Abstract
The sperm flagella 2 (SPEF2) gene is essential for development of normal sperm tail and male fertility. In this study, we characterized first the splice variants, promoter and its methylation, and functional single-nucleotide polymorphisms (SNPs) of the SPEF2 gene in newborn and adult Holstein bulls. Four splice variants were identified in the testes, epididymis, sperm, heart, spleen, lungs, kidneys, and liver tissues through RT-PCR, clone sequencing, and western blot analysis. Immunohistochemistry revealed that the SPEF2 was specifically expressed in the primary spermatocytes, elongated spermatids, and round spermatids in the testes and epididymis. SPEF2-SV1 was differentially expressed in the sperms of high-performance and low-performance adult bulls; SPEF2-SV2 presents the highest expression in testis and epididymis; SPEF2-SV3 was only detected in testis and epididymis. An SNP (c.2851G>T) in exon 20 of SPEF2, located within a putative exonic splice enhancer, potentially produced SPEF2-SV3 and was involved in semen deformity rate and post-thaw cryopreserved sperm motility. The luciferase reporter and bisulfite sequencing analysis suggested that the methylation pattern of the core promoter did not significantly differ between the full-sib bulls that presented hypomethylation in the ejaculated semen and testis. This finding indicates that sperm quality is unrelated to SPEF2 methylation pattern. Our data suggest that alternative splicing, rather than methylation, is involved in the regulation of SPEF2 expression in the testes and sperm and is one of the determinants of sperm motility during bull spermatogenesis. The exonic SNP (c.2851G>T) produces aberrant splice variants, which can be used as a candidate marker for semen traits selection breeding of Holstein bulls.
Introduction
The widespread use of dairy bull semen requires high sperm quality, which is economically important in the artificial insemination industry. During mammalian spermatogenesis, male germ cells undergo a series of differentiation steps that lead to the production of mature haploid spermatozoa. This complex physiologic process includes chromatin reorganization, cytoplasm elimination, acrosome formation, and flagellum development in the seminiferous tubules of the testis and epididymis (O'Donnell et al. 2001, Bettegowda & Wilkinson 2010). Normal sperm flagellum is essential for sperm motility and fertilization of the egg (Yanagimachi 1993). This process involves the coordinated expression of many genes with unique cellular and temporal specificities. Identifying the genes specifically expressed in newborn and adult testes and sperm is crucial to understand testicular development and function, as well as spermatogenesis (Huang et al. 2004).
The sperm flagella 2 (SPEF2) gene, also known as KPL2, is essential for normal sperm tail development and male fertility. Spef2 expression has previously been found to be stage specific and intensive in spermatocytes and round spermatids in the seminiferous tubules of rat testes (Ostrowski et al. 1999). SPEF2 has been detected in both germ cells and Sertoli cells. The intense expression is located in the manchette, in the tail of elongating spermatids, and in the tail of the mouse sperm (Sironen et al. 2010). The loss of the SPEF2 gene causes a decline in elongating spermatids during spermiogenesis and fault in the formation of sperm tail (Sironen et al. 2011). Previous studies have suggested that SPEF2 has an important role in the sperm tail formation and ciliary function involved in sperm motility. However, the molecular mechanisms of SPEF2 gene expression regulation remain unknown. The developmental processes that act on male germ cells that culminate in the production of functional spermatozoa are regulated by alternative splicing (AS) and methylation mechanisms at the transcriptional, post-transcriptional, and epigenetic levels (Zamudio et al. 2008, Bettegowda & Wilkinson 2010, Schagdarsurengin et al. 2012). AS provides a versatile means of regulating gene expression using different combinations of exons from the same primary transcript, resulting in the generation of different mature transcripts and coding the same, shorter, or even distinct proteins (Elliott & Grellscheid 2006). About 5–45% of multi-exon genes undergo AS in different eukaryotes (Brett et al. 2000). AS is particularly prevalent in the testes and it plays an important role in several developmental pathways. Several studies have shown that deviations in aberrant transcripts are one of the causal factors in the reduced reproductive performance of bulls, including sperm maturation and fertilization (Erikson et al. 2007, Brandenburger et al. 2011, Noda et al. 2013). Single-nucleotide polymorphisms (SNPs) in exonic splice enhancers (ESEs) or exonic splice silencers are associated with spliced pre-mRNA by causing aberrant splicing in or near cis-acting elements, including exon skipping and/or intron retention; the mutation increases the frequency of the skipping of exons in the transcription process and gives rise to aberrant transcripts (Liegel et al. 2011). The bovine SPEF2 gene is composed of 36 exons and 35 introns and is considered as a multi-exon gene. A retrotransposon inserts an SPEF2 intron that causes aberrant splicing, which leads to immotile short-tail sperm in Finnish Yorkshire boar (Sironen et al. 2006). Therefore, we hypothesize that the expression of bovine SPEF2 gene may be regulated by the AS mechanism and mutations of bovine SPEF2 can lead to aberrant splice variants, which has potential roles in testicular development and spermatogenesis in bulls.
In mammals, DNA methylation is one of the most stable epigenetic modifications and is an important regulator in a number of biological processes, including testicular development and spermatogenesis (Oakes et al. 2007). Genetically, DNA methylation usually occurs in sequences with a CpG island, which are located on the promoter regions of genes in differentially methylated regions or in imprinting control regions (Bird 2002, Jaenisch & Bird 2003). Correct DNA methylation, however, has an important role in sperm production because hypermethylation has been associated with poor sperm parameters, idiopathic male infertility, and even pregnancy failure (Schagdarsurengin et al. 2012). Several genes, namely MTHFR, PAX8, IGF2, KCNQ1OT1 (LIT1), and SNRPN, in the testes are regulated through epigenetic mechanisms and are involved in poor semen parameters or male infertility (Rajender et al. 2011). During the selection process of excellent bulls with elite genetic potential and high semen performance, some of the full-sib bulls present diverse semen phenotypes. We deduced that the difference might be caused by epigenetic regulation. Therefore, we analyzed the promoter methylation pattern of the candidate SPEF2 gene 5′-flanking region in the sperm cells from high-performance and low-performance adult bulls.
To confirm our hypotheses, the splice variants and their expression, as well as the localization of the SPEF2 gene in Holstein bull tissues, including the testes, epididymis, and sperm, were investigated; the functional SNPs that caused splice variants and associated with semen quality traits were also detected; the promoter and methylation pattern of the SPEF2 gene in ejaculated sperm and testis were also identified in full-sib paired Holstein bulls.
Subjects and methods
Ethics statement
All experiments were carried out according to the Regulations for the Administration of Affairs Concerning Experimental Animals published by the Ministry of Science and Technology, China in 2004 and approved by the Animal Care and Use Committee in Shandong Academy of Agricultural Sciences, Shandong, People's Republic of China. We obtained permission from the slaughterhouse and Shandong OX Biotech Co., Ltd. to use animal parts.
Semen and tissue samples
Experiment 1
Tissue samples from three newborn (2 days old) and three adult Holstein (3 years old), including testes, liver, heart, spleen, lungs, and epididymis, obtained from the Shandong commercial slaughterhouse were used to search the splice variants of the SPEF2 gene using RT-PCR and clone sequencing methods.
Experiment 2
To further investigate the expression of different transcripts of bovine SPEF2 gene in semen, fresh semen samples were obtained from 20 adult Holstein bulls (3–5 years old) in Shandong OX Biotechnology Co., Ltd. One ejaculate from each bull was collected using an artificial vagina, which was evaluated in terms of volume per ejaculate, sperm motility, sperm concentration, and percentage of abnormal sperm as described by Pan et al. (2013). Twenty bulls were assigned into two groups based on semen quality and age: a high-performance group and a low-performance group. According to the Frozen Bovine Semen Standard (GB/T 4143-2008, China), the semen that met the following criteria were included into the high-performance group (sperm motility 69.7±0.99%): sperm motility ≥65%, sperm concentration ≥6×108/ml, and abnormal sperm percentage ≤15%. The remaining bulls were included into the low-performance group (sperm motility 51.3±1.86%). The fresh semen samples were maintained at 37 °C and immediately returned to the laboratory for total RNA and DNA extraction. The tissue samples were immediately frozen in liquid nitrogen and used for total RNA and protein isolation. Fresh testicular and epididymal samples from two adult bulls were fixed in 4% paraformaldehyde for 48 h at room temperature, embedded in paraffin, and then cut into 7 μm sections for localization of bovine SPEF2 protein.
Experiment 3
To investigate the promoter methylation pattern of the SPEF2 gene, the core region of the SPEF2 promoter was identified and fresh semen DNA samples from four full-sib paired bulls (Table 1) as well as another four adult bulls' testicular tissues were treated with sodium bisulfite. The samples were treated the same as in Experiment 2.
Semen quality parameters and promoter methylation levels of the four full-sib bulls in Experiment 3.
Bull no. | Age | Volume of ejaculate (ml) | Sperm motility (%) | Sperm concentration (108/ml) | Percentage of abnormal sperm (%) | Percentage of promoter methylation (%) |
---|---|---|---|---|---|---|
1 | 4.5 | 7.08 | 55 | 14.67 | 19.42 | 8.02 |
2 | 4.5 | 8.96 | 73 | 17.06 | 9.05 | 5.56 |
3 | 6 | 6.47 | 53 | 10.29 | 20.33 | 2.78 |
4 | 6 | 9.09 | 72 | 12.41 | 8.26 | 5.56 |
Bulls 1 and 3 belong to the low-performance group; bulls 2 and 4 belong to the high-performance group; bulls 1 and 2 are full-sib, whereas bulls 3 and 4 are full-sib.
Experiment 4
A total of 109 Chinese Holstein bulls from three bull stations (Shandong OX Biotechnology Co., Ltd., (Jinan, Shandong, China) Beijing Dairy Cattle Center (Beijing, China), and Shanghai Bright Holstan Co., Ltd., Shanghai, China) were included in this study for the association analysis between genotypes and semen quality traits. The fresh semen quality traits, including semen volume per ejaculate (ml), and sperm concentration (×108/ml) and mobility were measured as mentioned in Experiment 2. After investigating the above traits, the fresh semen was diluted with glycerol–egg yolk–citrate mixture, packaged in 0.25 ml straws and cryopreserved. Two straws were randomly obtained from each sample, ejaculated and thawed at 38 °C for 20 s after storage in liquid nitrogen for 5–7 days, and immediately evaluated for the frozen/thawed sperm motility under light microscopy, according to the criteria entitled Frozen Bovine Semen standard (GB/T 4143-2008, China).
Sperm DNA isolation
Genomic DNA was salted out from the semen. Briefly, 80 ml of lysis solution (12.5 mM EDTA, pH 8.0; 12.5 mM Tris–HCl, pH 8.0; 0.4 M dithiothreitol; 0.4 M NaCl; and 12.5% SDS) with 20% SDS and proteinase K (20 mg/ml) were added to the sperm pellet. The mixed liquor proceeded at 55 °C for about 10 h. Then, the genomic DNA was separated using saturated NaCl solution and precipitated using precooled 100% ethanol. The DNA pellet was washed twice in 500 μl of 75% ethanol and air-dried for a few minutes at room temperature. Finally, the DNA was dissolved in 100 μl of TB buffer (0.1 M Tris–HCl and 1 mM EDTA). The DNA was then stored at −20 °C until subsequent analysis.
Total RNA extraction and cDNA synthesis
Total RNA was isolated from semen following the same protocol. Briefly, the somatic cells in semen were lysed in 1 ml of cell lysis solution (0.1% SDS and 0.5% Triton X-100) and then washed in 1 ml of rinsing solution (60% Tris–OH and 8.6% sucrose). The sperm cells were lysed in 1 ml of TRIzol (Invitrogen) and proteins were removed using 200 μl of chloroform. Total RNA was precipitated with an equal volume of isopropanol, and the RNA pellet was washed with 75% ethanol. Finally, the total RNA was dissolved in 20 μl of DEPC water. cDNA was synthesized using a transcript first-strand cDNA synthesis kit (TaKaRa, Dalian, China). The total RNA extraction from tissues and RT into cDNA were performed according to a previously published protocol (Hou et al. 2012).
Identification of SPEF2 splice variants
To search potential splice variants of the bovine SPEF2 gene, five primer pairs named S1, S2, S3, S4, and S5 (Table 2), which span the 5′-UTR and the 3′-UTR, were designed to amplify the full-length transcript and splice variants according to the bovine SPEF2 reference sequence (GenBank:
Primer information of the bovine SPEF2 gene.
Primer names | Sequences (5′–3′) | Annealing temperature (°C) | Length (bp) |
---|---|---|---|
S1 | Forward: AGGCAGGCGGGCTAGGTTTC | 60.0 | 1117 |
Reverse: CCTTATCTCTGAGGGCTTGT | |||
S2 | Forward: GAAGAACAAGCCCTCAGAGA | 58.0 | 1121 |
Reverse: GAAGAACAAGCCCTCAGAGA | |||
S3 | Forward: CCTCATAGAACTGGAGGGAA | 58.0 | 1229 |
Reverse: GCCTGCATCAGTGAGAAGAA | |||
S4 | Forward: GGAGCGTCTGGACATCATTA | 57.0 | 838 |
Reverse: CATTCAAGTACCTCTCACCG | |||
S5 | Forward: GATAGCTACACAGTTCCGAC | 60.0 | 1352 |
Reverse: GACCTCTCTTCATCACTTCC | |||
SV3 | Forward: AGATAAACAGTCATTGTGCC | 55.0 | 396 |
Reverse: CGGTGAAACATCTGTAGACT | |||
G20 | Forward: TAATCCTCCACCAGAATCTG | 59.0 | 584 |
Reverse: GCCTTATGATGTGGGTATGA | |||
SPEF2-1 | Forward: AGAAAACCAGCCAGCAGAAA | 50.0 | 635 |
Reverse: ACGTGAGTCCCAATGGCTAC | |||
Q-SPEF2 | Forward: GCTGGATTTGGTAACCCTGAA | 63.0 | 189 |
Reverse: GGTCTCCAGAAGCTCCTCCTC | |||
Q-SPEF2-As | Forward: AGATCCACCTCCTCCGGTACG | 59.0 | 233 |
Reverse: AACCACAGGCCAGCTTCAG | |||
β-actin | Forward: GCACAATGAAGATCAAGATCATC | 55.0 | 173 |
Reverse: CTAACAGTCCGCCTAGAAGCA | |||
M-SPEF2-outer | Forward: AAAATTGAGGTTATTGGAGTAGGGG | 55.3 | 285 |
Reverse: AAAATACCAAAACTCAACCCTAA | |||
M-SPEF2-inner | Forward: AGGTTATTGGAGTAGGGGTTTTTATAA | 55.3 | 271 |
Reverse: CAAAACTCAACCCTAAAAAACAAA | |||
pGL3-2004 | Forward: CGGGGTACC GATTTACAGCCAAGGAGTGC (KpnI) | 61.9 | 2004 |
Reverse: CCGCTCGAGCAGGGGCACACAACTAATAG (Xhol) | |||
pGL3-1515 | Forward: CGGGGTACCGGTCTGGGACTAAAAGTTGT (KpnI) | 60.1 | 1515 |
Reverse: CCGCTCGAGCAGGGGCACACAACTAATAG (Xhol) | |||
pGL3-1039 | Forward: CGGGGTACCCTAGGAGGACTTTTGTTGGC (KpnI) | 61.6 | 1039 |
Reverse: CCGCTCGAGATTCTAACTTGTCCAGGGGC (XhoI) | |||
pGL3-450 | Forward: CGGGGTACCAGGCCTAACCATGTCGGAGA (KpnI) | 65.0 | 450 |
Reverse: CCGCTCGAGCAGGGGCACACAACTAATAG (Xhol) | |||
pGL3-829 | Forward: CGGGGTACCCAAGAGTCTTCTCCAACACCA (KpnI) | 63.2 | 829 |
Reverse: CCGCTCGAGATCAGCTCGCCTCGTTCCA (Xhol) | |||
pGL3-463 | Forward: CGGGGTACC TCCCTTGCCTCTCCTCCTCCA (KpnI) | 68.5 | 463 |
Reverse: CCGCTCGAGATCAGCTCGCCTCGTTCCA (Xhol) | |||
pGL3-287 | Forward: CGGGGTACCGCACCCCTCTTCTCCCAGCCT (KpnI) | 66.2 | 287 |
Reverse: CCGCTCGAGATCAGCTCGCCTCGTTCCA (Xhol) |
Quantitative RT-PCR
Quantitative RT-PCR (RT-qPCR) was carried out to determine the mRNA relative expression of the SPEF2 splice variants in the high-performance and low-performance bull semen. To determine the differential expression between SPEF2-complete and SPEF2-SV1, we designed the specific PCR primers Q-SPEF2 and Q-SPEF2-AS (Table 2 and Fig. 1a and b) and used cDNA produced from sperm-derived templates to amplify SPEF2-complete and SPEF2-SV1 transcripts using the SYBR Green PCR Master Mix (Tiangen, Beijing, China) according to the manufacturer's protocol. The primers for the housekeeping internal control β-actin gene that amplifies a 173 bp fragment were referred from a previous report (Hou et al. 2012). The 20 μl RT-qPCR mixture contained 10.0 μl of SYBR Premix 2× ExTaq II, 0.4 μM of the forward and reverse primers, 2.0 μl of cDNA (∼100 ng) or plasmid DNA, and 6.4 μl of ddH2O. The RT-qPCR was performed under the following conditions: 94 °C for 5 min, 40 cycles at 94 °C for 15 s, 56 °C for 15 s, and 70 °C for 5 s. The last stage for the dissociation curve was as follows: 95 °C for 15 s, 60 °C for 15 s, and 95 °C for 15 s. The relative expression of the transcripts was calculated based on a previous report (Wang et al. 2012). Each sample was measured in triplicate and the experiment was repeated at least three times.
Prediction of promoter bioinformatics
The CpG islands were analyzed using the online software CpG Island Searcher (http://www.uscnorris.com/cpgislands2/cpg.html). We designed two bisulfite-modified specific PCR primer pairs, M-SPEF2-outer and M-SPEF2-inter (Table 2), for nested PCR amplification using the MethPrimer program (http://www.urogene.org/cgi-bin/methprimer/methprimer.cgi). The promoter of the bovine SPEF2 gene was predicted using the Genomatix Software (http://www.genomatix.de/applications/index.html). The presence of a TATA box was predicted using PROSCAN, version 1.7 (http://www-bimas.cit.nih.gov/cgi-bin/molbio/proscan). Transcription factors were predicted using Promoter Scan (http://www-bimas.cit.nih.gov/molbio/proscan/) and TFSEARCH (ver.1.3; http://www.cbrc.jp/research/db/TFSEARCH.html).
Sodium bisulfite treatment
Genomic DNA was treated using a BisulFlash DNA modification kit (Epigentek, Brooklyn, NY, USA) according to the protocol of the manufacturer. Modified DNA was maintained at −20 °C until PCR amplification.
PCR amplification, cloning, and bisulfite sequencing
PCR was carried out in a final volume of 20 μl. The nested PCR conditions were as follows: the reaction system for the first-round amplification consisted of 10 of 2× GC buffer I (5 mM Mg2+ Plus), 3.4 μl of 2.5 mM dNTP mixture, 0.2 μl of TaKaRa LATaq (5 U/μl), 1 μl of each 10 μM M-SPEF2-out primers, 3 μl of template DNA for the first round, 0.5 μl of template DNA (from the first round product), and M-SPEF2-inter primers for the second round amplification. The PCR program was as follows: initial denaturation at 94 °C for 3 min; 45 cycles at 94 °C for 40 s, 45 °C for 1 min, and 72 °C for 1 min; and final extension at 72 °C for 15 s. The PCR products were detected on 1.5% agarose gel, purified using a Gel/PCR extraction kit (Biomiga, Beijing, China), cloned into the pGEM-T Easy Vector (TransGen Biotech), and then transformed into E. coli DH5α cells for clone sequencing. Only the sequences derived from clones with >95% cytosine conversion were analyzed. The percentage of DNA methylation was calculated by counting the number of methylated CpGs out of the total number of CpG sites in individual clones. The bovine SPEF2 sequence (GenBank:
Construction of SPEF2 promoter-reporter plasmids
SPEF2 promoter activity was evaluated by dividing the region from g.−2064 to g.+440 into seven fragments, designated as pGL3-2004, pGL3-1515, pGL3-1039, pGL3-450, pGL3-829, pGL3-463, and pGL3-287. The primers contained the restriction enzyme site for KpnI and XhoI (Table 2). Each fragment was amplified, recovered, purified, and cloned into the pGL3-basic luciferase reporter vector according to a previously described protocol (Li et al. 2013). The sequences of these vectors were confirmed through direct sequencing on an ABI 3730xl sequencing platform.
Transient transfection and luciferase reporter assay
The murine Leydig tumor (MLTC-1) cell line was cultured in RPMI-1640 medium (Sigma) supplemented with 10% fetal bovine serum (Invitrogen) containing 10 mg/l of penicillin and streptomycin (Invitrogen) at 37 °C in a controlled humidified atmosphere with 5% CO2. The day before transfection, MLTC-1 cells were distributed in a 48-well plate. Upon reaching 75–85% confluence, the cells were cotransfected with 400 ng of the constructs or plasmid pGL3-basic and 50 ng of PRL-TK using Lipofectamine 2000 (Invitrogen). The plasmid pGL3-basic was used as the negative control and PRL-TK was used as the internal control to conform the difference in transfection. After 24 h of cultivation, the cells were washed and lysed in 1.5 ml tubes. A Dual-Luciferase Reporter Assay System (Promega) was used to analyze the luciferase activity according to the manufacturer's instructions. All transfections were performed in triplicate and repeated at least thrice in independent experiments. Promoter activity was analyzed relative to firefly luciferase activity normalized against Renilla luciferase activity.
Western blot analysis
The tissue samples from the newborn and adult bulls were homogenized using RIPA lysis buffer (Beyotime, Nantong, Jiangsu, China). After cooling the lysate on ice for 30 min, it was centrifuged at 10 000 g for 10 min at 4 °C. The proteins were separated after denaturation via 12% SDS–PAGE, wet transferred onto a PVDF membrane, blocked with blocking buffer (Beyotime), and rotated for 1 h at RT. The blots were incubated with monoclonal anti-SPEF2 (1:500; Abcam, Hong Kong, China) or polyclonal β-actin (1:500; Beyotime) for 2 h at RT. The SPEF2 peptide sequences were based on the following murine part of SPEF2 (AQEEAYREEQLINRLMRQSQQERRIAVQLMHVRHEKEVLWQNRIFREKQHEERRLKDFQDALDREAALAKQAKIDFEEQFLKEKRFHDQIAVERAQARY), which has 90.9% homology with the corresponding bovine SPEF2 protein. After washing the membranes three times with 0.1% Tween-20 in 1× TBS for 5 min each time, anti-mouse or anti-goat secondary antibodies (1:500; Beyotime) were incubated with the membranes to detect antigen–antibody complexes. The bound secondary antibodies were visualized using a 3,3′-diaminobenzidine tetrachloride (DAB) HRP color development kit (Beyotime) according to the manufacturer's instructions. The molecular mass of the proteins was measured using a prestained protein ladder (Thermo, Waltham, MA, USA).
Immunohistochemistry
Testicular and epididymal tissues from two adult bulls were fixed in 4% paraformaldehyde. All tissues were then embedded in paraffin and sectioned for immunohistochemistry (IHC). A total of 1 l of deionized water and 10 ml of citrate buffer solution were used to rehabilitate the antigen and washed with 0.01 M PBS (pH 7.4) twice every 3 min. Then, the immunoreaction slides were deparaffinized and hydrated. The slides were blocked with endogenous peroxidase for 10 min and were subsequently washed with PBS and incubated with monoclonal anti-SPEF2 antibodies (1:50; Abcam) for 60 min at RT. After washing with PBS, the slides were incubated with anti-mouse secondary antibodies for 10 min at RT. The antibodies were visualized with 0.6 mg/ml DAB (Cwbiochem) HRP color development kit (Cwbiochem) for brown staining under a microscope (Leica LB30T, Wetzlar, Germany) according to the manufacturer's instructions. The slides were then stained with hematoxylin (Cwbiochem), dried again, and photographed using a digital camera (Leica).
SNP screening and genotyping
The primer pairs spanning from the exons 17, 18, 19, and 20 of the SPEF2 gene were used to amplify the fragment including the deletion of splice variants and to screen potential SNPs. The PCR amplification fragments were sequenced for SNP screening using an ABI 3730xl sequencing platform in the commercial server (Invitrogen). Only the primer pairs named G20 (Table 2) screened one SNP. Genotyping of the SNP was performed using the PCR–restriction fragment length polymorphism. The restriction endonucleases TaqI was selected to digest PCR products and the digested fragments were separated on 2% agarose gel.
Statistical analysis
Data are presented as mean or mean±s.e.m. Statistical significance for gene expression and promoter activity was tested using a Student's t-test. The methylation patterns of the CpG sites of the SPEF2 promoter were analyzed using a χ2 test.
The association between SNP genotypes and sperm quality traits was analyzed using the general least-square model procedure from SAS 9.0 (Statistical Analysis Software, SAS Institute, Cary, NC, USA). The line model is Yijkml=μ+Gi+Ak+Pj+Hl+eijkl, where Yijkl is the observed value of each semen quality trait; μ is the overall mean; Gi is the fixed effect of genotype; Ak is the fixed effect of age; Pj is the fixed effect of the origin of bull; Hl is the effect of farm; and eijkl is the random residual error. Differences between groups were considered significant at P<0.05.
Results
Identification and expression of bovine SPEF2 splice variants
To analyze the possible splice variants of the SPEF2 gene among the different tissues from two developmental stages of bulls, primer S1, S2, S3, S4, and S5 was used to amplify the full-length coding region using cDNA as the template. We obtained the full-length transcript and the three novel transcripts through clone sequencing. To distinguish various transcripts using agarose gel electrophoresis, primers SPEF2-1, and SV3 spanning the deletion region were designed to amplify the shorter SPEF2 gene transcripts. The RT-PCR detected four amplicons in different tissues that were dependent on developmental stage (Fig. 1). SPEF2 mRNA was expressed predominantly in the testes of adult bulls and weakly expressed in other tissues. Relatively higher signals were also detected in the epididymis and the ejaculate. Intriguingly, only one 635 bp band was detected in the lungs, whereas two bands were detected in other tissues in newborn bulls (Fig. 1a). In adult bulls, we also detected SPEF2-complete and SPEF2-SV1 in the kidneys, lungs, spleen, liver, heart, testes, and semen (Fig. 1b). The two main bands, 635 and 452 bp, were purified and cloned into the pGEM-T Easy vector for sequencing. As shown in Fig. 2, the 635 bp fragment corresponds to the total transcripts, whereas the 452 bp fragment belongs to a novel SPEF2-SV1 splice variant that lack a 183 bp of exon 31. The 396, 222, and 147 bp fragments corresponded to the total SPEF2, novel SPEF2-SV2, and SPEF2-SV3 transcripts respectively (Fig. 1c). The expression level of SPEF2-SV2 transcript in testis was more abundant than in other tissues. Interestingly, the SPEF2-SV3 was only detected in testis and epididymis (Fig. 1c). SPEF2-SV2 and SPEF2-SV3 delete exon 18 and exon 18+exon 19 respectively (Fig. 2). Three novel transcripts were predicted to encode the truncated proteins with 1710, 1713, and 1688 aa, without an introduction of open reading frame shifting. As shown in Fig. 3, the complete bovine SPEF2 encodes a six-domain protein that includes two calponin homology domains, one actin-binding formin homology 2 domain (FH2), an adenylate kinase site, and UDF domains, whereas, the novel transcript SPEF2-SV1 (deposited in GenBank:
Expression and localization of bovine SPEF2 protein
The bull tissues were subjected to western blot analysis to validate the expression and localization of SPEF2. The results show that SPEF2 proteins ∼200, 190, 180, 100, 50, and 37 kDa were expressed in the newborn and adult bull tissues, including the testes, epididymis, spleen, kidneys, liver, heart, and lungs (Fig. 4). Five SPEF2 protein bands with different molecular weights were detected in the western blot analysis using antibodies against the 287 bp fragment from exons 6 to 8 of the bovine SPEF2 gene. Thus, a ∼200 kDa band was detected in the testicular samples and at lower levels in the epididymis (Fig. 4). Another four low-molecular-weight bands were also visible in the detected tissues, which may correspond to other possible bovine SPEF2 isoforms, degradation products of the long SPEF2 isoforms, or even cross-reactive products. Four bands were detected in the testes compared with other tissues. The 190 kDa band was predominantly expressed in the testes. Interestingly, 180 and 50 kDa tissue-specific bands were also detected in the heart. We detected a 100 kDa band in the liver of adult bulls. In newborn bulls, the full-length SPEF2 protein (200 kDa) was detected only in the testes and no band was found in the spleen (Fig. 4a). The expression pattern of SPEF2 in the heart was similar to that in adult bulls, and the 180 kDa band was absent in the liver and kidneys (Fig. 4b). This result shows that the SPEF2 protein is differentially expressed in the testes at two developmental stages and it may have a potential role in spermatogenesis in Holstein bulls. The two proteins, ∼200 and 190 kDa, are consistent with the predicted molecular weights of the SPEF2-complete (202 kDa) and SPEF2-AS protein (∼195 kDa). Although we were unable to distinguish the other proteins in the western blot analysis, we identified an AS variant of SPEF2 that is transcribed in the spermatogenic cells of the testes and epididymis of bulls.
To further understand SPEF2 protein expression during spermatogenesis, the testicular and epididymal morphologies of the bulls were analyzed via IHC. SPEF2 immunoreactivity was detected in the Sertoli cell and the seminiferous epithelium, including pachytene spermatocytes, primary spermatocytes, and spermatids (Fig. 4c). The immunoreactive pattern of the elongated spermatids was similar to that found in mice (Sironen et al. 2010). The IHC revealed SPEF2 protein expression in epithelial cells throughout the bull epididymis, especially in the corpus epididymis. The SPEF2 protein was located in the tall columnar epithelium with long microvilli in the corpus epididymis; however, a weak signal was detected in the caput epididymis and the cauda epididymis. We also observed abundant sperm in the lumen of adult cauda, which presented a feeble signal.
Relative mRNA expression patterns of SPEF2-SV2 in bull testes, epididymis, and semen
The RT-qPCR result shows that SPEF2 splice variants were abundant in the bull testes, epididymis, and mature sperm. The SPEF2-complete mRNA expression in the testicles, epididymis, and semen of adult bulls was significantly higher than that of the SPEF2-SV1 (P<0.05), which indicates that the long transcript is the main transcript in the three tissues. The total SPEF2 and SPEF2-SV1 mRNA expression in the high-performance bull semen was significantly upregulated compared with that in the low-performance group, while SPEF2-SV3 presents the inverse expression tendency in semen. The SPEF2 splice transcripts are therefore potentially involved in spermatogenesis.
Cloning and activity analysis of SPEF2 promoter
We found a promoter in the 5′-flanking region and the other six promoters in the bovine SPEF2 gene, using bioinformatics software (data not shown). We first focused on the promoter in the 5′-flanking region and amplified seven fragments through progressive deletion of nucleotides from the 5′-end. Each fragment was cloned into the pGL3-basic luciferase reporter vector and then transiently transfected into MLTC-1 cells respectively. The relative luciferase activity of the promoter pGL3-1039 fragment was upregulated by ∼10.3-fold compared with that of pGL3-1515 and by ∼5.4-fold compared with that of pGL3-450 (Fig. 5a). The result indicated negative regulatory elements in the region from g.−1564 to g.−586. Several inhibitory transcription element-binding sites, such as E2F and AML1a, which inhibit the transcriptional activity of genes (Uchida et al. 1997, Ousephe et al. 2012), were found using the TFSEARCH Online Software (http://www.cbrc.jp/research/db/TFSEARCH.html) in the region from g.−1066 to g.−586. To shorten the core promoter region, we divided the region from g.−699 to g.+130 into three fragments and cloned them into the pGL3-basic luciferase vector. These recombinant vectors were transiently transfected into MLTC-1 cells for luciferase activity analysis. Figure 5b shows that the promoter named pGL3-829 activity was significantly higher than that of pGL3-287 and pGL3-463 (P<0.05). This result indicates that the region from g.−586 to g.−157 is the core promoter responsible for most of the promoter activity in the bovine SPEF2 gene.
DNA methylation of the SPEF2 promoter
To test whether the SPEF2 promoter in the bull was transcribed normally, the DNA methylation patterns of SPEF2 in the semen of four full-sib bulls were detected using bisulfite sequencing. We detected a CpG-rich domain in the core promoter using the online software CpG Island Searcher. Using the quantitative bisulfite sequencing method, we amplified the 271 bp fragment with nine CpG sites. In this study, 60 clones were evaluated, with each clone containing nine CpG sites within the amplified fragment of promoter analyzed. The percentage of the analyzed sequences in all clones were hypomethylated (<50% of CpG sites on a given methylated strand). No differences in the sperm DNA methylation of the SFEF2 promoter were found between the high-performance (5.40%, n=30 clones) and low-performance (5.56%, n=30 clones) bulls (Table 1). However, the percentage of promoter methylation of bull 3 in the high-performance group was the lowest, which indicate individual differences among the four bulls.
Sperm nucleus is highly condensed, resulting in transcriptional repression. Thus, the transcription of the SEPF2 gene is expected to mainly take place in spermatogenic cells in the testis. We also detected the promoter methylation status of SPEF2 in the testis of four adult bulls. They also showed a hypomethylation level (mean value: 10.26%, n=50 clones) in the testis tissues.
Relationships among functional SNPs, aberrant splice variants, and bull semen quality traits
Several studies showed that AS could be caused by the exonic SNPs located in the potential ESE motif (Cartegni et al. 2002). Consequently, we identified the mutations of the exons that are close to the region producing the aberrant splicing. As expected, we found a synonymous SNP (c.2851G>T) in exon 20 of SPEF2 and a nonsynonymous SNP (c.3548T>C) in exon 25 in Chinese Holstein bulls. Using ESEfinder 3.0 Software (http://rulai.cshl.edu/cgi-bin/tools/ESE3/esefinder.cgi), we predicted the change in ESE using the c.2851G>T mutation for it near the splicing region of SPEF2-SV3 and found that it increased one binding site for the splicing factor SRp40 (Fig. 6).
To verify whether the SNP (c.2851G>T) is associated with the production of the novel splice variants, we genotyped six bulls and also identified their corresponding SPEF2-SV2 and SPEF2-SV3 splice variants. As a result, the SPEF2-SV3 transcript was not found in the bulls with the GG genotype.
To analyze the effect of the novel genetic variation of SPEF2 on sperm quality traits (ejaculate volume, sperm density, and thawed sperm motility), the association analysis was performed in a population of 109 Chinese Holstein bulls. The result showed that the bulls with the GG genotype had lower deformity rates than those of the GT and TT genotypes, and that the GG genotype had significantly higher thawed sperm motility when compared with the bulls with the GT and TT genotypes. However, no significant differences in ejaculate volume and fresh sperm density were found (Table 3 and Fig. 7).
Genetic effect of the SNP (c.2851G>T) on semen quality traits in Chinese Holstein bulls.
Genotype/sample | Allelic frequency | Ejaculate volume (ml) | Deformity rate (%) | Sperm density (×108/ml) | Thawed sperm motility (%) |
---|---|---|---|---|---|
GG/16 | G(0.42) | 5.93±0.45 | 15.60±0.02b | 10.46±1.78 | 43.32±3.02a |
TG/59 | 5.78±0.23 | 16.3±0.02b | 12.15±0.93 | 38.68±1.59a,b | |
TT/34 | T(0.58) | 5.62±0.31 | 20.42±0.02a | 13.11±1.23 | 35.42±2.10b |
Least-square mean indicated by different small letter superscripts within the same column differ significantly (P<0.05).
Discussion
SPEF2 splice variants are differentially expressed in the bovine testis, epididymis, and semen and are involved in sperm motility and function
We determined that the SPEF2 gene has three novel transcripts that lack one or two exons in Holstein bulls. Of which, the novel transcript SPEF2-SV1, generated through AS, was expressed in the sperm cells, epididymis, testes, heart, spleen, and kidneys of newborn and adult bulls, and in the lungs of newborn bulls. Moreover, it was differentially expressed in the high-performance and low-performance bull semen. Further, the localization of SPEF2 in the testes and epididymis suggests a role in spermatogenesis. These results indicate that SPEF2 splice variants are involved in the development of testis and spermatogenesis in dairy bulls. They can play roles by gene AS mechanisms. Its abundant expression in epididymal epithelium is consistent with its role in bovine sperm maturation. Two sequence variants in the gene encode mouse SPEF2 proteins, namely flagellar assembly and cilia function, which are involved in spermatogenesis (Sironen et al. 2011). The SPEF2 variants exhibited tissue-specific, phage-specific, and species-specific expression. In rats, SPEF2 is expressed in tissues containing cilia-like structures, such as the lungs, trachea, testes, and the brain, at lower levels in the kidneys and spleen, and is not expressed in the heart and liver, which suggest its role in ciliogenesis (Sironen et al. 2010). In pigs, the SPEF2 gene is differentially expressed in abnormal and healthy animals. Thus, the region coding for the C-terminal part of the protein appears to be expressed only in the testes and trachea, whereas the region coding for the N-terminal part is expressed in the lungs, liver, and kidneys (Sironen et al. 2006). SPEF2 genes participate in spermatogenesis through AS in several species. In this study, we identified three novel splice variants via RT-PCR and clone sequencing; other potential splice variants need to be confirmed in the next experiment. The western blot analysis results imply that the bovine SPEF2 gene is a multifunction gene that has broad functions even though the current study lacked specific antibodies for bovine isoforms. Previous research has reported that the presence of an insert retrotransposon within an intron causes immotile short-tailed sperm in Finnish Yorkshire pigs (Sironen et al. 2006). In rats, the testis-specific long SPEF2 variant and transcripts containing exons 6–43 were detected (Sironen et al. 2010). Fifteen ASs of the human SPEF2 gene were deposited in the Ensemble database (ENSG00000152582). They encode 12 different proteins through exon skipping and retained intron splicing patterns, and two splice variants were identified.
5′ Flanking promoter of SPEF2 is hypomethylated in ejaculated semen and its methylation pattern is unrelated to sperm quality
DNA methylation defects in genes are reportedly associated with impaired human sperm production and quality (Navarro-Costa et al. 2010, Krausz et al. 2012). Whether these differences in semen quality between full-sib bulls reflect differences in DNA methylation pattern has not been addressed yet. We determined the methylation status of the SPEF2 gene promoter. Methylation of the 5′ promoters of DNA suppresses gene expression. In this study, the 5′ promoter methylation pattern is consistent with the expression pattern of the SPEF2 gene in the semen of the full-sib bulls, which is a good animal model for epigenetic analysis. During germ cell development, mammalian cells undergo nearly complete reprograming of DNA methylation patterns. The majority of the promoters in sperm escape methylation, whereas the corresponding hypomethylated regions show substantial structural differences (Molaro et al. 2011). In mammals, tissue- and cell-specific methylation occurs in a small percentage of 5′ CpG island promoters, whereas a far larger proportion occurs across gene bodies. Intragenic methylation is involved in the regulation of cell context-specific alternative promoters in gene bodies (Maunakea et al. 2010). In this study, the DNA methylation profile of the SPEF2 promoter was unrelated to bull sperm quality. However, several promoters in the gene body were predicted in the bovine SPEF2; therefore, we cannot exclude the possible existence of other methylation patterns in bull sperm.
An exonic SNP (c.2851G>T) potentially produces aberrant splice variants and is associated with semen quality traits
Although the mechanisms of AS have been studied extensively, we have not understood fully the diversity and complexity of regulation. Increasing evidence shows that silent SNPs can affect mRNA splicing or mRNA levels/stability, leading to disease in humans (Cartegni et al. 2002). In our study, we identified three novel transcripts and an SNP that is located in the potential ESE motifs. The ESE can interact with specific serine/arginine-rich (SR) proteins, and they have diverse and critical functions in alternative pre-mRNA splicing. Using the ESEfinder 3.0 Software prediction, the T allele did not change the affinity with SF2/ASF, SF2/ASF (IgM-BRCA1), or SC35; however, it increased one potential binding site for the splicing factor SRp40. SRp40 being the splicing protein was reported to having a relationship with the unusual alternative splicing (Patel et al. 2005, Wang et al. 2012). We further found that the TT genotype of the SNP (c.2851G>T–G) could produce SPEF2-SV3 aberrant transcript, while the GG genotype did not. Therefore, we speculate that the mutation may participate in the splicing process of the SPEF2-SV3 splice variant. The overexpression of SPEF2-SV3 transcript lacking the FH2 domain may partially affect sperm cilia formation and flagella assembly, which is needed for further investigation. Moreover, the results of association analyse also suggested that the SNP have significant effects on deformity rates and thawed sperm motility. We will construct plasmids containing different genotypes and transfect them into cells using the exon capture mini-gene system and further verify whether the SNP can cause SPEF2-SV3 transcripts in the next study.
In summary, AS, rather than methylation, plays a role in the regulation of SPEF2 expression in the testes and sperm. The exonic SNP (c.2851G>T) potentially leads to aberrant splice variants and is associated with semen deformity rate and thawed sperm mobility. Analysis of the expression and regulation mechanism as well as SNP genetic effect of the SPEF2 gene will improve our understanding of testicular development and spermatogenesis at the molecular level and may provide insights into candidate genes that determine the semen quality of Holstein bulls.
Declaration of interest
The authors declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported.
Funding
This study was supported by the Support Program of the Ministry of Science and Technology, People's Republic of China (2011BAD19B02 and 2011BAD19B04), grants from the National Natural Science Foundation of China (31271328 and 31000543), the Major Project of National Transgene in China (2013ZX08007-001), and the Program of National Cow Industrial Technology System (CARS-37).
Acknowledgements
The authors thank Mr Yuanpei Zhang, Bo Han, Ming Li, and Ms Lingling Wang of the bull station for their assistance and support in semen collection of bulls.
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