Steroidogenic capacity of residual ovarian tissue in 4-vinylcyclohexene diepoxide-treated mice

Abstract

Menopause is an important public health issue because of its association with a number of disorders. Androgens produced by residual ovarian tissue after menopause could impact the development of these disorders. It has been unclear, however, whether the postmenopausal ovary retains steroidogenic capacity. Thus, an ovary-intact mouse model for menopause that uses the occupational chemical 4-vinylcyclohexene diepoxide (VCD) was used to characterize the expression of steroidogenic genes in residual ovarian tissue of follicle-depleted mice. Female B6C3F1 mice (age, 28 days) were dosed daily for 20 days with either vehicle or VCD (160 mg kg(-1) day(-1)) to induce ovarian failure. Ovaries were collected on Day 181 and analyzed for mRNA and protein. Acyclic aged mice were used as controls for natural ovarian senescence. Relative to cycling controls, expression of mRNA encoding steroidogenic acute regulatory protein (Star); cholesterol side-chain cleavage (Cyp11a1); 3beta-hydroxysteroid dehydrogenase (Hsd3b); 17alpha-hydroxylase (Cyp17a1); scavenger receptor class B, type 1 (Scarb1); low-density lipoprotein receptor (Ldlr); and luteinizing hormone receptor (Lhcgr) was enriched in VCD-treated ovaries. In acyclic aged ovaries, mRNA expression for only Cyp17a1 and Lhcgr was greater than that in controls. Compared to cycling controls, ovaries from VCD-treated and aged mice had similar levels of HSD3B, CYP17A1, and LHCGR protein. The pattern of protein immunofluorescence staining for HSD3B in follicle-depleted (VCD-treated) ovaries was homogeneous, whereas that for CYP17A1 was only seen in residual interstitial cells. Circulating levels of FSH and LH were increased, and androstenedione levels were detectable following follicle depletion in VCD-treated mice. These findings support the idea that residual ovarian tissue in VCD-treated mice retains androgenic capacity.

FIG. 1.

Relative abundance of mRNA encoding steroidogenic enzymes in follicle-depleted ovaries. RNA was obtained from whole ovaries from VCD-treated, follicle depleted mice (Day 181 after the onset of dosing), from age-matched cycling controls, and from acyclic aged mice (age, 2 yr). Expression of the genes of interest was determined by real-time PCR as described in Materials and Methods. A) Star (steroidogenic acute regulatory). B) Cyp11a1 (cholesterol side-chain cleavage). C) Hsd3b1 (3β-hydroxysteroid dehydrogenase type 1). D) Cyp17a1 (17α-hydroxylase/17,20-desmolase). Data are presented as mean relative expression ± SEM (n = 3–4 ovaries/treatment). *P < 0.05 versus cycling control, ^#P < 0.05 versus VCD-treated follicle-depleted.

FIG. 2.

Expression levels of HSD3B and CYP17A1 protein in follicle-depleted ovaries. Ovaries were collected from VCD-treated, follicle-depleted mice (Day 181 after the onset of dosing), from age-matched controls, and from acyclic aged mice (age, 2 yr) and then processed for Western blot analysis as described in Materials and Methods. A) Representative Western blots showing expression of HSD3B and CYP17A1 in Day-181 cycling control (lanes 1–3), VCD-treated (lanes 4–6), and acyclic aged ovaries (lanes 7 and 8). Peptidylprolyl isomerase B (PPIB) binding was used for evaluation of loading efficiency. B) Quantification of HSD3B and CYP17A1 protein expression (normalized to expression of PPIB) in VCD-treated and acyclic aged ovaries compared to that in cycling control ovaries (n = 2–3/group averaged from two experiments). Data are presented as mean relative intensity/relative intensity of PPIB ± SEM.

FIG. 3.

Morphological evaluation and localization of HSD3B and CYP17A1 protein in follicle-depleted ovaries. Ovaries were collected from VCD-treated, follicle-depleted mice (Day 181 after the onset of VCD dosing), from age-matched cycling controls, and from acyclic aged (age, 2 yr) mice and then processed for immunofluorescence or hematoxylin and eosin (H&E) staining as described in Materials and Methods. Sections were immunostained for HSD3B (red; A and B) or CYP17A1 (red; D and E) in cycling control ovaries (A and D, respectively) and in follicle-depleted ovaries (B and E, respectively). All sections (A–F) were stained with YOYO-1 (green) for genomic DNA visualization. Theca cells (Theca), interstitial cells (Inst), follicles (f), and corpora lutea (CL) are labeled when present. Primary antibody was preabsorbed with antigen before staining using sections from cycling control ovaries to evaluate specificity (HSD3B in C and CYP17A1 in F). Overlapping images of H&E-stained slides were taken and stitched to show whole ovaries from cycling (G), VCD-treated follicle-depleted (H), and acyclic aged (I) animals. Original magnification ×20 (A-C, E, and F), ×40 (D), and ×10 (G–I) Bars = 50 μm (immunofluorescence; A–F) and 500 μm (H&E stain; G–I).

FIG. 4.

Relative abundance of Scarb1, Ldlr, and Lhcgr mRNAs and LHCGR protein in follicle-depleted ovaries. RNA or protein was obtained from whole ovaries from VCD-treated, follicle depleted mice (Day 181 after the onset of dosing), from age-matched cycling controls, and from acyclic aged (age, 2 yr) mice. Expression of mRNA and protein was determined by real-time PCR (A–C) and Western blot analysis (D), respectively, as described in Materials and Methods. A) Scarb1 (scavenger receptor class B type 1). B) Ldlr (low density lipoprotein receptor). C) Lhcgr (luteinizing hormone/chorionic gonadotropin receptor). D) Representative Western blot showing expression of LHCGR in Day 181 cycling control (lanes 1 and 2), VCD-treated (lanes 3 and 4), and acyclic aged ovaries (lanes 5 and 6). Peptidylprolyl isomerase B (PPIB) binding was used for evaluation of loading efficiency. E) Quantification of LHCGR protein expression (normalized to expression of PPIB) in VCD-treated and acyclic aged ovaries compared to that in cycling control ovaries (n = 2 per group). Real-time PCR data are presented as mean relative expression ± SEM (n = 3–4 ovaries/treatment); Western blot data are presented as mean relative intensity/relative intensity of PPIB ± SEM. *P < 0.05 versus cycling control; ^#P < 0.05 versus VCD-treated follicle-depleted.

FIG. 5.

Circulating FSH (A), LH (B), and androstenedione (C) levels in follicle-depleted animals. Blood samples were collected from cycling controls and VCD-treated mice at various time points (Day 181 only for FSH and LH; throughout the study for androstenedione) or from acyclic aged animals (age, 2 yr; for FSH and LH) at the time of death. Levels of FSH, LH, and androstenedione were determined and compared as described in Materials and Methods. Data are presented as mean ± SEM (n = 3–8 animals/treatment group). *P < 0.05 versus cycling controls; ^#P < 0.05 versus baseline (Day 35).