Local effect of allopregnanolone in rat ovarian steroidogenesis, follicular and corpora lutea development.

Allopregnanolone (ALLO) is a known neurosteroid and a progesterone metabolite synthesized in the ovary, CNS, PNS, adrenals and placenta. Its role in the neuroendocrine control of ovarian physiology has been studied, but its in situ ovarian effects are still largely unknown. The aims of this work were to characterize the effects of intrabursal ALLO administration on different ovarian parameters, and the probable mechanism of action. ALLO administration increased serum progesterone concentration and ovarian 3ß-HSD2 while decreasing 20α-HSD mRNA expression. ALLO increased the number of atretic follicles and the number of positive TUNEL granulosa and theca cells, while decreasing positive PCNA immunostaining. On the other hand, there was an increase in corpora lutea diameter and PCNA immunostaining, whereas the count of TUNEL-positive luteal cells decreased. Ovarian angiogenesis and the immunohistochemical expression of GABAA receptor increased after ALLO treatment. To evaluate if the ovarian GABAA receptor was involved in these effects, we conducted a functional experiment with a specific antagonist, bicuculline. The administration of bicuculline restored the number of atretic follicles and the diameter of corpora lutea to normal values. These results show the actions of ALLO on the ovarian physiology of the female rat during the follicular phase, some of them through the GABAA receptor. Intrabursal ALLO administration alters several processes of the ovarian morpho-physiology of the female rat, related to fertility and oocyte quality.

Effect of peripheral neural stimulation with allopregnanolone on ovarian physiology.

Neuroactive steroids can rapidly regulate multiple physiological functions in the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in low nanomolar and high micromolar concentrations, can: (i) induce changes in the ovarian progesterone (P4) and estradiol (E2) release; (ii) modify the ovarian mRNA expression of Hsd3b1 (3ß-hydroxysteroid dehydrogenase, 3ß-HSD)3ß-, Akr1c3 (20α-hydroxysteroid dehydrogenase, 20α-HSD), and Akr1c14 (3α-hydroxy steroid oxidoreductase, 3α-HSOR)); and (iii) modulate the ovarian expression of progesterone receptors A and B, α and ß estrogenic receptors, luteinizing hormone receptor (LHR) and follicle-stimulating hormone receptor (FSHR). To further characterize ALLO peripheral actions, the effects were evaluated using a superior mesenteric ganglion-ovarian nervous plexus-ovary (SMG-ONP-O) and a denervated ovary (DO) systems. ALLO SMG administration increased P4 concentration in the incubation liquid by decreasing ovarian 20α-HSD mRNA, and it also increased ovarian 3α-HSOR mRNA expression. In addition, ALLO neural peripheral modulation induced an increase in the expression of ovarian LHR, PRA, PRB, and ERα. Direct ALLO administration to the DO decreased E2 and increased P4 concentration in the incubation liquid. The mRNA expression of 3ß-HSD decreased and 20α-HSD increased. Further, ALLO in the OD significantly changed ovarian FSHR and PRA expression. This is the first evidence of ALLO's direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, and these findings might help devise some of the pleiotropic effects of neuroactive steroids on female reproduction. Moreover, ALLO modulation of ovarian physiology might help uncover novel treatment approaches for reproductive diseases.

Superior mesenteric ganglion neural modulation of ovarian angiogenesis, apoptosis and proliferation by the neuroactive steroid allopregnanolone.

Allopregnanolone (ALLO), a potent neuroactive steroid, is synthesized and active in the peripheral nervous system. Previous studies have shown that ALLO participates in the central regulation of reproduction with effects on ovarian physiology, although there is little evidence for its ability to modulate peripheral tissues. The present study aimed to determine whether ALLO, administered to an ex vivo system that comprises the superior mesenteric ganglion (SMG), the ovarian nervous plexus (ONP) and the ovary (O), or to the denervated ovary (DO), was able to modify ovarian apoptosis, proliferation and angiogenesis. For this purpose, the SMG-ONP-O system and DO were incubated during 120 min at 37°C, in the presence of two ALLO doses (0.06 µm and 6 µm). The intrinsic and extrinsic pathways of apoptosis were analyzed. Incubation of the SMG-ONP-O system with ALLO 0.06 µm led to an increase in the BAX/BCL-2 ratio and a reduction of FAS-L mRNA levels. ALLO 6 µm induced a decrease of FAS-L levels. Incubation of DO with ALLO 0.06 µm reduced FAS-L, whereas ALLO 6 µm significantly increased it. Cyclin D1 mRNA was measured to evaluate proliferation. Treatment with ALLO 6 µm increased proliferation in both SMG-ONP-O and DO. ALLO 0.06 µm produced an increase of Cyclin D1 in DO only. Administration of either ALLO dose led to a higher ovarian expression of vascular endothelial growth factor in the SMG-ONP-O system, but a lower one in the DO system. ALLO 6 µm induced ovarian sensitization to GABA by increasing GABAA receptor expression. In conclusion, ALLO participates in the peripheral neural modulation of ovarian physiology. It can also interact directly with the ovarian tissue, modulating key mechanisms involved in normal and pathological processes in a dose-dependent manner.

"Rapid actions of the neurosteroid allopregnanolone on ovarian and hypothalamic steroidogenesis: Central and peripheral modulation".

The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3ß-hydroxysteroid dehydrogenase (3ß-HSD) enzymatic activity and gene expression in in vivo and ex vivo systems in pro-oestrus (PE) and dioestrus I (DI) rats. Animals were injected with vehicle, ALLO, bicuculline or bicuculline plus ALLO and were then killed. In the in vivo experiment, the hypothalamus, ovaries and serum were extracted and analysed. In the ex vivo experiment, the superior mesenteric ganglion - ovarian nerve plexus - ovary system was extracted and incubated during 120 minutes at 37 ºC. The serum and ovarian compartment fluids were used to determine progesterone by radioimmunoanalysis. In the in vivo experiments, ALLO caused a decrease in hypothalamic and ovarian 3ß-HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3ß-HSD activity and gene expression. The ovarian 3ß-HSD activity increased in both stages in the ex vivo system; gene expression increased only during DI. ALLO induced an increase in serum progesterone only in D1 and in the ovarian incubation liquids in both stages. All findings were reversed by an injection of bicuculline before ALLO. Ovarian steroidogenic changes could be attributed to signals coming from ganglion neurones, which are affected by the acute central neurosteroid stimulation. The i.c.v. administration of ALLO via the GABAergic system altered 3ß-HSD activity and gene expression, modulating the neuroendocrine axis. The present study reveals the action that ALLO exerts on the GABAA receptor in both the central and peripheral nervous system and its relationship with hormonal variations. ALLO is involved in the "fine tuning" of neurosecretory functions as a potent modulator of reproductive processes in female rats.

Allopregnanolone alters follicular and luteal dynamics during the estrous cycle.

BACKGROUND: Allopregnanolone is a neurosteroid synthesized in the central nervous system independently of steroidogenic glands; it influences sexual behavior and anxiety. The aim of this work is to evaluate the indirect effect of a single pharmacological dose of allopregnanolone on important processes related to normal ovarian function, such as folliculogenesis, angiogenesis and luteolysis, and to study the corresponding changes in endocrine profile and enzymatic activity over 4 days of the rat estrous cycle. We test the hypothesis that allopregnanolone may trigger hypothalamus - hypophysis - ovarian axis dysregulation and cause ovarian failure which affects the next estrous cycle stages. METHODS: Allopregnanolone was injected during the proestrous morning and then, the animals were sacrificed at each stage of the estrous cycle. Ovarian sections were processed to determine the number and diameter of different ovarian structures. Cleaved caspase 3, proliferating cell nuclear antigen, α-actin and Von Willebrand factor expressions were evaluated by immunohistochemistry. Luteinizing hormone, prolactin, estrogen and progesterone serum levels were measured by radioimmunoassay. The enzymatic activities of 3ß-hydroxysteroid dehydrogenase, 3α-hydroxysteroid oxidoreductase and 20α-hydroxysteroid dehydrogenase were determined by spectrophotometric assays. Two-way ANOVA followed by Bonferroni was performed to determine statistical differences between control and treated groups along the four stages of the cycle. RESULTS: The results indicate that allopregnanolone allopregnanolone decreased the number of developing follicles, while atretic follicles and cysts increased with no effects on normal cyclicity. Some cysts in treated ovaries showed morphological characteristics similar to luteinized unruptured follicles. The apoptosis/proliferation balance increased in follicles from treated rats. The endocrine profile was altered at different stages of the estrous cycle of treated rats. The angiogenic markers expression increased in treated ovaries. As regards corpora lutea, the apoptosis/proliferation balance and 20α-hydroxysteroid dehydrogenase enzymatic activity decreased significantly. Progesterone levels and 3ß-hydroxysteroid dehydrogenase enzymatic activity increased in treated rats. These data suggest that allopregnanolone interferes with steroidogenesis and folliculogenesis at different stages of the cycle. CONCLUSION: Allopregnanolone interferes with corpora lutea regression, which might indicate that this neurosteroid exerts a protective role over the luteal cells and prevents them from luteolysis. Allopregnanolone plays an important role in the ovarian pathophysiology.