Expression of repulsive guidance molecule b (RGMb) in the uterus and ovary during the estrous cycle in rats

Repulsive guidance molecule b (RGMb; a.k.a. Dragon), initially identified in the embryonic dorsal root ganglion, is the first member of the RGM family shown to enhance bone morphogenetic protein (BMP) signaling by acting as a BMP co-receptor. BMP signaling has been demonstrated to play an important role in the reproductive organs. Our previous study found that RGMb was expressed in the reproductive axis, but whether RGMb expression in reproductive organs changes across the estrous cycle remains unknown. Here, we show in the rat that RGMb mRNA expression in the uterus was significantly higher during metesterus and diestrus than during proestrus and estrus. Western blotting indicated that RGMb protein was significantly lower during estrus compared with the other three stages. Immunohistochemistry revealed that RGMb protein was mainly localized to the uterine luminal and glandular epithelial cells of the endometrium. RGMb mRNA and protein in the ovary remained unchanged during the estrous cycle. RGMb protein was expressed in the oocytes of all follicles. Weak staining for RGMb protein was also found in corpora lutea. RGMb was not detected in granulosa cells and stromal cells. Taken together, RGMb expression in the uterus and ovary across the estrus cycle demonstrate that RGMb may be involved in the regulation of uterine function, follicular development as well as luteal activity.     

Hormonal modification of epithelial differentiation and expression of cell surface heparan sulfate proteoglycan in the mouse vaginal epithelium. An immunohistochemical and electron microscopic study

Immunohistochemical staining of a cell surface antigen was evaluated in the adult mouse vaginal epithelium at different stages of the estrous cycle and in response to exogenous sex hormones and endocrine ablation. The antigen is recognized by a monoclonal antibody directed against the core protein of a heparan sulfate-rich proteoglycan from mouse mammary epithelial cells. Vaginal epithelium at estrus showed the most intense staining; cells of the basal and intermediate layers stained, but the more superficial parakeratotic, cornified, and sloughing layers did not. At metestrus and diestrus, immunostaining was limited to basal cells and some deeper intermediate cells. The staining was absent from the more superficial layers which were invaded by leukocytes. At late diestrus and proestrus, staining was primarily in the intermediate cells; staining was absent from parakeratotic and basal cell layers. There was no staining of submucosal cells throughout the estrous cycle. In ovariectomized mice, staining of the epithelium was reduced in intensity. Diethylstilbestrol treatment of ovariectomized mice increased the intensity and extent of epithelial staining and produced a state comparable to that seen at estrous. Administration of a combination of progesterone and estradiol to ovariectomized mice elicited vaginal stratification and mucification, a state comparable to that observed in diestrus in which basal and intermediate layers stained while the apical mucified cells did not. In animals expressing natural or diethylstilbestrol-induced estrus, electron microscopic immunoperoxidase staining revealed the presence of the antigen on the surface of cell processes in the intercellular spaces between vaginal epithelial cells. Cuprolinic blue staining for glycosaminoglycan using the critical electrolyte concentration method demonstrated filamentous structures on the epithelial surface in the same location to that of the antigen. The stained filaments were reduced by treatment with heparitinase, but not with chondroitinase ABC or heparin, suggesting that they contained heparan sulfate glycosaminoglycan. These data suggest that as vaginal epithelial differentiation fluctuates during the estrous cycle in response to changing levels of estrogens and progesterone, expression of a cell surface heparan sulfate proteoglycan undergoes dramatic changes spatially and quantitatively.     

Oestrogen receptor alpha and beta mRNA expression in human endometrium throughout the menstrual cycle.

We examined the localization of oestrogen receptor (ER) beta mRNA in the human endometrium throughout the menstrual cycle using non-radioactive in-situ hybridization with Brigati-tailed oligonucleotides. The findings were compared with those of ERalpha in order to examine the possible biological significance of ERbeta in the human endometrium. Both ERalpha and ERbeta mRNA expression were detected in all major human uterine cell types, including glandular epithelial cells, stromal cells and smooth muscle cells of the uterine wall, at every menstrual cycle stage. However, ERalpha mRNA expression was more prominent than that of ERbeta in all cell types throughout the menstrual cycle. In proliferative phase endometrium, ERalpha mRNA was expressed in both glandular epithelial and stromal cells, while ERbeta mRNA was expressed predominantly in glandular epithelial cells. Although the same pattern was observed in the secretory phase, both the ERalpha and ERbeta mRNA expression was relatively weaker. These results suggest that oestrogenic effects occur predominantly through ERalpha, but that ERbeta may also play a role in the modulation of oestrogenic action, especially on glandular epithelial cells in the human endometrium throughout the menstrual cycle.     

Endometrial connexin expression in the mare and pig: Evidence for the suppression of cell-cell communication in uterine luminal epithelium

This investigation examines the relationship between implantation strategy and gap junction protein expression in uterine endometrium. The pattern of gap junction and connexin protein expression was analyzed in porcine and equine endometrium from cycling and pregnant animals using electron microscopy and immunocytochemistry. Functional analysis of cell-cell communication was also monitored by laser cytometry in primary cultures of endometrial epithelial cells. Gap junctions were detected in endometrial stroma of cycling and pregnant animals, which was correlated with immunoreactive Cx43 within stromal fibroblasts and vascular elements. No Cx26, Cx32, or Cx43 immunostaining was detected in luminal endometrial epithelium in either the mare or the pig at any stage of the estrous cycle or pregnancy. In contrast, endometrial glands of the mare exhibited a spatiotemporal pattern of Cx43 expression in the apicolateral plasma membrane which, when present, colocalized with the tight junction–associated protein, ZO-1. Uterine glandular Cx43 expression in mares was present from day 3 postovulation through day 14 of diestrus and until day 23 of pregnancy, whereas Cx43 was absent within uterine glands during seasonal anestrus, estrus, and after day 30 of pregnancy. Primary cultures of equine endometrial epithelial cells expressed both immunoreactive Cx43 and significant gap junction–mediated intercellular communication (GJIC) which was rapidly upregulated by 1.0 mM 8-bromo-cAMP or blocked with 1.0 mM octanol. No GJIC or connexin protein was detected in cultured porcine epithelial cells despite incubation with a variety of agents, including 8-bromo-cAMP, steroid hormones, retinoic acid, and/or prolactin. Junctional communication in endometrial epithelium of domestic farm animals is different than that reported for species exhibiting invasive implantation. The absence of GJIC in uterine luminal epithelium of the gilt and mare may be involved in limiting trophoblast invasiveness. Anat. Rec. 251:277–285, 1998. © 1998 Wiley-Liss, Inc.     

Ultrastructural localization of gastrin-releasing peptide (GRP) in the uterine gland of cow

Gastrin-releasing peptide (GRP) is thought to act mainly as a neurotransmitter and localized almost exclusively to neurons and neuroendocrine cells. Recently, the localization of GRP in mammalian uterus and placenta has been demonstrated. Moreover, the exocrine manner of GRP release was deduced in ewes from the distribution of GRP on the uterine gland cells and its secretion as well as in the circulation. However, these reports have been examined at light-microscopic level. The present study was designed to make clear the localization of GRP in the uterine gland cells of nonpregnant and pregnant cows using an avidin-biotin-peroxidase complex (ABC) method at light-microscopic level and a pre-embedding immunogold with silver enhancement method at electron-microscopic level. The light-microscopic observation showed positive staining for GRP immunoreactivity in the supranuclear region and in the secreted materials of the uterine gland cells. At the electron-microscopic level, the supranuclear secretory granules and the secreted materials on the surface of the cell were labeled with immunogold particles representing GRP immunoreactivity in the uterine gland cells of nonpregnant and pregnant cows. Western blotting analysis showed a larger molecular form of GRP in the endometrial tissues taken from nonpregnant and pregnant cows. The present results revealed the localization of GRP in the uterine gland cells at light- and electron-microscopic levels and suggested the release of GRP from the cell into the lumen of the gland by exocrine manner.     

Expression of oestrogen receptor-alpha and -beta in ovarian endometriomata.

The contribution of oestrogen receptor (ER) isoforms, ER-alpha and ER-beta, in oestrogen-dependent development and growth of ovarian endometriomata, is unknown. Therefore, we examined the expression of ER-alpha and ER-beta in ovarian endometriomata and normal uterine endometrium. ER-alpha and ER-beta were shown to be dominantly expressed in the nuclei of the epithelial lining cells of ovarian endometrioma and of the glandular cells of normal uterine endometrium. ER-beta was expressed at a much lower level than ER-alpha in the glandular cells of normal uterine endometrium, while ER-beta was expressed at a slightly lower level than ER-alpha in the epithelial lining cells of ovarian endometrioma. In normal uterine endometrium, ER-beta mRNA was expressed at a much lower level than ER-alpha mRNA, and the expression pattern of ER-beta mRNA during the menstrual cycle was similar to that of ER-alpha mRNA. On the other hand, ER-beta mRNA expression was significantly higher and over a much greater range in ovarian endometriomata (P < 0.05) than in normal uterine endometrium during the menstrual cycle, while ER-alpha mRNA expression was relatively lower and more random. Therefore, in ovarian endometriomata, oestrogen action via ER-alpha cascades seems to be partially damaged, as the expression of ER-alpha mRNA does not respond to endocrinological alterations during the menstrual cycle, while the relative over-expression of ER-beta might be related to a unique oestrogen-dependent growth and spreading of ovarian endometriomata.     

GABAergic neurones in the rat periaqueductal grey matter express alpha4, beta1 and delta GABAA receptor subunits: plasticity of expression during the estrous cycle

Immunoreactivity for alpha4, beta1 and delta GABAA receptor subunits on neurones in the periaqueductal gray matter was investigated at different stages of the estrous cycle in Wistar rats. Immunostaining for alpha4, beta1 and delta GABAA receptor subunits was present on neurones throughout the periaqueductal gray matter. The numbers of subunit-immunoreactive neurones remained constant during the early phases of the estrous cycle (proestrus to early diestrus) but increased significantly in late diestrus. Dual immunolabeling for the GABA synthesizing enzyme glutamic acid decarboxylase revealed that almost 90% of the subunit-positive cells contained immunoreactivity for glutamic acid decarboxylase. During the early phases of the estrous cycle (proestrus to early diestrus), approximately one third of the glutamic acid decarboxylase-positive population co-localized alpha4, beta1 and delta GABAA receptor subunits. When the number of subunit positive cells increased during late diestrus, the proportion of the glutamic acid decarboxylase-containing population that expressed alpha4, beta1 and delta GABAA receptor subunits almost doubled. We propose that GABAA receptors with the alpha4beta1delta configuration are expressed by GABAergic neurones in the periaqueductal gray matter and that the numbers of cells expressing these subunits are increased in late diestrus in line with falling plasma progesterone levels. Changes in GABAA receptor expression may lead to changes in the excitability of the neural circuitry in the periaqueductal gray matter.     

Effect of the estrous cycle on uterine infection induced by Escherichia coli.

Escherichia coli was inoculated into the uterine lumen of rats and rabbits at different estrous stages; one uterine horn of each animal was ligated at the cervical end. In rats, a large number of E. coli were retained in the ligated horns regardless of the estrous stage. E. coli inoculated at diestrus or pseudopregnancy induced purulent endometritis, but when inoculated at proestrus-estrus the organism caused asymptomatic infection. In nonligated horns, few E. coli were recovered, and marked histopathological changes were not observed. Large numbers of E. coli were retained in the nonligated horn at proestrus as a result of physiological constriction of the cervix. E. coli inoculated at proestrus never caused purulent endometritis in either the ligated horn or the nonligated horn. In rabbits, E. coli infused into ligated horns brought about purulent inflammation irrespective of ovarian states. The number of recoverable E. coli was reduced rapidly at the follicular phase as compared with the luteal phase. These results suggest that the stage of the estrous cycle when animals are inoculated with E. coli influences the course of the uterine infection.     

Estrogen receptor alpha (ERα) and progesterone receptor (PR) in the mammary gland of bitches during different stages of estrous cycle

The objective of this study was to evaluate the estrogen receptor alpha (ERα) and progesterone receptor (PR) expressions in normal mammary tissues of bitches during different stages of the estrous cycle. Mammary tissues were collected from five beagle bitches at six different estrous stages for each bitch, which were: anestrus, proestrus, estrus, early diestrus, mid diestrus, and late diestrus. The expressions of ERα and PR were evaluated by avidin–biotin–peroxidase complex (ABC) method, and ERα and PR scores were calculated. The lowest scores of ERα and PR were found in mammary tissue collected during mid diestrus, whereas the ERα and PR scores were significantly higher during estrus and proestrus. A significantly higher score of the PR during anestrus compared to during mid diestrus was also observed, but this was not found for ERα score. In addition, positive correlation between ERα and PR scores were found which indicated that the presence of ERα and PR may be under the same regulatory mechanism. In conclusion, these findings suggested that ovarian steroid hormones, estrogen and progesterone, which differed during the stages of the estrous cycle may have a regulatory role in ERα and PR expression in bitch mammary gland.     

Follicle stimulating hormone receptor protein is expressed in ovine uterus during the estrous cycle and utero-placenta during early pregnancy: An immunohistochemical study

Follicle stimulating hormone (FSH) is a well characterized gonadotropin that controls primarily development and functions of ovarian follicles in mammalian species. FSH binds to a specific G protein-coupled receptor (FSHR) belonging to the glycoprotein hormone receptor family that plays an essential role in reproduction. Although the primary location of FSHR is in the gonads (mainly in ovarian follicles), FSHR protein and/or mRNA have also been detected in extragonadal female reproductive tissues including embryo, placenta, endometrium, cervix, ovarian cancer tissues, and/or endometriotic lesions in several species. To determine the pattern of FSHR expression in the uterus and placenta, uterine tissues were collected at the early, mid- and/or late luteal phases of the estrous cycle from non-treated or FSH-treated ewes, and utero-placental tissues were collected during early pregnancy followed by immunohistochemistry and image generation. FSHR was immunolocalized to several uterine and utero-placental compartments including luminal epithelium, endometrial glands and surrounding stroma, myometrium, and endothelium and vascular smooth muscle cells in endometrium, myometrium and mesometrium. Intensity of staining and distribution of FSHR in selected compartments differed and seems to depend on the stage of the estrous cycle or pregnancy, and FSH-treatment. These novel data demonstrate differential expression of FSHR protein indicating that FSH plays a specific role in regulation of uterine and utero-placenta functions in sheep.     

A method for in vitro cell culture of superficial bovine uterine endometrial epithelium

Summary Bovine epithelial cells were obtained for culture from the uterine endometrium of adult, cyclic cattle. Using the procedures described herein, cell-specific monolayers of uterine epithelial cells developed rapidly in culture and maintained a good level of viability for seven to eight subcultures. In addition, frozen-thawed uterine epithelial cells also maintained a respectable level of viability during postthaw subculture. Patterns of cell growth for uterine epithelial cells were determined by a growth curve. A growth curve of fresh epithelial cells over an 8-day interval revealed a short lag phase (24 h) followed by a log growth phase for 5 days and then a stationary phase starting on Days 6 or 7 of incubation. This method for isolation and culture of uterine epithelial cells provides a potential model for evaluating uterine epithelial cell secretory capacity during the estrous cycle. This culture system may offer benefits for in vitro culture of bovine embryos.     

The progressive rise in the expression of alpha crystallin B chain in human endometrium is initiated during the implantation window: modulation of gene expression by steroid hormones

Human endometrium undergoes sequential changes during the menstrual cycle and becomes receptive to implantation during a defined period in the secretory phase. We attempted to identify the genes expressed during this period by representational difference analysis (RDA). When the cDNAs of a proliferative endometrium were used as the driver and the cDNAs of a post-ovulatory day 5 endometrium were used as the tester, a number of bands were identified by RDA. DNA of the cloned RDA products revealed that the majority of the clones contained a fragment of a cDNA identical to that of a crystallin B chain. Northern blot analysis showed that the expression of the alpha crystallin B chain mRNA was absent during the proliferative phase. The expression of the mRNA of alpha crystallin B chain first appeared in the secretory phase, progressively increased during this phase and peaked in the late secretory endometria. The pattern of expression of alpha crystallin B chain mRNA in the endometrium of mature cycling baboons (Papio anubis) was similar to that seen in human endometrium. As revealed by Western blot analysis, the expression of the alpha crystallin B chain protein in human endometrium followed a pattern of expression similar to its mRNA. At the cellular level, the immunoreactive protein first appeared in the surface epithelial cells of human endometrium within the implantation window without significant immunoreactivity in the underlying glandular cells. During the mid- and late secretory phases, the intensity of staining in the epithelial cells was enhanced and an intense immunoreactivity was developed in the glandular epithelium, alpha crystallin B chain was virtually an epithelial product and no immunoreactivity for this protein was detectable in the stromal cells, endothelial cells or lymphoid cells. The expression of alpha crystallin B chain could be regulated, by medroxy progesterone acetate as well as by oestrogen withdrawal, in human endometrial carcinoma cells (EnCa- 101), transplanted to nude mice. Based on the data presented here, the known function of alpha crystallin B chain and its distinct pattern of expression in human endometrium, we suggest that this protein is an important factor within the molecular repertoire that makes endometrium receptive to implantation.      

Differential localization and expression of urokinase plasminogen activator (uPA), its receptor (uPAR), and its inhibitor (PAI-1) mRNA and protein in endometrial tissue during the menstrual cycle

Normal endometrium is a highly dynamic tissue, which responds to ovarian steroids with cyclic proliferation, differentiation (secretion), and degradation (menstruation). The urokinase plasminogen activator (uPA)-dependent proteolytic cascade as well as ligand activation of the uPA receptor (uPAR) is critically involved in physiological as well as pathophysiological aspects of tissue expansion and remodelling. Cyclic variation and distribution of uPA, uPAR and plasminogen activator inhibitor 1 (PAI-1) mRNA were examined by in situ hybridization, real-time PCR and northern blot in normal endometrium. Their corresponding proteins were localized with immunohistochemistry. uPA mRNA is exclusively expressed by stromal cells, whereas uPA protein is present in both epithelial and stromal cells. Immunostaining for uPA protein is reduced or undetectable at midcycle, thus coinciding with peak concentration of uPA in the uterine fluid. uPAR mRNA is expressed by epithelial cells in the proliferative phase and by stromal cells in the secretory phase. However, epithelial cells stain for uPAR protein throughout the cycle, suggesting that uPAR may detach from stromal cells and then bind to epithelial cells in the secretory phase. PAI-1 mRNA is located in vessel walls. The late secretory phase has greatly increased expression of all three mRNA and their proteins, mainly in pre-decidual cells in the superficial stroma. Discordant localization of the mRNA and proteins suggest that uPA is produced by stromal cells, released and bound to epithelial cells in both the proliferative and secretory phases, whereas uPAR is released from the stroma and bound to epithelial cells in the secretory phase. Also, the present data together with earlier reports suggest that uPA is released from the epithelial cells to the uterine fluid.     

Regulation of surfactant protein D in the mouse female reproductive tract in vivo

Surfactant protein D (SP-D) plays a role in innate immunity in the lung and is expressed at many other mucosal surfaces throughout the human body. In this study, we show that SP-D mRNA and protein are present in the murine female reproductive tract; i.e. in the vagina, cervix, uterus and oviduct. SP-D protein is primarily localized to epithelial cells lining the genital tract and is also present in secretory material within the lumen of the uterus and cervix. The levels of SP-D mRNA in the uterus vary by a factor of 10 during the estrous cycle with peak levels present at estrus and the lowest levels at diestrus. In contrast, SP-D mRNA levels in the lung do not change during the estrous cycle. Since SP-D is an innate host defense protein present in the mouse reproductive tract, we studied the influence of infection on SP-D levels in vivo. We found that Chlamydia muridarum infection caused an increase in the SP-D protein content of reproductive tract epithelial cells. These data are suggestive that SP-D may play a role in innate immunity in the female reproductive tract in vivo.     

The presence of interleukin-1beta in the bovine reproductive tract.

Interleukin-1 (IL-1) is a pleiotropic cytokine implicated in endometrial and embryonic physiology. Our objective was to determine the presence of IL-1 in the endometrium, oviduct, and uterine fluid of cows at days 0, 7, and 14 of the estrous cycle. Immunoreactive IL-1beta was identified in endometrial and oviductal tissues throughout the estrous cycle by immunohistochemistry. Both glandular and luminal endometrial epithelium exhibited intense IL-1beta staining. For luminal epithelium, staining was strongest at day 0 and least at day 7. Staining in glandular epithelium was similar at all stages of the estrous cycle examined. There was a diffuse immunostaining throughout the endometrial stroma, and some isolated stromal cells stained strongly, as did endothelial cells. Immunoreactive IL-1beta was detected in uterine flushings by Western blotting, and the frequency of positive samples and intensity of immunoreactive bands did not differ between days of the estrous cycle. In the oviduct, immunoreactive IL-1beta was found in the epithelium and stroma of ampulla and isthmus. The staining intensity score for the oviduct was not different between isthmus and ampulla or between days of the estrous cycle. The presence of IL-1beta in the bovine endometrium, oviduct, and uterine flushings supports the idea that this cytokine may play an important role in regulating embryonic and endometrial function in cattle.     

生殖周期中小鼠子宫巨噬细胞的分布

为了认识生殖周期中子宫巨噬细胞的分布，用Ｆ４／８０单克隆抗体，对小鼠子宫巨噬细胞进行了免疫组织化学标记。结果显示，间情期子宫内膜中有大量巨噬细胞，动情前期数量略有减少，但子宫系膜三角区出现较多巨噬细胞。动情期子宫内膜中有少量巨噬细胞，动情后期显著减少。在动情期和动情后期，子宫系膜三角区中均有较多巨噬细胞，妊娠１ｄ的子宫内膜中巨噬细胞数量很少，妊娠７ｄ的子宫蜕膜中则消失。妊娠１４ｄ子宫的壁蜕膜组织中有少量巨噬细胞，在子宫肌间偶见，基蜕膜与子宫腺区中未见巨噬细胞。妊娠１８ｄ子宫中，壁蜕膜中巨噬细胞明显增多，子宫腺区也有少量，同时子宫系膜基部可见许多，但基蜕膜中未见巨噬细胞。产后１ｄ子宫中，巨噬细胞在内膜中数量极多，子宫肌间可见成簇分布。产后３ｄ子宫内膜结构基本恢复正常，其内仍有许多巨噬细胞。实验结果提示：子宫巨噬细胞在妊娠早期数量减少可能与胚泡着床有关；分娩前后子宫巨噬细胞的增多可能与产后子宫的修复有关     

Expression of interleukin-11 during the human menstrual cycle: coincidence with stromal cell decidualization and relationship to leukaemia inhibitory factor and prolactin

Interleukin-11 (IL-11) is crucial in the decidualization response of the uterine stroma to the implanting blastocyst in the mouse. This study examined the localization and expression of IL-11 in human endometrium throughout the menstrual cycle and of prolactin and leukaemia inhibitory factor (LIF) in secretory phase endometrium. The mRNA expression of IL-11 receptor alpha and the signalling component, gp130, in endometrial tissue were also determined. Immunoreactive IL-11 was highest in the secretory phase and present in decidualized stromal cells, glandular epithelial cells, endothelial and smooth muscle cells, and the mRNA expression was verified by in-situ hybridization. Decidual cells showed the most intense staining. IL-11 receptor alpha and gp130 mRNA were detected throughout the cycle with minimal variation. Expression of IL-11 mRNA and protein preceded that of prolactin. While immunoreactive prolactin was found in stromal, decidual and glandular epithelial cells, prolactin mRNA was confined to decidual cells. In contrast, endometrial LIF expression preceded IL-11 but was largely confined to the glandular epithelium. The sequence of appearance of LIF, IL-11 and prolactin suggests a synchronized role for each in the differentiation of the endometrium. The cyclical changes and cell type specific expression of IL-11 suggests a potential role in the decidualization of stromal cells.