Follicular Atresia in Buffalo: Cocaine- and Amphetamine-Regulated Transcript (CART) and the Underlying Mechanisms.

Atresia is a process in ovarian follicles that is regulated by hormone-induced apoptosis. During atresia, granulosa cell (GC) apoptosis is a key mechanism orchestrated through diverse signaling pathways. Cocaine- and amphetamine-regulated transcript (CART) signaling within ovarian GCs has been demonstrated to play a key role in the regulation of follicular atresia in cattle, pigs, and sheep. The present work aimed to investigate the potential local regulatory role of CART in GC apoptosis-induced follicular atresia in buffalo, focusing on the modulation of the AKT/GSK3ß/ß-catenin signaling pathways, which are the intracellular signaling pathways involved in cell viability. Our findings revealed increased expression of CARTPT and BAX and decreased levels of AKT, ß-catenin, and CYP19A1 genes in atretic follicles compared to healthy follicles. Subsequently, CART treatment in the presence of FSH inhibited the FSH-induced increase in GC viability by reducing estradiol production and increasing apoptosis. This change was accompanied by an increase in the gene expression levels of both CARTPT and BAX. At the protein level, treatment with CART in the presence of FSH negatively affected the activity of AKT, ß-catenin, and LEF1, while the activity of GSK3ß was enhanced. In conclusion, our study shows how CART negatively influences buffalo GC viability, underlying the modulation of the AKT/GSK3ß/ß-catenin pathway and promoting apoptosis-a key factor in follicular atresia.

CX43 and oxidative stress are the targets of BCB staining to predict the developmental potential of buffalo oocytes.

This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.

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 (10-9 M) 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 10-9 M) improved the proliferation of granulosa cells. Moreover, preincubation of the cells for an hour by APLN receptor antagonist (ML221, 10 µM) 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 10-8 M 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.

Oocyte aneuploidy rates in river and swamp buffalo types (Bubalus bubalis) determined by Multi-color Fluorescence In Situ Hybridization (M-FISH).

Aneuploidy is one of the main causes of fetal and embryonic mortality in mammals. Nonetheless, its incidence in domestic ruminants has been investigated little. Indeed, no incidence data have ever been reported for water buffalo. To establish the incidence of aneuploidy in this species, we analysed in vitro matured metaphase II (MII) oocytes with corresponding first polar bodies (I PB) of the river (2n = 50) and swamp (2n = 48) buffaloes. For the first time, six river type probes (corresponding to chromosomes 1-5 and heterosome X), were tested on swamp buffalo metaphases using Multicolor-Fluorescent In Situ Hybridization (M-FISH) before their use on oocytes MII metaphases. Of the 120 total Cumulus Oocyte Complexes (COCs, 60 for each buffalo type) subjected to in vitro maturation, 104 reached the MII stage and were analysed by M-FISH. Haploid chromosome arrangement and visible I PB were observed in 89 of the oocytes (45 in river and 44 in swamp type). In the river type, the analysis revealed one oocyte was disomic for the chromosome X (2.22%). In the swamp type, one oocyte was found to be nullisomic for chromosome X (2.27%); another was found to be nullisomic for chromosome 5 (2.27%). We also observed one oocyte affected by a premature separation of sister chromatids (PSSC) on the chromosome X (2.27%). In both buffalo types, no abnormalities were detected in other investigated chromosomes. Based on merged data, the overall aneuploidy rate for the species was 3.37%. Oocytes with unreduced chromosomes averaged 1.92% across the two types, with 1.96% in river and 1.88% in swamp. The interspecies comparison between these data and cattle and pig published data revealed substantial difference in both total aneuploidy and diploidy rates. Reducing the negative impact of the meiotic segregation errors on the fertility is key to more sustainable breeding, an efficient embryo transfer industry and ex-situ bio-conservation. In this respect, additional M-FISH studies are needed on oocytes of domestic species using larger sets of probes and/or applying next generation sequencing technologies.

2-Mercaptoethanol promotes porcine oocyte maturation in vitro by maintaining autophagy homeostasis.

2-Mercaptoethanol (2-ME) is often used as an antioxidant to optimize culture systems for in vitro oocyte maturation in livestock. However, the relationship between 2-ME and autophagy has not yet been elucidated. In this study, we hypothesized that 2-ME can promote porcine oocyte maturation in vitro by maintaining autophagy homeostasis. To test this hypothesis, we explored the effects of 2-ME on the maturation of porcine oocytes exposed to an autophagy activator (rapamycin) or an autophagy inhibitor (3-methyladenine, i.e., 3-MA) in vitro. Rapamycin-induced autophagy over-activation significantly increased autophagy- and apoptosis-related gene expression, oxidative stress, apoptosis rates, abnormal mitochondrial redistribution, and significantly decreased oocyte first polar body extrusion (PBE) rates, spindle/chromosome integrity and developmental competence. 3-MA-mediated autophagy inhibition exerted similar effects on all these parameters except the expression of genes that promote autophagy and inhibit apoptosis. Importantly, 2-ME supplementation significantly attenuated the detrimental effects of rapamycin and 3-MA. Interestingly, we observed that 44 h of coincubation with rapamycin/3-MA and 2-ME restored autophagy homeostasis in vitro. In conclusion, our study confirmed that 2-ME promotes porcine oocyte maturation and embryo development in vitro by maintaining autophagy homeostasis and lays a foundation for further research on the underlying mechanism.

Apelin and Apelin Receptor in Follicular Granulosa Cells of Buffalo Ovaries: Expression and Regulation of Steroidogenesis.

Apelin (APLN), as a ligand for APJ (an orphan G-protein-coupled receptor), is an adipokine with pleiotropic effects in many physiological processes of the body. It has an important role in the control of reproduction particularly in females (mainly in control of ovarian function). This study was carried out to investigate the mRNA and protein amounts of APLN/APJ in granulose cells (GCs) of ovarian follicles with small (SF), medium (MF), and large (LF) sizes of buffalo (Bubalus bubalis) and the effect of IGF1 and follicle-stimulating hormone (FSH) on the expression levels of APLN/APJ. In addition, we evaluated the effect of various doses of APLN (isoforms -13 and -17) singly or in combination with IGF1 and FSH on estradiol (E2) and progesterone (P4) secretion in GCs. The mRNA and protein abundance of APLN was the highest in GCs of LF while the APJ expression enhanced with follicle enlargement in GCs (p-value <0.01). IGF1 and FSH elevated the mRNA and protein amounts of APLN and FSH, and IGF1 increased the expression of APJ in buffalo GCs (p-value <0.01). Both isoforms of APLN (-13/-17) singly or in the presence of IGF1 or FSH increased the secretion of E2 and P4 with or without preincubation of cells with APJ antagonist (ML221 10 µM), although we had some variation in the effects. Concurrently, APLN-13/-17 significantly increased the mRNA and protein expression of CYP19A1 and StAR (p-value <0.01). ML221 substantially diminished the secretion of E2 and P4 and also the expression of CY19A1 and StAR in buffalo GCs (p-value <0.01). We also revealed that APLN-13/-17 (10-9 M), singly or in response to IGF1 and FSH, increased the production of E2 and P4 in different times of stimulation. In conclusion, APLN may play a crucial role in steroidogenesis and follicular development in ovarian GCs of buffalo.

Curcumin mediates autophagy and apoptosis in granulosa cells: a study of integrated network pharmacology and molecular docking to elucidate toxicological mechanisms.

Curcumin (Cur) is a flavonoid derived from Curcuma longa L. that has been shown to have a variety of biological activities, but some previous studies have described its non-negligible negative effects on female reproduction and embryo development. To further explore the toxic stress effect, this study investigated apoptosis and autophagy of healthy buffalo (Bubalus bubalis) derived granulosa cells (GCs) exposed to Cur and/or autophagy inhibitors. Results showed that Cur declined viability of GCs in a concentration-dependent manner. Apoptosis was observed in Cur-treated GCs from 3 h. Meanwhile, under Cur stress, autophagosomes accumulated in cells, and the expression levels of autophagy key proteins LC3 and Beclin 1 were up-regulated, suggesting that Cur could induce autophagy in GCs. Early autophagy inhibitor 3-methyladenine (3-MA) increased the apoptosis rate of Cur exposed GCs, but the autophagosome degradation inhibitor chloroquine (CQ) had no effect on the apoptosis rate. The network pharmacological and molecular docking analysis indicated that the perturbation of IKK/NF-κB might be the cause of Cur toxicity toward GCs. This study unveiled another side of Cur pharmacological effects that programmed cell death can be induced by Cur in GCs, suggesting that it should be prudent to use Cur as a clinical drug for its side effects on the female reproductive system.

Reproductive roles of novel adipokines apelin, visfatin, and irisin in farm animals.

The adipose tissue has a substantial impact on reproduction in mammals, specifically in females. As an energy depository organ, it is precisely associated with the reproductive success of mammals. Adipose tissue secretes many single molecules that are called 'adipokines' which mainly act as endocrine hormones. Adipokines homeostasis is fundamental to energy regulation, metabolic and cardiovascular diseases. The endocrine function of adipokines is influential for the long-term control of energy metabolism and performs an important function in metabolic state and fertility modulation. During the last years, new roles for adipokines have been appearing in the field of fertility. The adipokines have functions in reproduction at levels of the hypothalamus, the pituitary, and the gonads in humans, rodents, and other animals. Normal levels of adipokines are indispensable to protect the integrity of the hypothalamus-hypophysis-gonadal axis, regular ovulatory processes, and successful embryo implantation. Leptin and adiponectin are the most studied adipokines, but also the novel adipokines; apelin, visfatin, and irisin are important adipokines having several functions within the reproductive tract. Due to the known and unknown effects of these novel adipokines in the reproduction of farm animals, in this review, we will highlight the reproductive functions of apelin, visfatin, and irisin and summarize the known reproductive effects in farm animals to introduce the gaps for future studies in farm animals.

Fucoidan from Laminaria japonica Inhibits Expression of GLUT9 and URAT1 via PI3K/Akt, JNK and NF-κB Pathways in Uric Acid-Exposed HK-2 Cells.

This work aimed to investigate the effect of fucoidan (FPS) on urate transporters induced by uric acid (UA). The results showed that UA stimulated the expression of glucose transporter 9 (GLUT9) and urate transporter 1 (URAT1) in HK-2 cells, and FPS could reverse the effect. Moreover, UA could activate NF-κB, JNK and PI3K/Akt pathways, but both pathway inhibitors and FPS inhibited the UA-induced activation of these three pathways. These data suggested that FPS effectively inhibited the expression induction of reabsorption transporters URAT1 and GLUT9 by UA, through repressing the activation of NF-κB, JNK and PI3K/Akt signal pathways in HK-2 cells. The in vitro research findings support the in vivo results that FPS reduces serum uric acid content in hyperuricemia mice and rats through inhibiting the expression of URAT1 and GLUT9 in renal tubular epithelial cells. This study provides a theoretical basis for the application of FPS in the treatment of hyperuricemia.

Molecular signatures of in vitro produced embryos derived from ovum pick up or slaughterhouse oocytes in buffalo.

This study was performed to investigate the difference in developmental competence of oocytes derived from ovum pick-up (OPU) and slaughterhouse ovaries (SLH), and its underlying mechanisms. The OPU and SLH oocytes were in-vitro maturated and fertilized to produce blastocysts, and these blastoycsts were collected to explore the expression of key genes for developmental potential and telomere (Oct-4, Sox2, Nanog, Cdx2, Gata3, E-cadherin, ß-catenin, TERT, TERF1 and TERF2). The results showed that both the cleavage and blastocyst rates were significantly higher for the OPU group (68.31%, 39.48%, respectively) than SLH group (57.59%, 26.50%, respectively) (P < 0.01). The relative mRNA abundances of Sox2, Oct-4, Nanog and E-cadherin were significantly higher in the OPU blastocysts than the SLH ones (P < 0.01). Protein expression analysis by Western blot and immunofluorescence also revealed that the expression of E-cadherin and Sox2 was significantly higher in OPU blastocysts than SLH ones. However, there was no significant differences between the two groups in the expression of Cdx2, ß-catenin, Gata3, TERT, TERF1, TERF2. These results imply oocyte sources modify the expression of development and adhesion related genes in blastocysts, which may elucidate a possible reasoning for the low development competence of buffalo SLH embryos.

Effect of zinc chloride and sodium selenite supplementation on in vitro maturation, oxidative biomarkers, and gene expression in buffalo (Bubalus bubalis) oocytes.

This study examined the effects of zinc chloride (ZnCl2) and sodium selenite (Na2SeO3) supplementation in maturation medium on in vitro maturation (IVM) rate, oxidative biomarkers and gene expression in buffalo oocytes. Ovaries from a slaughterhouse were aspirated and good quality cumulus-oocyte complexes (COCs) with at least four layers of compact cumulus cells and evenly granulated dark ooplasm were selected. COCs were randomly allocated during IVM (22 h) to one of four treatment groups: (1) control maturation medium (basic medium), or basic medium supplemented with (2) ZnCl2 (1.5 µg/ml), (3) Na2SeO3 (5 µg/l), or (4) ZnCl2 + Na2SeO3 (1.5 µg/ml + 5 µg/l, respectively). Oocytes were denuded after 22 h of IVM in the first four replicates. Specimens were fixed and stained to evaluate the stage of nuclear maturation. The spent medium was collected for biochemical assays of total antioxidant capacity (TAC), malondialdehyde (MDA) and hydrogen peroxide concentrations. A second four replicates were used for COCs for RNA extraction. The expression levels of antioxidant (SOD1, GPX4, CAT and PRDX1), antiapoptotic (BCL2 and BCL-XL) and proapoptotic (BAX and BID) genes were measured. Supplementation with ZnCl2 and Na2SeO3 during IVM increased the ratio of oocytes reaching metaphase II at 22 h, increased TAC and decreased MDA and H2O2 concentrations in the maturation medium (P < 0.05). Moreover, beneficial effects were associated with complementary changes in expression patterns of antioxidative, antiapoptotic and proapoptotic genes, suggesting lower oxidative stress and apoptosis. Supplementation medium with zinc chloride and sodium selenite improves the maturation rate, reduces oxidative stress and increases expression levels of antioxidative and antiapoptotic genes.

Transcriptome analysis revealed differences in the microenvironment of spermatogonial stem cells in seminiferous tubules between pre-pubertal and adult buffaloes.

The microenvironment in the seminiferous tubules of buffalo changes with age, which affects the self-renewal and growth of spermatogonial stem cells (SSCs) and the process of spermatogenesis, but the mechanism remains to be elucidated. RNA-seq was performed to compare the transcript profiles of pre-pubertal buffalo (PUB) and adult buffalo (ADU) seminiferous tubules. In total, 17,299 genes from PUB and ADU seminiferous tubules identified through RNA-seq, among which 12,271 were expressed in PUB and ADU seminiferous tubules, 4,027 were expressed in only ADU seminiferous tubules, and 956 were expressed in only PUB seminiferous tubules. Of the 17,299 genes, we identified 13,714 genes that had significant differences in expression levels between PUB and ADU through GO enrichment analysis. Among these genes, 5,342 were significantly upregulated and possibly related to the formation or identity of the surface antigen on SSCs during self-renewal; 7,832 genes were significantly downregulated, indicating that genes in PUB seminiferous tubules do not participate in the biological processes of sperm differentiation or formation in this phase compared with those in ADU seminiferous tubules. Subsequently, through the combination with KEGG analysis, we detected enrichment in a number of genes related to the development of spermatogonial stem cells, providing a reference for study of the development mechanism of buffalo spermatogonial stem cells in the future. In conclusion, our data provide detailed information on the mRNA transcriptomes in PUB and ADU seminiferous tubules, revealing the crucial factors involved in maintaining the microenvironment and providing a reference for further in vitro cultivation of SSCs.

Molecular, functional, and cellular alterations of oocytes and cumulus cells induced by heat stress and shock in animals.

Global warming is considered as the main environmental stress affecting ecosystems as well as physiological and biochemical characteristics, and survivability of living organisms. High temperature induces various stresses and causes reduction of fertility through reducing the oocyte developmental competence and alteration in surrounding cells' functions. This causes major economic loss to livestock creating a selective pressure on animals to the advantage of better adapted genotypes and to the detriment of others. In this review, a search in Science Direct, Google Scholar, PubMed, Web of Science, Scopus, and SID databases until 2020 was conducted. Keywords which include heat stress, shock, high temperature, oocyte, cumulus, and animals were investigated. Studies have exhibited that heat stress can disturb the development and function of oocyte and cumulus cells (CCs) concerning reproductive efficiency. Heat stress has deleterious consequences on oocyte maturation and development via reduced number of polar body extrusion, adenosine monophosphate, and guanosine monophosphate synthesis. Heat stress caused the alteration of cytoplasmic and nuclear features as well as trans-zonal projections and gap junctions. In addition, heat stress is accompanied with reduced mitochondrial activity (copy mDNA number, distribution, and membrane potential) in cumulus-oocyte complexes. This review targets the description of results in the most recent studies that aimed to call attention to the influences of heat stress on molecular, functional, and cellular changes in oocytes and CCs in animals to design evidence on the acting mechanisms as the core of this problem from a comparative review.

Effect of season on the in-vitro maturation and developmental competence of buffalo oocytes after somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) is a valuable technology tool with various uses in transgenic animals, regenerative medicine, and stem cell research. However, the efficiency of SCNT embryos appears to have poor developmental competency. Environmental issues may adversely affect SCNT embryos in buffalo. Thereafter, the present study aimed to explore the effect of season on the maturation of buffalo oocytes and subsequent developmental capability after parthenogenetic activation and SCNT in buffalo. Buffalo oocytes (n = 6353) were collected from local slaughterhouse at various seasons; spring (March-April), summer (May-August), autumn (September-November), and winter (December-January). A significant increase (p < 0.05) was recorded in the maturation rate (57.07%) at autumn compared with spring, summer, and winter (50.46, 50.93, and 50.66%, respectively). No significant differences were recorded in the fusion and the cleavage rates among all seasons. Blastocyst development rate was higher (p < 0.05) in autumn and winter (16.52 ± 8.45% and 15.98 ± 7.17%, respectively) than in spring and summer (9.47 ± 6.71% and 10.84 ± 6.58%, respectively) seasons. It could be concluded that the season had a significant effect on oocyte development competence which can be used for SCNT in buffalo.

Effects of laser zona thinning and artificial blastocoel collapse on the cryosurviving and hatching of buffalo (Bubalus bulalis) blastocysts of different ages.

The objectives of this study were to investigate whether blastocoel collapse before vitrification induced by laser improves the cryo-survivability of buffalo in-vitro-fertilized (IVF) blastocysts and whether laser assisted hatching (LAH) promotes hatchability of fresh and frozen-thawed IVF blastocysts. The expanded blastocysts were harvested on Days 6-9 and randomly allocated into five groups as follows: (1) blastocysts were vitrified and thawed without any treatment; (2) blastocysts were vitrified after 15-20 µm zona pellucida (ZP) thinning opposite to the inner cell mass, and blastocoels were also blotted in order to outflow the blastocoelic fluid before vitrification; (3) ZP thinning was made immediately after thawing; (4) fresh blastocysts underwent LAH; and (5) as a control, fresh blastocysts without treatment. Results of the present study showed that the cryosurvival rates of vitrified Day 8 and Day 9 blastocysts in Group 2 were significantly (P < 0.01) higher in Group 2 than Group 1. The hatching rates of Day 8 and Day 9 blastocysts in Group 2 and Group 3 were also significantly (P < 0.01) higher compared with Group 1. Moreover, the hatching rate of Day 9 blastocysts in Group 4 was notably (P < 0.05) higher than Group 5. In conclusion, LAH promotes the hatching rates of Day 9 fresh and Days 8-9 vitrified blastocysts, and artificial blastocoel collapse before vitrification improves the cryosurvival rate of Days 8-9 IVF buffalo blastocysts.

Characterization of Circular RNAs in Chinese Buffalo (Bubalus bubalis) Adipose Tissue: A Focus on Circular RNAs Involved in Fat Deposition.

Circular RNAs (circRNAs) have been identified as a novel type of regulators involved in multiple biological processes. However, circRNAs with a potential function in fat deposition in buffalo are poorly understood. In this study, six RNA libraries of adipose tissue were constructed for three young and three adult Chinese buffaloes with paired-ends RNA sequencing using the Illumina HiSeq 3000 platform. A total of 5141 circRNAs were computationally identified. Among them, 252 circRNAs were differentially expressed (DE) between the young and adult buffaloes. Of these, 54 were upregulated and 198 were downregulated in the adult group. Eight DE circRNAs were further identified by quantitative real-time-PCR (qRT-PCR) and Sanger sequencing. Co-expression analysis revealed that 34 circRNAs demonstrated a strong correlation with fat deposition-associated genes (|r| > 0.980). Among these, expressional correlation between two circRNAs (19:45387150|45389986 and 21:6969877|69753491) and PR/SET domain 16 was further verified using qRT-PCR, and a strong correlation was revealed (1 > |r| > 0.8). These results strongly suggest that circRNAs 19:45387150|45389986 and 21:6969877|69753491 are potential regulators of buffalo fat deposition. In summary, this study characterized the circRNA profiles of adipose tissues at different stages for the first time and revealed two circRNAs strongly correlated with fat deposition-associated genes, which provided new candidate regulators for fat deposition in buffalo.

Vitrification alters cell adhesion related genes in pre-implantation buffalo embryos: Protective role of ß-mercaptoethanol.

The objectives of the present study were to investigate the effect of vitrification on the expression of the key genes associated with blastocyst developmental potential (ß-catenin, E-cadherin, Oct-4, Cdx2, Gata3), and whether the presence of ß-mercaptoethanol (ß-ME, 100 µM) in in vitro culture (IVC) media will affect the expression of these genes. Buffalo pre-implantation embryos were divided into three groups: (1) fresh non-vitrified embryos were used as control, (2) vitrified embryos cultured with ß-ME (+), and (3) vitrified embryos cultured without (-) ß-ME. The results showed that all genes were affected by vitrification, however, the presence of ß-ME in IVC media significantly (P < 0.05) modified the expression level of ß-catenin, E-cadherin and Oct-4 in vitrified blastocyst compared to those cultured without ß-ME. Protein expression analysis by immunofluorescence and western blot also revealed that the expression level of ß-catenin and E-cadherin was significantly higher in vitrified embryos cultured with ß-ME than those cultured without ß-ME, which, in turn, was lower than fresh control group. However, there was no significant difference between vitrified groups in the expression level of Cdx2 and Gata3. Furthermore, the reduced rate of apoptosis in embryos cultured with ß-ME confirms its role in protecting vitrified blastocyst against stress. In summary, vitrification alters the expression of the adhesion related genes in vitrified blastocyst, which may explain, at least in part, the reason for the low pregnancy rate following transfer of such embryos into recipient animal, and the supplementation of IVC media with ß-ME significantly improved the quality of vitrified blastocyst evidenced by the modulation of the expression of blastocyst important genes, ß-catenin, E-cadherin and Oct-4, and the ability to protect vitrified blastocyst against apoptosis.

Developmental competence of buffalo (Bubalus bubalis) denuded oocytes cocultured with cumulus cells: Protective role of cumulus cells.

The objectives of the present study were to evaluate the developmental competence of buffalo denuded oocytes (DOs) cocultured with cumulus cells (CCs) during in vitro maturation, and to investigate the mechanisms by which CCs promote oocyte maturation and development. Buffalo oocytes were matured in vitro for 24 h in three groups: (1) intact cumulus-oocyte complexes (COCs) (2) DOs cocultured with CCs (DOsCC), and (3) DOs cultured alone (DOs). Matured oocytes were used to determine the relative mRNA abundance of Gdf-9, Bmp15, Zar1, Caspase-3, Bcl-2, Zp2, Zp3, Cd9 and Pde3a by Rt-qPCR and CASPASE-3 protein expression by immunofluorescence. The intracellular content of cGMP, cAMP and MPF activity and the rate of embryonic development were also assessed. Results of the present study showed that in DOs, the relative mRNA abundance of Gdf-9, Bmp15, and Cd9 significantly (P < 0.05) decreased, whereas Caspase-3 (mRNA and protein levels), Bcl-2, and Pde3a exhibited higher expression than DOsCC and COCs. However, there was no significant difference among the groups in the expression level of Zar-1, Zp2, and Zp3. The intracellular content of cAMP and MPF activity was notably higher (P < 0.05) in DOs compared to COCs and DOsCC. There was no significant difference between COCs and DOsCC in cGMP content, which was significantly lower (P < 0.05) in DOs. Moreover, the cleavage and blastocyst rates were 58.4 ±â€¯1.8%, 43.7 ±â€¯1.1%, 18.4 ±â€¯0.9% and 18.0 ±â€¯1.3%, 11.0 ±â€¯0.9% and 4.5 ±â€¯0.6% in COCs, DOsCC and DOs groups, respectively. In conclusion, the presence of CCs protects buffalo DOs from apoptosis and promotes maturation through regulation of the intracellular content of cAMP and MPF activity and improves the fertilizing capacity of oocytes through modulation of the gamete fusion gene, Cd9.

Buffalo oocyte-secreted factors promote cumulus cells apoptosis and the rate of cGMP production but not steroidogenesis.

The objectives of this study were to investigate the effect of buffalo oocyte-secreted factors (OSFs) on cumulus cells (CCs) functions, apoptosis and cGMP generation, and whether the direct contact between oocyte and CCs is essential for oocyte-mediated regulation of CCs functions. Buffalo CCs were cultured during IVM within three groups: (a) intact cumulus-oocyte complexes (COCs), (b) CCs cocultured with denuded oocytes (DOs) (CCs + DOs) and (c) CCs monolayer cultured alone (CCsM). After 24 hr of IVM, CCs were harvested for evaluation of the relative mRNA abundance of the genes encoding gap junction (GJA1), glycolysis (PFKP and LDHA), apoptosis (CASPASE-3 and BCL-2) and steroidogenesis (ER-ß and PGR) by QRT-PCR, and CASPASE-3 proteins, using western blot. Intracellular cGMP content was also assessed by ELISA. Results showed that the relative abundance of LDHA, PFKP and BCL-2 significantly increased (p < 0.05) in COCs, whereas GJA1 and CASPASE-3 exhibited lower expression (p < 0.05) compared to CCs + DOs and CCsM groups. However, the expression levels of CASPASE-3, both mRNA and protein, were significantly (p < 0.05) downregulated in CCs + DOs compared to CCsM. There was no significant difference in the expression level of PGR and ER-ß between the groups. The intracellular content of cGMP was notably (p < 0.05) higher in COCs compared to CCs + DOs and CCsM groups. In conclusion, this study demonstrated, for the first time, that buffalo OSFs protect CCs against apoptosis and stimulate their cGMP production; however, the regulation of cumulus glycolysis and gap junction is confined to those in close contact with the oocyte. Neither OSFs from COCs nor those from DOs have any effect on CCs steroidogenesis.

Inhibition of chemotherapy-induced apoptosis of testicular cells by squid ink polysaccharide.

The aim of this study was to determine the mechanisms driving the protective effects of squid ink polysaccharide (SIP) against cyclophosphamide (CP)-induced testicular damage, focusing on germ cells. In the testes of mice exposed to CP and/or SIP, the present study examined the levels of reactive oxygen species (ROS) and malondialdehyde, activity of superoxide dismutase levels, protein expression levels of B-cell lymphoma 2 (Bcl2), Bcl2-associated X protein (Bax), and total Caspase 3, activation of p-p38 and p-Akt proteins, and tissue morphology. The findings indicated that CP induced ROS production and oxidative stress, resulting in testicular damage. However, under administration of SIP, oxidative stress was impaired and the testicular toxicity induced by CP was weakened, which implied that SIP may have an important role in preventing chemotherapeutic damage to the male reproductive system via promoting antioxidant ability. Furthermore, the altered expression levels, including the upregulation of Bax and Caspase 3, downregulation of Bcl-2 and the increased Bax/Bcl-2 ratio, indicated that apoptosis occurred in CP exposed testes of mice; however, the alterations were reversed in mice treated with SIP. Moreover, in CP-exposed testes, p38 and Akt proteins were significantly phosphorylated (P<0.05), whereas in the testes of mice co-treated with SIP and CP, phosphorylation of the two proteins was inhibited, demonstrating that the two signalling pathways participated in the regulative processes of the deleterious effects caused by CP, and the preventive effects SIP mediated.