The modulation of prostaglandin production by coumestrol in the equine endometrium in vitro

https://doi.org/10.1016/j.jevs.2013.10.091Get rights and content

Introduction

Phytoestrogens are estrogenic compounds present in some forages fed to horses. The presence of phenolic rings in phytoestrogens structure makes them similar to 17β-estradiol (E2), and enables their binding to estrogen receptors [2], [8]. Thus, phytoestrogens may act as endocrine disrupters by competing with the endogenous estrogens, influencing the estrous cycle [7], [8]. Coumestrol, a phytoestrogen, has similar in vitro estrogenic potency to estradiol itself, and is the most potent phytoestrogen known [2], [6], which justifies evaluating its effect on reproduction. In the breeding season, even though the incidence of at least one period of persistence of the corpus luteum in mares is about 5 to 20% [5], its association to phytoestrogens effect has not been shown. However, in our recent study, we have shown the presence of coumestrol (Cou) and its metabolite methoxycoumestrol (Met) in plasma of mares fed phytoestrogen rich plants such as clover and alfalfa (Medicago sativa) [1]. To the best of our knowledge, this was the first study on the presence of phytoestrogens in mares' plasma and its biological effects. In a controlled experiment, mares eating up to 1Kg of alfalfa pellets added to their diet, showed increased plasma concentrations of conjugated and free forms of Cou and Met between 1h and 4h after ingestion [1]. As previously shown in others species [reviewed by Woclawek-Potocka et al., 8], in the present work, plasma concentrations of free forms of isoflavones (Cou) and its metabolite were lower than their conjugated ones, indicating that most of these compounds remain in blood in the form they were absorbed, and only a small portion is potentially active. A clinical case was also reported by us on mares fed a mixture of alfalfa and clover haylage for either 5 or 9 months, who presented very intensive signs of hyperestrogenicity with no ovulation from the beginning of the breeding season [1]. These animals showed hyperedema of the uterus, uterine fluid accumulation, excessive cervical mucus discharge, and estrous cycle disruption. These clinical signs were associated with high plasma concentrations of phytoestrogens Cou and Met, and E2, while progesterone (P4) level was low (less than 1 ng/mL). The temporary infertility observed in these mares was solved within 2-3 weeks after alfalfa clover based haylage withdrawal. In all mares, hyperestrogenicity signs subsided with resumption of normal ovarian cyclicity. This indicates a strong estrogenic activity of this fodder due to a long term intake of Cou and its metabolite Met, which were the highest in mares still ingesting alfalfa and clover haylage at the time of blood collection, than in the mares no longer fed haylage [1]. In summary, our recent in vivo study shows that in the mare, blood Cou and Met increase after estrogenic plants ingestion, mainly in alfalfa-based haylage, which seems to be estrogenic endocrine disruptors [1]. Therefore, in vitro studies were carried out to evaluate the potential effects and mechanisms of action of Cou on equine endometrium functions.

The specific aims of the study were to determine the influence of Cou on: (i) prostaglandin (PG) production and prostaglandin synthases mRNA transcription in equine epithelial and stromal cells; and (ii) endometrial cell proliferation.

Section snippets

Materials and Methods

Epithelial and stromal cells were isolated from the equine endometrium (early luteal phase; n=20) and treated after passage I [4]. Only cells derived from the endometria that were classified as I (no degenerative changes) according to Kenney classification [3] classification were assigned to our present study. In Experiment 1, epithelial (n=5 mares) and stromal cells (n=5 mares) were exposed to Cou (10-8M) and 17-β estradiol (E2; 10-9M) for 24h. Oxytocin (OT; 10-7M) was used as a positive

Results and Discussion

In epithelial cells, E2 as well as Cou, augmented PGE2 and PGF secretion compared to control group (P<0.05; Figure 1A,B). In contrast, in stromal cells, only Cou increased PGE2 secretion compared to control group (P<0.001). However, E2 as well as Cou stimulated PGF secretion compared to control group in stromal cells (P<0.001). Both 17-β estradiol and Cou up-regulated PTGS-2 mRNAs transcription in stromal and epithelial cells compared with the control group (P<0.05; Figure 1C,D). In

Acknowledgements

Granted by Maestro Project of NSC (No 2011/02/A/NZ5/00338). DJS, MB and GMF-D were supported by the joint Polish-Portugal project under Agreement of MSHE (Poland) and FCT (Portugal).

References (8)

There are more references available in the full text version of this article.

Cited by (0)

View full text