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Supatcharee Arun, Jaturon Burawat, Wannisa Sukhorum, Apichakan Sampannang, Chanwit Maneenin, Sitthichai Iamsaard,
Volume 14, Issue 7 (7-2016)

Background: Stress is a cause of male infertility. Although sex hormones and sperm quality have been shown to be low in stress, sperm physiology and testicular functional proteins, such as phosphotyrosine proteins, have not been documented. 
Objective: To investigate the acrosome status and alterations of testicular proteins involved in spermatogenesis and testosterone synthesis in chronic stress in rats. 
Materials and Methods: In this experimental study, male rats were divided into 2 groups (control and chronic stress (CS), n=7). CS rats were immobilized (4 hr/day) for 42 consecutive days. The blood glucose level (BGL), corticosterone, testosterone, acrosome status, and histopathology were examined. The expressions of testicular steroidogenic acute regulatory (StAR), cytochrome P450 side chain cleavage (CYP11A1), and phosphorylated proteins were analyzed. 
Results: Results showed that BGL (71.25±2.22 vs. 95.60±3.36 mg/dl), corticosterone level (24.33±4.23 vs. 36.9±2.01 ng/ml), acrosome reacted sperm (3.25±1.55 vs. 17.71±5.03%), and sperm head abnormality (3.29±0.71 vs. 6.21±1.18%) were significantly higher in CS group in comparison with control. In contrast, seminal vesicle (0.41±0.05 vs. 0.24±0.07 g/100g), testosterone level (3.37±0.79 vs. 0.61±0.29 ng/ml), and sperm concentration (115.33±7.70 vs. 79.13±3.65×106 cells/ml) of CS were significantly lower (p<0.05) than controls. Some atrophic seminiferous tubules and low sperm mass were apparent in CS rats. The expression of CYP11A1 except StAR protein was markedly decreased in CS rats. In contrast, a 55 kDa phosphorylated protein was higher in CS testes. 
Conclusion: CS decreased the expression of CYP11A, resulting in decreased testosterone, and increased acrosome-reacted sperm, assumed to be the result of an increase of 55 kDa phosphorylated protein.
Sitthichai Iamsaard, Wannisa Sukhorum, Supatcharee Arun, Nichapa Phunchago, Nongnuch Uabundit, Porntip Boonruangsri, Malivalaya Namking,
Volume 15, Issue 4 (6-2017)

Background: Valproic acid (VPA), an anti-epileptic drug, can cause male subfertility. However, the degree to which testicular and epididymal histopathologies and androgen receptor (AR) expression are changed under VPA treatment has never been reported.
Objective: To investigate the histopathological changes and AR protein levels of testis and epididymis in VPA-treated rats for every single day.
Materials and Methods: Sixty-four adult male Wistar rats were divided intocontrol and VPA-treated groups (n=8/ each). Treated rats were injected with 500mg/ kgBW, intraperitoneally, VPA for 10 consecutive days. At the end of everyexperimental day, all reproductive parameters including histology by hematoxylinand eosin staining and protein expression of AR by Immuno-Western blot in testisand epididymis were examined.
Results: VPA-treated rats showed dramatically changes in testicular and epididymal histopathologies compared to control group. The multinucleated giant cells and sloughing of germ cells were observed on day 6. The germ cell disintegration and increased intercellular spaces of seminiferous tubular epithelium appeared in days 7-10 of VPA treatment. Additionally, extensive multinucleated giant cells and complete exfoliation were clearly found from days 8-10. Such exfoliated germ cells were clearly seen in its epididymal lumen at day 10. The increasing rate of sperm concentration was approximately 32.31% of that in control group at day 10 (p=0.03). Moreover, the protein expressions of testicular and epididymal AR (% intensity/ 80 μg protein lysate) was decreased in VPA-treated rats compared with control.
Conclusion: VPA treatment induces histologic changes of germ cell epithelium in seminiferous tubules and decreases the expression of testicular and epididymal
Apichakan Sampannang, Supatcharee Arun, Jaturon Burawat, Wannisa Sukhorum, Sitthichai Iamsaard,
Volume 16, Issue 4 (April 2018)

Background: The streptozotocin (STZ)-induced diabetic model is widely used to evaluate the adverse effects of diabetes mellitus (DM) on spermatogenesis and testicular steroidogenesis. However, the actual mechanism of sub/infertility in DM males needs to be elucidated.
Objective: To conduct a detailed examination of the testicular histopathology, sperm acrosome reaction (AR) status, and tyrosine-phosphorylated protein expression in the testis of male mice induced with STZ.
Materials and Methods: Ten ICR mice were divided into two groups (n=5/each): control and diabetes induced by multiple low doses of streptozotocin (MLD-STZ). The control mice were intraperitoneally injected with citrate buffer, whereas MLD-STZ mice were injected with STZ at 40 mg/kg body weight for five consecutive days. At the end of the experiment (day 40), reproductive parameters, AR status, and the histopathology of the testis and epididymis were evaluated. The expression of testicular tyrosine phosphorylated proteins was examined.
Results: Blood glucose levels, AR percentages, and sperm abnormality of STZ group were significantly higher (p=0.003, 0.001, 0.000), while sperm concentration was significantly lower (p=0.001) compared to control. Histopathology of the seminiferous tubule was classified into 7 types. Additionally, abundant round cells were found in the epididymal lumen of the MLD-STZ mice. Moreover, the intensities of testicular phosphorylated proteins (170, 70, 36, 30, and 25 kDas) were markedly higher and a 120 kDa protein band was noticeably lower in the MLD-STZ mice.
Conclusion: MLD-STZ-induced DM causes many testicular histopathologies, precocious sperm AR, and increased expression of testicular phosphorylated proteins. These findings may clarify some mechanisms of sub/infertility in DM males.
Apichakan Sampannang, Supatcharee Arun, Jaturon Burawat, Wannisa Sukhorum, Sitthichai Iamsaard,
Volume 17, Issue 8 (August 2019)

Background: Types 1 and 2 diabetes mellitus (DM) are known to be the cause of sub/infertility. However, the comparisons of potential markers in spermatogenesis and steroidogenesis in DM males have never been elucidated.
Objective: This study aimed to examine the expressions of tyrosine-phosphorylated and steroidogenic acute regulatory (StAR) proteins in testis of DM mice.
Materials and Methods: Fifty-six male C57BL/6 mice were divided into four groups (n = 14/ each): control of MLD-STZ (multiple low doses of streptozotocin), MLD-STZ, control of HFD-STZ (high-fat diet with STZ), and HFD-STZ. MLD-STZ mice (type 1 DM) were intraperitoneally (i.p.) injected with STZ at 40 mg/kg BW for five days. HFD-STZ mice (type 2 DM) received an HFD for 14 days and i.p.-induced by STZ at 85 mg/kg BW and fed with HFD. At the end of the experiment (days 36 and 72), the expressions of phosphorylated proteins and StAR were examined.
Results: Tyrosine phosphorylated proteins were localized in late spermatids, luminal fluid, and Leydig cells. The intensities of phosphorylated 110, 85, 72, 60, and 55 kDas were lower in the 36 day-DM mice. Although such intensities were present in both groups, only 85 kDa in the MLD-STZ mice was higher in HFD mice at 72 days. StAR expressions in both groups were decreased than that of the controls.
Conclusion: Decreased expressions of StAR and tyrosine-phosphorylated proteins may be directly involved in low testosterone levels and impaired spermatogenesis. These findings support the notion that both DM types play a role in male infertility.
Wannisa Sukhorum, Jariya Umka Welbat, Suchada Ktutsri, Sitthichai Iamsaard,
Volume 18, Issue 5 (May 2020)

Background: Methotrexate (MTX) has been shown to affect the testes adversely, especially the seminiferous epithelium. As melatonin, an endocrine hormone, has been shown to normalize testicular function, its ability to prevent MTX-induced testicular damage should be considered.
Objective: Based on the antioxidant, anti-inflammatory, and antiapoptotic activities of melatonin, this study aimed to investigate its protective effect against testicular damage induced by MTX.
Materials and Methods: Forty adult male rats (200-230 g) were divided into five groups (n = 8/each). The rats in group I were injected with vehicle as a control. In group II, the rats were received intraperitoneal injections of melatonin (8 mg/kg) for 15 consecutive days. The rats in group III were intravenously injected with MTX (75 mg/kg) for 15 consecutive days. The remaining two groups received melatonin (8 mg/kgBW) for 15 (group IV) and 30 (group V) consecutive days, intraperitoneally, and then intravenously received MTX (75 mg/kgBW) on days 8 and 15 of the experimental period. Reproductive parameters, including epididymal sperm concentration, testicular tyrosine-phosphorylated protein expression, steroidogenic acute regulatory (StAR) protein expression, and caspase-3 and malondialdehyde levels, were examined.
Results: The sperm concentrations (×106/ml) of groups IV (58.75 ± 1.28) and V (55.93 ± 2.57) were improved significantly (p = 0.032) compared with that of group II (32.92 ± 2.14). The seminiferous epithelium in groups IV and V also increased, while caspase-3 expression decreased. In the melatonin-treated groups, the expression of tyrosine-phosphorylated proteins at 32 kDa was decreased and that of proteins at 47 kDa was increased compared with the MTX group. StAR protein expression was not altered in any of the groups.
Conclusion: Our results indicate that melatonin improves the epididymal sperm concentration by decreasing the expression of caspase-3 and increasing that of tyrosine-phosphorylated proteins in MTX-treated testes.

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