In vitro development of primordial follicles after long-term culture of goat ovarian tissue

This study aims to investigate the effects of follicle stimulating hormone (FSH) and fibroblast growth factor-2 (FGF-2) on the survival and growth of caprine preantral follicles. Ovarian tissues were cultured for 1, 7, 14, 21 or 28 days in medium supplemented with FSH (FSH-2d or FSH-7d, i.e., with replacement of the culture medium every 2 or 7 days, respectively) or FSH + FGF-2 (replacement of the medium every 2 days). Non-cultured (control) and cultured ovarian fragments were processed for histological and ultrastructural analysis. After 28 days of culture, the media supplemented with FSH-2d was the most effective in maintaining the percentage of normal follicles and in promoting follicular growth. Furthermore, both treatments with FSH increased the percentage of the primary follicles. However, ultrastructural studies did not confirm follicular integrity from 14 days of culture onward. In conclusion, culturing tissue for up to 7 days in medium containing FSH alone or combined with FGF-2 maintains caprine preantral follicle integrity and promotes their growth in vitro.     

Evaluation of in vitro culture systems for goat preantral follicles using reused ovaries from reproductive biotechniques: An alternative to maximize the potential of reproduction

This study aimed to examine the in vitro culture of secondary preantral follicles, using reused ovaries, to compare both the 2D and 3D methods of in vitro culture of preantral follicles, and the system of medium replacement. Twenty‐five pairs of ovaries from mixed‐breed goats were used for the experiment. Follicular puncture of antral follicles was performed for in vitro production. After this procedure, the secondary preantral follicles were submitted to a microdissection procedure. The isolated preantral follicles were randomly divided into three treatments: (a) Two‐dimensional culture with partial replacement of medium during culture (2D PR), (b) Three‐dimensional culture with addition of medium during culture (3D AD) and (c) Three‐dimensional culture with partial replacement of medium (3D PR). The culture period was 18 days. All treatments at the end of the in vitro culture period (18 days) presented a follicular survival rate which ranged from 59% to 70%, demonstrating that it was possible to perform an experiment with preantral follicles using ovaries that had previously been used in another reproductive biotechnique. The 3D AD treatment showed a survival percentage and follicular diameter higher than the 2D PR treatment, however, it did not differ from the 3D PR treatment. In conclusion, experiments employing the use of preantral follicles can be performed with success after the ovaries have been used for experiments with antral follicles. Moreover, the three‐dimensional system with the addition of medium is recommended for in vitro culture of preantral follicles, since this system is more practical and financially feasible.     

The effect of bpV(HOpic) on in vitro activation of primordial follicles in cultured swine ovarian cortical strips

The vanadate‐derivative dipotassium bisperoxo (5‐hydroxy‐pyridine‐2‐carboxylic) oxovanadate (V) (bpV(HOpic)), a pharmacological inhibitor of phosphatase and tensin homolog (PTEN), has been used in ovarian follicle culture systems for activation of follicular growth in vitro and suggested to be responsible for primordial follicle survival through indirect Akt activation. For pig ovarian tissue, it is still not clear which culture medium needs to be used, as well as which factors and hormones could influence follicular development; this also applies to bpV(HOpic) exposure. Therefore, ovarian cortical strips from pigs were cultured in 1 µM bpV(HOpic) (N = 24) or control medium (N = 24) for 48 hr. Media were then replaced with control medium and all tissue pieces incubated for additional 4 days. The strips were embedded in paraffin for histological determination of follicle proportions at the end of the culture period and compared to histological sections from tissue pieces without cultivation, which had been embedded right after preparation; comparison of healthy follicles for each developmental stage was performed to quantify follicle survival and activation. After 6‐day culture, follicle activation occurred in tissue samples from both cultured groups but significantly more follicles showed progression of follicular development in the presence of 1 µM bpV(HOpic). The amount of non‐vital follicles was not significantly increased during cultivation. BpV(HOpic) affects pig ovarian follicle development by promoting the initiation of follicle growth and development, similar as in rodent species and humans.     

Regional distribution and integrity of equine ovarian pre‐antral follicles

The goal of this study was to determine the distribution of pre‐antral follicles in the ovarian parenchyma of mares. For Experiment 1, each ovary was cut longitudinally at the greater curvature, performing two hemiovaries. After that, six fragments from each hemiovary were obtained, resulting in 12 fragments, which were divided into the innermost region of the parenchyma, the middle region and the outermost region. All the three obtained sections were cut transversally to obtain two fragments from each one. For Experiment 2, each ovary also submitted to a longitudinal cut on the greater curvature, forming two hemiovaries. Each hemiovary was sectioned into four symmetrical fragments, resulting in eight fragments per ovary. The fragments were related as being near to or far from the ovulatory fossa. The fragments of both experiments were immediately fixed in Carnoy for 12 hr and kept in 70% ethanol for 24 hr. Follicles were classified according to the stages of development and for morphological integrity according to oocyte morphology and granulosa cells. After the histological assessment, a total of 1,130 follicles were visualized from Experiment 1, being 1,054 (93.3%) primordial follicles and 76 (4.7%) follicles in development. The innermost region had the highest percentage of pre‐antral follicles compared to the other regions (p < .05). The middle and outermost regions showed higher percentages of intact primordial and developing follicles than the innermost region (p < .05). Considering Experiment 2, 938 follicles were found, being 894 (95.3%) primordial and 44 (4.7%) follicles in development. The region near the ovulatory fossa presented higher (58.7%; 551 of 938) follicular concentration compared to the region far from the ovulatory fossa (41.3%; 387 of 938; p < .05). As a conclusion, distribution of pre‐antral follicles in the equine ovary has a specific pattern through the parenchyma. Also, the follicular integrity differed in the studied ovarian areas.     

Effects of tumour necrosis factor‐alpha and interleukin‐1 beta on in vitro development of bovine secondary follicles

The objective of this study was to determine the effects of TNF‐α and IL‐1β on development and survival of bovine secondary follicle culture in vitro for 18 days. Secondary follicles (~0.2 mm) were isolated from ovarian cortex and individually cultured at 38.5°C, with 5% CO₂ in air, for 18 days, in TCM‐199⁺ alone (cultured control) or supplemented with 10 ng/ml IL‐1β, 10 ng/ml TNF‐α or both TNF‐α and IL‐1β. The effects of these treatments on growth, follicular survival, antrum formation, viability, ultrastructure and mRNA levels for GDF‐9, c‐MOS, H1foo and Cyclin B1 were evaluated. The results showed that addition of TNF‐α to culture medium increased follicular diameter and rate of antrum formation, whereas that of IL‐1β and a mixture of IL‐1β and TNF‐α did not do so. Ultrastructural analysis showed that, among the tested cytokine treatments, follicles cultured in the presence of TNF‐α had the best‐preserved oocytes and granulosa cells. The presence of TNF‐α, IL‐1β or both did not influence the expression of mRNAs analysed. In conclusion, in contrast to IL‐1β, TNF‐α promotes growth of and antrum formation in in vitro cultured bovine secondary follicles, while their ultrastructure and viability were maintained.     

Oxygen consumption rate of early pre-antral follicles from vitrified human ovarian cortical tissue

The study of human ovarian tissue transplantation and cryopreservation has advanced significantly. Autotransplantation of human pre-antral follicles isolated from cryopreserved cortical tissue is a promising option for the preservation of fertility in young cancer patients. The purpose of the present study was to reveal the effect of vitrification after low-temperature transportation of human pre-antral follicles by using the oxygen consumption rate (OCR). Cortical tissues from 9 ovaries of female-to-male transsexuals were vitrified after transportation (6 or 18 h). The follicles were enzymatically isolated from nonvitrified tissue (group I, 18 h of transportation), vitrified-warmed tissue (group II, 6 and 18 h of transportation) and vitrified-warmed tissue that had been incubated for 24 h (group III, 6 and 18 h of transportation). OCR measurement and the LIVE/DEAD viability assay were performed. Despite the ischemic condition, the isolated pre-antral follicles in group I consumed oxygen, and the mean OCRs increased with developmental stage. Neither the transportation time nor patient age seemed to affect the OCR in this group. Meanwhile, the mean OCR was significantly lower (P < 0.05) in group II but was comparable to that of group I after 24 h of incubation. The integrity of vitrified-warmed primordial and primary follicles was clearly corroborated by the LIVE/DEAD viability assay. These results demonstrate that the OCR can be used to directly estimate the effect of vitrification on the viability of primordial and primary follicles and to select the viable primordial and primary follicles from vitrified-warmed follicles.     

黄淮白山羊孤雌胚胎的体外培养

采用离子霉素和6-DMAP对黄淮白山羊体外成熟卵母细胞进行联合激活,分别在不同的培养体系进行体外培养,观察孤雌胚胎的发育情况。培养体系分别为:无共培养条件下,M199(10?S)、CR1aa和HTF P1;颗粒细胞共培养条件下,CR1aa、HTF P1和SOFaa。结果发现,无共培养条件下,CR1aa和HTF P1组孤雌胚胎的卵裂率显著高于M199组(P<0.05),但3组均未有囊胚;共培养条件下,HTF P1组的卵裂率显著高于CR1aa和SOFaa组(P<0.05),而CR1aa组的囊胚率却显著高于其他2组(P<0.05)。综合试验结果说明,CR1aa联合颗粒细胞共培养能够获得较好的培养效果,适用于山羊孤雌胚胎的体外培养。     

Effects of melatonin on morphology and development of primordial follicles during in vitro culture of bovine ovarian tissue

This study aims to investigate the effect of melatonin on activation, growth and morphology of bovine primordial follicles, as well as on stromal cells density in ovarian tissues after in vitro culture. Ovarian fragments were cultured in α‐MEM⁺ alone or supplemented with melatonin (250, 500, 1,000 or 2,000 pM) for a period of six days. Non‐cultured and cultured tissues were processed for histological analysis; according to developmental stages, follicles were classified as primordial or growing follicles. These follicles were further classified as morphologically normal or degenerated. Ovarian stromal cell density was also evaluated. The percentages of primordial and developing follicles, as well as those classified of normal follicles, were compared by Fisher's exact test, and the differences were considered significant when p < .05. The results showed that the presence of 1,000 and 2,000 pM melatonin in culture medium promoted a reduction in the percentage of primordial follicles and an increase in the percentage of development follicles, when compared to follicles cultured in control medium. On the other hand, the presence of 250 or 500 pM melatonin did not show a significant effect on the percentage of primordial and developing follicles. Besides that, the presence of 500, 1,000 and 2,000 pM melatonin maintained the percentage of normal follicles similar to those seen uncultured control. Moreover, tissues cultured in presence of 1,000 pM melatonin showed a higher percentage of normal follicles when compared to follicles cultured in the presence of 250 pM melatonin. It was observed a similar profile of stromal density in both uncultured tissues and those cultured in vitro in the presence of melatonin. In conclusion, melatonin (1,000 and 2,000 pM) promotes bovine primordial follicles activation and maintains the stromal cell density during in vitro culture of ovarian cortical tissue.     

Growth differentiation factor‐9 improves development,mitochondrial activity and meiotic resumption of sheep oocytes after in vitro culture of secondary follicles

This study analysed the effect of growth differentiation factor‐9 (GDF‐9) on the in vitro culture of isolated ovine secondary follicles. The follicles were cultured in α‐MEM supplemented with BSA, insulin, glutamine, hypoxanthine, transferrin, selenium, ascorbic acid and FSH (α‐MEM⁺—control medium) or α‐MEM⁺ supplemented with 1, 10, 50 or 100 ng/ml GDF‐9. Next, the oocytes were destined to in vitro maturation (IVM). After 12 days of culture, there were no differences regarding the percentage of normal follicles, antrum formation and follicle diameter between the treatments (p > 0.05). The rates of fully grown oocytes (≥110 µm) were higher (p < 0.05) in 100 ng/ml GDF‐9 than other treatments, except for 10 ng/ml of GDF‐9 (p > 0.05). Treatment containing 100 ng/ml GDF‐9 showed higher (p < 0.05) mitochondrial activity than the control group. Moreover, 100 ng/ml GDF‐9 showed more oocytes in MI than α‐MEM⁺, 1 or 50 ng/ml GDF‐9 (p < 0.05). In conclusion, 100 ng/ml GDF‐9 increased the growth, mitochondrial function and meiotic resumption of oocytes from in vitro grown sheep secondary follicles.     

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。     

奶山羊CIDEa基因的克隆、序列分析与组织表达

为获得奶山羊CIDEa基因序列并检测其在乳腺组织中的表达,采用RT-PCR技术从奶山羊乳腺组织扩增CIDEa基因序列,利用生物信息学软件对其进行预测分析,并采用荧光定量PCR检测CIDEa在干奶期和不同泌乳时期乳腺组织中的表达。结果表明:通过克隆测序得到奶山羊CIDEa基因CDS区660 bp,编码219个氨基酸,与GenBank公布的绵羊、牛、人和猪的核苷酸序列同源性分别为99%、96%、85%、85%。奶山羊CIDEa蛋白属于碱性、不稳定亲水蛋白,不存在跨膜结构和信号肽;蛋白主要定位在细胞质、线粒体和细胞核。CIDEa蛋白结构主要由α螺旋、β折叠和不规则卷曲组成。奶山羊CIDEa在泌乳前期、盛期和中期乳腺组织中表达量均显著高于干奶期(P<0.05)。说明CIDEa可能参与奶山羊乳腺的泌乳过程,为进一步研究其在奶山羊乳腺组织中的功能及对乳品质的调控作用奠定基础。     

影响山羊体外胚胎生产的主要因素

研究了不同成熟培养时间（21、242、7 h）、FSH剂量（0、0.02、0.04、0.06 AU/mL）和激素组合（FSH＋LH＋E2、FSH＋LH、LH＋E2、FSH＋E2）、氨基酸以及负压低氧条件（-300 mmHg2、%CO28、%～10%O2）对山羊卵母细胞体外成熟/体外受精（IVM/IVF）的影响。结果表明：山羊卵母细胞成熟培养以24～27 h较为适宜;添加FSH组的受精卵裂率显著高于未添加组（P〈0.01）,添加剂量以0.02～0.04 AU/mL效果为最佳;LH对山羊的IVM/IVF无显著影响;E2添加对山羊IVM/IVF有负面影响,使囊胚发育率显著降低（P〈0.05）。在成熟液中添加必需氨基酸（EAA）和非必需氨基酸（NEAA）均能显著提高受精卵裂率（P〈0.05）。负压低氧环境对山羊卵母细胞的体外成熟无显著影响,但是不利于山羊的体外受精,使受精卵裂率极显著降低（P〈0.01）;负压低氧环境对提高胚胎发育率的作用不明显。     

Effect of oxygen tension on in vitro viability and development of dog follicles enclosed within the ovarian cortex

Oxygen concentration has been shown to influence in vitro viability and growth of ovarian follicles. The present study examined the effect of oxygen tension on in vitro development of dog follicles enclosed within the ovarian cortex. Ovaries were obtained from domestic dogs (age, 8 months to 2 years), and cortical fragments were recovered. The cortices were then incubated on 1.5% (w/v) agarose gel blocks within a 4‐well culture plate containing Eagle Minimum Essential Medium (MEM). Ovarian follicles within the tissues were processed for histology and assessed for follicle density, viability and diameter immediately after collection (Control) or after 2 or 5 days of in vitro incubation. Apoptotic cells were assessed using TUNEL assay. Comparisons of follicular viability and diameter were performed using analysis of variance followed by Tukey's test (p < 0.05). Comparisons of follicle density and apoptosis among treatments were conducted using Non‐parametric Kruskal–Wallis test followed by Friedman's test (p < 0.05). No difference (p > 0.05) in follicle density was observed among groups at Day 2 of in vitro culture. However, the density of follicles within cortices cultured in 20% oxygen for 5 days significantly reduced compared to the Control and those incubated in 5% concentration. The viability of cultured follicles in all treatments decreased (p < 0.05) compared to the Control after 2 days incubation, and this value further reduced (p < 0.05) in 20% oxygen group at Day 5. There were no differences in the percentages of apoptotic follicles between the two treatment groups (p > 0.05). Nevertheless, after 5 days of culture, the percentage of TUNEL‐positive follicles increased significantly (p < 0.05) in cortices incubated in 20% oxygen environment. In conclusion, our findings demonstrated that 5% oxygen level was superior to 20% concentration in sustaining in vitro viability of dog follicles enclosed within the ovarian cortex.     

Early ovine preantral follicles have a potential to grow until antral stage in two‐step culture system in the presence of aqueous extract of Justicia insularis

The objective of this study was to determine whether preantral follicles cultured in vitro for 7 days within ovine ovarian cortical strips could be isolated at the secondary follicles (SF) and grown until antral stage during an additional 6 days period of in vitro culture in the presence of aqueous extract of Justicia insularis. Fresh ovarian fragments from 16 adult sheep were fixed for histological analysis (Control 1) or in vitro cultured individually in α‐MEM⁺ supplemented with 0.3 mg/ml J. insularis (Step 1) for 7 days. Part of the fragments then were fixed for histological analysis (in vitro culture group). Remaining fragments were exposed stepwise to increasing trehalose concentrations before immediate isolation of SF and viability assessment (Control 2) or after 6 days of culture in α‐MEM⁺⁺ supplemented with 0.3 mg/ml J. insularis (Step 2). In Step 1, percentage of follicular activation was 80%. In Step 2, a significant increase (p < 0.05) in follicular diameter and antrum formation within 6 days in vitro culture of isolated follicles was achieved. The total antioxidant capacity from both steps significantly increase (p < 0.05) from day 2 to day 6. Confocal analysis of oocytes showed 57.14% oocytes with homogeneous distribution and 42.86% with peri‐cortical distribution. In conclusion, SF can be successfully isolated from sheep ovarian cortex after 7 days of culture and are capable of surviving and forming an antral cavity if cultured in vitro for an additional 6 days in the presence of 0.3 mg/ml J. insularis.     

组织块再移法分离培养奶山羊乳腺上皮细胞

通过比较组织块贴壁培养法、组织块再移法、胰蛋白酶消化法、胶原酶Ⅱ消化法和胶原酶消化配合组织块贴壁法分离、培养、纯化山羊乳腺上皮细胞,绘制细胞生长曲线并计算细胞群体倍增时间,研究乳腺上皮细胞培养效果。结果显示,利用胶原酶消化配合组织块贴壁法不能获得正常生长的乳腺上皮细胞;利用组织块贴壁培养法、组织块再移法和胶原酶Ⅱ消化法获得大量的乳腺上皮细胞,所得到的上皮细胞生长曲线呈典型的＂S＂型,符合细胞生长的一般规律。组织块再移法不仅可快速获得大量纯化的乳腺上皮细胞,而且所得到的细胞经传代后,细胞群体倍增时间最短、增殖能力最强,表明,该法是获得山羊乳腺上皮细胞最适宜的方法。     

Interaction between melatonin and follicle-stimulating hormone promotes in vitro development of caprine preantral follicles

The aim of this study was to investigate the effects of melatonin and follicle-stimulating hormone (FSH) on the in vitro culture of goat preantral follicles. Ovarian fragments were cultured for 7 d in α-minimum essential medium (α-MEM⁺) containing melatonin (100, 250, 500, or 1,000 pM), FSH (50 ng/mL), or a combination of the 2 hormones and further analyzed by histology and transmission electron and fluorescent microscopy. The results showed that after 7 d of culture, tissues cultured in α-MEM⁺ alone or supplemented with FSH alone, melatonin (500 and 1,000 pM), or the combination of FSH and melatonin (1,000 pM) maintained percentages of normal preantral follicles similar to the fresh control. In contrast to the noncultured tissues, the percentage of developing follicles was increased under all culture conditions after 7 d (P < 0.05). The addition of 1,000 pM melatonin associated with FSH to the culture medium increased follicular and oocyte diameters compared with α-MEM⁺ alone after 7 d of culture (P < 0.05). Ultrastructural and fluorescent analyses confirmed the integrity of follicles cultured with 1,000 pM of melatonin plus FSH for 7 d. In conclusion, this study demonstrated that the interaction between melatonin and FSH maintains ultrastructural integrity and stimulates further growth of cultured caprine preantral follicles.     

The development and integrity of equine pre‐antral follicles cultured in vitro with follicle‐stimulating hormone (FSH) supplementation

This study investigated the effects of different concentrations of FSH (10, 50, 100 and 200 ng/ml) in supplemented MEM+ on the development of equine pre‐antral follicles that were cultured in vitro for 2 or 6 days. The ovaries (n = 5) from mares in seasonal anoestrus were collected from a local abattoir. Ten ovarian tissue fragments of approximately 3 × 3 × 1 mm were obtained from each animal. The fragments were cultured in situ for 2 days (D2) or 6 days (D6) in MEM+ or MEM+ supplemented with FSH at four different concentrations, establishing the following 11 groups: control (D0); MEM + (D2); MEM + (D6); MEM + 10 ng/ml of FSH (D2); MEM + 10 ng/ml of FSH (D6); MEM + 50 ng/ml of FSH (D2); MEM + 50 ng/ml of FSH (D6); MEM + 100 ng/ml of FSH (D2); MEM + 100 ng/ml of FSH (D6); MEM + 200 ng/ml of FSH (D2); and MEM + 200 ng/ml of FSH (D6). Follicles were observed in only 9.65% (388 of 4,018) of the histological sections. Of the 861 follicles evaluated, 488 were in the primordial stage, and 373 were in various developmental stages; 59.7% were morphologically normal. Regarding the integrity of the pre‐antral follicles, the groups with 100 ng/ml FSH of 2‐days culture as well as 50, 100 and 200 ng/ml FSH of 6‐days culture provided the best results. In conclusion, the in vitro culture of abattoir‐derived equine ovarian fragments presented better morphological integrity when supplemented with FSH for 6 days, in comparison with the MEM culture group. However, no clear effects were observed with FSH regarding the promotion of activation from a primordial to a developing follicle.     

多胎和单胎山羊品种Bcl-2和Bax基因的克隆及组织表达研究

以低繁藏山羊和高繁金堂黑山羊卵巢等组织为试验材料,通过RT-PCR,对Bcl-2和Bax基因c DNA进行克隆、序列分析,并采用Real-time PCR对其在发情前期母羊垂体、卵巢、子宫和输卵管等组织的表达进行检测。结果:山羊Bcl-2基因编码区全长为690 bp,共编码229个氨基酸,2个品种间有3处碱基差异,但未导致氨基酸的差异;Bax基因编码区全长为579 bp,共编码192个氨基酸,2个品种的同源性为100%。Bcl-2和Bax mRNA在2个品种山羊的卵巢、子宫、输卵管、垂体中均有表达,但品种间差异不显著(P0.05)。说明Bcl-2和Bax基因在动物进化中比较保守,与山羊多羔性状的相关性有待进一步研究。     

Expression of steroidogenic enzymes and synthesis of steroid hormones during development of ovarian follicles in prepubertal goats

Expression of mRNAs encoding cytochrome P450 side-chain cleavage (P450scc), cytochrome P450 17 -hydroxylase (P450c17), and cytochrome P450 aromatase (P450arom) were characterized by the RT-PCR technique and concentrations of progesterone (P4), testosterone (T0) and estradiol (E2) were measured by radioimmunoassay during follicular development of prepubertal goats. Synthesis of mRNAs encoding P450scc and P450c17 began in preantral follicles, but mRNA encoding P450arom was not detectable until early antral formation. While mRNA for P450scc was expressed in both theca and granulosa cells, mRNA for P450c17 was expressed only in theca cells while P450arom mRNA only in granulosa cells. In nonatretic follicles from prepubertal ovaries, the relative quantity of mRNA expression of all the three enzymes increased with follicle size; however, while the concentration of P4 and E2 increased, that of T0 decreased with follicle size. While expression of mRNA encoding P450scc was unaffected, that of P450c17 mRNA decreased to the lowest level and mRNA for P450arom became undetectable following atresia; accordingly, while the concentration of P4 increased in the atretic medium follicles, that of T0 and E2 decreased to the lowest level after atresia. While the adult follicular stage follicles showed a similar cytochrome expression as the nonatretic follicles of prepubertal goats, the former contained higher levels of E2 and P4 than the latter. The presence of corpus luteum in an ovary decreased expression of P450scc, significantly in large follicles while it increased concentration of P4. These findings indicated that (1) similar to other species, changes in follicular steroid production in goats were explained in large measure by changes in steroidogenic enzyme expression; (2) while mRNA expression was similar, activities of some of the steroidogenic enzymes may differ between sexually mature and immature goats.     

Expression and localization of adiponectin and its receptors in ovarian follicles during different stages of development and the modulatory effect of adiponectin on steroid production in water buffalo

Adiponectin is an adipocyte‐derived hormone regulating energy metabolism, insulin sensitivity and recently found to regulate reproduction. The current study was carried out to investigate gene and protein expression, immunolocalization of adiponectin and its receptors AdipoR1 and AdipoR2 in ovarian follicles of different developmental stages in water buffalo (Bubalus bubalis) and to investigate the effect of adiponectin on steroid production in cultured bubaline granulosa cells. qPCR, western blotting and immunohistochemistry were applied to demonstrate mRNA expression, protein expression and immunolocalization, respectively. The results indicate that adiponectin, AdipoR1 and AdipoR2 were present in granulosa cells (GC) and theca interna (TI) of ovarian follicles and the expression of adiponectin, AdipoR1, AdipoR2 in GC and AdipoR1 and AdipoR2 in TI increased with increase in follicle size (p < .05). Expression of adiponectin was high in small and medium size follicles in TI. The adiponectin and its receptors were immunolocalized in the cytoplasm of GC and TI cells. Further, in the in‐vitro study, GCs were cultured and treated with recombinant adiponectin each at 0, 1 and 10 µg/ml alone or with follicle stimulating hormone (FSH) at 30 ng/ml) or Insulin‐like growth factor I (IGF‐I) at 10 ng/ml for 48 hr after obtaining 75%–80%s confluency. Adiponectin at 10 µg/ml increased IGF‐I‐induced estradiol (E₂) and progesterone (P₄) secretion and FSH‐induced E₂ secretion from GC and also increased the abundance of factors involved in E₂ and P₄ production (cytochrome P45019A1 [CYP19A1] and 3‐beta‐hydroxysteroid dehydrogenase [3β‐HSD]). In conclusion, this study provides novel evidence for the presence of adiponectin and its receptors in ovarian follicles and modulatory role of adiponectin on steroid production in buffalo.