Leptin, hormonal product of the ob gene, is involved in energy metabolism and reproduction. Expression of leptin and its receptor in some reproductive organs suggest that in addition to endocrine effects of leptin, it has paracrine/autocrine effects on reproduction. Evidence, such as expression and secretion of leptin in human and pig spermatozoa, have pointed to a direct role of leptin in the physiologic functions of spermatozoa. The present study was the first to investigate the presence of the leptin mRNA transcript in ejaculated spermatozoa of Holstein cattle using RT-PCR analysis. Total RNA was extracted from sperm cells by TRizol procedure, and used to construct cDNA. The PCR with spermatozoa cDNA and outer leptin primer pairs resulted in amplification of the expected product. To confirm the first results, RT-PCR products were amplified with nested PCR using inner leptin primer pairs. Both outer and inner primer pairs, which were located in exon 2 and exon 3 be RNA specific, gave the expected PCR amplified products, 441 bp and 384 bp respectively, and ruled out the possibility of DNA contamination. The presence of leptin mRNA in the ejaculated spermatozoa of bulls suggests that leptin might act on physiological processes of bovine spermatozoa through autocrine/paracrine mechanisms.

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