Buffalo Ovarian Follicles Research Articles

Follistatin (FST) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development.

Follistatin (FST), a member of the transforming growth factor-β (TGF-β) superfamily, has been identified as an inhibitor of follicle-stimulating hormone. Previous studies showed that it plays an important role in animal reproduction. Therefore, this study aims to investigate its effect on the maturation of buffalo oocytes invitro, and the underlying mechanism of FST affecting oocyte maturation was also explored in buffalo cumulus cells. Results showed that FST was enriched in the ovary and expressed at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. The FST expression level was up-regulated in MII buffalo oocytes compared with the GV stage (p < .05). To study the effects of FST on buffalo oocytes' maturation and early embryonic development, we added the pcD3.1 skeleton vector and PCD3.1-EGFP-FST vector into the maturation fluid of buffalo oocytes, respectively. It was demonstrated that FST promoted the invitro maturation rate of buffalo oocytes and the blastocyst rate of embryos cultured invitro (p < .05). By interfering with FST expression, we discovered that FST in cumulus cells plays a crucial role in oocyte maturation. Interference with the FST expression during the buffalo oocyte maturation did not affect the first polar body rate of buffalo oocyte (p > .05). In contrast, the location of mitochondria in oocytes was abnormal, and the cumulus expansion area was reduced (p < .05). After parthenogenetic activation, the cleavage and blastocyst rates of the FST-interfered group were reduced (p < .05). Furthermore, RT-qPCR was performed to investigate further the underlying mechanism by which FST enhances oocyte maturation. We found that overexpression of FST could up-regulate the expression level of apoptosis suppressor gene Bcl-2 and TGF-β/SMAD pathway-related genes TGF-β, SMAD2, and SMAD3 (p < .05). In contrast, the expression levels of SMAD4 and pro-apoptotic gene BAX were significantly decreased (p < .05). The FST gene could affect buffalo oocyte maturation by regulating the oocyte mitochondria integrity, the cumulus expansion, cumulus cell apoptosis, and the expression levels of TGF-β/SMAD pathway-related genes.

The effects of apelin on IGF1/FSH-induced steroidogenesis, proliferation, Bax expression, and total antioxidant capacity in granulosa cells of buffalo ovarian follicles.

Apelin (APLN) was believed to be an adipokine secreted from adipose tissue. However, studies demonstrate that it is a pleiotropic peptide and has several effects on the female reproductive system. In this study, We examined the effects of different doses of IGF1 and FSH in the presence of APLN-13 on the production of progesterone in buffalo ovary granulosa cells. Furthermore, different doses of APLN isoforms (APLN-13 and APLN-17) were tested on proliferation, Bax protein expression, and antioxidant capacity in the same cells. Granulosa cells of buffalo ovaries were cultured in the presence of different doses of IGF1 and FSH with or without APLN-13 (10-9M) to evaluate its effect on the secretion of progesterone tested by ELISA assay. The WST-1 method was used to survey the effect of APLN on granulosa cell proliferation and cytotoxicity. In addition, the antioxidant capacity of the cells in the presence of APLN was assessed using the FRAP method. mRNA and Bax protein levels were measured in granulosa cells treated with APLN using real-time PCR and western blot techniques. APLN-13 (10-9) stimulated the effect of IGF1 on the production of progesterone, and its levels were affected by APLN-13 dose-dependently. However, it did not significantly stimulate the effect of FSH on the secretion of progesterone. APLN-13 (all doses) and APLN-17 (10-8 and 10-9M) improved the proliferation of granulosa cells. Moreover, preincubation of the cells for an hour by APLN receptor antagonist (ML221, 10µM) did not significantly affect the proliferation of cells induced by APLN. Neither APLN-13 nor APLN-17 were not cytotoxic for the cells compared to the control treatment. APLN-13 at the doses of 10-6 and 10-8M substantially up and down-regulated Bax protein expression; however, such effects were not observed when the cells were preincubated with ML221. In addition, APLN-17 did not influence the expression amount of Bax. Furthermore, both APLN-13 and -17 improved the total antioxidant capacity of the ovarian granulosa cells, but such effects were not seen when the cells were preincubated with ML221. According to these results, APLN enhanced the steroidogenesis induced by IGF1 but did not affect the steroidogenesis induced by FSH. APLN also enhanced the cell proliferation and antioxidant capacity of buffalo ovaries follicular granulosa cells; however, its effect on Bax expression was different.

Orexin system in buffalo ovarian follicles and effect of orexin on estradiol production.

Orexin is a ligand for orexin receptors OX1R and OX2R; it is a neuropeptide with pleiotropic functions, including regulation of reproduction. The current study was carried out to investigate the mRNA expression of the prepro-orexin gene (PPO) and orexin receptors (OX1R and OX2R) in ovarian follicles during different stages of their development in the ovary of water buffalo (Bubalus bubalis) and to determine the role of orexin on estradiol production. Ovarian follicles were classified into four groups based on size and estradiol (E2 ) level in the follicular fluid (FF) as follows: (i) small or F1, (ii) medium or F2, (iii) large or F3 and (iv) dominant/pre-ovulatory follicle or F4. In follicles, the mRNA expression of PPO and OX1R was greater in F3 and F4 follicles in granulosa cells (GC) and theca interna (TI) cells. OX2R expression did not vary amongst the different follicular stages in GC. Orexin A and orexin receptors were localized in the cytoplasm of GC and TI, and intensity was higher in F3 and F4 follicles. In addition, we cultured GC and treated them at 0.1,1.0, and 10 ng/ml orexin A alone or in the presence of FSH (30 ng/ml) or IGF-I (10ng/ml) for 48h. There was a significant (p < 0.05) increase in estradiol (E2 ) secretion and cytochrome P0450 family 19 subfamily A member 1 (CYP19A1) expression from GC at 1.0 and 10.0 ng/ml orexin A in the presence of 30 ng/ml follicle-stimulating hormone (FSH) or 10 ng/ml insulin-like growth factor-I (IGF-I). In conclusion, the present study provided evidence that the orexin system is expressed in buffalo ovarian follicles, and orexin-A in the presence of FSH and IGF-I has a stimulatory effect on estradiol secretion from the GC of water buffalo.

Expression and localization of apelin and apelin receptor (APJ) in buffalo ovarian follicles and corpus luteum and the in-vitro effect of apelin on steroidogenesis and survival of granulosa cells

Apelin is an adipose tissue-derived hormone with many physiological functions, including the regulation of female reproduction. It acts through an orphan G protein-coupled receptor APJ/APLNR. The present study aimed to investigate the expression of apelin and its receptor APJ in the ovarian follicles and corpus luteum (CL) and the role of apelin on steroidogenesis and cell survival. Ovarian follicles were classified into four groups based on size and estradiol (E2) level in the follicular fluid as follows: (i) F1 (4–6 mm; <0.5 ng/mL) (ii) F2 (7–9 mm; 0.5–5 ng/mL) (iii) F3 (10–13 mm; 5–40 ng/mL) and (iv) F4 (dominant/pre-ovulatory follicle) (>13 mm; >180 ng/mL). The corpora lutea (CL) were categorized into early (CL1), mid (CL2), late luteal (CL3), and regressing (CL4) CL stages. Expression of apelin increased with follicle size, with significantly greatest in the dominant or pre-ovulatory follicle (P < 0.05). Expression of APJ was greater in large and dominant follicles than in small and medium follicles (P < 0.05). In CL, the mRNA and protein abundance of apelin and apelin receptor was greater during mid (CL2) and late luteal (CL3) stages as compared to early (CL1) and regressing (CL4) stages (P < 0.05). Both the factors were localized in granulosa and theca cells of follicles and small and large luteal cells of CL. The pattern of the intensity of immunofluorescence was similar to mRNA and protein expression. Granulosa cells were cultured in vitro and treated at 1, 10, and 10 ng/mL apelin-13 either alone or in the presence of the follicle-stimulating hormone (FSH) (30 ng/mL) or insulin-like growth factor-I (IGF-I) (10 ng/mL) for 48 h. The luteal cells were treated with apelin-13 at 1, 10, and 100 ng/mL doses for 48 h. Apelin treatment at 10 and 100 ng/ml significantly (P < 0.05) increased E2 secretion, cytochrome P450 aromatase or CYP19A1 expression in GC. In luteal cells, apelin at 10 ng/mL and 100 ng/mL significantly (P < 0.05) increased progesterone (P4) secretion and HSD3B1 expression. In GCs, apelin, either alone or in combination, increased PCNA expression and inhibited CASPASE3 expression suggesting its role in cell survival. In conclusion, this study provides novel evidence for the presence of apelin and receptor APJ in ovarian follicles and corpora lutea and the stimulatory effect on E2 and P4 production and promotes GC survival in buffalo, suggesting the role of apelin in follicular and luteal functions in buffalo.

Cleavage Rate of in vitro Matured Oocytes Fertilized with Conventional Semen in Buffaloes

Background: In vitro embryo production in buffaloes has gained much importance in this current scenario due to ever increasing population and high demand of milk and meat. Slaughter house derived bubaline ovaries are a cheap and abundant source of cumulus oocyte complexes.Methods: Oocytes from the buffalo ovarian follicles were recovered by aspiration technique as it facilitates quick recovery. Total 155 ovaries were used in the present study. Surface follicles were measured using vernier calliper and categorized into three groups viz. less than 3 mm, 3-5 mm and greater than 5 mm based on follicular diameter and oocytes were processed for IVM, IVF and IVC using conventional non sorted semen.Result: Overall percentage of small, medium and large follicles in the ovaries were recorded as 16.29 ± 0.94%, 8.14±0.60%, 5.35 ± 0.76%, respectively. Overall recovery rate of COCs was 38%. The percentage of these oocytes were 16.74% (A), 15.25% (B), 25.26% (C), 18.33% (D) and 29.87% (E) respectively. Maturation rate of oocytes were 81.96 ± 2.70%. Fertilization rate was 74.98 ± 3.87%, Cleavage rate % was 40.84±2.51% and Blastocyst percentage was 21.57±1.75% respectively. Application of in vitro embryo production technique using slaughter house ovaries can salvage the genetic potential of bubaline species.

Association of taurine with ovarian follicular steroids and postpartum anestrus condition in Murrah buffaloes

Taurine is an abundant intracellular beta-amino acid majorly synthesized in the liver and transported through plasma. In mammals, taurine was reported to be involved in various physiological functions, including the enhancement of testosterone levels, the major estradiol precursor. Therefore, we hypothesize that taurine levels are associated with ovarian follicular steroids as well as with a reproductive problem called postpartum anestrus (PPA) in dairy buffaloes. To understand the taurine levels and its possible role in buffalo ovarian follicles, a correlation was established among taurine, estradiol, and testosterone levels in the ovarian follicular fluid. For this purpose, buffalo ovaries were obtained from the slaughterhouse, and follicular fluid samples were collected from small (<4 mm), medium (4–8 mm) and large (>8 mm) follicles. Taurine and steroid levels in the follicular fluid were analyzed by TLC and ELISA, respectively. Taurine and testosterone levels were significantly (P < 0.05) higher in the follicular fluid of small and medium follicles than large follicles, whereas the estradiol levels were significantly (P < 0.001) higher in the large follicles. Thus, taurine showed a positive correlation (r = 0.75) with testosterone and a negative correlation (r = −0.77) with estradiol in buffalo follicular fluid, indicating its possible role in testosterone function during follicular development. Interestingly, significantly (P < 0.001) lower plasma taurine levels in PPA (n = 50) than normal cyclic (n = 50) buffaloes represented its association with PPA. Therefore, our present study recommends the need for future nutrition studies on taurine supplementation to PPA buffaloes.

Brain-derived neurotrophic factor (BDNF) is expressed in buffalo (Bubalus bubalis) ovarian follicles and promotes oocyte maturation and early embryonic development

Brain-derived neurotrophic factor (BDNF) has been discovered and characterized for several decades, yet its expression pattern in non-neuronal tissues like ovary and potential mechanism during oocyte maturation are still poorly understood. Thus the present study was devised to determine the expression pattern and mechanism of BDNF during buffalo oocyte maturation. The results revealed that BDNF was presented at different stages of buffalo ovarian follicles as well as during oocyte maturation and early embryo development. BDNF’s receptor p75 was detected in granulosa cells, cumulus cells, oocytes, and early embryos, while another receptor neurotrophic tyrosine kinase receptor, type2 (NTRK2) was only identified in granulosa cells and cumulus cells. To determine the effect of BDNF on oocyte maturation and early embryo development, different concentrations (0, 1, 10, 100 ng/mL) of BDNF were added into the in vitro maturation media, respectively. It was divulged that 10 ng/mL BDNF promoted the in vitro maturation rate of buffalo oocytes and the blastocysts rate of embryos cultured in vitro (P < 0.05). Then through using NTRK2 inhibitor K-252a, we found BDNF and its receptor NTRK2 in cumulus cells played an essential role during oocyte maturation. Moreover, to further investigate the underlying mechanism by which BDNF enhances oocyte maturation, RT-qPCR was performed. 10 ng/mL BDNF treatment could decrease the expression level of apoptosis-related genes CCASP9, FAS, up-regulate the expression level of receptor gene NTRK2, cell proliferation-related genes CCNB1, PCNA, gap junction-related genes GJA4, GJA1 as well as cumulus cells expansion-related genes HAS2, PTX3 and TNFAIP6 (P < 0.05). Altogether, our results showed for the first time that BDNF was expressed throughout buffalo ovarian follicle development, oocyte maturation and early embryogenesis. Furthermore, BDNF treatment could improve the efficiency of buffalo oocyte maturation through regulating genes expression in cumulus cells and then promote early embryo development.

Expression and localization of fibroblast growth factor (FGF) family in buffalo ovarian follicle during different stages of development and modulatory role of FGF2 on steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the expression and localization of FGF and its functional receptors in the follicle of buffalo and the treatment of FGF2 on mRNA expression of CYP19A1 (aromatase), PCNA, and BAX (BCL-2 associated X protein) in cultured buffalo granulosa cells (GCs). Follicles were classified into four groups based on size and E2 level in follicular fluid (FF): F1, 4–6mm diameter, E2<0.5ng/ml of FF; F2, 7–9mm, E2=0.5–5ng/ml; F3, 10–13mm, E2=5–40ng/ml; F4, >14mm, E2>180ng/ml. The qPCR studies revealed that the mRNA expression of FGF1, FGF2 and FGF7 were maximum (P<0.05) in theca interna (TI) whereas the transcripts of FGFR1, FGFR2, FGFR2IIIB and FGFR2IIIC were up-regulated (P<0.05) in GCs of F4 follicles. Protein expression of most members were maximum (P<0.05) in F4 follicles except FGFR3 and FGFR4. All members were localized in GC and TI with a stage specific immunoreactivity. Primary culture of GCs with treatment of FGF2 at different dose-time combinations revealed that the mRNA expression and immunoreactivity of CYP19A1 and PCNA were maximum (P<0.05) whereas BAX was minimum (P<0.05) with 200ng/ml at 72h of incubation. The findings indicate that FGF family members are expressed in a regulated manner in buffalo ovarian follicles during different stages of development where FGF2 may promote steroidogenesis and GC survival through autocrine and paracrine manner.

Effect of growth differentiation factor-9 (GDF-9) on the progression of buffalo follicles in vitrified-warmed ovarian tissues.

To improve the reproductive performance of water buffalo to level can satisfy our needs, the mechanisms controlling ovarian follicular growth and development should be thoroughly investigated. Therefore, in this study, the expressions of growth differentiation factor-9 (GDF-9) in buffalo ovaries were examined by immunohistochemistry, and the effects of GDF-9 treatment on follicle progression were investigated using a buffalo ovary organ culture system. Frozen-thawed buffalo ovarian follicles within slices of ovarian cortical tissue were cultured for 14days in the presence or absence of GDF-9. After culture, ovarian slices were fixed, sectioned and stained. The follicles were morphologically analysed and counted. Expression pattern of GDF-9 was detected in oocytes from primordial follicles onwards, besides, also presented in granulosa cells. Moreover, GDF-9 was detected in mural granulosa cells and theca cells of pre-antral follicles. In antral follicles, cumulus cells and theca cells displayed positive expression of GDF-9. In corpora lutea, GDF-9 was expressed in both granulosa and theca lutein cells. After in vitro culture, there was no difference in the number of primordial follicles between cultured plus GDF-9 and cultured control that indicated the GDF-9 treatment has no effect on the primordial to primary follicle transition. GDF-9 treatment caused a significant decrease in the number of primary and secondary follicles compared with controls accompanied with a significant increase in pre-antral and antral follicles. These results suggest that a larger number of primary and secondary follicles were stimulated to progress to later developmental stages when treated with GDF-9. Vitrification/warming of buffalo ovarian tissue had a little remarkable effect, in contrast to culturing for 14days, on the expression of GDF-9. In conclusion, treatment with GDF-9 was found to promote progression of primary follicle that could provide an alternative approach to stimulate early follicle development and to improve therapies for the most common infertility problem in buffaloes (ovarian inactivity).

Expression and localization of angiopoietin family in buffalo ovarian follicles during different stages of development and modulatory role of angiopoietins on steroidogenesis and survival of cultured buffalo granulosa cells

The present study investigated the expression and localization of angiopoietin (ANPT) family members in buffalo ovarian follicles of different size. It also looked at the role of ANPTs in estradiol secretion and mRNA expression of phosphoinositide-3-kinase–protein kinase B signaling pathway cellular proliferation (phosphoinositide-dependant kinase and protein kinase B [AKT]) and proapoptotic (BAD) factors with caspase 3 in cultured buffalo granulosa cells (GCs). The mRNA and protein expression of ANPT-1 was greatest (P < 0.05), whereas ANPT-2 was reduced (P < 0.05) in preovulatory follicles as compared to F1 follicle. Tyrosine kinase with immunoglobulin-like and EGF-like domains 1 transcripts and protein expression did not change in all follicular groups, whereas tyrosine kinase with immunoglobulin-like and EGF-like domains 2 mRNA was highest (P < 0.05) in theca interna but not GC layer of preovulatory follicle. All members of ANPT family were localized in GC and theca interna showing a stage specific immunoreactivity. Cultured GCs were treated with ANPT-1 and ANPT-2 separately at doses of 1, 10, and 100 ng/mL and in combination at 100 ng/mL for three incubation periods (24, 48, and 72 hours). Estradiol secretion was highest (P < 0.05) at 100 ng/mL at 72 hours of incubation when GCs were treated with either protein alone. The mRNA expression of phosphoinositide-dependant kinase and AKT was highest (P < 0.05), and BAD with caspase 3 was lowest (P < 0.05) at 100 ng/mL at 72 hours of incubation, when cultured GCs were treated separately with each protein or in combination. The immuoreactivity of AKT, pAKT, and pBAD were maximal, whereas BAD was minimal with 100 ng/mL at 72 hours when cultured GCs treated with either protein alone. The findings indicate that ANPTs are expressed in a regulated manner in buffalo ovarian follicle during different stages of development where they may promote steroidogenesis and GC survival through autocrine and paracrine actions.

Kit Ligand Expression in granulosa cells during follicular culture and associated maturation rate of oocytes in buffalo (Bubalus bubalis)

The objectives of the study to associate the expression of Kit Ligand gene under different gonadotropins and growth factors supplementation with the maturation rate of oocytes in buffalo ovarian follicular cells during whole follicular culture at subsequent intervals of 24 hours. Intact ovarian follicles were dissected out of buffalo ovaries and cultured in Tissue Culture Media-199 supplemented with gonadotropins and growth factors for 24 hours. A semi-quantitative RT-PCR amplification was obtained in granulose cells of buffalo ovarian follicles and its expression was found to be restricted during initial stage of follicle culture. KL expression persistency in granulosa cells beyond 4 hours during follicular culture was found to be negatively correlated with maturation rate of oocytes. It was concluded that expression of Kit Ligand gene in granulose cells could be predictive role for assessing oocytes maturation rate and possibly the oocytes competence in buffalo.

Expression of mRNA Encoding IGF-I, IGF-II, Type-I, and II IGF-Receptors and IGF-Binding Proteins-1-4 during Ovarian Follicular Development in Buffalo (Bubalus bubalis)

The present study was designed to investigate the expression pattern of IGF-I, IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in different stages of buffalo ovarian preantral follicles (PFs), antral follicles (AFs), ovulatory follicles (OFs), and immature (IM) and in vitro matured (MO) oocytes. Buffalo ovaries were collected from local abattoir, PFs (200-250 µm), AFs (1-3 mm), and OFs (5-8 mm) were isolated by mechanical method. PFs, AFs, OFs, and oocytes were lysed to release mRNA, reverse transcribed, and then subjected to RT-PCR, whereas protein were localized through immunohistochemistry. Relative expression of mRNA transcripts was clearly seen for IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 in all the stages of developing follicles and oocytes. We were unable to detect mRNA and protein expression of IGF-1 in any of the oocytes or follicles at any stage of the development. IGF-II and both IGF receptors mRNA expression were found higher (P < 0.05) in PFs compared to AFs and OFs. Expression of IGFBP-1 and 2 in PFs, as well as IGFBP-3 and 4 in AFs, was found with higher (P < 0.05) levels. The expression results were further confirmed by localization of IGF-II, type-I and II IGF-receptors, and IGFBP-1-4 proteins. In conclusion, IGF-II appears to be the only ligand that is endogenously expressed by all the follicular stages and oocytes, which may act in an autocrine manner through the Type-1 IGF receptor. Expression of IGFBP-1-4 and IGF-II suggests the possible role of these genes in recruitment, growth, proliferation, and steroidogenic responses during developmental phases of buffalo ovarian follicles.

Expression and localization of ghrelin and its receptor in ovarian follicles during different stages of development and the modulatory effect of ghrelin on granulosa cells function in buffalo

Ghrelin, a hormone predominantly found in the stomach, was recently described as a factor that controls female reproductive function. The aim of our study was to investigate the expression and localization of ghrelin and its active receptor, growth hormone secretagogue receptor type 1a (GHS-R1a) in buffalo ovarian follicles of different follicular size and to investigate role of ghrelin on estradiol (E2) secretion, aromatase (CYP19A1), proliferating cell nuclear antigen (PCNA) and apoptosis regulator Bax gene expression on granulosa cell culture. Using real time PCR and western blot, we measured gene and protein expression of examined factors. Localization was done with immunofluorescence method. Expression of ghrelin increased with follicle size with significantly highest in dominant or pre-ovulatory follicle (P<0.05). Expression of GHS-R1a was comparable in medium and large follicle but was higher than small follicles (P<0.05). Both the factors were localized in granulosa and theca cells. Pattern of intensity of immunofluorescence was similar with mRNA and protein expression. In the in vitro study granulosa cells (GCs) were cultured and treated with ghrelin each at 1, 10 and 100ng/ml concentrations for two days after obtaining 75–80 per cent confluence. Ghrelin treatment significantly (P<0.05) inhibited E2 secretion, CYP19A1 expression, apoptosis and promoted cell proliferation. In conclusion, this study provides novel evidence for the presence of ghrelin and receptor GHS-R1a in ovarian follilcles and modulatory role of ghrelin on granulosa cell function in buffalo.

Interrelationships between follicular size, estradiol-17β, progesterone and testosterone concentrations in individual buffalo ovarian follicles

Interrelationships between follicular size, estradiol-17β, progesterone and testosterone concentrations in individual buffalo ovarian follicles