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ZJUS-B
Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology)  2016, Vol. 17 Issue (1): 21-29    DOI: 10.1631/jzus.B1500174
Article     
Changes of testicular phosphorylated proteins in response to restraint stress in male rats
Supatcharee Arun1,Jaturon Burawat1,Wannisa Sukhorum1,Apichakan Sampannang1,Nongnut Uabundit1,Sitthichai Iamsaard1,2,?()
Department of Anatomy, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand
Center for Research and Development of Herbal Health Products, Faculty of Pharmaceutical Sciences, Khon Kaen University, Khon Kaen 40002, Thailand
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Abstract  

Objective: To investigate male reproductive parameters via changes of potential testicular protein markers in restraint-stress rats. Methods: Male Sprague-Dawley rats were divided into two groups (non-immobilized control and restraint-immobilized/stress groups, n=8 each group). The stress animals were immobilized (12 h/d) by a restraint cage for 7 consecutive days. All reproductive parameters, morphology and histology were observed and compared between groups. In addition, the expression of steroidogenic acute regulatory (StAR) and phosphotyrosine proteins (previously localized in Sertoli and late spermatid cells) in testicular lysate was assayed by immuno-Western blotting. Results: Testosterone level, sperm concentration and sperm head normality of stress rats were significantly decreased while the corticosterone level was increased as compared with the control (P<0.05). Histologically, stress rats showed low sperm mass in epididymal lumen and some atrophy of seminiferous tubules. Although the expression of testicular StAR protein was not significantly different between groups, changed patterns of the 131, 95, and 75 kDa testicular phosphorylated proteins were observed in the stress group compared with the control group. The intensity of a testicular 95-kDa phosphorylated protein was significantly decreased in stress rats. Conclusions: This study has demonstrated the alteration of testicular phosphorylated protein patterns, associated with adverse male reproductive parameters in stress rats. It could be an explanation of some infertility in stress males.

Key words： Restraint-stress rats      Steroidogenic acute regulatory (StAR) protein      Testicular phosphorylated protein     
Received: 16 July 2015      Published: 01 January 2016
Fund:  Project supported by the Postgraduate Study Support Grant and Invitation Research Grant, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand(IN59134)
Corresponding Authors: Sitthichai Iamsaard     E-mail: sittia@kku.ac.th
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Supatcharee Arun
Jaturon Burawat
Wannisa Sukhorum
Apichakan Sampannang
Nongnut Uabundit
Sitthichai Iamsaard
Cite this article:

Supatcharee Arun,Jaturon Burawat,Wannisa Sukhorum,Apichakan Sampannang,Nongnut Uabundit,Sitthichai Iamsaard. Changes of testicular phosphorylated proteins in response to restraint stress in male rats. Journal of Zhejiang University-SCIENCE B (Biomedicine & Biotechnology), 2016, 17(1): 21-29.

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http://www.zjujournals.com/xueshu/zjus-b/10.1631/jzus.B1500174     OR     http://www.zjujournals.com/xueshu/zjus-b/Y2016/V17/I1/21

Fig. 1 Body weights (a) and comparative morphologies of testis, penis, epididymis plus vas deferens, and seminal vesicle (b) between the control and restraint-stress groups Each data point in (a) is represented as mean±SD (n=8 rats each group). * P<0.05, vs. restraint-stress group
Fig. 2 Photographs showing sperm head morphologies observed in the control and restraint-stress groups Normal sperm head with normal hook (arrow) (a); abnormal sperm heads without hook (arrow head) (b), pinhead (c), and crooked neck (d)
Group Absolute weight (g)
 Relative weight (g/100 g)
 c s (106 cells/ml) Sperm head abnormality (%) c c (ng/ml) c t (ng/ml)
Testis Epididymis plus vas deferens Seminal vesicle Testis Epididymis plus vas deferens Seminal vesicle
Control 1.60±0.09 0.40±0.01 0.64±0.04 0.54±0.04 0.14±0.01 0.22±0.01 167.00±2.53 1.38±0.86 389.00±23.00 1.08±0.46
RS 1.26±0.15* 0.38±0.01 0.63±0.07 0.49±0.06* 0.15±0.00 0.25±0.03 69.50±8.98* 2.75±0.66* 506.20±57.47* 0.47±0.26*
Table 1 Effect of stress on the weights of male reproductive organs, sperm concentration, sperm head abnormality, and corticosterone levels in rats
Fig. 3 Photomicrographs showing histologies of testis (a, b), caudal epididymis (c, d), and penis (e, f) stained by H&E and penile section stained by Masson’s trichrome (g, h) of the control and restraint-stress groups, respectively Asterisks: atrophic seminiferous tubules; S: sheath of tunica albugenia; Cv: corpora cavernosa; Cs: corpus spinosum; U: urethra
Fig. 4 Representative Western-blot analysis of StAR protein expression in testicular lysates (a), relative intensity of testicular StAR protein (b), and ratio of StAR/β-actin (c) in the control and restraint-stress groups StAR lysate was used as a positive control. Data are represented as mean±SD (n=4 rats each group)
Fig. 5 Representative Western-blot analysis of tyrosine protein phosphorylation in testicular lysates (a), protein profiles (SDS-PAGE) (b), and relative intensity of the testicular phosphorylated 95-kDa protein (c) in the control and restraint-stress (RS) groups Bovine serum albumin (BSA) and epidermal growth factor (EGF)-like growth factors were used as negative and positive controls, respectively. Data were represented as mean±SD (n=4 rats each group). * P<0.05, compared with the control group
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