Date Published: January 20, 2017
Publisher: Public Library of Science
Author(s): Shenqiang Hu, Raj Duggavathi, David Zadworny, Bin He.
Prolactin (PRL) has both pro- and anti-gonadal roles in the regulation of avian ovarian functions through its interaction with the receptor (PRLR). However, neither the pattern of expression of PRLR nor its regulatory mechanisms during follicle development have been clearly defined. The objective of the present study was to investigate mechanisms of PRLR expression in chicken granulosa cells. Levels of PRLR transcript were highest in the stroma and walls of follicles < 2 mm in diameter and progressively declined with the maturation of follicles. In preovulatory follicles, PRLR was expressed at higher levels in granulosa than theca layers. FSH exerted the greatest stimulatory effect on PRLR and StAR expression in cultured granulosa cells of the 6–8 mm follicles but this effect declined as follicles matured to F1. In contrast, LH did not alter the expression of PRLR in granulosa cells of all follicular classes but increased levels of StAR in F2 and F1 granulosa cells. Both non-glycosylated- (NG-) and glycosylated- (G-) PRL upregulated basal PRLR expression in granulosa cells of the 6–8 mm, F3 or F1 follicles but had little effect in F2 follicles. Furthermore, FSH-stimulated PRLR expression was reduced by the addition of either isoform of PRL especially in F2 granulosa cells. These results indicate that PRLR is differentially distributed and regulated by FSH or PRL variants independently or in combination in the follicular hierarchy. By using activators and inhibitors, we further demonstrated that multiple signaling pathways, including PKA, PKC, PI3K, mTOR and AMPK, are not only directly involved in, but they can also converge to modulate ERK2 activity to regulate FSH-mediated PRLR and StAR expression in undifferentiated granulosa cells. These data provide new insights into the regulatory mechanisms controlling the expression of PRLR in granulosa cells.
In chickens, ovarian follicles go through initial (activation of cortical follicles) and cyclic (follicle selection) recruitment before ovulation. These events are tightly coupled with the morphological and functional changes in granulosa cells . In follicles prior to selection, granulosa cells are undifferentiated and steroidogenically inactive  due to low levels of expression of the two key genes required for steroidogenesis, steroidogenic acute regulatory protein (StAR)  and cytochrome P450 side chain cleavage (P450scc) enzyme . Subsequent to selection, granulosa cells are differentiated and become steroidogenically active . The process of follicle selection is mainly under the control of follicle stimulating hormone (FSH) [5, 6]. Within the cohort of prehierarchical 6–8 mm follicles, a single follicle showing the highest expression of FSH receptor (FSHR) in the granulosa layer is likely to be next in line to enter the preovulatory hierarchy . FSH signaling leads to the differentiation of granulosa cells by controlling the expression of several steroidogenic genes such as StAR, P450scc and luteinizing hormone receptor (LHR), which is achieved via modulation of multiple intracellular signaling cascades, including protein kinase A (PKA), protein kinase C (PKC), phosphatidylinositol 3-kinase (PI3K) and extracellular signal-regulated kinases (ERKs) and AMP-activated protein kinase (AMPK) [1, 5, 8, 9]. In differentiated granulosa cells, LHR substitutes for the dominant role of FSHR in further promoting LH-induced steroidogenesis which is largely mediated by the PKA pathway .
In chickens, PRL has been shown to have direct effects on the ovary to modulate steroidogenesis yet the distribution of its receptor PRLR within the follicular hierarchy has not been quantified. In the current study we show that maximal expression of PRLR transcript was observed in the stroma and walls of follicles < 2 mm in diameter before a progressive decline with follicle maturation (Fig 1A). Since the smaller (< 2 mm) follicles represent the largest follicular class in the ovary and are thought to be the major source of estrogen  and exogenous PRL could suppress both basal and gonadotropin-stimulated estrogen production by cultured hen these small follicles [20, 41], it is likely that PRL may have a dominantly negative influence on steroidogenesis in the smaller follicles. However, such inhibitory effects by PRL may be attenuated or even become stimulatory dependent on its concentration and the stage of follicle development. Divergent effects of PRL upon steroidogenesis in porcine granulosa cells was demonstrated to be associated with the degree of cell differentiation . Furthermore, in chickens, a stimulatory role for PRL in recruiting large white follicles into small yellow ones was suggested through immunizations against PRL or PRLR . Dependent on the dose of PRL, the stage of follicle development and the stage of the ovulatory cycle, PRL could be either stimulatory or inhibitory on estradiol secretion by the theca layers or progesterone production by the granulosa layers of F3-F1 follicles . Notably, in the preovulatory hierarchy, the PRLR transcript was more abundant in granulosa than theca layers (Fig 1B), implying a relatively more important role of PRL signaling in regulating progesterone production and progesterone-induced ovulation. Indeed, activity of 3β-hydroxysteroid dehydrogenase (3βHSD), a key enzyme in the progesterone biosynthetic pathway, was shown to be regulated by gonadotropins and PRL in granulosa cells of chicken F3-F1 follicles , and PRL tended to suppress LH-induced premature ovulation in chickens . Accordingly, we speculated that varying levels of PRLR transcript during follicle development are related to changes in the process of follicular cell steroidogenesis as a result of their different responsiveness to gonadotropins and PRL. Source: http://doi.org/10.1371/journal.pone.0170409