Prenatal hyperandrogenization induces metabolic and endocrine alterations which depend on the levels of testosterone exposure

Abstract

Prenatal hyperandrogenism is able to induce polycystic ovary syndrome (PCOS) in rats. The aim of the present study was to establish if the levels of prenatal testosterone may determine the extent of metabolic and endocrine alterations during the adult life. Pregnant Sprague Dawley rats were prenatally injected with either 2 or 5 mg free testosterone (groups T2 and T5 respectively) from day 16 to day 19 day of gestation. Female offspring from T2 and T5 displayed different phenotype of PCOS during adult life. Offspring from T2 showed hyperandrogenism, ovarian cysts and ovulatory cycles whereas those from T5 displayed hyperandrogenism, ovarian cysts and anovulatory cycles. Both group showed increased circulating glucose levels after the intraperitoneal glucose tolerance test (IPGTT; an evaluation of insulin resistance). IPGTT was higher in T5 rats and directly correlated with body weight at prepubertal age. However, the decrease in the body weight at prepubertal age was compensated during adult life. Although both groups showed enhanced ovarian steroidogenesis, it appears that the molecular mechanisms involved were different. The higher dose of testosterone enhanced the expression of both the protein that regulates cholesterol availability (the steroidogenic acute regulatory protein (StAR)) and the protein expression of the transcriptional factor: peroxisome proliferator-activated receptor gamma (PPAR gamma). Prenatal hyperandrogenization induced an anti-oxidant response that prevented a possible pro-oxidant status. The higher dose of testosterone induced a pro-inflammatory state in ovarian tissue mediated by increased levels of prostaglandin E (PG) and the protein expression of cyclooxygenase 2 (COX2, the limiting enzyme of PGs synthesis). In summary, our data show that the levels of testosterone prenatally injected modulate the uterine environment and that this, in turn, would be responsible for the endocrine and metabolic abnormalities and the phenotype of PCOS during the adult life.

Figure 1. Prenatal hyperandrogenization and body weight.

Pregnant Sprague Dawley rats were subcutaneous injected with 2 or 5 mg free testosterone from 16 to 19 day of pregnancy. Female offspring prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). Panel (A) represents the weight at 21 days of age and panel (B) represents the weight at 60 days of age. Panel (C) represents the growth rates of three groups control, T2 and T5; a vs b P<0.01; b vs c P<0.0001; a vs c P<0.001 by ANOVA test.Each column represents the mean+SEM from ten different animals, N = 20 animals/group.

Figure 2. Prenatal hyperandrogenization and glucose homeostasis.

As a measurement of glucose homeostasis, dynamic studies were performed in female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). Blood sample followed by itraperitoneal injection of 2 g dextrose/kg body weight was collected at 0, 30, 60, 90 and 120 post-injection. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.

Figure 3. Prenatal hyperandrogenization and ovarian steroidogenesis.

(A) Serum progesterone levels, (B) Serum estradiol levels and (C) Serum testosterone levels from female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). CP: rats from the control group at proestrous stage of the estrous cycle, T2P: rats from the T2 group of treatment at proestrous stage of the estrous cycle CD: rats from the control group at diestrous stage of the estrous cycle, T2D: rats from the T2 group of treatment at diestrous stage of the estrous cycle, T5: rats from the T5 group of treatment. a vs b; a vs c and b vs c P<0.0001 by ANOVA test. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.

Figure 4. Prenatal hyperandrogenization and ovarian morphology.

(A) A representative ovarian tissue section from rats prenatally injected with vehicle (Control group), ×200. (B) Detail of granulosa (GC), theca cells (TC) and luteinized cells (LC) of ovarian tissue from rats prenatally injected with vehicle (Control group), ×400. (C) Magnification of conserved oocyte of ovarian tissue from rats in the Control group, ×1000. (D) Detail of conserved oocytes (O) and distribution of granulosa (GC) and theca cells (TC) of ovarian tissue from rats in the Control group, ×400. (E) Detail of luteinized cells (LC) of ovarian tissue from rats in the Control group, ×400 (F) Magnification of conserved luteinized cells (LC) of ovarian tissue from rats in the Control group, ×1000. (G) A representative ovarian tissue section from rats prenatally injected with 2 mg testosterone (T2 group), PF = primary follicle, SF = secondary follicle, ×400. (H) A representative atretic oocyte (AO) of ovarian tissue from rats in the T2 group, ×400. (I) A representative follicular cyst (FC) of ovarian tissue from rats in the T2 group, ×400. (J) Magnification of granulosa (GC) and theca cell (TC) of the follicular cyst, ×1000. (K) Aspect of luteinized cells (LC) of ovarian tissue from rats in the T2 group, ×1000. (L) A representative section of ovarian tissue from rats prenatally injected with 5 mg testosterone (T5 group), AF = antral follicle, ×200. L.C. = luteal cells (M) Follicular cysts (FC) in ovarian tissue from rats in the T5 group, ×400. (N) A detail of altered organization of granulosa (GC) and theca cells (TC) and atretic oocytes (AO) of ovarian tissue from rats in the T5 group, N = ten ovaries per group.

Figure 5. Percentages of follicles (primary, secondary, antral and atretic) per group of treatment: control, T2 and T5.

Ten ovaries from each group were fixed in 4% (w/v) paraformaldehyde, included in paraffin, cut 4 µm per section, placed on gelatin-coated slides and stained with haematoxylin and eosin.

Figure 6. Prenatal hyperandrogenization and ovarian inflammatory status.

Ovarian prostaglandin E content from female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). CP: rats from the control group at proestrous stage of the estrous cycle, T2P: rats from the T2 group of treatment at proestrous stage of the estrous cycle CD: rats from the control group at diestrous stage of the estrous cycle, T2D: rats from the T2 group of treatment at diestrous stage of the estrous cycle, T5: rats from the T5 group of treatment. a vs b P<0.0001 by ANOVA test. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.

Figure 7. Prenatal hyperandrogenization and ovarian oxidant-antioxidant balance.

(A) Lipid peroxidation, (B) Nitric oxide synthase (NOS) activity and (C) Content of anti-oxidant gluthatione (GSH) of ovarian tissue from female offspring of Sprague Dawley rats prenatally injected with 2 mg testosterone (T2 group), 5 mg testosterone (T5) or vehicle (C). CP: rats from the control group at proestrous stage of the estrous cycle, T2P: rats from the T2 group of treatment at proestrous stage of the estrous cycle CD: rats from the control group at diestrous stage of the estrous cycle, T2D: rats from the T2 group of treatment at diestrous stage of the estrous cycle, T5: rats from the T5 group of treatment. a vs b P<0.0001 by ANOVA test. Each column represents the mean+SEM from ten different animals, N = 20 animals/group.

Figure 8. Mechanisms involved in the prenatal hyperandrogenization on ovarian tissue.

Western blotting for the expression of the steroidogenic acute regulatory protein (StAR) in ovarian tissue from rats prenatally injected with vehicle (Control group), 2 mg testosterone (T2 group) or 5 mg testosterone (T5 group). Bands correspond to 30 kDa, (A) A representative Western blot, (B) actin as control protein and (C) integrated optical density of the bands. Each column represent mean+SEM of ten different animals. a vs b P<0.0001 by ANOVA test. Western blotting for the expression of the nuclear peroxisome proliferator- activated receptor (PPAR) gamma in ovarian tissue from rats prenatally injected with vehicle (Control group), 2 mg testosterone (T2 group) or 5 mg testosterone (T5 group). Bands correspond to 67 kDa, (D) A representative Western blot, (E) actin as control and (F) integrated optical density of the bands. Each column represent mean+SEM of ten different animals. a vs b P<0.0001 by ANOVA test. Western blotting for the expression of cyclooxygenase 2 (COX2) in ovarian tissue from rats prenatally injected with vehicle (Control group), 2 mg testosterone (T2 group) or 5 mg testosterone (T5 group). Bands correspond to 62 kDa, (G) A representative Western blot, (H) actin as control and (I) integrated optical density of the bands. Each column represent mean+SEM of ten different animals. a vs b P<0.0001 by ANOVA test, N = 20 animals/group.