Altered Expression of 3β‐HSD,CYP17 and 17β‐HSD in the Foetal Porcine Gonads in Response to Anti‐androgen Flutamide Exposure

We investigated whether the limited access to androgens during late prenatal period alters expression of steroidogenic enzymes involved in androgen production: 3β‐hydroxysteroid dehydrogenase/Δ5‐Δ4 isomerase (3β‐HSD), cytochrome P450 17α‐hydroxylase/17,20‐lyase (CYP17) and 17β‐hydroxysteroid dehydrogenase type 1 (17β‐HSD1) or type 3 (17β‐HSD3) in the foetal porcine gonads. Pregnant gilts were injected with anti‐androgen flutamide (for seven days, 50 mg/day/kg bw) or corn oil (control) starting at 83 (GD90) or 101 (GD108) gestational day. To assess 3β‐HSD, CYP17 and 17β‐HSD1 or 17β‐HSD3 expression, real‐time PCR and immunohistochemistry were performed. In testes from flutamide‐treated foetuses, increased 3β‐HSD and CYP17 mRNA expression was observed in the GD90 group, while decreased 3β‐HSD and 17β‐HSD3 mRNA expression and increased CYP17 mRNA expression were found in the GD108 group. CYP17 and 17β‐HSD3 were localized in Leydig cells. Following flutamide administration, the intensity of CYP17 immunostaining was higher in both treated groups, while 17β‐HSD3 intensity was lower in the GD108 group. In ovaries from flutamide‐treated foetuses in the GD90 group, mRNA level for 3β‐HSD was elevated, but it was diminished for CYP17 and 17β‐HSD1. In the GD108 group, flutamide treatment led to lower mRNA level for 3β‐HSD but higher for CYP17. 3β‐HSD was found in granulosa cells, while CYP17 was localized within egg nests and oocytes of forming follicles. Following flutamide treatment, the intensity of 3β‐HSD and CYP17 immunostaining was higher in the GD90 and GD108 groups, respectively. Immunohistochemical staining for 3β‐HSD was restricted to the ovary. Concluding, diminished androgen action in the porcine foetal gonads during late gestation induces changes in steroidogenic enzymes expression, which may led to changes in gonadal function. However, it seems that androgens exert diverse biological effects depending on the gestational period.     

Altered vitamin D metabolic system in follicular cysts of sows

This study aimed to examine 25OHD₃ concentration in the fluid of follicular and follicular lutein cysts of sows in comparison with preovulatory follicles as well as immunolocalize vitamin D metabolic enzymes (CYP27B1 and CYP24A1) and determine their protein abundances in the cyst wall. We have shown for the first time that 25OHD₃ level in the fluid of both cyst types was significantly lower than in preovulatory follicles. Furthermore, we have demonstrated CYP27B1 and CYP24A1 protein immunolocalization and abundance in follicular and follicular lutein cysts. The abundance of protein for both metabolic enzymes was decreased in ovarian cysts when compared to preovulatory follicles. We propose that altered VD metabolism in ovarian cyst might associate with their formation in sows.     

Expression of messenger ribonucleic acid encoding for steroidogenic acute regulatory protein and enzymes, and luteinizing hormone receptor during the spring transitional season in equine follicles

The period of spring transition, from the anovulatory to the ovulatory season, is characterized in many mares by cyclical growth and regression of large dominant follicles. These follicles produce only low concentrations of estradiol and it is thought that acquisition of steroidogenic competence by large follicles during spring transition is prerequisite in stimulating LH prior to first ovulation. In situ hybridization was used to localize and quantify expression of factors that play a key role in follicular steroidogenesis: StAR, P450scc (CYP11A1), P450c17 (CYP17), P450arom (CYP19), and LH receptor (LHr). One ovary was obtained from mares on the day after detection of an actively growing 30 mm transitional anovulatory follicle (defined as the transitional follicle), and the remaining ovary was removed at the third estrus of the breeding season on the day after the preovulatory follicle reached 30 mm in diameter (defined as the preovulatory follicle). Messenger RNAs encoding StAR, CYP11A1, and CYP17 were detected only in theca cells and CYP19 mRNA was confined to the granulosa layer. There was significantly lower expression of mRNAs for the steroidogenic enzymes, StAR (P<0.001) and LHr (P<0.05) in transitional follicles than in preovulatory follicles. In conclusion, large equine follicles during spring transition have low levels of mRNA encoding steroidogenic enzymes, StAR and LHr which will contribute to the steroidogenic incompetence of dominant follicles during spring transition and their subsequent regression.     

Subclinical,chronic intramammary infection lowers steroid concentrations and gene expression in bovine preovulatory follicles

Chronic, subclinical intramammary infection depresses fertility. We previously found that 30% of subclinical mastitic cows exhibit delayed ovulation, low circulating estradiol levels, and delayed luteinizing hormone surge. We examined the function of preovulatory follicles of cows experiencing subclinical mastitis or a past event of acute clinical mastitis. Cows were diagnosed for mastitis by somatic cell count and bacteriological examination. All clinical infections were caused by Escherichia coli, and most subclinical infections were caused by Streptococcus dysgalactiae and coagulase-negative staphylococci. On day 6 of the cycle, cows received PGF2α; 42 h later, follicular fluids and granulosa cells or theca cells were aspirated from preovulatory follicles in vivo or following slaughter, respectively. Overall, follicular estradiol and androstenedione concentrations in the subclinical group (n = 28) were 40% lower (P < 0.05) than those in uninfected cows (n = 24) and lower than in past clinical mastitic cows (n = 9). Distribution analysis revealed a clear divergence among subclinical cows: one-third (9/28) exhibited low follicular estradiol; the other two-thirds had normal levels similar to all uninfected (P < 0.01) and most clinical cows (P < 0.08) that had normal follicular estradiol levels. Subclinical normal-estradiol cows had twofold higher (P < 0.05) circulating estradiol concentrations and sevenfold and fourfold higher (P < 0.05) follicular androstenedione levels and estradiol-to-progesterone ratio, respectively, than subclinical low-estradiol cows. Follicular progesterone level was not affected. Reduced expression (P < 0.05) of LHCGR in theca and granulosa cells, CYP11A1 (mRNA and protein) and CYP17A1 in theca cells, and CYP19A1 in granulosa cells may have contributed to the lower follicular steroid production in the subclinical low-estradiol subgroup. StAR and HSD3B1 in theca cells and FSHR in granulosa cells were not affected. Mastitis did not alter follicular growth dynamics, and no carryover effect of past clinical mastitis on follicular function was detected. These data indicate that a considerable proportion (one-third) of subclinical mastitic cows have abnormal follicular steroidogenesis, which can explain the reproductive failure associated with this disease.     

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.     

Absence of Sirtuin 1 impairs the testicular development in cattleyak by inactivating SF-1

Cattleyak, which are interspecific hybrids between cattle and yak, display much higher growth performances than yak. However, F1 male cattleyak are infertile due to defective testicular development. Sirtuin 1 (SIRT1) is a histone deacetylase that is essential for various biological processes, while the roles of testicular SIRT1 in yak and cattleyak are still poorly understood. Here, we found that SIRT1 was localized in various kinds of yak testicular cells except elongated spermatids while it was deficient in cattleyak testis. Further studies indicated that cattleyak testis exhibited decreased histone acetylation levels on H3 and H4. One of SIRT1 co-factors, steroidogenic factor-1 (SF-1), was lost in cattleyak testis at protein level. Expressions of several SF-1 target genes responsible for Sertoli cell development and steroidogenesis, including STAR, CYP11A1, CYP26B1, FDX1 and HSD3B, decreased significantly in cattleyak testis. In addition, SIRT1-mediated P53 acetylation was not responsible for the cell apoptosis in cattleyak testis. Taken together, our results suggested the deficiency of SIRT1 in yak testis caused inactivation of SF-1 and the impairment of testicular development. This research provides theoretical bases for understanding the mechanism of cattleyak sterility and gives new insights in revealing the roles of SIRT1 in regulating yak testicular development.     

Possible role of IGF2 receptors in regulating selection of 2 dominant follicles in cattle selected for twin ovulations and births

Abundance of IGF-2 receptor (IGF2R), FSH receptor (FSHR), and LH receptor (LHCGR) mRNA in granulosa cells (GCs) or theca cells (TCs) or both cells as well as estradiol (E₂), progesterone (P₄), and androstenedione concentrations in follicular fluid were compared in cows genetically selected (Twinner) or not selected (control) for multiple ovulations and twin births. Cows were slaughtered at day 3 to 4 (day 3) and day 5 to 6 (day 5) of an estrous cycle, and ovaries, follicular fluid, GCs, and TCs were collected. The two largest (F1 and F2) E₂-active (EA) and E₂-inactive (EI) follicles were selected according to their E₂-to-P₄ ratio and diameter. Androstenedione levels in EA F1 and F2 follicles were 5-fold greater (P < 0.05) in Twinner cows than in control cows on day 3 but did not differ on day 5. Twinner cows also had greater (P < 0.05) E₂ and P₄ concentrations, whereas steroid levels in EI follicles did not differ (P > 0.10) between genotypes. In EA F2 follicles, IGF2R levels in GCs were greater (P < 0.05) in control cows than in Twinner cows on day 3 and day 5, whereas IGF2R mRNA in TCs did not differ (P > 0.10). On day 3, FSHR mRNA levels were greater (P < 0.05) in GCs of EA F1 and EI F2 follicles of control cows than of Twinner cows. LH receptor mRNA expression was less in GCs and greater in TCs of EA F2 follicles in control cows than in Twinner cows (P < 0.05). We hypothesize that reduced GC IGF2R expression in F2 follicles of Twinner cows may play a role in the development of 2 or more dominant follicles.     

Quantitative Echotexture Analysis for Prediction of Ovulation in Mares

The aim of this study was to predict the ovulation in mares by quantitative analysis of the echotextural changes of preovulatory follicular walls. Four mares of breeding age with 32 preovulatory follicles and 11 anovulatory follicles were observed by ultrasonography. The slope of the regression line of the follicular wall and the echogenicity score of granulosa layer (GL) and anechoic layer (AL) were measured from the images on Days -3 (Day 0 = ovulation), -2, and -1, respectively. GL was scored from 1 (anechoic) to 3 (echoic), and prominence of AL was recorded from 1 (gray and thin) to 3 (black and thick). The results indicated that the regression line of the follicular wall for 81.3% (26/32) of preovulatory follicles had the slope value ≥19.0 on Day -1, in which 4 of the 26 preovulatory follicles were ≥19.0 on Day -2 already. Mean slope value on Day -1 (21.9 ± 1.5) was significantly greater (P < .01) than on Day -2 (15.0 ± 1.4) and Day -3 (14.0 ± 1.1). All of the slope values for the 11 anovulatory follicles were <19.0 on any given day. GL and AL scores of preovulatory follicles were significantly greater (P < .01) than in anovulatory follicles on Days -3, -2 and -1; nevertheless, only 28.1% (9/32) of preovulatory follicles scored 3 for both GL and AL simultaneously on Day -1. All anovulatory follicles scored <2 for both GL and AL on Day -1. It was concluded that the slope of the regression line of the follicular wall is useful in predicting preovulatory follicles within 48 hours of ovulation when the value is ≥19.0. Of these follicles (N = 26), 84.6% (22/26) were predicted to ovulate within 24 hours, and 15.4% (4/26) within 24 to 48 hours.

Introduction

Insemination in mares by accurately predicting the time of ovulation may obtain maximum fertility with minimum use of semen, and therefore would definitely be a profitable advantage in the horse farming business. The optimal time for insemination with frozen-thawed semen usually include a shorter interval than if fresh semen or natural breeding is used. To achieve the maximal pregnancy rates with frozen-thawed semen, it is necessary to inseminate mares during a period between 12 hours pre- and 6 hours post-ovulation.[1] Therefore, if the timing of ovulation could be predicted, it would be helpful for the veterinarian to inseminate a mare only once per cycle if performed very close to the time of ovulation. [2] In recent years, many indicators have been reported for predicting impending ovulation in mares, including measurement of electrical resistance of the vaginal mucus, [3] the distinguishable endometrial folding pattern of uterus in estrus, [4] changes in size and shape of the preovulatory follicles, [5, 6 and 7] and the echotexture changes in the preovulatory follicular wall. [8] The latter has been more efficient for predicting the imminence of ovulation; nevertheless, their assessment of criterions was scored subjectively. The hypothesis for this study was based on the published report from Gastal et al in 1998 [8]; they found that 2 echotexture changes of the preovulatory follicle-increasing echogenicity of the granulosa layer and increasing prominence of an anechoic layer beneath the granulosa, were detected in the follicular wall as ovulation approached in mares. Computer-assisted image analysis is an advanced technology for diagnostic ultrasonography to improve the reproductive management of patients. [9, 10 and 11] The purpose of this study is to quantify the echotextural changes in the preovulatory follicular wall as ovulation approaches using computer-assisted image analysis, so that the quantified echotexture changes could serve as an indicator for prediction of ovulation in mares.

Materials and Methods

Animals and Ultrasonography

Four non-lactating and nonpregnant mixed mares between 4 and 14 years of age and weighing between 450 and 550 kg were studied from January to December 2001. The geographic area of the mares in this study was in subtropical Taiwan of the northern hemisphere. All mares were maintained on alfalfa/grass hay and had access to water and mineralized salt. A teaser stallion was introduced to detect the estrus signs of mares about 2 weeks after the end of the last estrus. Follicular changes were monitored with a real-time B-mode linear assay ultrasound scanner, equipped with a 7.5-MHz transrectal probe (Model Scanner 200 Vet, Pie Medical, The Netherlands). Upon detection of a preovulatory follicle, ultrasound examination was performed daily and continued until ovulation. A total of 32 preovulatory follicles and 11 anovulatory follicles were identified from a retrospective determination.Ultrasonographic images were recorded on Hi-8 MP videotape with a Sony DCR-TRV 120 Digital-8 camera. The brightness and contrast controls of the monitor and the time-gain compensation of the scanner were standardized to constant settings throughout the observation period.

Image Analysis

Still images were subsequently captured and saved as TIF files by computer using a digital image analysis program (Image-Pro Express V4.0 for Windows, Media Cybernetics, L.P., USA) with a resolution of 640 × 480 pixels and 256 shades of gray. Echotexture of the regions of interest was defined in terms of pixel intensity ranging from 0 (black) to 255 (white). Three ultrasonographic images of each preovulatory follicle at its distinctly discernible cross section were subsequently selected. To avoid the enhancement of through-transmission, sampling regions were located within the 10 or 2 o'clock position for measurement of pixel values (Fig 1). The pixel values were measured with the “Line Profile” tool, which involved sampling pixel values along a line traversing the follicle wall from the peripheral antrum, GL, AL, to the stroma. A graph of the pixel intensities along the line was produced ( Fig 2). The GL was defined as the highest pixel after which there was a sequential fall in gray-scale values. The pixel values along the curve (P0, P1, P2) were obtained as an average of 9 measurements (3 images per follicle and 3 lines per image) and were used to measure the slope of a regression line of the fall segment ( Fig 2).     

Follicle Dynamics and its Relation with Plasma Concentrations of Progesterone, Luteinizing Hormone and Estradiol during the Egg-Laying Cycle in Ostriches

The aims of this study were (i) to describe the changes in the volume of large ovarian follicles (diameter >3 cm) during the 48 h egg laying cycle in farmed ostriches, and (ii) to quantify factors affecting the volume of the largest measured follicle and the plasma concentrations of progesterone (P₄) and estradiol‐17β (E₂β). In eight egg‐producing birds, which all ovulated during the study period, transcutaneous ultrasound scanning and blood sampling was performed at 3 h intervals. The average volume of the total number of visualized large follicles (V_total), the largest measured follicle (V_F1), the second largest follicle (V_F2) and of all follicles smaller than F2 (V_F3–Fn) were each higher before than after oviposition. V_total, V_F2 and V_F3–Fn nearly doubled in the 24‐h period before oviposition, while V_F1 remained at an equal, rather high level until oviposition. Immediately after oviposition V_total, as well as the volume of the other follicle categories, decreased within 6 h, i.e. around the moment of ovulation. By performing statistical analysis on the basis of linear mixed‐effects modelling, we quantified that: (i) V_F1 was 13.2% higher before than after oviposition and increased with 6.5% when LH increased with 1 ng/ml; (ii) P₄ levels were 93.2% higher before than after oviposition and increased with 43.1% for every 3 h closer to oviposition; when LH and E₂β levels and V_F1 increased with 1 ng/ml, 10 pg/ml and 10 ml, respectively, P₄ increased with 116.6%, 50% and 6.1%; and (iii) E₂β levels were 35.6% higher before than after oviposition, increased with 2.7% for every 3 h closer to oviposition and increased with 14.6% when LH increased with 1 ng/ml. It is concluded that during the egg‐laying cycle in ostriches: (i) follicular mass, as estimated by the volume of visualized follicles larger than 3 cm, increases before and decreases after ovulation, and (ii) follicular dynamics and its accompanying endocrine plasma hormone profiles during the egg‐laying cycle in ostriches follow a pattern similar to that in chickens.     

Effect of Oestrus Synchronization with PGF2α/eCG/hCG on Luteal P4 Synthesis in Early Pregnant Gilts

Administration of hormones to synchronize oestrus is a useful tool in animal breeding. However, exogenous ovarian stimulation may be detrimental to reproductive function. This study was aimed to examine whether an oestrus synchronization with PGF2α/eCG/hCG could affect luteal P4 synthesis in early pregnant gilts. Corpora lutea (CLs) were collected on days 9, 12 and 16 of pregnancy from gilts with natural (n = 16) and synchronized (n = 18) oestrus and analysed for (i) the expre‐ssion of steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A polypeptide (CYP11A1), and 3β‐hydroxysteroid dehydrogenase (3βHSD); (ii) the concentration of P4 in the luteal tissue and blood; and (iii) the expression of luteinizing hormone receptors (LHR) and oestrogen receptors (ERα and ERβ). Additionally, the effect of LH on P4 secretion from CL slices collected from synchronized and naturally ovulated animals has been studied in vitro. PGF_2α/eCG/hCG administration increased mRNA expression of StAR, CYP11A1, 3βHSD, and LHR on day 9 and CYP11A1 and LHR on day 12 of pregnancy compared with the control group (p < 0.05). CYP11A1, 3βHSD, LHR, ERα and ERβ proteins were not affected by synchronization; only StAR protein increased in hormonally treated animals (p = 0.017). The concentration of P4 in luteal tissue was greater on day 9 (p < 0.01), but lower on day 16 (p < 0.05) in gilts with hormonally induced oestrus compared with control animals. Blood serum levels of P4 were lower in synchronized than control gilts (p < 0.001). Synchronization did not affect LH‐stimulated P4 secretion from luteal slices; however, greater basal concentration of P4 in incubation medium was detected for CLs collected from synchronized than control gilts (p < 0.05). In conclusion, synchronization of oestrus with PGF2α/eCG/hCG protocol in gilts did not impair the expression of luteal P4 synthesis system, although decreased P4 concentration in the blood.     

大豆黄酮对伊莎鸡卵泡发育及其P450arom基因表达的影响

实验探讨了大豆黄酮(DAI)对伊莎鸡卵泡发育及其芳香化酶(P450arom)mRNA表达的影响。实验选取16只产蛋后期伊莎鸡,等分为对照组和DAI处理组。对照组饲喂基础日粮,实验组在基础日粮中添加10 mg/kgDAI。实验持续7周后,分离排卵前卵泡(F1、F2、F3……)的颗粒层及小黄卵泡和大白卵泡,通过RT-PCR法检测P450arom mRNA表达的相对丰度。结果表明:DAI明显提高了伊莎鸡小黄卵泡和大白卵泡的数量,P450arommRNA在伊莎鸡不同发育阶段卵泡中的表达存在差异,部分卵泡P450arom mRNA表达的相对丰度显著增加。因此,在产蛋后期伊莎鸡基础日粮中添加DAI可增加不同发育阶段卵泡的数目,上调部分卵泡中与发育相关的基因表达以促进卵泡发育。     

Effect of metyrapone on the timing of oviposition and ovarian steroidogenesis in the laying hen

1. The purpose of this study was to observe the effects of metyrapone on the time of oviposition and LH-stimulated steroidogenesis by granulosa cells and small yellow follicles. 2. In experiment 1, White Leghorn hens were injected for 11 d with 240 mg metyrapone 5 h before 'lights off'. Control hens were injected with 1 ml of vehicle (PEG-400). Metyrapone treatment resulted in a 28% decrease in the rate of lay and the modal frequency of the time of oviposition was phase-shifted by 15 h. 3. In experiment 2, hens were injected with 240 mg metyrapone 5 h before 'lights off' or at 'lights on'. While metyrapone treatment reduced the rate of lay, a clear phase-shift in the distribution of oviposition was not observed. Basal and LH-stimulated progesterone synthesis by the granulosa cells of the largest follicle and oestradiol synthesis by small yellow follicles was significantly reduced. 4. Metyrapone treatment significantly reduced basal, but not LH-stimulated output of androstenedione by whole small yellow follicles compared to that observed in control hens. 5. The addition of metyrapone in vitro to isolated granulosa cells from the three largest preovulatory follicles inhibited LH-stimulated progesterone production in a dose-specific manner. 6. The results of this study suggest that the ability of metyrapone to perturb the open-period is a pharmacological effect mediated through inhibition of ovarian and adrenal steroidogenesis.     

Gossypol inhibits LH‐induced steroidogenesis in bovine theca cells

Gossypol, a polyphenolic aldehyde found in cottonseed, has been shown to perturb steroidogenesis in granulosa and luteal cells of rats, pigs and cattle. However, little is known about the direct effect of gossypol on theca cell functions in any species. The present study was conducted to investigate the effect of gossypol on the steroidogenesis and the expression of genes involved in it in cultured bovine theca cells. Theca cells were isolated from healthy preovulatory follicles and were cultured in the presence of luteinizing hormone (LH) for up to 7 days. During the culture period, main steroid products of the theca cells shifted from androstenedione (A4) at day 1 to progesterone (P4) from day 2 onward. At days 1 and 7, theca cells were treated with gossypol (0‐25 μg/mL) for 24 h. Gossypol inhibited LH‐stimulated theca cell A4 and P4 production in a dose‐dependent manner at both occasions. The viability of theca cells was not affected by gossypol at any doses used. Gossypol down‐regulated expressions of steroidogenic enzymes CYP11A1, HSD3B1 and CYP17A1, but not that of LHR. These results indicate that gossypol inhibits thecal steroidogenesis through down‐regulating gene expressions of steroidogenic enzymes but without affecting cell viability in cattle.     

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.     

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.