Microscopical analysis of the uterus of Saidi sheep during the follicular phase of the estrous cycle revealed that the uterine wall was composed of three layers: the inner endometrium, middle myometrium and the outer perimetrium. The endometrium had mucosal folds, caruncles and narrow uterine lumen. The endometrium formed of lamina epithelialis of pseudostratified columnar epithelium with intraepithelial lymphocytes and connective tissue lamina propria contained uterine glands, fibroblasts, collagen fibers, blood vessels and leucocytic infiltrations especially lymphocytes and plasma cells. The uterus (horns and body) in ruminants was lined with the simple columnar to pseudostratified columnar epithelium. The mean height of the epithelium was less in follicular phase [23–25]. In buffalo the endometrium was lined with three types of columnar cells, i.e. ciliated, non-ciliated cells and basal cells [25]. The endometrial stroma (propria submucosa) consisted of fibro-reticular connective tissue, stromal cells and blood vessels. Its cellular components comprised of stromal cells, fibroblasts, mesenchymal cells, neutrophils and lymphocytes. The stromal cells' nuclei were elliptical, oval, or circular in shape. In the follicular phase, the stroma was very crowded and swollen [24, 25]. The endometrium showed a period of growth preceded by vascularization during the follicular phase of the estrous cycle in sheep[26]. Leukocytes invaded the functional layer on Day 7 of estrous cycle in cow [27]. The bovine luminal epithelium changes during the estrous cycle through a remodeling process [28].
The endometrium in adult ruminants (sheep, goat, buffalo and cattle) consists of a glandular caruncles and glandular intercaruncular areas [10]. The caruncles were non glandular, highly cellular and highly vascular endometrial elevation or projections. The locations of superficial implantation and placentation are occurred in caruncular regions. Interdigitation and branching morphogenetic growth of placental cotyledons with endometrial caruncles creates placentomes in synepitheliochorial placentation observed in ruminants. Placentomes are primarily involved in fetal-maternal gas exchange and the placenta's absorption of micronutrients for hemotrophic nutrition of the fetus [10].
The current study showed that the follicular phase of estrous cycle in Saidi sheep was characterized by epithelial proliferation and many epithelial invaginations which form the uterine glands (uterine gland adenogenesis). Herein, the uterine glands during the follicular phase of estrous cycle were highly branched and highly coiled and formed of columnar epithelium which surrounded by myoepithelial cells and had intraepithelial lymphocytes. Similar results were obtained by [23, 24] in goat during follicular phase of estrous cycle. While uterine glands in sheep and pigs, are tightly coiled, heavily branched tubular glands, uterine glands in mice are comparatively simple tubes with little branching [29]. These glands have occasionally penetrated and reached the stratum vascularis. Proliferation of the endometrial glands were observed in the follicular phase [23] as a result of glandular epithelium mitoses [27].
Uterine gland development, or adenogenesis, is uniquely a postnatal event in sheep and pigs and involves differentiation and budding of glandular epithelium from luminal epithelium, followed by invagination and extensive tubular coiling and branching morphogenesis throughout the uterine stroma to the myometrium. Uterine adenogenesis is regulated by both intrinsic transcription factors and extrinsic factors from the pituitary, ovary, and mammary gland (lactocrine) [30, 31]. To support the effects of certain hormones and growth factors, this mechanism necessitates site-specific changes in cell proliferation and extracellular matrix (ECM) remodeling in addition to paracrine cell-cell and cell-ECM interactions. According to studies on uterine development in newborn ungulates, prolactin, estradiol-17b, and their receptors are implicated in mechanisms controlling endometrial adenogenesis. When the functionalis is rebuilt from the basalis endometrium during menstruation, these hormones also seem to control endometrial gland development in menstrual primates and humans [30].
The endometrium of all mammalian uteri contains glands that produce, transport, and release chemicals necessary for the conceptus's survival and development (the embryo/fetus and related extraembryonic tissues). Adult ruminants' endometrium is composed of several a glandular caruncular regions and intercaruncular areas, each of which contained hundreds of glands per uterine wall cross-section [10, 30, 32]. The establishment of uterine receptivity, blastocyst implantation, and stromal cell decidualization all depend on uterine glands and their secretions. Similar to this, in humans, uterine glands and the secretory products they produce are probably important regulators of the uterine receptivity, blastocyst implantation and growth and development of the conceptus throughout the first trimester [32, 33]. The survival and development of the periimplantation conceptus depend on the endometrial glands and their secretions [34, 35].
The myometrium was formed of a thick, inner circular layer and a thin, outer longitudinal layer of smooth muscle fibers. A well vascularized stratum vascularis with many branches of the uterine artery was observed in the outer most layer of inner circular myometrium [10, 24].
The main blood supply to the uterus is provided by the uterine arteries, which are found inside the myometrium. During the proliferative phase, the subepithelial capillary plexus has the highest vascular length density. Endothelial proliferation is the main mechanism of endometrial angiogenesis during the proliferative phase influenced by estrogen. Estradiol stimulates vascular permeability, angiogenesis, and endothelial cell proliferation in response to VEGF[36]. The estrous cycle is regulated in large part by the uterine blood supply. The two main hormones influencing blood flow in the arteries feeding the uterus are estrogens and progesterone. Vasodilation and vasoconstriction are regulated, complemented, and supported by the following factors: PGE2, LH, oxytocin, cytokines, neurotransmitters, and other local blood flow regulators [37].
The process of endometrial angiogenesis is strictly regulated. The endometrium and the macrophages that reside there can produce most of the key cytokines and factors that are currently known to be involved in the regulation of angiogenesis [38]. Some of these factors which expressed throughout the menstrual cycle include: vascular endothelial growth factor (VEGF) [39, 40], fibroblast growth factor (FGF) [41] transforming growth factor-α (TGF-α) [42], interleukin (IL)-1 and IL-6 [43], epidermal growth factor (EGF) [44] and IL-8 [45]. Uterine vascular remodeling is important to the cycling and early pregnant endometrium. These vascular changes are strongly mediated by maternal regulatory factors, including ovarian hormones, VEGF, angiopoietins, Notch, and uterine natural killer cells [36].
We found that the endometrium had PAS positive epithelial basement membrane of the surface lamina epithelialis and uterine glands. This agree with the finding of [23] in Bakerwali Goat. No PAS positive secretion in the uterine glands could be observed during the follicular phase of estrous cycle. The intense PAS positivity was seen at supranuclear zone of the secretory glandular epithelium during the luteal phase [23].
The current study showed that few elastic fibers were observed in the endometrium (in the sub epithelial connective tissue and between the uterine glands) and between the smooth muscle fibers in the myometrium. However many elastic membranes were observed in the internal and external elastic lamina of the blood vessels of the tunica vascularis. Similar finding were observed in human [46] and mice [47] uterus. Elastic fibers in the uterus are mainly located in the myometrium and perimetrium while the endometrium contains few elastic fibers[47]. The uterine elasticity is likely maintained without excess stress being placed on the developing fetus by these thin sheets of elastic membranes and elastic fibrils [48]. Elastic fibers are resistant to tensile stresses and have persistently variable functions based on the needs of the microenvironment in which they are found [49].
Our findings revealed that the collagen fibers were more thickly distributed in the lamina propria of the uterine endometrium close to the endometrial glands and were located between the muscles [49]. While the intercellular matrix of the endometrial stroma contained a moderate amount of collagen fibers [25]. It had been suggested that the variability of the connective tissue thread distribution in the uterus may have a role in the fertilization process [49]. Because collagen fibers are found in the stroma and between the muscles, they enable the uterus to contract and stretch [49]. Collagen fiber visualization could make it easier to assess the thickness of the connective tissue that envelops endometrial glands. Elevated density may lead to degeneration and loss of function by impairing the flow of nutrients and endocrine signaling molecules from blood arteries to the glandular epithelium [50].
Our results showed slight GR immunostaining in the lamina epithelialis and in the stroma cells of lamina propria. Furthermore, slight GR immunostaining in the smooth muscle fibers of the blood vessels of the stratum vascularis. Whereas, slight SOD2 immunoexpression were observed in the lamina epithelialis, stroma cells and the smooth muscle fibers of the blood vessels of the lamina propria. Slight SOD2 immunostaining in the smooth muscle fibers of blood vessels of stratum vascularis were also noticed. Estradiol and progesterone control uterine glutathione reductase, which may be crucial in preserving the uterus's lowered glutathione levels. This molecule may be necessary in detoxification reactions involving H2O2 and electrophylic chemicals as well as for the regulation of the redox state of thiol groups. Glutathione reductase is stimulated by estrogens, which contributes to their antioxidant properties[51]. The uterus and fallopian tube contain antioxidants that aid in removing excess reactive oxygen species (ROS), creating the ideal environment for embryonic growth. To get rid of the ROS that cytokines and inflammation produce in mitochondria, SOD2 content is raised [52]. In addition there was a close relation between increased ROS and apoptosis. SOD2 and GR expression help to control apoptosis in the uterus during estrous cycle [53].
Our finding by using TUNEL assay immunofluorescence in the sheep uterus during the follicular phase of estrous cycle explored some apoptotic endometrial glandular epithelial cells and some apoptotic endometrial stromal cells. While the inner circular layer of myometrium showed few apoptotic cells. Apoptotic endometrial stromal cells in the caruncles were also observed in addition to apoptotic smooth muscle fibers of the blood vessels of the stratum vascularis. Apoptotic cell death had been demonstrated in hamster and rat uterine epithelium during the estrous cycle. There was an inverse correlation between cell death and cell proliferation in rat uterine and vaginal epithelial cells during the estrous cycle. Uterine epithelial cell proliferation, differentiation, and death are regulated by estrogen and progesterone [54, 55]. In human uterus apoptotic uterine cells were scattered in the functional layer of the early proliferative endometrium [55]. In contrast, in the dog uterus a high apoptotic index was not detected in the surface epithelium and there was no significant correlation between the apoptotic index in any cell group and progesterone concentrations [56]. It was postulated that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone [57]. Dynamic changes in the porcine endometrium during the estrous cycle are a type of homeostasis through control of cell proliferation and exclusion. Homeostasis of the uterus is closely related to apoptosis and involving many hormones and cyctokines [58]. Our results indicate that apoptosis might have crucial role in the regulation of the estrous cycle in Saidi sheep.
The current study revealed PRA immunolocalization in the lamina epithelialis, stroma cells, endothelial cells, columnar epithelial cells of the uterine glands and in the smooth muscle fibers of the myometrium. Moreover, the endothelium and the smooth muscle fibers of the blood vessels of the stratum vascularis showed also PRA immunolocalization. On the other hand, there was PRA immunolocalization in the perimetrial mesothelial cells and in the perimetrial endothelial cells. Similar findings were obtained in rabbit uterus during psedopregnancy [59]. Progesterone, a critical steroid hormone in the reproductive system, plays vital roles during the follicular phase of the estrous cycle [60]. Although traditionally associated with the luteal phase, emerging research highlights its significant functions in the follicular phase [61]. Progesterone receptors (PRs), which exist in two main isoforms, PR-A and PR-B, are differentially expressed in ovarian follicles and uterus [62]. Their expression is dynamically regulated by fluctuating hormone levels throughout the cycle. During the follicular phase, PR-A predominantly mediates progesterone’s inhibitory effects on follicular atresia, promoting the survival of developing follicles. Conversely, PR-B is implicated in the regulation of ovulatory processes. Studies have shown that PR knockout models exhibit impaired follicular development and ovulation, underscoring the importance of these receptors. The PR-A is also essential for uterine decidualization and implantation [62]. The interaction between PRs and other intracellular signaling pathways, such as the PI3K/AKT pathway, further illustrates the complexity of progesterone's role in folliculogenesis [63]. Progesterone, via acting through PR-A control the development and function of the endometrium and modifies cells essential for implantation and the establishment and maintenance of pregnancy. During pregnancy, progesterone via the PRs stimulats myometrial relaxation and cervical closure [64, 65].
Progesterone modulates follicular development and ovulation through its interactions with progesterone receptors (PRs) [66]. During the follicular phase, low levels of progesterone and its receptors are necessary to prime the ovarian follicles for growth and maturation. Progesterone (P4) works synergistically with estrogen to regulate the expression of genes involved in follicle-stimulating hormone (FSH) and luteinizing hormone (LH) receptors, crucial for follicular responsiveness to gonadotropins [67].
It was discovered that the plane of nutrition, the estrous cycle phase, and/or FSH all had an impact on the percentage of PR-positive uterine cells and/or staining intensity [68]. Changes in endometrial functions in superovulated models may arise from direct or indirect FSH action pathways. FSH exerts its indirect effects through binding to ovarian receptors and stimulating the synthesis of the estrogen and P4 [68, 69] which in turn control endometrial activities [70–72]. Remarkably, during the menstrual cycle, the endometrial lining has high expression of the functional FSH receptor (FSHR). Increased expression of FSHR in the human endometrium can shed light on the potential direct effects of FSH on endometrial regeneration, which is primarily sustained by tissue-resident endometrial stem cells [73–76]. Through a complicated paracrine signaling network, the progesterone receptor controls glandular growth, decidualization, implantation, and the maintenance of a healthy uterus [70–72].
The role of progesterone and its receptors during the follicular phase is crucial for maintaining the delicate hormonal balance required for successful ovulation. By modulating the expression of enzymes like matrix metalloproteinases (MMPs), which are involved in follicular rupture, progesterone ensures that the dominant follicle reaches full maturation and is capable of releasing a viable oocyte [77]. We suggested that proper ovarian functions during follicular phase are necessary for proper uterine functions during both follicular phase and implantation. Additionally, other findings suggest that progesterone's actions extend beyond the ovaries and uterus, influencing the hypothalamic-pituitary-gonadal axis to fine-tune gonadotropin release, further demonstrating its integral role in reproductive physiology [78].
Interestingly, tryptase-positive immunostaining mast cells were recruited in the deep lamina propria of the caruncles during the follicular phase of the estrous cycle. Some degranulated mast cells were seen close to the macrophage in the endometrial lamina propria. Mast cells were also observed in-between uterine glands and smooth muscle fibers of the myometrium. Moreover they were noticed around the blood vessels of the stratum vascularis. MCs during the follicular phase of the estrous cycle play a crucial role in secreting substances that promote tissue remodeling [79]. Histamine is one of these important substances released from uterine mast cells, influencing ovulation, embryo implantation, and myometrium contractility leading to successful implantation and ultimately to parturition [80–82]. However, spatiotemporally expression of MCs in the female reproductive tract has been reported in other studies [82–92]. In the uterus, they change in number and structure depending on the hormonal level variations during the menstrual or estrous cycle [84, 93–95]. An investigation under a light microscope has revealed alterations in the characteristics and location of MCs in mice, rats, hamsters, cows, and guinea pigs uterine tissues during pregnancy and estrous cycle [96–99]. In human endometrium, MCs were decreased in the stromal tissue [100, 101]. Notably, the number and activity of MCs are correlated with estrogen concentrations in the uterine tissue of sows and rats [96, 102], while they are correlated with progesterone concentrations in the canine uterus [82].
Nevertheless, to preserve regular reproductive processes and create the best environment for potential implantation, all uterine cell types interact with one another through junctacrine, paracrine, or endocrine pathways [68].