards the oocyte showed CD36‐positive endothelial cells. In conclusion, a fine balance between angiogenic and anti‐angiogenic processes assures that a dense net of blood vessels develops during the rapid growth of a selected follicle. Anti‐angiogenic molecules, such as TSP‐1 and its receptor CD36 may, after the oocyte has reached its final size, inhibit further angiogenesis and limit the transport of yolk material to the mature oocyte. By this mechanism, the growth of the megalecithal oocyte during folliculogenesis may cease.     

Xenotransplantation of goat ovary as an alternative to analyse follicles after vitrification

The aim of this study was to evaluate the caprine preantral follicles enclosed on vitrified/warmed ovarian cortex grafted to nude BALB/mice during 1 month. The ovarian cortex from goats was fragmented (3 × 3 × 0.5 mm) and divided into four groups: fresh control, vitrified control, fresh transplant and vitrified transplant. Follicular morphology, development and density, fibrosis as well as apoptosis, and tissue revascularization were evaluated. It was also observed a significant decrease in morphologically normal preantral (primordial, transition, primary and secondary) follicles in both vitrified control and vitrified transplant treatments when compared with both fresh control and fresh transplant. However, fresh control and fresh transplant exhibited a similar percentage of developing follicles. Additionally, Vitrified control showed a significant increase in developing follicles in comparison with both fresh control and fresh transplant. Follicular density significantly decreased in all treatments in comparison with fresh control. We observed high fibrosis in both fresh transplant and vitrified transplant. The mRNA expression of caspase 3 was lower in both fresh transplant and vitrified transplant in comparison with vitrified control. In conclusion, xenotransplantation is an excellent strategy to maintain normal preantral follicle morphology after vitrification/warming of goat ovarian tissue. Yet, in order to ensure the survival and development of these follicles, it is essential to improve the revascularization of the graft.     

黄体期短期优饲对道寒杂交羊卵泡发育的影响

为研究母羊黄体期短期优饲对其卵泡发育的影响。选择60只道寒杂交羊随机分为试验组和对照组,每组30只。试验羊均先饲喂基础日粮(DE 11.72 MJ/d,DP 79.71 g/d),并对试验羊进行同期发情处理(肌注PG 0.1 mg,3 d后阴道埋置CIDR 12 d,撤栓再次肌注PG 0.1 mg),同期发情处理埋栓2 d后试验组羊饲喂试验日粮(DE 18.75 MJ/d,DP 108.44 g/d),饲喂期10 d,并于饲喂开始第1,2,4,6,8,10天分别采集试验组和对照组羊颈静脉血,测定血浆中葡萄糖,胰岛素,瘦素等含量。在撤栓注射PG后,埋栓第13,14,15天每组随机选择6只羊进行屠宰,采集卵巢,按小卵泡(≤3.0 mm)、中等卵泡(3.0～5.0 mm)和大卵泡(≥5.0 mm)进行卵泡数量统计及卵泡液、颗粒细胞收集。采用实时定量PCR技术,分析不同大小卵泡颗粒细胞中OB-R和IGF-1基因mRNA表达水平。结果显示,与对照组相比,试验组小卵泡数量显著下降(P<0.01),中等卵泡和大卵泡数量显著升高(P<0.01),同时血浆和大卵泡中葡萄糖(P<0.01)、胰岛素(P<0.01)和瘦素(P<0.05)的平均浓度均显著升高,中等卵泡和大卵泡颗粒细胞中OB-R和IGF-1基因的表达显著上升(P<0.05)。结果表明,母羊黄体期短期优饲可提高血浆中葡萄糖、胰岛素、瘦素浓度和颗粒细胞中OB-R和IGF-1基因表达量,促进卵泡期的卵泡优势化数量。     

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P₄) and estradiol (E₂)) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P₄ secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.     

Involvement of vascular endothelial growth factor in the formation of the thecal layer and vasculature during follicular development in the ovaries of neonatal female rats

Vascular endothelial growth factor (VEGF) is an important angiogenic factor in the ovary, but the localization of VEGF in the ovary of neonatal animals is poorly understood. A clear understanding of the relationship between the formation of the thecal layer and the cell‐specific expression of the VEGF system during follicular development in the neonatal ovary is still lacking. Immature female Wistar‐Imamichi rats used in this study were killed by decapitation 5, 7, 9 and 11 days after birth, and their ovaries were removed and subjected to histological and immunohistochemical observation. The number of primordial follicles had decreased in the ovaries at day 11 compared with that at day 5. The number of secondary follicles significantly increased with age. In the morphological observation of secondary follicles, we found that the theca layer (70 µm in diameter of follicles) began to form at day 9 and was completely formed at day 11. An endothelial cell marker, CD31, VEGF and Flk‐1 were located in the stromal tissues in the ovaries on each day examined after birth. In particular, in the ovaries at day 9 and day 11, when the secondary follicles appeared, CD31, VEGF and Flk‐1 were expressed in the theca layer. Flt‐1 was expressed in the oocytes of the ovaries at day 5 and day 7, and the sites of its expression changed to stromal and thecal tissues at day 9 and day 11. In conclusion, we provide the first evidence that the theca layer of secondary follicles begin to form at day 9 after birth and that VEGF and Flk‐1 may be able to stimulate the differentiation of stromal‐interstitial cells into thecal cells and the formation of the thecal vasculature in the neonatal rat ovaries, suggesting that the VEGF system may be involved in the formation of the thecal layer and vasculature during folliculogenesis in the neonatal rat.     

Follicular development in livestock: Influencing factors and underlying mechanisms

Livestock farming development has become increasingly important in recent years. It not only provides us with meat nutrition and pet feeding but also increases the economic value by providing numerous employment opportunities, which improves our life quality. The livestock farming development depends on successful animal reproduction. As a vital process in animal reproduction, folliculogenesis and its influencing factors as well as their underlying mechanisms need to be understood thoroughly. This review is aimed at summarizing the factors such as cellular processes, gene regulation, noncoding RNAs and other endocrine or paracrine regulatory factors that affect follicular development, and their underlying mechanisms of action in livestock in order to provide novel insights for future studies. The above factors were found as significant determinants influencing the follicular development in livestock through various signaling pathways.     

雌激素对哺乳动物卵泡发育的影响研究进展

雌激素在卵泡发育过程中所起的作用,已经得到人们的普遍认可。雌二醇(E2)及其类似物在卵泡的体细胞的增殖和分化中起重要作用,但其作用机理目前还不完全清楚。一些最新研究结果雌激素受体β(ERβ)的发现、雌激素受体基因敲除以及雌激素缺失动物模型的建立等,使我们能够进一步探讨雌激素在卵泡发育过程中的作用机理。结合这些最新研究动态,探讨雌激素在卵泡发育过程中的作用及雌激素对卵巢內的体细胞表型分化的影响。     

Amino acids profile within peripheral blood and follicular fluid based on high‐performance liquid chromatography methods may explain differences in folliculogenesis between short‐term under/over‐fed treatments during luteal phase of Hu sheep

The nutritional alteration of amino acids (AAs) profile in physiological fluid was poorly characterized in livestock. After oestrus synchronization, 24 ewes were randomly assigned to two groups based on the nutrient requirement recommended for maintenance (M): the feed‐supplemented group (S, 1.5 × M, N = 12) and feed‐restricted group (R, 0.5 × M, N = 12) on days 6–12 of their oestrous cycle, which occurred shortly before ovulation. The concentration of 30 AAs in peripheral blood (PB) and follicular fluid (FF) was quantified to calculate the PB‐to‐FF concentration gap for each AA and determine its correlation with metabolites and hormones in PB and FF. Results showed that the feed restriction enlarged the oestrous cycle length, decreased the number of follicles 2.5–3.5 mm, increased the number of follicles >3.5 mm and augmented the volume of follicles >2.5 mm. Nineteen AAs from PB were significantly different between the groups. The phosphoethanolamine (PEtN) and ration of essential AAs to nonessential AAs (EAA/NEAA) in FF significantly (p < 0.05) increased and decreased in the R group, respectively. Most AAs, except aspartate (Asp) and carnosine (Car) in the R group and alanine (aAla) in both groups, were significantly lower within FF than those within PB. The correlation of AAs with FSH and progesterone (P₄) was more significant than that of AAs with other endocrine milieu characteristics. In conclusion, our results revealed that the influence of short‐term nutritional manipulation during luteal phase on folliculogenesis might not be due to the variation of intrafollicular AAs profile but rather attribute to the peripheral blood AAs profile alteration.     

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.