Elsevier

Theriogenology

Volume 88, 15 January 2017, Pages 50-60
Theriogenology

Research article
Expression and localization of matrix metalloproteinases (MMP-2, -7, -9) and their tissue inhibitors (TIMP-2, -3) in the chicken oviduct during pause in laying induced by tamoxifen

https://doi.org/10.1016/j.theriogenology.2016.09.051Get rights and content

Abstract

Induced pause in egg laying simulates natural molting events in which the hen's reproductive organs regress and rejuvenate. Such processes require extracellular matrix remodeling that is maintained, at least in part, by the action of proteolytic enzymes known as matrix metalloproteinases (MMPs). Nevertheless, information concerning the expression and hormonal regulation of MMP system members in chickens is scarce. Therefore, MMP-2, -7, and -9 and their tissue inhibitors (TIMP-2, -3) expression and localization were investigated in all segments of the domestic hen oviduct (infundibulum, magnum, isthmus, shell gland, vagina) during a pause in egg laying induced by tamoxifen (TMX)—an estrogen receptor modulator. Hy-Line Brown hens were treated daily with TMX (n = 6) at a dose of 6 mg/kg of body weight or a vehicle (n = 6) until complete cessation of egg laying (for 7 days). Chickens were decapitated on Day 7 of the experiment. Real-time polymerase chain reaction and Western blotting revealed section-dependent expression of MMP-2, -7, -9 and TIMP-2 and -3. Immunohistochemistry found tissue and cell-dependent localization of examined proteins in the wall of the oviduct. The MMP-2, TIMP-2, and TIMP-3 were localized mainly in the luminal epithelium, MMP-7 in the luminal and glandular epithelium, whereas MMP-9 was detected only in the connective tissue. Treatment of chickens with TMX markedly elevated the relative expression of MMP-7 and MMP-9 mRNA in the oviduct, but did not affect MMP-2, TIMP-2, and TIMP-3 mRNA levels. However, TMX increased the MMP-2 protein level in the infundibulum, shell gland, and vagina as well as activity of MMP-2 evaluated by gelatin zymography. The results obtained indicate that MMP-2, MMP-7, and MMP-9 are involved in chicken oviduct regression. Moreover, changes in the expression and activity of chosen MMPs after TMX treatment may indicate a contribution of estrogen in the regulation of transcription, translation, and/or the activity of selected elements of the MMP system.

Introduction

The avian oviduct has been recognized as an outstanding model for investigating the biological actions of estrogen [1]. In the chicken oviduct, estrogen induces cell proliferation and differentiation and exhibits antiapoptotic effects [2], [3]. Besides stimulation of the oviduct growth, estrogen induces synthesis of several egg proteins in the oviduct and liver due to both transcriptional increase and mRNA stabilization [1], [4].

The physiological actions of estrogen are mediated by its nuclear receptors, estrogen receptor alfa, and estrogen receptor beta, although evidence of rapid cytoplasmic responses to estrogen is also increasing. These receptors function primarily as ligand-activated transcription factors and bind directly with target genes at estrogen response elements or indirectly via tethering to the AP-1 or Sp1 transcription factors. Cytoplasmic signaling pathways can also activate estrogen receptor (ER) in a ligand-dependent or ligand-independent manner by phosphorylation of ER directly or by phosphorylation of its coactivators. The changes in conformation of ER caused by ligands likely determine its interaction with specific coactivators or corepressors resulting in the stimulation or inhibition of transcription at a given promoter site of an estrogen-responsive gene [1].

The triphenylethylene antiestrogens, including tamoxifen (TMX), are very potent antagonists of estrogen action in the chicken and manifest little agonist activity compared to their action in other species. TMX is thought to bind to ER receptors and work against the effect of estrogen in the target tissue [5], [6]. Therefore, it has been frequently used to investigate estrogen action in birds. This study was designed to evaluate the influence of TMX treatment on the expression, localization and activity of selected matrix metalloproteinase (MMP) system members in the chicken oviduct.

The MMP comprise a family of proteolytic enzymes that include four broad classes: the collagenases, gelatinases, stromelysins, and membrane-type enzymes. The gelatinases (MMP-2 and MMP-9) contain a fibronectin-like sequence within their catalytic domain, which results in a potent ability for these MMPs to cleave gelatin. The stromelysin enzymes (e.g., MMP-7) act on a broad and diverse array of extracellular matrix (ECM) substrates. Both the gelatinases and stromelysins are capable of degrading major constituents of basement membranes, including type IV collagen, laminin, and fibronectin. In addition to degrading the ECM, the MMPs and especially the stromelysins exhibit activity toward other MMPs, growth factors, cytokines, chemokines, and other growth regulators that are matrix bound or present on the cell surface [7], [8].

Expression of MMPs is controlled at the level of transcription in response to proto-oncogenes and exogenous signals, including hormones. At the posttranslational level, MMPs are regulated by both serum-borne and tissue-derived endogenous inhibitors termed tissue inhibitors of metalloproteinases (TIMPs). TIMPs are widely expressed in all tissues, and they are regulated jointly with the MMPs in most cases. In addition to inhibition of MMPs, the TIMPs participate in several essential biological functions. For example, TIMP-2 stimulates fibroblast growth [9], and TIMP-3 induces apoptosis and regulates steroidogenesis and growth of ovarian follicles [10]. MMP-2 is primarily regulated by TIMP-2, whereas MMP-9 is regulated by TIMP-3 [11]. Furthermore, most MMPs are secreted as zymogens, requiring the cleavage of a specific propeptide domain for activation. However, the mechanisms orchestrating pro-MMP activation have not been fully elucidated.

The activity of MMPs in the reproductive system of mammals is regulated by the sex steroids, growth factors, and cytokines that control reproductive function [12], [13], [14]. However, despite the fact that the MMP system has been described in the mammalian oviduct and uterus [14], [15], [16], [17], its role and regulation in the avian oviduct is poorly understood. First, the mRNAs of MMP-2, TIMP-2, and TIMP-3 were found in the chicken oviduct by Réhault-Godbert et al. [18], and our previous studies revealed changes in the expression and activity of some MMP system members in the chicken oviduct during sexual maturation [19] as well as during pause in egg laying induced by food deprivation [20]. In the current study, our aim was to investigate the expression and localization of selected MMPs (MMP-2, MMP-7, MMP-9) and TIMPs (TIMP-2, TIMP-3) as well as MMPs activity in the particular segments of the chicken oviduct during pause in laying induced by TMX (ER modulator) treatment. Additionally, concentration of ovarian steroids was measured in blood plasma.

Section snippets

Birds and experimental design

The animal experiment was conducted according to research protocol approved by the Local Animal Ethics Committee in Kraków, Poland. Laying Hy-Line Brown hens were purchased from a commercial farm, H&P2 s.c. (Czarków, Poland). Chickens were caged individually under a photoperiod of 14L:10D with commercial food and water ad libitum.

The 12 laying hens at 25 weeks of age (average body weight 1.86 ± 0.036) were divided into two groups. The experimental chickens (n = 6) were treated daily (for

Changes in the oviduct weight after TMX treatment

On day six of the experiment the hens treated with TMX entirely stopped egg laying.

The weight of the oviduct in the TMX-treated chickens was 17% lower than in the control hens (P < 0.001).

Sex hormones concentrations after TMX treatment

Concentrations of steroids in the blood plasma of control and experimental chickens are shown in Figure 1A–C. Treatment with TMX significantly decreased the progesterone level (by 66% on Day 7 of experiment in comparison with control), while it did not influence testosterone and estradiol concentrations.

Messenger RNA expression of MMPs and TIMPs in the chicken oviduct after TMX treatment

Discussion

Alterations in the expression of MMPs and TIMPs and activity of MMPs in the chicken oviduct during sexual maturation and pause in egg laying observed in previous studies [19], [20] are accompanied by changes in the concentrations of steroid hormones in the blood plasma of birds [22], [23], [24], [25], which may indicate the involvement of steroids in the regulation of MMP system components. The main hormones that control the development and functioning of the oviduct are estrogens, which

Acknowledgments

This work was financially supported by the National Science Center, Poland (grant PRELUDIUM No. UMO-2012/07/N/NZ4/00165 dedicated to A.L.-W). Results presented in part at the 18th Annual Conference of the European Society for Domestic Animal Reproduction (ESDAR), September 11–13 Helsinki, Finland.

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