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We previously demonstrated that in the CD-1 mouse, which exhibits a high incidence of age-related adenomyosis, neonatal exposure to tamoxifen induced premature uterine adenomyosis and was associated with abnormal development particularly of the inner myometrium. In the present study, we examined the effect of neonatal tamoxifen administration upon uterine development in the C57/BL6J mouse strain that is not known to develop uterine adenomyosis. Female C57/BL6J pups (n=20) were treated with oral tamoxifen (1 mg/kg) from age 1 to 5 days. Uteri from control and treated mice were obtained on days 5, 10, 15 and 42 of age. We examined sections histologically using image analysis and immunohistochemistry for alpha-smooth muscle actin (ACTA2, alpha-SMA), desmin, vimentin, laminin, fibronectin and oestrogen receptor-alpha (ESR1). Following tamoxifen exposure, all uteri showed inner myometrium thinning, lack of continuity, disorganisation and bundling. However, adenomyosis was not seen in any uterus. ACTA2 immunostaining was less in the circular muscle layer of treated mice. The temporal pattern of desmin immunostaining found in control mice was absent in tamoxifen-treated mice. There was no difference in the localisation of laminin or fibronectin between control and tamoxifen-treated groups. However, laminin immunostaining was reduced in the circular muscle layer of treated mice. Vimentin could not be detected in either group. In conclusion, our results demonstrate that the development of the inner myometrium is particularly sensitive to oestrogen antagonism, and is affected by steroid receptor modulation. Although tamoxifen induces inner myometrial changes including that of ACTA2, desmin, ESR1 and laminin expression in C57/BL6J neonatal mice similar to those induced in CD-1 mice, C57/BL6J mice did not develop premature adenomyosis. Thus, disruption of the development and differentiation of the inner myometrium cannot alone explain the development of tamoxifen-associated adenomyosis, and this must be dependent upon its interaction with strain-dependent factors.

We used a neonatal mouse model to examine the histogenesis of uterine adenomyosis, and to test whether adenomyosis is due to an abnormality in myometrial differentiation, or in extracellular matrix proteins expression. We also studied the effects of tamoxifen and estradiol on uterine development, myometrial differentiation, and organization. Female CD1 pups were treated with oral tamoxifen (1 mg/kg) (n=27) or estradiol (0.1 mg/kg) (n=24) from age 1 to 5 days. Uteri from control (n=27) and treated mice were obtained on days 2, 5, 10, 15, and 42 of age. We examined the sections histologically, using image analysis and immunohistochemistry for alpha-smooth muscle actin (alpha-SMA), desmin, vimentin, laminin, fibronectin, and estrogen receptor-alpha. Following tamoxifen exposure, all uteri showed adenomyosis by 6 weeks of age (seen as early as day 10). The inner myometrium showed thinning, lack of continuity, disorganization, and bundling. alpha-SMA expression was normal. Desmin expression normally showed a wave of maturation that was absent in tamoxifen-treated mice. In the estradiol group, adenomyosis was not observed. All uterine layers were normally developed, but hypertrophied. The inner myometrium retained its circular arrangement. There was no difference in the localization of laminin or fibronectin between groups (laminin expression was reduced in the tamoxifen treated uteri). Vimentin could not be detected in all groups. Our results suggest that the development of the inner myometrium is particularly sensitive to estrogen antagonism, and can be affected by steroid receptors modulation. Disruption of the inner myometrium may play a role in the development of uterine adenomyosis.

Controversy still exists regarding the involvement of relaxin (RLX) in cervical reorganization throughout parturition in the human, despite its well-known role in facilitating extensive extracellular matrix (ECM) remodeling in diverse organs. Therefore, the aim of the present study was to examine the influence of RLX and estrogen (E2) on the cervical tissue of the common marmoset monkey. Two experimental designs were used: 1) in vivo analysis of the intracervical diameter under locally applied RLX and 2) ovariectomized (ov) marmosets were treated systemically with either recombinant human (rh) RLX, E2 or rhRLX+E2 to examine their action on the cervix. In vivo-locally applied rhRLX induced a distinct and significant widening of the cervix (before: 4.8+/-1.1 mm versus after: 5.7+/-0.9 mm in diameter; P<0.030, MV+/-S.E.M.). This widening effect was most pronounced in animals without previous pregnancies. In vitro investigation of cervical tissue showed significantly increased wet weights after all three hormone treatments (E2: 0.27+/-0.07 g, RLX: 0.25+/-0.04 g, E2+RLX: 0.30+/-0.11 g; all P<0.05; MV+/-S.E.M.) versus controls (0.10+/-0.04 g). Furthermore, morphological changes such as loosening of the connective tissue structure and decline in collagen content, an increase in the number of eosinophils, increased the expression of matrix metalloproteinases (MMP1) and MMP2, as well as gene and protein expression of the RLX receptor RXFP1 could be detected in the cervical tissue after all hormone treatments, compared with controls. In summary, RLX has a potent widening effect on the cervix of the common marmoset monkey. Although E2 is not required for this RLX effect, a combined application of E2 and RLX induced the most prominent cervical ripening.

Many women using progestogen (P)-only contraceptives experience uterine bleeding problems. In clinical trials, a single low dose of mifepristone, given to Implanon users at the beginning of a bleeding episode reduced the number of bleeding days by approximately 50% compared with controls. In this study, a single dose of mifepristone was administered to etonogestrel (ENG)-exposed pseudo-pregnant mice, 5 days after artificial decidualization was induced when the endometrium showed signs of bleeding. Control mice received vehicle alone. Mice were culled 12-, 18-, 24- and 48-h post-treatment. In the continued presence of ENG, a single dose of mifepristone stimulated tissue breakdown followed by very rapid repair: most treated tissues were fully restored to the pre-decidualized state by 48 h post-treatment. During repair, proliferating cells (Ki67 immunostained) were localized to a band of cells around the basal area in breaking down tissues and to the repairing luminal epithelium and glands. Progesterone receptor-positive cells were largely localized to the basal area of the breaking down tissue in treated mice compared with decidual cells in controls. Oestrogen receptor-positive cells were observed in the repairing luminal epithelium and glands compared with the decidua and the basal region in control tissues. It is concluded that mifepristone treatment stimulates rapid restoration of luminal epithelial integrity: such action may be a key event in reducing the number of bleeding days observed in women using Implanon who were treated with a single dose of mifepristone.

The Japanese quail (Coturnix japonica) is a widely used model species for studying the roles of steroid hormones in avian sex differentiation. The aim of the present study was to elucidate the significance of estrogen receptors alpha and beta (ERalpha and ERbeta) in normal sex differentiation of the reproductive organs in the Japanese quail and in xenoestrogen-induced disruption of reproductive organ differentiation. Real-time PCR indicated that ERalpha (ESR1) mRNA is expressed in both right and left gonads and Müllerian ducts (MDs) in both sexes during early morphological differentiation. ERbeta (ESR2) transcripts were also detected in gonads and MDs, but at very low levels. Both receptor subtypes were expressed in the liver and may therefore mediate the expression of estrogen-regulated egg-yolk proteins. Aromatase mRNA was expressed at much higher levels in female than male gonads as early as embryonic day 5, indicating early sex differences in estrogen synthesis. Treatment with the ERalpha-selective agonist propyl pyrazole triol showed that frequently reported xenoestrogen effects, such as ovotestis formation, abnormal MD development, and hepatic expression of egg-yolk proteins, were induced by selective activation of ERalpha. Taken together, our results suggest that activation of ERalpha is crucial for estrogen-dependent sex differentiation of the reproductive organs and that ERalpha mediates xenoestrogen-induced toxicity during reproductive development in birds.

The molecular mechanisms underlying the control of corpus luteum lifespan in women are not fully understood. Oestradiol has various luteolytic, or luteotrophic, functions in some species, and as it is synthesised within the human corpus luteum, it is an excellent candidate molecule to be a paracrine regulator of luteal function. This study aimed to comprehensively investigate the expression, regulation and effects of oestrogen receptors (ER) in human luteal cells. Genomic oestrogen receptors ERalpha, ERbeta1 and ERbeta2 were immunolocalised in human corpora lutea from throughout the luteal phase. mRNA expression was investigated throughout the luteal phase and after luteal rescue with exogenous human chorionic gonadotrophin (hCG). The regulation of ER expression and oestradiol action was investigated in cultures of luteinised granulosa cells. ER subtypes ERbeta1 and ERbeta2 were localised throughout the luteal phase to steroidogenic cells in the human corpus luteum and cells of the surrounding stroma. Unlike follicular granulosa cells, steroidogenic cells in the corpus luteum showed minimal ERalpha immunostaining. The presence of endothelial cells in the granulosa cell layer with ERbeta1 and ERbeta2 positive nuclei was noted. ERbeta1 and ERbeta2 were differentially regulated across the luteal phase with ERbeta1 maximally expressed in the mid-luteal phase, while ERbeta2 expression was maximal in the early luteal phase. In vivo and in vitro, hCG had no long-term effect on ER expression, although in vitro hCG and oestradiol acutely down-regulated ERs. Treatment with oestradiol in vitro down-regulated 11beta-hydroxysteroid dehydrogenase type 1 and inhibin betaA subunit confirming a functional oestradiol response. These data highlight functional and differentially regulated oestradiol reception in human luteal cells.

The study presented herein was designed to test the hypothesis that reduced endogenous estrogen in the boar alters efferent duct morphology, epididymal morphology, and steroid receptor expression. Twenty-eight littermate pairs of boars were treated with Letrozole, an aromatase inhibitor, or with vehicle from 1 week of age until castration at 2 through 8 months. Efferent ducts and epididymides were examined for morphological development and steroid receptor expression. Efferent duct morphology was not different between control and Letrozole-treated animals at any examined age. Androgen receptor (AR), estrogen receptor alpha (ERalpha), and beta (ERbeta) were expressed in the epithelial cells of the efferent ducts at all ages; expression was similar in control and treated animals. Morphological development of the caput and corpus was delayed in Letrozole-treated animals, but this delay was transient since morphology was similar between control and treated animals at 8 months. The cauda did not show a delay in development, but was more developed in treated animals at 2 months. AR, ERalpha, and ERbeta were expressed in all three epididymal regions; no difference was observed between control and treated animals. In summary, estrogen appears to be important for development of the epididymis; however, the cauda may be regulated differently than the caput and corpus. Results for the efferent ducts suggest that the normally high endogenous estrogens are not required for regulation of fluid reabsorption in the boar. It also suggests that any ER activation required for maintenance of efferent duct morphology and function is normal in Letrozole-treated boars.

Marmosets are widely used, but detailed studies on localisation of endometrial oestrogen receptors alpha and beta (ER alpha and ER beta ), and the progesterone receptor (PR) are lacking. These receptors were localised and semi-quantitatively analysed throughout the ovulatory cycle, weeks 2, 3 and 4 of pregnancy and after treatment with GnRH antagonist, vascular endothelial growth factor (VEGF) Trap or ovariectomy. The PR in epithelial cells increased markedly between the mid- and late proliferative phases before declining in the mid-secretory phase and pregnancy. PR in stromal cells was present throughout the cycle and levels were maintained in pregnancy. ER alpha was present at the mid-proliferative phase and increased in glands at the late proliferative and early secretory phases, before declining at the late secretory phase and week 4 of pregnancy. Stromal ER alpha showed a similar trend, but decreased earlier, by the mid-secretory phase. ER beta was highly expressed in epithelial cells throughout the cycle and in pregnancy. In stroma, increases in ER beta expression were observed at the late proliferative phase with the staining index decreasing by half as the secretory phase progressed and in pregnancy. GnRH antagonist, VEGF Trap or ovariectomy caused significant reductions in PR and ER beta expression, but not in ER alpha when compared with the late proliferative phase of the normal cycle. Endothelial cells expressed ER beta , but not ER alpha or PR. It is concluded that the steroid receptor profile in the marmoset endometrium is generally similar to the human and should provide a useful model for studies on hormonal manipulation of the endometrium.

Breakthrough bleeding (BTB), a major side effect of long-acting progestogen (p)-only contraceptives in women, is the main reason for discontinuation of their use. To understand the mechanisms of BTB, a mouse model of endometrial breakdown and repair was adapted to evaluate the effects of long-term progestogens on the endometrium. Appropriately prepared mice received either an etonogestrel (ENG)- or levonorgestrel (LNG)-releasing subdermal implant. Forty eight hours after decidualization was induced in one uterine horn the majority of tissues were highly decidualized, designated 0 day (0d). Uteri were collected subsequently at 5-day intervals (to 45d) and both decidualized and non-decidualized horns were analysed for morphological changes, leukocyte infiltration and matrix metalloproteinase expression (MMP). In decidualized horns, large blood vessels (BV) developed and disturbance of tissue integrity was observed at 5d with substantial stromal breakdown by 10d, progressing until 25d when re-epithelialization was initiated. By 45d, the tissue was restored to its pre-decidualized state but with considerable tortuosity of the luminal epithelium. Tissue remodelling was not apparent in the non-decidualized horns before 35d, when hyperproliferation of the luminal epithelium resulted in tortuosity. Changes in morphology were similar with the two progestogens, but occurred more rapidly with LNG. Apart from macrophages, few leukocytes were present in non-decidualized horns but large infiltrates of neutrophils and uterine natural killer cells (uNK) were associated with tissue breakdown in decidualized tissue, many of these cells were MMP9-positive. MMP7 was primarily associated with tissue repair. Therefore, this model mimics some of the changes observed in endometria of women using p-only contraceptives and provides an opportunity for functional studies.

It has been widely accepted that mammalian females are born with a non-renewing, finite pool of oocytes that will be continuously cleared by atresia, with only a small proportion of them reaching ovulation. Apoptosis regulates this mass germ cell death, especially through the balance between pro- and anti-apoptotic proteins encoded by the BCL-2 gene family. The caviomorph rodent Lagostomus maximus, the South American plains viscacha, displays the highest ovulation rate known for a mammal releasing 400-800 eggs per cycle. We tested the hypothesis that in L. maximus massive polyovulation is a consequence of reduced apoptosis resulting in suppressed follicular atresia. We found that anti-apoptotic BCL-2 gene is markedly expressed in all kind of follicles from primordial to fully mature antral stages in the adult ovary of L. maximus. On the other hand, pro-apoptotic BAX gene showed weak signals or was undetectable by immunohistochemical examination. Western blot against both proteins confirmed immunohistochemical results. Screening for DNA fragmentation by TUNEL assay was conspicuously negative in ovaries from both pregnant and non-pregnant females. In addition, alpha-oestrogen receptor also showed an enhanced expression from primordial stage to fully mature antral follicles. Our results show that natural preferential expression of BCL-2 and restricted BAX expression greatly suppresses apoptosis in the ovary of L. maximus. This prevents the decline of the oocyte reserve by abolishing follicular atresia and enables the highest ovulation rate known for a mammal, 400-800 or more eggs per cycle.

A first step to elucidating the roles that steroids may play in the processes of ovarian development and early follicular growth is to identify the cell types that are likely to be receptive to steroids. Thus, cell types expressing receptors for oestrogen (alpha and beta form; ERalpha and ERbeta respectively), androgen (AR) and progesterone (PR) were determined by in situ hybridisation and immunohistochemistry in ovine ovarian tissues collected during ovarian development and follicular formation (days 26-75 of fetal life) as well as during the early stages of follicular growth. Expression of ERbeta was observed early during ovarian development and continued to be expressed throughout follicular formation and also during the early stages of follicular growth. ERbeta was identified in germ cells as well as in the granulosa cells. At the large preantral stage of follicular growth, expression of ERalpha was also consistently observed in granulosa cells. AR was first consistently observed at day 55 of fetal life in stroma cells throughout the ovary. Within the follicle, expression was observed in granulosa and thecal cells from the type-2 to -3 stage of follicular growth. PR mRNA did not appear to be expressed during ovarian development (days 26-75 of gestation). However, PR (mRNA and protein) was observed in the theca of type-3 (small preantral) and larger follicles, with mRNA -- but not protein -- observed in granulosa cells of some type-4 and 5 follicles. Expression of ERbeta, ERalpha and AR, as well as PR, was also observed in the surface epithelium and ovarian stroma of the fetal, neonatal and adult ovary. Thus, in sheep, steroid hormones have the potential to regulate the function of a number of different ovarian cell types during development, follicular formation and early follicular growth.

Uterine function is primarily controlled by the combined actions of oestrogen and progesterone working through their cognate nuclear receptors. The mechanism of establishment of pregnancy in the mare is of interest because it involves prolonged pre-attachment and conceptus migration phases, and both invasive and non-invasive placental cell types, and as such has been an important comparative model. This study characterised regulation of oestrogen (ER) and progesterone (PR) receptors in the endometrium of the mare during the oestrous cycle and early pregnancy. Endometrial tissues collected during the oestrous cycle and early pregnancy were analysed for steady-state levels of ER and PR mRNA and protein. Steady-state levels of ER and PR mRNA were highest on days 0, 17 and 20 in cyclic mares and lowest on days 11 and 14. A day-by-status interaction was detected, indicating that day 17 and day 20 pregnant mares exhibited low levels of ER and PR compared with the corresponding days of the oestrous cycle. In situ hybridisation analyses showed receptor mRNA localisation primarily in the luminal epithelium (LE), glandular epithelium (GE) and stroma around oestrus. During dioestrus and early pregnancy, receptors were not detected in the LE, and were lower in the stroma and deeper GE. Changes in hybridisation intensity in these cell types were consistent with changes in mRNA levels detected by slot-blot hybridisation. ER and PR proteins were detected in the nuclei of LE, GE and stromal cells. Consistent with results from in situ hybridisation, levels of ER and PR immunoreactivity were higher around oestrus, declined to low levels during dioestrus and remained low during early pregnancy. Results described here for temporal and spatial changes in steroid receptor gene expression in mares show the greatest similarities with those described for cattle and sheep.

The ovarian surface epithelium (OSE) plays pivotal roles during ovulation and postovulatory wound repair. In this paper we describe the proliferative activity of the OSE through the estrous cycle in adult cycling rats, by immunohistochemical detection of DNA-incorporated bromodeoxyuridine (BrdU). Immunohistochemical detection of estrogen receptor alpha (ERalpha) and progesterone receptor was also performed. The cycle of the OSE consists of a proliferative phase (that lasts for two consecutive estrous cycles) and a quiescent phase of variable duration. Cyclic changes in the OSE were related to the underlying ovarian structure. OSE areas covering growing follicles entered into the proliferative phase during the transition from proestrus to estrus, with the appearance of fast-growing class 1 follicles, destined to ovulate at the end of the current estrous cycle. A labeling index (after pulse-labeling BrdU treatment) of about 7% was maintained throughout the estrous cycle in parallel to follicle growth. Cumulative BrdU-labeling (after daily BrdU treatment) indicated that about 1/3 of the total OSE cell proliferation was related to follicle growth. Following ovulation, OSE cells covering newly-formed corpora lutea showed a labeling index of about 50% that decreased through metestrus and diestrus (about 13% and 3%, respectively), returning to basal levels by proestrus. Cumulative BrdU-labeling indicated that about 2/3 of the total proliferative activity was related to ovulation repair/luteinization. The remaining OSE covering ovarian stroma or structurally regressing corpora lutea of previous cycles showed negligible BrdU labeling. The equivalent proliferative activity found in the OSE covering newly-formed corpora lutea in indomethacin-treated rats lacking rupture of the OSE at the apex, demonstrated that ovulation-triggered proliferation was not dependent on the loss of integrity of the OSE at the ovulation site. OSE cells expressed ERalpha throughout the cycle, but no differential expression was found between proliferating and quiescent OSE areas. On the contrary, OSE cells did not express PR at any time of the cycle. These data indicate the existence of a cycle of the OSE, related to the cyclic changes in the underlying ovarian structure and strongly suggest that the proliferative activity of the OSE is regulated by local microenvironmental rather than by systemic factors.