Comprehensive Proteomics Analysis of In Vitro Canine Oviductal Cell-Derived Extracellular Vesicles

Dogs (Canis lupus familiaris) have unique and peculiar reproductive characteristics. While the interplay between in vitro oviductal cell-derived extracellular vesicles (OC-EVs) and cumulus-oocyte complexes in dogs has begun to be elucidated, no study has yet provided extensive information on the biological content and physiological function of OC-EVs and their role in canine oocyte development. Here, we aimed to provide the first comprehensive proteomic analysis of OC-EVs. We identified 398 proteins as present in all OC-EVs samples. The functional enrichment analysis using Gene Ontology terms and an Ingenuity Pathway Analysis revealed that the identified proteins were involved in several cellular metabolic processes, including translation, synthesis, expression, and protein metabolism. Notably, the proteins were also involved in critical canonical pathways with essential functions in oocyte and embryo development, such as ERK/MAPK, EIF2, PI3K/AKT, and mTOR signaling. These data would be an important resource for studying canine reproductive physiology and establishing a successful in vitro embryo production system in dogs.

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

One of the endogenous estrogens, 17β-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. The oviduct response to E2 is virtually unknown in an in vivo environment. 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 are differentially regulated by E2 and support gene expression changes that are required for normal embryo development and transport in mouse models.

Effects of spermatozoa–oviductal cell coincubation time and oviductal cell age on spermatozoa–oviduct interactions

The oviduct plays a crucial role in sperm storage, maintenance of sperm viability and sperm transport to the site of fertilisation. The aim of the present study was to investigate the effects of oviductal cell culture passage number, oviductal cell age and spermatozoa–oviduct coincubation times on gene expression in oviductal cells. Immortalised oviductal epithelial cells (OPEC) obtained from two different cell passages (36 and 57) were subcultured three times with and without spermatozoa for 24 h (control group). In a second study, OPEC were cocultured with spermatozoa for different time intervals (0, 4, 12 and 24 h). Expression of adrenomedullin (ADM), heat shock 70 kDa protein 8 (HSPA8) and prostaglandin E synthase (PGES) in OPEC was measured by quantitative polymerase chain reaction. The expression of ADM and HSPA8 was decreased significantly in OPEC cells from Passage 57, particularly in the later subculture group. These effects on HSPA8, but not ADM, expression in OPEC were further altered after coculture with spermatozoa for 24 h. We also demonstrated that spermatozoa–oviduct coculture for 12 and 24 h resulted in significantly higher expression of ADM, HSPA8 and PGES in OPEC. Overall, the data suggest that the OPEC lose some of their properties as a result of oviductal cell aging and that there are spermatozoa–oviduct interactions leading to increased oviductal cell gene expression.

Expression of Adrenomedullin in Human Oviduct, Its Regulation by the Hormonal Cycle and Contact with Spermatozoa, And Its Effect on Ciliary Beat Frequency of the Oviductal Epithelium

Context: Adrenomedullin (ADM) has been found expressed in the mouse oviduct and might play a role in reproduction. Objective: To demonstrate the expression of adrenomedullin (ADM) in the human oviduct, investigate its regulation by steroidal hormones and spermatozoa contact, and study its effect on ciliary beat frequency (CBF) in the human oviduct. Design, Setting, Patients and Interventions: Oviducts from women undergoing hysterectomy for benign diseases in a university hospital were collected. The oviducts were treated with estradiol and/or progesterone to simulate different phases of the ovarian cycle. ADM expression was studied at the peptide and mRNA levels by immunohistochemistry and RT-PCR respectively. CBF was measured after treatment with graded concentrations of ADM and its antagonists. Cells from OE-E6/E7, an immortalized oviductal cell line, as well as oviductal tissue were co-cultured with and without direct contact with capacitated human spermatozoa to compare oviductal cell ADM expression levels. CBF was also analyzed in oviductal tissue after spermatozoa-oviduct co-incubation. Results: ADM expression was the highest in the isthmic region and in a hormonal environment simulating the early luteal phase. CBF was increased by ADM in a dose-dependent manner which was blocked by ADM and calcitonin-gene-related peptide receptor antagonists. Direct contact with spermatozoa in co-culture resulted in higher ADM expression in OE-E6/E7 cell line and oviductal tissue, and higher CBF in oviductal epithelium. Conclusions: ADM expression in the human oviduct is hormone-dependent and is up-regulated by sperm contact. ADM stimulates ciliary motility of the human oviduct.

Isolation and culture of bovine oviductal epithelial cells for use in the anatomy and physiology laboratory and undergraduate research

This article presents methods for the isolation and culture of epithelial cells from the bovine oviduct for use in both research and the teaching laboratory and provides examples of ways that an oviductal cell culture can be incorporated into an undergraduate research program. Cow reproductive tracts are readily available from area butchers, and the procedure for isolation of the epithelium is simple and inexpensive. The cells can be observed immediately after isolation or can be cultured for up to 72 h under simple conditions for observation over several days. For experimental use, epithelial cells are cultured in standard cell culture medium, where they continue to divide and actively secrete substances into the medium. The ease with which the tissue can be collected and cells isolated makes the oviductal epithelium ideal for use in both the teaching laboratory and research projects in which undergraduates serve as investigators.