ABSTRACT
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.
Subject(s)
Aromatase/genetics , Buffaloes/genetics , Buffaloes/metabolism , Ovary/enzymology , 5' Untranslated Regions , Animals , Base Sequence , Buffaloes/physiology , Cloning, Molecular , DNA Primers/genetics , DNA, Complementary/genetics , Female , Granulosa Cells/enzymology , Molecular Sequence Data , RNA, Messenger/genetics , RNA, Messenger/metabolism , Reproduction/genetics , Reproduction/physiology , Sequence Homology, Nucleic Acid , Species SpecificityABSTRACT
The cytochrome P450 aromatase (aromP450) deficient mice are infertile due to an impairment of spermatogenesis associated with a decrease in sperm motility and inability to fertilize oocytes. The sperm analysis showed decreased sperm motility in humans, having Cyp19 gene mutations. Further, in human, it was hypothesized that aromatase could be used as marker of sperm quality, particularly in the acquisition of its motility. However, there is no information regarding the expression of aromP450 in spermatozoa of farm animals including cattle and buffalo. In the present study, the expression of aromP450 in ejaculated buffalo spermatozoa and its relationship with sperm motility of ejaculated spermatozoa was studied by RT-PCR using total RNA isolated from buffalo-ejaculated spermatozoa. The results showed that conventional RT-PCR could not amplify aromatase transcript, while a nested PCR detected the presence of P450arom mRNA in buffalo-ejaculated spermatozoa. RT reaction followed by nested PCR was performed to compare the expression of aromatase transcripts in buffalo-ejaculated spermatozoa of two category semen graded on the basis of mass motility and motile and non-motile spermatozoa separated by swim-up. A higher (P<0.01) expression of aromP450 transcript was found in spermatozoa obtained from the good quality semen (higher mass motility) to that in spermatozoa of poor quality semen (low mass motility). Similarly, higher (P<0.01) expression of aromP450 mRNA was observed in the motile spermatozoa as compared to non-motile spermatozoa separated from good quality semen by swim-up. It is concluded that the present study demonstrates a positive relation between aromatase transcript and mass motility of buffalo-ejaculated spermatozoa, which could be a putative marker for the quality of semen in farm animals, particularly the acquisition of sperm motility.
Subject(s)
Aromatase/genetics , Buffaloes/physiology , RNA, Messenger/analysis , Sperm Motility/physiology , Spermatozoa/enzymology , Animals , Electrophoresis, Polyacrylamide Gel , Magnesium Chloride/administration & dosage , Male , Polymerase Chain Reaction/methods , Reverse Transcriptase Polymerase Chain ReactionABSTRACT
Rapid isolation of DNA from goat blood using different brands of detergents available in Indian market, is reported. The integrity and efficiency of these DNA preparations were compared with genomic DNA isolated by a standard kit (Flexi gene DNA kit), using amplification of exon 2 of CYP19 (aromatase) gene. The similar and significant amplification of this gene was obtained using genomic DNA isolated by kit and various detergents. However, among the detergents used, the Rin and Ezee were found to be the best to get DNA of high purity comparable to that obtained by kit.
