Potential testicular toxicity of sodium nitrate in adult rats

Nitrate is a common contaminant in groundwater aquifers. Current study aimed at evaluating the potential testicular toxicity of sodium nitrate in rats. Sodium nitrate was given orally to rats at doses of 50, 100 or 200 mg/kg/day for 60 consecutive days. Sperm count and motility, daily sperm production and testis weight were significantly decreased specially at high doses. Testicular activity of lactate dehydrogenase-X, glucose-6-phosphate dehydrogenase, and acid phosphatase were inhibited in a dose-related manner. Lipid peroxides and hydrogen peroxide production were significantly increased in all treated animals. This was accompanied by inhibition of testicular activities of superoxide dismutase and glutathione peroxidase. Fifty mg/kg of sodium nitrate did not significantly alter catalase or glutathione reductase activity. Glutathione was significantly decreased by sodium nitrate in a dose-related manner. The decrease in sperm count and motility and daily sperm production was confirmed by histopathological studies which indicated chromatolysis, pyknosis and necrosis in spermatocytes. In conclusion, subchronic exposure of rats to sodium nitrate results in testicular toxicity as evidenced by decreased sperm count and motility, daily sperm production and testis weight, inhibited activity of enzyme markers of spermatogenesis and induction of histopathological changes. These effects are attributed, at least partly, to testicular oxidative stress.     

Differential Changes in Expression of a and b FGF,IGF-1 and -2, and TGF-α During Seasonal Growth and Involution of Roe Deer Testis

Growth factors are involved in the regulation of testicular growth and involution of seasonal breeders. Therefore, we studied the seasonal expression of several growth factors in roe deer: aFGF, bFGF, IGF-1, IGF-2, and TGF- &#33. Total RNA from testis tissue was extracted monthly and analyzed using quantitative RT-PCR. Localization of mRNAs was examined by in situ -hybridization. Levels of expression differed by more than three orders of magnitude. Expression also showed different seasonal patterns. IGF-1, IGF-2 and bFGF were maximally transcribed during testis recrudescence in spring. In contrast, the mRNA amount of aFGF reached its maximum between July (breeding season) and January. TGF- &#33 mRNA-levels were very low and showed poor seasonal variation. Each growth factor showed its own typical expression localization in testicular tissues and cell types. The results suggest the specific role of different growth factors in the paracrine control of spermatogenesis and its seasonal regulation.     

T3 fails to restore mitochondrial thiol redox status altered by experimental hypothyroidism in rat testis

Oxidative stress impaired sperm function might lead to infertility. The objective of this study was to evaluate the effects of altered thyroid hormone levels on regulation of mitochondrial glutathione redox status and its dependent antioxidant defense system in adult rat testis and their correlation with testicular function. Adult male Wistar rats were rendered hypothyroid by administration of 6-n-propyl-2-thiouracil in drinking water for six weeks. At the end of the treatment period, a subset of the hypothyroid rats was treated with T₃ (20 μg/100 g body weight/day for 3 days). Mitochondria were isolated from euthyroid, hypothyroid and hypothyroid + T₃-treated rat testes, and sub-fractionated into sub-mitochondrial particles and matrix fractions. Mitochondrial respiration, oxidative stress indices and antioxidant defenses were assayed. The results were correlated with daily testicular sperm production and epididymal sperm viability. Increased pro-oxidant level and reduced antioxidant capacity rendered the hypothyroid mitochondria susceptible to oxidative injury. The extent of damage was more evident in the membrane fraction. This was reflected in higher degree of oxidative damages inflicted upon membrane lipids and proteins. While membrane proteins were more susceptible to carbonylation, thiol residue damage was evident in matrix fraction. Reduced levels of glutathione and ascorbate further weakened the antioxidant defenses and impaired testicular function. Hypothyroid condition disturbed intra-mitochondrial thiol redox status leading to testicular dysfunction. Hypothyroidism-induced oxidative stress condition could not be reversed with T₃ treatment.     

哺乳动物血睾屏障相关细胞连接及连接蛋白的研究进展

在雄性哺乳动物的睾丸中,紧密连接是血睾屏障中的主要结构,为相邻Sertoli细胞之间的一种联系,并与基部外胞质特殊结构和细胞桥粒连接共同存在.血睾屏障在生理上将生精上皮分为两个腔室:基底小室和近腔小室.基底小室内主要有精原细胞和早期精母细胞,而近腔小室有精子发生过程中各个阶段的生精细胞.前细线期精母细胞为了能够进入近腔小室必须穿过血睾屏障,该过程涉及多种分子以及相关的信号传导通路.分析目前与血睾屏障相关的细胞连接及连接蛋白的研究结果,进一步探讨这些细胞连接及连接蛋白对前细线期精母细胞穿越血睾屏障过程的作用.     

High-resolution image cytometry of rat sperm nuclear shape, size and chromatin status. Experimental validation with the reproductive toxicant vinclozolin

Recent studies have shown that the complex inter-related processes of sperm chromatin organization and nuclear morphogenesis, both of which are important fertility determinants, may be disrupted by chemicals. A high-resolution image cytometry method has been developed, using the fluorescent dye bisbenzimide, for the measurement of 20 features of the sperm nucleus related to size, form and chromatin status in the rat. For the complete set of features measured and from a total of 150 spermatozoa assessed per sample, the overall coefficient of reproducibility was 5%. Then, an experimental validation of the method was carried out in rats chronically exposed to the antiandrogenic reproductive toxicant vinclozolin and control animals. Univariate statistics revealed significant vinclozolin-induced changes for 19 out of 20 morphometric and chromatin features. Stepwise linear discriminant analysis classified correctly 84.3% of the sperm nuclei with only four features selected. The accuracy and reproducibility of the cytometry assessment of the sperm nuclei together with the results of the experimental validation suggest this method may be a new powerful tool for use in reproductive toxicology.     

Combined administration of low-dose gossypol acetic acid with desogestrel/mini-dose ethinylestradiol/testosterone undecanoate as an oral contraceptive for men

To evaluate the efficacy and feasibility of a new regimen of low-dose gossypol acetic acid (GA) combined with desogestrel/ethinylestradiol and testosterone undecanoate (DSG/E/TU) as a male contraceptive, adult male rats were fed orally with GA (12.5 mg/kg/day) and DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg) daily for 8 weeks as loading dose until infertility, and a similar low dose of GA alone for infertility maintenance. Control animals were administered a single low dose of GA (12.5 mg/kg/day) or DSG (0.125 mg/kg)/E (0.025 mg/kg)/TU (100 mg/kg), and vehicle, respectively. Results demonstrated that the combined dosage regimen could damage epididymal sperm motility and density, and induce infertility within 8 weeks in rats; the infertility could be consistently sustained by giving single GA (12.5 mg/kg/day), and was reversible in about 8 weeks following withdrawal of gossypol. The regimen rendered treated male rats with spermiation failure within a period of 6-20 weeks of treatment. Also, the serum luteinizing hormone, follicle-stimulating hormone and testicular interstitial fluid testosterone levels showed a transient decrease at the end of 6 or 8 weeks, which returned to control levels after 8 weeks of recovery phase. No hypokalemia or other adverse effects in viscera were observed. These results provide a promising approach to using the new regimen for the development of an effective and reversible oral male contraceptive.     

The eyestalk-androgenic gland-testis endocrine axis in the crayfish Cherax quadricarinatus

In decapod crustaceans, a number of neurohormones regulating a variety of physiological processes, including reproduction, are to be found in the X-organ-sinus gland complex of the eyestalk. Bilateral eyestalk ablation was thus performed in mature males of the Australian red claw crayfish Cherax quadricarinatus with the aim of studying the role of eyestalk-borne hormones on spermatogenic activity in the testis and on the androgenic gland (AG). The latter gland controls the differentiation and functioning of male sexual characteristics in crustaceans. Eyestalk ablation caused hypertrophy of the AG, as indicated by an increase in gland weight (3.9 +/- 0.44 mg vs < 0.1mg in intact males) and by overexpression of AG polypeptides. In the testes of eyestalk-ablated males, empty spermatogenic lobules were common, while lobules containing primary spermatocytes were infrequent. These findings were reflected in decreased amounts of DNA in these testes and a consequent increase in the relative weights of the sperm ducts. Since it was found that eyestalk ablation affected both the AG and the reproductive system, in vitro experiments were conducted to study the direct effects of the sinus gland on the AG and testes and of the AG on the testes. Sinus gland extracts inhibited by 30% the incorporation of radiolabeled amino acids into AG polypeptides and almost totally inhibited the secretion of radiolabeled AG polypeptides into the culture medium. However, sinus gland extracts had no significant effects on testicular tissue. On the other hand, AG extracts affected the in vitro phosphorylation of a testicular polypeptide (of 28 kDa), in a time- and dose-dependent manner, suggesting a direct effect of AG-borne hormones on the testes. The above findings, together with the evidence for direct inhibition by the sinus gland on the AG, suggest an endocrine axis-like relationship between the sinus gland, the AG, and the male reproductive system in decapod crustaceans.     

Reexamining the polyadenylation signal: were we wrong about AAUAAA?

Polyadenylation is the process by which most eukaryotic mRNAs form their 3′ ends. It was long held that polyadenylation required the sequence AAUAAA and that 90% of mRNAs had AAUAAA within 30 nucleotides of the site of poly(A) addition. More recent studies, aided by computer analysis of sequences made available in GenBank and expressed sequence tag (EST) databases, have suggested that the actual incidence of AAUAAA is much lower, perhaps as low as 50–60%. Reproductive biologists have long recognized that a large number of mRNAs in male germ cells of mammals lack AAUAAA but are otherwise normally polyadenylated. Recent research in our laboratory has uncovered a new form of an essential polyadenylation protein, τCstF-64, that is most highly expressed in male germ cells, and to a smaller extent in the brain, and which we propose plays a significant role in AAUAAA-independent mRNA polyadenylation in germ cells.     

Specific expression of the mRNA for 25 kDA heat-shock protein in the spermatocytes of mouse seminiferous tubules

 The 25 kDa heat-shock protein (Hsp25) is a member of the family of small heat-shock proteins. We investigated the expression and cellular localization of Hsp25 mRNA in the testis of adult and developing mice using Northern blotting and in situ hybridization techniques. In the early postnatal days, i.e., before the onset of spermatogenesis, no Hsp25 mRNA was detected in the testis. At around 10 days postpartum, Hsp25 mRNA began to be expressed in the testis in coincidence with the onset of the first wave of spermatogenesis and increased in amount progressively toward adulthood. Throughout the testis development, the signal for Hsp25 mRNA was localized exclusively to germ cells and was not detected in Sertoli or interstitial cells. The testis of W/Wv mutant mice, which lack the germ cell line, exhibited no Hsp25 mRNA expression. In the testis of normal adult mice, the abundance of Hsp25 mRNA differed among the seminiferous tubules in different stages of spermatogenesis. The most intense signal for Hsp25 mRNA was localized to the spermatocytes at leptotene, zygotene and early pachytene phases, which are present in the tubules of stages I–III and IX–XII. The signal decreased in intensity in the late pachytene and diplotene spermatocytes and was not detected in spermatids. Spermatogonia were also devoid of the signal. These results suggested that Hsp25 plays some specific role in the meiotic prophase of the testicular germ cell. Accepted: 27 Oct 1998     

Continued reproductive potential in aging platyfish as demonstrated by the persistence of gonadotropin,luteinizing hormone releasing hormone and spermatogenesis

Male platyfish, Xiphophorus maculatus, ranging in age from 12 to 54 months (normal life span is 30 months), were examined for age-related changes in the distribution of immunoreactive gonadotropin (ir-GTH) and luteinizing hormone releasing hormone (ir-LHRH) in their pituitary glands, and for changes in the histology of their testes. Males sacrificed at 13 and 16 months of age served as controls. Immunocytochemical methods demonstrate that even in the oldest fish, some almost twice the average platyfish life span, ir-GTH and ir-LHRH are localized in the same pituitary cell types in the caudal pars distalis and pars intermedia as in younger sexually mature fish. The testes of old fish continue to contain all stages of spermatogenesis; however, there are age-related increases in the amount of intertubular connective tissue and the relative number of spermatozeugmata, and distortions in the organization of acini. Our observations indicate that GTH and LHRH production and spermatogenesis continue, and thus a potential for reproductive capability persists, in male platyfish up to the time of their death resulting from old age.