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. 2023 Jan 3:101:skad082.
doi: 10.1093/jas/skad082.

Sheep with ovarian androgen excess have fibrosis and follicular arrest with increased mRNA abundance for steroidogenic enzymes and gonadotropin receptors

Mohamed A Abedal-Majed  1 , Mohannad Abuajamieh  1 , Mohmmad Al-Qaisi  1 , Kevin M Sargent  2 , Hosam H Titi  1 , Mufeed A Alnimer  1 , Anas Abdelqader  1 , Ahmad I Shamoun  1 , Andrea S Cupp  3
Affiliations

Sheep with ovarian androgen excess have fibrosis and follicular arrest with increased mRNA abundance for steroidogenic enzymes and gonadotropin receptors

Mohamed A Abedal-Majed et al. J Anim Sci. .

Abstract

An androgen excess ovarian micro-environment may limit follicle progression in sheep. Two populations of ewes with divergent follicular fluid androstenedione (A4) were identified in a flock in Jordan: High A4; (A4) ≥ 30 ng/mL, (N = 12) or Control A4 (Control); A4 ≤ 15 ng/mL; (N = 12). We hypothesized High A4 ewes would have increased steroidogenic enzyme mRNA abundance, inflammation, and follicular arrest. Messenger RNA abundance for steroidogenic enzymes StAR, CYP17A1, CYP11A1, and HSD3B1 were increased in theca cells while CYP17A1, CYP19A1, and HSD3B1 were increased in granulosa cells in High A4 ewes compared to Control. Gonadotropin receptor mRNA expression for LHCGR was increased in theca and FSHR in granulosa in High A4 ewes. Messenger RNA expression of FOS when reduced, increases expression of CYP17A1 which was observed in High A4 granulosa cells compared to Control. Furthermore, High A4 ewes had greater numbers of primordial follicles (P < 0.001) and fewer developing follicles compared to Control before, and after 7 d of culture, indicating follicular arrest was not alleviated by cortex culture. Increased fibrosis in the ovarian cortex was detected in High A4 ewes relative to Control (P < 0.001) suggesting increased inflammation and altered extracellular matrix deposition. Thus, this High A4 ewes population has similar characteristics to High A4 cows and women with polycystic ovary syndrome suggesting that naturally occurring androgen excess occurs in multiple species and may be a causative factor in follicular arrest and subsequent female sub- or infertility.

Keywords: androgen excess; ewes; follicle arrest; ovarian cortex culture; ovarian fibrosis; steroidogenic enzymes.

Plain language summary

Excess androgen (androstenedione; A4) in ewes can result in ovarian follicular arrest and fibrosis contributing to anovulation in sheep. We have identified a naturally occurring ovarian A4 excess in a sheep population with similar characteristics to High A4 cows, both of which are similar to that in women with polycystic ovary syndrome indicating that several mammalian species experience naturally occurring androgen excess resulting in infertility or follicle arrest. Somatic cells, theca and granulosa, surrounding the egg in High A4 ewes had increased expression of steroidogenic enzymes, similar to that seen in High A4 cows, permitting more ovarian cells to manufacture androgens, which may be the cause of androgen excess. Thus, naturally occurring androgen-excess in domestic livestock females can be utilized as models to research the causes of androgen excess and determine the mechanisms that result in follicular arrest and sub- or infertility.

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Conflict of interest statement

The authors declare no real or perceived conflicts of interest.

Figures

Graphical Abstract
Graphical Abstract
Figure 1.
Figure 1.
Steroid hormone concentrations in follicular fluid from pooled largest growing follicles (A–C) compared between Control and High A4 lambs. (A) Progesterone (P4), (B) Androstenedione (A4), and (C) Estradiol (E2).
Figure 2.
Figure 2.
Average numbers of follicles at different stages (A) before culture, (B) after culture, (C) fold change(D7/D0) of follicles per field, and (D) representative H and E staining for histology in Control and High A4 ewes, demonstrating follicle stages present in each lamb classification.
Figure 3.
Figure 3.
Collagen staining (PSR) for Control and High A4 ovarian cortex (A,B) before culture and (D,E) after in vitro culture. Graph comparing the average area of PSR-positive staining per ovarian cortex field (pixels/μm2) between High A4 and Control ewes (C) before culture and (F) after in vitro culture.
Figure 4.
Figure 4.
Quantitative RT-PCR results for StAR (A), LHCGR (B), CYP17A1 (C), CYP11A1 (D), and HSD3B1 (E) in theca cells collected from largest growing follicles of Control (white bars, N = 6) and High A4 (black bars, N = 6) ewes. The geometric mean of GAPDH was used as an endogenous control. Graphs represent a fold change in mRNA abundance with Control set to (1). The mean SEM normalized values are presented.
Figure 6.
Figure 6.
Illustration of steroidogenesis in High A4 sheep ovary. Arrows show increased mRNA abundance in genes regulating steroidogenesis within theca and granulosa cells.
Figure 5.
Figure 5.
Quantitative RT-PCR results for StAR (A), LHCGR (B), FSHR (C), CYP17A1 (D), CYP11A1 (E), HSD3B1 (F), CYP19A1 (G), and FOS (H) in granulosa cells collected from largest growing follicles of Control (white bars, N = 6) and High A4 (black bars, N = 6) ewes. The geometric mean of GAPDH was used as an endogenous control. Graphs represent a fold change in mRNA abundance with Control set as normal (1). The mean SEM normalized values are presented.
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