Hari O. Goyal

Neonatal Estrogen Exposure of Male Rats Alters Reproductive Functions at Adulthood

Abstract The effects of neonatal exposure to different doses of diethylstilbestrol (DES) on the reproductive functions of male rats at adulthood were evaluated. Sprague-Dawley rats (5–8/group) received sc injections of 25 µl olive oil containing DES (Sigma Chemical Co., St. Louis, MO) at a dose of 10 µg, 1 µg, 100 ng, 10 ng, or 1 ng per rat on alternate days from Postnatal Days 2–12. Control animals received olive oil only. All animals were allowed to develop until 83–91 days of age; however, when they were 70 to 80 days old, four male rats each from the 10 µg, 1 µg, 100 ng, and control groups were cohabited with untreated 60- to 70-day-old females (1:1) for 12 days. At the end of cohabitation, both mated and unmated male rats were weighed, and blood and tissue samples were collected and processed. Results revealed that although sperm motility patterns and sperm morphology were adversely affected in the 10-µg group, other reproductive parameters, including 1) daily sperm production (DSP)/testis; 2) absolute and relative weights of the testis, epididymis, and seminal vesicle; and 3) sperm numbers in both regions of the epididymis declined significantly in a dose-dependent manner in the 10- and 1-µg groups. Conversely, in the <1-µg groups, none of these parameters (except DSP/testis and weight of the epididymis in the 100-ng group, and sperm numbers in the epididymis of the 100- and 10-ng groups) was different from controls. Generally, plasma testosterone levels decreased in the 10- and 1-µg groups, FSH level increased in the 10-µg group, and prolactin and LH levels were unaltered. In the fertility study, although each male in the 1-µg, 100-ng, and control groups produced a copulatory plug and impregnated a female, none could do so in the 10-µg group. The mean number of pups per litter was reduced to eight in the 1-µg group, in contrast to 15 each in the 100-ng and control groups. In conclusion, exposure of neonatal male rats to DES altered sperm motility patterns, sperm fertility (as evident from the reduced number of pups in the 1-µg group), and sexual behavior (as evident from the absence of copulatory plugs in the 10-µg group) and reduced weights of reproductive organs, DSP/testis, and sperm numbers in the epididymis. Whether these alterations/reductions persist in older rats (6–8 mo of age) is under investigation.

Diethylstilbestrol-Treated Adult Rats with Altered Epididymal Sperm Numbers and Sperm Motility Parameters, but Without Alterations in Sperm Production and Sperm Morphology

Abstract In this study, we characterized estrogenic effects of diethylstilbestrol (DES) on reproductive parameters in male rats to identify a minimal dose level that alters epididymal and sperm functions but has little or no effect on sperm production and/or spermatogenesis. Adult rats (five animals/group) received s.c. injections of 0.2 ml of corn oil containing DES at a rate of 1.0 mg, 200 μg, 40 μg, 8 μg, 1.6 μg, or 320 ng rat−1 day−1 for 12 days. The control group received corn oil only. DES effects were similar in the 8-μg group and higher dose groups and included significant (P ≤ 0.05) reductions in 1) absolute and relative weights of the head and body of the epididymis (EP), tail of the EP, and seminal vesicle, 2) numbers of sperm in both regions of the EP, and 3) motility characteristics in sperm collected from the tail of the EP. Conversely, no significant changes were observed in relative testis weight, daily sperm production, spermatogenesis, seminiferous epithelial height in stage VII, and sperm morphology. All of the above parameters in the 1.6-μg group (except seminal vesicle weight) and 320-ng group were comparable to those of controls. Plasma testosterone (T) level was reduced to an almost undetectable level in the ≥8-μg groups and to a very low level in the 1.6-μg group (0.35 vs. 2.36 ng/ml in controls or 320-ng group), but LH level was unaltered. In a parallel fertility study, males received DES at a rate of 40, 8, or 1.6 μg rat−1 day−1 for 12 days prior to and 12 days during cohabitation (1:1) with untreated females. Of the 15 females cohabited with treated males (5 females/dose), none in the 40-μg and 8-μg groups and 1 in the 1.6-μg group formed a copulatory plug and delivered 8 pups, in contrast to 5/5 copulatory plugs and 13–15 pups/litter in the controls. DES at a rate of 8 μg rat−1 day−1 for 12 days reduced EP weights, sperm numbers in the EP, and sperm motility patterns but caused minimal to no alterations in daily sperm production, spermatogenesis, or sperm morphology. Factors other than T, or in addition to lower T, may be responsible for DES-induced reproductive disorders (despite lower T, sperm contents and sperm motility patterns in the EP were normal in the 1.6-μg group). Deficits in EP sperm functions and/or sexual behavior (as evident from absence of copulatory plugs) probably accounted for reduced fertility in treated males.

IMMUNOLOCALIZATION OF ANDROGEN RECEPTOR AND ESTROGEN RECEPTOR IN THE DEVELOPING TESTIS AND EXCURRENT DUCTS OF GOATS

Because of the significance of androgens and estrogens in prenatal and postnatal differentiation of the testis and excurrent ducts, it is important to understand the developmental pattern of androgen receptor (AR) and estrogen receptor (ER) in these organs.

Regulation of androgen and estrogen receptors in male excurrent ducts of the goat: An immunohistochemical study

Since androgens and/or estrogens must bind with specific receptors in order to elicit a response at the target organ(s), it is important to understand factors that regulate expression of androgen receptors (AR) and estrogen receptors (ER). Hence, the objective of the study is to determine the relative significance between circulating androgen (CA) and luminal androgen (LA) in maintaining normal expression of AR and ER in male excurrent ducts.

Abnormal Morphology of the Penis in Male Rats Exposed Neonatally to Diethylstilbestrol Is Associated with Altered Profile of Estrogen Receptor-α Protein, but Not of Androgen Receptor Protein: A Developmental and Immunocytochemical Study

Abstract Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)α in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 μg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERα-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERα.

Role of estrogen in induction of penile dysmorphogenesis: a review.

In this review, we report permanent dysmorphogenesis of the penis and loss of fertility in adult rats treated neonatally with estrogen. Specifically, we report replacement of smooth muscle cells and cavernous spaces by fat cells in the corpus cavernosum penis, but not in the adjoining corpus spongiosum. Induction of these novel, region-specific phenotypes is dose-dependent, requires a critical window of exposure and associated with decreased testosterone and up-regulation of estrogen receptor alpha (ER alpha). The resistance of ER alpha knockout mice to develop these abnormalities implies an unequivocal role for ER alpha in mediating maldevelopment of the penis. Additionally, the prevention of estrogen-inducible penile abnormalities by ER antagonist ICI 182 780 implies that a functional ER-mediated pathway is essential for inducing penile abnormalities. Likewise, the ability of testosterone or dihydrotestosterone to negate these abnormalities suggests a role for an androgen receptor (AR)-mediated pathway. Taken together, these observations led us to hypothesize that neonatal estrogen exposure, via an ER-mediated pathway (direct action) or an AR-mediated pathway (indirect action through decreased testosterone) or both pathways, up-regulates ER alpha expression in stromal cells of the penis, which are then reprogrammed such that their differentiation into smooth muscle cells is inhibited and their differentiation into adipocytes is stimulated.

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.

Effects of Androgen Deprivation in the Goat Epididymis

With the deprivation of both circulating androgen (CA) and luminal androgen (LA; orchiectomized goats), the epithelial height (EH) in regions I-IV of the epididymis was reduced to 28, 67, 58 and 56% of that of controls, respectively, but it was increased to 109% of that of controls in region V. Similarly, the volume density of epithelium (VDE) in regions I-V was reduced to 33, 49, 45, 41 and 70% of that of controls, respectively. Conversely, in the absence of LA only (extratesticular-rete-ligated goats), while both EH and VDE were reduced to almost 50% of those of controls in region I, they remained similar to those of controls in other regions. The morphological changes in the epithelium such as cytoplasmic regression, loss of stereocilia and disorderly arrangement of epithelial cells were maximal in region I, moderate in regions II-IV and minimal in region V. Testosterone treatment appreciably reduced the degenerative changes caused by orchiectomy in all regions except region I where the restorations were marginal at best. Hence, the results suggest a differential epididymal response to androgen deprivation. Whereas the LA and/or other rete fluid components seem essential for maintaining the epithelial structure of region I, the CA alone can maintain, at least partially, the epithelial structure of regions II-IV and almost completely that of region V.

Estrogen-Induced Developmental Disorders of the Rat Penis Involve Both Estrogen Receptor (ESR)- and Androgen Receptor (AR)-Mediated Pathways

This study tested the hypothesis that the estrogen receptor (ESR) pathway, androgen receptor (AR) pathway, or both mediate estrogen-induced developmental penile disorders. Rat pups received diethylstilbestrol (DES), with or without the ESR antagonist ICI 182,780 (ICI) or the AR agonist dihydrotestosterone (DHT) or testosterone (T), from Postnatal Days 1 to 6. Testicular T concentration, penile morphology and morphometry, and/or fertility was determined at age 7, 28, or 150 days. DES treatment alone caused 90% reduction in the neonatal intratesticular T surge; this reduction was prevented by ICI coadministration, but not by DHT or T coadministration. Unlike the T surge, coadministration of ICI and coadministration of DHT or T mitigated penile deformities and loss of fertility. Generally, ICI, DHT, or T treatment alone did not alter penile morphology; however, fertility was 20% that of controls in ICI-treated rats vs. 70%–90% in DHT- or T-treated rats. The lower fertility in the rats treated with ICI alone could be due to altered sexual behavior, as these males did not deposit vaginal plugs. In conclusion, observations that both an ESR antagonist and AR agonists prevent penile deformities and infertility suggest that both pathways are involved in estrogen-induced penile disorders. Observations that coadministration of ICI, but not DHT or T, prevents the DES-induced reduction in the neonatal T surge suggest that, although ICI exerts its mitigating effect both at the level of Leydig cells and penile stromal cells, DHT and T do so only at the level of stromal cells.

Exposure of neonatal rats to anti-androgens induces penile mal-developments and infertility comparable to those induced by oestrogens

We previously reported that oestrogen exposure in neonatal rats induced permanent infertility and malformed penis characterized by fat accumulation, which replaced most of the smooth muscle cells and cavernous spaces in the body of the penis, structures essential for erection. The objective of this study was to determine if reduced androgen production/action in the neonatal period, in the absence of exogenous oestrogen exposure, induces penile deformities similar to those caused by oestrogen. Male rats were treated from postnatal days 1-6 with GnRH antagonist antide (A, 10 mg/kg) or androgen receptor (AR) antagonist flutamide (F, 50 mg/kg) or F + A, with or without AR agonist dihydrotestosterone (DHT, 20 mg/kg). For comparison, pups received diethylstilbestrol (DES, 0.1 mg/kg), with or without DHT. Tissues were collected at ages 7 and 12 days and at adulthood. Flutamide alone decreased penile length and weight significantly (p < 0.05), but it caused neither fat accumulation, nor affected fertility (80% vs. 87% in controls). Antide alone reduced penile length and weight significantly, and induced fat accumulation in 4/11 rats and infertility in 13/14 rats. Conversely, all 11 F + A-treated rats, similar to all nine DES-treated rats, had fat accumulation and loss of smooth muscle cells and cavernous spaces in the body of the penis and were infertile. In addition, reductions in penile length and weight were higher than in rats treated with F or A alone. DHT co-administration mitigated penile deformities in the DES group, but did not in the F + A group. Testicular testosterone was reduced by 70-95% at 7 or 12 days of age in all treated groups, except in the F group, which had threefold higher testosterone than controls. Collectively, data unequivocally show that reduced androgen production/action in the neonatal period, in the absence of oestrogen exposure, induces permanent infertility and malformed penis similar to that caused by oestrogen.