Prepubertal Male Sprague-Dawley Rats Research Articles

Dose-Dependent Degeneration of Leydig Cells Following Kisspeptin-10 Administration: An Ultrastructural Study.

The discovery of kisspeptin signaling as a key regulator of gonadotropin- releasing hormone (GnRH) secretion from the hypothalamus enhanced our understanding of the neuroendocrine regulation of mammalian reproduction. Effects of central and peripheral administration of kisspeptin on plasma gonadotropins, testosterone, and spermatogenesis are studied in detail. The present study was conducted to check the ultrastructure of Leydig cells in prepubertal male rats in response to the administration of a range of kisspeptin doses. We administered a range of kisspeptin-10 doses (1 μg, 1 ηg, and 10 ρg) intraperitoneally to prepubertal male Sprague-Dawley rats (PND 35) twice daily after every 12 hours. Control rats were injected with physiological saline in parallel. At the end of the treatment, plasma concentrations of testosterone were measured by competitive binding radioimmunoassay, and small pieces of rat testicular tissue were processed for electron microscopy to examine the ultrastructure of Leydig cells. Plasma testosterone concentration was reduced significantly at 1ηg (P<0.05) and 1μg (P<0.01) doses as compared to control. Distinct ultrastructural changes categorized as dilatation of cytoplasmic organelles, irregularly shaped nuclei with nuclear membrane invaginations, reduced nuclear sizes, degeneration, and vacuolation were observed in the kisspeptin-10 treated Leydig cells as compared to control. Quantification of the data showed reduced Leydig cell indices and hyperplasia of the interstitial cells. It is concluded that chronic intermittent administration of kisspeptin-10 has a dose-dependent degenerative effect on the plasma testosterone levels and Leydig cells ultrastructure in prepubertal male rats.

Delayed Puberty by Ziram Is Associated with Down Regulation of Testicular Phosphorylated AKT1 and SIRT1/PGC-1α Signaling.

Ziram is a dimethyldithiocarbamate fungicide, which may influence the male reproductive system as a potential endocrine disruptor. We interrogated the disruption of ziram on rat progenitor Leydig cell development. Prepubertal male Sprague-Dawley rats were orally treated with 0, 2, 4, or 8 mg/kg ziram for 2 weeks. We investigated the effects of ziram on serum testosterone levels, Leydig cell number, and Leydig and Sertoli cell gene and protein expression, SIRT1/PGC-1α levels, and phosphorylation of AKT1, ERK1/2, and AMPK in vivo. We also interrogated the effects of ziram on reactive oxidative species (ROS) level, apoptosis rate, and mitochondrial membrane potential of progenitor Leydig cells in vitro. Ziram decreased serum testosterone and follicle-stimulating hormone levels, the down-regulated Leydig cell-specific gene ( Lhcgr, Scarb1, Star, Cyp17a1, and Hsd17b3), and their protein expression. However, ziram stimulated anti-Müllerian hormone production. Ziram lowered SIRT1/PGC-1α and phosphorylated protein levels of AKT1. Ziram induced ROS and apoptosis and lowered the mitochondrial membrane potential of progenitor Leydig cells in vitro. In conclusion, ziram disrupts Leydig cell development during the prepubertal period potentially through the SIRT1/PGC-1α and phosphorylated AKT1 signaling.

Different exercise modalities have distinct effects on the integrin-linked kinase (ILK) and Ca2+ signaling pathways in the male rat bone.

Mechanical loading is essential to maintain optimal skeletal health. Despite the fact that early-life exercise has positive, long-lasting effects on the musculo-skeletal system, the response of the musculo-skeletal system to spontaneous low-impact exercise has been poorly studied. Previously, we identified subtle morphological changes in the femoral diaphysis of exercised animals compared to nonexercised controls. We hypothesized that significant changes in gene expression of cells should precede significant measurable phenotypic changes in the tissues of which they are part. Here, we employed RNA-Seq to analyse the transcriptome of the cortical bone from the femoral mid-diaphysis of prepubertal male Sprague-Dawley rats that were assigned to control (CON); bipedal stance (BPS); or wheel exercise (WEX) groups for 15 days. We identified 808 and 324 differentially expressed transcripts in the BPS and WEX animals respectively. While a number of transcripts change their levels in an exercise-specific manner, we identified 191 transcripts that were differentially expressed in both BPS and WEX. Importantly, we observed that the exercise mode had diametrically opposite effects on transcripts for multiple genes within the integrin-linked kinase (ILK) and Ca2+ signaling pathways such that they were up-regulated in BPS and down-regulated in WEX. The findings are important for our understanding of possible ways in which different exercise regimens might affect bone when normal activities apply mechanical stimuli during postnatal growth and development.

Detrimental effects of bisphenol A on development and functions of the male reproductive system in experimental rats.

Bisphenol A (BPA) is widely used in manufacturing industries. It is commonly detected in the environment and was reported to exert oestrogenic effects which may be harmful to the reproductive system. The present study was carried out to observe the effects of oral administration of BPA on the development of the reproductive organs and plasma sex hormone levels in prepubertal male Sprague-Dawley (SD) rats. Prepubertal male SD rats (n=8 in each group) were administered BPA in the doses of 1, 5, 10 and 100 mg/kg BW (body weight) via oral gavage for a period of 6 weeks. The control animals received the vehicle for BPA (Tween 80 in distilled water). Following 6 weeks of BPA exposure, the rats exhibited less evidence of spermatogenesis. There was seminiferous epithelial damage which included disruption of intercellular junctions and sloughing of germ cells into the seminiferous tubular lumen. Furthermore, the lumina of the seminiferous tubules and the epididymis of these animals were filled with immature germ cells and cellular debris. This damage may lead to the significant reduction in the seminiferous tubular diameter in BPA-treated animals. These findings were associated with the significant lower plasma testosterone and 17β-oestradiol levels. There was no significant difference between the body weight gain, the absolute as well as relative testis weight or epididymal weight of BPA-treated animals when compared to the control animals. The findings provided further evidence of the detrimental effects of BPA on the male reproductive system.

Prepubertal exposure to genistein alleviates di-(2-ethylhexyl) phthalate induced testicular oxidative stress in adult rats.

Di-(2-ethylhexyl) phthalate (DEHP) is the most widely used plastizer in the world and can suppress testosterone production via activation of oxidative stress. Genistein (GEN) is one of the isoflavones ingredients exhibiting weak estrogenic and potentially antioxidative effects. However, study on reproductive effects following prepubertal multiple endocrine disrupters exposure has been lacking. In this study, DEHP and GEN were administrated to prepubertal male Sprague-Dawley rats by gavage from postnatal day 22 (PND22) to PND35 with vehicle control, GEN at 50 mg/kg body weight (bw)/day (G), DEHP at 50, 150, 450 mg/kg bw/day (D50, D150, D450) and their mixture (G + D50, G + D150, G + D450). On PND90, general morphometry (body weight, AGD, organ weight, and organ coefficient), testicular redox state, and testicular histology were studied. Our results indicated that DEHP could significantly decrease sex organs weight, organ coefficient, and testicular antioxidative ability, which largely depended on the dose of DEHP. However, coadministration of GEN could partially alleviate DEHP-induced reproductive injuries via enhancement of testicular antioxidative enzymes activities, which indicates that GEN has protective effects on DEHP-induced male reproductive system damage after prepubertal exposure and GEN may have promising future in its curative antioxidative role for reproductive disorders caused by other environmental endocrine disruptors.

Kisspeptin‐10 induces dose dependent degeneration in prepubertal rat prostate gland

Kisspeptin peptides mediate their actions through the GnRH loop system. How kisspeptins affect prostate gland in prepubertal male mammals remains elusive. To address this kisspeptin was administered as subchronic (12 days) twice daily i.p. dose at three different dosage regimens: 10 pg, 1 ng and 1 µg, to prepubertal male Sprague-Dawley rats (PND 35). Control rats were maintained in parallel. At the end of the experiment prostate gland was dissected out and processed for light and electron microscopy. DNA damage was also estimated by DNA ladder assay and DNA fragmentation assay. Prostate weights decreased significantly (P < 0.05) at 1 µg treatment dose of kisspeptin. The epithelial height of secretory acini of prostate decreased at 10 pg (P < 0.05), 1 ng, and 1 µg doses (P < 0.001). Histomorphology and ultrastructure demonstrated, decrease in epithelial cell height, epithelial folding and dilatation of the organelles with kisspeptin treatment. Percent DNA damage to the prostatic tissue was 20.74 ± 2.18, 43.60 ± 2.39, and 58.18 ± 2.59 at 10 pg, 1 ng and 1 µg doses, respectively. The study reveals that continuous administration of kisspeptin does not lead to an early maturation but instead severe degeneration of prepubertal prostate gland. Wiley Periodicals, Inc.

Preventive effect of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury in rat

To investigate the preventive effects of d-psicose, one of rare ketohexoses, on di-(2-ethylhexyl) phthalate (DEHP)-induced testicular injury, prepubertal male Sprague–Dawley rats were exposed to DEHP via their diet or orally, while under treatment with d-psicose. The rats given a diet-containing 1% DEHP alone for 7–14 days showed severe testicular atrophy accompanied by aspermatogenesis. On the other hand, those given the diet plus 2% but not 1% d-psicose-supplemented water for 14 days did not develop testicular atrophy, and exhibited an almost complete spermatogenesis. There was no significant difference in plasma mono-(2-ethylhexyl) phthalate (MEHP) levels between the d-psicose-free and d-psicose-treated groups. The testicular malondialdehyde (MDA) level after a single oral administration of 2 g/kg of DEHP showed a similar pattern of increase to the plasma MEHP level and peaked in 24 h suggesting a close and dose-dependent relation between plasma MEHP and testicular reactive oxygen species (ROS) levels. Pretreatment with d-psicose at a concentration of 2% and 4% resulted in an almost complete but not absolute suppression of testicular MDA production among rats administered 2 g/kg of DEHP. The microarray analysis showed the induction of oxidative stress related genes including the thioredoxin, glutathione peroxidase 1 and 2, glutaredoixn 1 after 24 h of the DEHP treatment in the testis. These results show that d-psicose prevents DEHP-induced testicular injury by suppressing the generation of ROS in the rat testis. This effect may be due to the direct scavenging by d-psicose of ROS generated in the testis.

EFFECTS OF TESTICULAR FIXATION ON OXIDATIVE STRESS-RELATED PARAMETERS IN PREPUBERTAL RAT TESTIS

The goal of orchiopexy is to provide adequate scrotal fixation to prevent recurrent torsion of the testis and the spermatic cord or ascent of the testis, to be achieved with minimal trauma to the testis. Forty-two prepubertal male Sprague-Dawley rats were equally distributed into 6 groups (1 SHAM + 5 Operation). In operational groups, two lateral sutures were placed near the upper and lower pole of the testis passing through the tunica albuginea with 4-zero polyglactin suture in the transverse axis of the testis. At the 1st-3rd-7th-15th and 30th postoperative days, bilateral orchiectomy was performed, then malondialdehyde (MDA), nitrite, superoxide dismutase (SOD) and glutathion peroxidase (GPx) values were measured in testicular tissue homogenates. MDA and nitrite levels increased, while GPx and SOD levels diminished, statistically significant in both ipsilateral and contralateral testicular tissue homogenates. Prepubertal testicular fixation causes overproduction of the lipid peroxidation end product and free radicals while heavy consuming anti-oxidant reserve systems in both ipsilateral and contralateral testicular tissue. These results clearly reveal the extent of the testicular deterioration due to suture fixation technique.

The effect of zinc therapy on damaged testis in pre-pubertal rats.

This experimental study was instituted to evaluate whether or not there is an effect of zinc aspartate administration on injured testes. Sixty prepubertal male Sprague-Dawley rats (weighing 180-240 g) were divided into six equal groups each containing ten rats. Group I was the control group. Rats in group II and group III which were blunt testicular trauma groups were subjected to right blunt testicular trauma to rupture the tunica albuginea. Just after this, animals in group III were given 30 mg/kg zinc aspartate intraperitoneally, and this treatment was continued for 30 days at a dose of 10 mg/kg per day. Animals in group IV and group V which were thermal injury groups were subjected to right thermal testicular injury with injection of boiling normal saline. After that, animals in group V were also treated with zinc aspartate as described in group III. Group VI was used as a sham group. After 30 days, both testes were removed and examined. Damage in ipsilateral testes was evaluated with histological scoring methods. Contralateral testes were evaluated with measurement of tubular diameter and percentage of spermatogenesis histologically. Ipsilateral testes from non-treated groups had much greater histological injury than treated groups ( p>0.05). Additionally, contralateral testes had no evidence of injury. As a consequence, after acute testicular injury that takes form in a variety of ways, immediate administration of zinc aspartate and its continuation for some period may prevent the progression of the injury and improves the healing process.

Protective effect of vasoactive intestinal peptide on testicular torsion-detorsion injury: association with heparin-containing mast cells

Objectives To elucidate the action of vasoactive intestinal peptide (VIP) on detorsion injury and the heterogeneity of mast cells in the testes of rats. Methods Prepubertal male Sprague-Dawley rats were used in six groups. Group 1 was the control group (sham operation); group 2 had 2 hours of torsion; group 3, 2 hours of torsion and 1 hour of detorsion after administration of saline; group 4 had 2 hours of torsion and 4 hours of detorsion after administration of saline; group 5, 2 hours of torsion and 1 hour of detorsion after administration of intraperitoneal VIP (25 ng/kg); and group 6, 2 hours of torsion and 4 hours of detorsion after intraperitoneal VIP. The 2 hours of torsion was created by rotating the right testis 720° in a clockwise direction. VIP (25 ng/kg) was injected intraperitoneally 1 minute before the 1 and 4 hours of detorsion. At the end of the experiment, catalase enzyme activity was measured polarographically, and superoxide dismutase, malondialdehyde, and protein were measured spectrophotometrically. Nitric oxide was measured by capillary electrophoresis in the testicular tissue. Routine histologic examination of testicular mast cells was done under light microscopy; the histochemistry was also analyzed. Results Torsion significantly induced oxidative stress, mast cell degranulation, and tissue damage. Detorsion attenuated oxidative stress without any diminution of the histologic damage to the tissue. VIP significantly protected the testicular tissue from detorsion injury. It also inhibited mast cell activity while increasing the heparin content. Conclusions VIP can protect testicular tissue from detorsion injury. Heparin-containing mast cells seem to be important mediator cells for this protection.

The effect of zinc aspartate pretreatment on ischemia-reperfusion injury and early changes of blood and tissue antioxidant enzyme activities after unilateral testicular torsion-detorsion

Background/purpose The aim of this study is to investigate the effect of zinc aspartate (ZA) pretreatment on ischemia-reperfusion (I/R) injury and blood and tissue antioxidant enzyme activity early after unilateral testicular torsion-detorsion (T/D). Methods Forty prepubertal male Sprague-Dawley rats (weight 160 to 220 g) were divided into 4 groups each containing 10 rats. Surgery was conducted under intraperitoneal 1-shot ketamine (50 mg/kg) anesthesia. The scrotum was entered through a midline incision. Rotating the left testis 720° in a clockwise direction was the model of the testicular torsion. Group 1 was for the basal values. Group 2 had 4 hours T/D. Group 3 also had 4 hours T/D and pretreated with 50 mg/kg intraperitoneal ZA injection half an hour before detorsion. Group 4 was designed as a sham group. In the Group 2 and Group 3, the tunica vaginalis was opened, and left testicles were rotated clockwise 720° and maintained in this torsion position by fixing with a silk suture to the scrotal wall. The scrotum was closed and 4 hours later reentered for testicular detorsion. After spermatic cord detorsion, the scrotum was closed. At the end of 4 hours detorsion period, bilateral orchiectomies were performed, and 5-mL intracardiac blood samples were taken. Blood and tissue superoxide dismutase (SOD), catalase (CAT) activities, and malondialdehyde (MDA) levels were measured, and histopathologic examination was performed. Results Group 2 and group 3 had decreased blood SOD and CAT activities and elevated MDA levels indicating I/R injury. The 2 groups were also different from each other for these parameters reflecting the beneficial effect of ZA pretreatment ( P < .05). The decreased ipsilateral tissue SOD and CAT activities in group 2 were different from the other groups including group 3 ( P < .05). Ipsilateral tissue MDA levels of both group 2 and group 3 were elevated. Group 2 had higher values than group 3 ( P < .05). In addition, specimens from group 2 had a significantly greater histologic injury than group 3 ( P < .05). These findings were also supporting the beneficial effect of ZA pretreatment. All measurements of contralateral tests were similar to the basal values for all groups ( P > .05). Conclusions ZA pretreatment reduces I/R injury by its antioxidant effects after unilateral testicular T/D and affects the antioxidant enzyme systems.

Effects of the Nitric Oxide Donor Molsidomine on the Early Stages of Liver Damage in Rats with Bile Duct Ligation: A Biochemical and Immunohistochemical Approach

This study aimed to evaluate the effects of the nitric oxide donor molsidomine on the early stages of liver damage and biochemical changes in rats with bile duct ligation (BDL). Forty prepubertal male Sprague-Dawley rats weighing 125–140 g were studied. Group 1 rats (sham-control, n = 10) were not subjected to any surgical manipulation. Group 2 rats (BDL/untreated, n = 10) were subjected to BDL but no drug was administered. Group 3 rats (BDL/L-NAME, n = 10) received a daily dose of N^G-nitro-L-arginine methyl ester (L-NAME) intraperitoneally for 7 days after BDL. Group 4 rats (BDL/molsidomine, n = 10) received a daily dose of molsidomine by gastric tube for 7 days after BDL. After 1 week, biochemical and histological evaluations were performed and the liver hydroxyproline content was measured. Serum bilirubin and liver enzymes were significantly increased in the BDL/untreated, BDL/L-NAME and BDL/molsidomine groups in comparison with the sham-control group 1 week after BDL. However, the liver enzymes were significantly decreased in the BDL/molsidomine group in comparison with the BDL/untreated and BDL/L-NAME groups. In the BDL/L-NAME group, proliferation of portal and periportal biliary ductules with disorganization of the hepatocyte plates, dilated portal spaces and areas of polymorphonuclear leukocyte infiltration, fibrosis and hepatocyte necrosis were observed. In the BDL/molsidomine group, polymorphonuclear leukocyte infiltration, hepatocyte necrosis and fibrosis were rarely seen. The hydroxyproline content in the liver was increased 1 week after obstruction in the BDL/untreated and BDL/L-NAME groups when compared to BDL/molsidomine group. Collagen type-IV expression was not observed in the BDL/molsidomine group in contrast to the BDL/untreated and BDL/L-NAME groups. In conclusion, during 1 week of treatment, the nitric oxide donor molsidomine improved hepatic fibrosis in the hepatic parenchyma and did not affect serum bilirubin values, but positively affected the serum aspartate aminotransferase and alanine aminotransferase values.

Thymocyte Development in Male Fetal Alcohol‐Exposed Rats

We previously reported altered responses of thymocytes to mitogen stimulation after fetal alcohol exposure (FAE) in prepubertal male Sprague-Dawley rats. The purpose of this study was to examine the effect of FAE on the developmental pattern of thymocyte subsets. In the first experiment, we found that the proportion of double-labeled CD4+CD8+ thymocytes is identical in fetal alcohol-exposed (E) and control (C) animals at 34 and 45 days of age. In the second experiment--at 20, 28, 35, and 48 days of age--we examined the proportion of CD4+ and CD8+ thymocytes that express or are devoid of the maturational markers, the alpha/beta configuration of the T-cell receptor (TcR), and the restriction fragment C of the common leukocyte antigen (CD45RC). We found significant age-dependent effects on the numbers of total double-positive CD4-TcR and CD8-TcR or CD45RC thymocytes, and significantly lower numbers of total CD4+ and CD8+ cells in E than in C rats throughout this period--a finding consistent with the significantly lower total number of thymocytes in E than in C rats. The developmental patterns for both markers were similar in E and C groups, in both the rising (days 20 to 28) and declining (days 35 to 48) phases. However, on day 35, E rats had significantly lower numbers of double-positive CD8-TcR and CD8-CD45RC cells than C rats. It therefore seems that FAE tends to accelerate the decline of double-positive CD8-TcR and CD8-CD45RC cells. The contribution of this phenotypic change to the thymic functional alterations induced by FAE remains to be determined.

The effects of an LHRH agonist on testicular function in the cryptorchid rat.

To assess the effects of an LHRH analog (LHRH-ethylamide des gly10 D-Leu6) on the function of the cryptorchid testis, an animal model was created, using prepubertal male Sprague-Dawley rats (21 days old). The animals were divided into six groups: 1) sham operated; 2) sham + LHRH treated; 3) cryptorchid; 4) cryptorchid + LHRH treated; 5) cryptorchid and orchidopexed; 6) cryptorchid, LHRH treated and orchidopexed. Two weeks post cryptorchidism, the animals were treated with either saline or LHRH-analog (one microgram./rat/day) subcutaneously for a total of twenty five days. Orchidopexy was performed following hormonal treatment. The effects of treatment were assessed in terms of body and reproductive organ weights, serum testosterone and LH levels, sperm counts and motility and fertility. The results demonstrated that experimental cryptorchidism impaired reproductive function, which was restored by orchidopexy, alone. Treatment of cryptorchid rats (groups 4 and 6) with LHRH did not produce any lasting improvement in spermatogenesis or fertility.