Anogenital Distance In Male Pups Research Articles

Gestational Monosodium Glutamate Exposure Effects on Anogenital Distance of Male Rat Pups

High-dose Monosodium Glutamate (MSG) expo sure increases the estrogen level in pregnant rats. However, there are limited data available on whether the MSG-related maternal hormonal effects can affect male litters' genitalia phenotype. This study aimed to analyze the impact of MSG on estrogen level in pregnant rats and anogenital distance in male pups. Experiment for this study was performed at the animal facility of Biomedical Laboratory at the Faculty of Medicine, Universitas Andalas, from April 2019 to February 2020. Pregnant Wistar rats were given MSG orally at 2 and 4 mg/g body weight (BW) for 20 days. On day 21, pregnant rats were sacrificed and blood was drawn intracardially. Estradiol serum level was measured by ELISA. Male pups were counted, and the anogenital distance (AGD) was measured. Maternal serum estradiol levels were statistically analyzed by One-Way ANOVA and the AGD of male litters were analyzed by the Kruskal-Wallis test. Results showed that perinatal MSG exposure increased the estradiol level (26.3±4.5 pg/ml; 37.5±6.7 pg/ml; 62.1±8.2 pg/ml in control, 2 mg/g BW, and 4 mg/g BW group, respectively [mean±SD; p=<0.001]) and decreased the AGD (4 mm; 3 mm; 1.5 mm in control, 2 mg/g BW, and 4 mg/gBW group, respectively [median; p=<0.01]) in a dose-dependent manner. Thus, MSG exposure during pregnancy is a risk factor for male rat feminization.

Perfluorotridecanoic acid inhibits fetal Leydig cell differentiation after in utero exposure in rats via increasing oxidative stress and autophagy.

Perfluorotridecanoic acid (PFTrDA) is a long-chain perfluoroalkyl substance, and its effect on the differentiation of fetal Leydig cells remains unclear. The objective of this study is to explore the effect of in utero PFTrDA exposure on the differentiation of fetal Leydig cells and investigate its underlying mechanisms. Pregnant Sprague-Dawley female rats were daily administered by gavage of PFTrDA at doses of 0, 1, 5, and 10 mg/kg from gestational day 14 to 21. PFTrDA had no effect on the body weight of dams, but significantly reduced the body weight and anogenital distance of male pups at birth at a dose of 10 mg/kg. PFTrDA significantly decreased serum testosterone levels as low as 1 mg/kg. PFTrDA did not affect fetal Leydig cell number, but promoted abnormal aggregation of fetal Leydig cells at doses of 5 and 10 mg/kg. PFTrDA down-regulated the expression of Insl3, Lhcgr, Scarb1, Star, Hsd3b1, Cyp17a1, Nr5a1, and Dhh as well as their proteins. PFTrDA lowered the levels of antioxidants (SOD1, CAT, and GPX1), induced autophagy as shown by increased levels of LC3II and beclin1, and reduced the phosphorylation of mTOR. In conclusion, PFTrDA inhibits the differentiation of fetal Leydig cells in male pups after in utero exposure mainly through increasing oxidative stress and inducing autophagy.

Isolation and molecular characterization of spermatogonia from male Sprague-Dawley rats exposed in utero and postnatally to dibutyl phthalate or acrylamide

The increased incidence of testicular disorders in young men and the possible influence of environmental chemicals, such as dibutyl phthalate (DBP) and acrylamide (AA), requires experimental models for identifying modes of action. Most published reproductive toxicologic studies use RNA samples from the total testis to evaluate testicular gene expression; however, analyses of isolated cell types could provide a more specific tool. Among testicular germ cells, spermatogonia are critical since they represent the onset of spermatogenesis. This study aimed, (1) to establish a technique for spermatogonia isolation; (2) to apply this isolation technique to verify possible gene expression alterations (Pou5f1, Kitlg, Mki-67, Bak1 and Spry4) in prepubertal post-natal day, (PND24) and pubertal (PND45) testes after in utero and postnatal exposure to DBP or AA. The technique was efficient for isolation of a majority of spermatogonia. In utero DBP exposure led to reduced litter body weight at birth, reduced anogenital distance of male pups on PND4, and increased frequency of male nipple retention on PND14 compared to controls. DBP-exposed relative testes weights were reduced only at PND24 compared to control but they did not differ at PND45. DBP-exposed animals showed reduced expression levels of Pou5f1 and Mki67 on PND24, and reduced expression of Pou5f1 and Spry4 on PND45. AA exposure reduced expression of Pou5f1, Mki67, and Spry4 at PND45 although not significantly. Our results suggest that DBP acts by reducing cell proliferation and impairing differentiation in prepubertal and pubertal testes.

In utero exposure to both high- and low-dose diethylstilbestrol disrupts mouse genital tubercle development.

Exposure to estrogenic endocrine disrupting chemicals (EDCs) during in utero development has been linked to the increasing incidence of disorders of sexual development. Hypospadias, the ectopic placement of the urethra on the ventral aspect of the penis, is one of the most common DSDs affecting men, and can also affect women by resulting in the misplacement of the urethra. This study aimed to comprehensively assess the resulting hypospadias phenotypes in male and female mice exposed in utero from embryonic day 9.5 to 19.5 to the potent estrogenic endocrine disruptor, diethylstilbestrol, at a high, clinically relevant dose, and a low, previously untested dose, administered via water. The anogenital distance of male pups was significantly reduced and hypospadias was observed in males at a high frequency. Females exhibited hypospadias and urethral-vaginal fistula. These results demonstrate the ability of an estrogen receptor agonist to disrupt sexual development in both male and female mice, even at a low dose, administered via drinking water.

Effects of maternal and lactational exposure to 2-hydroxy-4-methoxybenzone on development and reproductive organs in male and female rat offspring.

2-Hydroxy-4-methoxybenzophenone (HMB) is an ultraviolet (UV) absorbing compound used in many cosmetic products as a UV-protecting agent and in plastics for preventing UV-induced photodecomposition. HMB has been detected in over 95% of randomly collected human urine samples from adults and from premature infants, and it may have estrogenic potential. To determine the effects of maternal and lactational exposure to HMB on development and reproductive organs of offspring, time-mated female Harlan Sprague-Dawley rats were dosed with 0, 1000, 3000, 10,000, 25,000, or 50,000 ppm HMB (seven to eight per group) added to chow from gestation day 6 until weaning on postnatal day (PND) 23. Exposure to HMB was associated with reduced body and organ weights in female and male offspring. No significant differences were observed in the number of implantation sites/litter, mean resorptions/litter, % litters with resorptions, number and weights of live fetuses, or sex ratios between the control and HMB dose groups. Normalized anogenital distance in male pups at PND 23 was decreased in the highest dose group. Spermatocyte development was impaired in testes of male offspring in the highest dose group. In females, follicular development was delayed in the highest dose group. However, by evaluating levels of the compound in rat serum, the doses at which adverse events occurred are much higher than usual human exposure levels. Thus, exposure to less than 10,000 ppm HMB does not appear to be associated with adverse effects on the reproductive system in rats.

Perinatal Exposure to Low Levels of the Environmental Antiandrogen Vinclozolin Alters Sex-Differentiated Social Play and Sexual Behaviors in the Rat

In this study we examined the effects of exposure to the antiandrogenic fungicide vinclozolin (Vz) on the development of two sex-differentiated behaviors that are organized by the perinatal actions of androgens. Pregnant Long-Evans rats were administered a daily oral dose of 0, 1.5, 3, 6, or 12 mg/kg Vz from the 14th day of gestation through postnatal day (PND)3. The social play behavior of juvenile offspring was examined on PND22 and again on PND34 during play sessions with a same-sex littermate. After they reached adulthood, the male offspring were examined with the ex copula penile reflex procedure to assess erectile function. Vz did not produce any gross maternal or neonatal toxicity, nor did it reduce the anogenital distance in male pups. We observed no effects of Vz on play behavior on PND22. However, the 12-mg/kg Vz dose significantly increased play behavior in the male offspring on PND34 compared with controls. The most dramatic increases were seen with the nape contact and pounce behavior components of play. The Vz effect was more pronounced in male than in female offspring. As adults, male offspring showed a significant reduction of erections at all dose levels during the ex copula penile reflex tests. The 12-mg/kg dose was also associated with an increase in seminal emissions. These effects demonstrate that perinatal Vz disrupts the development of androgen-mediated behavioral functions at exposure levels that do not produce obvious structural changes or weight reductions in androgen-sensitive reproductive organs.

Administration of potentially antiandrogenic pesticides (procymidone, linuron, iprodione, chlozolinate, p,p'-DDE, and ketoconazole) and toxic substances (dibutyl- and diethylhexyl phthalate, PCB 169, and ethane dimethane sulphonate) during sexual differentiation produces diverse profiles of reproductive malformations in the male rat.

Antiandrogenic chemicals alter sexual differentiation by a variety of mechanisms, and as a consequence, they induce different profiles of effects. For example, in utero treatment with the androgen receptor (AR) antagonist, flutamide, produces ventral prostate agenesis and testicular nondescent, while in contrast, finasteride, an inhibitor of 5 alpha-dihydrotestosterone (DHT) synthesis, rarely, if ever, induces such malformations. In this regard, it was recently proposed that dibutyl phthalate (DBP) alters reproductive development by a different mechanism of action than flutamide or vinclozolin (V), which are AR antagonists, because the male offsprings display an unusually high incidence of testicular and epididymal alterations--effects rarely seen after in utero flutamide or V treatment. In this study, we present original data describing the reproductive effects of 10 known or suspected anti-androgens, including a Leydig cell toxicant ethane dimethane sulphonate (EDS, 50 mg kg-1 day-1), linuron (L, 100 mg kg-1 day-1), p,p'-DDE (100 mg kg-1 day-1), ketoconazole (12-50 mg kg-1 day-1), procymidone (P, 100 mg kg-1 day-1), chlozolinate (100 mg kg-1 day-1), iprodione (100 mg kg-1 day-1), DBP (500 mg kg-1 day-1), diethylhexyl phthalate (DEHP, 750 mg kg-1 day-1), and polychlorinated biphenyl (PCB) congener no. 169 (single dose of 1.8 mg kg-1). Our analysis indicates that the chemicals discussed here can be clustered into three or four separate groups, based on the resulting profiles of reproductive effects. Vinclozolin, P, and DDE, known AR ligands, produce similar profiles of toxicity. However, p,p'-DDE is less potent in this regard. DBP and DEHP produce a profile distinct from the above AR ligands. Male offsprings display a higher incidence of epididymal and testicular lesions than generally seen with flutamide, P, or V even at high dosage levels. Linuron treatment induced a level of external effects consistent with its low affinity for AR [reduced anogenital distance (AGD), retained nipples, and a low incidence of hypospadias]. However, L treatment also induced an unanticipated degree of malformed epididymides and testis atrophy. In fact, the profile of effects induced by L was similar to that seen with DBP. These results suggest that L may display several mechanisms of endocrine toxicity, one of which involves AR binding. Chlozolinate and iprodione did not produce any signs of maternal or fetal endocrine toxicity at 100 mg kg-1 day-1. EDS produced severe maternal toxicity and a 45% reduction in size at birth, which resulted in the death of all neonates by 5 days of age. However, EDS only reduced AGD in male pups by 15%. Ketoconazole did not demasculinize or feminize males but rather displayed anti-hormonal activities, apparently by inhibiting ovarian hormone synthesis, which resulted in delayed delivery and whole litter loss. In summary, the above in vivo data suggest that the chemicals we studied alter male sexual differentiation via different mechanisms. The anti-androgens V, P, and p,p'-DDE produce flutamide-like profiles that are distinct from those seen with DBP, DEHP, and L. The effects of PCB 169 bear little resemblance to those of any known anti-androgen. Only in depth in vitro studies will reveal the degree to which one can rely upon in vivo studies, like those presented here, to predict the cellular and molecular mechanisms of developmental toxicity.

Critical Developmental Periods for Effects on Male Rat Genitalia Induced by Finasteride, a 5α-Reductase Inhibitor

The conversion of testosterone to 5α-dihydrotestosterone by the enzyme 5α-reductase is inhibited by finasteride. In a study in which maternal dosing with finasteride commenced on Gestational Day 15 and terminated on Postpartum Day 21, there were 13 and 27% decreases in anogenital distance of male pups on Postnatal Day 1 at 0.03 and 3 mg/kg/day, respectively. These decreases were largely reversed by Postnatal Day 22 even though treatment of the dams continued. Treatment at 3 mg/kg/day also resulted in hypospadias with cleft prepuce and a 5-day delay in the separation of the prepuce from the glans penis in those animals without hypospadias. A second study in which 20 mg/kg/day finasteride was administered on successive 2-day periods during late gestation in rats demonstrated that the period of Gestational Days 16 to 17 was the most sensitive (critical period) for finasteride-induced hypospadias, cleft prepuce, decreased anogenital distance, reduced prostate weight, and nipple formation in F1 male offspring. This critical period is just prior to the appearance on Day 18 of gestation of a midline mesenchymal plate between the urogenital sinus and the rectum in normal male fetuses. This midline plate does not appear in finasteride-exposed fetuses destined to have hypospadias as demonstrated in a previous study. Based on these observations. we hypothesize that finasteride causes hypospadias by preventing the formation of the medial mesenchymal plate which is necessary for assisting the movement of the urogenital sinus from the base to the tip of the genital tubercle.