Neonatal Exposure to Agonists and Antagonists of Sex Steroid Receptors Affects AMH and FSH Plasma Level and Their Receptors Expression in the Adult Pig Ovary

Abstract

In this study piglets were injected with testosterone propionate (TP, an androgen), flutamide (FLU, an antiandrogen), 4-tert-octylphenol (OP, an estrogenic compound), ICI 182,780 (ICI, an antiestrogen) or corn oil (controls) between postnatal days 1 and 10 (N = 5/group). Then plasma anti-Müllerian hormone (AMH) and follicle stimulating hormone (FSH) concentration and the expression of their receptors were examined in the adult pig ovary. TP and FLU decreased plasma AMH and FSH concentration. In preantral follicles, TP resulted in upregulation of AMHR2 and FSHR expression, but decreased AMH protein abundance. FLU upregulated AMHR2 expression, while OP increased FSHR mRNA. In small antral follicles, OP upregulated ACVR1 and BMPR1A expression, while FLU increased BMPR1A mRNA. FLU and ICI resulted in upregulation of AMHR2 expression. TP and FLU upregulated AMH expression, while it was downregulated in response to OP or ICI. Moreover, OP and ICI resulted in downregulation of FSHR expression, while FLU decreased FSHR protein abundance. In conclusion, neonatal exposure to either agonist or antagonist of androgen receptor affected AMH and FSH signalling systems in preantral follicles. In small antral follicles these systems were influenced by compounds with estrogenic, antiestrogenic, and antiandrogenic activity. Consequently, these hormonal agents may cause an accelerated recruitment of primordial follicles and affect the cycling recruitment of small antral follicles in pigs.

Keywords: AMH; FSH; endocrine active compounds; ovary; pig.

Figure 1

Plasma anti-Müllerian hormone (AMH) (a) and follicle stimulating hormone (FSH) (b) concentrations in control (CTR) and testosterone propionate—(TP), flutamide—(FLU), 4-tert-octyl-phenol—(OP), and ICI 182,780-treated (ICI) adult pigs. Data are expressed as the mean ± standard error of the mean (N = 5). Asterisks denote significant differences between control and treated animals, * p < 0.05, ** p < 0.01, Mann-Whitney U test.

Figure 2

ACVR1 (a,a’), BMPR1A (b,b’), AMHR2 (c,c’), AMH (d,d’), and FSHR (e,e’) mRNA expression and protein abundance in primordial, primary, and early secondary follicles obtained from control (CTR), testosterone propionate—(TP), flutamide—(FLU), 4-tert-octylphenol—(OP), and ICI 182,780-treated (ICI) pigs. The mRNA expression was determined using quantitative real-time PCR and presented relative to glyceraldehyde-3-phosphate dehydrogenase as mean ± standard error of the mean (a–e). Relative abundance of protein was evaluated densitometrically and expressed as the ratio relative to β-actin abundance (mean ± standard error of the mean; (a’–e’)). (f) The fragment of membrane with bands corresponding to predicted molecular weights are shown. Asterisks denote significant differences between the control and treated animals, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney U test.

Figure 2

ACVR1 (a,a’), BMPR1A (b,b’), AMHR2 (c,c’), AMH (d,d’), and FSHR (e,e’) mRNA expression and protein abundance in primordial, primary, and early secondary follicles obtained from control (CTR), testosterone propionate—(TP), flutamide—(FLU), 4-tert-octylphenol—(OP), and ICI 182,780-treated (ICI) pigs. The mRNA expression was determined using quantitative real-time PCR and presented relative to glyceraldehyde-3-phosphate dehydrogenase as mean ± standard error of the mean (a–e). Relative abundance of protein was evaluated densitometrically and expressed as the ratio relative to β-actin abundance (mean ± standard error of the mean; (a’–e’)). (f) The fragment of membrane with bands corresponding to predicted molecular weights are shown. Asterisks denote significant differences between the control and treated animals, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney U test.

Figure 3

ACVR1 (a,a’), BMPR1A (b,b’), AMHR2 (c,c’), AMH (d,d’) and FSHR (e,e’) mRNA expression and protein abundance in small antral follicles obtained from control (CTR), testosterone propionate—(TP), flutamide—(FLU), 4-tert-octylphenol—(OP), and ICI 182,780-treated (ICI) pigs. The mRNA expression was determined using quantitative real-time PCR and presented relative to glyceraldehyde-3-phosphate dehydrogenase as mean ± standard error of the mean (a–e). Relative abundance of protein was evaluated densitometrically and expressed as the ratio relative to β-actin abundance (mean ± standard error of the mean; (a’–e’)). (f) The fragment of membrane with bands corresponding to predicted molecular weights are shown. Asterisks denote significant differences between control and treated animals, * p < 0.05, ** p < 0.01, *** p < 0.001, Mann-Whitney U test.

Figure 4

ACVR1 immunostaining in the preantral (a–e) and small antral (a’–e’) follicles obtained from control gilts (a,a’) and treated with testosterone propionate (b,b’), flutamide (c,c’), 4-tert-octylphenol (d,d’), and ICI 182,780 (e,e’). Immunopositivity was detected in oocytes (asterisks) and granulosa cells (arrows) of preantral follicles (a–e) as well as in both granulosa (arrows) and theca (arrowheads) cells of small antral follicles (a’–e’). Hematoxylin QS was used for counterstaining sections. Control sections showed no positive staining ((a’) inset). Bars = 50 µm.

Figure 5

BMPR1A immunostaining in the preantral (a–e) and small antral (a’–e’) follicles obtained from control gilts (a,a’) and treated with testosterone propionate (b,b’), flutamide (c,c’), 4-tert-octylphenol (d,d’), and ICI 182,780 (e,e’). Immunopositivity was detected in oocytes (asterisks) and granulosa cells (arrows) of preantral follicles (a–e) as well as in both granulosa (arrows) and theca (arrowheads) cells of small antral follicles (a’–e’). Hematoxylin QS was used for counterstaining sections. Control sections showed no positive staining ((a’) inset). Bars = 50 µm.

Figure 6

AMHR2 immunostaining in the preantral (a–e) and small antral (a’–e’) follicles obtained from control gilts (a,a’) and treated with testosterone propionate (b,b’), flutamide (c,c’), 4-tert-octylphenol (d,d’), and ICI 182,780 (e,e’). Immunopositivity was detected in oocytes (asterisks) and granulosa cells (arrows) of preantral follicles (a–e) as well as in both granulosa (arrows) and theca (arrowheads) cells of small antral follicles (a’–e’). Hematoxylin QS was used for counterstaining sections. Control sections showed no positive staining ((a’) inset). Bars = 50 µm.

Figure 7

FSHR immunostaining in the preantral (a–e) and small antral (a’–e’) follicles obtained from control gilts (a,a’) and treated with testosterone propionate (b,b’), flutamide (c,c’), 4-tert-octylphenol (d,d’), and ICI 182,780 (e,e’). Immunopositivity was detected in oocytes (asterisks) and granulosa cells (arrowheads) of preantral follicles (a–e) as well as granulosa cells (arrowheads) of small antral follicles (a’–e’). Hematoxylin QS was used for counterstaining sections. Control sections showed no positive staining ((a’) inset). Bars = 50 µm.