Elsevier

Theriogenology

Volume 83, Issue 2, 15 January 2015, Pages 276-284
Theriogenology

Research article
Temporal analysis of prostaglandin F2α receptor, caspase 3, and cyclooxygenase 2 messenger RNA expression and prostaglandin F2α receptor and cyclooxygenase 2 protein expression in endometrial tissue from multiparous Nelore (Bos taurus indicus) cows treated with cloprostenol sodium during puerperium

https://doi.org/10.1016/j.theriogenology.2014.09.022Get rights and content

Abstract

The use of cloprostenol sodium in puerperium is questionable, as both favorable and unfavorable responses during the uterine involution process have been reported in the literature. This study is based on the hypothesis that cloprostenol sodium promotes modifications in the prostaglandin F2α receptor (FP), caspase 3 (CASP-3), and cyclooxygenase 2 (COX-2) mRNA expression that may favor the process of postpartum uterine involution in multiparous Nelore (Bos taurus indicus) females. Additionally, we aimed to describe the presence and immunolocalization of the FP and COX-2 protein in endometrial tissue at different postpartum time points in these females. Multiparous Nelore cows (n = 24) were treated with cloprostenol sodium (n = 12) or saline solution (n = 12) on postpartum Days 1 and 4 (Day 0 = birth), and endometrial biopsies were performed with a Yomann biopsy instrument and collected on Days 1, 7, 14, 28, and 42 postpartum. The mRNA expression from samples on the Days 1, 7, 14, 28, and 42 and the protein expression from samples on the Days 1, 14, 28, and 42 were then analyzed. The treated cows had altered FP and CASP-3 mRNA expression, and FP and COX-2 protein were observed in the endometrial surface epithelium, the stroma, and the glandular epithelium, with cytoplasmic immunolocalization. Although we attribute the change in CASP-3 mRNA expression to physiological phenomena, the results obtained for FP mRNA expression opens new doors for the study of hormonal protocols associated with cloprostenol sodium in the puerperium of Zebu females.

Introduction

During the postpartum period, the uterus undergoes modifications that restore the environment required to sustain a new pregnancy. Ymer et al. [1] proposed that in cows, a 65-day puerperium period is optimal, and conception within 85 to 90 days is desirable. Therefore, the production of one calf per year is economically feasible.

The postpartum uterus produces prostaglandin F2α (PGF2α), which can assist in the process of uterine involution by stimulating myometrial contraction [2], [3]. Cloprostenol sodium is a synthetic analog of PGF2α that is used for estrous synchronization, disease treatment, and to reduce uterine involution during the postpartum period [4], [5], [6], [7].

Prostaglandin F2α binds to the G-protein–coupled prostaglandin F2α receptor (FP), which mediates the release of intracellular calcium (Ca+) to promote muscular contraction [6], [8]. The mature form of the FP is highly expressed in luteal [8], [9], [10], [11], [12] and follicular cells [13] and in fetal membranes and uterine tissue during gestation [14], [15].

During estrus, the mammalian endometrium undergoes a series of morphologic and functional changes [16]. Furthermore, during the postpartum period, the endometrial tissue undergoes a series of morphologic changes to restore the histologic features that were altered during pregnancy and delivery.

Apoptosis is physiological programmed cell death, which ensures the balance between cellular death and proliferation. Caspases are a group of cysteine proteases that degrade proteins by recognizing aspartic acid residues [17]. These proteases are involved in apoptosis, are synthesized as zymogens, and belong to two classes: the initiators (caspase 2, caspase 8, caspase 9, and caspase 10) and the executioners (caspase 3 [CASP-3], caspase 6, and caspase 7). The initiator caspases exist as monomers and are activated by dimerization. The executioner caspases exist as dimers and are activated by cleavage of their interdomain linkers by initiator caspases [17], [18], [19]. Caspase-3 function is present during the final step of apoptosis and can be activated by intrinsic (mitochondrial) or extrinsic (death receptor) pathways [18]. Also, CASP-3 mRNA expression has been observed in the bovine placenta and uterus during the estrous cycle and during gestation [16], [19].

Cyclooxygenase (COX) is the enzyme required for the biosynthesis of prostaglandins from arachidonic acid [20], and it exists in two forms, namely COX-1 and COX-2. Cyclooxygenase 1 is a constitutive enzyme that is present in most cells. Cyclooxygenase 2 gene expression is induced by stimuli or mediators [6]. Also, COX-2 is important for cell growth and during inflammation [20], and it may produce prostaglandin during inflammation [21]. The COX expression in different tissues differs among species [22], but COX-2 is expressed in bovine uterine tissue during the estrous cycle, gestation, and the postpartum period [22], [23], [24], [25].

This study is based on the hypothesis that cloprostenol sodium promotes modifications in FP, COX-2, and CASP-3 mRNA expression that may favor the process of postpartum uterine involution in multiparous Nelore (Bos taurus indicus) females. Additionally, we aimed to describe the presence and immunolocalization of the FP and the COX-2 protein in endometrial tissue at different postpartum time points in these females. We examined the effect of serial administration of cloprostenol sodium (Days 1 and 4, Day 0 = birth) during the puerperal period of multiparous Nelore cows. We used quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR) to monitor mRNA expression (on Days 1, 7, 14, 28, and 42) and immunohistochemical analysis to monitor tissue-specific protein expression (on Days 1, 14, 28, and 42). The results of this study may provide a better understanding of the molecular mechanisms involved in the postpartum period and may direct future studies toward the more effective use of cloprostenol sodium in hormonal protocols during the postpartum period of Nelore females.

Section snippets

Ethical aspects

The study was performed according to the ethical guidelines recommended by the National Council for Control of Animal Experimentation and the College of Animal Experimentation, and it was approved by the institution's Animal Care and Experimentation Ethics Committee (protocol number 50/2012).

Animals

A total of 24 multiparous Nelore (B. taurus indicus) females with calves were used. The parity of the animals was between two and six (median = 3), their age was between 4 and 13 years (median = 9), and

Results

Differences (P ≤ 0.05) were observed between the patterns of mRNA expression in the PG and TG only for CASP-3 and for the FP. The FP, CASP-3, and COX-2 mRNA expression differed among all time points during the postpartum period in the endometrial samples from Nelore cows. Immunohistochemical analyses revealed cytoplasmic COX-2 and FP localization in the endometrial surface epithelium, stroma, and glandular epithelium.

Discussion

During the puerperium period, the uterus undergoes modifications that restore an environment able to maintain a new pregnancy. In cows, a postpartum period of 65 days is recommended, and new conception within 85 to 90 days is desired, according to a review [1].

Because most studies are performed during gestation or in the first few hours after delivery, it is difficult to compare our study with previous results. To our knowledge, this is the first study of the spatiotemporal expression patterns

Acknowledgments

We are grateful to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) for a scholarship (Proc.2011/15330-0), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (Capes (Proc./096/2010) for financial support, and to Fazenda Marino Avaré.

References (34)

  • M. Takagi et al.

    Messenger RNA expression of angiotensin-converting enzyme, endothelin, cyclooxigenase-2 and prostaglandin synthases in bovine placentomas during gestation and the postpartum period

    Vet J

    (2008)
  • N. Ymer et al.

    Ovarian activity in beef and dairy cows with prolonged postpartum period and heifers that fail to conceive

    Trop Anim Health Prod

    (2010)
  • L.A. Guilbault et al.

    Source of F series prostaglandins during the early postpartum period in cattle

    Biol Reprod

    (1984)
  • C.A.C. Fernandes et al.

    Avanços na utilização de prostaglandinas na reprodução de bovinos

    Rev Bras Reprod Anim

    (2007)
  • B.A. Tenhagen et al.

    Serum progesterone levels in post-partum dairy cows after repeated application of prostaglandin F2α analogue D (+) cloprostenol sodium

    J Vet Med A

    (2000)
  • T. Nakao et al.

    Postpartum plasma PGF metabolite profile in cows with dystocia and/or retained placenta, and effect of fenprostalene on uterine involution and reproductive performance

    J Vet Med Sci

    (1997)
  • C.M. Barros et al.

    Farmacologia veterinária

    (2012)
  • Cited by (6)

    View full text