Effect of heat exposure on prostaglandin production and expression of COX-2, PGES, PGFS, ITGAV and LGALS15 mRNAs in endometrial epithelial cells of buffalo (Bubalus bubalis).

BACKGROUND: Early embryonic mortality is one of the major intriguing factors of reproductive failure that causes considerable challenge to the mammalian cell biologists. Heat stress is the major factor responsible for reduced fertility in farm animals. The present study aimed to investigate the influence of heat stress on prostaglandin production and the expression of key genes, including COX-2, PGES, PGFS, ITGAV and LGALS15, in buffalo endometrial epithelial cells. METHODS AND RESULTS: Buffalo genitalia containing ovaries with corpus luteum (CL) were collected immediately post-slaughter. The stages of the estrous cycle were determined based on macroscopic observations of the ovaries. Uterine lumens of the mid-luteal phase (days 6-10 of the estrous cycle) were washed and treated with trypsin to isolate epithelial cells, which were then cultured at control temperature (38.5 °C for 24 h) or exposed to elevated temperatures [38.5 °C for 6 h, 40.5 °C for 18 h; Heat Stressed (HS)]. The supernatant and endometrial epithelial cells were collected at various time points (0, 3, 6, 12, and 24 h) from both the control and treatment groups. Although heat stress (40.5 °C) significantly (P < 0.05) increased COX-2, PGES, and PGFS transcripts in epithelial cells but it did not affect the in vitro production of PGF2α and PGE2. The expression of ITGAV and LGALS15 mRNAs in endometrial epithelial cells remained unaltered under elevated temperature conditions. CONCLUSION: It can be concluded that elevated temperature did not directly modulate prostaglandin production but, it promoted the expression of COX-2, PGES and PGFS mRNA in buffalo endometrial epithelial cells.

Effect of α-tocopherol in the vitrification medium on the viability, lipid peroxidation, expression of key developmental, apoptotic and stress-related genes in ovine secondary follicles.

The present study aimed to evaluate the effect of α-tocopherol on viability, lipid peroxidation and the expression of apoptosis, stress and development-related genes in the vitrified sheep secondary follicles. Ovarian secondary follicles (200-300 µm) were isolated and distributed separately to the vitrification treatment and supplemented with 5 mM, 10 mM, 20 mM and 30 mM of α-tocopherol (while the control fresh group was without vitrification and supplementation of α-tocopherol). After a week, the follicles were thawed and evaluated for follicular viability by trypan blue dye exclusion method, lipid peroxidation and gene expression studies. The results showed that the vitrification with 10 and 20 mM of α-tocopherol positively affected (p < .05) the viability of vitrified follicles in comparison with vitrified ones without α-tocopherol but the higher concentration of α-tocopherol, i.e., 30 mM negatively affected the viability (p < .05) in comparison with the 10 and 20 mM of α-tocopherol groups. The malondialdehyde (MDA) levels were significantly (p < .05) higher in the vitrified without α-tocopherol group in comparison to the vitrified with 20 mM of α-tocopherol group. The expression of apoptotic-related gene, BCL2L1 was significantly higher in 10 mM α-tocopherol group compared to the control fresh and CASPASE 3, 9 expressions were significantly higher in the vitrified group when compared to the vitrified with 10 mM α-tocopherol group. Expressions of BAX, BAD, BAK, BMP-15 and GDF-9 showed no significant difference among the groups. The mRNA expression of SOD1 was significantly higher in the vitrified without α-tocopherol group when compared to other groups. We conclude that the supplementation of 10 and 20 mM α-tocopherol in vitrification solution was the efficient vitrification procedure for the vitrification of ovine secondary follicles.

Antioxidants supplementation improves the quality of in vitro produced ovine embryos with amendments in key development gene expressions.

The present study assessed the effects of supplementation of different antioxidants on oocyte maturation, embryo production, reactive oxygen species (ROS) production and expression of key developmental genes. In this study, using ovine as an animal model, we tested the hypothesis that antioxidant supplementation enhanced the developmental competence of oocytes. Ovine oocytes aspirated from local abattoir-derived ovaries were subjected to IVM with different concentrations of antioxidants [(Melatonin, Ascorbic acid (Vit C), alpha-tocopherol (Vit E), Sodium selenite (SS)]. Oocytes matured without any antioxidant supplementation were used as controls. The oocytes were assessed for maturation rates and ROS levels. Further, embryo production rates in terms of cleavage, blastocysts and total cell numbers were evaluated after performing in vitro fertilization. Real-Time PCR analysis was used to evaluate the expression of stress related gene (SOD-1), growth related (GDF-9, BMP-15), and apoptosis-related genes (BCL-2 and BAX). We observed that maturation rates were significantly higher in alpha-tocopherol (100 µM; 92.4%) groups followed by melatonin (30 µM; 89.1%) group. However, blastocyst rates in ascorbic acid (100 µM; 19.5%), melatonin (30 µM; 18.4%), alpha-tocopherol (100 µM; 18.2%), and sodium selenite (20 µM; 16.9%) groups were significantly higher (P 0.05) than that observed in the control groups. Total cell numbers in blastocysts in the melatonin, ascorbic acid and alpha-tocopherol groups were significantly higher than those observed in sodium selenite and control groups. ROS production was reduced in groups treated with melatonin (30 µM), vitamin C (100 µM), sodium selenite (20 µM) and α-tocopherol (200 µM) compared with that observed in the control group. Supplementation of antioxidants caused the alterations in mRNA expression of growth, stress, and apoptosis related gene expression in matured oocytes. The results recommend that antioxidants alpha-tocopherol (200 µM), sodium selenite (40 µM), melatonin (30 µM) and ascorbic acid (100 µM) during IVM reduced the oxidative stress by decreasing ROS levels in oocytes, thus improving embryo quantity and quality.

Regulatory role of Wnt signal in the oestradiol synthesis of different size categories of ovarian follicles in buffalo (Bubalus bubalis).

The aim of the present study was to understand the role of Wnt signal in ovarian oestradiol synthesis in various size categories of ovarian follicles. A six-day cell culture system was adopted to test the effect of a Wnt inhibitor i.e. Inhibitor of Wnt response (IWR) on the ovarian granulosa cell oestradiol synthesis and associated genes related to oestradiol synthesis and Wnt signalling (CYP19A1, CCND2, WNT2, FZD6, DVL1, APC, AXIN2, CTNNB1) in buffalo. It was conducted with four groups: Group 1: control, Group 2: control + FSH, Group 3: IWR, Group 4: IWR + FSH. No significant effect of IWR was observed on the ovarian granulosa cell proliferation. No significant difference in the oestradiol levels was found in the spent media harvested after six days of in vitro culture among different groups in small and large-sized ovarian follicles. However, the oestradiol level varied significantly (p < .05) among different treatment groups in medium-sized follicles. The oestradiol level was significantly lower (p < .05) in IWR group compared with the control group and was also significantly lower in IWR + FSH group compared with the FSH group. The Wnt inhibitor had significantly (p < .05) reduced the gene expression of CYP19A1 in large ovarian follicles. Varied effects of IWR-1 and FSH on the expression of other genes were observed. The results indicated that there is a positive role of Wnt signal in oestradiol synthesis in buffalo, but the positive role was more discernible in medium- and large-sized follicles.

Effect of different vitrification protocols on post thaw viability and gene expression of ovine preantral follicles.

The aim of the present study was to establish a vitrification protocol for ovine preantral follicles, which can retain viability after thawing and to evaluate the impact of different vitrification treatments on apoptosis and development-related gene expression. Preantral follicles were isolated from cortical slices of ovaries by the mechanical method of isolation. The isolated preantral follicles (200-300 µm) were randomly assigned into four groups. Group1 - Control Fresh preantral follicles (256 follicles); Group 2- Vitrification treatment A (259 follicles) (Vitrification solution 1 (VS1) - Fetal bovine serum (FBS)10%, Ethylene glycol (EG):1.8 M, Dimethyl sulfoxide (DMSO): 1.4 M, Sucrose-0.3 M for 4 min; VS2- FBS10%, EG:4.5 M, DMSO: 3.5 M, Sucrose:0.3 M for 45 s), Group 3 - Vitr. treatment B (235 follicles) (VS1-FBS 20%, EG:1.3 M, DMSO1.05 M for 15 min, VS2- FBS 20%, EG:2.7 M, DMSO:2.1 M for 5 min) and Group 4-Vitrification treatment C (248 follicles) (VS1-Glycerol(Gly):1.2 M for 3 min, VS2- Gly:1.2 M, EG:3.6 M for 3 min, VS3- Gly3M, EG: 4.5 M for 1 min). Preantral follicles were placed in corresponding vitrification treatments and later plunged immediately into liquid nitrogen (-196 °C). After a week, the follicles were thawed and analyzed for follicular viability by trypan blue dye exclusion method as well as for gene expression. The results showed that the low concentration of cryoprotectants (vitrification treatment B) negatively affected the viability of preantral follicles in comparison with control follicles. There was no significant difference in the viability rates among the Control (87%), Treatment A (79%) and Treatment C (75%). The percentage of viable preantral follicles (73%) derived from Treatment B was significantly decreased (P<0.05%) in comparison to that of control. The expression of apoptotic gene BAK was higher in the vitrification treatment B group. Expressions of the other apoptosis-related genes i.e. Bcl2L1, BAD, BAX, Caspase 3, and Annexin showed no significant difference among the groups. The expression pattern of development competence genes GDF-9 and BMP-15 were higher (P < 0.05) in vitrification treatment A and C, respectively. Expression of NOBOX gene was significantly increased in preantral follicles with Vitrification treatment B compared to the control group. We conclude that both the Vitrification treatment A and Treatment C were the efficient vitrification treatment methods for the vitrification of ovine preantral follicles.

Copper and Selenium stimulates CYP19A1 expression in caprine ovarian granulosa cells: possible involvement of AKT and WNT signalling pathways.

The role of copper and selenium on activation of estradiol synthesis pathways viz. PKA/AKT/WNT is not clearly elucidated. On this background we attempt to elcuiated the role of copper and selenium on mRNA expression of genes associated with estradiol synthesis in caprine ovarian granulose cell models. Ovarian granulosa cells from medium (3-5 mm) sized follicles were aspirated and distributed separately to different groups. Group I: control, Group II: cupric chloride (Cu: 0.5 mM), Group III: sodium selenite (Se: 100 ng/ml), Group IV: Cu + Se. The cells (105/well) were cultured in 96 well plate in the base culture medium of MEMα comprising of nonessential amino acids (1.1 mM), FSH (10 ng/mL), transferrin (5 µg/mL), IGF-I (2 ng/mL), androstenedione (10-6 M), penicillin (100 IU/mL), streptomycin (0.1 mg/mL) and fungizone (0.625 µl/mL) and insulin (1 ng/mL). The cells were incubated in a carbondioxide incubator (38 °C, 5% CO2, 95% RH). The medium was changed on alternate days and cells were harvested on day 6. Day 6 media was used for estimation of estradiol. The RNA isolated form harvested cells was used for qPCR assay. There was no significant (p > 0.05) difference in estradiol concentration between groups. The mRNA expression of AKT1, CYP19A1, WNT2 & 4, FZD6 and APC2 were significantly (p < 0.05) higher in Cu and Cu + Se groups compared to control. Whereas, the mRNA transcript of DVL1 and CSNK1 was significantly (p < 0.05) higher in Cu + Se group compared to control. Incontrast, no significant difference in mRNA expression of PRKAR1A and CTNNB1 was noticed. Our study support a key role of copper and selenium in activation of AKT and WNT signalling pathway that further lead to increase in the mRNA expression of CYP19A1.

In Vitro Embryo Production in Sheep.

The recent advances in biotechnological research have led to development of many advanced reproductive techniques and biological tools which are set to revolutionize the productive efficiency of livestock species. The development of technology for sequencing of whole genomes and mass screening of gene regulation has widened our approach to genetic profiling and mapping, as well as furthering our understanding of underlying physiological mechanisms. The newer biotechnologies of gene transfer, in vitro fertilization and embryo production, cloning, and stem cell technology have been developed and are being refined with efficiencies suitable for use in animal farming. Efficient in vitro systems for maturing oocytes, fertilizing, and developing embryos have resulted in commercial in vitro production of embryos. Here we describe in vitro maturation, in vitro fertilization, embryo production, embryo culture, and quantitation of gene expression in sheep embryos.

Nutritional and metabolic stressors on ovine oocyte development and granulosa cell functions in vitro.

The present study was undertaken to study the effect of ammonia, urea, non-esterified fatty acid (NEFA), and ß-hydroxybutyric acid (ß-OHB) on oocyte development and granulosa cell (GC) growth parameter of ovine (Ovis aries). Ovine oocytes were matured in vitro in the presence of different concentration of ammonia, urea, NEFA, and ß-OHB for 24 h, in vitro inseminated and evaluated for cleavage and blastocyst yield. Same concentrations of ammonia, urea, NEFA, and ß-OHB were examined on growth parameters and hormone secretion activity of granulosa cells in vitro. Real-time reverse transcription polymerase chain reaction was used to evaluate the expression of steroidogenic genes (steroidogenic cytochrome P-450 (CYP11A1, CYP19A1)), cell proliferation-related genes (GDF9, FSHr), and apoptosis-related genes (BCL-2 and BAX). The maturation, cleavage, and blastocyst production rates were significantly lowered in media containing either 200 µM ammonia or 5 mM urea or high combo NEFA or 1 µM ß-OHB. Exposure of granulosa cell to 400 µM ammonia or 1 µM ß-OHB or very high combo or 6 mM urea significantly decreased all the parameters examined compared to lower levels of all nutritional and metabolic stressors. Elevated concentration of metabolic stressors induced GC apoptosis through the BAX/BCL-2 pathway and reduced the steroidogenic gene messenger RNA (mRNA) expression and cell proliferation gene mRNA expression. These results suggested that the decreased function of GCs may cause ovarian dysfunction and offered an improved understanding of the molecular mechanism responsible for the low fertility in metabolic stressed condition.

Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes.

The present study was undertaken to study the effect of metabolic stressors like elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and ß-hydroxybutyric acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles, hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100-250 µm) and large pre-antral follicles (LPFs, 250-450 µm) were isolated from slaughterhouse ovaries by a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7 days respectively and examined for their growth, survival and growth rates of enclosed oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300 and 400 µM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70 µM): stearic acid, SA (10 µM)+Palmitic acid, PA(20 µM)+oleic acid, OA(40 µM), b) Medium combo (140 µM): SA (20 µM)+ PA(40 µM)+ OA(80 µM), c) High combo (210 µM): SA (30 µM)+PA(60 µM)+OA(120 µM), d) Very high Combo (280 µM): SA(40 µM)+PA(80 µM)+OA(160 µM)] and BHB (0, 0.5, 0.75, and 1 µM). Results indicated that ammonia, urea, NEFA and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes at the levels of 300 µM, 8 mM, high combo level of NEFA and 0.75 µM respectively. Our study may contribute to the identification of the mechanisms involved in decline of fertility due to metabolic and nutritional stress in ruminants.

L-carnitine Mediated Reduction in Oxidative Stress and Alteration in Transcript Level of Antioxidant Enzymes in Sheep Embryos Produced In Vitro.

The objective of this study was to find out the effect of L-carnitine on oocyte maturation and subsequent embryo development, with L-carnitine-mediated alteration if any in transcript level of antioxidant enzymes (GPx, Cu/Zn-SOD (SOD1) and Mn-SOD (SOD2) in oocytes and developing sheep embryos produced in vitro. Different concentrations of L-carnitine (0 mm, 2.5 mm, 5 mm, 7.5 mm and 10 mm) were used in maturation medium. Oocytes matured with 10 mm L-carnitine showed significantly (p < 0.05) higher cleavage (66.80% vs 39.66, 41.76, 44.64, 64.31%), morula (48.50% vs 20.88, 26.01, 26.99, 44.72%) and blastocyst (33.22% vs 7.66, 9.19, 10.71, 28.57%) percentage as compared to lower concentrations (0 mm, 2.5 mm, 5 mm and 7.5 mm). Cleavage percentage between 10 mm and 7.5 mm L-carnitine were not significantly different. Maturation rate was not influenced by supplementation of any experimental concentration of L-carnitine. There was a significant (p < 0.05) decrease in intracellular ROS and increase in intracellular GSH in 10 mm L-carnitine-treated oocytes and embryos than control group. Antioxidant effect of L-carnitine was proved by culturing oocytes and embryos with H2O2 in the presence of L-carnitine which could be able to protect oocytes and embryos from H2O2-induced oxidative damage. L-carnitine supplementation significantly (p < 0.05) upregulated the expression of GPx and downregulated the expression of SOD2 genes, whereas the expression pattern of SOD1 and GAPDH (housekeeping gene) genes was unaffected in oocytes and embryos. It was concluded from the study that L-carnitine supplementation during in vitro maturation reduces oxidative stress-induced embryo toxicity by decreasing intracellular ROS and increasing intracellular GSH that in turn improved developmental potential of oocytes and embryos and alters transcript level of antioxidant enzymes.

Ammonia concentrations in different size classes of ovarian follicles of sheep (Ovis aries): Possible mechanisms of accumulation and its effect on oocyte and granulosa cell growth in vitro.

The present study investigated the concentrations and the mechanisms of accumulation of ammonia in different sizes of ovarian follicles and the effect of ammonia on oocyte and granulosa cell growth and functions in vitro with sheep (Ovis aries) as an animal model. The effects of cyclicity, seasonality, phases of the estrous cycle, and seasons (environmental) on ammonia concentrations in follicular fluid were also investigated. The effect of ammonia on in vitro development of oocytes (maturation rate, viability rate, cleavage rate, morulae/blastocysts yield) recovered from different sizes of follicles was examined at the levels of 0, 50, 100, 150, 250, 300, and 500 µM. Same concentrations of ammonia were examined on growth parameters (metabolic activity, viability, cell number increment, monolayer formation, apoptosis rate) and hormone (progesterone, estrogen) secretion activity of granulosa cells in vitro. Results suggested as the follicle size increased, ammonia concentrations decreased. The ammonia concentrations in ovine follicular fluid were found to be 261.5 ± 32.4, 157.7 ± 19.2, and 42.9 ± 8.3 µM, respectively, for small, medium, and large follicles. The corresponding ranges were 290 to 238 µM, 184 to 142 µM, and 70 to 22 µM. The differences were due to more accumulation of fluid, less metabolic activity of granulosa cells, and elevation of protein, potassium, and chloride as the follicle size increased. The seasonality and phases of the estrous cycle did not have any effect on ammonia level in ovarian follicles. Ammonia concentrations in all size classes of follicles examined were significantly reduced in ewes during hot seasons compared to cold seasons and in acyclic animals compared to cyclic ones. Ammonia impaired oocyte development at 300 µM when the oocytes were isolated from small follicles and at 250 µM when the oocytes were isolated from medium and large follicles. In contrast, ammonia caused the negative impact on granulosa cells growth and secretary activity at 250 µM when the cells were isolated from small and medium follicles and at 150 µM when the cells were isolated from large follicles.

Effect of ammonia-generating diet on ovine serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell functions.

This study was undertaken to elucidate the effect of ammonia-generating diet on serum and follicular fluid ammonia and urea levels, serum oestrogen and progesterone concentrations and granulosa cell growth and secretion parameters in ewes (Ovis aries). Ewes were fed with 14% CP diet (control) or ammonia-generating diet or ammonia-generating diet plus soluble sugar. The serum and follicular fluid ammonia and urea level, serum oestrogen and progesterone levels and granulosa cell (obtained from ovaries of slaughtered ewes) growth parameters and secretory activities were estimated. Ammonia-generating diet (high-protein diet) increased the serum ammonia and urea concentration. Supplementation of soluble sugar significantly reduced the ammonia concentration in serum with comparable levels as in control group; however, the urea level in the same group was higher than that observed in control group. Supplementation of soluble sugar significantly reduced the follicular fluid ammonia concentration; however, the level was significantly higher compared to control group. Supplementation of soluble sugar brought down the follicular fluid urea level comparable to that observed in control group. Oestrogen and progesterone levels remained unchanged in ewes fed with different types of diet. Oestrogen and progesterone secretion were significantly lowered from granulosa cells recovered from ewes fed with high ammonia-generating diet. Low metabolic activity and high incidence of apoptosis were observed in granulosa cells obtained from ovaries of ewes fed with ammonia-generating diet.

Maturation timing and fetal bovine serum concentration for developmental potential of sheep oocytes in vitro.

The success of in vitro embryo production (IVEP) in animals has improved over time, employing a variety of culture media. Here, we assessed the maturation timing and developmental potential of sheep oocytes in vitro at different concentrations of fetal bovine serum (FBS): Cumulus oocyte complexes (COCs) were aspirated from follicles (2-6 mm) of sheep ovaries collected from local slaughter house. COCs were randomly divided into two groups and matured at 38.5'C, 5% CO2 for 24 h (Group I) and 27 h (Group II). Oocytes cultured for 27 h showed significantly (P <0.05) more maturation than those cultured for 24 h (82 vs. 76%) followed by more cleavage (35 vs. 30%), morula (53 vs. 39%) and blastocyst (17 vs. 11%) percentage. In the second experiment, oocytes were randomly divided into two groups and matured with 10% FBS (Group I) and 20% FBS (Group II) for 27 h supplemented with pyruvate, glutamine, LH, FSH and estradiol. After maturation, oocytes were fertilized by fresh semen for 18 h. Presumptive zygotes in both the groups were again divided into two groups and culturedin 10 and 20% FBS during post fertilization period, respectively. Different FBS concentration in maturation medium did not influence maturation percentage (82 vs. 79%) significantly. Out of culture groups, presumptive zygotes matured in 20% FBS and cultured in 20% FBS during post fertilization period showed significant increase in cleavage percentage (44 vs. 39, 35 and 27%) as compared to other groups but subsequent development to morula (55 vs. 53, 43 and 40%) and blastocyst (20 vs. 17, 16 and 15%) percentage were more in the group matured in 10% FBS and cultured in 20% FBS during post fertilization period.

Regulation and regulatory role of WNT signaling in potentiating FSH action during bovine dominant follicle selection.

Follicular development occurs in wave like patterns in monotocous species such as cattle and humans and is regulated by a complex interaction of gonadotropins with local intrafollicular regulatory molecules. To further elucidate potential mechanisms controlling dominant follicle selection, granulosa cell RNA harvested from F1 (largest) and F2 (second largest) follicles isolated at predeviation (PD) and onset of diameter deviation (OD) stages of the first follicular wave was subjected to preliminary RNA transcriptome analysis. Expression of numerous WNT system components was observed. Hence experiments were performed to test the hypothesis that WNT signaling modulates FSH action on granulosa cells during follicular waves. Abundance of mRNA for WNT pathway members was evaluated in granulosa cells harvested from follicles at emergence (EM), PD, OD and early dominance (ED) stages of the first follicular wave. In F1 follicles, abundance of CTNNB1 and DVL1 mRNAs was higher and AXIN2 mRNA was lower at ED versus EM stages and DVL1 and FZD6 mRNAs were higher and AXIN2 mRNA was lower in F1 versus F2 follicle at the ED stage. Bovine granulosa cells were treated in vitro with increasing doses of the WNT inhibitor IWR-1+/- maximal stimulatory dose of FSH. IWR-1 treatment blocked the FSH-induced increase in granulosa cell numbers and reduced the FSH-induced increase in estradiol. Granulosa cells were also cultured in the presence or absence of FSH +/- IWR-1 and hormonal regulation of mRNA for WNT pathway members and known FSH targets determined. FSH treatment increased CYP19A1, CCND2, CTNNB1, AXIN2 and FZD6 mRNAs and the stimulatory effect on CYP19A1 mRNA was reduced by IWR-1. In contrast, FSH reduced CARTPT mRNA and IWR-1 partially reversed the inhibitory effect of FSH. Results support temporal and hormonal regulation and a potential role for WNT signaling in potentiating FSH action during dominant follicle selection.

Stage-specific expression and effect of bone morphogenetic protein 2 on bovine granulosa cell estradiol production: regulation by cocaine and amphetamine regulated transcript.

Members of the bone morphogenetic protein (BMP) family regulate follicular development and granulosa cell function. However, changes in expression of BMP2 and its receptors during follicular waves in cattle and ability of BMP2 to modulate bovine granulosa cell estradiol production are not well understood. The objectives of this study were to determine temporal regulation of mRNA for BMP2 and its type I and II receptors (BMPR1A and BMPR2) in bovine follicles collected at specific stages of a follicular wave (predeviation, early dominance, mid dominance, preovulatory), ability of BMP2 to modulate bovine granulosa cell steroidogenesis, and whether effects of BMP2 on granulosa cell estradiol production are influenced by cotreatment with cocaine- and amphetamine-regulated transcript (CART), an intrafollicular regulatory peptide shown to inhibit estradiol production in response to other trophic hormones (FSH and IGF1). Relative abundance of mRNAs for Bmp2 and Bmpr2 was elevated at the mid dominance stage relative to earlier stages of the follicular wave and further increased at the preovulatory stage. Abundance of mRNA for Bmpr1a was lowest at early dominance stage and highest at preovulatory stage relative to other stages of the follicular wave examined. Treatment of bovine granulosa cells in vitro with BMP2 increased estradiol but decreased progesterone concentrations. Co-incubation with CART reduced the BMP2-stimulated increase in granulosa cell estradiol production. Results suggest that BMP2 may play a regulatory role in development of bovine follicles to the preovulatory stage and that CART can inhibit granulosa cell estradiol production in response to multiple hormones/growth factors, including BMP2.

Isolation and culture of preantral follicles for retrieving oocytes for the embryo production: present status in domestic animals.

The development of efficient ovarian preantral follicle (PF) isolation and culture systems provide a large number of oocytes for the manipulation and embryo production. It also helps for understanding the mechanisms of follicle and oocyte development. Isolation and culture protocols for PFs were developed for many domestic species like cattle, buffalo, sheep, goat, pig, horse, camel, dog and cats; however, embryo production from oocytes derived from in vitro grown PFs was reported only in pigs, buffalo, sheep and goat. The rate of oocyte maturation from PFs grown in vitro is low and requires considerable research. This paper presents an overview of isolation and culture systems of PFs that have been developed for domestic species (cattle, buffalo, sheep, goat, pigs, horse, camel, dog and cat) along with the current status of progress achieved in the direction of producing embryos using PFs as the source of oocyte in these species.

Effects of heavy metals and pesticides on buffalo (Bubalus bubalis) spermatozoa functions in vitro.

Industrial toxic metals, pollutants and bio-accumulative pesticides interfere with the male reproductive functions in farm animals. Frozen-thawed semen samples were incubated with heavy metals (cadmium and lead) and pesticides (chlorpyrifos and endosulfan) of different concentrations (0, 0.005, 0.05, 0.02, 0.1, 0.5, 1.0, 2.0 and 4.0 µg/ml) for 1 h, and various spermatozoa functional parameters and in vitro fertilization rates were assessed. Any significant effect was assessed by comparing the 1 h data between the control and treatment groups. Progressive forward motility was significantly (p < 0.05) reduced in spermatozoa exposed to lower concentrations (0.05-0.5 µg/ml) of toxic substances. The straight-line velocity (µm/s) and the average path velocity (µm/s) were significantly (p < 0.05) reduced in spermatozoa exposed to 1.0 and 0.5 µg/ml of cadmium (11.6 ± 1.9 and 16.3 ± 1.9) and chlorpyrifos (10.4 ± 1.5 and 17.1 ± 1.3), respectively, when compared to control (20.4 ± 1.4 and 28.1 ± 1.7). The acrosomal integrity was also significantly (p < 0.05) reduced at 0.05 µg/ml of chlorpyrifos (33.3 ± 1.9), 1.0 µg/ml of cadmium (36.8 ± 3.7), 1.0 µg/ml of lead (39.4 ± 2.8) and 0.5 µg/ml of endosulfan (38.3 ± 3.2), respectively. The spermatozoa chromatin decondensation was significantly (p < 0.05) affected at higher concentrations (>0.5 µg/ml) of these chemicals. The mitochondrial membrane potential (%) was significantly (p < 0.05) reduced at 0.05 µg/ml of cadmium (3.2 ± 0.2) and chlorpyrifos (4.3 ± 0.4), 0.1 µg/ml of lead (3.8 ± 0.3) and 0.5 µg/ml of endosulfan (3.2 ± 0.3) when compared to control (6.7 ± 1.0). The in vitro fertilization capabilities (cleavage percentage) of spermatozoa were significantly reduced at 1.0 µg/ml of cadmium (28.3 ± 2.4) and 2.0 µg/ml of lead (31.1 ± 2.7), chlorpyrifos (29.4 ± 2.2) and endosulfan (32.6 ± 2.5) when compared to control (59.4 ± 4.4). This study suggested that the mitochondrial membrane potential was primarily affected even with lowest doses of toxic chemicals. Cadmium when compared to lead and chlorpyrifos when compared to endosulfan were found to be more toxic to the spermatozoa.

Chlorpyrifos and endosulfan affect buffalo oocyte maturation, fertilization, and embryo development in vitro directly and through cumulus cells.

This study was undertaken to examine the effect of 10 different levels (0, 0.005, 0.01, 0.02, 0.05, 0.1, 0.5, 1.0, 2.0, and 4.0 µg/mL) of two pesticides (chlorpyrifos and endosulfan) on buffalo oocyte viability, maturation, fertilization, and developmental competences in vitro. Studies were conducted to test the development of oocytes cultured with pesticides during maturation, fertilization, and during different embryo development stages. We also conducted experiments to test the hypotheses that the effects of these pesticides are hormones and somatic cells mediated. We observed a dose dependent decline in viability and developmental competence rates of oocytes. Chlorpyrifos and endosulfan had a negative impact on oocytes at 0.02 and 0.1 µg/mL levels, respectively. These pesticides reduced the oocyte nuclear maturation by a direct effect on oocytes, cumulus cell-mediated action, and by blocking the action of hormones. Chlorpyrifos was found to be more ovotoxic and embryotoxic than endosulfan. This study will provide information on dose-response relationship and risk assessment in domestic buffaloes.

Effects of exposure to heavy metals on viability, maturation, fertilization, and embryonic development of buffalo (Bubalus bubalis) oocytes in vitro.

The aim of the present study was to examine the effect of heavy metals, cadmium and lead, on buffalo oocyte viability and in vitro development. Oocytes were aspirated from ovaries of slaughtered buffaloes. Only viable and metabolically active oocytes with more than three layers of cumulus cell layers and homogeneous ooplasm were selected. Effects of nine concentrations (0, 0.005, 0.05, 0.5, 1.0, 1.5, 2.5, 5, and 10 microg/mL) of cadmium or lead on buffalo oocyte viability, morphological abnormities, maturation, and embryonic development in vitro were studied. Oocytes were cultured for 24 h and then checked for viability (0.05% trypan blue staining for 2 min), morphological abnormalities, and reduction assay by MTT test in experiment 1. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were determined (experiment 2). In experiment 3, viable oocytes were matured in vitro in media containing different levels of cadmium or lead and then inseminated in vitro with frozen-thawed spermatozoa, and the resultant cleaved embryos were cultured in a control embryo culture medium for 8 days. In experiment 4, oocytes were cultured in control oocyte maturation medium, then fertilized, and the resultant embryos were cultured in media containing different levels of cadmium or lead for 8 days. The number of cells in the trophectoderm and inner cell mass (ICM) and the total cell counts (TCN) of blastocysts derived by in vitro culture of two- to four-cell-stage embryos (produced in control medium) in media containing 0, 0.005, 0.05, 0.5, and 1.0 microg/mL of cadmium or lead were analyzed by differential staining technique (experiment 5). Cadmium and lead were found to have a dose-dependent effect on viability, morphological abnormities, maturation, cleavage and morula/blastocyst yield, and blastocyst hatching. A significant decline in viability of oocytes was observed at 1.0 mg/mL cadmium or lead compared to the control group. The doses of cadmium and lead causing 100% oocyte death (1-day culture) were 18 and 32 microg/mL, respectively. Cadmium and lead at 1.0 and 2.5 microg/mL, respectively, caused a significant reduction of maturation of oocytes compared to the lower concentrations. No cleavage or morulae/blastocysts were produced when the oocytes/embryos were cultured in media containing 2.5 and 5.0 mg/mL of either cadmium or lead, respectively. Similarly, no morulae/blastocysts were produced from cleaved embryos cultured in media containing 2.5 and 5.0 microg/mL cadmium and lead, respectively. The developmental block, degeneration, and asynchronous divisions were higher in embryos exposed to cadmium than in those exposed to lead. TCN and number of cells in ICM were significantly lower in blastocysts derived from two- to four-cell-stage embryos cultured in media containing heavy metals. In conclusion, cadmium and lead lowered the viability and development of buffalo oocytes but at a concentration higher than that estimated in the body fluids and environment. Cadmium was found to be more ovotoxic than lead.

Viability and growth of buffalo preantral follicles and their corresponding oocytes in vitro: effect of growth factors and beta mercaptoethanol.

The present study was undertaken to isolate buffalo preantral follicles (PFs), to test the viability and sizes of buffalo PFs and to examine the effect of various growth factors (insulin-like growth factor, fibroblast growth factor) and an antioxidant (beta mercaptoethanol) on the in vitro growth, survival and antrum formation rates of buffalo PFs and growth rates of oocytes in cultured PFs. Preantral follicles from slaughtered buffalo ovaries were recovered by a combined mechanical and enzymatic method. The recovery rates of >40-100, 101-200, 201-300, 301-400 and 401-500 microm PFs were 5.1, 3.2, 3.1, 6.3 and 5.1 per ovary, respectively. The corresponding viability rates were 76.1%, 78.1%, 85.2%, 92.5% and 92.6%, respectively. There was a positive correlation (r = 0.73) between oocyte size and the follicular size. However, there was no significant correlation between the size of oocyte and its viability at the time of its retrieval from ovary. Insulin-like growth factor and fibroblast growth factor improved the survival of buffalo PFs and regulated their growth in culture. The growth factors and beta mercaptoethanol in association synergically improved the growth and survival of buffalo PFs.