Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.     

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.     

Sex steroid receptor expression in the oviduct and uterus of sheep with estrus synchronized with progestagen or prostaglandin analogues

The objective of this study was to investigate differences in the expression of estrogen receptor-alpha (ERalpha), progesterone receptor (PR) and the proliferative indexes (Ki-67), in the uterus and oviduct of sheep with estrus synchronized either by prostaglandin analogues (Group PA, n=27) or by treatment with progestagens (Group P, n=29) on days 4 and 7 (day 0=estrus), when the embryos were collected. Immunohistochemical methods were used to quantify ERalpha, PR and Ki-67 in six superficial and deep compartments in the uterus and oviduct. The expression of ERalpha was significantly (P<0.01) lower in progestagen treated ewes than in prostaglandin analogues treated group in the luminal epithelium, superficial glands and superficial stroma in the uterus on day 4. The expression of PR was significantly lower in progesterone treated ewes than in the PA Group in the superficial gland (P<0.05) in both days studied. The lowest expression of PR was observed in the luminal caruncular epithelium and superficial glands in both treatments, obtaining the lowest levels on day 4 (P<0.05). There were significant differences between days 4 and 7 in the Ki-67 immunostaining in the luminal epithelium (P<0.01) and superficial glands (P<0.05). A higher cell proliferation was observed in the uterine epithelium (P<0.05) on day 4 in the animals treated with progestagens. Results indicate that sheep with synchronization of estrus with progestagens showed a reduction of ERalpha and PR protein expression in most of oviductal and uterine cells.     

Actions of progesterone on uterine immunosuppression and endometrial gland development in the uterine gland knockout (UGKO) ewe

In ewes, the uterine gland knockout (UGKO) phenotype is caused by neonatal exposure to norgestomet to arrest uterine gland development and produce an adult which has a uterus characterized by the lack of endometrial glands. Since endometrial glands in the sheep produce the lymphocyte-inhibitory protein, ovine uterine serpin (OvUS), an experiment was conducted with ewes of the UGKO phenotype to evaluate whether the inhibitory actions of progesterone on tissue rejection responses in utero are dependent upon the presence of endometrial glands. Control and UGKO ewes were ovariectomized and subsequently treated with either 100 mg/day progesterone or corn oil vehicle for 30 days. An autograft and allograft of skin were then placed in each uterine lumen and treatments were continued for an additional 30 days before grafts were examined for survival. All autografts survived and had a healthy appearance after histological analysis. Allografts were generally rejected in ewes treated with vehicle but were present for hormone-treated ewes, regardless of uterine phenotype. Analysis of the histoarchitecture and protein synthetic capacity of the uterus revealed that progesterone induced differentiation of endometrial glands and synthesis and secretion of OvUS in UGKO ewes. The UGKO ewes had reduced density of CD45R+ lymphocytes in the endometrial epithelium and there was a tendency for progesterone to reduce this effect in luminal epithelium. Taken together, results confirm the actions of progesterone to inhibit graft rejection response in utero. Responses of UGKO ewes to progesterone indicate that the hormone can induce de novo development and differentiation of endometrial glands, at least when skin grafts are in the uterus.     

Estrogen Responsiveness and the Estrogen Receptor during Development of the Murine Female Reproductive Tract

The developing uterus, vagina, and cervix of mice whose age ranged from 16 days of gestation to 90 days postnatal were examined for nuclear estrogen receptors (ERs) by autoradiographic and whole cell uptake techniques. ERs were present within mesenchymal cells of these organs throughout the entire period of development and maturation. By contrast, nuclear ER first became detectable by autoradiography in the epithelium of vagina and uterus at 5 and 6 days postnatal, respectively.
As a result of administration of the synthetic estrogen, diethylstilbestrol (DES), consecutively from 16 to 18 days of gestation, uterine and vaginal epithelial cell height was increased and epithelial secretory activity was elevated during the first 48 hr of postnatal life. Also, a single does of DES administered on the 2nd day after birth stimulated epithelial proliferation in the uterus as determined by ³H-thymidine incorporation. These typical estrogenic effects occurred in the absence of nuclear ER within the epithelium. Prenatal DES treatment accelerated the onset of ER activity within the epithelium by 2 to 3 days relative to controls. The possibility that certain effects of estrogen on epithelial differentiation may be mediated indirectly via ER positive mesenchymal cells is discussed.     

Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.     

Junctional adhesion molecule 2 mediates the interaction between hatched blastocyst and luminal epithelium: induction by progesterone and LIF

Background

Junctional adhesion molecule 2 (Jam2) is a member of the JAM superfamily. JAMs are localized at intercellular contacts and participated in the assembly and maintenance of junctions, and control of cell permeability. Because Jam2 is highly expressed in the luminal epithelium on day 4 of pregnancy, this study was to determine whether Jam2 plays a role in uterine receptivity and blastocyst attachment in mouse uterus.

Methodology/Principal Findings

Jam2 is highly expressed in the uterine luminal epithelium on days 3 and 4 of pregnancy. Progesterone induces Jam2 expression in ovariectomized mice, which is blocked by progesterone antagonist RU486. Jam2 expression on day 4 of pregnancy is also inhibited by RU486 treatment. Leukemia inhibitory factor (LIF) up-regulates Jam2 protein in isolated luminal epithelium from day 4 uterus, which is blocked by S3I-201, a cell-permeable inhibitor for Stat3 phosphorylation. Under adhesion assay, recombinant Jam2 protein increases the rate of blastocyst adhesion. Both soluble recombinant Jam2 and Jam3 can reverse this process.

Conclusion

Jam2 is highly expressed in the luminal epithelium of receptive uterus and up-regulated by progesterone and LIF via tyrosine phosphorylation of Stat3. Jam2 may play a role in the interaction between hatched blastocyst and receptive uterus.     

Uterine gland development begins postnatally and is accompanied by estrogen and progesterone receptor expression in the dog

During neonatal and juvenile life, mammalian uteri undergo extensive structural and functional changes, including uterine gland differentiation and development. In sheep and mice, inhibition of neonatal uterine gland development induced by progestin treatment led to a permanent aglandular uterine phenotype and adult infertility, suggesting that this strategy might be useful for sterilizing dogs and other companion animals. The goal of this study was to define temporal patterns of adenogenesis (gland development), cell proliferation, and progesterone and estrogen receptor expression in uteri of neonatal and juvenile dogs as a first step toward determining whether neonatal progestin treatments might be a feasible contraceptive approach in this species. Uteri obtained from puppies at postnatal wk 1, 2, 4, 6, or 8 were evaluated histologically and immunostained for MKI67, a marker of cell proliferation, estrogen receptor-1, and progesterone receptor. Adenogenesis was under way at 1 wk of age, as indicated by the presence of nascent glands beginning to bud from the luminal epithelium, and rapid proliferation of both luminal epithelial and stromal cells. By Week 2, glands were clearly identifiable and proliferation of luminal, glandular, and stromal cells was pronounced. At Week 4, increased numbers of endometrial glands were evident penetrating uterine stroma, even as proliferative activity decreased in all cell compartments as compared with Week 2. Whereas gland development was most advanced at Weeks 6 to 8, luminal, glandular, and stromal proliferation was minimal, indicating that the uterus was nearly mitotically quiescent at this age. Both estrogen receptor-1 and progesterone receptor were expressed consistently in uterine stromal and epithelial cells at all ages examined. In summary, canine uterine adenogenesis was underway by 1 wk of age and prepubertal glandular proliferation was essentially complete by Week 6. These results provided information necessary to facilitate development of canine sterilization strategies based on neonatal progestin treatments designed to permanently inhibit uterine gland development and adult fertility.     

Progesterone inhibits uterine gland development in the neonatal mouse uterus

Uterine glands and their secretions are required for conceptus (embryo/fetus and associated placenta) survival and development. In most mammals, uterine gland morphogenesis or adenogenesis is a uniquely postnatal event; however, little is known about the mechanisms governing the developmental event. In sheep, progestin treatment of neonatal ewes permanently ablated differentiation of the endometrial glands. Similarly, progesterone (P4) inhibits adenogenesis in neonatal mouse uterus. Thus, P4 can be used as a tool to discover mechanisms regulating endometrial adenogenesis. Female pups were treated with sesame vehicle alone as a control or P4 from Postnatal Day 2 (PD 2) to PD 10, and reproductive tracts were examined on PD 5, 10, or 20. Endometrial glands were fully developed in control mice by PD 20 but not in P4-treated mice. All other uterine cell types appeared normal. Treatment with P4 stimulated proliferation of the stroma but suppressed proliferation of the luminal epithelium. Microarray analysis revealed that expression of genes were reduced (Car2, Fgf7, Fgfr2, Foxa2, Fzd10, Met, Mmp7, Msx1, Msx2, Wnt4, Wnt7a, Wnt16) and increased (Hgf, Ihh, Wnt11) by P4 in the neonatal uterus. These results support the idea that P4 inhibits endometrial adenogenesis in the developing neonatal uterus by altering expression of morphoregulatory genes and consequently disrupting normal patterns of cell proliferation and development.     

Lectin binding sites as markers of neonatal porcine uterine development.

To identify lectin binding sites and to determine if lectin binding patterns change with age in developing neonatal porcine uterine tissues, gilts (n = 3/day) were hysterectomized on Day 0 (birth), 7, 14, 28, 42, or 56. Lectin binding was visualized in Bouin's-fixed uterine tissues with seven biotinylated lectins (ConA, DBA, PNA, RCA-I, SBA, UEA-I, and WGA) and avidin-peroxidase staining procedures. Lectin specificities were demonstrated by pre-incubating lectins with appropriate inhibitory sugars (0.2 M). Staining intensity was evaluated visually (absent, weak, moderate, or strong) for three endometrial tissues; luminal epithelium, glandular epithelium, and stroma. Staining intensities for DBA, PNA, SBA, and WGA were not affected by neonatal age. Staining with these lectins was greater in uterine epithelium (moderate or strong) than in stroma (weak). In contrast, binding patterns for ConA, UEA-I, and RCA-I were affected by neonatal age. Strong epithelial staining associated with ConA binding was observed on all days, whereas stromal ConA staining decreased in intensity from moderate to weak after Day 14. Epithelial staining with UEA-I increased from moderate to strong after Day 28, whereas stromal UEA-I staining decreased from moderate to weak after day 28. Staining with RCA-I was homogeneous for luminal epithelium and stroma but variegated for glandular epithelium on and after Day 7. These observations indicate that a variety of lectin binding sites are present in developing neonatal porcine endometrial tissues and that developmentally related alterations in the distribution and/or orientation of glycoconjugates containing alpha-D-mannose, beta-D-galactose, beta-D-acetyl-N-galactosamine, and alpha-L-fucose residues occur between birth and Day 56 as these tissues mature.     

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.     

Implantation-induced changes in uterine arylamidase localization in the rabbit, rat, hamster and guinea-pig

Localization of uterine arylamidase activity varied between species: arylamidase was found primarily in the apical aspect of uterine epithelial cells in the rabbit, hamster and non-pregnant rat; only moderate staining was observed in these animals in the endometrial stroma. By contrast, arylamidase localization was primarily stromal in the guinea-pig at all stages studied while the luminal epithelium was devoid of reactivity. In all species, uterine enzyme activity increased before implantation but decreased in the vicinity of the blastocyst once implantation had begun. A generalized increase over the entire length of the uterus was seen during the preimplantation phase in the uterine epithelium of the rabbit and in the endometrial stroma of the guinea-pig. Increase in stromal activity appeared to indicate predecidual transformations which were embryo-dependent (i.e. localized to the implantation site) in the rat, or embryo-independent (i.e. occurring throughout the uterus) in the guinea-pig. A subsequent decrease in enzyme activity occurred in the vicinity of the implanting embryo irrespective of the cell type involved (epithelium in the rabbit, stroma/decidua in the rat and guinea-pig). Since arylamidases of the type studied here are integrated membrane proteins, the uniformity of changes observed in different species may reflect profound changes in membrane properties of endometrial cells as an element of the implantation reaction.     

Relationship between cellular DNA synthesis,PCNA expression and sex steroid hormone receptor status in the developing mouse ovary,uterus and oviduct

The proliferative activities of the different cellular compartments of the developing mouse ovary, uterus, and oviduct were studied by radioautographic assessment of DNA synthesis with [³H]-thymidine labeling and by immunohistochemical staining of proliferating cell nuclear antigen (PCNA). The distributions of estrogen and progesterone receptors (ER and PR) were studied by immunohistochemical staining. The values of the PCNA positive staining indices were a little higher than that of the radioautographic labeling indices. However, linear relations were shown for the two indices. The proliferative activities were high from postnatal day 1–7 and decreased from day 14 in the different cellular compartments of the ovary. The proliferative activities were high on days 1, 3 and decreased from day 7 in the uterus and oviduct. Staining of ER and PR was very weak in the surface epithelium, stroma and large follicles of the ovary. Positive staining for ER occurred from day 14 in the uterine epithelium and from day 7 in oviductal epithelium. Positive staining for PR was observed from day 1 in both the uterine and oviductal epithelium. However, the positivity of both ER and PR occurred from postnatal day 1 in the stromal cells of the uterus and oviduct. These results suggest that the appearance of the steroid receptors differ between the different cellular compartment of the reproductive organs. The proliferative activities have an inverse relation with the expression of the steroid hormone receptors in the female reproductive organs during developmental stages. Therefore, we propose that there is an autonomous proliferation mechanism in the development of the reproductive organs or that the proliferation is moderated by factors other than steroid hormones.     

Expression of c-fos and c-jun protooncogenes in the uteri of immature mice neonatally exposed to diethylstilbestrol

We studied the cell-type-specific and temporal expression of c-fos and c-jun protooncogenes after 17beta-estradiol (E2) stimulation in the uteri of immature 3-week-old mice neonatally exposed to diethylstilbestrol (DES), DES-mice, and the ontogenic expression of these genes in the uteri of DES-mice using immunohistochemistry and in situ hybridization. A single E2 injection induced the transient and rapid expression of c-fos mRNA and c-Fos protein in the endometrial epithelium and endothelial cells of the blood vessels in both 3-week-old vehicle-treated controls and DES-mice; a peak of mRNA expression was 2 hours after E2 injection and that of protein expression was 2 to 3 hours after the injection. The expression of c-fos mRNA and protein after E2 stimulation was lower in the DES-mice than in the control animals. There were no significant differences in the c-jun expression patterns in both experimental groups before and after the E2 injection. The E2 injection transiently down-regulated the c-jun expression in the epithelium and up-regulated it in the stroma and myometrium. The uterine epithelium of DES-mice showed much stronger c-Jun immunostaining on days 4 and 10, compared with those of controls. Neonatal DES treatment reduced c-Jun immunoreactivity in the uterine epithelium on days 4 and 10, and increased the reaction in the stroma on day 4. These results suggested that the neonatal DES treatment induces permanent changes in the c-fos expression pattern independent of the postpuberal secretion of ovarian steroids. The changes in the expression of c-fos and c-jun protooncogenes, particularly during postnatal development, are likely to play important roles in the production of uterine abnormalities in the DES-mice.     

Oviductal morphology and eggshell formation in the lizard,Sceloporus woodi

Despite a great deal of work in recent years on the structure of reptilian eggshells, few studies have examined the structure and regulation of the female reproductive tract in the formation of eggshell components, and none have examined the entire process from ovulation to oviposition. In this study, we examined oviductal structure in the oviparous lizard, Sceloporus woodi, followed changes in oviductal structure during gravidity, and determined uterine function in the formation of eggshell components. The endometrial glands of the uterus produce the proteinaceous fibers of the eggshell membrane mainly during the first 24 hours following ovulation, and the fibers are secreted intact and subsequently wrapped around the in utero eggs. Eggshell fibers of different thicknesses are layered around each egg, ranging from an inner layer of thick fibers that gradually become thinner medially and finally forms an outer layer of densely packed particulate matter. These changes in the fibrous layer are reflected by the thickness and length of fibers released from the endometrial glands. Calcium deposition occurs from 3 days following ovulation through day 14 (oviposition) and is accompanied by cellular changes in the luminal epithelium suggestive of secretory activity. Deposition of the eggshell components within the uterus occurs on all eggs simultaneously, rather than sequentially. © 1993 Wiley-Liss, Inc.     

Epidermal growth factor (EGF) in the goat uterus: immunohistochemical localization of EGF and EGF receptor and effect of EGF on uterine activity in vivo

This study examined the distribution of immunoreactive epidermal growth factor (EGF) and EGF receptor (EGF-R) in the uterus and the effects of EGF on uterine activity in goats. Immunohistochemistry of EGF and EGF-R in the uteri showed distinct staining in the luminal and glandular epithelium and slight to moderate staining in the stromal and myometrial cells. To examine possible roles of the EGF system in the regulation of uterine activity, pressure changes in the intrauterine balloon were determined after intraluminal infusion of EGF into the uterine horn. Either at estrus or diestrus (9 to 14 days after the first day of estrus), treatment with 1 or 5 microg of EGF gradually reduced uterine activity, whereas infusion of the vehicle alone had no effect. The maximum reduction in uterine activity was seen 4 h after the treatment with 1 microg of EGF (40% to 45% reduction in the area surrounded by the contraction curve and its baseline), and the activity slowly returned thereafter. These results suggest that EGF in the uterus may play a role in regulating uterine activity in goats.