Intratesticular Testosterone Levels Research Articles

Effect of chronic oestrogen administration on androgen receptor expression in reproductive organs and pituitary of adult male rat

Following chronic (15 or 30 days) treatment with oestradiol 3-benzoate (75 microg rat(-1) day(-1) in 100 microl of olive oil) to adult rats, androgen receptor (AR) expression was analysed simultaneously in testis, epididymis, seminal vesicle, prostate and pituitary utilising three independent tools i.e. immunohistochemistry, Western blotting and RT-PCR. All the five organs showed higher AR transcriptional activity gradually increasing from 15 to 30 days of oestrogen treatment. However, the AR protein expression either through immunostaining or Western blotting demonstrated a significant decline in all the reproductive organs. In the pituitary, on the other hand, the decline coincided with a distinct breakdown of the AR protein into two bands with increasing duration of treatment. Serum and intra-testicular testosterone levels were found significantly lowered. Spermatogenesis was adversely affected with concurrent decrease in weights of testis and accessory sex organs. Decrease in testis weight was consistent with the reduction in the number of maturing germ cells per tubule. Despite the decrease in weight, accessory sex organs like epididymis, seminal vesicle and prostate were completely devoid of any apoptotic cells which were characterised only in testis and pituitary. Seminiferous epithelium demonstrated a marked increase in the number of germ cells undergoing apoptosis. However, the rate of cell apoptosis was much higher in the pituitary than in the testis at the end of 30 days treatment. It is therefore concluded that degradation of AR protein expression after oestrogen treatment is probably directly linked to an increase in cell apoptosis both in testis and pituitary.

The effects of an in utero exposure to 2,3,7,8‐tetrachloro‐dibenzo‐p‐dioxin on male reproductive function: identification of Ccl5 as a potential marker

2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and dioxin-like compounds are widely encountered toxic substances suspected of interfering with the endocrine systems of humans and wildlife, and of contributing to the loss of fertility. In this study, we determined the changes in testicular gene expression caused by in utero exposure to TCDD along with the intra-testicular testosterone levels, epididymal sperm reserves, daily sperm production (DSP) and testis histology. To this purpose, female pregnant Sprague–Dawley rats orally received TCDD (10, 100 or 200 ng/kg body weight) or vehicle at embryonic day 15, and the offspring was killed throughout development. Hepatic Cyp1a1 gene expression was measured in the offspring to confirm the exposure to TCDD. The gross histology of the testes and intra-testicular testosterone levels were normal among the studied groups. Sperm reserves were altered in 67-day-old rats of the TCDD-200 group, but not in 145-day-old animals or in the other TCDD-exposed groups. Nonetheless, fertility was not altered in males of the TCDD-200 group, and the F2 males generated had normal sperm reserves and DSP. Microarray analysis permitted the identification of eight differentially expressed genes in the 4-week-old testes of the TCDD-200 compared with that of the control group (cut-off value ± 1.40), including the down-regulated chemokine Ccl5/Rantes. Inhibition of Ccl5/Rantes gene expression was observed throughout development in the TCDD-200 group, and at 67 and 145 days in the TCDD-100 group (animals of younger ages were not examined). Ccl5/Rantes gene expression was mostly confined in Leydig cells. F2 males generated from males of the TCDD-200 group had normal levels of Ccl5/Rantes in testis and Cyp1a1 in liver, which might indicate that Ccl5/Rantes is a marker of TCDD exposure in testis such as Cyp1a1 in liver. In conclusion, we demonstrated a decrease in Ccl5/Rantes RNA levels and a transitory decline in sperm reserves in the testes of rats of TCDD-dosed dams.

Intratesticular testosterone levels are high in rats maturing in the below the sea level enviroment of the jordan valley

intratesticular testosterone levels are high in rats maturing in the below the sea level enviroment of the jordan valley

Vitamin C and Resveratrol Supplementation to Rat Dams Treated with Di(2-ethylhexyl)phthalate: Impact on Reproductive and Oxidative Stress End Points in Male Offspring

This study was carried out to assess the influence of di(2-ethylhexyl)phthalate (DEHP) alone or associated with antioxidants on the male reproductive system in newborn rats, emphasizing the implications of oxidative stress and hormonal balance during prenatal and early postnatal periods. Wistar females were exposed by oral route to DEHP alone or associated with antioxidants from gestational day 7 to lactational day 2 according to the following treatment regimens: (C) vehicle control (canola oil + 1% Tween-80); (V) vitamin C (200 mg/kg) + canola oil; (R) resveratrol (10 mg/kg) + canola oil; (D) DEHP (500 mg/kg) + 1% Tween-80; (DV) DEHP (500 mg/kg) + vitamin C (200 mg/kg); and (DR) DEHP (500 mg/kg) + resveratrol (10 mg/kg). Two male pups per litter were randomly selected and necropsied on postnatal day 2. The brain and liver were removed and weighed and anogenital distance (AGD) was measured. Additionally, the testes were removed for assessment of intratesticular testosterone levels and histopathology; the liver was used to measure biomarkers of oxidative stress. Vitamin C and resveratrol alone did not affect the reproductive end points and did not induce oxidative stress. Exposure of dams to DEHP alone and associated with antioxidants resulted in hepatomegaly in offspring and significantly increased the incidence of multinucleated gonocytes in seminiferous cords. Testosterone and AGD presented a trend to decrease in DEHP-exposed groups. Catalase activity increased only in groups exposed to DEHP associated with antioxidants, although GST (gluthatione-S-transferase) activity decreased in all DEHP-exposed groups. The levels of hydroperoxides increased only in group exposed to DEHP associated with vitamin C. These results indicate that the association of DEHP with antioxidants was unable to ameliorate DEHP-induced reproductive changes, and the coadministration of DEHP and these antioxidants might even contribute to an overall increase in oxidative stress.

Estrogen Enhances Recovery From Radiation‐Induced Spermatogonial Arrest in Rat Testes

Irradiation of LBNF(1) rat testes induces spermatogonial differentiation arrest, which can be reversed by gonadotropin-releasing hormone (GnRH) antagonist-induced suppression of intratesticular testosterone (ITT) and follicle-stimulating hormone (FSH). Although exogenous estrogen treatment also enhanced spermatogenic recovery, as measured by the tubule differentiation index (TDI), it was not clear whether estrogen stimulated spermatogonial differentiation only by further suppressing ITT or by an additional independent mechanism as well. To resolve this question, we performed the following experiments. At 15 weeks after irradiation, rats were treated with GnRH antagonist; some also received 17beta-estradiol (E2) and were killed 4 weeks later. GnRH antagonist treatment increased the TDI from 0% to 8%, and addition of E2 further increased the TDI to 39%. However, E2 addition further reduced ITT from 7 ng/g testis, observed with GnRH antagonist to 3 ng/g testis, so decreased ITT levels might have contributed to recovery. Next GnRH antagonist-treated rats were given exogenous testosterone and flutamide to stabilize ITT levels and block its action. This increased TDI slightly from 8% to 13%, but the further addition of E2 significantly raised the TDI to 27%, indicating it acted by a mechanism independent of ITT levels. Plots of TDI for all treatment groups compared with ITT, FSH, or a linear combination of ITT and FSH showed that treatments including E2 produced higher TDI values than did treatments without E2. These results indicate that there was an effect of E2 on spermatogonial differentiation because of an additional direct action on the testis that is unrelated to its suppression of testosterone or gonadotropins.

Hormonal Regulation of Rat Testicular γ-Glutamyl-Transpeptidase “In Vivo” and “In Vitro”/Die hormonelle Regulation von testikulärer γ-Glutamyl-Transpeptidase bei Ratten “in vivo” und “in vitro”

The hormonal regulation of gamma-glutamyl transpeptidase (gamma-GTP), an enzyme marker of Sertoli cells, was studied in immature rats that received 50 micrograms/day of testosterone propionate (TP) during 6 days to suppress pituitary LH and FSH. Suppression of LH was monitored indirectly by the determination of intratesticular levels of testosterone and suppression of FSH by radioimmunoassay of serum FSH. Enzyme activity in the testis decreased in parallel to intratesticular testosterone suppression, and it did recover up to control values when animals received 500 micrograms/day of TP, a dose that was able to maintain intratesticular testosterone at normal levels. beta-glucuronidase, another enzyme marker of Sertoli cells, was not affected by these treatments. A significant decrease in gamma-GTP was detected 24h after significant suppression of intratesticular testosterone and it returned to control levels 2 days after increasing the dose of TP to 500 micrograms/day. Administration of FSH to rats with depletion of intratesticular testosterone was able to maintain testicular gamma-GTP at control levels. An stimulatory action of FSH could also be demonstrated in primary Sertoli cell cultures. It is concluded that testicular gamma-GTP is under the regulation of both androgens and FSH while beta-glucuronidase is not. Eventhough the function of gamma-GTP in the testis is not known, the key role that it plays in other tissues suggests that it might be important in the regulation of Sertoli cell-germ cell interactions.

Inhibition of Rat and Human Steroidogenesis by Triazole Antifungals

Environmental chemicals that alter steroid production could interfere with male reproductive development and function. Three agricultural antifungal triazoles that are known to modulate expression of cytochrome P450 (CYP) genes and enzymatic activities were tested for effects on steroidogenesis using rat in vivo (triadimefon), rat in vitro (myclobutanil and triadimefon), and human in vitro (myclobutanil, propiconazole, and triadimefon) model systems. Hormone production was measured in testis organ cultures from untreated adult and neonatal rats, following in vitro exposure to 1, 10, or 100 μM of myclobutanil or triadimefon. Myclobutanil and triadimefon reduced media levels of testosterone by 40–68% in the adult and neonatal testis culture, and altered steroid production in a manner that indicated CYP17-hydroxylase/17,20 lyase (CYP17A1) inhibition at the highest concentration tested. Rat to human comparison was explored using the H295R (human adrenal adenocarcinoma) cell line. Following 48 h exposure to myclobutanil, propiconazole, or triadimefon at 1, 3, 10, 30, or 100 μM, there was an overall decrease in estradiol, progesterone, and testosterone by all three triazoles. These data indicate that myclobutanil, propiconazole, and triadimefon are weak inhibitors of testosterone production in vitro. However, in vivo exposure of rats to triazoles resulted in increased serum and intra-testicular testosterone levels. This discordance could be due to higher concentrations of triazoles tested in vitro, and differences within an in vitro model system lacking hepatic metabolism and neuroendocrine control.

169. SERTOLI CELL-SPECIFIC DISRUPTION OF THE ANDROGEN RECEPTOR DNA-BINDING DOMAIN REVEALS DIFFERENTIAL TEMPORAL CONTROL OF DISTINCT ANDROGEN-REGULATED GENES

Androgen receptor (AR) actions are vital for spermatogenesis. However, in postnatal development male germ cells do not express AR, highlighting its key role in testicular somatic cells. We recently used a Cre-loxP strategy to determine the in vivo requirement of AR DNA-binding in Sertoli cell (SC) function. Transgenic (Tg) mice with Cre expression targeted by SC-specific AMH or Abp promoters were crossed with floxed-Ar (Arflox) mice for Cre-loxP inframe deletion of Ar exon 3, which encodes a zinc finger essential for the DNA-binding domain (DBD). SC-specific mutated ARΔex3 (SCARΔex3) produced infertile AMH.SCARΔex3 and Abp.SCARΔex3 males. Testes from adult homozygous TgCre(+/+) AMH.SCARΔex3 or Abp.SCARΔex3 males were 30% of normal size and exhibited meiotic arrest, whereas testes from hemizygous TgCre(+/–) Abp.SCARΔex3 males were larger (47% normal) with more postmeiotic germ cell development. Despite marked Leydig cell hypertrophy, testicular expression of the adult Leydig marker Hsd3b6 (RT-PCR) and normal intratesticular testosterone levels (LC-MS/MS) in SCARΔex3 males indicated the presence of morphologically distinct but functional adult Leydig cells. SC-specific mutated AR Δex3 was predicted to disrupt classical AR-regulated pathways via loss of direct DNA interaction. Androgen-repressed testicular Ngfr expression (known to be via non-classical AR pathways) was not upregulated in SCARΔex3 testes, suggesting maintenance of a non-classical mechanism independent of AR-DBD. In contrast, SC-specific Rhox5 and Eppin transcription, regulated by divergent or classical androgen-response elements respectively, were both decreased in postnatal SCARΔex3 vs. control testes, demonstrating SC-specific AR function as early as postnatal day 5. However, Rhox5 expression declined dose-dependently, whereas Eppin expression increased, in adult TgCre(+/−) and TgCre(+/+) SCARΔex3 testes, revealing differential temporal control for distinct AR-regulated transcripts. Thus, our SCARΔex3 paradigm displayed dose-dependent TgCre-disruption of meiotic competence and post-meiotic development as well as gene expression, and represents a unique model to selectively differentiate AR-regulated genes.

The Histone Deacetylase SIRT1 Controls Male Fertility in Mice Through Regulation of Hypothalamic-Pituitary Gonadotropin Signaling1

Sirtuins (SIRTs) are class-III NAD-dependent histone deacetylases (HDACs) that regulate various physiological processes. Inactivation of SIRT1 in the mouse leads to male sterility, but the molecular mechanisms responsible for this phenotype have not been determined. Here we show that fetal testis development appears normal in Sirt1(-/-) mice. In contrast, the first round of spermatogenesis arrests before the completion of meiosis with abundant apoptosis of pachytene spermatocytes, abnormal Leydig and Sertoli cell maturation, and strongly reduced intratesticular testosterone levels. We show that this phenotype is the consequence of diminished hypothalamic gonadotropin-releasing hormone expression and strongly reduced luteinizing hormone levels. Rather than having an intrinsic effect on male germ cells per se, our results show that SIRT1 regulates spermatogenesis at postnatal stages by controlling hypothalamus-pituitary gonadotropin (HPG) signaling. In addition to its well studied role in control of metabolism and energy homeostasis, our results thus reveal a novel and critical function of SIRT1 in controlling HPG signaling. This phenotype is more severe than those previously described using mice bred on different genetic backgrounds, and highlights the fact that SIRT1 function is strongly modified by other genetic loci.

Suppression of Insulin-Like 3 by Androgen Depletion in Hormone-Treated Irradiated Rat Testis.

Irradiation causes spermatogenic arrest in adult rats and this arrest can be reversed by reduction of intratesticular testosterone by GnRH-antagonist, whereas further testosterone treatment attenuates the spermatogenesis recovery. To identify hormonally regulated genes correlating with the spermatogenic differentiation status, gene expression profiles were investigated by microarray with Rat 230 2.0 GeneChips (Affymetrix), comparing normal control rats, irradiated rats with no treatment, irradiated rats treated with GnRH-antagonist and either flutamide (androgen receptor antagonist) or testosterone, for 2 weeks, at which time there were minimal changes in number of germ cells. Insulin-like 3 (Insl3), a member of the relaxin-insulin hormone family was the second most strongly down-regulated gene by testosterone suppression. Real-time RT-PCR confirmed microarray observations: after irradiation, relative Insl3 mRNA level increased about 9-fold above the normal control; suppression of testosterone by GnRH-antagonist and flutamide repressed Insl3 level by 20-fold compared to the irradiated-only group; adding testosterone together with GnRH antagonist elevated Insl3 level so that it was only 6-fold reduced from the irradiated only group. Characterization of the changes in INSL3 protein levels by immunoblot supported the mRNA results: irradiation caused over 10-fold increase in relative prepro-(or pro-)INSL3 protein levels compared to that of normal controls; treatment of GnRH-antagonist and flutamide down-regulated INSL3 protein to an undetectable level; supplementation with testosterone restored INSL3 protein levels that were 7-fold reduced from the irradiated-only group. As a first step to determining if the down-regulation of Insl3 mRNA level by androgen suppression is direct or indirect, irradiated rats were treated with GnRH-antagonist + flutamide for 1, 3, and 7 days, respectively. One-day androgen suppression induced only a 1.3-fold reduction in Insl3 mRNA level compared to irradiated-only rats; 3 days of androgen suppression resulted in 4-fold Insl3 mRNA reduction; and 7 days androgen depletion caused a 10-fold reduction in Insl3 mRNA level. Meanwhile, one day GnRH-antagonist + flutamide reduced INSL3 protein 1.7-fold, and both 3 days and 7 days androgen depletion suppressed INSL3 protein to undetectable levels. Measurement of intratesticular testosterone levels will give us more insight on whether Insl3 is directly or indirectly regulated by androgen. Similar regulation of Insl3 mRNA by androgen has been observed in germ cell depleted (e.g. jsd) mice treated with GnRH-antagonist plus flutamide or testosterone. Thus INSL3 levels are associated with spermatogenesis arrest/recovery in at least two different pathogenic models. Although INSL3 plays a major role in testicular descent during embryonic development by acting on the gubernaculum, little is known about its function in the adult. Further characterization of INSL3 regulation will help us understand the role of INSL3 in the recovery of spermatogenesis after toxicant treatment of adult mammals. (Supported by Grant ES-08075 from NIEHS and HD-40397 from NICHD)

The effect of sodium valproate on the biochemical parameters of reproductive function in male albino Wistar rats

Objective:To assess the effects of sodium valproate on intratesticular testosterone and lactic dehydrogenase level in rats.Methods:Male Wistar rats (12 weeks old) were treated with sodium valproate and sacrificed at the end of the 2nd, 4th, 5th, 7th, 10th and 15th week, after the last exposure to sodium valproate. The testes were removed, weighed and processed for biochemical analysis.Results:The intratesticular testosterone level was significantly (P<0.001) reduced in 200 mg/kg and 400 mg/kg treated rats. The intratesticular lactate dehydrogenase (LDH) level was significantly (P<0.001) increased by valproate in a time dependent manner.Conclusion:Valproate causes reversible change in intratesticular testosterone and LDH level.

Transcripts of testicular gonadotropin-releasing hormone, steroidogenic enzymes, and intratesticular testosterone levels in infertile men

To investigate the expressions of testicular GnRH and steroidogenic enzymes and their correlations with intratesticular T levels (ITT) and serum hormonal parameters in infertile men. Prospective case study. University reproductive laboratory and clinics. Thirty-four azoospermic men. The mRNA transcript levels of GnRH-I, GnRH-II, GnRH-R, and five steroidogenic enzymes in the testes of azoospermic men were determined by quantitative real time polymerase chain reaction. The ITT level was determined by radioimmunoassay. Transcript levels of genes and ITT determination. The mRNA transcript levels of GnRH-I, GnRH-II, GnRH-R, cytochrome P450 side-chain cleavage (CYP11A1), and 3beta-hydroxy-steroid dehydrogenase type 2 enzyme (HSD3B2) as well as the ITT levels were significantly increased in patients with spermatogenic failure, especially in men with Sertoli cell-only syndrome. GnRH-I and -II mRNA transcript levels positively correlated with HSD3B2 mRNA transcript levels, ITT levels, and serum FSH levels. Increased testicular GnRH transcripts, steroidogenic enzyme transcripts, and ITT levels are associated with spermatogenic failure in infertile men. Testicular GnRH is involved in the regulation of human spermatogenesis and steroidogenesis.

Estrogen receptor-α mediates estrogen-inducible abnormalities in the developing penis

Previously, we reported an association between estrogen receptor-alpha (ERalpha) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERalpha knockout (ERalphaKO) mouse model to test the hypothesis that ERalpha mediates DES effects in the developing penis. ERalphaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 microg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERalphaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wild-type/DES group. ERalphaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERalphaKO mice when compared with controls, although testosterone concentration was much higher in the ERalphaKO mice. Hence, the resistance of ERalphaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERalpha in mediating the harmful effects of neonatal DES exposure in the developing penis.

Role of Androgens in Fetal Testis Development and Dysgenesis

This study sought to establish whether reduced androgen levels/action in the fetal rat testis induced by di(n-butyl) phthalate (DBP) contributes to dysgenetic features, namely reduced Sertoli cell number, occurrence of multinucleated gonocytes (MNG), and Leydig cell aggregation. Pregnant rats were administered treatments or cotreatments designed to manipulate testosterone levels [DBP, testosterone propionate (TP)] or action [flutamide, 7,12-dimethyl-benz[a]anthracene (DMBA)]. The aforementioned end points were analyzed and related to intratesticular testosterone (ITT) levels and peripheral androgen action (anogenital distance). Dysgenetic features were also evaluated in mice with inactivation of the androgen receptor (testicular feminized or ARKO mice). Exposure to DBP alone, or combined with flutamide, DMBA, or TP, resulted in reduced Sertoli cell number and ITT levels, as did exposure to TP alone; coadministration of DBP + TP caused the most severe reduction in both parameters. A positive correlation between ITT levels and Sertoli cell number was found (r = 0.791; P = 0.019). Similarly, exposure to DBP alone, or as a cotreatment, significantly increased occurrence of MNG and Leydig cell aggregation, and these were negatively correlated with ITT levels. Exposure to flutamide or DMBA alone had no significant effect on these dysgenetic end points. These findings suggest that reduced ITT decreases fetal Sertoli cell numbers and might be involved in Leydig cell aggregation and MNG. However, of these three end points, only Sertoli cell number was affected significantly in ARKO/testicular feminized mice with absent androgen action. Therefore, induction of MNG and Leydig cell aggregation might result from DBP-induced effects other than suppression of ITT levels.

Effects of Monobutyl and Di( n -butyl) Phthalate in Vitro on Steroidogenesis and Leydig Cell Aggregation in Fetal Testis Explants from the Rat: Comparison with Effects in Vivo in the Fetal Rat and Neonatal Marmoset and in Vitro in the Human

BackgroundCertain phthalates can impair Leydig cell distribution and steroidogenesis in the fetal rat in utero, but it is unknown whether similar effects might occur in the human.ObjectivesOur aim in this study was to investigate the effects of di(n-butyl) phthalate (DBP), or its metabolite monobutyl phthalate (MBP), on testosterone production and Leydig cell aggregation (LCA) in fetal testis explants from the rat and human, and to compare the results with in vivo findings for DBP-exposed rats. We also wanted to determine if DBP/MBP affects testosterone production in vivo in the neonatal male marmoset.MethodsFetal testis explants obtained from the rat [gestation day (GD)19.5] and from the human (15–19 weeks of gestation) were cultured for 24–48 hr with or without human chorionic gonadotropin (hCG) or 22R-hydroxycholesterol (22R-OH), and with or without DBP/MBP. Pregnant rats and neonatal male marmosets were dosed with 500 mg/kg/day DBP or MBP.ResultsExposure of rats in utero to DBP (500 mg/kg/day) for 48 hr before GD21.5 induced major suppression of intratesticular testosterone levels and cytochrome P450 side chain cleavage enzyme (P450scc) expression; this short-term treatment induced LCA, but was less marked than longer term (GD13.5–20.5) DBP treatment. In vitro, MBP (10−3 M) did not affect basal or 22R-OH-stimulated testosterone production by fetal rat testis explants but slightly attenuated hCG-stimulated steroidogenesis; MBP induced minor LCA in vitro. None of these parameters were affected in human fetal testis explants cultured with 10−3 M MBP for up to 48 hr. Because the in vivo effects of DBP/MBP were not reproduced in vitro in the rat, the absence of MBP effects in vitro on fetal human testes is inconclusive. In newborn (Day 2–7) marmosets, administration of a single dose of 500 mg/kg MBP significantly (p = 0.019) suppressed blood testosterone levels 5 hr later. Similar treatment of newborn co-twin male marmosets for 14 days resulted in increased Leydig cell volume per testis (p = 0.011), compared with co-twin controls; this is consistent with MBP-induced inhibition of steroidogenesis followed by compensatory Leydig cell hyperplasia/hypertrophy.ConclusionsThese findings suggest that MBP/DBP suppresses steroidogenesis by fetal-type Leydig cells in primates as in rodents, but this cannot be studied in vitro.

Obesity and Male Reproductive Potential

Obesity and Male Reproductive Potential

Dacarbazine induces genotoxic and cytotoxic germ cell damage with concomitant decrease in testosterone and increase in lactate dehydrogenase concentration in the testis

Treatment of cancers with cytotoxic agents such as alkylating drugs often, but not always results in transient to permanent testicular dysfunction. The present study was planned to investigate the effects of dacarbazine [5-(3,3-dimethyltriazeno) imidazole-4-carboxamide] on testicular function in mice. Swiss albino mice (9–12 weeks old) were treated with 0, 5, 25, 50, or 100 mg/kg body weight/day dacarbazine (i.p.) for 5 days at intervals of 24 h between treatments. Mice were sacrificed on days 7, 14, 21, 28, 35, 49, and 70 after the last treatment (6 mice/dose/sample time), and the epididymal sperm count, sperm motility, sperm morphology, testicular histopathology (qualitative histopathology, seminiferous tubular diameter and epithelial height), and intra-testicular levels of testosterone and lactate dehydrogenase were assessed. Dacarbazine decreased the body weight only on day 28 at 25 mg/kg dose-level, but increased the paired testes weights at 50 mg/kg on day 7, at 25–100 mg/kg on day 14, and at 25 and 50 mg/kg on day 21 ( P < 0.05–0.01; one-way ANOVA and Bonferroni's post hoc test). The sperm count was decreased on all sampling days except at 5 and 25 mg/kg dose-levels on day 70, but with severe oligospermia on days 28 and 35 ( P < 0.05–0.001). The sperm motility was decreased at 100 mg/kg on days 14 and 21, at 5, 25, and 100 mg/kg on day 28, and at all dose-levels on day 35 ( P < 0.05–0.001). Dacarbazine induced both head and tail abnormalities and some sperms with cytoplasmic droplets, but significant increase was seen in all dose groups on days 14 and 21, and at 100 mg/kg dose-level on day 35. Drug-induced epithelial sloughing was seen on days 14–35 and other histopathological changes observed were vacuoles and abnormal cells. The STD was increased at 25–100 mg/kg on day 7, at all dose-levels on day 14, at 50–100 mg/kg on days 21 and 28, but without any effects on days 35–70 ( P < 0.05–0.001), and the tubular lumen was found dilated. The SE was increased on days 7, 21 and 28 at 100 mg/kg and on day 14 at 50–100 mg/kg. Dacarbazine reduced the intra-testicular testosterone level at 100 mg/kg on day 7, at 5, 50 and 100 mg/kg on day 14, at all dose-levels on days 21, 28, and 35, and at 50 mg/kg on day 49 ( P < 0.05–0.001). The intra-testicular lactate dehydrogenase concentration increased at all dose-levels up to day 35, but without any effect on days 49 and 70 ( P < 0.05–0.001). There was no particular dose–response of dacarbazine on any parameters tested. The sperm count (except on day 7-positive correlation; Pearson product moment correlation) or sperm motility did not have any relation but increase in abnormal sperms showed negative correlation with decrease in testosterone level on days 7, 21 and 28. Decrease in sperm count was in negative correlation on days 14 and 35, and increase in abnormal sperms showed positive correlation on day 35 with increase in LDH level. Finally, the decrease in sperm motility had no correlation with increase in abnormal sperm shapes. We conclude that dacarbazine is genotoxic and cytotoxic to the mouse testis in a transient fashion, and these effects are exerted along with decrease in testosterone and increase in lactate dehydrogenase levels in the testis.

Estradiol treatment induces testicular oxidative stress and germ cell apoptosis in rats

In order to understand the pathogenesis of estradiol induced effects in the seminiferous epithelium, studies were undertaken in adult rats with estradiol-3-benzoate administered for different durations. After 30 d of treatment, a significant rise in lipid peroxidation with concomitant fall in the activities of superoxide dismutase and catalase was observed. Both, serum and intra-testicular testosterone levels were found severely depleted. Seminiferous epithelium was devoid of elongated spermatids and spermatozoa by 30 d of treatment. Number of spermatocytes and round spermatids were significantly (p < 0.001) reduced. Flowcytometric analysis confirmed a drastic reduction of the haploid cell population (1c peak). Beginning from day 10 of treatment, there was a consistent rise in the number of pyknotic/apoptotic germ cells in the seminiferous epithelium. A gradual increase in Bax protein expression was observed with the duration of treatment. The shift in Bax immunostaining from the cytoplasm and nucleus of germ cells (at 10 d of treatment) to only nuclei of cells by 30 d of treatment was also noticed. By this time testicular tissue showed three-fold increase in caspase-8 enzyme activity. Viable testicular cells isolated in vitro decreased drastically subsequent to different periods of estradiol treatment. The above findings substantiate the fact that the testicular pathogenesis of estradiol benzoate treatment may be primarily because of altered reproductive hormone levels and high oxidative stress leading to germ cell apoptosis and subsequent germ cell loss in the seminiferous epithelium.

AN ORGANOPHOSPHATE INSECTICIDE METHYL PARATHION (O- O- DIMETHYL O-4-NITROPHENYL PHOSPHOROTHIOATE) INDUCES CYTOTOXIC DAMAGE AND TUBULAR ATROPHY IN THE TESTIS DESPITE ELEVATED TESTOSTERONE LEVEL IN THE RAT

Methyl parathion (MP) is an organophosphate pesticide used in agriculture, although quite often illegally used indoors to contain insects. The present study was planned to investigate the effects of MP on rat testis. Adult male Wistar rats (13-14 weeks) were treated with MP as follows. Experiment 1-0, 1.75, 3.5 or 7 mg/kg i.p. for 5 days and sacrificed on Day 14; experiment 2 and 3- 0, 0.5, or 1 mg/kg i.p. for 12 days, and sacrificed on Days 130 and 77, respectively; experiment 4- 0, 0.75, or 1.5 mg/kg i.p. for 25 days, and sacrificed on Day 17; experiment 5- 0 or 3.5 mg/kg po for 25 days, and sacrificed on Day 17, after the last exposure. MP decreased the body weight and the testis weight in experiments 4 and 5 (p<0.05-0.001) due to decreased food intake and tubular atrophy respectively. MP increased the intra-testicular testosterone level and decreased the LH level in experiments 4 and 5. The seminiferous epithelium showed sloughing of germ cells, vacuoles, focal necrosis, and formation of multinucleated giant cells, cellular degeneration (nuclear pyknosis, halo appearance and shrinkage of nuclei) and tubular atrophy, especially in experiment 4. The degree of testicular damage was higher in experiment 4>5>1>3>2 indicating more effect of prolonged i.p. treatment. Homogenization-resistant spermatid count was decreased in experiments 1, 4 and 5, and MP also decreased the tubular diameter, and epithelial height (p<0.05-0.001). Incidences of stage XIV tubules, number of meiotic figures and elongating spermatids were also decreased, whereas the incidence of tubules showing epithelial sloughing increased (p<0.05-0.001). We conclude that MP is a reproductive toxicant in male rats which causes significant testicular damage in the testis.

The Androgen Microenvironment of the Human Testis and Hormonal Control of Spermatogenesis

It is well established for both rat and man that the total testosterone concentration within the testis is far higher than that in serum. We know for the rat that intratesticular testosterone can be reduced by 50-60% without an adverse effect on spermatogenesis but that the required intratesticular testosterone concentration is still 10-fold greater than serum testosterone concentration. This kind of information, if available for the human, could prove invaluable for understanding and treating select men with infertility and in the development of male hormonal contraceptives. Unfortunately, we know little about the androgen content of intratesticular fluid within the human testis and nothing about the relationship between intratesticular androgens and human spermatogenesis. Using a newly developed minimally invasive technique for repetitive testicular sampling, our recent studies of the human have demonstrated that, as in the rat, there is a gradient between the concentration of testosterone in serum and within the testis; intratesticular testosterone levels were found to be 100-fold higher than serum testosterone levels in normal men. Using liquid chromatography tandem mass spectroscopy, we have shown that intratesticular 5alpha-dihydrotestosterone (DHT) levels are only 2% that of testosterone and, thus, despite greater affinity for the androgen receptor, intratesticular DHT is not significant in normal men. In order to assess how much of the testosterone within the human testis is bioactive, we adapted a highly sensitive recombinant protein mammalian cell-based bioassay to measure androgen bioactivity. The androgen bioactivity in the normal human testis is roughly two-thirds that of the total testosterone measurable by radioimmunoassay, despite the fact that the concentrations of the major androgen-binding proteins (sex hormone-binding globulin- and androgen-binding protein) are insufficient to account for this difference. This finding suggests that androgens may bind to other, as-yet-unknown molecules in the human testis. How, or if, this relates to spermatogenesis in the rat, or to man-to-man differences in the response to hormonal contraceptives, is not clear. We do not yet know how much testosterone is required within the human testis to either maintain or restore quantitatively normal spermatogenesis because, as yet, experimental studies comparable to those performed in the rat have not been feasible for the human.