World J Urol DOI 10.1007/s00345-015-1516-5
ORIGINAL ARTICLE
Low‑expressed testis‑specific calcium‑binding protein CBP86‑IV (CABYR) is observed in idiopathic asthenozoospermia Shulin Shen · Jinzi Wang · Jihong Liang · Chunhui Zhu
Received: 20 October 2014 / Accepted: 11 February 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Objectives To investigate the expression level of testisspecific calcium-binding protein CBP86-IV in normal and asthenozoospermic human sperm. Methods The total RNA was extracted from human sperm, and target cDNA was obtained by reverse transcription–polymerase chain reaction. Then the cDNA was used for quantitative PCR analysis and cloned into the prokaryotic expression vector pET-28a, respectively. The fusion protein was induced and expressed as inclusion body which was used to produce the polyclonal antibody against TSCBP86-IV. The protein expression level of TSCBP86-IV from normal human sperm and idiopathic asthenozoospermic samples was detected by the purified antibody. Results The experimental results showed that the protein expression of TSCBP86-IV was reduced in idiopathic asthenozoospermia and consistent with the transcriptional changing tendency which was detected by quantitative PCR analysis. Conclusions The stable and reliable change of TSCBP86IV may be taken as a new molecular marker for clinical diagnosis of idiopathic asthenozoospermia. Keywords Idiopathic asthenozoospermia · Molecular marker · Testis-specific calcium-binding protein CBP86-IV S. Shen (*) · J. Liang (*) · C. Zhu Department of Andrology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China e-mail: [email protected] J. Liang e-mail: [email protected] J. Wang Agricultural College, State Key Laboratory for Conservation and Utilization of Subtropical Agro‑bioresources, Guangxi University, Nanning 530004, China
Introduction Testis-specific calcium-binding protein CBP86 (TSCBP86) is a member of calcium-binding protein (CBP) family which could be found in all eukaryotic organisms and plays an important role in calcium activation and modulation. A large number of CBPs have EF-hands structural domain which consists of two alpha helices and joins as a calcium-binding loop. CBPs regulate concentration of calcium ions to carry out cellular functions through transmembrane transport and signal transduction [1]. The intracellular CBPs with calcium-binding domain could be generally classified into parvalbumin, calmodulin, S100 proteins, and calcineurin [2]. Meanwhile, the extracellular and membrane-binding CBPs were also found to perform important functions such as G-protein coupled receptors which modulate extracellular calcium signal transduction and protein kinase C with intracellular calcium store function [3, 4]. In recent years, abnormally expressed CBPs were taken as potential molecular targets in several human diseases for the function of cellular signaling as voltage-gated calcium channels [5, 6]. The localization and processing forms of some CBPs were also essential for physiological functions. For example, calmyrin is present in human forebrain and observed to be different distribution in Alzheimer’s as compared with normal aging brains [7]. TSCBP86 was also known as calcium-binding tyrosine phosphorylation-regulated protein (CABYR) in mammals which originally isolated from human spermatozoa. Several isoforms of this protein have been shown to exist at protein level. An acidic 86-kDa isoform of TSCBP86 was found to be increased during capacitation and lose the function of calcium binding by dephosphorylation, which proves the regulation of cAMP/ PKA pathway on sperm protein tyrosine phosphorylation and capacitation [8]. Recently,
13
Table 1 Comparison with semen parameters both patients and controls
Data from thirty-five asthenozoospermic patients and thirty-five healthy donors * a: rapid progressive, b: Slow or sluggish, c: non-progressive %
World J Urol
Asthenozoospermic patients Age of patients (year) Semen volume (mL) Sperm count (×106 sperm/mL) Motility a + b + c* Control Age of patients (year) Semen volume (mL) Sperm count (×106 sperm/mL) Motility a + b + c*
TSCBP86 isoforms were also found in other human tissue and various cancer cells [9, 10]. Because of high similarity of TSCBP86 isoforms, the detection results of Western blot in human sperm samples were very consistent but discriminating when used in diverse cancer cells [10]. Calcium ion is considered as a key regulator for normal functions of human sperm. It displays dual character when modulating sperm motility. Calcium was essential for sperm maturation. But on the other hand, it suppressed sperm movement in ejaculated semen. The changed calcium content in the process of sperm maturation coupled with binding proteins and inhibitors [11]. TSCBP86 has been studied for a long time, and several protein isoforms were found with different functions and interaction relationship [12–14]. In this study, the expression level of TSCBP86-IV between normal motility human sperm and idiopathic asthenozoospermic samples was detected by Western blot and qPCR. Combined with previous studies, the experimental results implied a new molecular marker for clinical diagnosis of asthenozoospermia.
Mean
Standard deviation Maximum
Minimum
33.5 2.6 85.6 44.2
5.4 0.9 42.3 16.1
46 6 158.2 56
19 2 20.4 14.7
31.4 2.7 94.6
4.3 1.0 49.5
42 5.8 171.3
21 2 21.4
84.2
8.5
96.3
71.8
both donors and asthenozoospermic samples was more than 30 %. All donors provided informed consent for participation in the current study in accordance with hospital policy and applicable ethical guidelines. The data concerning the number of patients and complete ejaculate analysis are listed in Table 1 according to MartinezHeredia et al. [16]. Construction of the prokaryotic expression vector of TSCBP86‑IV The total RNA and cDNA of human sperm were obtained according to a previous report [17]. Primers containing cleavage sites for the restriction enzyme EcoRI (5′-gcgggatccatgatttcttcaaagccc-3′) and SalI (5′-gcagaaactgaaaactgaaagcttgcg-3′) were used to amplify the ORF sequence of TSCBP86-IV gene (GI = 10,444,393). The PCR product was digested with restriction enzymes and cloned into the expression vector pET28a and sequenced (Fig. 1). Expression of TSCBP86‑IV fusion protein
Materials and methods Sperm sample collection Patients and donors were recruited for this study at the Department of Andrology of the First Affiliated Hospital of Guangxi Medical University. A computer-assisted semen analyzer (WLJY-9000, Beijing Weili New Century Science & Tech. Deve. Co. Ltd) was used to analyze semen samples. The collected samples were screened according to the published World Health Organization (WHO) guidelines [15]. Samples with progressive sperm motility
ORIGINAL ARTICLE
Low‑expressed testis‑specific calcium‑binding protein CBP86‑IV (CABYR) is observed in idiopathic asthenozoospermia Shulin Shen · Jinzi Wang · Jihong Liang · Chunhui Zhu
Received: 20 October 2014 / Accepted: 11 February 2015 © Springer-Verlag Berlin Heidelberg 2015
Abstract Objectives To investigate the expression level of testisspecific calcium-binding protein CBP86-IV in normal and asthenozoospermic human sperm. Methods The total RNA was extracted from human sperm, and target cDNA was obtained by reverse transcription–polymerase chain reaction. Then the cDNA was used for quantitative PCR analysis and cloned into the prokaryotic expression vector pET-28a, respectively. The fusion protein was induced and expressed as inclusion body which was used to produce the polyclonal antibody against TSCBP86-IV. The protein expression level of TSCBP86-IV from normal human sperm and idiopathic asthenozoospermic samples was detected by the purified antibody. Results The experimental results showed that the protein expression of TSCBP86-IV was reduced in idiopathic asthenozoospermia and consistent with the transcriptional changing tendency which was detected by quantitative PCR analysis. Conclusions The stable and reliable change of TSCBP86IV may be taken as a new molecular marker for clinical diagnosis of idiopathic asthenozoospermia. Keywords Idiopathic asthenozoospermia · Molecular marker · Testis-specific calcium-binding protein CBP86-IV S. Shen (*) · J. Liang (*) · C. Zhu Department of Andrology, The First Affiliated Hospital of Guangxi Medical University, Nanning 530021, China e-mail: [email protected] J. Liang e-mail: [email protected] J. Wang Agricultural College, State Key Laboratory for Conservation and Utilization of Subtropical Agro‑bioresources, Guangxi University, Nanning 530004, China
Introduction Testis-specific calcium-binding protein CBP86 (TSCBP86) is a member of calcium-binding protein (CBP) family which could be found in all eukaryotic organisms and plays an important role in calcium activation and modulation. A large number of CBPs have EF-hands structural domain which consists of two alpha helices and joins as a calcium-binding loop. CBPs regulate concentration of calcium ions to carry out cellular functions through transmembrane transport and signal transduction [1]. The intracellular CBPs with calcium-binding domain could be generally classified into parvalbumin, calmodulin, S100 proteins, and calcineurin [2]. Meanwhile, the extracellular and membrane-binding CBPs were also found to perform important functions such as G-protein coupled receptors which modulate extracellular calcium signal transduction and protein kinase C with intracellular calcium store function [3, 4]. In recent years, abnormally expressed CBPs were taken as potential molecular targets in several human diseases for the function of cellular signaling as voltage-gated calcium channels [5, 6]. The localization and processing forms of some CBPs were also essential for physiological functions. For example, calmyrin is present in human forebrain and observed to be different distribution in Alzheimer’s as compared with normal aging brains [7]. TSCBP86 was also known as calcium-binding tyrosine phosphorylation-regulated protein (CABYR) in mammals which originally isolated from human spermatozoa. Several isoforms of this protein have been shown to exist at protein level. An acidic 86-kDa isoform of TSCBP86 was found to be increased during capacitation and lose the function of calcium binding by dephosphorylation, which proves the regulation of cAMP/ PKA pathway on sperm protein tyrosine phosphorylation and capacitation [8]. Recently,
13
Table 1 Comparison with semen parameters both patients and controls
Data from thirty-five asthenozoospermic patients and thirty-five healthy donors * a: rapid progressive, b: Slow or sluggish, c: non-progressive %
World J Urol
Asthenozoospermic patients Age of patients (year) Semen volume (mL) Sperm count (×106 sperm/mL) Motility a + b + c* Control Age of patients (year) Semen volume (mL) Sperm count (×106 sperm/mL) Motility a + b + c*
TSCBP86 isoforms were also found in other human tissue and various cancer cells [9, 10]. Because of high similarity of TSCBP86 isoforms, the detection results of Western blot in human sperm samples were very consistent but discriminating when used in diverse cancer cells [10]. Calcium ion is considered as a key regulator for normal functions of human sperm. It displays dual character when modulating sperm motility. Calcium was essential for sperm maturation. But on the other hand, it suppressed sperm movement in ejaculated semen. The changed calcium content in the process of sperm maturation coupled with binding proteins and inhibitors [11]. TSCBP86 has been studied for a long time, and several protein isoforms were found with different functions and interaction relationship [12–14]. In this study, the expression level of TSCBP86-IV between normal motility human sperm and idiopathic asthenozoospermic samples was detected by Western blot and qPCR. Combined with previous studies, the experimental results implied a new molecular marker for clinical diagnosis of asthenozoospermia.
Mean
Standard deviation Maximum
Minimum
33.5 2.6 85.6 44.2
5.4 0.9 42.3 16.1
46 6 158.2 56
19 2 20.4 14.7
31.4 2.7 94.6
4.3 1.0 49.5
42 5.8 171.3
21 2 21.4
84.2
8.5
96.3
71.8
both donors and asthenozoospermic samples was more than 30 %. All donors provided informed consent for participation in the current study in accordance with hospital policy and applicable ethical guidelines. The data concerning the number of patients and complete ejaculate analysis are listed in Table 1 according to MartinezHeredia et al. [16]. Construction of the prokaryotic expression vector of TSCBP86‑IV The total RNA and cDNA of human sperm were obtained according to a previous report [17]. Primers containing cleavage sites for the restriction enzyme EcoRI (5′-gcgggatccatgatttcttcaaagccc-3′) and SalI (5′-gcagaaactgaaaactgaaagcttgcg-3′) were used to amplify the ORF sequence of TSCBP86-IV gene (GI = 10,444,393). The PCR product was digested with restriction enzymes and cloned into the expression vector pET28a and sequenced (Fig. 1). Expression of TSCBP86‑IV fusion protein
Materials and methods Sperm sample collection Patients and donors were recruited for this study at the Department of Andrology of the First Affiliated Hospital of Guangxi Medical University. A computer-assisted semen analyzer (WLJY-9000, Beijing Weili New Century Science & Tech. Deve. Co. Ltd) was used to analyze semen samples. The collected samples were screened according to the published World Health Organization (WHO) guidelines [15]. Samples with progressive sperm motility
