Research articleEffects of cell storage and passage on basal and oxytocin-regulated prostaglandin secretion by equine endometrial epithelial and stromal cells
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 PGF2α 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 PGF2α secretion by equine endometrium showed a positive correlation between PGF2α release, endometrial OT receptor (OTR) density, and plasma OT concentrations, suggesting that PGF2α 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 PGF2α 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 PGF2α release by endometrial cells
The release of PGE2 and PGF2α 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).
References (38)
- et al.
Seasonal reproduction in the mare: possible role of plasma leptin, body weight and immune status
Dom Anim Endocrinol
(2005) - et al.
Activation and expression of urokinase-type plasminogen activator are modulated by freezing/thawing process through activation of redox signal pathway in primary porcine endometrial cells
Cryobiology
(2010) - et al.
Expression of estrogen receptor and maintenance of hormone-responsive phenotype in bovine fetal uterine cells
Dom Anim Endocrinol
(1998) - et al.
Concentration of prostaglandins F in uterine venous plasma of anesthetized mares during the estrous cycle and early pregnancy
Prostaglandins
(1976) - et al.
A light microscopic and ultrastructural study on the presence and location of oxytocin in the equine endometrium
Theriogenology
(2003) - et al.
Intracellular free calcium in response to oxytocin in pig endometrial cells
Mol Cell Endocrinol
(1999) - et al.
Oxytocin stimulates prostaglandin F2α secretion from porcine endometrial cells through activation of calcium-dependent protein kinase C
Prostaglandins Other Lipid Mediat
(2001) - et al.
Sparing effects of intrauterine treatment with prostaglandin E2 on luteal function in cycling gilts
Prostaglandins
(1986) - et al.
Prostaglandin E2 counteracts the effects of PGF2 alpha in indomethacin treated cycling gilts
Prostaglandins
(1988) - et al.
Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum
Prostaglandins Other Lipid Mediat
(2003)
Evaluation of genes involved in prostaglandin action in equine endometrium during estrous cycle and early pregnancy
Anim Reprod Sci
A passage and storage system for isolated bovine endometrial epithelial and stromal cells
J Reprod Dev
Effects of storage and passage of bovine luteal endothelial cells on endothelin-1 and prostaglandin F2alpha production
J Reprod Dev
Freezing equine semen: the effect of combinations of semen extenders and glycerol on post-thaw motility
Aust Vet J
Cryopreservation of mouse and human oocytes using 1,2-propanediol and the configuration of the meiotic spindle
Hum Reprod
Mouse embryo cryopreservation utilizing a novel high-capacity vitrification spatula
BioTechniques
Bovine endometrial epithelial cells as a model system to study oxytocin receptor regulation
Hum Reprod Update
Isolation and culture of bovine endometrial epithelial cells in a serum-free culture system
J Reprod Dev
Oxytocin-stimulated phosphoinositide hydrolysis and prostaglandin F secretion by luminal epithelial, glandular epithelial, and stromal cells from pig endometriumI. Response of cyclic pigs on day 16 postestrus
Biol Reprod
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