The authors regret an error in Figure (n = 6; *P < 0.05, **P < 0.01 and ***P < 0.001).
Effect of progesterone and first evidence about allopregnanolone action on the progression of epithelial human ovarian cancer cell lines.
The present study aimed to determine whether an i.c.v. administration of allopregnanolone (ALLO) rapidly modifies the hypothalamic and ovarian 3β‐hydroxysteroid dehydrogenase (3β‐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 3β‐HSD enzymatic activity during PE. During DI, ALLO increased hypothalamic and ovarian 3β‐HSD activity and gene expression. The ovarian 3β‐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 3β‐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.
BackgroundAllopregnanolone 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.MethodsAllopregnanolone 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 3β-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.ResultsThe 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 3β-hydroxysteroid dehydrogenase enzymatic activity increased in treated rats. These data suggest that allopregnanolone interferes with steroidogenesis and folliculogenesis at different stages of the cycle.ConclusionAllopregnanolone 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.
Allopregnanolone, a progesterone metabolite, is one of the best characterized neurosteroids. In a dose that mimics serum levels during stress, allopregnanolone inhibits sexual receptivity and ovulation and induces a decrease in luteinizing hormone levels. The aim of this work was to examine the effect of an intracerebroventricular administration of allopregnanolone on ovarian morphophysiology, serum and tissue levels of progesterone and estrogen, and enzymatic activity of 3β-hydroxysteroid dehydrogenase, 20α-hydroxysteroid dehydrogenase and 3α-hydroxysteroid oxido-reductase in the ovary and in the medial basal hypothalamus on the morning of estrus. Ovarian morphology was analyzed under light microscopy. The hormone assays were performed by radioimmunoassay. The enzymatic activities were measured by spectrophotometric analysis. The morphometric analysis revealed that, in allopregnanolone-treated animals, the number of secondary and Graafian follicles was decreased while that of atretic follicles and cysts was significantly increased. Some cysts showed luteinized unruptured follicles. There were no differences in the number of tertiary follicles or corpora lutea in comparison with the corresponding control groups. In allopregnanolone-treated animals, progesterone serum levels were increased, while ovarian progesterone levels were decreased. Moreover, 3β-HSD and 3α-HSOR enzymatic activities were increased in the medial basal hypothalamus while ovarian levels were decreased. The enzyme 20α-hydroxysteroid dehydrogenase showed the opposite profile. The results of this study showed that allopregnanolone interferes on ovarian steroidogenesis and ovarian morphophysiology in rats, providing a clear evidence for the role of this neurosteroid in the control of reproductive function under stress situations.
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.
Neuroactive steroids can rapidly regulate multiple physiological functions on the central and peripheral nervous systems. The aims of the present study were to determine whether allopregnanolone (ALLO), administered in a low nanomolar and a high micromolar concentrations, can: a) induce changes in the ovarian progesterone (P4) and estradiol (E2) release, b) modify the ovarian mRNA expression of 3 β-HSD, 20 α-HSD and 3 α-HSD, c) modulate the ovarian expression of progesterone receptors A and B, α and β estrogenic receptors, LH receptor (LHR) and FSH 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, 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 3β-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 direct effect on ovarian steroidogenesis. Our results provide important insights about how this neuroactive steroid interacts both with the PNS and the ovary, 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.
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