Potential biological functions emerging from the different estrogen receptors.

Technological advances and new tools have brought about tremendous advances in elucidating the roles of estradiol and the estrogen receptors (ERs) in biological processes, especially within the female reproductive system. Development and analysis of multiple genetic models have provided insight into the particular functions of each of the ERs. This article reviews the insights into ER biology in female reproduction gained from the development and use of new types of experimental models.

Comparative developmental biology of the mammalian uterus.

The uterus is an essential organ for reproduction in mammals. Despite the importance of the uterus for the fertility and health of women and their offspring, relatively little is known about the hormonal, cellular, and molecular mechanisms that regulate development of the uterus in either the fetus or neonate. Disruption of uterine development in the fetus and neonate by genetic defects or exposure to endocrine disruptors can program the function of the uterus in the adult and lead to infertility, cancer, and even death. The intent of this chapter is to review the current knowledge of regulatory factors and pathways governing prenatal organogenesis and postnatal morphogenesis of the uterus in mammals, with a particular focus on laboratory and domestic animals. Prenatal organogenesis, postnatal morphogenesis, and adult functional differentiation of the uterus are complex, multifactorial processes. Although conservation of some factors and pathways are observed between species, it is clear that mutation of candidate genes in the mouse does not always recapitulate the same defects observed in the human. Therefore, comparative biology of the mechanisms regulating uterine development in other species may be useful to identify candidate genes and pathways to understand congenital abnormalities in humans. This knowledge is necessary to develop rational therapies to prevent and treat infertility and to enhance fertility in humans and domestic animals.

The IGF system in the neonatal ovine uterus.

Postnatal development of the ovine uterus primarily involves uterine gland morphogenesis or adenogenesis. Adenogenesis involves the budding differentiation of the glandular epithelium (GE) from the luminal epithelium (LE) and then GE proliferation and coiling/branching morphogenetic development within the stroma between birth (postnatal day or PND 0) and PND 56. Insulin-like growth factor (IGF)-I and IGF-II mRNAs were previously found to be expressed only in the endometrial stroma, whereas the IGF receptor (IGF-1R) mRNA was most abundant in epithelia and in stroma, suggesting that an intrinsic IGF system regulates postnatal development of the uterus. Given that the biological activities of IGFs are modulated by a family of six IGF binding proteins (IGFBPs) and specific proteases, the objective was to determine the effects of age and estrogen disruption on expression of IGFs, IGFBPs and pregnancy-associated plasma protein A (PAPP-A or IGFBP-4 protease) in the ovine uterus. In Study One, circulating levels of IGF-I and IGF-II in the serum of neonatal ewes did not change between PND 0 and PND 56. Levels of immunoreactive IGF-I, IGF-II and IGF-1R protein were most abundant on the apical surface of the endometrial LE and GE. RT-PCR analyses detected expression of IGFBPs (3, 4, 5 and 6) as well as PAPP-A mRNAs in the uterus, but not IGFBP-1 and IGFBP-2 mRNAs. IGFBP-3 and IGFBP-4 mRNAs were expressed specifically in the endometrial stroma and myometrium and increased after birth. PAPP-A mRNA was expressed specifically in the endometrial stroma and increased after birth. In Study Two, ewes were treated from birth with estradiol-17beta valerate (EV), which reduces uterine growth and inhibits endometrial adenogenesis. On PNDs 14 and 56, IGFBP-3 mRNA was decreased in the uterus of EV-treated ewes, but IGF-1R and IGFBP-4 mRNAs were not affected. PAPP-A mRNA was increased by EV treatment on PND 14, but decreased on PND 56. These results support the hypothesis that an intrinsic IGF system in the uterus regulates epithelial-stromal interactions important for postnatal uterine growth and endometrial gland morphogenesis in the sheep.

Neonatal estrogen exposure disrupts uterine development in the postnatal sheep.

Postnatal development of the ovine uterus between birth and postnatal day (PND) 56 involves budding differentiation of the endometrial glandular epithelium from the luminal epithelium (LE) followed by extensive coiling and branching morphogenesis of the tubular glands. To determine the short- and long-term effects of estrogen on neonatal ovine uterine development after PND 14, neonatal sheep were randomly assigned at birth (PND 0) to be treated daily with estradiol-17beta benzoate (EB; 0, 0.01, 0.1, 1, or 10 microg/kg body weight.d) during one of two developmental periods (PND 14-27 or 42-55). All ewes were hemiovariohysterectomized at the end of EB treatment on either PND 28 or 56, and the remaining uterine horn and ovary removed on PND 112. Immediate responses to EB treatment included dose- and age-dependent increases in uterine wet weight, thickness of the endometrium, myometrium, and LE, but decreases in endometrial glands on PND 28 and 56. Transient exposure to EB decreased gland number and thickness of the endometrium and LE on PND 112 but did not affect extrauterine reproductive tract structures. The mechanism of estrogen inhibition of uterine development did not involve effects on cell proliferation. Real-time PCR analyses found that EB exposure disrupted normal patterns of growth factor (IGF-I, IGF-II, fibroblast growth factor-7, fibroblast growth factor-10, and hepatocyte growth factor) and receptor mRNA expression in the uterus. Transient exposure of the neonatal ewe to estrogens during critical periods specifically alters growth factor networks that perturb normal development of the uterus, leading to permanent alterations in uterine structure and function.

The activin-follistatin system in the neonatal ovine uterus.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding and tubulogenesis followed by coiling and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (Postnatal Day [PND] 0) and PND 56. Activins, which are members of the transforming growth factor beta superfamily, and follistatin, an inhibitor of activins, regulate epithelial branching morphogenesis in other organs. The objective of the present study was to determine effects of postnatal age on expression of follistatin, inhibin alpha subunit, betaA subunit, betaB subunit, activin receptor (ActR) type IA, ActRIB, and ActRII in the developing ovine uterus. Ewes were ovariohysterectomized on PND 0, 7, 14, 21, 28, 35, 42, 49, or 56. The uterus was analyzed by in situ hybridization and immunohistochemistry. Neither inhibin alpha subunit mRNA or protein was detected in the neonatal uterus. Expression of betaA and betaB subunits was detected predominantly in the endometrial LE and GE and myometrium between PND 0 and PND 56. In all uterine cell types, ActRIA, ActRIB, and ActRII were expressed, with the highest levels observed in the endometrial LE and GE and myometrium. Between PND 0 and PND 14, follistatin was detected in all uterine cell types. However, between PND 21 and PND 56, follistatin was only detected in the stroma and myometrium and not in the developing GE. Collectively, the present results indicate that components of the activin-follistatin system are expressed in the developing neonatal ovine uterus and are potential regulators of endometrial gland morphogenesis.

Ovarian regulation of endometrial gland morphogenesis and activin-follistatin system in the neonatal ovine uterus.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. Previous results indicated that ovariectomy of ewes at birth did not affect uterine growth or initial stages of endometrial gland genesis on PND 14 but did affect uterine growth after PND 28. Available evidence from a number of species supports the hypothesis that the ovary does not affect endometrial gland morphogenesis in the postnatal uterus. To test this hypothesis in our sheep model, ewes were assigned at birth to a sham surgery as a control or bilateral ovariectomy (OVX) on PND 7. Uteri were removed and weighed on PND 56. Ovariectomy did not affect circulating levels of estradiol-17beta. Uterine weight was 52% lower in OVX ewes. Histomorphological analyses indicated that the thickness of the endometrium and myometrium, total number of endometrial glands, and endometrial gland density in the stratum spongiosum stroma was reduced in uteri of OVX ewes. In contrast, the number of superficial ductal gland invaginations and gland density in the stratum compactum stroma was not affected by ovariectomy. The uteri of OVX ewes contained lower levels of betaA subunit, activin receptor (ActR) type IA, ActRIB, and follistatin protein expression but higher levels of betaB subunit. In the neonatal ovary, follistatin, inhibin alpha subunit, betaA subunit, and betaB subunit were expressed in antral follicles between PNDs 0 and 56. These results led to rejection of the hypothesis that the ovary does not influence endometrial adenogenesis. Rather, the ovary and, thus, an ovarian-derived factor regulates, in part, the coiling and branching morphogenetic stage of endometrial gland development after PND 14 and expression of specific components of the activin-follistatin system in the neonatal ovine uterus that appear to be important for that critical process.

Estrogen and antiestrogen effects on neonatal ovine uterine development.

Postnatal development of the ovine uterus between birth and Postnatal Day (PND) 56 involves differentiation of the endometrial glandular epithelium from the luminal epithelium followed by tubulogenesis and branching morphogenesis. These critical events coincide with expression of estrogen receptor alpha (ERalpha) by nascent endometrial glands and stroma. To test the working hypothesis that estrogen and uterine ERalpha regulate uterine growth and endometrial gland morphogenesis in the neonatal ewe, ewes were treated daily from birth (PND 0) to PND 55 with 1) saline and corn oil as a vehicle control (CX), 2) estradiol-17 beta (E2) valerate (EV), an ERalpha agonist, 3) EM-800, an ERalpha antagonist, or 4) CGS 20267, a nonsteroidal aromatase inhibitor. On PND 14, ewes were hemihysterectomized, and the ipsilateral oviduct and ovary were removed. The remaining uterine horn, oviduct, and ovary were removed on PND 56. Treatment with CGS 20267 decreased plasma E2 levels, whereas EM-800 had no effect compared with CX ewes. Uterine horn weight and length were not affected by EM-800 or CGS 20267 but were decreased in EV ewes on PND 56. On PND 14 and PND 56, treatment with EV decreased endometrial thickness but increased myometrial thickness. The numbers of ductal gland invaginations and endometrial glands were not affected by CGS but were lower in EM-800 ewes on PND 56. Exposure to EV completely inhibited endometrial gland development and induced luminal epithelial hypertrophy but did not alter uterine cell proliferation. Exposure to EV substantially decreased expression of ERalpha, insulin-like growth factor (IGF) I, and IGF-II in the endometrium. Results indicate that circulating E2 does not regulate endometrial gland differentiation or development. Although ERalpha does not regulate initial differentiation of the endometrial glandular epithelium, results indicate that ERalpha does regulate, in part, coiling and branching morphogenesis of endometrial glands in the neonatal ewe. Ablation of endometrial gland genesis by EV indicates that postnatal uterine development is extremely sensitive to the detrimental effects of inappropriate steroid exposure.

Prolactin regulation of neonatal ovine uterine gland morphogenesis.

Uterine gland development or adenogenesis in the neonatal ovine uterus involves budding, proliferation, and branching morphogenesis of the glandular epithelium (GE) from the luminal epithelium (LE) between birth (postnatal day or PND 0) and PND 56. This critical developmental event is coincident with increases in serum PRL and expression of long and short PRL receptors specifically in the nascent and proliferating GE. In study one, ewes were treated with a placebo pellet as a control (CX) or a bromocryptine mesylate pellet from PNDs 0-56. On PND 56, the endometrium of bromocryptine mesylate ewes contained fewer glands, particularly in the stratum spongiosum that contained numerous coiled and branched glands in CX uteri. In study two, ewes were treated with saline as a CX or recombinant ovine PRL from PNDs 0-56. Treatment with PRL increased gland number and density on PND 14 and PND 56. In study three, expression of signal transducers and activators of transcription (STAT) 1, 3, and 5 proteins was detected in the developing glands from PNDs 7-56. In study four, Western blot analyses indicated that PRL increased levels of phosphorylated STATs 1 and 5, but not STAT 3, and phosphorylated ERK 1 and 2 MAPKs and c-Jun N-terminal kinase/stress-activated protein kinase proteins in explanted PND 28 ovine uteri. Collectively, results indicate that PRL regulates endometrial adenogenesis in the neonatal ovine uterus.