Molecular Cloning Of Several Pig Sperm Functional Genes And Characterization Of Their MRNA Expression Profiles In The Genital Tracts And Ejaculated Sperm

In the current study, we cloned four Cation channel of sperm (Catsper 1-4) genes and zona pellucida binding protein 2 (ZPBP2) gene by using PCR and classical RACE techniques and obtained the full length of their cDNA. Then we characterized the spatial and temporal expression profiles of mRNA of several pig sperm functional gene families in the genital tracts of Meishan boar and gilt, and ejaculated sperm by RT-PCR and semi-quantitative RT-PCR. These functional gene families included spermadhesin gene family (AQN1, AQN3, AWN, PSPI, PSPII), cystein-rich secretory protein (CRISP) gene family (CRISP1, CRISP2, CRISP3), fibronectin type II (Fn-2) gene family (Epididymal sperm binding protein 1, ELSPBP1 and seminal plasma protein 1, pB1), zona pellucida binding protein (ZPBP) gene family (ZPBP1/2), and sperm motility inhibitor (SPMI) gene. Furthermore we analyzed the association of mRNA expression levels of spermadhesin genes (AQN1, AQN3, AWN, PSPI and PSPII) and sperm motility inhibitor (SPMI) gene in ejaculated sperm with sperm motility, capacitation and acrosome reaction status. The main results were summarized in the following parts:1. The full lengths cDNA, and 5'and 3'5′-terminal untranslated regions (UTR) of pig Catsper1, Catsper2, Catsper3 and Catsper4 genes were obtained. The mRNA of Catsper1 was 2452bp, and comprised with 2169bp open reading frame (ORF), flanked by a 99bp 5′UTR and a 184bp 3′UTR; The mRNA of Catsper2 was 2054bp, and comprised with 1599bp ORF, flanked by a 333bp 5′UTR and a 133bp 3′UTR; The mRNA of Catsper3 was 1340bp, and comprised with 1200bp ORF, flanked by a 121bp 5′UTR and a 19bp 3′UTR; The mRNA of Catsper4 was 1799bp, and comprised with 1350bp ORF, flanked by a 47bp 5′UTR and a 402bp 3′UTR. Homology analysis of protein seuqence revealed that, pig Catsper1 showed high identify with that of horse (72% ) and dog (78.7%), moderate with cattle (64%) and human (53.9%), low with rat (47.5%) and mouse (48.8%); the identifity of pig Catsper2 with that of human (78.1%), horse (88%), dog (88.5%) and cattle (87.8%) was high, it was low with that of rat (25.2%) and mouse (24.3%); While the identifity of pig Catsper3 with that of cattle (84%), dog (85.3%) and human (71.6%) was very high, and moderate with that of rat (63.7%) and mouse (64.1%); the identifity of pig Catsper4 with that of human (77.4%), cattle (81.4%), dog (77.4%), rat (71.7%) and mouse (68.8%) was relative higher among all species compared with Catsper1-3. The amino acid sequence alignment among species revealed that, the C terminal of Catsper1 was conservative and the homology of N terminal was low; While the N and C terminals of Catsper2 were conservative and the identity of the middle sequence near to C terminal was low; And the N and C terminals of Catsper 3 and 4 was not conservative and the identity of the middle sequence was relatively high. The protein domains prediction revealed that all four Catpser proteins contain 6 transmembrane domains and a coiled coil motif, the 6 transmembrane domains were located in the C terminal of Catsper1, while these domains were located in the N terminals of Catsper2, Catsper3 and Catsper4; The coiled coil motif was located in the C terminals of Catspers.2. The RT-PCR analysis of Catsper1-4 genes in the genital tracts of boar and gilt revealed that, Catsper1-4 genes were expressed mainly in testis, but Catsper1 was expressed in caput epididymes, corpus epididymes, caudal epididymes, cervix and uterine horn, weak expression of Catsper3 in protaste was observed as well, Catsper2 and Catsper4 expression were testis specific. The temporal expression of Catsper1-4 in testis during Meishan boar development showed that, totally the mRNA expression of Catsper1-4 was improved with the age increase, but different genes showed variant pattern. The expression of Catsper1 and Catsper4 initiated on day 1, but maintained at low level, then Catsper1 increaed slightly, while Catpser4 decreased significantly (P<0.05) on day 30. Both Catsper1 and Catsper4 expression increased significantly on days 60 and 90 (P<0.05), and Catsper1 increased until day 150 (P<0.05), whereas Catsper4 decreased slightly on day 150. The expression of Catsper2 and Catsper3 was detectable until day 60, but kept at high level and increased significantly on day 90 (P<0.05), then Catsper2 kept the increase until day 150 (P<0.05), while Catsper3 represented obvious drop at that time (P<0.05).3. The sequences of two mRNA variants of pig ZPBP2 (1488bp and 1269bp) were obtained. The two mRNA sequences comprised 981 bp and 762bp ORF respectively, flanked by a 131 bp 5′UTR and a 376 bp 3′UTR. The 981 bp ORF encoded a 326 amino acids precursor peptide, while the 762bp ORF encoded a 253 amino acids precursor peptide. The homology analysis revealed pig ZPBP2 showed 82%, 76%, 75% identity with human, rat and mouse respectively. Sequence alignment of mammal ZPBP2 revealed that the ZPBP2 have a relative conservative C-terminal region and a higher degree of diversity in the N-terminal region including signal peptide. 15 cysteine residues in the mature peptide region are strict conserved among ZPBP2 proteins. Motifs search by PredictProtein web server showed that there were some conserved motifs among mammal ZPBP2 proteins. A putative immunoglobulin-like (IG-like) domain and two overlap epidermal growth factor-like (EGF-like) domains were predicted by SMART web server. The two EGF-like domains are located in the C-terminal cysteine-rich domain (CRD).4. The RT-PCR analysis of ZPBP1 and ZPBP2 genes in the genital tracts of boar and gilt revealed that, in all sampled tissues, strong signal of ZPBP1 mRNA was detected in testis and it was not detectable in other tissues; while the mRNA transcript of ZPBP2 could be detected in testis, seminal vesicle and caudal epididymes but with significant variation of expression level. ZPBP2 showed strongest expression in testis, modest in seminal vesicle and very weak in caudal epididymis. Additionally weak ZPBP2 transcript variant 2 was observed in seminal vesicle. The temporal expression of ZPBP1 and ZPBP2 in pig testis and seminal vesicle indicated that, the expression of ZPBP1 in testis was not detectable until day 60, but kept significant increase to 90 day (P<0.05), and the expression level was dramatically enhanced from day 90 to 150 (P<0.05). Whereas the expression of ZPBP2 in testis initiated on day 1 and decreased to very low level on day 30 (P<0.05), then there was a significant increase in the relative abundance of ZPBP2 mRNA on day 60 (P<0.05) and then remained upregulation until 90 days (P<0.05) and evaluated sharply from 90 to 150 days (P<0.05). The expression of ZPBP2 in seminal vesicle was not detected until 30 days and remained low level until 60 days, then displayed considerable increase on day 90 (P<0.05) and sharp growth on day 150 (P<0.05).5. The RT-PCR analysis of spermadhesin (AQN1, AQN3, AWN, PSPI and PSPII) genes in the genital tracts of boar and gilt revealed that, AWN, PSPI and PSPII were detected in all sampled tissues, no signal of AQN1 was detected in the corpus epididymes, oviduct and ovary, whereas AQN3 transcript was not detected in the corpus epididymes, testis and ovary. In general, all spermadhesin transcripts were highest in the seminal vesicle, but moderate in the prostate and caput epididymes. Of the five spermadhesin genes, most were weakly expressed, or not expressed, in the corpus epididymes and testis, whereas only AWN was strongly expressed in the testis. Temporal expression for spermadhesins in seminal vesicle, prostate and bulbourethral gland from birth to Day 150 revealed that, in the seminal vesicle, the expression of AWN gene was constitutive at all ages studied, whereas expressions for AQN1, AQN3, PSPI and PSPII genes were elevated from days 1 to 150. AQN1 and PSPI increased significantly from days 1 to 30, then the expression level of PSPI maintained a slow growth until day 90, with a sharp up-regulation day 150 (P<0.05). Expression of AQN3 and PSPII significantly increased from days 60 to 150, whereas expression of AQN1 were dramatically up-regulated on days 60 and 150 (P<0.05); In the prostate, AWN, PSPI and PSPII had considerable expression soon after birth, peaked at day 60, decreased significantly at day 90, and continued to decrease until day 150. The genes of AQN1 and AQN3 had a very low level of expression at day 1. However, expression of AQN3 increased dramatically from days 1 to 60, then started to decrease until day 150. Expression of AQN1 was low at days 1, 30, and 60, but was very high at days 90 (P<0.05) and 150. In the bulbourethral gland, PSPII was weak at Day 1 and significantly increased at Day 30, whereas all other genes were not detectable. Expression of PSPII increased sharply at days 60, 90 and 150 (P<0.05). Expression of AQN1, AQN3, and AWN was detected at day 60 and greatly increased at days 90 and 150 (P<0.05). While PSPI gene was expressed at a constantly high level but without obvious increase on days 90 and 150.6.The RT-PCR analysis of CRISP (CRISP1, CRISP2, CRISP3) and Fn-2 (ELSPBP1 and pB1) genes in the genital tracts of boar and gilt revealed that, CRISP1 exhibited very strong signal in whole epididymides (caput, corpus and cauda), moderate in seminal vesicle and weak in prostate. CRISP2 mRNA represented more extensive distribution along male and female reproductive tracts with highest abundance in the testis. CRISP3 gene was expressed with high level in the prostate and bulbourethral gland, but weakly in testis and ovary. ELSPBP1 gene was expressed with highest abundance in cauda epididymides, moderate in corpus epididymides, and weak in seminal vesicle and prostate. pB1 mRNA expression was also abundant along reproductive tracts. During the sexual development of boars after birth, these genes showed different expression patterns. CRISP1 and CRISP3 gene expression was high on day 1 of age and this high expression level was maintained until day 150 of age, while CRISP2 expression was low on day 1, started to increase on day 60, kept this high level until day 90 and dropped on day 150. ELSPBP1 showed low expression on day 1 and increased significantly on day 30 (P<0.05) and then kept static until day 150. pB1 gene displayed moderate expression from birth to day 30 and increased significantly on day 60 (P<0.05) and maintained high level until day 150.7. The RT-PCR analysis of SPMI gene in the genital tracts of boar and gilt revealed that, the mRNA expression of SPMI was detected in seminal vesicle, prostate, bulbourethral gland, cervix, uterine horn and ovary with different abundance. The highest abundance of SPMI expression was in seminal vesicle, moderate in bulbourethral gland and uterine horn, lowest in prostate cervix and ovary. The temporal expression of SPMI revealed the expression level of SPMI in seminal vesicle and bulbourethral gland initiated on day 1 and improved until day 150 with age increase. The expression of SPMI in seminal vesicle increased significantly on days 60, 90 and 150 (P<0.05); while the expression of SPMI in bulbourethral gland increased significantly on days 90 and 150 (P<0.05).8. The mRNA expression analysis of pig sperm functional genes in ejaculated sperm by RT-PCR revealed that, except for ELSPBP1, the mRNA expression of three major seminal plama protein gene families were detectable but with variant abundance. The spermadhesin (AQN1, AQN3, AWN, PSPI and PSPII) genes, CRISP2 gene of CRISP gene family and pB1 of fibronectin type II (Fn-2) gene family represented high abundance, while CRISP1 and CRISP3 genes presented weak expression. SPMI gene also maintained a high mRNA expression in ejaculated sperm, whereas ZPBP1/2 and Catsper1-4 didn't display any mRNA signal in ejaculated sperm.9. The association analysis of the mRNA expression of spermadhesins (AQN1, AQN3, AWN, PSPI, PSPII) and SPMI genes with sperm motility, capacitation and acrosome reaction showed that, the expression level of spermadhesins (AQN1, AQN3, AWN, PSPI, PSPII) and SPMI genes in low motility sperm were significantly higher than that in high motility sperm (P<0.05); Capacitation in vitro has significantly effected on the mRNA expression level of spermadhesin genes (AQN1, AQN3, PSPI, PSPII) in sperm (P<0.05), acrosome reaction induced in vitro has significantly effected on the mRNA expression level of AWN, PSPI and PSPII genes in sperm (P<0.05) as well.Overall, in general, most sperm functional genes (Catsper1, Catsper3, ZPBP2, AQN1, AQN3, AWN, PSPI, PSPII, CRISP1, CRISP2, CRISP3, ELSPBP1, pB1and SPMI) detected in this study represented more extensively distributation along genital tracts of Meishan boar and gilt compared with previous reports. This could be related with the speciality of Meishan breed. Except for CRISP1 and CRISP3 genes, in general, the developmental expression of most sperm functional genes, including Catsper1-4, ZPBP1/2, AQN1, AQN3, AWN, PSPI, PSPII, CRISP2, ELSPBP1, pB1 and SPMI, displayed age dependent pattern, the mRNA expression levels were parralled with the sexual development, they increased significantly from days 60 to 90 during purbtey and maintained high level from days 90 to 150 during sexual maturity. The mRNA expression of major seminal plasma gene families in sperm was reported firstly; the mRNA expression level of spermadhesins (AQN1, AQN3, AWN, PSPI and PSPII) and SPMI genes were significantly associated with sperm motility, capacitation and acrosome reaction status of sperm; and the mRNA expression of these genes could be used as the markers of semen quality and fertility.