Association of increased type I collagen expression and relative stromal overgrowth in mouse epididymis neonatally exposed to diethylstilbestrol

The aim of this study was to investigate the molecular changes that underlie morphological changes in the epididymis following neonatal exposure to potent synthetic estrogen, namely diethylstilbestrol (DES). Newborn male mice were subcutaneously injected with DES or endogenous estrogen, namely 17 beta-estradiol (E2) (5 microg/mouse/day), for the first 5 days. At the age of 2, 4, and 8 weeks, epididymides of the mice were dissected. Characteristic morphological abnormality, such as relative stromal overgrowth, was observed at the age of 2 weeks in the epididymis of DES-treated mice, but not in E2-treated mice. Microarray and real-time RT-PCR analyses revealed that the expression levels of procollagen type I alpha 1 (col1a1) and col1a2 genes were markedly upregulated at the age of 2 weeks in the epididymis of DES-treated mice in comparison with the control. Western blot analysis revealed that type I collagen protein expression level in epididymis of DES-treated mice was elevated at the age of 2 weeks. In situ hybridization analysis revealed that the signals of col1a1 mRNA were detected similarly throughout the stromal tissue of epididymis at the age of 2 weeks in control and DES- and E2-treated mice. The gene expression level of epididymal type III collagen (col3a1), which is found in many stromal connective tissues as well as type I collagen, did not change at the age of 2 weeks in all groups. These results suggest that the increased type I collagen expression is associated with the relative stromal overgrowth in the epididymis of DES-treated mice.     

Expression of aquaporin 9 in rat liver and efferent ducts of the male reproductive system after neonatal diethylstilbestrol exposure.

Aquaporins (AQP) have important solute transport functions in many tissues including the epididymal efferent ducts (ED) and in the liver. We investigated the effect of neonatal exposure to diethylstilbestrol (DES) on AQP9 expressions in the ED and in the liver of rats. DES was administered from day 2 to day 20 postnatally at a dose of 4,8 microg/day, and AQP9 protein and mRNA were measured by immunoblotting and real-time PCR, respectively, along with immunohistochemistry. DES caused hepatic downregulation of AQP9 at both the protein and mRNA level; however, decreased AQP9 labeling was only observed in the periportal zone. In the ED, AQP9 protein expression was increased in the DES-treated animals by 300% that could be ascribed to a widening of the ED lumen, whereas no difference was observed in AQP9 mRNA expression. Immunohistochemical findings revealed that AQP9 expression was confined to the epithelial cells of the ED. In conclusion, neonatal DES exposure appears to upregulate AQP9 channels in the ED in male rats, whereas a downregulation in the hepatic expression was observed, particularly in the periacinous area.     

Estrogen receptor-alpha knockout mice exhibit resistance to the developmental effects of neonatal diethylstilbestrol exposure on the female reproductive tract.

Data indicate that estrogen-dependent and -independent pathways are involved in the teratogenic/carcinogenic syndrome that follows developmental exposure to 17beta-estradiol or diethylstilbestrol (DES), a synthetic estrogen. However, the exact role and extent to which each pathway contributes to the resulting pathology remain unknown. We employed the alphaERKO mouse, which lacks estrogen receptor-alpha (ERalpha), to discern the role of ERalpha and estrogen signaling in mediating the effects of neonatal DES exposure. The alphaERKO provides the potential to expose DES actions mediated by the second known ER, ERbeta, and those that are ER-independent. Wild-type and alphaERKO females were treated with vehicle or DES (2 microg/pup/day for Days 1-5) and terminated after 5 days and 2, 4, 8, 12, and 20 months for biochemical and histomorphological analyses. Assays for uterine expression of the genes Hoxa10, Hoxa11, and Wnt7a shortly after treatment indicated significant decreases in DES-treated wild-type but no effect in the alphaERKO. In contrast, the DES effect on uterine expression of Wnt4 and Wnt5a was preserved in both genotypes, suggesting a developmental role for ERbeta. Adult alphaERKO mice exhibited complete resistance to the chronic effects of neonatal DES exposure exhibited in treated wild-type animals, including atrophy, decreased weight, smooth muscle disorganization, and epithelial squamous metaplasia in the uterus; proliferative lesions of the oviduct; and persistent vaginal cornification. Therefore, the lack of DES effects on gene expression and tissue differentiation in the alphaERKO provides unequivocal evidence of an obligatory role for ERalpha in mediating the detrimental actions of neonatal DES exposure in the murine reproductive tract.     

Effects of perinatal exposure to a synthetic estrogen and progestin on mammary tumorigenesis in mice

The effects of perinatal exposure to synthetic estrogens and progestins on mammary tumorigenesis were studied in female C3H/HeN/MTV + mice. Mice were treated neonatally with 0.001 microgram/day diethylstilbestrol (DES), with 15 micrograms/day 17 alpha-hydroxyprogesterone caproate (HPC), or with oil on days 1-5 of life (birth = day 1). As adults, neonatally hormone-treated mice received long-term treatment with a synthetic estrogen and progestin combination or vehicle. Animals were palpated weekly for mammary gland tumors. The effect of treatment on the probability of tumor development was examined. Neonatal treatment with a low dose of DES increased the probability of mammary-gland tumor formation, whereas neonatal treatment with HPC had a slightly protective effect on tumorigenesis. Subsequent treatment of adult mice with synthetic steroids did not affect mammary gland tumorigenesis in neonatally DES-treated or oil-treated animals. There was a significant interaction between the effect of neonatal HPC treatment and subsequent steroid treatment on mammary tumorigenesis but examination of the data indicated that this interaction was due to the protective effect of HPC in the absence of subsequent exposure to synthetic steroids and the probability of tumor appearance in mice treated with both HPC and synthetic steroids as adults did not differ from that of neonatally oil-treated controls.     

Failure to detect a second-generation effect in female mice after neonatal treatment with an estrogen (diethylstilbestrol).

Inbred female mice of the NMRI strain were treated subcutaneously with 5 micrograms diethylstilbestrol (DES) in olive oil or vehicle only for the first 5 days after birth. One group of DES-treated females was killed at the age of 8-12 weeks, and the uterine cervix and adjacent parts of the vagina and uterine horns prepared for histological studies. In all preparations, the cervical epithelial lining contained regions with heterotopic columnar epithelium (HCE) along 69-100% of the length of the common cervical canal. Ovaries from neonatally DES-treated females were grafted to 8-week-old ovariectomized control hosts and these hosts were mated to control males 2 weeks later. The hosts gave birth to normal-sized litters. The female offspring from these litters had a normal cervical epithelial lining and, in turn, gave birth to normal-sized litters. These results indicate that treatment of neonatal female mice with DES does not affect the female germ cells as far as concerns factors associated with the development of HCE or reduced fertility in the next generation.     

Persistent trefoil factor 1 expression imprinted on mouse vaginal epithelium by neonatal estrogenization

Exposure of female mice to estrogenic substances during the neonatal period induces developmental defects in the reproductive tract such as estrogen-independent persistent proliferation of the vaginal epithelium, which often leads to carcinogenesis in adulthood. In this study, several estrogen-regulated genes have been identified in the neonatal mouse vagina by DNA microarray hybridization analysis. Among the genes up-regulated in the developing vagina by a high dose of estrogen, trefoil factor 1 (TFF1), a mucin-associated gastrointestinal growth factor, showed a unique expression pattern in accordance with the irreversible changes induced by neonatal estrogenization in the vagina. Vaginal expression of TFF1 mRNA was markedly increased by estrogen in neonatal mice but not in adults, and pronouncedly intensified expression of the gastrointestinal gene was observed in the vagina of neonatally estrogenized mice even at adulthood. The specific localization of TFF1 protein in the epithelium of neonatally estrogenized vagina was confirmed by immunohistochemistry. Moreover, without any obvious alteration in the expression of gel-forming mucin genes, the lumen of the neonatally estrogenized vagina became filled with periodic-acid-Schiff-stained mucinous gel, which was possibly caused by the overexpression of TFF1. Thus, estrogen acts directly on the developing vagina in the permanent induction of TFF1 gene expression, and the gene induction does not appear to be related to hypermethylation of the cis-promoter of the TFF1 gene. TFF1 may be a useful marker for developmental estrogenization syndrome of the mouse vagina. This work was supported by a Grant-in-Aid for Encouragement of Young Scientists from the Ministry of Education, Science, Sports, and Culture, Japan, and grants from the University of Tsukuba to M. M.     

Structural and functional changes in ovaries from adult mice treated with diethylstilboestrol in the neonatal period

Ovaries from 8-week-old female NMRI mice in different stages of the oestrous cycle, or from females neonatally treated with the synthetic oestrogen diethylstilboestrol (DES; 5-10(-6) micrograms daily for 5 days), were studied histologically and for the ability to synthesize steroids from [3H]pregnenolone in vitro. Daily doses of 10(-4) micrograms DES or higher resulted in absence of corpora lutea. In ovaries lacking corpora lutea, the interstitial tissue dominated and the cells in this compartment were large with a clear cytoplasm. The steroids synthesized in ovarian homogenates were separated with thin-layer chromatography. The homogeneity of the steroids was checked in recrystallization experiments. Daily doses of 5-10(-4) micrograms DES in the neonatal period resulted in pronounced deviations in the pattern of ovarian steroids synthesized as compared with control ovaries. In DES-exposed ovaries, the synthesis of androstenedione and, above all, progesterone was high while the synthesis of 17 alpha-hydroxyprogesterone and testosterone was reduced compared with controls. These results could argue for a difference in activities of 17 alpha-hydroxylase and 17 beta-ol-dehydrogenase in ovaries from DES-treated females compared with controls. After transplantation of DES-exposed ovaries to ovariectomized control females, the steroid pattern changed to that typical for control ovaries. Control ovaries transplanted to DES-treated females had a steroid pattern similar to that of DES-exposed ovaries.     

Ovarian reproductive function after exposure to diethylstilbestrol in neonatal life

Inbred and random-bred NMRI mice were treated with diethylstilbestrol (DES, 5 micrograms per day) or vehicle (olive oil) on Days 1-5 after birth. At the age of 8 wk, females were treated with saline or eCG and hCG to induce ovulation. Ova never occurred in the ampulla of the uterine tube of saline-treated, DES-treated females when these mice were not mated. After gonadotropin treatment, ova were found in the ampulla of all olive oil-treated females and in approximately 80% of DES-treated females. The number of ovulated ova was similar in both groups. Twenty percent of gonadotropin-treated, DES-treated females had ova in the ampulla and a vaginal plug after being caged with males but none became pregnant. Ovaries from inbred control or DES-treated females were grafted to the ovarian bursa of control or DES-treated ovariectomized hosts. DES-treated hosts, carrying control or DES-exposed ovaries, never became pregnant. Control females, with control ovaries or DES-exposed ovaries, became pregnant; pregnancy rate and litter size were similar for control mice regardless of whether they were supporting DES-exposed or control ovaries. Oocytes from ovaries exposed neonatally to DES can thus give rise to apparently normal offspring. The results also indicate DES-induced nonovarian disturbances, e.g. tubal and/or endometrial function, both of which are important for fertility. In the grafting experiments, a high mortality rate was found in inbred DES-exposed females caged with males. All deaths were associated with vaginal concrements (vaginal stones) and intestinal complications.     

ADAM7 is associated with epididymosomes and integrated into sperm plasma membrane

During epididymal transit, mammalian sperm acquire selected proteins secreted by the epididymis. We previously showed that a disintegrin and metalloprotease (ADAM) 7 is expressed specifically in the epididymis and transferred to the sperm surface during epididymal transit. Here, we show that mouse ADAM7 secreted to the epididymal lumen is associated with membranous vesicles known as epididymosomes. Furthermore, we found that ADAM7 can be transferred directly from epididymal vesicles to sperm and that it is an integral plasma membrane protein in sperm. Thus, our study provides new information regarding the unique mode of secretion and interaction of ADAM7 during the epididymis-to-sperm transfer process.     

Diethylstilbestrol-induced cervical and vaginal adenosis using the neonatal mouse model

The relevance of diethylstilbestrol (DES) administration to neonatal mice as a model for human pathology attributed to the use of DES in high-risk pregnancies has been investigated, particularly with respect to cervical and vaginal changes in female offspring. Neonatal DES treatment of mice results in tonic pituitary gonadotropin release and continuous estrogen secretion by the ovary. Studies were designed to determine the effect of this altered ovarian endocrine activity on cervical and vaginal histopathology. Ovariectomy of DES-treated mice, with or without estradiol replacement, did not eliminate the lesions, nor did estrogen and progesterone administered in a regimen intended to mimic estrous cycle changes. Induction of the constant estrus state by neonatal estradiol benzoate or testosterone propionate administration or by exposure to constant light did not produce the type of vaginal or cervical changes seen in DES mice. Thus, altered ovarian function is apparently not required for the vaginal and cervical changes appearing in later life. A role for endogenous (or exogenous) ovarian hormones in the developmental progression toward normality is suggested.     

Localization and expression of ADAM2 in the dromedary camel testis,epididymis and sperm during rutting season

ADAM2 (fertilin β) is a sperm surface protein reported in several mammalian species. However, the presence of ADAM2 in the male reproductive system and sperm of the camel is not well known. The present study was to clarify the localization and expression of ADAM2 in the dromedary camel testis, epididymis and spermatozoa during rutting season using immunohistochemistry (IHC) and the quantitative real-time polymerase chain reaction (qPCR). Tissue samples were obtained from the testis (proximal and distal) and epididymis (caput, corpus, and cauda) from eight mature male camels. Epididymal and ejaculated sperms were collected from four other fertile camels. IHC analysis clearly showed the localization of ADAM2 protein in the spermatocytes and the round and elongated spermatids of the testis, in the epithelial cells along the epididymis tract, on the posterior head of the sperm within the cauda epididymis, and on the acrosomal cap of both the epididymal and ejaculated sperm. The expression of camel ADAM2 mRNA was significantly higher (P < 0.05) in the testis when compared with the epididymis. These findings may suggest an important role of ADAM2 in the fertility of male dromedary camels.     

Polyovular follicles in mouse ovaries exposed neonatally to diethylstilbestrol in vivo and in vitro

In 35-day-old C57BL/Tw female mice given daily injections of 1 microgram diethylstilbestrol (DES) for 5 days from the day of birth, a significantly higher incidence of polyovular follicles (PF) were found in the ovaries than in those of age-matched control mice. Ovaries of prepubertal mice treated neonatally with oil or DES (DES mice) showed an enhancement of ovulation and luteinization following a combined treatment with eCG and hCG. Tubal ova in DES mice treated with eCG plus hCG were surrounded by many granulosa cells. Incidence of PF in control mice was not changed by eCG plus hCG treatment. In contrast, PF incidence in DES mice was reduced by prepubertal injections of eCG plus hCG. A high incidence of PF was also found in newborn mouse ovaries transplanted for 30 days into ovariectomized adult hosts given DES injections, but not in ovaries transplanted into intact or ovariectomized DES-untreated hosts. When neonatal ovaries were cultivated in a serum-free medium containing DES for 5 days and then transplanted into ovariectomized hosts, PF were formed in the grafts, but not in DES-unexposed grafts. Oocytes from PF in DES mice were found to have a smaller capacity for fertilization when examined in vitro. The present study also demonstrated that neonatal ovaries exposed to estrogen in vivo or in vitro (which produces PF in prepubertal hosts) are capable of responding to gonadotropins given later, resulting in a reduction of PF incidence, and that exogenous estrogen acts directly on neonatal ovaries to induce PF.     

Effects of neonatal diethylstilbestrol exposure on c-fos and c-jun protooncogene expression in the mouse uterus

Quantitative and cell-type-specific expression of c-fos and c-jun genes after 17beta-estradiol (E2) stimulation, was investigated in the uteri of neonatally diethylstilbestrol (DES)-exposed and ovariectomized adult mice (neoDES-mice), employing Northern blot analysis, immunohistochemistry and in situ hybridization. The c-fos mRNA level before E2 injection (at baseline) was about 2.2-fold higher in neoDES-mice than in vehicle-treated control mice. In controls, E2 treatment transiently increased c-fos mRNA levels, showing a peak value (15.8-fold relative to the baseline) after 2 hours. In neoDES-mice, c-fos mRNA level reached a peak showing a 2.1-fold increase compared with its baseline value 1 hour after E2 injection. Immunohistochemistry and in situ hybridization revealed that c-fos protein (Fos) and mRNA are induced in the epithelium and vascular endothelium in both groups. Most uterine epithelia of neoDES-mice revealed low sensitivity to the c-fos expression after E2 administration compared with those of vehicle-treated controls, whereas few epithelia showed high c-fos mRNA expression even at baseline. The c-jun mRNA concentration in the neoDES-mice uteri at baseline was 70% of that in vehicle-treated controls. At 1 hour after E2 injection, c-jun mRNA levels increased 1.8-fold in controls and 1.3-fold in the neoDES-mice relative to each baseline value. There were no significant differences in the distribution pattern of c-jun protein (Jun) and mRNA in the uteri of either groups; E2 stimulated c-jun mRNA expression in the stromal and myometrial cells but suppressed it in the epithelial cells, whereas intensity of c-jun immunostaining increased in the three cell types. The permanent changes in the expression of estrogen-regulated protooncogenes, c-fos and c-jun genes, by neonatal DES exposure may be responsible for the wide range of abnormalities in the genital tract of mature animals.     

Histologic, morphometric, and immunocytochemical analysis of myometrial development in rats and mice: II. Effects of DES on development

The effects of diethylstilbestrol (DES) treatment on myometrial development from the prenatal to adult period were examined in rats and mice by histologic and immunocytochemical methods using anti-actin, -vimentin, and -laminin to assess cytodifferentiation of smooth muscle and fibroblastic cells, and by morphometric procedures to assess quantitatively the effect of DES on the expression of cellular orientation in the emerging inner circular myometrial layer. Neonatal rats and mice were treated with DES from day 0 (day of birth) to day 2 with dosages known to perturb myometrial development. Neonatal treatment with DES increased the degree of circular orientation within the uterine mesenchyme, an effect detectable following as little as 24 hr of DES treatment. This effect on spatial organization of the mesenchyme was followed by an increase in the thickness of the actin-positive middle layer (prospective circular myometrium) of uterine mesenchyme during days 3-15; from day 15 onward, however, the circular myometrial layer began to fragment into irregular bundles of smooth muscle, and the longitudinal myometrial layer became thinner and more irregularly organized than controls. Vimentin localization in rats treated with DES neonatally was more intense than in controls within the circularly orientated uterine mesenchyme at 5 days. By 60 days the circular and longitudinal myometrial layers of DES-treated animals showed strands and bundles of vimentin-positive cells, which were not present in controls. Both rats and mice show comparable effects of DES treatment.     

Effects of neonatal exposure to diethylstilbestrol on early mouse embryo development in vivo and in vitro

Eight-week-old female mice of the NMRI strain that had been treated neonatally with diethylstilbestrol (DES, 5 micrograms/day for five days) or not (controls) were treated with gonadotropins to induce ovulation and then were artificially inseminated. Ova or young embryos were recovered from the oviducts on the morning after insemination and on Days 2, 3, and 4. In other experiments, ova were obtained from inseminated females on the morning after ovulation and cultured in vitro. In DES-treated females, a few zygotes developed to the 4-cell stage, but no more advanced stages were seen. Under in vitro conditions, zygotes from DES-treated females developed into blastocysts and to the implantation stage, but the incidence of these stages was lower than with zygotes from controls. Our results point to an abnormal oviductal function in DES-treated females that is not compatible with early embryo survival, even though an additional zygote factor contributing to degeneration of early cleavage stages cannot be excluded.