Edward V. Younglai

Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo(a)pyrene☆

Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase I enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) (n = 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52-71.5); BaP 213 (192-248), P = 0.01 compared to control; BaP plus resveratrol 83 (70-90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161-223) versus control 83 (57-93), P < 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99-121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules.

Canadian semen quality : an analysis of sperm density among eleven academic fertility centers

OBJECTIVE To determine whether sperm quality has declined among Canadian men during the past 13 years and whether there are regional differences in sperm quality. DESIGN Retrospective temporal series of cross-sectional studies. SETTING University fertility centers across Canada. PATIENT(S) Men being investigated as part of the normal infertility work-up. MAIN OUTCOME MEASURE(S) Sperm concentrations among all the samples were compared on an annual basis to assess any changes over 13 years from 1984 through 1996. RESULT(S) There were regional differences and trends in both up and down directions. Linear regression analysis of the means of each center for each year showed no significant trend. However, when all the samples were analyzed by regression analysis there was a significant downward trend. CONCLUSION(S) Linear regression analysis showed a significant downward trend in sperm concentration among 48,968 samples from Canadian men obtained from 1984 through 1996. A significant difference was seen in the mean concentrations between centers, ranging from 48.6 to 104.5 X 10(6)/mL. Secular trends in sperm density are dependent on the statistical method used for analysis.

Melatonin receptor mRNA expression in human granulosa cells.

We have shown that the melatonin receptor agonist, 2-[125I] iodomelatonin, binds to high-affinity guanine nucleotide-sensitive sites on human granulosa (HG) cell membranes. In order to confirm the presence of melatonin receptors in HG cells, we have now used a reverse transcriptase-polymerase chain reaction (RT-PCR) procedure to examine receptor subtype expression. RT-PCR studies revealed the presence of the mt1 (Mel1alpha) melatonin receptor subtype in ten single or pooled HG cell samples which were obtained from 14 patients. In contrast, expression of MT2 ( Mel1b) mRNA was observed in only two of these HG samples. DNA sequencing of the mt1 PCR product confirmed its identity with the reported human mt1 melatonin receptor. The expression of mt1 and MT2 receptor mRNA in HG cells and the reported presence of melatonin in human follicular fluid indicate a potentially important role for this hormone in regulating human ovarian and reproductive function.

Cigarette smoking and the outcomes of in vitro fertilization: measurement of effect size and levels of action

OBJECTIVE To assess whether cigarette smoking in women or men affects the outcomes of IVF-ET and at what functional levels smoking is active. INTERVENTIONS Demographic and smoking data were collected by questionnaire at the onset of consecutive treatment cycles (n = 462) and at the time of ET. In addition to routine endocrine and clinical data, circulating immunoreactive inhibin, follicular fluid E2 endometrial thickness, and morphology were assessed. Reported exposure to cigarettes was validated using a serum cotinine assay. RESULTS Serum cotinine level at the onset of treatment correlated strongly with the number of cigarettes reported (r = 0.68). The duration and dose of gonadotropin treatment was greater among active smokers than never smokers: 10.2 versus 9.2 days and 24.7 versus 19.8 ampules, respectively. Fertilization, pregnancy, and abortion rates were similar between groups. Multivariate analyses demonstrated negative correlation between female age, but no such effect was seen with female or male smoking. Sperm concentration was significantly reduced in male smokers (prewash: 108 versus 130 x 10(6); postwash: 17.1 versus 21.6 x 10(6)), although fertilization rate was unaffected (66% versus 62%). Follicular function, assessed using serum inhibin and E2, as well as follicular fluid E2 levels showed no significant difference between active smokers and never smokers. Endometrial thickness and morphology also were similar between groups. CONCLUSIONS These data suggest that among couples undergoing IVF neither female nor male smoking has a measurable deleterious effect on conception rate. Female age remains a far more potent prognostic factor in the current study. However, when all the published data are combined, a significant deleterious effect of smoking on conception is suggested, with a common odds ratio of 0.540 (95% confidence interval 0.385 to 0.757).

Up‐regulation of microRNA‐145 promotes differentiation by repressing OCT4 in human endometrial adenocarcinoma cells

MicroRNA‐145 (miR‐145) has been reported to be a tumor‐suppressing agent in several studies. It can repress pluripotency and control human embryonic stem cell differentiation by regulating the core pluripotency factor OCT4. However, it is not known whether miR‐145 can play a role in inducing tumor cell differentiation and repressing growth of tumors.

Reproductive endocrine effects of chronic lead exposure in the male cynomolgus monkey

The reproductive endocrine effects of chronic lead exposure were investigated in 9 year old male (n = 16) cynomolgus monkeys, orally dosed with lead acetate (1500 micrograms/kg/day) according to the following dosing regimens: continuous exposure from birth onward (lifetime, n = 4), beginning at postnatal day 300 (post-infancy, n = 5) and postnatal days 0 to 400 (infancy, n = 4), or vehicle only (control, n = 3). Altered Sertoli cell function was shown by a significant (P = 0.0286) decrease in the inhibin/follicle stimulating hormone (INH/FSH) ratio in both the lifetime and post-infancy groups compared to the control group. Gonadotropin releasing hormone (GnRH) stimulated levels of luteinizing hormone (LH) were significantly (P = 0.0370) lower in the lifetime group compared to the control group as determined by comparisons of the area under the curve. These data suggest that chronic lead exposure exerts a subtle effect on the pituitary as well as on Sertoli cell function.

Reproductive Toxicology of Environmental Toxicants: Emerging Issues and Concerns

Environmental toxicants comprise a number of man-made organic chemicals which may resist metabolism or their metabolites may persist in the environment and accumulate in the food chain. Some of these persistent chemicals are carried over long distances via the atmospheric transport and can have biological effects in fish, wildlife and humans. In this review the relationship between structure of these chemicals, their mode of action and their possible roles in adverse developmental and reproductive processes in humans will be discussed. The focus will be on model polychlorinated biphenyls and dioxins, organochlorines, phthalates, a constituent of cigarette smoke (benzo-a-pyrene), synthetic polymers (polybrominated diphenyl ethers and polyfluorinated compounds), and a fungicide (vinclozolin).

The short-term reproductive toxicity of cyclophosphamide in the female rat

Cyclophosphamide (CTX) is a potent ovarian toxicant. Previous studies of the acute effects of CTX in the rat have demonstrated widespread ovarian follicle atresia, reduced serum estradiol, and progesterone with normal serum LH and FSH. The present investigations demonstrate that a single injection of CTX induces ovarian toxicity that reflects the loss of growing ovarian follicles. CTX induces a sensitization of serum FSH in response to GnRH within 24 h; this sensitization is lost by 7 days, and after 14 days the animals are capable of normal mating behavior. The observed protection of primordial follicles from the acute administration of CTX under these experimental circumstances may be related to the stage of the granulosa cell cycle of these follicles.

Alterations in circulating ovarian steroids in hexachlorobenzene-exposed monkeys

Hexachlorobenzene (HCB) is a global pollutant that has been identified in human serum and ovarian follicular fluid, and its effect on ovarian function has not been adequately defined. Thus, the effects of HCB on ovarian steroidogenesis and menstrual cycle characteristics were investigated in cynomolgus monkeys (n = 16) orally dosed by gelatin capsule (0.0, 0.1, 1.0, and 10.0 mg HCB/kg b.wt./d) for 90 d (approximately three menstrual cycles). Analysis of change in menstrual cycle length for each animal revealed a dose-dependent increase (P = 0.02) in cycle length. Ovulatory levels of estradiol (E2) were significantly reduced (P = 0.02) in the highest treatment group. During ovulation induction, the area under the E2 concentration curve (AUC) was significantly (P = 0.03) suppressed in the highest treatment group. Our data demonstrate that HCB treatment, under the conditions of the present study, alters both ovarian function and menstrual cycle characteristics with a no observable adverse effect level of 1.0 mg/kg.

Dichlorodiphenylchloroethylene Elevates Cytosolic Calcium Concentrations and Oscillations in Primary Cultures of Human Granulosa-Lutein Cells

Abstract 1,1-Dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), a metabolite of DDT (1,1-dichlorodiphenyltrichloroethane), is a persistent hormonally active environmental toxicant that has been found in human serum and follicular fluid. The objective of this study was to determine whether DDE can alter free calcium ion concentrations in the cytosol ([Ca2+]cyt) of human granulosa cells. Changes in [Ca2+]cyt in single cells loaded with Fura-2 were studied using a dynamic digital Ca2+ imaging system. At a concentration of 100 ng/ml, DDE stimulated small elevations of [Ca2+]cyt accompanied by Ca2+ oscillations. At 1 μg DDE/ml, there was a biphasic Ca2+ response with marked elevations of [Ca2+]cyt over time. In Ca2+-free medium, cells showed an initial small elevation of [Ca2+]cyt, which was magnified after addition of Ca2+ to the medium. Washing the cells after DDE treatment failed to remove the elevated [Ca2+]cyt and oscillations, both of which were eliminated by addition of EGTA. ATP also induced [Ca2+]cyt elevations and oscillations, and these effects were potentiated when DDE was added. FSH induced transient [Ca2+]cyt elevations, whereas hCG caused a prolonged elevation and marked oscillations in [Ca2+]cyt. These results suggest that DDE at concentrations normally found in human tissues induces elevations in [Ca2+]cyt in granulosa-lutein cells. Our data therefore highlight a novel mechanism through which DDE can alter endocrine homeostasis and possibly act as an endocrine toxicant.