Ovarian sex steroid and epithelial control of immune responses in the uterus and oviduct: human and animal models†.

The female reproductive tract (FRT), including the uterus and oviduct (Fallopian tube), is responsible for maintaining an optimal microenvironment for reproductive processes, such as gamete activation and transportation, sperm capacitation, fertilization, and early embryonic and fetal development. The mucosal surface of the FRT may be exposed to pathogens and sexually transmitted microorganisms due to the opening of the cervix during mating. Pathogens and endotoxins may also reach the oviduct through the peritoneal fluid. To maintain an optimum reproductive environment while recognizing and killing pathogenic bacterial and viral agents, the oviduct and uterus should be equipped with an efficient and rigorously controlled immune system. Ovarian sex steroids can affect epithelial cells and underlying stromal cells, which have been shown to mediate innate and adaptive immune responses. This, in turn, protects against potential infections while maintaining an optimal milieu for reproductive events, highlighting the homeostatic involvement of ovarian sex steroids and reproductive epithelial cells. This article will discuss how ovarian sex steroids affect the immune reactions elicited by the epithelial cells of the non-pregnant uterus and oviduct in the bovine, murine, and human species. Finally, we propose that there are regional and species-specific differences in the immune responses in FRT.

Sperm selection by the oviduct: perspectives for male fertility and assisted reproductive technologies†.

The contribution of sperm to embryogenesis is gaining attention with up to 50% of infertility cases being attributed to a paternal factor. The traditional methods used in assisted reproductive technologies for selecting and assessing sperm quality are mainly based on motility and viability parameters. However, other sperm characteristics, including deoxyribonucleic acid integrity, have major consequences for successful live birth. In natural reproduction, sperm navigate the male and female reproductive tract to reach and fertilize the egg. During transport, sperm encounter many obstacles that dramatically reduce the number arriving at the fertilization site. In humans, the number of sperm is reduced from tens of millions in the ejaculate to hundreds in the Fallopian tube (oviduct). Whether this sperm population has higher fertilization potential is not fully understood, but several studies in animals indicate that many defective sperm do not advance to the site of fertilization. Moreover, the oviduct plays a key role in fertility by modulating sperm transport, viability, and maturation, providing sperm that are ready to fertilize at the appropriate time. Here we present evidence of sperm selection by the oviduct with emphasis on the mechanisms of selection and the sperm characteristics selected. Considering the sperm parameters that are essential for healthy embryonic development, we discuss the use of novel in vitro sperm selection methods that mimic physiological conditions. We propose that insight gained from understanding how the oviduct selects sperm can be translated to assisted reproductive technologies to yield high fertilization, embryonic development, and pregnancy rates.

Dynamics of gametes and embryos in the oviduct: what can in vivo imaging reveal?

In brief: In vivo imaging of gametes and embryos in the oviduct enables new studies of the native processes that lead to fertilization and pregnancy. This review article discusses recent advancements in the in vivo imaging methods and insights which contribute to understanding the oviductal function. Abstract: Understanding the physiological dynamics of gametes and embryos in the fallopian tube (oviduct) has significant implications for managing reproductive disorders and improving assisted reproductive technologies. Recent advancements in imaging of the mouse oviduct in vivo uncovered fascinating dynamics of gametes and embryos in their native states. These new imaging approaches and observations are bringing exciting momentum to uncover the otherwise-hidden processes orchestrating fertilization and pregnancy. For mechanistic investigations, in vivo imaging in genetic mouse models enables dynamic phenotyping of gene functions in the reproductive process. Here, we review these imaging methods, discuss insights recently revealed by in vivo imaging, and comment on emerging directions, aiming to stimulate new in vivo studies of reproductive dynamics.

Cell-type specific analysis of physiological action of estrogen in mouse oviducts.

One of the endogenous estrogens, 17ß-estradiol (E2 ) is a female steroid hormone secreted from the ovary. It is well established that E2 causes biochemical and histological changes in the uterus. However, it is not completely understood how E2 regulates the oviductal environment in vivo. In this study, we assessed the effect of E2 on each oviductal cell type, using an ovariectomized-hormone-replacement mouse model, single-cell RNA-sequencing (scRNA-seq), in situ hybridization, and cell-type-specific deletion in mice. We found that each cell type in the oviduct responded to E2 distinctively, especially ciliated and secretory epithelial cells. The treatment of exogenous E2 did not drastically alter the transcriptomic profile from that of endogenous E2 produced during estrus. Moreover, we have identified and validated genes of interest in our datasets that may be used as cell- and region-specific markers in the oviduct. Insulin-like growth factor 1 (Igf1) was characterized as an E2 -target gene in the mouse oviduct and was also expressed in human fallopian tubes. Deletion of Igf1 in progesterone receptor (Pgr)-expressing cells resulted in female subfertility, partially due to an embryo developmental defect and embryo retention within the oviduct. In summary, we have shown that oviductal cell types, including epithelial, stromal, and muscle cells, are differentially regulated by E2 and support gene expression changes, such as growth factors that are required for normal embryo development and transport in mouse models. Furthermore, we have identified cell-specific and region-specific gene markers for targeted studies and functional analysis in vivo.

Sperm migration in the genital tract-In silico experiments identify key factors for reproductive success.

Sperm migration in the female genital tract controls sperm selection and, therefore, reproductive success as male gametes are conditioned for fertilization while their number is dramatically reduced. Mechanisms underlying sperm migration are mostly unknown, since in vivo investigations are mostly unfeasible for ethical or practical reasons. By presenting a spatio-temporal model of the mammalian female genital tract combined with agent-based description of sperm motion and interaction as well as parameterizing it with bovine data, we offer an alternative possibility for studying sperm migration in silico. The model incorporates genital tract geometry as well as biophysical principles of sperm motion observed in vitro such as positive rheotaxis and thigmotaxis. This model for sperm migration from vagina to oviducts was successfully tested against in vivo data from literature. We found that physical sperm characteristics such as velocity and directional stability as well as sperm-fluid interactions and wall alignment are critical for success, i.e. sperms reaching the oviducts. Therefore, we propose that these identified sperm parameters should be considered in detail for conditioning sperm in artificial selection procedures since the natural processes are normally bypassed in reproductive in vitro technologies. The tremendous impact of mucus flow to support sperm accumulation in the oviduct highlights the importance of a species-specific optimum time window for artificial insemination regarding ovulation. Predictions from our extendable in silico experimental system will improve assisted reproduction in humans, endangered species, and livestock.

Cyclic activity of signal transduction pathways in fimbrial epithelium of the human fallopian tube.

INTRODUCTION: The local environment of the fallopian tube represents the optimal conditions for reproductive processes. To maintain tissue homeostasis, signal transduction pathways are thought to play a pivotal role. Enhancing our understanding of functional signal transduction pathway activity is important to be able to clarify the role of aberrant signal transduction pathway activity leading to female subfertility and other tubal diseases. Therefore, in this study we investigate the influence of the hormonal cycle on the activity of key signal transduction pathways in the fimbrial epithelium of morphologically normal fallopian tubes. MATERIAL AND METHODS: We included healthy pre- (n = 17) and postmenopausal (n = 8) patients who had surgical interventions for benign gynecologic conditions. Histologic sections of the fallopian tubes were reviewed by two pathologists and, for the premenopausal patients, hormone serum levels and sections of the endometrium were examined to determine the hormonal phase (early follicular [n = 4], late follicular [n = 3], early luteal [n = 5], late luteal [n = 5]). After laser capture microdissection, total mRNA was extracted from the fimbrial epithelium and real-time quantitative reverse transcription-PCR was performed to determine functional signal transduction pathway activity of the androgen receptor (AR), estrogen receptor (ER), phosphoinositide-3-kinase (PI3K), Hedgehog (HH), transforming growth factor-beta (TGF-ß) and canonical wingless-type MMTV integration site (Wnt) pathways. RESULTS: The early luteal phase demonstrated high AR and ER pathway activity in comparison with the late luteal phase (p = 0.016 and p = 0.032, respectively) and low PI3K activity compared with the late follicular phase (p = 0.036), whereas the late luteal phase showed low activity of HH and Wnt compared with the early follicular phase (both p = 0.016). Signal transduction pathway activity in fimbrial epithelium from postmenopausal patients was most similar to the early follicular and/or late luteal phase with regard to the AR, ER and PI3K pathways. Wnt pathway activity in postmenopausal patients was comparable to the late follicular and early luteal phase. We observed no differences in HH and TGF-ß pathway activity between pre- and postmenopausal samples. The cyclic changes in signal transduction pathway activity suggest a stage-specific function which may affect the morphology and physiology of the human fallopian tube. CONCLUSIONS: We demonstrated cyclic changes in activity of the AR, ER, PI3K, HH and Wnt pathways throughout the hormonal cycle.

Identification of multiple male reproductive tract-specific proteins that regulate sperm migration through the oviduct in mice.

CRISPR/Cas9-mediated genome editing technology enables researchers to efficiently generate and analyze genetically modified animals. We have taken advantage of this game-changing technology to uncover essential factors for fertility. In this study, we generated knockouts (KOs) of multiple male reproductive organ-specific genes and performed phenotypic screening of these null mutant mice to attempt to identify proteins essential for male fertility. We focused on making large deletions (dels) within 2 gene clusters encoding cystatin (CST) and prostate and testis expressed (PATE) proteins and individual gene mutations in 2 other gene families encoding glycerophosphodiester phosphodiesterase domain (GDPD) containing and lymphocyte antigen 6 (Ly6)/Plaur domain (LYPD) containing proteins. These gene families were chosen because many of the genes demonstrate male reproductive tract-specific expression. Although Gdpd1 and Gdpd4 mutant mice were fertile, disruptions of Cst and Pate gene clusters and Lypd4 resulted in male sterility or severe fertility defects secondary to impaired sperm migration through the oviduct. While absence of the epididymal protein families CST and PATE affect the localization of the sperm membrane protein A disintegrin and metallopeptidase domain 3 (ADAM3), the sperm acrosomal membrane protein LYPD4 regulates sperm fertilizing ability via an ADAM3-independent pathway. Thus, use of CRISPR/Cas9 technologies has allowed us to quickly rule in and rule out proteins required for male fertility and expand our list of male-specific proteins that function in sperm migration through the oviduct.

Sperm-oviduct interaction: Differential gene expression of growth factors induced by sperm DNA fragmentation.

The present study investigated the effects of DNA fragmentation of spermatozoa on the growth factors expression by a human oviduct epithelial cell line (OE-E6/E7). Two separate groups were examined in this study. The cell line was cultured in the presence of spermatozoa with normal DNA fragmentation index (DFI) or abnormal DFI. Total RNA from the cell line in each group was isolated, and relative expression of objective genes was analysed using PCR array. Also, the concentration of VEGF, BMP-2, BMP-7 and MSTN in the supernatant of cell culture was analysed by the ELISA method. The PCR array analysis revealed that most of the growth factors had been upregulated in the abnormal group. However, the differences between groups were statistically significant (p < 0.05) for five genes, including VEGF-A, BMP-2, BMP-6, BMP-7 and OSM. Furthermore, MSTN was the only gene that down-regulated significantly under the influence of the spermatozoa with abnormal DFI. Moreover, the results of ELISA analysis were in agreement with the data of the PCR array. It has been concluded that DNA fragmentation in human spermatozoa can probably change regular events throughout the oviducts. Consequently, the genes of interest may change sperm function and probably its fate in the female reproductive tract.

In vivo three-dimensional tracking of sperm behaviors in the mouse oviduct.

Mammalian sperm evolutionarily acquired complex mechanisms to regulate their behaviors, which are thought to be crucial in navigating through the female reproductive tract toward fertilization. However, all current knowledge of this process is largely extrapolated from in vitro and ex vivo studies, because in vivo analysis of sperm in their native fertilization environment has not been possible. Here, we report a functional optical coherence tomography approach that allows, for the first time, in vivo three-dimensional tracking of sperm behaviors in the mouse oviduct. Motile sperm are identified with their intrinsic dynamic characteristics. Sperm trajectories are reconstructed in three dimensions with a ∼5 µm spatial resolution, allowing for quantitative analysis of the sperm velocity and location relative to the oviduct. Using this method, we found different behavior patterns, including sperm collection by the oviduct epithelium, spatial dependence of sperm velocity, and sperm grouping and separation as the first in vivo evidence of sperm cooperation in the ampulla, the site of fertilization. This approach opens new avenues to study sperm-oviduct interactions in vivo toward a more complete understanding of fertility and reproductive disorders.

Biophysical optimization of preimplantation embryo culture: what mechanics can offer ART.

Owing to the rise of ART and mounting reports of epigenetic modification associated with them, an understanding of optimal embryo culture conditions and reliable indicators of embryo quality are highly sought after. There is a growing body of evidence that mechanical biomarkers can rival embryo morphology as an early indicator of developmental potential and that biomimetic mechanical cues can promote healthy development in preimplantation embryos. This review will summarize studies that investigate the role of mechanics as both indicators and promoters of mammalian preimplantation embryo development and evaluate their potential for improving future embryo culture systems.

Roles of steroid hormones in oviductal function

The oviduct (known as the fallopian tube in humans) is the site for fertilization and pre-implantation embryo development. Female steroid hormones, estrogen and progesterone, are known to modulate the morphology and function of cells in the oviduct. In this review, we focus on the actions of estrogen and progesterone on secretory, ciliated, and muscle cell functions and morphologies during fertilization, pre-implantation embryo development, and embryo transport in humans, laboratory rodents and farm animals. We review some aspects of oviductal anatomy and histology and discuss current assisted reproductive technologies (ARTs) that bypass the oviduct and their effects on embryo quality. Lastly, we review the causes of alterations in secretory, ciliated, and muscle cell functions that could result in embryo transport defects.

Early embryo-maternal communication in the oviduct: A review.

An intact embryo-maternal communication is critical for the establishment of a successful pregnancy. To date, a huge number of studies have been performed describing the complex process of embryo-maternal signaling within the uterus. However, recent studies indicate that the early embryo communicates with the oviductal cells shortly after fertilizationand that this is important for the successful establishment of pregnancy. Only if the early embryo is capable to signal the mother within a precise timeframe and to garner a response, will the embryo be able to survive and reach the uterus. This review will give an overview of all the experimental designs which have investigated embryo-maternal interaction in the oviduct. In addition to that, it will provide a comprehensive analysis of the findings to date elucidating the morphological and molecular changes in the oviduct which are induced by the presence of the early embryo highlighting how the tubal responses affect embryo development and survival.

Human fallopian tube epithelial cells exhibit stemness features, self-renewal capacity, and Wnt-related organoid formation.

BACKGROUND: Fallopian tube epithelial cells (FTEC) were thought to be the origin of high-grade serous ovarian carcinoma (HGSOC). Knowledge of the stemness or initiating characteristics of FTEC is insufficient. Previously, we have characterized the stemness cell marker of FTEC, this study aims to further characterize the clonogenicity and spheroid features of FTEC. METHODS: We successfully derived FTECs from the epithelial layer of the human fallopian tubes. We examined the morphology, proliferation rate, doubling time, and clonal growth of them. At passage 3, the sphere formations on gelatin-coated culture, suspension culture, and matrigel culture were observed, and the expression of LGR5, SSEA3, SSEA4, and other stemness markers was examined. Furthermore, tissue-reconstituted organoids from coculture of FTEC, fallopian stromal cells (FTMSC) and endothelial cells (HUVEC) were examined. RESULTS: FTEC exhibited cuboidal cell morphology and maintained at a constant proliferation rate for up to nine passages (P9). FTEC could proliferate from a single cell with a clonogenic efficiency of 4%. Flow cytometry revealed expressions of normal stem cell markers (SSEA3, SSEA4, and LGR5) and cancer stem cell markers (CD24, CD44, CD117, ROR1, and CD133). FTEC formed spheres and colonies when cultured on low attach dish. In the presence of Matrigel, the stemness and colony formation activity were much enhanced. In co-culturing with FTMSC and HUVEC, FTEC could form organoids that could be blocked by Wnt inhibitor DKK1. Expressions of LGR5 and FOXJ1 expression were also decreased by adding DKK1. CONCLUSION: We demonstrated abundantly presence of stem cells in human FTECs which are efficient in forming colonies, spheres and organoids, relying on Wnt signaling. We also reported for the first time the generation of organoid from reconstitutied cell lineages in the tissue. This may provide a new model for studying the regneration and malignant transformation of the tubal epithelium.

Does Maternal Stress Affect the Early Embryonic Microenvironment? Impact of Long-Term Cortisol Stimulation on the Oviduct Epithelium.

Maternal stress before or during the sensitive preimplantation phase is associated with reproduction failure. Upon real or perceived threat, glucocorticoids (classic stress hormones) as cortisol are synthesized. The earliest "microenvironment" of the embryo consists of the oviduct epithelium and the oviductal fluid generated via the epithelial barrier. However, to date, the direct effects of cortisol on the oviduct are largely unknown. In the present study, we used a compartmentalized in vitro system to test the hypothesis that a prolonged stimulation with cortisol modifies the physiology of the oviduct epithelium. Porcine oviduct epithelial cells were differentiated at the air-liquid interface and basolaterally stimulated with physiological levels of cortisol representing moderate and severe stress for 21 days. Epithelium structure, transepithelial bioelectric properties, and gene expression were assessed. Furthermore, the distribution and metabolism of cortisol was examined. The polarized oviduct epithelium converted basolateral cortisol to cortisone and thereby reduced the amount of bioactive cortisol reaching the apical compartment. However, extended cortisol stimulation affected its barrier function and the expression of genes involved in hormone signaling and immune response. We conclude that continuing maternal stress with long-term elevated cortisol levels may alter the early embryonic environment by modification of basic oviductal functions.

Identification of Rabbit Oviductal Fluid Proteins Involved in Pre-Fertilization Processes by Quantitative Proteomics.

Oviductal fluid (ODF) proteins modulate and support reproductive processes in the oviduct. In the present study, proteins involved in the biological events that precede fertilization have been identified in the rabbit ODF proteome, isolated from the ampulla and isthmus of the oviduct at different time points within 8 h after intrauterine insemination. A workflow is used that integrates lectin affinity capture with stable-isotope dimethyl labeling prior to nanoLC-MS/MS analysis. In total, over 400 ODF proteins, including 214 lectin enriched glycoproteins, are identified and quantified. Selected data are validated by Western blot analysis. Spatiotemporal alterations in the abundance of ODF proteins in response to insemination are detected by global analysis. A subset of 63 potentially biologically relevant ODF proteins is identified, including extracellular matrix components, chaperones, oxidoreductases, and immunity proteins. Functional enrichment analysis reveals an altered peptidase regulator activity upon insemination. In addition to protein identification and abundance changes, N-glycopeptide analysis further identifies 281 glycosites on 199 proteins. Taken together, these results show, for the first time, the evolving oviductal milieu early upon insemination. The identified proteins are likely those that modulate in vitro processes, including spermatozoa function.

Influence of metabolic status and genetic merit for fertility on proteomic composition of bovine oviduct fluid†.

The oviduct plays a crucial role in fertilization and early embryo development providing the microenvironment for oocyte, spermatozoa, and early embryo. Since dairy cow fertility declined steadily over the last decades, reasons for early embryonic loss have gained increasing interest. Analyzing two animal models, this study aimed to investigate the impact of genetic predisposition for fertility and of metabolic stress on the protein composition of oviduct fluid. A metabolic model comprised maiden Holstein heifers and postpartum lactating (Lact) and non-lactating (Dry) cows, while a genetic model consisted of heifers from the Montbéliarde breed and Holstein heifers with low- and high-fertility index. In a holistic proteomic analysis of oviduct fluid from all groups using nano-liquid chromatography tandem-mass spectrometry analysis and label-free quantification, we were able to identify 1976 proteins, among which 143 showed abundance alterations in the pairwise comparisons within both models. Most differentially abundant proteins were revealed between low fertility Holstein and Montbéliarde (52) in the genetic model and between lactating and maiden Holstein (19) in the metabolic model, demonstrating a substantial effect of genetic predisposition for fertility and metabolic stress on the oviduct fluid proteome. Functional classification of affected proteins revealed actin binding, translation, and immune system processes as prominent gene ontology (GO) clusters. Notably, Actin-related protein 2/3 complex subunit 1B and the three immune system-related proteins SERPIND1 protein, immunoglobulin kappa locus protein, and Alpha-1-acid glycoprotein were affected in both models, suggesting that abundance changes of immune-related proteins in oviduct fluid play an important role for early embryonic loss.

Effects of selective serotonin reuptake inhibitors on motility of isolated fallopian tube.

Selective serotonin reuptake inhibitors (SSRIs) affect the smooth muscle cells acting on voltage-dependent channels for Na+ , K+ and Ca2+ , but their action is tissue and species specific. The aim of our study was to investigate effects of selective serotonin reuptake inhibitors on motility of the isolated fallopian tubes. Isolated preparations of isthmus and ampoule were taken from fallopian tubes of 20 women during hysterectomy due to uterine fibroids and then tested for reactivity on increasing concentrations of selective serotonin reuptake inhibitors. Escitalopram (from 0.9 × 10-9  M/L to 1.4 × 10-6  M/L) produced concentration-dependent increase of spontaneous contractions of the isolated ampulla (EC50 = 1.20 ± 1.06 × 10-8  M/L, r = 0.580, P < 0.05) (F = 2.980, df1  = 6, df2  = 28, P < 0.05). Paroxetine (from 1.2 × 10-9  M/L to 5.1 × 10-5  M/L) produced concentration-dependent increase of spontaneous contractions of the isolated isthmus (EC50 = 7.01 ± 3.50 × 10-8  M/L, r = 0.500, P < 0.05) (F = 2.350, df1  = 9, df2  = 40, P < 0.05). The SSRIs differ among themselves in regard to their potential to affect motility of the fallopian tubes. Escitalopram and paroxetine have clear stimulating effect which may interfere with functioning of the fallopian tubes, and potentially impair fertility if taken by women in reproductive period of life. The other SSRIs tested in the study did not produce significant effect throughout the concentration range used in the experiments.

Myosalpinx Contractions Are Essential for Egg Transport Along the Oviduct and Are Disrupted in Reproductive Tract Diseases.

Oviducts (also called fallopian tubes) are smooth muscle-lined tubular organs that at one end extend in a trumpet bell-like fashion to surround the ovary, and at the other connect to the uterus. Contractions of the oviduct smooth muscle (myosalpinx) and the wafting motion of the ciliated epithelium that lines these tubes facilitate bidirectional transport of gametes so that newly released ovum(s) are transported in one direction (pro-uterus) while spermatozoa are transported in the opposite direction (pro-ovary). These transport processes must be temporally coordinated so that the ovum and spermatozoa meet in the ampulla, the site of fertilization. Once fertilized, the early embryo begins another precisely timed journey towards the uterus for implantation. Myosalpinx contractions facilitate this journey too, while luminal secretions from secretory epithelial cells aid early embryo maturation.The previous paradigm was that oviduct transport processes were primarily controlled by fluid currents generated by the incessant beat of the ciliated epithelium towards the uterus. More recently, video imaging and spatiotemporal mapping have suggested a novel paradigm in which ovum/embryo transport is highly dependent upon phasic and propulsive contractions of the myosalpinx. A specialized population of pacemaker cells, termed oviduct interstitial cells of Cajal (ICC-OVI), generate the electrical activity that drives these contractions. The ionic mechanisms underlying this pacemaker activity are dependent upon the calcium-activated chloride conductance, Ano1.This chapter discusses the basis of oviduct pacemaker activity, its hormonal regulation, and the underlying mechanisms and repercussions when this activity becomes disrupted during inflammatory responses to bacterial infections, such as Chlamydia.

Composing the Early Embryonic Microenvironment: Physiology and Regulation of Oviductal Secretions.

The oviductal fluid is the first environment experienced by mammalian embryos at the very beginning of life. However, it has long been believed that the oviductal environment was not essential for proper embryonic development. Successful establishment of in vitro embryo production techniques (which completely bypass the oviduct) have reinforced this idea. Yet, it became evident that in vitro produced embryos differ markedly from their in vivo counterparts, and these differences are associated with lower pregnancy outcomes and more health issues after birth. Nowadays, researchers consider the oviduct as the most suitable microenvironment for early embryonic development and a substantial effort is made to understand its dynamic, species-specific functions. In this review, we touch on the origin and molecular components of the oviductal fluid in mammals, where recent progress has been made thanks to the wider use of mass spectrometry techniques. Some of the factors and processes known to regulate oviductal secretions, including the embryo itself, as well as ovulation, insemination, endogenous and exogenous hormones, and metabolic and heat stress, are summarized. Special emphasis is laid on farm animals because, owing to the availability of sample material and the economic importance of fertility in livestock husbandry, a large part of the work on this topic has been carried out in domestic animals used for dairy and/or meat production.

Immunohistochemical localization of estrogen receptor alpha (ERα) in the oviduct of Indian buffalo during follicular and luteal phases of estrous cycle.

The localization and distribution of estrogen receptor alpha (ERα) in different segments of oviduct of buffalo during follicular and luteal phases of estrous cycle were investigated using immunohistochemistry. Tissue samples from the different segments of oviduct from 12 buffaloes (six each during follicular and luteal phases of estrous cycle) were collected from slaughter house after assessing the gross morphology of ovaries. In addition, blood samples were collected from the animals before slaughter to estimate levels of estrogen and progesterone hormones. The tissue distribution of estrogen receptor was determined by immunohistochemical technique using one-step polymer HRPO staining system. The estrogen receptor was localized in the lamina epithelialis, propria submucosa, tunica muscularis, and tunica serosa. The maximum localization was observed in the lamina epithelialis, where both ciliated and secretory cell types were positive for ERα. Percentage of positive cells varied during the follicular and luteal phases of estrous cycle. The lining epithelium of oviductal glands was also intensely positive for ERα. No immunostaining was observed in any tunic of the oviduct when the primary antibody was replaced by antibody diluent or buffer, and it served as negative control. The data showed that highest immune positive cells were observed in the ampulla region of the oviduct and these cells were lowest in the utero-tubal junction (p < 0.05). Infundibulum, ampulla, and isthmus showed a higher percentage of ERα-positive cells during follicular phase of estrous cycle as compared with those of the luteal phase of estrous cycle (p < 0.05). There was no significant difference in the percentage positive cells during the two phases of estrous cycle in the utero-tubal junction. Immunogold labeling with anti-ERα antibody confirmed the findings of immunohistochemical study at subcellular level. The higher expression during the follicular phase was directly correlated with the level of estrogen hormone.