Differential expression of CTGF in pre- and post-ovulatory granulosa cells in the hen ovary is regulated by TGFβ1 and gonadotrophins

Abstract

Connective tissue growth factor (CTGF) is a cysteine-rich, matrix-associated heparin-binding protein that is important in many cell types as a regulator of cell proliferation, angiogenesis, cell remodelling and other cellular processes. CTGF is necessary for normal follicle growth and luteinisation in mammals. The avian follicular hierarchy provides an excellent experimental model to study developmental events, particularly the role of cellular remodelling factors in the process of folliculogenesis. In this study, we examined CTGF expression and regulation in the hen ovary. CTGF expression was increased considerably as follicular development proceeds in pre-ovulatory follicles, peaking in expression at the time of ovulation. Immunohistochemistry revealed that CTGF protein was concentrated in the cytoplasm of follicular granulosa cells throughout the ovulation cycle. We isolated granulosa cells from the follicles at two key stages of the ovulation cycle (in terms of cellular alteration): during pre-ovulatory growth and during post-ovulatory regression. Follicle-stimulating hormone (FSH) and luteinising hormone (LH) inhibited CTGF expression in pre-ovulatory granulosa cells but stimulated CTGF expression in post-ovulatory granulosa cells. Moreover, TGFβ1 stimulated CTGF expression in both pre- and post-ovulatory granulosa cells. Nevertheless, TGFβ1 could rescue the inhibition of gonadotrophins on pre-ovulatory granulosa CTGF expression but could not further stimulate CTGF expression in gonadotrophin-treated post-ovulatory granulosa cells. The results of this study indicate that CTGF expression in avian granulosa cells is modulated by a combination of gonadotrophins and TGFβ1 according to the different stages of follicle maturation and degradation. The results also suggest that the gonadotrophic action on post-ovulatory follicles in the avian ovary differs from the gonadotrophin-induced luteinisation in mammals.

Introduction

Connective tissue growth factor (CTGF) is a cysteine-rich heparin-binding growth factor that is a member of the CTGF/cysteine-rich 61/nephroblastoma (CCN) overexpressed family of genes [19]. CTGF is expressed in various tissues and is particularly abundant in the ovary of rat tissues [11]. CTGF is thought to take part in diverse cellular events, such as stimulation of proliferation, differentiation, attachment, migration, and, most importantly, extracellular matrix (ECM) remodelling [26]. Ovarian follicle development requires continuous formation and degradation of the ECM to provide the intercellular microenvironment necessary for normal oocyte growth and release. In the mammalian ovary, CTGF mRNA is expressed predominantly in the granulosa layer, which undergoes continuous dramatic alteration in both proliferation and differentiation. In pigs [31] and rats [9], CTGF expression levels increase in preantral and early antral follicles, reach peak expression in mid-antral follicles and decrease in pre-ovulatory follicles. After ovulation, there is a marked increase of CTGF mRNA in granulosa-lutein cells of the corpus luteum in pigs [32], but little or no expression is observed in rats [11], humans [5] and mice [10]. CTGF conditional knockout mice exhibit disrupted follicle development, decreased ovulation and enhanced corpus luteum formation [21].

Therefore, subsequent studies have focused on how peripheral endocrine signals, such as gonadotrophins, regulate local CTGF production and other related cellular signals in granulosa cells to control normal folliculogenesis and luteogenesis. Follicle-stimulating hormone (FSH) strongly inhibits the CTGF expression in undifferentiated granulosa cells isolated from preantral and early antral follicles of immature rat [9], [10], [27]. This FSH-induced downregulation is consistent with evidence that CTGF is overexpressed in the granulosa cells of FSHß knockout mice [10]. CTGF is also downregulated by FSH and luteinizing hormone (LH) in isolated human luteinised granulosa cells [17], [23] and by human chorionic gonadotrophin (hCG) during luteal rescue in in vivo models of early pregnancy [5]. Therefore, it is possible to infer that granulosa CTGF is tightly controlled by gonadotrophins throughout the ovulation cycle in mammals.

The domestic fowl provides a unique model for studying cellular mechanisms during follicular development and differentiation. Unlike mammalian counterparts, the single left ovary of the hen contains follicles of various sizes and developmental stages. The resting primordial follicles, pre-hierarchical growing follicles, pre-ovulatory follicles and the post-ovulation follicles are represented simultaneously in one reproductively active ovary. After the daily selection of a single follicle into the pre-ovulation hierarchy, the follicle size increased from 6–8 mm to 40 mm in 5–9 days until the final ovulation, therefore the growth rate likely approached the record for an animal cell [35]. Unlike corpus luteum formation in mammals, the post-ovulation follicles disappear within days, as it is difficult to find the 5th or 6th post-ovulation follicles in the experience of our group and others [12]. With this record rate of cell growth and degradation, there are extensive cellular alterations in ovarian follicles, including widespread cell and tissue remodelling, throughout the ovulation cycle in hens. The whole process also requires more finely controlled endocrine, paracrine and autocrine regulations in the streamlined hierarchical follicles of all different stages within the same ovary.

Given the paucity of studies of ECM characteristics in the follicles of the avian ovary, we studied the gonadotrophic regulation of CTGF during the ovulatory cycle as an indicator for its potential role in ECM restructuring during follicle development and regression in avian species. We first characterised the spatio-temporal expression of CTGF mRNA and protein in the hen ovary. Given the evidence that TGFβ interact with CTGF in a variety of cells [1], [19], we also studied the involvement of TGFβ in gonadotrophin-regulated CTGF expression in chicken granulosa cells. Our results revealed a stage-specific regulation that is distinct from mammalian regulation.