Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis in bovine ovarian granulosa cells

Steroid hormones are required for normal reproductive function of female. The aim of this study was to investigate the role of Raf‐ERK1/2 on steroid hormone synthesis in bovine ovarian granulosa cells. Immunohistochemistry assay showed that both B‐Raf and C‐Raf were expressed in granulosa cells, theca cells and Sertoli cells. The protein expression of Raf or ERK1/2 was clearly decreased by Raf inhibitor GSK2118436 or ERK1/2 inhibitor SCH772984, respectively (p < 0.05). In addition, western blotting was performed for investigating the crosstalk between Raf and ERK1/2, the data showed that Raf positively regulated ERK1/2, whereas ERK1/2 had a negative feedback effect on Raf. The biosynthesis of oestradiol or testosterone was significantly decreased by treatment with GSK2118436 or SCH772984 (p < 0.05). Conversely, the progesterone biosynthesis was clearly increased by treatment with those inhibitors (p < 0.05). Furthermore, the mRNA expression of STAR, aromatase and CYP17 was blocked by Raf‐ERK1/2 signalling inhibition, which oppositely induced the mRNA expression of CYP11. Together, these findings suggested that Raf‐ERK1/2 signalling pathways mediate steroid hormone synthesis via affecting the expression of steroidogenic enzymes.     

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.     

Natriuretic peptide system regulation in granulosa cells during follicle deviation and ovulation in cattle

Natriuretic peptides (NPs) are known to regulate reproductive events in polyovulatory species, but their function and regulation in monovulatory species remain to be fully characterized. Using a well‐established in vivo model, we found that bovine granulosa cells from follicles near the deviation stage express mRNA for the three NP receptors (NPR1, NPR2 and NPR3), but not for NP precursors (NPPA, NPPB and NPPC). The abundance of NPR3 mRNA was higher in dominant compared to subordinate follicles at the expected time of follicular deviation. After deviation, mRNA for all NP receptors was significantly more abundant in the dominant follicle. Intrafollicular inhibition of oestrogen receptors downregulated NPR1 mRNA in dominant follicles. In granulosa cells from preovulatory follicles, NPPC mRNA increased at 3 and 6 h after systemic GnRH treatment, but decreased at 12 and 24 h to similar levels observed in samples collected at 0 h. After GnRH treatment, NPR1 mRNA was upregulated at 24 h, NPR3 mRNA gradually decreased after 3 h, while NPR2 mRNA was not regulated. The mRNA expression of the enzyme FURIN increased at 24 h after GnRH treatment. These findings revealed that the expression of mRNA encoding important components of the NP system is regulated in bovine granulosa cells during follicular deviation and in response to GnRH treatment, which suggests a role of NP system in the modulation of these processes in monovulatory species.     

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.     

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).     

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...     

Changes of serum anti‐Müllerian hormone in a mare with granulosa cell tumour following surgery and reinitiation of follicular activity

Anti‐Müllerian hormone (AMH) has been reported to be elevated in mares with granulosa cell tumour (GCT). An 8‐year‐old Thoroughbred mare was presented for not being observed in oestrus after the beginning of the breeding season. Rectal palpation and ultrasonography revealed enlargement and cystic appearance of the left ovary while the right ovary was small with an anoestrous‐like appearance. The AMH concentration was 694.9 ng/ml. Presumptively diagnosed with GCT, the mare was subjected to tumour removal. Histopathology confirmed GCT. To evaluate changes of AMH concentration following surgery, blood samples were collected immediately prior to surgery, and on Days 1, 2, 4, 8, 16 and 32 after surgery. Thereafter, blood samples were collected monthly and also at the time the mare was observed in oestrus (148 days after tumour removal). The AMH concentrations decreased over the first 2 months after surgery (from 721.2 ng/ml to 0.1 ng/ml). Subsequently, AMH concentration increased and peaked at the time of oestrus expression (0.7 ng/ml). The mare then became anoestrous, and AMH concentration decreased and reached 0.2 ng/ml, which was not significantly different from the mean concentration of AMH in normal anoestrous mares (n = 5; 0.26 ± 0.07 ng/ml). In conclusion, the present report implies the potential use of AMH for determination of the time of follicular resumption in mares after GCT removal.     

Characterization of anti‐Müllerian hormone in a case of bovine male pseudohermaphroditism

The current report aimed to characterize plasma anti‐Müllerian hormone (AMH) in bovine male pseudohermaphroditism. The blood AMH concentration in a Japanese Black male pseudohermaphrodite calf was compared with pre‐ and post‐pubertal male and female calves and castrated calves. The concentration in the case was higher than in post‐pubertal males, castrated males, and pre‐ and post‐pubertal female calves (p < .05), but similar to that in pre‐pubertal male calves. After extraction of the testes, the concentration in the case dropped to a certain extent. The extracted testes expressed AMH, as detected by immunohistochemistry. This study is the first to show the characterization of AMH in a male pseudohermaphrodite calf. AMH levels in peripheral blood might be useful to diagnose male pseudohermaphroditism in cattle.     

Determination of serum anti‐Müllerian hormone concentrations for the diagnosis of granulosa‐cell tumours in mares

Reasons for performing study: Endocrinological assays are important for evaluation of mares with granulosa‐cell tumours (GCTs), and our research in mares indicates that anti‐Müllerian hormone (AMH) may be a good biomarker for this type of ovarian tumour. Objectives: To evaluate the use of serum AMH concentrations for endocrine diagnosis of GCTs in mares. Methods: Archived serum samples (n = 403) previously assayed for determination of serum inhibin, testosterone and progesterone concentrations (GCT panel) were assayed for serum AMH concentrations using a heterologous enzyme‐linked immunosorbent assay previously validated by our laboratory. For a subset (n = 44) of these samples, a clinical diagnosis of GCT was confirmed by histopathology. Results: Overall, the sensitivity of AMH (98%) for detection of histologically confirmed GCTs was significantly (P<0.05) greater than that of either inhibin (80%) or testosterone (48%) or the combination of inhibin and testosterone (84%). Conclusions: Determination of serum AMH concentrations is a useful biomarker for detection of GCTs in the mare. Potential relevance: Measurement of serum AMH concentrations can be used for diagnosis of GCTs in the mare. As serum AMH concentrations do not vary significantly during the oestrous cycle or pregnancy, interpretation of these results is not confounded by these physiological states.     

Co-culture model reveals the characteristics of theca cells and the effect of granulosa cells on theca cells at different stages of follicular development

Theca cells (TCs) play an important role in follicular development, which cannot be separated from granulosa cells (GCs). However, compared with mammals, the TCs and the effects of GCs on TCs at different follicular development stages (FDSs) have specific characteristics in avian species, but none of them have been clearly defined. In this study, we established an in vitro co-culture (with GC at the corresponding stage) model of goose TCs at different FDSs (pre-hierarchical, hierarchical and F1) by using a transwell system. The properties of TCs in co-culture at the three FDSs, including cell morphology, activity and intracellular lipid content, as well as the expression of key genes involved in de novo lipogenesis, steroidogenesis, proliferation and apoptosis, were examined and defined. We further compared the mono-culture and co-culture groups. After co-culture, the activity of TCs showed significant (p < .01) increases in all stages; moreover, in pre-hierarchical TCs, the expression levels of FAS, SREBP, 3β-HSD and CCND1 were promoted, and PPARγ, CYP19, BCL2 and CAS3 were inhibited (p < .05); in the hierarchical TCs, the expression levels of PPARγ, FAS, CYP19, CCND1 and BCL2 were promoted, and SREBP, STAR, 3β-HSD and CAS3 were inhibited (p < .05), whereas in the F1 TCs, the expression levels of PPARγ, FAS, 3β-HSD, CYP19 and CCND1 were promoted, and STAR and CAS3 were inhibited (p < .05). These results suggested that GCs at the three FDSs have dynamic and complex influences on the physiological characteristics of TCs, and the influences on TCs at the three FDSs were varied.     

Histological and immunohistochemical evaluation of duodenal and colonic biopsies after oral bovine lactoferrin supplementation in beagle puppies

Lactoferrin is a natural compound in the milk of mammals and was shown to influence the intestinal micro‐flora and the immune system in mice, calves, dogs and man. The present study was carried out to investigate the effect of orally administered bovine lactoferrin (0, 30, 60 and 120 mg/kg DM feed) on the intestinal morphology and lymphocyte colonization in 36 motherless raised puppies. Endoscopic biopsies from duodenum and colon, taken in week 14, were scored histologically after staining with haematoxylin and eosin (H&E) and lymphocytes (CD3⁺, CD4⁺, CD8⁺) and plasma cells (IgA⁺, IgG⁺, IgM⁺) were enumerated after immunohistochemical staining by computer‐aided quantification. Histological scoring revealed no significant differences amongst the groups. IgG⁺ plasma cells were reduced (p < 0.05) in the lamina propria of the colon of the 30 and the 60 mg group. The number of CD8⁺ lymphocytes was higher (p < 0.05) in the epithelium of the colon of the lactoferrin groups. In conclusion, this study indicated only minimal effects of bovine lactoferrin on the population of selected immune cells in the gut mucosa of puppies. More investigations are needed to describe the impact of lactoferrin on the digestive physiology of puppies.     

醋酸铅对小鼠卵巢组织结构和卵巢颗粒细胞凋亡的影响

将40只20日龄雌性昆明系小鼠随机分成3个处理组和1个对照组,每组10只。给3个处理组小鼠分别连续2d腹腔按体质量注射醋酸铅10,20,40mg/kg,对照组小鼠注射等体积的生理盐水。于注射后24,72h分离卵巢,用原位末端标记法(TUNEL)测定卵巢颗粒细胞的凋亡率,研究卵巢组织结构及卵巢颗粒细胞凋亡的变化。结果显示,醋酸铅可使小鼠卵巢组织结构发生病变,加速卵巢颗粒细胞的凋亡,且凋亡率随着攻毒剂量的增加和时间的延长而升高,与对照组相比,差异极显著(P0.01);表明醋酸铅对小鼠卵巢具有毒性作用,可诱导卵巢颗粒细胞发生凋亡,并呈现一定的剂量-时间依赖关系。     

Effects of leptin on the release of luteinizing hormone, growth hormone and prolactin from cultured bovine anterior pituitary cells

The effects of leptin on the release of luteinizing hormone (LH), growth hormone (GH) and prolactin (PRL) were studied in cultured bovine anterior pituitary (AP) cells in vitro. The AP cells were obtained from fully‐fed Japanese Black steers and were incubated for 3 h with 10^?13 to 10^?7 mol/L of leptin after incubating in Dulbecco's modified Eagle's Medium for 3 days. Leptin significantly increased the concentration of LH in the culture medium by 45 and 44% at doses of 10^?8 and 10^?7 mol/L, respectively, compared with the controls (P < 0.05). Leptin significantly increased the concentration of GH in the culture medium by 14 and 12% at doses of 10^?8 and 10^?7 mol/L, respectively (P < 0.05). Leptin also significantly increased the concentration of PRL in the culture medium by 26% compared with the controls at a dose of 10^?7 mol/L (P < 0.05). These results show that leptin stimulates the release of LH, GH and PRL by acting directly on bovine AP cells from fully‐fed steers.     

The effects of phospholipase C on oestradiol and progesterone secretion in porcine granulosa cells cultured in vitro

Granulosa cells play important roles in the regulation of ovarian functions. Phospholipase C is crucial in several signalling pathways and could participate in the molecular mechanisms of cell proliferation, differentiation and ageing. The objective of this study was to identify the effects of phospholipase C on the steroidogenesis of oestradiol and progesterone in porcine granulosa cells cultured in vitro. Inhibitor U73122 or activator m‐3M3FBS of phospholipase C was added to the in vitro medium of porcine granulosa cells, respectively. The secretion of oestradiol decreased after 2 hr, 8 hr, 12 hr, 24 hr and 48 hr of treatment with 500 nM U73122 (p < .05) and decreased after 2 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The secretion of progesterone increased after 4 hr of treatment with 500 nM U73122 (p < .05) and increased after 2 hr and 8 hr of treatment in the 500 nM m‐3M3FBS addition group (p < .05). The ratio of oestradiol to progesterone decreased at each time point, except 8 hr after the addition of 500 nM U73122 (p < .05). The ratio of oestradiol to progesterone decreased after 2 hr (p < .05) of treatment with 500 nM m‐3M3FBS. In genes that regulate the synthesis of oestradiol or progesterone, the mRNA expression of CYP11A1 was markedly increased (p < .05), and the mRNA expression of other genes did not change significantly in the U73122 treatment group, while the addition of m‐3M3FBS did not change those genes significantly despite the contrary trend. Our results demonstrated that phospholipase C can be a potential target to stimulate the secretion of oestradiol and suppress progesterone secretion in porcine granulosa cells cultured in vitro, which shed light on a novel biological function of phospholipase C in porcine granulosa cells.     

Characterization of mesenchymal stem cells in bovine endometrium during follicular phase of oestrous cycle

Stem cells have been postulated as responsible for cell regeneration in highly and continuously regenerative tissues such as the endometrium. Few studies in cattle have identified and specified the presence of stem cells in the endometrium during the oestrous cycle. The aim of this study was to investigate the presence of mesenchymal stem cells (MSCs) in the bovine endometrium during the follicular phase (FP) of the oestrous cycle. Uterine tissue was collected in the time‐frame comprising day 18 of the cycle and ovulation (day 0). We isolated, cultured and expanded four primary cell lines from endometrium and identified byRT‐qPCR the expression of OCT4, SOX2 but not NANOG (undifferentiated/embryonic markers), CD44 (MSCs marker) and c‐KIT (stem cell marker) genes; and the encoded Oct4, Sox2 and Cd44 proteins by Western blot or immunostaining of paraffin‐embedded tissue in endometrium. We demonstrated that cells isolated from bovine endometrium displayed essentially the same gene expression pattern; however, at the protein level, Oct4 and Cd44 were not detected. Besides, they showed typical functional characteristics of MSCs such as fibroblast‐like morphology, plastic adherence, high proliferative capacity, clone formation in vitro and the ability to differentiate into chondrogenic, osteogenic and adipogenic lineages. We obtained for the first time an extensive characterization of undifferentiated cells populations contained in the bovine endometrium during the FP of the oestrous cycle.     

Evaluation of steroidogenic capacity after follicle stimulating hormone stimulation in bovine granulosa cells of Revalor 200~ implanted heifers

Background： Heifers not used as breeding stock are often implanted with steroids to increase growth efficiency thereby altering hormone profiles and potentially changing the environment in which ovarian follicles develop. Because bovine granulosa cell culture is a commonly used technique and often bovine ovaries are collected from abattoirs with no record of implant status, the objective of this study was to determine if the presence of an implant during bovine granulosa cell development impacts follicle stimulating hormone-regulated steroidogenic enzyme expression. Paired ovaries were collected from 16 feedlot heifers subjected to 1 of 3 treatments： non-implanted （n = 5）, Revalor 200 for 28 d （n = 5）, or Revalor 200 for 84 d （n = 6）. Small follicle （1 to 5 mm） granulosa cells were isolated from each pair and incubated with phosphate buffered saline （n = 16） or 100 ng/mL follicle stimulating hormone （n = 16） for 24 h. Results： Granulosa cells of implanted heifers treated with follicle stimulating hormone produced medium concentrations of progesterone similar （P = 0.22） to non-implanted heifers, while medium estradiol concentrations were increased （P 〈 0.10） at 28 and 84 d compared to non-implanted heifers indicating efficacy of treatment. Additionally, real-time PCR analysis in response to follicle stimulating hormone treatment demonstrated a decrease in steroidogenic acute regulatory protein （P = 0.05） mRNA expression in heifers implanted for 84 d and an increase in P450 side chain cleavage mRNA in granulosa cells of heifers implanted for 28 （P 〈 0.10） or 84 d （P 〈 0.05） compared to non-implanted females. However, no difference in expression of 3-beta-hydroxysteroid dehydrogenase （P= 0.57） and aromatase （P = 0.23） were demonstrated in implanted or non-implanted heifers. Conclusions： These results indicate follicles which develop in the presence of high concentrations of androgenic and estrogenic steroids via an implant tend to demonstrate an alter     

Histological and immunohistochemical investigations of ovarian interstitial glands during non‐breeding season in camels (Camelus dromedarius)

The aim of this was to investigate the histology and immunohistochemistry of interstitial glands during non‐breeding season in camel ovaries. A total of 21 mature, non‐pregnant and apparently healthy camels aged between 8 and 12 years were slaughtered. The ovaries were removed within 15 min, cleaned from adipose tissue, weighted and examined grossly. The histological preparation was made, and then, the blocks were cut at 3–5 microns thickness and stained by H&E for histological examinations. Moreover, some sections were stained with Sudan Black for lipid detection. Immunohistochemical analysis of paraffin‐embedded ovarian tissues was performed to detect the localization of S‐100, vimentin, progesterone receptors (PR) and oestrogen receptors (ER). Immunoreactive signals were detected using UltraVision Detection System. The results revealed that the interstitial glands were located in the cortical region and they were arranged in various arrangements either single, in couple or in groups rich in lipid droplet. All interstitial gland arrangements were enclosed by connective tissue capsules containing fibroblasts and collagenous fibres separated them from the surrounding ovarian structures. Both interstitial glands and their surrounding CT were penetrated by several blood vessels. There was a strong immunoreactive signal for S‐100 in the nuclei of interstitial cells, and no signals were detected either in cells of the interstitial glands or their connective tissue with PR. We could conclude that the interstitial gland is distinct in ovary of camel and further studies are needed to elucidate its rule in steroid synthesis.