Domestic Animal Endocrinology 34 (2008) 238–249 Available online at www.sciencedirect.com Expression of cytochrome P450 aromatase transcripts in buffalo (Bubalus bubalis)-ejaculated spermatozoa and its relationship with sperm motility Ashutosh Tiwari, Dheer Singh ∗ , O. Suneel Kumar, M.K. Sharma Molecular Endocrinology Lab., Division of Animal Biochemistry, National Dairy Research Institute, Karnal 132001, Haryana, India Received 11 March 2007; received in revised form 16 July 2007; accepted 20 July 2007 Abstract 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. © 2007 Elsevier Inc. All rights reserved. Keywords: Buffalo; Cytochrome P450 aromatase; Ejaculated spermatozoa; Motility; RT-PCR 1. Introduction The estrogens are considered as specific female hor- mones and the source of these hormones in the male genital tract has been extensively investigated during last decade [1–3]. The generation of knockout mice for estro- ∗ Corresponding author. Tel.: +91 184 2259135; fax: +91 184 2250042. E-mail address: drdheer.singh@gmail.com (D. Singh). gen receptors (ER)  and  as well as for cytochrome P540 aromatase has provided evidence for a significant and crucial role of estrogens in maintaining normal sper- matogenesis [4]. Moreover, aromatase deficient mice (ArKO mice) are infertile due to an impairment of sper- matogenesis associated with a decrease in sperm motility and inability to fertilize oocytes [5–7]. In rat testis, there is an age-related change in the cellular localization of the aromatase activity, mainly in Sertoli cells in imma- ture animals, whereas it is located in Leydig cells in adults [8]. The P450arom mRNA is more abundant in 0739-7240/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.domaniend.2007.07.003