Uptake of tritiated uteroglobin by the endometrium of the rabbit during peri-implantation

Summary Uteroglobin, labelled with N-succinimidyl-(2–3³H)-propionate, was applied in vivo for 3 h to pregnant rabbit uteri 7 and 9 days after mating. Light- and electronmicroscopic autoradiographs showed that the endometrial epithelium, both ciliated and non-ciliated cells, is able to take up³H-uteroglobin, however, with differing intensity. Large areas of labelling were found in the luminal epithelium, whereas the glandular epithelium contained fewer silver grains. Moreover, intensively labelled single cells or symplasms occurred in both luminal and glandular epithelium. They were identified as degenerating or dead cells. After internalization by pinocytosis or phagocytosis, the tritiated uteroglobin was observed in multivesicular bodies or in lysosomes with floccular content. Later, radioactivity was either found within residual bodies or distributed throughout the entire epithelium and the subepithelial stroma, i.e., the silver grains could no longer be assigned to specific cell organelles.     

Distribution of progestin-binding cells in estrogen-treated and untreated neonatal mouse uterus and oviduct: autoradiographic study with [125I]progestin

Summary Progestin-binding sites in uteri and oviducts of estrogen-treated and untreated 8-day-old mice were studied by thaw-mount autoradiography with [¹²⁵I]progestin. In the untreated uteri, nuclear concentration of radiolabelled progestin was observed in all tissues of the uterus, with strongest nuclear labelling in luminal and glandular epithelia and in stroma. In the estrogen-treated uteri, the degree of labelling was markedly augmented in stroma and muscle, but much reduced in the luminal and glandular epithelia, compared to untreated uteri. In untreated oviducts, nuclear labelling was observed in stroma and muscle in all regions and in epithelium in the isthmic and uterine regions. The epithelium in infundibular and ampullar regions was only scarcely labelled. The estrogen-treatment augmented the labelling in stroma and muscle of the oviduct as in the uterus, but the labelling in epithelium was not affected. These results indicate that estrogen-treatment induces progesteron receptor differentially among tissue compartments both in the uterus and oviduct.     

Progestagen-concentrating cells in the brain, uterus, vagina and mammary glands of the galago (Galago senegalensis)

Progestagen-concentrating cells were localized in the oestrogen-primed ovariectomized galago by radioautography after injection of [3H]promegestone (R5020). In the brain, radioactivity was concentrated in the nuclei of neurones in the preoptic region and in the mediobasal hypothalamus. Labelled cells were also observed in the anterior pituitary. In the uterus (uterine horns and cervix), the muscle and stromal cells showed greater labelling than did the glandular and luminal epithelia. Labelled cells were present in the different cell layers of the vagina. The majority of glandular epithelial cells of the mammary glands exhibited a high degree of labelling. Pretreatment with an excess of unlabelled promegestone but not with an excess of nonradioactive testosterone reduced the nuclear concentration of radioactivity in these target tissues. These results show that there are no major differences in the distribution of progestagen-concentrating cells in rodents and galago.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

Distribution of progestin-binding cells in estrogen-treated and untreated neonatal mouse uterus and oviduct: autoradiographic study with [125I]progestin

Progestin-binding sites in uteri and oviducts of estrogen-treated and untreated 8-day-old mice were studied by thaw-mount autoradiography with [125I]progestin. In the untreated uteri, nuclear concentration of radiolabelled progestin was observed in all tissues of the uterus, with strongest nuclear labelling in luminal and glandular epithelia and in stroma. In the estrogen-treated uteri, the degree of labelling was markedly augmented in stroma and muscle, but much reduced in the luminal and glandular epithelia, compared to untreated uteri. In untreated oviducts, nuclear labelling was observed in stroma and muscle in all regions and in epithelium in the isthmic and uterine regions. The epithelium in infundibular and ampullar regions was only scarcely labelled. The estrogen-treatment augmented the labelling in stroma and muscle of the oviduct as in the uterus, but the labelling in epithelium was not affected. These results indicate that estrogen-treatment induces progesterone receptor differentially among tissue compartments both in the uterus and oviduct.     

Steroidal control mechanism of cell proliferation in mouse uterine epithelium

The percentage of labeled cells in the uterine luminal epithelium of cycling mice showed the different zonal distributions at each stage of estrous cycle after cumulative labeling with 3H-thymidine for 36 hr. It was estimated that the proliferating fraction in the epithelium at proestrus, estrus, metestrus, and diestrus was 100%, 100%, 40% and 5%, respectively. The percentage of labeled cells in the uterine luminal epithelium of cycling mice treated with progesterone remained below 10% level for at least 20 hr after injections of progesterone. Total labeling was attained in the uterine epithelium of castrated mice by the administration of estradiol-17beta. On the other hand, the cell proliferation in the uterine epithelium of castrated mice treated with estradiol and progesterone was markedly suppressed and the percentage of labeled cells remained approximately at 35%. The remaining cell population, however, still showed the mitotic potency when mice received estradiol. It is suggested from this study that the effect of progesterone is to suppress the epithelial cell proliferation and transfer cells into resting cell fraction which is still evoked to proliferate as the effect of estradiol and that a key factor controlling epithelial proliferation in mouse uterus during the estrous cycle is proliferating fraction rather than cell cycle time.     

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.     

KINETICS OF CELL RENEWAL, CELL MIGRATION AND CELL LOSS IN THE HAIRLESS MOUSE DORSAL EPIDERMIS

Forty hairless mice were given injections of tritiated thymidine every 4th hour during 10 days. At 24 hr intervals groups of four mice were killed. The numbers of labelled basal and differentiating cells were determined by autoradiography with a stripping film technique. To determine the background activity skin sections from uninjected control mice were subjected to the same stripping film procedure. Another group of hairless mice was given one single pulse labelling with tritiated thymidine. The number of labelled mitoses was scored for 12 hr after the injection. At 10, 12 and 15 hr after the injection, the numbers of labelled basal and differentiating cells were also determined. A mathematical model of cell population kinetics in the epidermis has been suggested. The results of different simulations on this model were compared with the observed results. The curve of mean grain counts under continuous labelling increased from day to day with two well-defined plateaux. The percentage of all labelled cells increased rapidly up to the 3rd day, and thereafter the curves gradually flattened off. When basal cells and differentiated cells were considered separately the labelling index of the basal cells increased rapidly for the first 3 days and then flattened off at the 100% level on the 5th day. The labelling index of the differentiating cells was low during the first 3–4 days. Then a steep increase in the percentage of labelled differentiating cells was seen, but the curve flattened off again close to the 100 % level after the 7th day. The labelled mitosis curve had its maximum 5 hr after the thymidine injection. The curve fell again to almost zero at 12 hr. Ten, 12 and 15 hr after the injection, 6, 7 and 7% respectively of the labelled cells were found in the spinous layer. It was concluded that three grains over each nucleus could be used as lower limit for considering a cell as labelled. On this basis, tritiated thymidine injections every 4th hour can be considered as continuous labelling.     

Immunohistochemical investigation of secretory component and immunoglobulin A in the genital tract of the female rat

There was intense labelling of secretory component (sc) in the glandular and luminal epithelia of the uterine horns at pro-oestrus, oestrus and Day 1 of pregnancy, but at other stages labelling was weak or undetectable. There was also intense labelling of sc in the superficial layer of cells in the stratified epithelia of the cervix and vagina at pro-oestrus and Days 4-7 of pregnancy, but not at other stages. Plasma cells containing immunoglobulin A (IgA) were not observed in any region of the genital tract at any of the times studied. The presence of sc coupled with an absence of IgA-containing plasma cells suggest that IgA in genital tract secretions of the female rat may be derived mainly from serum.     

Expression of cellCAM-105 in the apical surface of rat uterine epithelium is controlled by ovarian steroid hormones

Affinity-purified antibodies to cellCAM-105, an adhesive cell surface glycoprotein, were used in immunohistochemical investigations of rat uteri at various functional stages: (i) the oestrous, pro-oestrous, metoestrous, and dioestrous stages of the oestrous cycle, (ii) Days 1-8 of normal pregnancy, (iii) delayed implantation, (iv) 18 h after oestrogen reactivation from delay of implantation, and (v) juvenile rats, and normal ovariectomized adults, respectively, before and after experimental injection of progesterone and/or oestrogen. CellCAM-105 was present in the apical zones of the luminal and glandular epithelium cells in a stage-specific and hormone-dependent manner. The results indicate that: (1) steroid hormones are essential for the expression of cellCAM-105 in the uterine epithelial cells; (2) progesterone induces cellCAM-105 expression in the glandular epithelium, and oestrogen induces cellCAM-105 expression in the luminal epithelium; (3) progesterone induces down-regulation of cellCAM-105 from the surface of the uterine luminal epithelium of juvenile rats; (4) cellCAM-105 is absent in the luminal epithelial cells but present in the glandular epithelial cells of the rat uterus at the time of blastocyst implantation.     

Progesterone and placentation increase secreted phosphoprotein one (SPP1 or osteopontin) in uterine glands and stroma for histotrophic and hematotrophic support of ovine pregnancy

Secreted phosphoprotein one (SPP1, osteopontin) may regulate conceptus implantation and placentation. We investigated effects of progesterone (P(4)) and the conceptus on expression and localization of SPP1 in the ovine uterus. Steady-state levels of SPP1 mRNA in the endometrium of unilaterally pregnant ewes did not differ significantly between nongravid and gravid horns within their respective days of pregnancy; however, levels did increase as pregnancy progressed. SPP1 mRNA was detectable in the glandular epithelium (GE) of both nongravid and gravid horns via in situ hybridization. SPP1 protein was localized to the apical surface of the luminal epithelium of both nongravid and gravid uterine horns. Gravid horns exhibited extensive stromal SPP1 on Days 40 through 120, whereas SPP1 was markedly lower in the stroma of nongravid uterine horns through Day 80 of pregnancy. By Day 120, stromal expression of SPP1 between nongravid and gravid horns was similar. Long-term P(4) treatment of ovariectomized ewes induced SPP1 in the uterine stroma and GE. A bioactive 45-kDa SPP1 fragment was purified from uterine secretions and promoted ovine trophectoderm cell attachment in vitro. Interestingly, increased stromal cell expression of SPP1 was positively associated with vascularization as assessed by von Willebrand factor staining. Finally, ovine uterine artery endothelial cells produced SPP1 during outgrowth into three-dimensional collagen matrices in an in vitro model system that recapitulates angiogenesis. Collectively, P(4) induces and the conceptus further stimulates SPP1 in uterine GE and stroma, where SPP1 likely influences histotrophic and hematotrophic support of conceptus development.     

STUDY OF CELL DEATH IN FRIEND LEUKAEMIA

Cell death of splenic Friend leukaemic cells has been studied in vivo, using ¹²⁵I-UdR and ³H-TdR pulse labelling. The evolution of the splenic specific activity has been measured by autoradiography and external counting during 40 hr after injection of the labelled precursor. These two techniques show the existence of a large reutilization of ³H-TdR (50%), which is measurable as soon as 7 hr after the injection. The DNA turnover rate is rapid, 83-8 % of the splenic cellular DNA being renewed per day. These results confirm that most of the cells produced in the Friend leukaemic spleen are rapidly lost; they also demonstrate that this cell loss is mainly due to a massive death, which occurs in proerythroblastic and erythroblastic compartments after one or two cell divisions. Friend leukaemic cells, which are characterized by a limited capacity of proliferation and a short lifespan, do not appear to be malignant.     

CIRCADIAN RHYTHMS OF PRESUMPTIVE STEM CELLS IN THREE DIFFERENT EPITHELIA OF THE MOUSE

Variation in the percentage of labelled cells (LI), mitoses (MI) and apoptosis (AI: i.e. shrinkage necrosis) have been studied throughout a 24 hr period (40 min after labelling with ³H-TdR) for tongue epithelium, epidermis and intestinal epithelium in the mouse. A room with reversed light cycle was used to obtain data for half of the 24 hr period. All three tissues showed marked variations in LI with peak values between 24.00 and 03.00 hours. In the intestine a maximum value for MI was observed 3-6 hr after that for LI and with a maximum value for AI slightly later. In all three epithelia the circadian rhythm was most striking in cells at positions which can be correlated with presumptive stem cell activity; e.g. in the crypts the labelling and mitotic peaks reflecting a circadian rhythm were most clearly distinguishable at the basal part of the crypts. These observations are discussed in relation to the validity of various proliferative models.     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Ovarian hormones regulate expression of the focal adhesion proteins,talin and paxillin,in rat uterine luminal but not glandular epithelial cells

During early pregnancy in the rat, focal adhesions disassemble in uterine luminal epithelial cells at the time of implantation to facilitate their removal so that the implanting blastocyst can invade into the underlying endometrial decidual cells. This study investigated the effect of ovarian hormones on the distribution and protein expression of two focal adhesion proteins, talin and paxillin, in rat uterine luminal and glandular epithelial cells under various hormone regimes. Talin and paxillin showed a major distributional change between different hormone regimes. Talin and paxillin were highly concentrated along the basal cell surface of uterine luminal epithelial cells in response to oestrogen treatment. However, this prominent staining of talin and paxillin was absent and also a corresponding reduction of paxillin expression was demonstrated in response to progesterone alone or progesterone in combination with oestrogen, which is also observed at the time of implantation. In contrast, the distribution of talin and paxillin in uterine glandular epithelial cells was localised on the basal cell surface and remained unchanged in all hormone regimes. Thus, not all focal adhesions are hormonally dependent in the rat uterus; however, the dynamics of focal adhesion in uterine luminal epithelial cells is tightly regulated by ovarian hormones. In particular, focal adhesion disassembly in uterine luminal epithelial cells, a key component to establish successful implantation, is predominantly under the influence of progesterone.     

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.     

EFFECTS OF UTERINE DISTENSION AND OESTRADIOL ON CELL KINETICS IN THE ENDOMETRIAL EPITHELIUM OF OVARIECTOMIZED RATS

Cell kinetics in the uterine epithelium of ovariectomized rats were studied after uterine distension and/or an oestradiol injection, by cumulative ³H-TdR labelling and percentage of labelled mitoses (PLM). With both methods it was found that distension shortens the total cell cycle at the expense of G₁ more than does oestradiol. Both treatments act in a cumulative manner since the greatest reduction in T_c is observed after distension plus oestradiol. PLM curves showed that distension and/or oestradiol induce a 30% reduction in S phase duration. The evolution of percentages of labelled cells and colchicine-blocked mitoses after these treatments confirms their additive effects and indicates that the mitogenic action of oestradiol is delayed compared to that of distension. It is suggested that these factors stimulate epithelial cell division in the uterus through partly different metabolic channels.     

Autoradiographic localization of [3H]hydroxytamoxifen to uterine oestrogen- and antioestrogen-binding sites

Summary Immature rats were injected subcutaneously with 0.36 g of [³H]hydroxytamoxifen ([³H]TAM(OH)) or 0.24 g of [³H]oestradiol in oil, and 4 h later uteri were processed for thaw-mount autoradiography. The specificity of [³H]TAM(OH) localization was determined by injecting a 200-fold excess of unlabelled TAM(OH) or a 20-, 200- or 2000-fold excess of oestradiol 1 h before injection of [³H]TAM(OH). After injection of [³H]TAM(OH) or [³H]oestradiol, autoradiograms showed concentration of radioactivity in nuclei of stromal, epithelial and myometrial cells, but this labelling varied among the cell types depending upon which compound was injected. After [³H]TAM(OH) injection, the decreasing order of labelling intensity was stroma, myometrium, epithelium; after [³H]oestradiol injection the decreasing order was stroma, epithelium, myometrium. Injection of TAM(OH) before [³H]TAM(OH) eliminated nuclear labelling in all the uterine cell types. Injection of oestradiol before [³H]TAM(OH) decreased nuclear labelling and resulted in the concentration of label in the cytoplasm of luminal epithelium which was not present when [³H]TAM(OH) was injected alone. Cytoplasmic labelling increased initially as the oestradiol competition dose increased, but the increase in labelling did not continue with increasing concentrations of oestradiol. The results indicate that antioestrogen and oestrogen localize to nuclei of the same uterine cell types, but that cellular uptake differs among the tissue compartments. The results also suggest that a high concentration of antioestrogen-binding sites exist in the cytoplasm of the uterine luminal epithelium.     

Mating modifies apoptosis pattern in epithelial cells of the rat uterus

Estrous cycle in mammals includes marked epithelial changes in reproductive tract, regulated by sex steroid hormones. In the present work we studied the activation of caspases and apoptotic pattern in uterine epithelial cells during proestrus and estrus, and the effect of mating in this process. In addition, we investigated the role of seminal vesicle secretions on apoptosis of uterine epithelia. Apoptotic index was evaluated by TUNEL assay, caspases‐8, ‐9, and ‐3 activation was detected by Western blot and active caspase‐3 expression was detected by immunohistochemistry. Our results show that mating during proestrus and estrus transition induced changes in the apoptotic pattern of uterine luminal epithelium during estrus, characterized by a delay in the onset of apoptosis as compared with that observed in nonmated rats. No differences in the apoptotic pattern in the glandular epithelium between mated and nonmated rats were observed. Seminal vesicle secretions inhibited luminal epithelium apoptosis, while no changes in glandular epithelium apoptosis were observed. We also demonstrate that activation of caspases‐8, ‐9, and ‐3 occurred in both mated and nonmated rats. Active caspase‐3 was detected in the luminal and glandular epithelium in both nonmated and mated rats. The overall results indicate that mating delays but does not prevent the cellular death of the rat uterine luminal epithelium and seminal vesicle secretions are involved in this delay. Finally, the activation of both the mitochondrial and the membrane receptor pathways of cell death are implicated in the molecular mechanism of uterine apoptosis. Mol. Reprod. Dev. 76: 564–572, 2009. © 2008 Wiley‐Liss, Inc.