Effect of allopregnanolone over the female rat reproductive physiology

ANTONELLA ROSARIO RAMONA CÁCERES GIMENEZ; LACONI, MYRIAM RAQUEL

Conferencia; Virtual International Meeting Steroids and Nervous System - Torino 2021.; 2021

Allopregnanolone (ALLO, 3α-hydroxi-5α-pregnan-20-one) is an active metabolite derived from progesterone (P4), synthesized in the central nervous system, ovary, placenta, and adrenal glands. Its levels vary throughout the estral and menstrual cycle in a similar profile as P4. ALLO, is a neurosteroid mostly known for its central inhibitory effects at the level of RGABAA and has recently been approved by the FDA (brexanolone), as a first in-class medication for postpartum depression. Previous studies in our laboratory have provided evidence that ALLO produces significant changes in the hypothalamic [1] and striatal [2] dopaminergic systems. An intracerebroventricular administration of ALLO suppresses the hypothalamic GnRH release, sexual receptivity, and LH preovulatory surge via the modification of GABA and dopamine activity [3,4,5]. Other related behavioural changes are anxiolysis and memory potentiation [6]. As we found previously, ALLO affected several important parameters that might alter reproduction; therefore, we aim to study the effects of ALLO on the reproductive physiology of the female rat. We proposed four experimental approaches at relevant points to the regulation of reproductive function. In this way, we were able to differentiate the central, peripheral, local, and cellular effects. The main focus was to analyse the possible alterations in ovarian morphometry, apoptosis, proliferation and steroidogenesis. For the experiments, we used adult virgin female rats of the Sprague Dawley and Holtzman strains, which were cycled daily. Only those animals exhibiting two or more consecutive 4 or 5-day cycle were used. All protocols were previously approved by the Institutional Committee for Care and Use of Experimental Animals (CICUAL N° 141021) and conducted according to the National Institutes of Health Guide for the Care and Use of Laboratory Animals of the National Research Council (National Academies, U.S.A., 8th Edition, 2011). To elucidate a central effect, ALLO was administered using an intracerebroventricular (i.c.v) cannula stereotaxically inserted into the right lateral ventricle. After the surgery, the animals were housed singly in cages and maintained for a week undisturbed to recover the oestrous cycle. On the proestrus morning, the experimental groups received a single i.c.v. injection of ALLO at increasing concentrations: 0.06, 6, 60 and 600 μM. Control animals were injected with KREBS solution. The animals were euthanized the following oestrous morning (24h later). The ovaries, HMB and truncal serum were stored at -80°C until analysis. To study the peripheral effect, we performed the extraction and incubation of an ex vivo superior mesenteric ganglion - ovarian nervous plexus - ovary (SMG-ONP-Ovary) system and the contralateral ovary as a denervated control. The systems were incubated in KREBS solution, and maintained in a metabolic bath for up to 120 minutes. ALLO at increasing concentrations: 0.06, 6 μM, (a physiological dose and the dose most effective at central level) was administered in the SMG compartment and to the denervated ovaries. At the end of the incubation time, the ovaries and incubation liquid were stored at -80°C until analysis. To assess the local effect, we designed a paired experiment. On the proestrus morning, ALLO at increasing concentrations: 0.06, 0.6, and 6 μM was administered intrabursa to the left ovary and saline solution was injected to the contralateral ovary. After 24h, the animals were euthanized, and the ovaries and truncal serum were stored at -80°C until analysis. Finally, to study in vitro the cellular effect, we obtained a primary culture of ovarian granulosa cells from pre-pubertal rats stimulated with dietilstilestrol. The granulosa cells were cultured in DMEM:F12 medium in presence or absence of ALLO at increasing concentrations: 0.06, 0.6 and 6 μM. After 24h, the cells were stored at -80°C until analysis. ALLO treatment induced an imbalance in the processes of ovarian apoptosis/proliferation and steroidogenesis. At a central level, ALLO altered the secretion of pituitary gonadotropins, in a RGABAA-dependent manner, it was also capable of modifying the reproductive ovarian functionality. ALLO 6µM on one hand, augmented the number of atretic follicles and cystic structures, and on the other hand, promoted corpora lutea angiogenesis, increased the number of old corpora lutea, and its diameter. ALLO also increased ovarian angiogenesis and altered some critical steps in ovarian and hypothalamic steroidogenesis. Ovarian P4 receptors were not modified significantly. At a peripheral level, the treatment modified the secretion of noradrenaline and P4. ALLO also induced an antiapoptotic ovarian effect. At local level, ALLO induced alterations of morphophysiological parameters, in a similar way to those that occurred at the central level, evidencing a paracrine ovarian modulation. The in vitro experiments revealed the direct action of ALLO on the ovarian granulosa cells, in a dose-dependent manner increasing apoptosis and decreasing proliferation markers. Based on these findings, ALLO could be postulated as a molecule with high potential in clinical pharmacology, intimately involved in regulation of the female reproductive physiology, modulating important processes such as ovarian apoptosis and steroidogenesis. ALLO is a molecule with a high therapeutic potential with great versatility, for the diagnosis or treatment of reproductive pathologies, including ovarian cancer. Recently, we published the first findings about the ALLO effects on ovarian epithelial cancer cell lines, where ALLO treatment at high doses increase migration and proliferation in IGROV-1 and SKOV-3 cells [7]. More studies are needed to clarify the relevance of ALLO actions on ovarian pathologies.