Elsevier

Theriogenology

Volume 77, Issue 8, May 2012, Pages 1698-1708
Theriogenology

Research article
Effects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells

https://doi.org/10.1016/j.theriogenology.2011.12.015Get rights and content

Abstract

Cell cultures are useful for determining the responses of specific cell types to various factors under controlled conditions and for obtaining a better understanding of in vivo physiologic processes. The aims of the present study were (i) to establish methodologies for isolation, culture and cryopreservation of equine endometrial epithelial and stromal cells; and (ii) to determine the effect of passage and cryopreservation on endometrial cell physiology, based on their basal and oxytocin (OT)-stimulated prostaglandin (PG) release. Epithelial and stromal cells were obtained by enzymatic digestion of equine endometrium collected from Days 2–5 of the estrous cycle (n = 16). Primary epithelial and stromal cells, as well as cryopreserved cells were stimulated with OT (10−7 m) for 24 h. The concentrations of PGE2 and PGF in the culture medium were measured by enzyme-linked immunosorbent assay (EIA). Oxytocin increased PGE2 and PGF release by primary cultures of unfrozen epithelial cells until passage I (P < 0.01) and by the primary culture of unfrozen and cryopreserved/thawed stromal cells until passage IV (P < 0.01). Cryopreserved/thawed stromal cells cultured up to passage IV and unfrozen epithelial cells derived from passage I have physiological properties similar to those observed in primary culture and may be successfully used for in vitro studies of PG secretion.

Introduction

The mare is a long-day seasonal breeder [1]. In the northern hemisphere, ovarian periodicity lasts from late March until late September [1], so in vitro studies of primary equine endometrial cells are presently restricted to this period. The development of a method for cryopreserving endometrial cells would allow such studies to take place throughout the year, as well as reducing the frequency of tissue collection which is tedious. For each cell type, there is a set of optimal conditions for cryopreservation [2], [3]. Several studies have described procedures for cryopreservation of cells derived from reproductive tracts e.g., bovine endometrial epithelial and stromal cells [2], bovine endothelial cells from the corpus luteum (CL) [3] or porcine endometrial epithelial cells [4]. Cryopreservation is still the most important method for storage of semen [5], [6] and embryos [7], both in humans and domestic animals.

Cryopreservation and thawing processes can have many deleterious effects on cells. There is an inherent variability in survival after freezing and thawing processes among different cell types and within individual cells of a given population. The production of prostaglandin (PG) in response to oxytocin (OT) is a useful parameter for determining the physiology and endocrine status of culture bovine endometrial epithelial cells [8], [9]. Murakami, et al. [2] evaluated the functional properties of endometrial cells by their production of PGF in response to OT and tumor necrosis factor-α (TNF). Thus, we suggest that the ability of endometrial epithelial and stromal cells to produce PG in response to OT may be used as an indicator of the cellular properties after freeze/thaw processes. Although OT stimulates endometrial cells to release PG in many species [10], [11], the effect of OT on secretion by specific types of the endometrial cells e.g., stromal and/or epithelial cells, in the mare is still unknown. In vivo studies of OT action on PGF secretion by equine endometrium showed a positive correlation between PGF release, endometrial OT receptor (OTR) density, and plasma OT concentrations, suggesting that PGF may be induced by OT [12]. However, there are no studies either in vivo or in vitro showing directly whether OT can stimulate endometrial PGE2 in the mare.

The aim of the present study was to develop a reliable method for isolation and culture of equine epithelial and stromal cells. The properties of cells after passage and cryopreservation were evaluated by their ability to secrete PG in response to OT and by their morphology, compared to unfrozen primary cells. Furthermore, the influence of OT on PGE2 and PGF release by equine epithelial and stromal cells was determined.

Section snippets

Animals and tissue collection

Uteri were collected post-mortem from cyclic mares from the end of March until the end of August at a local abattoir. All procedures for animal treatment and tissue collection were approved by the Local Animal Care and Use Committee in Olsztyn, Poland (Agreements Number 51/2011). The mares were healthy as stated by the official governmental veterinary inspection. The whole uterus was collected within 5 min after the death of an animal, placed in sterile, incomplete (Ca2+-and Mg2+-free) Hank's

Preliminary study: dose-dependent effect of OT on PGE2 and PGF release by endometrial cells

The release of PGE2 and PGF by stromal and epithelial cells increased in response to OT in a dose-dependent manner (P < 0.05; Fig. 2A–D). Based on these results, OT at a dose of 10−7M was chosen for the main Experiment. In addition, OT increased stromal cell viability to approximately 27% compared to the control (P < 0.05; Fig. 3B).

The effect of passage and cryopreservation on secretory function and morphology of epithelial and stromal cells

In stromal cells, OT increased PGE2 release in primary cultures and at passages I, II, III and IV of unfrozen stromal cells (P < 0.05) (Fig. 4A). Moreover, OT

Discussion

The present study describes a method of isolation, culture, cryopreservation and storage of pure populations of equine epithelial and stromal endometrial cells. In this work, it was feasible to culture cryopreserved/thawed stromal cells until passage IV. During long-term culture, the cells maintained their characteristic morphology and responded to OT like cells in primary cultures. Moreover, basal and OT-stimulated PG secretion by passaged unfrozen cells did not differ from passaged

Acknowledgments

Supported by Grants in Aids: the International Project of Polish Ministry of Science and Higher Education (DPN/N5/COST/2010) and PTDC/CVT/121805/2010 from FCT, Portugal. A. Z. Szóstek was supported by the European Union within the European Social Fund (DrINNO). A. Galvão was supported by FCT (SFRH/BD/29765/2006).

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