The key action of estradiol and progesterone enables GnRH delivery during gestation in the South American plains vizcacha, Lagostomus maximus

Highlights

• Pharmacological doses of progesterone (P4) and estradiol (E2) inhibited hypothalamic GnRH expression.

• Physiological doses of P4 and E2 differentially altered GnRH pulsatile release frequency or genomic expression.

• The modulation of GnRH synthesis and release is subjected to different levels of action of steroid hormones.

• E2 displayed a rapid effect on GnRH release frequency and a long-term effect on its mRNA expression.

• The fine action of E2 and P4 enables the hypothalamic activity during the pregnancy of this mammal.

Abstract

The South American plains vizcacha, Lagostomus maximus, is the only mammal described so far that shows expression of estrogen receptors (ERs) and progesterone receptors (PRs) in gonadotropin-releasing hormone (GnRH) neurons. This animal therefore constitutes an exceptional model for the study of the effect of steroid hormones on the modulation of the hypothalamic-pituitary-ovarian (HPO) axis. By using both in vivo and ex vivo approaches, we have found that pharmacological doses of progesterone (P4) and estradiol (E2) produced an inhibition in the expression of hypothalamic GnRH, while physiological doses produced a differential effect on the pulsatile release frequency or genomic expression of GnRH. Our ex vivo experiment indicates that a short-term effect of E2 modulates the frequency of GnRH release pattern that would be associated with membrane ERs. On the other hand, our in vivo approach suggests that a long-term effect of E2, acting through the classical nuclear ERs-PRs pathway, would produce the modification of GnRH mRNA expression during the GnRH pre-ovulatory surge. Particularly, P4 induced a rise in GnRH mRNA expression and protein release with a decrease in its release frequency. These results suggest different levels of action of steroid hormones on GnRH modulation. We conclude that the fine action of E2 and P4 constitute the key factor to enable the hypothalamic activity during the pregnancy of this mammal.

Introduction

Pubertal development and adult reproductive function depend on the activation of the hypothalamic-pituitary-ovarian (HPO) axis. In most species, gonadotropin-releasing hormone (GnRH), a decapeptide involved in the modulation of the HPO axis, is synthesized in the hypothalamus by a discrete specialized group of neurons scattered throughout the preoptic area (POA), the ventromedial nucleus (VMN) and the arcuate nucleus (ARC) [[1], [2], [3]]. The majority of GnRH neurons project their processes towards the median eminence (ME), releasing GnRH into the hypothalamic-pituitary portal circulation, that transports the hormone to the anterior pituitary gland where it binds to its specific receptor and modulates gonadotropin synthesis and delivery [[3], [4], [5], [6], [7], [8]]. As the central regulator of fertility in mammals, GnRH is released in discrete pulses separated by periods of little to no secretion, from puberty up to menopause, except during pregnancy [9]. This mode of secretion sensitizes the pituitary gonadotrophs to GnRH stimulation and regulates gonadotropin gene expression [10].Variations in the pulsatile pattern of GnRH release differentially modulates the synthesis and secretion of the two pituitary gonadotropins, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), that influence gonadal gametogenesis, folliculogenesis and steroidogenesis [11,12]. Low GnRH pulse frequency favors FSH release whereas high pulse frequency stimulates the release of LH [11,[13], [14], [15]]. Although the pulsatile secretion of GnRH is an intrinsic property of hypothalamic GnRH neurons, attributed to specific mechanisms of spontaneous electrical activity, its pulsatile delivery frequency and amplitude is under modulation of a complex network of molecules [16]. One of the classical pathways of GnRH modulation includes the feedback produced by the gonadal steroid hormones progesterone (P4) and estradiol (E2) [7,8,[17], [18], [19], [20], [21]].

Although most mammals show inhibition of the HPO axis during gestation, we have recently described that the South American plains vizcacha (Lagostomus maximus), a hystricognathe caviomorph rodent inhabiting the southern area of the Neotropical region, especially the Pampean region of Argentina [22], displays reactivation of the reproductive axis at mid-gestation [[23], [24], [25]] among other exceptional reproductive traits such as the highest ovulation rate, so far recorded for a mammal, up to 800 oocytes per estrous cycle [26,27], natural selective and sequential resorption of the anteriorly implanted fetuses [26], and suppression of apoptosis-dependent follicular atresia driven through an over-expression of the anti-apoptotic BCL2 gene and a basal or absent expression of pro-apoptotic BAX gene, both in the developing and adult ovary [[28], [29], [30]]. We hypothesized that the reactivation of the HPO axis during gestation is enabled by a fine equilibrium in the neuroendocrine environment of the pregnant vizcacha that makes possible follicular maturation and development of a new set of secondary corpora lutea that provides the hormonal boost necessary to get pregnancy to term. This event correlates with an increased expression of hypothalamic GnRH, estrogen receptor alpha (ERα) and progesterone receptors (PRs), despite increased and sustained levels of serum P4, E2 and LH [[23], [24], [25],31]. Finally, we have also shown that GnRH neurons of POA and supraoptic nucleus (SON) express ERα and PRs, suggesting a direct action of E2 and P4 to assure GnRH synthesis and delivery during pregnancy [23,25].

The aim of this study was to evaluate the involvement of E2 and P4 in the modulation of hypothalamic GnRH synthesis and release in this species with this particular reproductive strategy. In order to elucidate this matter, we employed both in vivo and ex vivo approaches, exposing the hypothalamus to physiological and pharmacological doses of E2 and P4, and agonists and antagonists of their specific receptors.