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. 2024 Mar 16;14(1):6402.
doi: 10.1038/s41598-024-57102-1.

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

Antonella Rosario Ramona Cáceres  1   2 , Daniela Alejandra Cardone  1 , María de Los Ángeles Sanhueza  1 , Ignacio Manuel Bosch  3 , Fernando Darío Cuello-Carrión  4 , Graciela Beatriz Rodriguez  5 , Leopoldina Scotti  6 , Fernanda Parborell  6 , Julia Halperin  7 , Myriam Raquel Laconi  8   9
Affiliations

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

Antonella Rosario Ramona Cáceres et al. Sci Rep. .

Abstract

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 3β-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.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Experimental design. On the day of proestrus the rats were subjected to an intrabursal administration surgery. On the day of estrous, 24 h later, they were euthanized by decapitation. Blue lines show the anatomical references used for the surgery: the greater trochanter of the femur, the vertebral column and the last rib. The red line shows the area of incision.
Figure 2
Figure 2
Gene expression of ovarian 3β-HSD2, 20α-HSD and 3α-HSOR after ALLO 6 µM intrabursal treatment. Results are expressed as mean ± SEM. Paired Student’s t test (*p < 0.05; n = 4 animals/experimental group).
Figure 3
Figure 3
Effect of intrabursal administration of ALLO 6 µM on serum progesterone (A), estrogen (B) and FSH concentration. Results are expressed as mean ± SEM. (A) and (C) Unpaired Student’s t test, (B) Mann–Whitney test (**p < 0.01, ***p < 0.001; n = 5 animals/experimental group).
Figure 4
Figure 4
Ovarian weight in grams (A) and number of ovulated oocytes (B) in the ampullae after ALLO 6 µM intrabursal treatment. Results are expressed as mean ± SEM. Paired Student’s t test (ns > 0.05; n = 5 animals/experimental group).
Figure 5
Figure 5
Effect of intrabursal administration of ALLO 6 µM on the number of structures per ovary. Primary follicles (P.F), secondary follicles (S.F), tertiary follicles (T.F), de Graaf follicles (G.F), atretic follicles (AT.F.), cysts, total number of corpora lutea (T.CL.), new corpora lutea (N.CL.) and old corpora lutea (O.CL.). Results are expressed as mean ± SEM. Two-way ANOVA with Bonferroni post hoc (n = 4 animals/experimental group, **p < 0.01).
Figure 6
Figure 6
Effect of intrabursal administration of ALLO 6 µM on tertiary follicle (T.F.) and corpora lutea (CL) diameter. Results are expressed as mean ± SEM. Paired Student’s t-test and Wilcoxon signed ranked test (n = 5 animals/experimental group, ***p < 0.0001).
Figure 7
Figure 7
Effect of intrabursal administration of ALLO 6 µM on the expression of BAX, BCL-2, FAS, FAS-L and the BAX/BCL-2 and FAS/FAS-L ratio (A–F). (G) ALLO effect on cyclin D1 gene expression. Results are expressed as mean ± SEM. Paired Student’s t test (* < 0.05; n = 4 animals/experimental group).
Figure 8
Figure 8
Upper panel: Representative microphotographs of tertiary follicles present in control (left panel) and ALLO 6 µM-treated (right panel), PCNA-labeled (A and B) and TUNEL-labeled (C and D) ovaries. Light microscopy, 100 x, bar = 200 µm. Lower panel: Proliferation (E, F) and apoptosis (H, I) rates of granulosa and theca cells of ovarian follicles. Number of positively immunolabeled cells per field. Results are expressed as mean ± SEM. Paired Student’s t test and Wilcoxon matched pairs signed rank test (granulosa PCNA) (*p < 0.05/**p < 0.01/***p < 0.001; PCNA: n = 16 sections/ovary for granulosa cells, n = 12 sections/ovary for theca cells, TUNEL: n = 10 sections/ovary for granulosa cells, n = 10 sections/ovary for theca cells, total of 5 ovaries/group).
Figure 9
Figure 9
Rate of proliferation (upper panel) and apoptosis (lower panel) of luteal cells. Number of positively immunolabeled cells per field. Results are expressed as mean ± SEM. Wilcoxon matched pairs signed rank test (PCNA) and Paired Student’s t test (***p < 0.001). Representative microphotographs of CL present in control (left panel) and ALLO 6 µM-treated (right panel) ovaries, stained with PCNA (A and B) and TUNEL (C and D). Light microscopy, 100 x, bar = 200 µm (PCNA: n = 27 sections/ovary, TUNEL: n = 35 sections/ovary, total of 5 ovaries/group).
Figure 10
Figure 10
Relative periendothelial area labeled with α-actin (αA) and relative vascular area labeled with von Willebrand factor (VW). Results are expressed as mean ± SEM. Paired Student’s t test (*p < 0.05/**p < 0.01; n = 5 ovaries/group). Representative microphotographs of sections from control (A and C) and ALLO 6 µM-treated (B and D) ovaries, stained with α-actin (A and B) and Von Willebrand factor (C and D). Light microscopy, 400 x, bar = 200 µm.
Figure 11
Figure 11
Upper panel: Representative microphotographs of sections from control (A) and ALLO 6 µM-treated (B) ovaries, stained with lectin BS-1. Light microscopy, 40 x, bar = 200 µm; inset 400 x, bar = 50 µm. Lower panel: Vascular area labeled with lectin BS-1. Results are expressed as mean ± SEM. Paired Student’s t test (*p < 0.05/**p < 0.01; n = 3 animals/group, 16 ovarian sections/group).
Figure 12
Figure 12
Effect of intrabursal ALLO 6 µM administration on the ovarian expression of the GABAA receptor. Representative microphotographs of sections from control (left panel) and ALLO 6 µM-treated (right panel) ovaries, stained with anti GABAA receptor Rα1-6. Atretic follicles (A and F), antral follicles (B and G), preantral follicles (C and H), corpora lutea section (D and I), stroma with blood vessels (E and J). Light microscopy, 400 x, bar = 100 µm.
Figure 13
Figure 13
Effect of intrabursal ALLO 6 µM and BIC 10 µM administration on the number of atretic follicles (A) and on the diameter (µm) of CL (B). Results are expressed as mean ± SEM. (A) ANOVA I, post hoc Tukey; (B) Kruskal–Wallis with Dunn’s multiple comparison test (**p ≤ 0.01, ***p ≤ 0.0001; n = 5 animals/group, 51 CL/group).
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