The Presence of Prolactin and Tyrosine Hydroxylase Messenger Ribonucleic Acid in Bovine Testis and Epididymis

Examples of the extra-pituitary expression of prolactin (PRL) have been reported in several species, and recently PRL was detected in bovine seminal fluid by using RIA; however, the source of production for PRL present in bovine seminal fluid has yet to be determined. The objectives of these experiments were to identify the presence or absence of the mRNA for bovine PRL (bPRL) and its rate-limiting enzyme, tyrosine hydroxylase (bTH) in bull testis and epididymis. Testis tissues were obtained from bulls 16 to18 months of age at time of slaughter. The tissues were frozen in liquid nitrogen and kept at -80° C until use for analysis. Total RNA was isolated from frozen testis using the mirVana mRNA isolation kit (Ambion, Austin, Texas,USA). Copy DNA was generated by reverse transcription reaction using Superscript II Frist-Strand kit (Invitrogen, Carlsbad,CA, USA). Primers for bTH and bPRL were generated through the PrimerQuest Tool (XXIDT, Coralville, Iowa, USA) for use in polymerase chain reaction (PCR), specific for bTH (accession number: NM_173884.2) and bPRL (accession number: NM_173953.2) corresponding to nucleotide positions 146−165 forward primer and 411−431 reverse primer of the mRNA bPRL sequence; 1263−1283 forward primer and 1360 −1381 reverse primer of the mRNA bTH sequence. End-point PCR was performed and the resulting amplified cDNA was subjected to slab gel electrophoresis in 2.5% agarose gels. The PCR products of the predicted size for both bPRL (285 bp) and bTH (118 bp). To further verify the identity of the PCR products, the cDNA for bPRL and bTH was subjected to an overnight ligation reaction at 4° C using the Qiagen PCR Plus Cloning kit and used to transform E. coli (EZ competent; Qiagen, Germantown, MD). Transformation reactions were plated on LB agar plates containing Ampicilin (100 mg/ml) and IPTG/Xgal for blue-white screening, and incubated over night at 37^o C. Colonies were selected, used to inoculate LB-Broth and grown to saturation overnight at 37° C shaking at 250 rpm. Resulting DNA was purified using the Wizard Mini-Prep Plus (Promega, Madison, WI) and subjected to restriction enzyme digest and dideoxy-sequencing. Plasmid DNA was digested with EcoR1 (Promega, Madison, WI, USA) and products of the expected sizes were released for the putative bPRL and bTH clones. Sequencing confirmed the identity of the subloned cDNA as bPRL and bTH in both testis and epididymis. These data indicate that PRL present in bull seminal fluid could be produced in male reproductive tissues.