Buffalo Ovary Research Articles

Molecular Cloning and Characterization of cDNA Encoding Buffalo (Bubalus bubalis) Cytochrome P450 Aromatase in the Ovary

One of the predominant causes of poor reproduction in buffaloes is low levels of ovarian estrogens. A rate limiting enzyme in estrogen biosynthesis is cytochrome P450 aromatase (P450 AROM), the product of CYP19 gene. In the present study CYP19 cDNA was cloned and its 5′UTR was characterized by 5′RACE in granulosa cells of large follicles. CYP19 transcripts with four different 5′UTRs (206, 114, 90 and 3 bases) were found in buffalo granulosa cells of large ovarian follicles. Interestingly, a predominant aromatase transcript with short 5′UTR (3 nucleotides) was found. Further studies are required to understand the relevance of these transcripts and their translational efficiency in granulosa cells of large follicles during folliculogenesis of buffalo ovary.

Different promoter usage for CYP19 gene expression in buffalo ovary and placenta

Aromatase is the key enzyme for estrogen biosynthesis and is coded by CYP19 gene. The expression of CYP19 gene is regulated in tissue-specific manner by alternate use of different promoters. In this study, we have analyzed tissue-specific expression and their regulation of CYP19 gene in preovulatory and postovulatory stages of buffalo ( Bubalus bubalis) ovary and placenta. RT-PCR analysis showed that the CYP19 gene expression was significantly ( p < 0.05) higher in granulosa cells of large follicles as compared to the other tissues. The transcript analysis and transcriptional start site identified by 5′-RLM RACE for CYP19 expression indicated different transcriptional start sites within the different sized follicles during folliculogenesis. Sequence analysis showed that the transcription start site of transcript isolated from buffalo granulosa cells of small follicles was 37 bases upstream of the transcript isolated from granulosa cells of large follicles. However, both the transcripts were found to be derived from proximal promoter II. Difference in the transcriptional start site indicates the different promoter sequence usage in granulosa cells of different sized follicles. Further, in silico analysis of the difference in promoter sequence based on the 5′-UTRs isolated from granulosa cells of small follicles (151 bases) and large follicles (114 bases) showed that consensus sequence for certain important trans-elements (viz., TATA binding protein, E2F and CAAT binding protein) would lie in the promoter sequence isolated from the granulosa cells of large follicles. These transcription factors may be involved in regulation of CYP19 gene expression in ovary, either directly or indirectly. The difference in the size of 5′-UTR among the granulosa cells of ovary reflects the possible mechanism for the differential regulation of CYP19 gene during development. The transcripts isolated from buffalo corpus luteum and placental cotyledons were having same 5′-UTR comprises of 168 bases and found to be derived from PI.1. Estimates of CYP19 gene transcript concentration in the different tissues revealed that the CYP19 gene expression in granulosa cells is predominantly regulated by PII and to a minor extent by PI.1. However, PI.1 is almost exclusively responsible for CYP19 gene expression in placenta and residual expression in corpus luteum. In order to understand the complex CYP19 gene regulation in these tissues, further studies are needed to elucidate the activity of different promoters and define regulatory elements for binding of transcription factors.

Molecular characterization of follicle stimulating hormone receptor (FSHR) gene in the Indian river buffalo ( Bubalus bubalis)

Follicle stimulating hormone (FSH) plays a central role in regulation of ovarian function in mammals. The actions of follicle stimulating hormone are mediated through receptors present on the granulosa cells of the ovary. In the present study we have cloned and characterized the FSHR gene of buffalo. Sequence analysis indicated that the buffalo FSHR cDNA sequence comprised of an open reading frame of 2085 bp encoding a 695 amino acid protein. Its nucleotide sequence showed more than 80% similarity to the homologous genes of mammalian species. At amino acid level buffalo FSHR exhibited a high percentage (84–96.7%) of identity with the corresponding mammalian homologs. This is the first isolation and characterization of FSHR cDNA from buffalo ovary.

Advanced reproductive technology in the water buffalo

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.

Cloning, Expression and Hormonal Regulation of Steroidogenic Acute Regulatory Protein Gene in Buffalo Ovary

In mammalian ovary, steroidogenic acute regulatory (StAR) protein mediates the true rate-limiting step of transport of cholesterol from outer to inner mitochondrial membrane. Appropriate expression of StAR gene represents an indispensable component of steroidogenesis and its regulation has been found to be species specific. However, limited information is available regarding StAR gene expression during estrous cycle in buffalo ovary. In the present study, expression, localization and hormonal regulation of StAR mRNA were analyzed by semi-quantitative RT-PCR in buffalo ovary and partial cDNA was cloned. Total RNA was isolated from whole follicles of different sizes, granulosa cells from different size follicles and postovulatory structures like corpus luteum and Corpus albicans. Semi-quantitative RT-PCR analyses showed StAR mRNA expression in the postovulatory structure, corpus luteum. No StAR mRNA was detected in total RNA isolated from whole follicles of different size including the preovulatory follicle (>9 mm in diameter). However, granulosa cells isolated from preovulatory follicles showed the moderate expression of StAR mRNA. To assess the hormonal regulation of StAR mRNA, primary culture of buffalo granulosa cells were treated with FSH (100 ng/ml) alone or along with IGF-I (100 ng/ml) for 12 to 18 h. The abundance of StAR mRNA increased in cells treated with FSH alone or FSH with IGF-I. However, effect of FSH with IGF-I on mRNA expression was found highly significant (p<0.01). In conclusion, differential expression of StAR messages was observed during estrous cycle in buffalo ovary. Also, there was a synergistic action of IGF-I on FSH stimulation of StAR gene.

Recovery of large preantral follicles from buffalo ovary: Effect of season and corpus luteum

Preantral follicle can be considered as an alternative source of oocyte for in vitro production of embryos. The objective of the present study was to standardize a procedure for the isolation of large preantral follicles (>150–500 μm) from buffalo ovaries and to determine the effect of season and the presence of corpus luteum on the recovery rate of the large preantral follicles. A combined enzymatic cum mechanical approach was adopted to recover the large preantral follicles. In the first experiment, the ovarian cortical pieces were suspended in trypsin (1000–1500 BAEE units for milligrams of solid) and incubated at various temperatures for different periods, i.e. (1) trypsin (1%), 37 °C for 10 min; (2) trypsin (1%), 37 °C for 10 min + 4 °C for 3 h; (3) trypsin (0.5%), 37 °C for 20 min; (4) trypsin (0.25%), 37 °C for 20 min. Although there was no significant difference ( P > 0.05) among the different protocols, the first protocol yielded more follicles (3.2, 2.6, 1.8 and 1.5 per ovary, respectively). Hence, the first protocol was selected and used in the second and third experiments. In the second experiment, the effect of season, i.e. peak breeding season (October–March) versus low breeding season (April–September) was evaluated on the recovery rate of the large preantral follicles. The recovery rate of large preantral follicles from the ovaries during the peak breeding season was significantly ( P < 0.05) greater (9.92 ± 0.85 per ovary) than that of the low breeding season (4.95 ± 0.27 per ovary). In the third experiment, effect of the presence of corpus luteum on the recovery rate of large preantral follicles was studied. There was a significantly ( P < 0.05) higher yield of large preantral follicles from the ovaries with corpus luteum (8.05 ± 0.88 per ovary) than for the ovaries without corpus luteum (4.57 ± 0.43 per ovary). This study confirms that the large preantral follicles can be isolated from buffalo ovaries using a combination of enzymatic cum mechanical methods and that more large preantral follicles can be recovered during the peak breeding season and from the ovaries having corpus luteum.

Immunolocalization of von Willebrand Factor and Vascular Endothelial Growth Factor during Follicular Atresia in the Swamp Buffalo Ovary

The aim of this study was to investigate the distribution pattern of von Willebrand factor (vWF) and vascular endothelial growth factor (VEGF) in the healthy antral and atretic follicles of Philippine swamp buffaloes (SB) in comparison with Holstein-Friesian cows (HF). Paraffin sections of healthy follicles and atretic follicles at various stages were immunostained with vWF antibody and VEGF antibody. The density of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in SB, whereas the density significantly decreased in late atretic follicles compared with advanced ones in HF. On the other hand, the area of vWF-positive capillary vessels in the theca interna significantly increased as atresia progressed in both SB and HF. Immunoreactions of VEGF in the granulosa cells (in all follicle types) were observed in both SB and HF. In the granulosa layer, a reduction in the VEGF immunoreaction was noted as follicles progressed from healthy to advanced atretic follicles in both animals. Granulosa cells (in both SB and HF) showed a higher immunopositive staining than theca cells. In the theca interna, VEGF immunostaining diminished as follicles progressed to the late atretic follicles in both animals. These results indicate that during atresia, changes of vWF expression are the opposite of VEGF expression in SB. Both vWF and VEGF are suggested to be associated with follicular atresia in SB.

Effect of Follicular Fluid Proteins and Gonadotropins on Progesterone Secretion by Buffalo Granulosa Cells In vitro

In the mammalian ovary the follicular fluid contains proteins and peptides which play an important role in growth, development and maturation of oocytes. The gonadotropins and some other factors work synergistically and regulate ovarian functions. In the present study the effect of follicular fluid proteins (FFP) and gonadotropins on progesterone secretion by granulosa cells (GC) from buffalo ovary, was investigated during culture. The follicular fluid was collected from small (<5 mm), and medium (5-8 mm) follicles obtained from buffalo ovaries. The follicular fluid from medium follicles was fractionated with ammonium sulphate at 80% saturation. The precipitated protein fraction was further resolved in to minor (peaks I, III) and major (peak II) proteins using gel filtration (Sephadex G-200). The FFP from small follicles and major FFP (peak II) at a dose of 200 μg/well, significantly stimulated progesterone secretion by pooled GC (3×10 5 cells/2 ml medium/well). The minor FFP did not show any stimulatory effect. There was a significant increase in progesterone secretion by pooled GC in presence of FFP and LH (10 ng/well), however, FSH (20 ng/well) with FFP exhibited an inhibitory effect. The major FFP and gonadotropins were also studied for their effect on progesterone production by GC isolated from medium and large size follicles. The GC from medium follicles were more responsive to FSH and FFP whereas GC from large follicles exhibited enhanced progesterone secretion with LH and FFP. These results indicated that FFP have their own stimulatory effect and also act synergistically with gonadotropins. The significantly different response shown by GC, for steroid hormone secretion, is based on their stage of growth and differentiation. The purification and characterization of such steroidogenic proteins may help in elucidating their role in growth and differentiation of granulosa cells.

Reproductive Biotechnologies for Improvement of Buffalo: The Current Status

Reproductive biotechnologies continue to be developed for genetic improvement of both river and swamp buffalo. Although artificial insemination using frozen semen emerged some decades back, there are still considerable limitations. The major problem appears to be the lack of efficient methods for estrus detection and timely insemination. Controlled breeding experiments in the buffalo had been limited and similar to those applied in cattle. Studies on multiple ovulation and embryo transfer are essentially a replica of those in cattle, however with inherent problems such as lower number of primordial follicles on the buffalo ovary, poor fertility and seasonality of reproduction, lower population of antral follicles at all stages of the estrous cycle, poor endocrine status and a high incidence of deep atresia in ovarian follicles, the response in terms of transferable embryo recovery has remained low with 0.51 to 3.0 per donor and pregnancy rates between 15 to 30%. In vitro production of buffalo embryos is a valid alternative to recovery of embryos by superovulation. This aspect received considerable attention during the past decade, however the proportion of embryos that develops to the blastocyst stage is still around 25-30% and hence the in vitro culture procedures need substantial improvement. Embryo cryopreservation procedures for direct transfer post thaw need to be developed for bubaline embryos. Nuclear transfer and embryo cloning is a technique that has received attention in various species during recent years and can be of immense value in buffaloes as they have a low rate of embryo recoveries by both in vitro and in vivo procedures. Gender pre-selection, genome analysis, gene mapping and gene transfer are a few of the techniques that have been studied to a limited extent during recent years and are likely to be included in future studies on buffaloes. Very recently, reproductive biotechnologies have been applied to feral buffaloes as well, but the results obtained so far are modest. When fully exploited they can play an important role in the preservation of endangered species.

Production of PCR mimics for any semiquantitative PCR application.

BioTechniquesVol. 22, No. 6 BenchmarksOpen AccessProduction of PCR Mimics for Any Semiquantitative PCR ApplicationStuart Alvaro Ali, Isabella Sarto & Alexander SteinkassererStuart Alvaro Ali*Address correspondance to Stuart A. Ali, Department of Oncogenic Viruses, Immuno A.G., Benatzkygasse 2-6, A-1220 Vienna, Austria. Internet: E-mail Address: stuali@immunology.co.atSandoz Research InstituteImmuno A.G., Vienna, AustriaBrunel University, Uxbridge, Middlesex, England, UKSearch for more papers by this author, Isabella SartoSandoz Research InstituteSearch for more papers by this author & Alexander SteinkassererSandoz Research InstituteImmuno A.G., Vienna, AustriaSearch for more papers by this authorPublished Online:7 Aug 2018https://doi.org/10.2144/97226bm10AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail FiguresReferencesRelatedDetailsCited ByMechanisms of intrinsic resistance to antimicrobial peptides of Edwardsiella ictaluri and its influence on fish gut inflammation and virulenceMicrobiology, Vol. 159, No. Pt_7PAPP-A in Indian Water Buffalo (Bubalus bubalis) Ovary: Phylogeny, Expression, Hormonal Regulation, and Sequence Characterization1 May 2013 | Agricultural Research, Vol. 2, No. 2Expression of cytochrome P450 aromatase transcripts in buffalo (Bubalus bubalis)-ejaculated spermatozoa and its relationship with sperm motilityDomestic Animal Endocrinology, Vol. 34, No. 3Normalization of Transfection Efficiency Using the β-Lactamase Gene of the pGL3 Luciferase Vector in Primary Anterior Pituitary CellsSeong W. Kang, Seungkwon You, Eric A. Wong & Mohamed E. El Halawani17 September 2018 | BioTechniques, Vol. 33, No. 2Semiquantitative RT-PCR analysis to assess the expression levels of multiple transcripts from the same sampleBiological Procedures Online, Vol. 3, No. 1Synaptic Activity-Dependent Developmental Regulation of NMDA Receptor Subunit Expression in Cultured Neocortical Neurons4 January 2002 | Journal of Neurochemistry, Vol. 75, No. 4An antigenic HIV-1 peptide sequence engineered into the surface structure of transferrin does not elicit an antibody response5 October 1999 | FEBS Letters, Vol. 459, No. 2Transferrin Trojan Horses as a Rational Approach for the Biological Delivery of Therapeutic Peptide DomainsJournal of Biological Chemistry, Vol. 274, No. 34Molecular analysis of AMPA-specific receptors: subunit composition, editing, and calcium influx determination in small amounts of tissueBrain Research Protocols, Vol. 3, No. 2 Vol. 22, No. 6 Follow us on social media for the latest updates Metrics Downloaded 170 times History Published online 7 August 2018 Published in print June 1997 Information© 2018 Author(s)PDF download

Follicle aspiration prior to superovulation in cattle: A field study

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.

Structural and functional analysis of the insulin-like growth factor I receptor gene promoter.

The insulin-like growth factor I receptor (IGF-I-R) gene is expressed in most body tissues. The levels of IGF-I-R mRNA, however, are regulated by a number of physiological conditions (development, differentiation, and hormonal milieu) as well as in certain pathological states (diabetes and tumors). To understand the molecular mechanisms which control the transcription of the IGF-I-R gene, we have cloned the promoter of the rat receptor gene and have characterized its activity by transient expression assays. Different fragments of the 5'-flanking region (subcloned upstream of a luciferase reporter gene) were transfected into buffalo rat liver 3A cells (a cell line with a low number of IGF-I binding sites) and Chinese hamster ovary cells (a cell line with a higher number of cell-surface receptors). In both cell lines, most of the promoter activity was located in the proximal 416 base pairs of 5'-flanking region. However, further dissection of this proximal fragment revealed a cell type-specific pattern of promoter activity. Thus, in buffalo rat liver 3A cells, subfragments of this region each contributed to total activity, suggesting that contiguous cis-elements can act together to activate transcription. In Chinese hamster ovary cells, on the other hand, subfragments of the proximal promoter region partially substituted for the proximal 416 base pairs of 5'-flanking region. Coexpression studies using an IGF-I-R promoter reporter construct together with an Sp1 expression vector (under the control of an ADH promoter) were performed in SL2 cells, a Drosophila cell line which lacks endogenous Sp1. The results obtained showed that Sp1 can trans-activate the IGF-I-R promoter in vivo. Transient transfection assays were complemented with gel-retardation assays and DNase I footprinting experiments, which showed that transcription factor Sp1 is potentially an important regulator of IGF-I-R gene expression.

Milk progesterone concentration in superovulated cows

Embryo transfer techniques in water buffalo were derived from those in cattle. However, the success rate is much lower in buffaloes, due to their inherent lower fertility and poor superovulatory response. The buffalo ovary has a smaller population of recruitable follicles at any given time than the ovary of the cow (89% fewer at birth). In addition, estrus detection is problematic. Progress in the field of embryo transfer in water buffalo has been slow, and is primarily due to a poor response to superovulation. The average yield of transferable embryos is less than one per superovulated donor. In vitro embryo production could considerably improve the efficacy and logistics of embryo production. The technique of Ovum Pick Up is superior to superovulation; it can yield more transferable embryos per donor on a monthly basis (2.0 versus 0.6). The feasibility of intergeneric embryo transfer between buffalo and cattle has been investigated. No pregnancy resulted after transfer of 13 buffalo embryos to synchronized Holstein heifers. Preliminary successes with nucleus transfer of Bubalus bubalis fetal and adult somatic nuclei into enucleated bovine oocytes and subsequent development to the blastocyst stage have been reported.