Evaluation of a single dose of intra-testicular insulin treatment in heat-stressed mice model.

Insulin plays important role in testicular functions such as germ cell proliferation and steroidogenesis, despite its conventional role as a hypoglycaemic agent. It is also well known that testicular activity is severely get affected by heat stress and heat stress induces testicular pathogenesis. The effect of insulin on heat-induced testicular impairment has not been investigated. Thus, it is hypothesized that insulin might modulate testicular activity in a heat-stressed model. Experimental mice were separated into 4 groups; the first group was the normal control (CN), and the second group was subjected to heat stress (HS) by submerging the lower body part in a thermostatically controlled water bath maintained at 43°C for 15 min. The third and fourth groups were treated with a single dose of intra-testicular insulin (0.6 IU/mice) before and after heat stress. Animal tissue samples were collected after 14 days of heat treatment. Insulin treatment did not improve the sperm parameters; however, both insulin pre and post-treatment improved the markers of spermatogenesis such as Johnsen score, germinal epithelium height and the number of stages VII/VIII. The histoarchitecture of testis also showed amelioration from heat-induced pathogenesis in the insulin-treated groups. Insulin treatment has also increased the proliferation of germ cells (increased PCNA and GCN), survival (Bcl2), and decreased apoptosis (active caspase-3). Furthermore, insulin treatment decreased MDA levels, without pronounced effects on the activities of antioxidant enzymes. Heat stress also decreased the circulating testosterone and oestrogen levels, and insulin treatment significantly increased oestrogen levels only. Although testosterone showed an increasing trend, it was insignificant. The expression of aromatase, AR, ER-α, and ER-ß was down regulated by heat-stress and insulin treatment up regulated these markers. In conclusion, our results showed the amelioration of heat-induced testicular impairment by pre and post-intra-testicular insulin treatments. Insulin-associated improvements in the pre-and post-treatment groups suggested a preventive mechanism of insulin against heat stress in the testis.

Effects of metformin on the uterus of d-galactose-induced aging mice: Histomorphometric, immunohistochemical localization (B-cell lymphoma 2, Bcl2-associated X protein, and active capase3), and oxidative stress study.

d-galactose (DG)-induced rodent aging model has widely been used for the study of age-related dysfunctions of various organs, including gonads and uterus. Antidiabetic drug metformin has gained an attention as antiaging drug in model organism and human but its effect on uterus has not been studied in relation to induced aging. Therefore, we investigated the effect of metformin on uterus of DG-induced aging mice model. Mice were randomly divided into three groups, that is, control (CN), DG-induced aging model and aging model treated with metformin. Histomorphometric results showed significantly decreased number of uterine glands, endometrial thickness, and increased luminal epithelium height in aging model. Furthermore, metformin resumed the number of uterine glands, endometrial thickness, and luminal epithelium height up to CN group. Metformin has also significantly decreased the age-associated oxidative stress (malondialdehyde and lipid hydroperoxide). Superoxide dismutase was significantly decreased in both treated groups compared to the CN group. However, catalase and glutathione peroxidase enzymes were significantly increased by metformin compared to the aging model. Immunostaining of active caspase3 and BAX were intense in the endometrium of aging model compare to CN- and metformin-treated groups. Localization of B-cell lymphoma 2 (Bcl2) showed intense immunostaining in the uterus of CN- and metformin-treated groups, with mild immunostaining in aging model. Our observations suggested that metformin treatment might be helpful for management of age-associated uterine dysfunctions. Moreover, it may be concluded that metformin might ameliorate uterine dysfunctions by reducing oxidative stress, suppressing apoptosis, and increasing the survival/antiapoptotic protein Bcl2.

Synthetic leptin c-fragment peptide minimises heat-induced impairment of spermatogenesis in mice via Stat3 signalling.

Mammalian spermatogenesis is a temperature-sensitive process, and an increase in testicular temperature impairs spermatogenesis. Leptin modulates testicular activity, but the effect of leptin or its synthetic analogue on heat-induced testicular impairment is unclear. We investigated the effects of synthetic leptin peptide (116-130 amides) on testicular activity in heat-stressed mice model. 15 adult mice (25.54 ± 1.43 g) were selected for the study. Ten mice were subjected to a single heat stress treatment (HS) at 43 °C for 15 min by submerging the lower half of the body in a thermostatic water bath. After heat treatment, mice were divided into two groups, the heat-stressed HS group (n = 5) and the second group as HSL, treated with leptin peptide (116-130 amide) for 14 days. The HS group showed a significant (p < 0.05) decline in the GSI (0.25 ± 0.018), Johnsenscore (4.5 ±.19), seminiferous tubule diameter (160.75 ± 10.18 µm), germinal epithelium height, (GEH) (37.5 ± 1.59 µm) compared to the CN (GSI-0.37 ± 0.015; Johnsen score-7.9 ± 0.20; GEH- 73.25 ± 1.29 µm; tubule diameter-230.25 ± 1.39 µm) and the HSL groups (GSI-0.38 ± 0.014; Johnsen' score-8.0 ± 0.32; GEH- 37.5 ± 1.59 µm; tubule diameter-160.75 ± 10.18 µm) groups. Heat treatment significantly (p < 0.05) increased the intra-testicular levels of leptin (HS-20.11 ± 2.1 pg/mg protein; CN-10.50 ± 0.17 pg/mg protein; HSL-12.99 ± 0.52 pg/mg protein) with a reduced level of pStat3, suggesting leptin resistance during testicular hyperthermia. Furthermore, heat treatment was associated with significantly (p < 0.05) decreased germ cell proliferation and reduced circulating testosterone levels (HS-2.69 ± 2.01 ng/mL; CN-7.69 ± 0.32 ng/mL; HSL-5.36 ± 0.73 ng/mL). However, the circulating androstenedione levels showed a significant (p < 0.05) increase in the HS group (0.75 ± 0.03 ng/mL) compared to the CN (0.51 ± 0.02 ng/mL) and HSL (0.57 ± 0.07 ng/mL) groups. Immunolocalisation of 3ß-HSD showed moderate to faint staining in the Leydig cells in the HS group compared to the CN and HSL groups. Treatment with leptin peptide resulted in decrease in the intra-testicular leptin levels with increased phosphorylation of Stat3, suggesting improved leptin resistance, which was positively associated with increased germ cell proliferation, elevated testosterone levels, and improved testicular histoarchitecture. Testicular hyperthermia may cause leptin resistance and impaired leptin signalling, decreased testosterone biosynthesis and suppressed spermatogenesis, which could be a manifestation of leptin resistance. Treatment with leptin peptide improves leptin signalling and testicular activity in heat-stressed mice, but the underlying mechanism is still unclear.

Co-treatment of testosterone and estrogen mitigates heat-induced testicular dysfunctions in a rat model.

The two gonadal steroid hormones, testosterone and estrogen, regulate spermatogenesis by proliferation, differentiation, and apoptosis of testicular cells. It has been reported that heat stress or increased scrotal temperature impairs spermatogenesis in many mammals. Moreover, testicular heat stress has also been shown to suppress testosterone and estrogen biosynthesis. Furthermore, it is well known that testosterone and estrogen are important for testicular activity. Therefore, we hypothesised that exogenous testosterone and estrogen, alone or in combination, might alleviate the testicular activity in a heat-stressed rat model. To the best of our knowledge, this will be the first report of the exogenous treatment of both testosterone and estrogen in the heat-stressed rat. Our results showed that a combined testosterone and estrogen treatment significantly increased sperm concentration. The histopathological analysis also exhibited a normal histoarchitecture in the combined treatment group along with decreased oxidative stress. The improved spermatogenesis in the combined treatment group was also supported by the increase in PCNA, GCNA, tubule diameter, germinal epithelium height, and Johnsen score in the combined treatment group. Furthermore, the combined treatment also increased the expression of Bcl2, pStat3, and active caspase-3 and decreased expression of Bax. Thus, increased proliferation, apoptotic and anti-apoptotic markers, along with improved histology in the combined treatment group suggest that estrogen and testosterone synergistically act to stimulate spermatogenesis by increasing proliferation and differentiation of germ cells and may also remove the heat-induced damaged germ cells by apoptosis. Overall, the final mechanism of testosterone- and estrogen-mediated improvement of testicular activity could be attributed to amelioration of oxidative stress.

Heat-induced changes in the expression and localisation of PGC-1α in the mice testis.

The functions of mammalian testis are temperature-sensitive. There are various testicular factors, which express in response to heat as a mechanism of defence. PGC-1α and HSP70 have poetical role in the protection from oxidative stress in various tissues, including testis. The expression of PGC-1α and HSP70 has been shown in the testis, and it has also been documented that heat modulates the expression of PGC-1α and HSP70. However, heat-dependent changes in the localisation and expression of PGC-1α have not been investigated so far. Thus, we studied the expression and localisation pattern of PGC-1α in the testis of heat-treated mice along with marker of proliferation (PCNA, GCNA), serum testosterone levels, MDA levels and HSP70. The results showed a significant increase in PGC-1α and HSP70 and MDA levels in the testis of heat-treated mice along with a decrease in PCNA, GCNA and serum testosterone levels. The immunolocalisation study showed intense immunostaining of PGC-1α in the Leydig cell and germ cells of the heat-treated testis, with pronounced damaged in the histoarchitecture. The results showed that increase expression of PGC-1α in germ cells and Leydig cells of testis could be a counter mechanism to cope up with oxidative stress in coordination with HSP70.

Effect of metformin on testicular expression and localization of leptin receptor and levels of leptin in the diabetic mice.

Diabetes mellitus impairs testicular activity and leads to infertility. Leptin is one of the endogenous regulators of the male reproductive functions, but the role of leptin and its receptor (LEPR/Ob-R) in the control of testosterone production and testicular proliferation has not been investigated so far, especially in the Type 1 diabetes mellitus (DM1). Metformin is an anti-hyperglycemic drug which is beneficial for treating the both DM2 and DM1. The aim of this work was to study the possible role of leptin and Ob-R in the regulation of steroidogenesis and proliferation in the testes of mice with streptozotocin-induced DM1 (75 mg/kg/day, 4 days) and to estimate the restoring effect of metformin treatment (500 mg/kg, 2 weeks) on the diabetic testes. In the diabetic testes, the plasma and intratesticular leptin levels and plasma testosterone levels were reduced and completely restored by metformin treatment. Metformin also restored the expression of the steroidogenic transport protein steroidogenic acute regulatory protein reduced in DM1. In the diabetic testes, the expression of Ob-R was downregulated and the immunolocalization of Ob-R showed weak staining in the Leydig cells, the primary spermatocytes and the round spermatids. The germ cell proliferation was also reduced in DM1, as noticed with proliferating cell nuclear antigen (PCNA) expression. Metformin increased the Ob-R expression and immunostaining in the different cell types and improved the PCNA expression. Thus, DM1 impairs the testicular steroidogenesis and proliferation by inhibiting the leptin signaling, causing a decrease in leptin levels and Ob-R expression in the testes of diabetic mice, while metformin improves the leptin signaling and restores testosterone production and testicular proliferation.

Vitamin D3 regulates apoptosis and proliferation in the testis of D-galactose-induced aged rat model.

The age-associated imbalances between proliferation and apoptosis lead to impaired spermatogenesis and infertility. The age-associated decline in vitamin D3 levels has been reported and suggested the anti-aging potential of vitamin D3. However, the age-associated decline levels of vitamin D3 has not been studied in relation to the testicular activity. Thus, we investigated the effect of vitamin D3 on the expression of testicular proliferation markers, apoptotic markers, antioxidants system and oxidative stress in a D-gal-induced aged rat model. The present study investigated the levels of vitamin D3 and AGE in serum and testes along with the expression of the AGE-receptor (AGER) in the testis. Vitamin D3 treatment significantly increases cell proliferation and decreases apoptosis in a D-gal-induced aged rat testis. Furthermore, vitamin D3 significantly decreases oxidative stress in aged rat testis by improving the antioxidant defense systems. The expression of AGER was down-regulated by vitamin D3 treatment in aged testis. The circulating and intra-testicular AGE was higher in aged groups, however, only circulating vitamin D3 levels decreased in aged groups. The immunolocalization of VDR showed increased immunostaining in the testis by vitamin D3 treatment. Thus, it can be concluded that vitamin D3 delays testicular senescence by regulating proliferation and apoptosis.

Vitamin D3 treatment regulates apoptosis, antioxidant defense system, and DNA integrity in the epididymal sperm of an aged rat model.

The present study aimed to investigate the effects of vitamin D3 in the epididymal sperm cells of D-gal-induced aged rats. It is well known that during aging sperm quality and quantity declines and leads to age-related infertility problems in males. The results of the present study showed that there were elevated levels of oxidative stress and poor DNA integrity of sperm of aged rats. The expression of BCL2 also showed a significant decline in the sperm of aged rats, however, the expression of BAX and active caspase-3 did not show significant change compared with the control group. The treatment of vitamin D3 at lower doses to aged rats showed increased expression of BAX and active caspase-3 in the sperm, this finding suggests that increased apoptosis may be responsible for removal of poor quality sperm during aging. Vitamin D3 treatment at both doses showed improvement in the oxidative stress and DNA integrity in the sperm of aged rats. We also investigated the expression of AGER, visfatin, and HSPA1A in the epididymal sperm. It has been found that expression of AGER, visfatin, and HSPA1A increased in the sperm aged rats and vitamin D3 treatments at both doses decreased its expression. Thus, it might be suggested that during aging vitamin D3 treatment would be important for managing the sperm quality by regulating the apoptosis, antioxidant system and DNA integrity via modulation of visfatin and HSPA1A.

Vitamin D3 mediated regulation of steroidogenesis mitigates testicular activity in an aged rat model.

Testicular aging leads to a decrease in spermatogenesis and steroidogenesis. Vitamin D plays an important role in reproduction by modulating testicular steroidogenesis. As the role of vitamin D3 in testicular steroidogenesis during aging has not been explored, the aim of this study was to evaluate the effects of vitamin D3 on testicular functions in d-gal-induced aged rats. Vitamin D3 treatment on d-gal-induced aged rats resulted in significant improvement in sperm parameters, histoarchitecture, serum testosterone, and rostenedione and estrogen levels. The results of both in vivo and in vitro studies showed that vitamin D3 directly regulates testicular steroidogenic markers. Vitamin D3 treatment also increased CYP19A1 and decreased AR expression in the testes of d-gal-induced aged and normal rats. These results suggest that estrogen-mediated action may be responsible for an improvement in spermatogenesis in aged testis. Furthermore, it may be suggested vitamin D3 has a protective role in the aged testis and unaffected spermatogenesis in normal rats treated with vitamin D3 could be due to a balance between estrogen and androgen action.

Localization pattern of visfatin (NAMPT) in d-galactose induced aged rat testis.

Aging is a complex irreversible process which leads to decline in body physiology including reproductive activity. Neurological and brain functions defects have been studied in the d-gal induced aging rodent model. However, there is dearth of literature on reproductive aging induced by d-gal treatment. Visfatin is an adipokine which regulates testicular steroidogenesis and its level increases under stress conditions to cope and extend longevity. To the best of our knowledge the expression and localization pattern of visfatin and histological evaluation of d-gal induced aged testis have not been investigated. Thus, we hypothesized that the expression pattern and histoarchitecture of d-gal induced aged testis are impaired. Therefore, the aim of the present study was to elucidate the histopthological, immunohistochemical localization and expression of visfatin in d-gal induced aged testis along with serum testosterone level, sperm count and daily sperm production. The western blot and immunohistochemical results of the present study showed that d-gal treatment decreases visfatin expression in the testis, particularly in the Leydig cell, and decreases serum testosterone level. Further, d-gal treatment decrease in testosterone levels was positively correlated with decreases in Johnsen's score, mean seminiferous tubule diameter, germinal epithelium height, sperm count and daily sperm production. The multinucleated giant cells showed strong immunostaining for visfatin and suggest the role of visfatin as pro/anti-apoptotic factor. Thus, it can be suggested that visfatin may play an important role in testicular aging by regulating testicular steroidogenesis and spermatogenesis.