Turkish Journal of Medical Sciences Turk J Med Sci (2015) 45: 1285-1291 http://journals.tubitak.gov.tr/medical/ © TÜBİTAK Research Article doi:10.3906/sag-1406-27 miRNA-mediated regulation of heat shock proteins in human ejaculated spermatozoa 1, 1 1 1 2 Tang WENHAO *, Liu DE-FENG , Hong KAI , Zhao LIAN-MING , Mao JIA-MING , 2, 1 1, Zhuang XIN-JIE **, Ma LU-LIN , Jiang HUI ** 1 Department of Urology, Third Hospital of Peking University, Beijing, P.R. China 2 Center of Reproductive Medicine, Third Hospital of Peking University, Beijing, P.R. China Received: 07.06.2014 Accepted/Published Online: 11.09.2014 Printed: 31.12.2015 Background/aim: Heat shock proteins (HSPs) are expressed in human spermatozoa and play a role in sperm function. MicroRNAs (miRNAs) regulate gene expression, and the possible involvement of miRNAs in the regulation of HSP gene expression in sperm was investigated in this study. Materials and methods: miRNAs differentially expressed in 8 copies of an oligoasthenozoospermic semen group (OA) were identified by comparison with a normal male semen control group (NC) using microarray technology. Potential targets of HSP proteins among the differentially expressed miRNAs were further investigated. Results: HSP40, HSP60, HSP70, and HSP90 were all found to be expressed in human ejaculated spermatozoa. A total of 32 miRNAs showed significant differences in expression between the OA and NC groups. Ten of these miRNAs encoded potential targets of HSPs. Conclusion: These results show that specific miRNAs are expressed in human ejaculated spermatozoa. These miRNAs appear to be involved in regulating the expression of HSP40, HSP70, and HSP90, and this in turn affects sperm function. Key words: Heat-shock proteins, microRNAs, microarray, spermatozoa, infertility 1. Introduction can lead to defensive system disorder, which eventually Since the identification of the first protein in human sperm, leads to a decrease in sperm concentration and abnormal researchers have investigated sperm protein function. morphology. However, the regulatory roles of HSPs in Sperm proteins are essential for important functions, human sperm are still poorly understood. including the regulation of sperm concentration, sperm In recent years, it was found that microRNAs (miRNAs) motility, and aspects of fertilization such as the fusion of participate in the regulation of male gametes (10,11). Small sperm and egg membranes. Heat shock proteins (HSPs) RNA clone library sequencing of human and mouse testes are widely expressed in the male reproductive system, showed that miRNAs are specifically and/or abundantly particularly in the testes and epididymis (1–5). HSPs have expressed (12). Genetically modified mouse models been shown to be particularly abundant on the cell surface have demonstrated the importance of miRNA pathways of mouse, rat, bull, boar, stallion, dog, cat, and human for normal spermatogenesis, and clinical studies have sperm (1–9). exploited the discovery of well-defined miRNA expression HSPs are associated with stress, and the expression profiles. Furthermore, miRNAs in human spermatozoa of these proteins is upregulated when cells are exposed and seminal plasma have been explored as potential to elevated temperatures or other stresses such as biomarkers for male factor infertility (10,12). Of the 1373 pathogens, cytokines (interleukin-1, interleukin-2, etc.) miRNAs present in the human clone library, miRNAs 449, (9), and physical and chemical factors. HSPs are present 449a, 892b, and 202 are expressed specifically in testes in virtually all living organisms from bacteria to man, (13). miRNAs regulate the expression of more than half of and are highly conserved at the structural and sequence all human protein-coding genes, and individual miRNAs level across the tree of life. Because of their crucial role can regulate multiple proteins. The proper functioning in male reproduction, the abnormal expression of HSPs of miRNAs is crucial for various physiological processes, * Correspondence: [email protected] ** These authors contributed equally to this work. 1285 WENHAO et al. / Turk J Med Sci as well as for preventing cancers and other diseases. the absorbance at 260 nm and 280 nm (A260/280 ratio) was Human sperm contains a stable population of miRNAs measured to determine RNA quality and concentration, that are potentially associated with embryogenesis and and this was checked by agarose gel electrophoresis. spermatogenesis (14,15). Recent research confirmed the 2.4. miRNA microarray analysis role of miRNAs in regulating the expression of HSPs in miRNAs were labeled using an miRNA Complete Labeling human sperm (6,8,16), and the possible mechanism of and Hyb Kit (Agilent). The RNeasy Mini Kit (QIAGEN, this regulation in human ejaculated spermatozoa was p/n 217004) was used to concentrate the sample, prior investigated in this study using a microarray approach. to dyeing, eluting, and hybridization according to the manufacturer’s instructions. The Agilent Human Catalog 2. Materials and methods Plex 8-15K miRNA microarray (AMADID 016436) with 2.1. Literature analysis one-color hybridizations was used for gene expression Using the terms ‘heat shock proteins/HSP’ and profiling. Arrays contained 470 human and 64 viral ‘spermatozoa/sperm’, the PubMed, Medline, Wanfang, miRNA probes from the Sanger database v9.1. Plates were and CNKI bibliographic databases were searched. Studies scanned with an Agilent G2565BA microarray scanner involving human semen and HSPs were included, while (p/n G2505B), and images were saved as TIF files and literature involving animal semen was excluded. This analyzed using GeneSpring GX with Microsoft Excel. study was approved by the Ethics Committee of Peking University Third Hospital (protocol study number 2.5. HSP target gene selection IRBSZ00000009-2012009). Putative miRNA gene targets were predicted using the microRNA.org (http://www.microrna.org/), Pictar (http:// 2.2. Samples With approval from the ethics committee, semen samples pictar.mdc-berlin.de/), and TargetScan 5.1 (http://www. were obtained from the Reproductive Center of Peking targetscan.org/) algorithm tools. miRNAs that were University Third Hospital. Human semen was collected by differentially expressed in human ejaculated spermatozoa masturbation and allowed to liquefy for 30 min. According were screened for HSP sequences, and only genes identified to semen analysis and fertility history, 8 pairs of samples in at least 2 different databases were included. were selected and divided into 2 groups, with half of the samples placed in the oligoasthenozoospermic group (OA) 3. Results and half in the normal control group (NC). After analysis, The semen parameters of the OA and NC groups were semen was immediately washed 3 times with phosphate- measured according to the World Health Organization buffered saline, and RNase Inhibitor (Epicentre, USA) laboratory manual (fifth edition) for the examination and was added according to the manufacturer’s instructions. processing of human semen (Table 1). HSP40, HSP60, Following storage at 4 °C overnight, the depressor was HSP70, and HSP90 were identified in human ejaculated removed and the sample was stored at –80 °C until needed. spermatozoa (Table 2). Semen parameters were measured using an SQA-V device The quantity and quality of the RNA samples in the OA (TECHNOPATH, Ballina, Ireland) according to the World group and the NC group were confirmed by agarose gel Health Organization laboratory manual (fifth edition) for electrophoresis and A260/280 absorbance ratio. A total of the examination and processing of human semen. 30 differentially expressed miRNAs were identified in the 2.3. miRNA isolation miRNA microarray, and 10 were confirmed as HSP targets Total RNA was extracted using TRIzol (Invitrogen, USA) in human semen, with 5 miRNAs downregulated and 8 according to the manufacturer’s instructions. The ratio of upregulated (Tables 3 and 4). Table 1. The semen parameters of the oligoasthenozoospermic semen group (OA) and normal male semen control group (NC). Sperm concentration Morphology Group Progressive motility (PR, %) ( ×106 cells/mL) (%) OA 6.8 ± 2.3 14.5 ± 5.0 5.3 ± 0.7 NC 31.9 ± 9.9 39.3 ± 4.6 8.9 ± 2.3 1286 WENHAO et al. / Turk J Med Sci Table 2. HSP expressed in human ejaculates. HSPs identified in previous studies References HSP60, Grp78 [10] HSP70 [11] HSP70, HSP90 [12] HSP90 [13] HSP-90, HSP-70 [14] HSP-90 [15] HSP 90-alpha,-beta, HSP-70, HSP60, HSP A2, HSPD1, ERp99 [16] HSPE1, DNAJB1, HSPD1, HSPA1A, HSPCA, HSPH1, HSPA5 [17] DnaJA1 [18] HSPH1 [19] HSP70, HSP70-2 [20] Table 3. miRNAs differentially expressed in human sperm expressing the HSP gene. miRNA names Target gene Gene name microRNA.org PicTar TargetScan 21 DNAJA2 DnaJ (Hsp40) homolog, subfamily A, member 2 ● ● 24 DNAJB12 DnaJ (Hsp40) homolog, subfamily B, member 12 ● ● 19b DNAJC16 DnaJ (Hsp40) homolog, subfamily C, member 16 ● ● DNAJA2 DnaJ (Hsp40) homolog, subfamily A, member 2 ● ● 27a DNAJC13 DnaJ (Hsp40) homolog, subfamily C, member 13 ● ● DNAJB9 DnaJ (Hsp40) homolog, subfamily B, member 9 ● ● DNAJC5B DnaJ (Hsp40) homolog, subfamily C, member 5 beta ● ● 23a DNAJC6 DnaJ (Hsp40) homolog, subfamily C, member 6 ● ● DNAJC12 DnaJ (Hsp40) homolog, subfamily C, member 12 ● ● HSPA12A Heat shock protein 70 kDa 12A ● ● HSP90B1 Heat shock protein 90kDa beta (Grp94), member 1 ● ● 449a DNAJB1 DnaJ (Hsp40) homolog, subfamily B, member 1 ● Does not exist ● 15a DNAJB4 DnaJ (Hsp40) homolog, subfamily B, member 4 ● ● 132 DNAJA2 DnaJ (Hsp40)