Characterization of Glutathione Peroxidase 4 in Rat Oocytes, Preimplantation Embryos, and Selected Maternal Tissues during Early Development and Implantation

This study aimed to describe glutathione peroxidase 4 (GPx4) in rat oocytes, preimplantation embryos, and female genital organs. After copulation, Sprague Dawley female rats were euthanized with anesthetic on the first (D1), third (D3), and fifth days of pregnancy (D5). Ovaries, oviducts, and uterine horns were removed, and oocytes and preimplantation embryos were obtained. Immunohistochemical, immunofluorescent, and Western blot methods were employed. Using immunofluorescence, we detected GPx4 in both the oocytes and preimplantation embryos. Whereas in the oocytes, GPx4 was homogeneously diffused, in the blastomeres, granules were formed, and in the blastocysts, even clusters were present mainly around the cell nuclei. Employing immunohistochemistry, we detected GPx4 inside the ovary in the corpus luteum, stroma, follicles, and blood vessels. In the oviduct, the enzyme was present in the epithelium, stroma, blood vessels, and smooth muscles. In the uterus, GPx4 was found in the endometrium, myometrium, blood vessels, and stroma. Moreover, we observed GPx4 positive granules in the uterine gland epithelium on D1 and D3 and cytoplasm of fibroblasts forming in the decidua on D5. Western blot showed the highest GPx4 levels in the uterus and the lowest levels in the ovary. Our results show that the GPx4 is necessary as early as in the preimplantation development of a new individual because we detected it in an unfertilized oocyte in a blastocyst and not only after implantation, as was previously thought.

Histomorphometric analysis of goat uterine tissue on in vitro exposure with ovarian hormones and mifepristone

Uterus, the largest reproductive tract organ in female mammals, is the site of implantation of fertilised egg and foetus development. Uterus is a dynamic reproductive organ; its morphology alters with reproductive phase and steroidal cues. The aim of the present study was to assess the effects of progesterone (P4), estrogen (E2) and antiprogestogen i.e., mifepristone on goat’s uterine histoarchitecture in in vitro short term culture. Uterine tissue slices were cultured in the presence of E2, P4 and mifepristone at the dose of 10–9 M, 10–7 M and 10–6 M respectively for 24 hours. Uter-ine morphology of E2- and P4-treated groups did not reveal marked changes from that of control group. Mifepristone treatment caused conspicuous changes in uterine histoarchitecture, led to congested endometrium, regressed uterine glands and constricted blood vessels. The changes ob-served in morphometry after E2 and P4 exposure included increased uterine gland diameter (47.00 and 45.95 µm respectively) and glandular epithelial cell height (18.37 and 17.43 µm respectively) while the mifepristone treatment resulted in significant reduction of gland diameter (34.95 µm) as well as epithelium height (14.25 µm) as compared to those in control group (39.9 and 15.56 µm respectively). These morphometrical changes revealed prominent regressive changes in anti-progestin treated group while E2 and P4 showed prolific effects in in vitro culture. Thus it is envis-aged that E2 and P4 induced characteristic progressive changes in the histologic structure especially in endometrial glands of the goat uterus while anti-steroidogenic formulation i.e. mifepristone severely reduced the normal histoarchitecture of the uterus which is a prerequisite for implanta-tion.

Histochemical Study of the Rat Uterine Glycoconjugate Alteration following Treatment with Exogenous Gonadotropic Hormones during the Implantation Period

One of the female causes of infertility is anovulation which is treatable with gonadotropin hormones. These hormones affect the molecular organization of the uterus such as glycoconjugates that are the first site of contact between the blastocyst and the uterus. The objective of this project was to study the alteration of glycoconjugates on the uterine apical, Golgi zone, and basement membrane of epithelial cells and the uterine gland after hyperstimulation with pregnant mare serum gonadotropin (PMSG) (4, 8, 16, 24, and 40 IU), during the implantation period. Injection of PMSG (in experimental groups) and injection of distilled water (in the control group) were followed by HCG administration (10 IU), mating, isolation of positive vaginal plug rats, and killing at 5.5 days of pregnancy. Histochemistry was done on the pregnant uterine horns with the use of WGA, DBA, PNA, ConA, SBA, and UEA lectins. The intensity of the immunohistochemical staining was scored, and quantitative data were generated. 4 IU did not show any significant differences with the control, 8 IU had less effect on the alteration of the Golgi zone, and apical and basement membrane glycoconjugates and 40 IU had the least effects on the alteration of uterine gland glycoconjugates. Also, 24 IU had the most effect on the alteration of uterine glycoconjugates. Understanding of the effects of gonadotropin hormones at the uterine level in implantation time helps to optimize hormonal manipulation for improving the outcome of assisted reproductive procedures. It seems that the optimal dose for superovulation and less alteration in uterine glycoconjugates of rats at implantation time were induced by the administration of 8 IU PMSG.

Endometrial epithelial ARID1A is critical for uterine gland function in early pregnancy establishment

Though endometriosis and infertility are clearly associated, the pathophysiological mechanism remains unclear. Previous work has linked endometrial ARID1A loss to endometriosis-related endometrial non-receptivity. Here, we show in mice that ARID1A binds and regulates transcription of the Foxa2 gene required for endometrial gland function. Uterine specific deletion of Arid1a compromises gland development and diminishes Foxa2 and Lif expression. Deletion of Arid1a with Ltf-iCre in the adult mouse endometrial epithelium preserves gland development while still compromising gland function. Mice lacking endometrial epithelial Arid1a are severely sub-fertile due to defects in implantation, decidualization, and endometrial receptivity from disruption of the LIF-STAT3-EGR1 pathway. FOXA2 is also reduced in the endometrium of women with endometriosis in correlation with diminished ARID1A, and both ARID1A and FOXA2 are reduced in non-human primates induced with endometriosis. Our findings describe a role for ARID1A in the endometrial epithelium supporting early pregnancy establishment through the maintenance of gland function.

Subchronic and Low Dose of Tributyltin Exposure Leads to Reduced Ovarian Reserve, Reduced Uterine Gland Number, and Other Reproductive Irregularities in Female Mice

Tributyltin (TBT) chloride is an endocrine disrupting chemical associated with reproductive complications. Studies have shown that TBT targets the reproductive tract, impairing ovarian folliculogenesis, and uterine morphophysiology. In this investigation, we assessed whether subchronic and low dose of TBT exposure results in abnormal ovarian follicular reserve and other irregularities in female mice. TBT was administered to female mice (500 ng/kg/day for 12 days via gavage), and reproductive tract morphophysiology was assessed. We further assessed reproductive tract inflammation and oxidative stress. Improper functioning of the reproductive tract in TBT mice was observed. Specifically, irregular estrous cyclicity and abnormal ovarian morphology coupled with reduction in primordial and primary follicle numbers was observed, suggesting ovarian reserve depletion. In addition, improper follicular development and a reduction in antral follicles, corpora lutea, and total healthy ovarian follicles together with an increase in cystic follicles were apparent. Evidence of uterine atrophy, reduction in endometrial gland number, and inflammation and oxidative stress were seen in TBT mice. Further, strong negative correlations were observed between testosterone levels and primordial, primary, and total healthy ovarian follicles. Thus, these data suggest that the subchronic and low dose of TBT exposure impaired ovarian follicular reserve, uterine gland number, and other reproductive features in female mice.

Epithelial morphogenesis in the perinatal mouse uterus

BackgroundThe uterus is the location where multiple events occur that are required for the start of new life in mammals. The adult uterus contains endometrial or uterine glands that are essential for female fertility. In the mouse, uterine glands are located in the lateral and anti-mesometrial regions of the uterine horn. Previous 3D-imaging of the adult uterus, its glands, and implanting embryos has been performed by multiple groups, using fluorescent microscopy. Adenogenesis, the formation of uterine glands, initiates after birth. Recently, we created a 3D-staging system of mouse uterine gland development at postnatal time points, using light sheet fluorescent microscopy. Here, using a similar approach, we examine the morphological changes in the epithelium of the perinatal mouse uterus.ResultsThe uterine epithelium exhibits mesometrial-antimesometrial (dorsoventral) patterning as early as three days after birth (P3), marked by the presence of the mesometrially-positioned developing uterine rail. Uterine gland buds are present beginning at P4. Novel morphological epithelial structures, including a ventral ridge and uterine segments were identified.ConclusionsThe perinatal mouse uterine luminal epithelium develops mesometrial-antimesometrial (dorsal-ventral) morphologies at 3-4 days post-partum. Between 5-6 days post-partum uterine epithelial folds form, defining alternating left-right segments.Bullet pointsMorphological patterning events in the perinatal uterine epithelium are not well described.Light sheet microscopy was used to generate volumetric reconstructions of the perinatal mouse uterine epithelium.At postnatal day 3 (P3), the uterine epithelium shows the first signs of dorsoventral pattern, with the presence of the forming mesometrially-positioned uterine rail.The first morphological indication of uterine adenogenesis begins at P4.Novel morphological structures were identified from volumetric reconstructions, including the presence of a ventral ridge (another sign of dorsoventral pattern) and uterine segmentation.

Neonatal Wnt-dependent Lgr5 positive stem cells are essential for uterine gland development

Wnt signaling is critical for directing epithelial gland development within the uterine lining to ensure successful gestation in adults. Wnt-dependent, Lgr5-expressing stem/progenitor cells are essential for the development of glandular epithelia in the intestine and stomach, but their existence in the developing reproductive tract has not been investigated. Here, we employ Lgr5-2A-EGFP/CreERT2/DTR mouse models to identify Lgr5-expressing cells in the developing uterus and to evaluate their stem cell identity and function. Lgr5 is broadly expressed in the uterine epithelium during embryogenesis, but becomes largely restricted to the tips of developing glands after birth. In-vivo lineage tracing/ablation/organoid culture assays identify these gland-resident Lgr5high cells as Wnt-dependent stem cells responsible for uterine gland development. Adjacent Lgr5neg epithelial cells within the neonatal glands function as essential niche components to support the function of Lgr5high stem cells ex-vivo. These findings constitute a major advance in our understanding of uterine development and lay the foundations for investigating potential contributions of Lgr5+ stem/progenitor cells to uterine disorders.

Development and Function of Uterine Glands in Domestic Animals

All mammalian uteri contain glands that synthesize or transport and secrete substances into the uterine lumen. Uterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation of glandular epithelium from luminal epithelium, followed by invagination and coiling morphogenesis throughout the stroma. Intrinsic transcription factors and extrinsic factors from the ovary and pituitary as well as the mammary gland (lactocrine) regulate uterine adenogenesis. Recurrent pregnancy loss is observed in the ovine uterine gland knockout sheep, providing unequivocal evidence that glands and their products are essential for fertility. Uterine gland hyperplasia and hypertrophy during pregnancy are controlled by sequential actions of hormones from the ovary and/or pituitary as well as the placenta. Gland-derived histotroph is transported by placental areolae for fetal growth. Increased knowledge of uterine gland biology is expected to improve pregnancy outcomes, as well as the health and productivity of mothers and their offspring.