Abstract
Hyperthermia and oxidative stresses are the two central elements contributing to varicocele-related sperm damage. Growing evidence indicates that microRNAs (miRNAs) are involved in the regulation of the heat and oxidative stress responses. In this study, we analyzed the expressions of several stress-related miRNAs in the sperm and found that the expression of miR-15a was significantly decreased in patients with varicocele compared with the control. Furthermore, miR-15a repressed the expression of HSPA1B, which is a typical stress-induced chaperone protein, through directly binding its 3′-UTR. The expressions of miR-15a and HSPA1B exhibited an inverse correlation in sperm. Our results provide a valuable insight into the varicocele-related sperm impairment and male infertility, and may help to develop potential therapeutic targets and novel biomarkers for male infertility.
Introduction
Varicocele is characterized by abnormal dilated, elongated, and tortuous veins in the pampiniform plexus with a reflux of blood. Approximately, 15% of general population has a varicocele, with 40% of them having fertility disorders (Will et al. 2011). Varicocele is common in infertile men and therefore it has been considered as one of the most important factors of male infertility. It can lead to testicular and epididymal malfunction, impaired spermatogenesis, and decreased semen quality. The etiology and pathophysiology of varicocele seem complicated and multifactorial (Fretz & Sandlow 2002, Will et al. 2011). Numerous hypothetical mechanisms have been put forward, such as increased temperature in scrotum, oxidative stress, altered blood flow, hormone disturbance, and back flow of gonadotoxic metabolites (Naughton et al. 2001, Will et al. 2011).
It appears that heat and oxidative stresses are the two central elements contributing to infertility in men with varicocele (Naughton et al. 2001, Fretz & Sandlow 2002, Ferlin et al. 2010, Will et al. 2011). More than 1 °C temperature elevation and increased reactive oxygen species (ROS) production in the testis are reported in men with varicocele (Goldstein & Eid 1989, Mostafa et al. 2009). A growing body of literature demonstrates that increased testicular temperature and ROS can reduce sperm quality (Smith et al. 2006, Jung & Schuppe 2007, Hamada et al. 2012). Testicular hyperthermia-induced oxidative stress builds up of ROS and can cause impaired sperm function through the oxidation of fatty acids in spermatozoa membranes or through direct damage of sperm DNA (Twigg et al. 1998). However, the underlying molecular mechanisms of varicocele-associated male infertility are not fully understood.
MicroRNAs (miRNAs) are noncoding, endogenous, small RNAs that regulate gene expression by binding to the 3′-UTR of target mRNA. By either degrading mRNA or translational repression, miRNAs regulate a variety of developmental and physiological processes including cellular differentiation, proliferation, and apoptosis (Landgraf et al. 2007, Inui et al. 2010). Numerous miRNAs are exclusively or preferentially expressed in the mouse testis (Ro et al. 2007). DICER1 is a cytoplasmic RNase III endonuclease that is essential for miRNA biogenesis as it cleaves the precursor miRNAs to produce mature miRNAs (Inui et al. 2010). Loss of Dicer1 in mouse postnatal male germ cells leads to infertility due to disruptions in the meiotic and post-meiotic stages (Korhonen et al. 2011, Romero et al. 2011, Greenlee et al. 2012), suggesting that miRNAs play important roles in spermatogenesis. Moreover, altered miRNAs expressions of the ejaculated spermatozoa in patients with different spermatogenic impairments have been demonstrated by microarray (Lian et al. 2009, Abu-Halima et al. 2013). But little is known about the molecular role of miRNAs in male infertility, especially in varicocele-related male infertility.
Recently, emerging data suggest that miRNAs could help to restore homeostasis upon cellular stresses. The miRNAs-mediated stress responses play important roles in plenty of human diseases, such as infection, cardiovascular disease, and cancer (Leung & Sharp 2010, Mendell & Olson 2012). Previous studies have shown that miR-15a, miR-16, miR-18, miR-192, and miR-320 are involved in hyperthermia or oxidative cellular stresses (Li et al. 2009, Ren et al. 2009, Wilmink et al. 2010). However, the roles of miRNAs on varicocele-related stress environment remain unclear. In this study, we examined the expressions of stress-related miRNAs in the ejaculated spermatozoa of patients with varicocele and tried to understand the molecular mechanisms of the miRNAs-mediated stress in varicocele.
Materials and methods
Subjects
The study groups consisted of 58 patients with Grade II or III varicocele and 24 healthy donors with normal semen quality and without varicocele recruited consecutively at the Peking University Shenzhen Hospital. Exclusion criteria were seminal infection, cryptorchidism, orchitis, or testicular atrophy. Varicocele was graded according to international clinical classification guidelines: Grade I, no visible or palpable distension except when the man performs the Valsalva maneuver; Grade II, palpable but not visible; and Grade III, easily visible through the scrotal skin (Will et al. 2011). Fresh ejaculate samples were collected from the patients and donors presenting to the andrology unit for routine semen analysis after given informed consent. Normal sperm parameters were defined according to World Health Organization (2010) criteria. The study was approved by the Local Ethics Committee. Written consent was obtained from the patients in the study.
Semen samples
Semen samples were collected by masturbation after 3–7 days of sexual abstinence. The samples were allowed to liquefy at 37 °C for 30 min before standard semen analysis was performed according to World Health Organization (2010) criteria for sperm concentration, motility, morphology, and vitality. Semen samples were subjected to a Percoll (GE Healthcare, Uppsala, Sweden) density-gradient centrifugation to recover sperm for subsequent RNA extraction. Briefly, 1 ml of semen was layered on discontinuous two-layer (40 and 80%) Percoll gradients in 15 ml conical tubes and centrifuged at 800 g for 30 min. The medium used to dilute the Percoll was PBS. Spermatozoa collected from the bottom layer were washed three times in PBS and immediately frozen in liquid nitrogen until RNA extraction.
RNA extraction, RT, and quantitative real-time PCR
Total RNA was extracted from the sperm pellets using the miRNeasy Mini Kit (Qiagen). The quantity and quality of RNA were measured using a Nano-Drop Spectrophotometer.
RT reaction was carried out by All-in-One miRNA First-Strand cDNA Synthesis Kit (GeneCopoiea, Inc., Rockville, MD, USA) for miRNAs and by RevertAid First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) for mRNA. Generally, 50 ng total RNA was used for RT reaction.
The quantitative real-time PCR (qRT-PCR) for miRNAs was performed using the All-in-One miRNA qRT-PCR Detection Kit (GeneCopoiea, Inc.). Amplification reactions were performed in a 20 μl final volume and were done for 40 cycles (15 s at 95 °C, 20 s at 60 °C, and 30 s at 70 °C). U6 small nuclear RNA was selected as the endogenous control. Primers of miRNAs were obtained from GeneCopoiea, Inc. The expressions of mRNAs were quantified by qRT-PCR using the SYBR Premix Ex Taq (Takara, Shiga, Japan). Amplification reactions were performed in a 20 μl final volume and were done for 40 cycles (15 s at 95 °C, 20 s at 55 °C, and 30 s at 70 °C). The following primers were used in this study: HSPA1B (sense, 5′-TGGACTGTTGGGACTCAAGGAC-3′ and antisense, 5′-GGAACGAAACACCCTTACAGTATCA-3′), RAD51C (sense, 5′-CCTGTCTCTTCGTACTCGGTT -3′ and antisense, 5′-TCCCAATGAAAGATTAGCCGTA-3′), TIA1 (sense, 5′-CATTTGTTCGGTTCAATTCCC-3′ and antisense, 5′-GTTGTGCATTTCCATACCACT-3′), JUN (sense, 5′-CGCAGCCCAAACTAACCTC-3′ and antisense, 5′-GTAGCCATAAGGTCCGCTCT-3′), and HSP90B1 (sense, 5′-ATTAATCCCAGACACCCGCTGA-3′ and antisense, 5′-ATACCCTGACCGAAGCGTTG-3′). To normalize the amount of expressed mRNAs, the internal housekeeping gene GAPDH (sense, 5′-AGAAGGCTGGGGCTCATTTG-3′ and antisense, 5′-AGGGGCCATCCACAGTCTTC-3′) was used. Comparative CT method for relative quantification was used to calculate data, which describes the expressions of the target gene in the tested samples relative to a calibrator sample from the control group and provides accurate comparison among each sample.
Cell culture and transfection
HEK 293T cell lines were cultured in DMEM supplemented with 10% fetal bovine serum, 1% penicillin, and streptomycin in 5% CO2 at 37 °C. The cells were transfected with the indicated amounts of miR-15a mimics, inhibitor, or a negative control (RIBOBIO, Guangzhou, Guangdong, China) using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions.
Western blot
The cells were lysed in RIPA buffer (Sigma–Aldrich) supplemented with Protease Inhibitor Cocktail I (Millipore, Billerica, MA, USA). Protein concentration was measured using the BCA Protein Assay Kit (Pierce, Rockford, MA, USA). The cells lysates (20 μg) were electrophoresed on 10% polyacrylamide gels and then transferred to PVDF membrane (Millipore). The membranes were blocked with 5% skimmed milk and then incubated with the primary antibodies, followed by anti-mouse or anti-rabbit IgG HRP-conjugated secondary antibodies (Santa Cruz Biotechnology), and were visualized with Immobilon Western (Millipore). The abundance of reaction bands was determined using the Quantity One Software (Bio-Rad, Hercules, CA, USA). The intensity of the test proteins was normalized to that of β-actin, which was used as an internal control. The primary antibodies used were anti-HSPA1 antibody and anti-β-actin (Santa Cruz Biotechnology) antibody.
Luciferase reporter assay
For the production of a reporter construct, a 360-nucleotide-long 3′-UTR of human HSPA1B was amplified using the primers: sense, 5′-CCGCTCGAGGGGCCTTTGTTCTTTAGTA-3′ and antisense, 5′-ATTTGCGGCCGCTTTCATATTTAAATAAACAAATTA-3′. The PCR fragment was digested with XhoI and NotI and then cloned into psiCHECK-2 luciferase vector (Promega). As for WT, two short fragments of HSPA1B 3′-UTR which contains the potential binding sites for miR-15a were chemically synthesized. Then, we manually changed the potential binding sites by exchanging the G and T, A and C as the mutant type. These four short fragments were all cloned into psiCHECKTM-2 luciferase vector respectively. The constructs were verified by sequencing. Then the luciferase reporter constructs, together with miR-15a mimics or a negative control, were transfected into HEK 293T cells. Luciferase activity at 48 h after transfection was detected using the dual luciferase assay system (Promega) according to the manufacturer's instructions. The Renilla luciferase activity was normalized to the firefly luciferase activity. The experiments were performed in duplicate and repeated at least three times.
Statistical analysis
Statistical significance was determined using Student's t-test or Mann–Whitney rank sum test, depending on whether the data was accepted by normal distribution with Kolmogorov–Smirnov test. Spearman's correlation was used to measure the association between miR-15a and HSPA1B. P<0.05 was considered statistically significant.
Results
Semen analysis
Table 1 shows the baseline sperm characteristics of varicocele group and control group. No differences were noted in the mean age, volume, or pH between the two groups. However, the sperm count, motility, and vitality of varicocele group were significantly decreased compared with the control, indicating that patients with varicocele have impaired spermatogenesis and poor semen quality. The spermatozoa were then purified by centrifugation through discontinuous density gradient for RNA extraction and qRT-PCR.
Patients and semen characteristics.
| Characteristics | CN (n=24) | VC (n=58) | P value* |
|---|---|---|---|
| Age (years) | 30.08±3.69 | 28.81±5.94 | 0.081 |
| Volume (ml) | 3.05±0.89 | 3.20±1.43 | 0.841 |
| pH | 7.38±0.20 | 7.38±0.21 | 0.959 |
| Count (106/ml) | 131.77±76.49 | 68.81±56.34 | 0.001 |
| Percentage of motility (grades a+b)* | 61.77±9.65 | 35.67±20.50 | 0.001 |
| Percentage of vitality | 80.53±8.28 | 50.69±22.45 | 0.001 |
CN, control group; VC, varicocele group. *P value was calculated using Student's t-test.
Expressions of stress-related miRNAs in the ejaculated spermatozoa
As hyperthermia and oxidative stresses are the two major mechanisms for the varicocele-related spermatogenesis damage, we first selected five miRNAs (namely miR-15a, miR-16, miR-18a, miR-192, and miR-320) which were reported to be implicated in hyperthermia or oxidative cellular stresses (Li et al. 2009, Ren et al. 2009, Wilmink et al. 2010). The expressions of these miRNAs in the ejaculated spermatozoa of ten pairs of patients with varicocele and the healthy donors were investigated by qRT-PCR. The results showed no significant difference in the expressions of miR-16, miR-18a, miR-192, or miR-320. However, the expression of miR-15a in the ejaculated sperm revealed significant decrease in patients with varicocele compared with healthy donors (Fig. 1A). The downregulation of miR-15a in varicocele was further verified in larger study group (Fig. 1B). Therefore, we focused on miR-15a in the following study.
Expressions of miRNAs in the ejaculated spermatozoa. (A) QRT-PCR analysis of the expressions of five miRNAs in the ejaculated spermatozoa of varicocele group or the healthy control. (B) The expression of miR-15a in the ejaculated spermatozoa was significantly decreased in the varicocele group. The expressions of miRNAs in the tested samples were normalized by a calibrator sample from the control group. The horizontal dashed line shows the mean value. *P<0.05.
Citation: REPRODUCTION 147, 5; 10.1530/REP-13-0656
Regulation of HSPA1B by miR-15a
Previous study has identified numerous target genes of miR-15a by microarray (Calin et al. 2008). Among these genes, we selected five targets reported to be involved in hyperthermia or oxidative cellular stress pathways, namely HSPA1B, HSP90B1, RAD51C, TIA1, and JUN (Maki et al. 1990, Kyriakis et al. 1994, Kedersha et al. 2000, Qian et al. 2006, Sage et al. 2010). Moreover, all of these five genes have the putative binding sites of miR-15a on the 3′-UTR. After miR-15a mimics or inhibitor transfection in human HEK 293T cells, qRT-PCR and western blot were carried out. As shown in Fig. 2A and B, only the expression of HSPA1B was significantly decreased when transfected with miR-15a mimics, and was significantly increased when transfected with miR-15a inhibitor. These results showed that the expression of HSPA1B was regulated by miR-15a.
miR-15a-regulated HSPA1B expression. (A) Analysis of the expressions of five putative target genes after miR-15a mimics or inhibitor transfection by qRT-PCR. Human HEK 293T cells were transfected with miR-15a mimics or inhibitor. The concentration of miR-15a mimics and inhibitor was 50 and 100 nM respectively. (B) miR-15a-regulated HSPA1B expression in protein level. The right panel shows the ratios of HSPA1B:β-actin protein that were quantified by Quantity One Software. Each experiment was performed three times. *P<0.05. Error bars indicate s.d.
Citation: REPRODUCTION 147, 5; 10.1530/REP-13-0656
miR-15a directly binds to the 3′-UTR of HSPA1B
As illustrated in Fig. 3A, alignment of human HSPA1B with miR-15a revealed two putative target sites at positions 45–66 and 83–104 of the HSPA1B 3′-UTR and with an exact match in the seed region at positions 59–65 and 97–103 of the HSPA1B 3′-UTR (Fig. 3A). In order to investigate whether HSPA1B is a target gene of miR-15a, we generated a reporter construct containing the whole 360 nucleotides 3′-UTR of HSPA1B downstream of the luciferase gene (Fig. 3B). The luciferase reporter construct was co-transfected with 50 nM miR-15a mimics or a negative control into HEK 293T cells. The result demonstrated that the luciferase activity was inhibited by miR-15a (Fig. 3C). To further define the miRNA-binding site, two short fragments of HSPA1B 3′-UTR were cloned into the luciferase vector, namely seed1 and seed2 respectively. As shown in Fig. 3D and E, the luciferase activity was significantly inhibited by miR-15a in both seed1 and seed2. Seed1 had the greater inhibitory effect than seed2. Then, we manually changed the potential binding sites by exchanging the G and T, A and C, which abolished the inhibitory effect of miR-15a on the luciferase activity (Fig. 3D and E). Given these results, we confirmed HSPA1B as a target gene of miR-15a and identified two binding sites of miR-15a in the 3′-UTR of HSPA1B mRNA.
Identification of the miR-15a binding sites. (A) Putative duplex formation of miR-15a and HSPA1B 3′-UTR. There are two putative bindings sites on HSPA1B 3′-UTR. (B) Schematic diagram of luciferase reporter construct. (C) HEK 293T cells were transfected with the reporter together with 50 nM of miR-15a mimics or a negative control and the luciferase reporter assays were performed. (D and E) Two short fragments of HSPA1B 3′-UTR were cloned into the luciferase vector, namely seed1 and seed2 respectively. WT sequence containing the putative binding site of miR-15a in bold and underlined. The mutant type (MT) sequence has the altered nucleotides of the binding site. Samples were assayed as in (C). Each experiment was performed three times. *P<0.05. Error bars indicate s.d.
Citation: REPRODUCTION 147, 5; 10.1530/REP-13-0656
Expression of HSPA1B in the ejaculated spermatozoa
The data of qRT-PCR showed that the expression of HSPA1B mRNA was significantly increased in the sperm of patients with varicocele compared with the control (Fig. 4A). We then collected another ten pairs of sperm samples to perform western blot. As shown in Fig. 4B, the HSPA1B protein was increased in the sperm of patients with varicocele compared with the healthy control. Moreover, the result of Spearman's correlation analysis showed that the expressions of miR-15a and HSPA1B mRNA were significantly correlated. The scatter plot of miR-15a and HSPA1B mRNA expressions was shown in Fig. 4C.
Expression of HSPA1B in the ejaculated spermatozoa. (A) Analysis of the expression of HSPA1B mRNA in the ejaculated spermatozoa of varicocele group or the healthy control by qRT-PCR. The expression of HSPA1B mRNA in the tested samples was normalized by a calibrator sample from the control group. (B) Analysis of the expression of HSPA1B protein in the ejaculated spermatozoa of varicocele group or the healthy control by western blot. Another 20 samples of control group (C1–C10) and varicocele group (V1–V10) were collected and western blotting was carried out. Representative images of ten samples (C1–C5 and V1–V5) are shown in the upper panel. The lower panel shows the ratios of HSPA1B:β-actin protein that were quantified by Quantity One Software. (C) Inverse correlation of miR-15a and HSPA1B mRNA in sperm. The scatter plot of the expressions of miR-15a and HSPA1B mRNA was shown. The correlation coefficient was −0.545. The expression of miR-15a is listed on the x-axis, and the expression of HSPA1B mRNA is listed on y-axis. *P<0.05. Error bars indicate s.d.
Citation: REPRODUCTION 147, 5; 10.1530/REP-13-0656
Discussion
It is well accepted that varicocele can impair spermatogenesis and result in infertility through the induction of hyperthermia and oxidative stresses (Naughton et al. 2001). However, the mechanisms of the stress responses in testis remain to be elucidated. Recently, growing evidence has suggested that stress conditions can alter the biogenesis of miRNAs, the expressions of target mRNAs, and the activities of miRNA–protein complexes (Leung & Sharp 2010). In turn, several lines of genetic evidence demonstrate that miRNAs play critical roles in the mediation of stress responses (Leung & Sharp 2007). But whether miRNAs are implicated in varicocele-related stress environment remains unknown. In this study, we analyzed the expressions of five stress-related miRNAs in the sperm and found that the expression of miR-15a was significantly downregulated in patients with varicocele compared with the control. Furthermore, miR-15a repressed the expression of HSPA1B, which is a typical stress-induced chaperone protein, through directly binding its 3′-UTR. These data show that miRNAs are involved in the regulation of the cellular stress responses in the sperm, and extend our current understanding of varicocele-related infertility.
Since first discovered in 1993, miRNAs have been implicated in various human regulatory pathways, including cell growth, proliferation, apoptosis, and differentiation and therefore participate in the process of development and diseases (He et al. 2009). It has been estimated that miRNAs may regulate up to 30% of all genes in the human genome (Lewis et al. 2005). The miR-15a/16-1 cluster is located in the host gene DLEU2 at chromosome 13q14.3, a genomic region frequently deleted in B-cell chronic lymphocytic leukemia, myeloma, and mantle cell lymphoma (Lerner et al. 2009). The cluster is highly conserved among mammalian species (Yue & Tigyi 2010), and these two miRNAs of the cluster are cotranscribed and downregulated in more than two-thirds of chronic lymphocytic leukemia (CLL) patients (Calin et al. 2002, Cimmino et al. 2005). Recently, several studies have demonstrated that miR-15a was involved in the regulation of stress responses. The expression of miR-15a was significantly downregulated in human cells after oxidative stress (Li et al. 2009). The members of miR-15 family act as mediators of stress signaling pathways that regulate cardiomyocyte proliferation and survival in response to stresses (Mendell & Olson 2012). Inhibition of miR-15 family members provides protection against cardiomyocyte apoptosis after myocardial infarction in rodents (Hullinger et al. 2012). Therefore, the decreased expression of miR-15a in varicocele may protect the sperm from hyperthermia or oxidative stress damage.
In order to find the putative mechanisms, we chose five stress-related genes and only HSPA1B was identified as a direct target of miR-15a. The heat-shock proteins (HSPs), which are regulated by heat-shock factors (HSFs), could stabilize proteins against aggregation and mediate the correct folding of nascent proteins so that the proteins could maintain the precise three-dimensional conformation (Akerfelt et al. 2010). Their expressions are increased when cells are exposed to elevated temperatures or other stresses and thus are crucial for regulation of cellular homeostasis (Tavaria et al. 1996, Katschinski 2004). HSPA1B belongs to HSP70 family and it is one of the most extensively studied heat inducible HSPs (Kampinga et al. 2009, Stetler et al. 2010, Scieglinska et al. 2011). Recent studies have demonstrated that HSF1, HSF2, SP1, and RNA polymerase II are bound to the Hspa1b promoter in the mature spermatozoa of mice, allowing the rapid expression of this cytoprotective gene if the cells encounter stress (Xing et al. 2005, Wilkerson et al. 2008, Wilkerson & Sarge 2009). In this study, we elucidate the novel post-transcriptional regulation of HSPA1B by miR-15a, extend our knowledge of stress-induced heat-shock response pathway.
Previous studies have shown that HSP70, HSP90, HSPA4, HSF1, and HSF2 were upregulated in the sperm of men with varicocele (Ferlin et al. 2010, Chan et al. 2013). Results of our present qRT-PCR analysis are consistent with these studies. HSPA1B plays a crucial role in the promotion of cell survival through refolding of damaged and unfolded proteins generated by stressful stimuli (Jäättelä 1999, Akerfelt et al. 2007) and protects cells against DNA damage (Kotoglou et al. 2009). Moreover, HSPA1B is involved in lightening the damage of hyperthermia on spermatogenesis and post-ejaculatory function of sperm (Meyerhoeffer et al. 1985). It can protect cells from various stresses by inhibiting stress-induced apoptosis. The mechanisms for the anti-apoptosis effect of HSPA1B include inhibiting cytochrome c release from the mitochondria (Li et al. 2000), inhibiting translocation of the Bcl-2 family member Bax (Stankiewicz et al. 2005), preventing the activation of caspase-3 through modulation of the apoptosome (Matsumori et al. 2006), and being the effector of the antiapoptotic Akt/PKB kinase (Barati et al. 2006). Thus, the upregulation of HSPA1B observed in the sperm of varicocele men may be one of the mechanisms that contribute to the protection of hyperthermia or oxidative-induced sperm damage.
Apart from the stress pathway, miR-15a and HSPs are also involved in germ cells development due to their roles in the early stages of spermatogenesis. By targeting cyclin T2, miR-15a had a negative effect on the early steps of spermatogenesis (Teng et al. 2011). Specifically, HSPs are critically important during spermatogenesis and the post-testicular maturation of mammalian spermatozoa that aberrant expression is associated with spermatogenesis arrested and defects in sperm function (Dun et al. 2012). The stage-specific expression pattern displayed by HSPs during spermatogenesis indicates that HSPs are involved in germ cell development (Feng et al. 2001, Huo et al. 2004, Asquith et al. 2005, Terada et al. 2005, Held et al. 2006, 2011, Adly et al. 2008, Walsh et al. 2008, Lachance et al. 2010, Ji et al. 2012). In addition, HSPA1B has been implicated in fertilization and embryo development (Neuer et al. 2000, Matwee et al. 2001). It can protect embryos from hyperthermia-induced congenital defects, possibly through reducing hyperthermia-induced apoptosis (Barrier et al. 2009). Taken together, the regulation of HSPA1B by miR-15a might play a vital protective role against cellular stresses throughout the processes of spermatozoa maturation, fertilization, and embryo development.
In conclusion, we revealed the altered levels of miR-15a and HSPA1B in the spermatozoa of varicocele patients and described miR-15a-mediated stress regulation of HSPA1B in sperm. Our study may help to better understand the mechanisms involved in varicocele-related sperm damage, and to provide a potential therapeutic target and novel biomarkers for male infertility.
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 work was supported by the National Key Scientific Program of China (grant number 2011CB944303), the National Natural Science Foundation of China (grant numbers 31271244 and 81200465), and Shenzhen Foundation of Science and Technology (grant numbers CXB201104220045A, 201202001, and 201302053).
Acknowledgements
The authors thank the patients and the family members for their cooperation during the study.
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(Z Ji and R Lu contributed equally to this work)
