SUMMARY

The metabolism of [4-¹⁴C]4-androstene-3,17-dione was studied in the 105000 g microsomal and supernatant fractions of liver from developing rats of both sexes. The following enzyme activities were measured: 5β-reductase (supernatant fraction) and 5α-reductase, 17α- and 17β-hydroxysteroid reductases, 6β-, 7α- and 16α-hydroxylases (microsomal fraction). The activities of the 3α- and 3β-hydroxysteroid reductases were estimated by calculating the ratios of 3α-:5α- and 3β-: 5α-reduced metabolites formed, respectively.

Most enzyme activities present at birth (i.e. 5β-reductase, 5α-reductase, 17β-hydroxysteroid reductase, 6β- and 7α-hydroxylase) increased until 20 days of age in both male and female rats. Between 20 and 30 days of age a number of masculine metabolic characteristics appeared in both sexes, i.e. the 16α-hydroxylase and the 17α-hydroxysteroid reductase were induced, the 5β-reductase activity rapidly increased and the 5α-reductase activity slightly decreased. During a third period beginning 30 days after birth the adult male enzyme activity pattern was completed by the induction of 3β-hydroxysteroid reductase and a further increase in the activity of 16α-hydroxylase. After 30 days of age a feminine type of liver metabolism also rapidly developed in female rats; the 16α-hydroxylase and the 17α-hydroxysteroid reductase activities disappeared, the 6β-hydroxylase and the 5β-reductase activities decreased and the 5α-reductase activity increased six times.

The developmental patterns of enzyme activities in the rat liver are consistent with a first developmental phase (0–30 days of age) independent of hypophysial control and probably determined primarily by the genome of the liver cell and a second phase (from 30 days onwards) with increasing sexual differentiation under hypophysial control. This control is mediated by some kind of feminizing factor in female rats and possibly by some kind of androgen-elicited secretion of masculinizing factor(s) in male rats.

The metabolism of [4-¹⁴C]4-androstene-3,17-dione was also studied during different times of the day and during different phases of the oestrous cycle. The 16α-hydroxylase activity showed a diurnal variation with higher values at noon than at midnight. The 5β-reductase activity reached a maximal activity during metoestrus.

SUMMARY

The metabolism of [4-¹⁴C]4-androstene-3,17-dione and [4-¹⁴C]5α-androstane-3α,17β-diol were studied in the microsomal fraction and the metabolism of [4-¹⁴C]4-androstene-3,17-dione was studied in the 105000 g supernatant fraction of liver from adult male rats castrated at birth or at 14 days of age. Some of these rats were adrenalectomized 6 weeks after castration and given dexamethasone substitution for 14 consecutive days and some were not adrenalectomized and were treated with adrenocorticotrophin for 14 days. Untreated, castrated control rats were also investigated. Adrenalectomy combined with dexamethasone substitution was found to abolish the masculine, imprinted character of the activities of 16α-hydroxylase, 17α- and 17β-hydroxysteroid reductases and 5β-reductase active on 4-androstene-3,17-dione and 2α- and 2β-hydroxylases active on 5α-androstane-3α,17β-diol in liver from male rats castrated at 14 days of age. The type of androgenic regulation characterizing these enzymes was called 'less stable' imprinting. In contrast to these findings, the activities of 5α-reductase and 3β-hydroxysteroid reductase retained their masculine character in male rats castrated 14 days after birth even after adrenalectomy combined with glucocorticoid substitution. The type of programming regulating these enzyme activities was called 'more stable' imprinting.

'Less stable' imprinting could be explained by the increased androgen responsiveness of the neonatally androgenized liver which thus responds more promptly to the enzyme-inducing or suppressing effects of adrenal androgens. Adrenalectomy combined with dexamethasone substitution results in elimination of these effectors and consequently loss of the masculine character of the enzyme activities regulated by 'less stable' imprinting. The activity of 5β-reductase, however, seems to be regulated by unknown central factors. 'More stable' imprinting may be explained by a specific, autonomous, irreversible enzyme induction in liver independent of postpubertal hormonal stimuli.

Corticotrophin treatment generally led to similar but less significant effects upon the hepatic enzyme activities than adrenalectomy combined with dexamethasone substitution. It is speculated that these effects may be attributable to increased glucocorticoid levels in blood possibly through secondary effects on central control mechanism(s) regulating hepatic enzyme activities.

The reduction of 4-[1,2-³H]androstene-3,17-dione (androstenedione) in vitro by scrotal skin was measured in samples from nine men (16–34 years old) with hypospadias and from ten male control subjects. The reduction of androstenedione was also studied in axillary and upper arm skin of seven control subjects. Androstenedione was reduced to material with chromatographic characteristics of 5α-androstane-3,17-dione and to 3α- and 3β-hydroxy-5α-androstan-17-one.

No difference in 5α-reductase activity (defined as the sum of these three metabolites formed) was found in scrotal skin from hypospadic and control men.

The mean concentration of 5α-dihydrotestosterone in serum from men with hypospadias was lower than that in serum from control subjects (P< 0·01). The mean ratio of the serum concentrations of testosterone and 5α-dihydrotestosterone was higher in hypospadic men than in control subjects (P< 0·05). No differences between the two groups were found in the mean serum concentrations of LH, FSH, prolactin, dehydroepiandrosterone, androstenedione, testosterone or testosterone-binding globulin.

The metabolism of 4-[1,2-³H]androstene-3,17-dione in the prepuce, axillary skin and skin from the arm was investigated in 27 boys operated for phimosis (controls) and 13 unselected boys with hypospadias (a congenital defect of the male urethra). In all types of skin investigated, androstenedione was metabolized to 5α-androstane-3,17-dione, 3α-hydroxy-5α-androstan-17-one, 3β-hydroxy-5α-androstan-17-one and testosterone. Conversion to testosterone was found in the prepuce of two out of 11 boys with hypospadias.

Mild forms of hypospadias in the age group 1–4 years had a higher level of 5α-reductase activity in the prepuce than controls in the same age group (P < 0·05); no such differences were found in the few severe cases of hypospadias in this group. No other differences in 5α-reductase activity were found between hypospadic boys and controls. The ratio of 5α-reductase activity in the prepuce: 5α-reductase activity in skin from the arm was significantly higher (P<0·05) in hypospadic boys than in controls in the age group 1–4 years.

Serum levels of LH and FSH were the same in normal and hypospadic boys but the concentration of prolactin in the serum was lower in boys with hypospadias compared with control subjects in the age group 1–4 years (P<0·005). No differences were found in serum concentrations of androstenedione, testosterone, oestradiol and testosterone-binding globulin.

SUMMARY

The metabolism of [4-¹⁴C]4-androstene-3, 17-dione, [4-¹⁴C]5α-androstane-3α, 17β-diol and 1,2-³H]5α-androstane-3α, 17β-diol 3,17-disulphate was studied using the microsomal fraction and the metabolism of [4-¹⁴C]4-androstene-3, 17-dione was studied using the 105 000 g supernatant fraction of liver from male and female rats aged 5 months that had been treated with cyproterone acetate before (from day 13 of pregnancy) and after birth (until 3 weeks of age). Nearly all sex-dependent enzyme activities in the treated male rats were changed in a direction characteristic of female rats: 5α-reductase active on 4-androstene-3, 17-dione increased in activity whereas 3β- and 17α-hydroxysteroid reductases and 6β- and 16α-hydroxylases active on 4-androstene-3, 17-dione and 2α-, 2β- and 18-hydroxylases active on 5α-androstane-3α, 17β-diol decreased in activity. Enzyme activities not under gonadal control, i.e. 3α- and 17β-hydroxysteroid reductases active on 4-androstene-3, 17-dione and 7α-hydroxylase active on both 4-androstene-3, 17-dione and 5α-androstane-3α, 17β-diol, were not affected by cyproterone acetate. The liver enzyme activities in treated female rats were generally not affected although significant effects were noted in two cases; in one of these (17α-hydroxysteroid reductase) a testosterone-like effect was observed.

The results obtained are probably best explained in the following way: treatment with the anti-androgen during the neonatal period results in less efficient imprinting of the hypothalamo-hypophysial system leading to less pronounced masculine setting of sex-dependent enzyme levels and also to a relative androgen unresponsiveness. It is suggested that the biochemical methods used in the present investigation may be used for more exact estimation of the degree of neonatal sexual differentiation of the hypothalamo-hypophysial system than biological and psychological methods previously available.

SUMMARY

The concentrations of LH and FSH were measured by radioimmunoassay in sera from immature male and female rats of various ages. Fairly high levels of FSH were found in both sexes at birth but LH was not detected. FSH peaks appeared in the male at 13 and 19 days of age and in the female at 13 and 17–19 days of age. LH was undetectable in the male before 12 days of age, rose to a peak (440 ± 60 (s.d.) ng/ml) at 13 days of age and fell below the detection level again between 15 and 25 days of age. A further increase then occurred which almost reached adult levels. LH was first detectable in the female rat at 11 days of age with a peak value of 130 ± 35 ng/ml at 12 days. The hormone was undetectable on days 14 and 15, rose to a second peak on day 18 (148 ± 56 ng/ml), and was again absent between 19 and 25 days of age. The concentration rose, as in the male, between days 25 and 28 to a level similar to that of the adult. The results show sexual differences in prepubertal gonadotrophin surges.

The LH peak at 12–13 days in both sexes appears to be light-dependent. The FSH peak at this time was affected by light but was not strictly light-dependent.

There are well known differences between males and females in hearing. In the present study, the role of estrogen receptor-β (ER-β; listed as ESR2 in the MGI Database) in hearing was investigated by comparing hearing and morphology of the inner ear in ER-β knock-out mice (ER-β^−/−) with that of wild-type (WT) littermates. Hearing was analyzed with auditory brainstem response audiometry at 3 and 12 months. The ER-β^−/− mice were deaf at 1 year of age, and the morphological analysis showed absence of hair cells and loss of the whole organ of Corti initiated in the basal turn of the cochlea. Furthermore, in ER-β^−/−, but not in WT mice, the spiral ganglion was lacking many of its neurons. Immunostaining showed the presence of both ER-α (listed as ESR1 in the MGI Database) and ER-β in the nuclei of some neurons in the inner ear in WT mice, but no ER-β was found in the ER-β^−/− mice as expected. ER-α staining was predominant in the nuclei of large neurons and ER-β in nuclei of small neurons and fibroblasts. These results reveal that both ERs are present in the inner ear at specific localizations suggesting subtype-specific functions. It is concluded that ER-β is important for the prevention of age-related hearing loss. These findings strengthen the hypothesis that estrogen has a direct effect on hearing functions.