Buck (Capra hircus) genes encode new members of the spermadhesin family

Abstract

Spermadhesins are the major proteins of boar seminal plasma and form a group of polypeptides probably involved in reproduction. In previous work, a member of the spermadhesin family from buck seminal plasma, called BSFP, was characterized by mass spectrometry and N-terminal sequencing. The present study aimed to clone and characterize the BSFP gene and investigate its expression along the genital tract using real-time polymerase chain reaction (PCR). The cDNAs of the seminal vesicle, testis, epididymis, bulbourethral gland, and ductus deferens were prepared from a buck. Following 3'- and 5'-end amplifications using seminal vesicle cDNA, we cloned and sequenced four highly similar (97-98%) nucleotide sequences encoding spermadhesins, which were named Bodhesin-1(Bdh-1), Bdh-2, Bdh-3, and Bdh-4. All deduced amino acid sequences contained the CUB domain signature and were 49-52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment. By using real-time PCR, it was verified specific amplifications for all Bdh in the seminal vesicle, testis, epididymis, and bulbourethral gland, with the exception of Bdh-2 in epididymis. The amplicons had a melting temperature and size of approximately 78 degrees C and 130 bp, respectively. Bdh expression was higher in the seminal vesicle when compared to the other tissues. The present work confirms that goat is the fifth mammalian species, after pig, cattle, horse, and sheep, in which spermadhesin molecules are found. To the best of our knowledge, this is the first report on buck spermadhesin genes using molecular cloning and expression profile.

Analysis of protein expression and a new prokaryotic expression system for goat (Capra hircus) spermadhesin Bdh-2 cDNA

“…In earlier studies, our group cloned and characterized cDNAs encoding the four main isoforms of buck spermadhesins, designated Bodhesin-1, -2, -3, and -4 (Melo et al, 2008(Melo et al, , 2009). In the present study, we subcloned the cDNA of Bdh-2, which was the most similar to BSFP, previously purified from seminal plasma by Teixeira et al (2002).…”

“…In the present study, we subcloned the cDNA of Bdh-2, which was the most similar to BSFP, previously purified from seminal plasma by Teixeira et al (2002). We used as PCR template the clones produced by Melo et al (2008), which contain cDNA inserts of 607 bp. In particular, the Bdh-2 cloned fragments comprised a 318-bp ORF (open reading frame) including the stop codon and a 278-bp 3'-untranslated region.…”

“…cDNA coding for spermadhesin Bdh-2 was produced according to the method described by Melo et al (2008). Briefly, total RNA was isolated from a single sexually mature buck of undefined breed using the Trizol reagent (Invitrogen, Carlsbad, CA, USA), following manufacturer instructions.…”

“…However, unlike the boar heparin-binding spermadhesins AQN-1, AQN-3 and AWN, BSFP is not able to bind heparin, resulting in low purification efficiency (Teixeira et al, 2006). Only after the above proteins had been described, were some spermadhesin cDNAs cloned (Wempe et al, 1992;Kwok et al, 1993;Ekhlasi-Hundrieser et al, 2002;Melo et al, 2008). In two earlier studies, we cloned and characterized four cDNAs encoding the main isoforms of buck spermadhesins, collectively called Bodhesins or Bdhs (Melo et al, 2008(Melo et al, , 2009).…”

“…All deduced amino acid sequences contained the CUB domain signature of the spermadhesin family and were 49 to 52% similar to boar AWN. Among the four Bdh amino acid sequences, Bdh-2 was the most similar to the BSFP N-terminal fragment (Melo et al, 2008). After the investigation of the expression profile of Bdh genes in several tissues of the buck reproductive tract (seminal vesicle, testis, epididymis, bulbourethral gland, ductus deferens, and prostate) by real-time quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR), we found that the seminal vesicle is the major site of Bdh production (Melo et al, 2009).…”