Proliferation and Apoptosis in Fetal Membranes and Endometrium During Placental Retention in Heavy Draft Mares

Abstract

Placental retention (retained placenta [RP]) is a serious and common peripartum disease in mares, but the etiology and pathogenesis of RP still remain unclear. The alteration of cell proliferation and apoptosis is considered to be an important factor in RP. Fetal membranes and endometrial biopsies were collected from mares with RP (n = 8) and from control mares (n = 10). The proliferation and apoptosis levels in the chorionic and the endometrial epithelia were assessed by proliferating cell nuclear antigen immunostaining and the terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling assay, respectively. The study revealed that there was an insignificant decrease in proliferation and a significant increase in apoptosis in the chorionic epithelium from mares with RP. This result excludes a proliferation imbalance from the possible causes of RP. In the area of the nonpregnant horn of the placenta, proliferation was negatively correlated and apoptosis was positively correlated with the degree of fetomaternal anchorage. It was observed that, in all mares with placental retention, the endometrial epithelium (both luminal and glandular) showed decreased proliferation and increased apoptosis, which may indicate a delay in postpartum uterine regeneration.

Introduction

The fetal membranes together with the endometrium constitute a complex organ of fetomaternal communication. In mares, the fetal chorionic epithelium adheres to the endometrial epithelium, creating microplacentomes diffused all over the placenta [1]. Retained placenta (RP) is a serious and common peripartum disease in heavy draft mares [2], often resulting in a delay in the involution of the uterus [3], and a decreased pregnancy rate after breeding at foal heat [4]. RP is defined as a failure to expel all fetal membranes within 3 hours after delivery of a foal [5]. The causes of the RP are unclear [2]. Boos et al. [6] suggested that placental homeostasis, which involves growth, maturation, release, or retention, is determined by proliferation and apoptosis. Apoptosis is essential for placental maturation and the proper release of the fetal membranes in cows [6], and is suspected to be important in horses. Histopathological examination of RPs revealed intense proliferation of the chorionic epithelium, and apoptotic bodies were frequently seen in this area (our own unpublished data). Routine analysis does not enable the exact determination of the proliferation–apoptosis balance in the RP. Thus, we decided to evaluate proliferation and apoptosis using immunohistochemical methods.

The cell proliferation is connected with DNA replication and therefore with specific enzymes called cell cycle markers [7]. The quantitative evaluation of cell proliferation can be performed by immunohistochemical staining for proliferating cell nuclear antigen (PCNA), a cell cycle marker organized as a ring that encircles DNA and works as a sliding clamp for DNA polymerase delta [8]. PCNA is maximally elevated in the late G1 and S phases of the cell cycle [9].

Apoptosis is physiological cell death and serves as a way to eliminate cells without inducing a local inflammatory response. The most characteristic feature of apoptosis is DNA fragmentation [10]. DNA strand breaks in formalin-fixed, paraffin-embedded tissues are detectable by the modified terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) method [11].

The aim of this study was to evaluate the dynamics of proliferation and apoptosis in the placenta during placental retention in mares and their possible roles in placental retention pathogenesis. We also investigated the influence of placental retention on postpartum endometrial regeneration.