Critical androgen-sensitive periods of rat penis and clitoris development

Abstract

Androgen control of penis development/growth is unclear. In rats, androgen action in a foetal 'masculinisation programming window' (MPW; e15.5-e18.5)' predetermines penile length and hypospadias occurrence. This has implications for humans (e.g. micropenis). Our studies aimed to establish in rats when androgen action/administration affects development/growth of the penis and if deficits in MPW androgen action were rescuable postnatally. Thus, pregnant rats were treated with flutamide during the MPW +/- postnatal testosterone propionate (TP) treatment. To assess penile growth responsiveness, rats were treated with TP in various time windows (late foetal, neonatal through early puberty, puberty onset, or combinations thereof). Phallus length, weight, and morphology, hypospadias and anogenital distance (AGD) were measured in mid-puberty (d25) or adulthood (d90) in males and females, plus serum testosterone in adult males. MPW flutamide exposure reduced adult penile length and induced hypospadias dose-dependently; this was not rescued by postnatal TP treatment. In normal rats, foetal (e14.5-e21.5) TP exposure did not affect male penis size but increased female clitoral size. In males, TP exposure from postnatal d1-24 or at puberty (d15-24), increased penile length at d25, but not ultimately in adulthood. Foetal + postnatal TP (e14-postnatal d24) increased penile size at d25 but reduced it at d90 (due to reduced endogenous testosterone). In females, this treatment caused the biggest increase in adult clitoral size but, unlike in males, phallus size was unaffected by TP during puberty (d15-24). Postnatal TP treatment advanced penile histology at d25 to more resemble adult histology. AGD strongly correlated with final penis length. It is concluded that adult penile size depends critically on androgen action during the MPW but subsequent growth depends on later androgen exposure. Foetal and/or postnatal TP exposure does not increase adult penile size above its 'predetermined' length though its growth towards this maximum is advanced by peripubertal TP treatment.

Figure 1

Schematic diagram to show timings of rat treatments in the current studies and their relation to comparable timings in humans. The masculinisation programming window in rats occurs between e15.5–e18.5 and is predicted to occur in humans between weeks 8–14.

Figure 2

Penile length (means ± SEM; top left), incidence of hypospadias (top right) and the relationship between penile length and anogenital distance (AGD; bottom) in adult male rats that had been exposed in utero (e15.5–e18.5) to vehicle (controls) or various doses of flutamide (left) and the effects of postnatal administration of testosterone propionate (TP) from d1 to d24 (right) in a group of animals prenatally exposed to 10 mg/kg flutamide. Values in the upper left panel are means ± SEM for 9–20 animals per group (overall n = 88). Solid horizontal line shows the mean penile length for control males and the dashed horizontal line shows the mean for males exposed in utero to 10 mg/kg flutamide. **p< 0.01, ***p< 0.001, in comparison with the control group. Postnatal TP treatment had no significant effect (NS) on penile length in comparison with animals prenatally exposed to flutamide alone.

Figure 3

(a) Penile length (top panels) and weight/length (lower panels) at mid-puberty (d25) or in adulthood (d90) in male rats exposed in utero (e14.5–e21.5) or postnatally from d1 to d24 or from d15 to d24, or a combination of prenatal and postnatal exposure (e14.5–d24), to testosterone propionate (TP). Data are shown for vehicle-treated males (controls) and females for comparison. Solid horizontal line shows the mean penile length for control males at d25 and the dashed horizontal line shows the mean for d90 male controls. *p< 0.05, **p< 0.01, ***p< 0.001, in comparison with the respective control group. Values are means ± SEM for the number of animals shown in Table 1. (b) Representative penile cross-sectional morphology in the control rats at d25 (left) and d90 (right) in comparison to a d25 rat treated postnatally from d1 to d24 with TP (middle). In the latter, note the increased penile diameter, preputial separation (small arrows) and ossification of the os penile bone (*blue-green staining) in comparison with the d25 control, and its advancement towards the adult phenotype. Sections were stained with Goldners as described elsewhere (Welsh et al., 2008). Scale bar shows 0.5 mm. U, urethra.

Figure 4

Clitoral length (top panels) and weight/length (lower panels) at mid-puberty (d25) or in adulthood (d90) in female rats exposed in utero (e14.5–e21.5) or postnatally from d1 to d24 or from d15 to d24, or a combination of prenatal and postnatal exposure (e14.5–d24), to testosterone propionate (TP). Data are shown for vehicle (controls)-treated males and females for comparison. Solid horizontal line shows the mean clitoral length for control females at d25 and the dashed horizontal line shows the mean for d90 female controls. *p< 0.05, ***p< 0.001, in comparison with the respective control group. Values are means ± SEM for the number of animals shown in Table 1.