Prenatal exposure to low levels of androgen accelerates female puberty onset and reproductive senescence in mice

Abstract

Sex steroid hormone production and feedback mechanisms are critical components of the hypothalamic-pituitary-gonadal (HPG) axis and regulate fetal development, puberty, fertility, and menopause. In female mammals, developmental exposure to excess androgens alters the development of the HPG axis and has pathophysiological effects on adult reproductive function. This study presents an in-depth reproductive analysis of a murine model of prenatal androgenization (PNA) in which females are exposed to a low dose of dihydrotestosterone during late prenatal development on embryonic d 16.5-18.5. We determined that PNA females had advanced pubertal onset and a delay in the time to first litter, compared with vehicle-treated controls. The PNA mice also had elevated testosterone, irregular estrous cyclicity, and advanced reproductive senescence. To assess the importance of the window of androgen exposure, dihydrotestosterone was administered to a separate cohort of female mice on postnatal d 21-23 [prepubertal androgenization (PPA)]. PPA significantly advanced the timing of pubertal onset, as observed by age of the vaginal opening, yet had no effects on testosterone or estrous cycling in adulthood. The absence of kisspeptin receptor in Kiss1r-null mice did not change the acceleration of puberty by the PNA and PPA paradigms, indicating that kisspeptin signaling is not required for androgens to advance puberty. Thus, prenatal, but not prepubertal, exposure to low levels of androgens disrupts normal reproductive function throughout life from puberty to reproductive senescence.

Fig. 1.

VO was advanced in PNA and PPA but not in PNA progeny. A, Diagram illustrates the time line for DHT administration in the PNA and PPA paradigms and the time frame for monitoring pubertal onset by VO. B, Wild-type females were subjected to the PNA paradigm and were monitored daily for VO. ***, P < 0.001 by Student's t test. C, Wild-type females were subjected to the PPA paradigm and were monitored daily for VO. ***, P < 0.001 by Student's t test. D, Adult PNA females were mated to wild-type males and the female progeny were monitored daily for VO (P > 0.05 by Student's t test).

Fig. 2.

VO was advanced in PPA AR/Syn-Cre and Kiss1r-null, and in PNA Kiss1r-null mice. A, AR/Syn-Cre females were mated to AR flox males. Female AR flox (Cre-) and AR/Syn-Cre (Cre+) offspring were subjected to the PPA paradigm and were monitored daily for VO. ***, P < 0.001 vs. control by two-way ANOVA. B, Heterozygous Kiss1r animals were mated. Female homozygous WT and KO Kiss1r-null offspring were subjected to the PPA paradigm and were monitored daily for VO. ***, P < 0.001 vs. control by log-rank χ². VO was not recorded after PND 35. C, Heterozygous Kiss1r dams were administered PNA. Female homozygous WT and Kiss1r-null (KO) offspring were subjected to the PNA paradigm and were monitored daily for VO. *, P < 0.05 vs. control by log-rank χ². ***, P < 0.001 vs. oil-treated knockout by log-rank χ². VO was not recorded after PND 35.

Fig. 3.

PNA exposure resulted in irregular estrus cycles and increased T in females. A, Estrous stages for 3-month-old PNA and PPA females were tested daily for 15–20 d. Representative cycle traces are shown. M, Metestrus; E, estrus; P, proestrus; D, diestrus. B, The proportion of time spent in each cycle stage was quantified for the PNA females (n = 8 oil and n = 7 DHT). ***, P < 0.001 by Student's t test. C, Four-month-old PNA and PPA females were killed in diestrus and sera were analyzed for T levels. *, P = 0.05 by Student's t test.

Fig. 4.

Testosterone implantation resulted in disrupted estrous cycling in wild-type females. Estrous stages were tested in wild-type females (2–3 months old) daily for 15 d both before (A) and after (B) T or cholesterol implantation, and the proportion of time spent in each cycle stage was quantified (n = 8 cholesterol and n = 8 T). *, P < 0.05 by Student's t test.

Fig. 5.

PNA females had an initial delay in fertility. PNA females were pair housed continuously with single wild-type males for 6 months. A, The number of days until birth of the first litter was measured. *, P < 0.05 by Student's t test. B, The total number of litters born during each month of the fertility assessment was quantified. *, P < 0.05 by Student's t test.

Fig. 6.

PNA females exhibited ovarian morphology consistent with the ability to produce litters. PNA females were killed in diestrus at 2 (A), 3 (B), 7 (C), and 10 (D) months of age. Ovaries were fixed, sectioned, and examined for normal follicles, Graffian follicles (GF), and corpora lutea (CL) (n = 3 for 2, 3, and 10 months; n = 2 for 7 months). CL, preantral follicles, and antral follicles were quantified and no differences were found.

Fig. 7.

Advanced reproductive senescence in aging PNA females. PNA females were cycled daily for 25 d at 8 and 10 months of age, and the incidence of PVC was quantified. **, P < 0.01 by χ².