Allopregnanolone alters follicular and luteal dynamics during the estrous cycle

Abstract

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 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.

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 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.

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.

Keywords: Allopregnanolone; Angiogenesis; Apoptosis; Corpora lutea; Folliculogenesis; Luteolysis; Steroidogenesis.

Fig. 1

Experimental design of the study. A: ALLO treated group, C: control, E: estrous, ICV: intracerebroventricular, D1: diestrous 1, D2: diestrous 2, MBH: medial basal hypothalamus, PE: proestrous, RIA: radioimmunoassay

Fig. 2

Effect of ALLO on the apoptotic index (cleaved CASP3 positive immunostained cells over the total number of cells) in follicles (a) and corpora lutea (b). Control groups follicles (1A upper panel), ALLO treated follicles (1A lower panel), control groups CL (1B upper panel), ALLO treated groups CL (1B lower panel); E, D1, D2, PE from left to right. Representative photomicrographs of follicles and corpora lutea immunostained for cleaved CASP3 at each stage of the estrous cycle. Values are expressed as mean ± S.E.M. Two-way ANOVA followed by Bonferroni’s post hoc, (n=6), *p<0.05, ***p<0.001. Magnification: follicles 200x, CL 400x

Fig. 3

Effect of ALLO on the proliferation index (PCNA positive immunostained cells over the total number of cells) in follicles (a) and corpora lutea (b). Control groups follicles (a upper panel), ALLO treated follicles (a lower panel), control groups CL (b upper panel), ALLO treated groups CL (b lower panel); E, D1, D2, PE from left to right. Representative photomicrographs of follicles and corpora lutea immunostained for PCNA at each stage of the estrous cycle. Black arrow indicate proliferating endothelial cells, red arrow indicate proliferating small luteal cells. Values are expressed as mean ± S.E.M. Two-way ANOVA followed by Bonferroni’s post hoc, (n=6), *p<0.05, ***p<0.001. Magnification: follicles 200x, CL 400x

Fig. 4

Effect of ALLO on ovarian relative vascular area. Representative photomicrographs of ovaries immunostained for α-actin (a) and von Willebrand factor (b) at each stage of the estrous cycle. α-actin control groups (a upper panel), α-actin ALLO treated groups (a lower panel); von Willebrand factor control groups (b upper panel), von Willebrand factor ALLO treated groups (b lower panel). Values are expressed as mean ± S.E.M of the relative vascular area. Two-way ANOVA followed by Bonferroni’s post hoc (n=6), *p<0.05, ***p<0.001. Bar = 50 μm. (c) Representative photomicrographs of von Willebrand factor immunostaining in corpora lutea of control (c, upper panel) and ALLO treated animals (c, lower panel). Inset shows higher magnification images (40x). Arrows indicate von Willebrand factor staining of endothelial cells. Bar = 50 μm

Fig. 5

LH (a), PRL (b), E2 (c) and PG (d) serum levels from control (white bars) and ALLO treated (black bars) groups along the estrous cycle. E: estrous, D1: diestrous 1, D2: diestrous 2, PE: proestrous. Values correspond to mean ± S.E.M. of hormones serum levels per experimental group. Two-way ANOVA followed by Bonferroni’s post hoc (n=6), *p<0.05, **p<0.01 and ***p<0.001

Fig. 6

Enzymatic activities of 3β-HSD (a–d), 3α-HSOR (b–e) and 20α-HSD (c–f) in the medial basal hypothalamus (MBH, left panel) and in the ovaries (right panel) at each stage of the estrous cycle. e: estrous, D1: diestrous 1, D2: diestrous 2, PE: proestrous. White bars correspond to control groups and black bars to ALLO treated groups. Values correspond to mean ± S.E.M. Two-way ANOVA followed by Bonferroni’s post hoc (n=6), ***p<0.001