Neuroendocrine consequences of androgen excess in female rodents

Abstract

Androgens exert significant organizational and activational effects on the nervous system and behavior. Despite the fact that female mammals generally produce low levels of androgens, relative to the male of the same species, increasing evidence suggests that androgens can exert profound effects on the normal physiology and behavior of females during fetal, neonatal, and adult stages of life. This review examines the effects of exposure to androgens at three stages of development--as an adult, during early postnatal life and as a fetus, on reproductive hormone secretions in female rats. We examine the effects of androgen exposure both as a model of neuroendocrine sexual differentiation and with respect to the role androgens play in the normal female. We then discuss the hypothesis that androgens may cause epigenetic modification of estrogen target genes in the brain. Finally we consider the clinical consequences of excess androgen exposure in women.

Figure 1. Serum LH levels in female rats treated with androgens in adulthood

Cycling female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT on metestrus, diestrus, proestrus or estrus. After four consecutive days of treatment, animals were sacrificed on presumptive metestrus (a), diestrus (b), proestrus (c), or estrus (d), respectively (n=5 for all groups). Trunk blood was collected and serum LH levels were determined by RIA (*p<0.05, ***p<0.001, as compared to the Control group). The data are represented as mean ± SEM.

Figure 2. Androgen treatment in adulthood suppresses serum LH levels in ovariectomized rats

Ovariectomized female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT (n=5 for all groups). Seven days later, trunk blood was collected and serum LH levels were determined by RIA. Animals treated with T and DHT showed a significant reduction (p<0.001) serum LH levels compared to oil-treated controls.

Figure 3. Serum FSH levels in female rats treated with androgens in adulthood

Cycling female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT on metestrus, diestrus, proestrus or estrus. After four consecutive days of treatment, animals were sacrificed on presumptive metestrus (a), diestrus (b), proestrus (c), or estrus (d), respectively (n=5 for all groups). Trunk blood was collected and serum FSH levels were determined by RIA (*p<0.05, **p<0.01, as compared to the Control group). The data are represented as mean ± SEM.

Figure 4. Androgen treatment in adulthood blocks the EB-induced LH surge

Adult female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT. Three days later on diestrus, at 0900h, the female rats were ovariectomized (OVX) and treated with estradiol benzoate (EB; 30 μg sc). At the same time, the rats were fitted with jugular catheters. The following day, blood samples were collected every 30 minutes from 1200h to 2100h to assess the release of the LH.. Statistically significant LH surges were detected in control females but not in T- or DHT-treated females (n=4 per group). The data are represented as mean ± SEM. (From Foecking and Levine, 2005).

Figure 5. GnRH and LH surges are absent in ovariectomized female rats treated with a PR antagonist

Representative GnRH and LH release profiles of OVX, E₂-primed rats (A) and OVX, E₂-primed rats treated with ZK98299 (B). A, E₂-priming stimulated significant increases in GnRH pulse amplitude commencing at approximately 1600 h, accompanied by a concomitant 3- to 5-fold increase in plasma LH. B, Treatment of E₂-primed, OVX rats with ZK98299 prevented this E₂-stimulated rise in both GnRH and LH release without affecting pulse frequency. Asterisks represent significant pulses as determined by the ULTRA pulse analysis program. (From Chappell et.al., 2000).

Figure 6. Androgen treatment in adulthood suppresses progesterone receptor mRNA expression in the hypothalamus

Cycling female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT on proestrus. After four consecutive days of treatment, animals were sacrificed on presumptive proestrus. The hypothalamus was dissected out of each brain. RNA was isolated from the hypothalamus, reverse transcribed, and processed for PR_A+B mRNA expression via semi-quantitative PCR. PR_A+B mRNA expression is significantly decreased (*p<0.05) in the hypothalamus of DHT-treated rats compared to controls (n=5 for all groups). The semi-quantitative RT-PCR results for PR_A+B are normalized to the housekeeping gene RPL19 and are represented as mean ± SEM.

Figure 7. Androgen treatment in adulthood suppresses EB-induced progesterone receptor mRNA expression in the POA-hypothalamus

Adult female rats were either empty Silastic capsules or capsules filled with either crystalline T, or DHT for four days, ovariectomized (OVX) and treated with estradiol benzoate (EB; 30 µg sc) or sesame oil vehicle (oil). The POA-hypothalamus was dissected out of each brain. RNA was isolated from the POA-hypothalamus, reverse transcribed, and processed for PR_A+B mRNA expression via semi-quantitative PCR. PR_A+B mRNA expression is significantly increased in the POA-hypothalamus of EB-treated, ovariectomized control rats (n=5) compared to their oil-treated (n=4) counterparts (p=0.0026). PR_A+B mRNA expression is not induced by EB in testosterone-treated females (OVX, n=4, OVX/EB, n=3). A significant interaction (**p=0.016) exists between the 4-day testosterone treatment and control group. The semi-quantitative RT-PCR results for PR_A+B are normalized to the housekeeping gene RPL19 and are represented as mean ± SEM. (From Foecking and Levine, 2005).

Figure 8. Androgen treatment in adulthood suppresses progesterone receptor mRNA expression in the pituitary

Cycling female rats were implanted with either empty Silastic capsules or capsules filled with either crystalline T, or DHT on proestrus. After four consecutive days of treatment, animals were sacrificed on presumptive proestrus and the pituitary of each animal was dissected. RNA was isolated from the pituitary, reverse transcribed, and processed for PR_A+B mRNA expression via semi-quantitative PCR. PR_A+B mRNA expression is significantly decreased in the pituitary of both the T- and DHT-treated rats compared to controls (n=5 for all groups). The semi-quantitative RT-PCR results for PR_A+B are normalized to the housekeeping gene RPL19 and are represented as mean ± SEM.

Figure 9. Neonatal androgen treatment blocks LH responses to ovariectomy in adulthood

Female rat pups were subcutaneously injected with oil or 2.5 mg of testosterone propionate on the day of birth. On PND 60, rats were either ovariectomized (OVX) or sham-operated and left intact. Six days later, these rats were fitted with jugular catheters. The following day, blood samples were collected every 30 minutes for 3 hours from 1200h to 2100h. Serum LH levels were determined by RIA. Both mean LH levels (ng/ml) and mean pulse amplitude of LH was significantly increased (*p<0.001) by ovariectomy in control-treated females as compared to the SHAM control group (SHAM, n=3; OVX, n=5). Ovariectomy did not alter LH levels or pulse amplitude in the T-treated (n=5 for both groups) females.

Figure 10. Prenatal androgen exposure accelerates LH pulsatility in adulthood

Pregnant female Sprague-Dawley rats were subcutaneously injected with oil vehicle (V), 5mg of T, or 5mg of DHT daily on embryonic days 16–19. Female offspring were ovariectomized in adulthood (approximately PND 60–70). Six days later, these rats were fitted with jugular catheters. The following day, blood samples were collected every 5 minutes for 3 hours from 1200h to 1500h. LH pulsatility is accelerated in pT (B; n=3) and pDHT (C; n=6) rats compared to pV females (A; n=6) (*significant pulse by ULTRA pulse analysis program). Prenatal exposure to DHT and T results in a significant increase (*p<0.05) in LH pulse frequency (D) compared to pV animals. Mean pulse amplitude (E) and mean LH levels (F) were significantly increased in the pDHT rats compared to pV animals. The data are represented as mean ± SEM. (From Foecking et.al., 2005)

Figure 11. Prenatal androgen exposure blocks the EB-induced preovulatory LH surge in adulthood

Pregnant female Sprague-Dawley rats were subcutaneously injected with oil vehicle (V), 5mg of T, or 5mg of DHT daily on embryonic days 16–19. Female offspring were ovariectomized (OVX) in adulthood at 0900h and treated with estradiol benzoate (EB; 30 μg sc). At the same time, these rats were fitted with jugular catheters. Twenty-four hours later, blood samples were collected every 30 minutes from 1200h to 2100h to capture the release of the LH surge. Serum LH levels were determined via RIA. Statistically significant LH surges were detected in females treated prenatally with vehicle (n=4) but not in pT-(n=3) or pDHT-treated (n=3) females. The data are represented as mean ± SEM. (From Foecking et.al., 2005)

Figure 12. Prenatal androgen exposure suppresses EB-induced PR mRNA expression in the POA of adult female rats

Pregnant female Sprague-Dawley rats were subcutaneously injected with oil vehicle (V), 5mg of T, or 5mg of DHT daily on embryonic days 16–19.Female offspring were ovariectomized (OVX) in adulthood and treated with estradiol benzoate (EB; 30 μg sc) or sesame oil vehicle (oil). The POA was dissected from the brain of each animal, and RNA was isolated, reverse transcribed, and processed for PR_A+B mRNA expression via semi-quantitative PCR. PR_A+B mRNA expression is significantly increased in the POA of EB-treated, ovariectomized control rats (pV, n=6 for both groups) compared to their oil-treated counterparts (*p<0.05). PR_A+B mRNA expression is not induced by EB in pT- (n=7 for both groups) or pDHT-treated (OVX/oil, n=7; OVX/EB, n=9) females. The semi-quantitative RT-PCR results for PR_A+B are normalized to the housekeeping gene RPL19 and are represented as mean ± SEM. (From Foecking et.al., 2005)