Feminization imprinted by developmental growth hormone

Abstract

Previously, we identified early developmental exposure to growth hormone (GH) as the requisite organizer responsible for programming the masculinization of the hepatic cytochromes P450 (CYP)-dependent drug metabolizing enzymes (Das et al., 2014, 2017). In spite of the generally held dogma that mammalian feminization requires no hormonal imprinting, numerous reports that the sex-dependent regulation and expression of hepatic CYPs in females are permanent and irreversible would suggest otherwise. Consequently, we selectively blocked GH secretion in a cohort of newborn female rats, some of whom received concurrent GH replacement or GH releasing factor. As adults, the feminine circulating GH profile was restored in the treated animals. Two categories of CYPs were measured. The principal and basically female specific CYP2C12 and CYP2C7; both completely and solely dependent on the adult feminine continuous GH profile for expression, and the female predominant CYP2C6 and CYP2E1 whose expression is maximum in the absence of plasma GH, suppressed by the feminine GH profile but more so by the masculine episodic GH profile. Our findings indicate that early developmental exposure to GH imprints the inchoate CYP2C12 and CYP2C7 in the differentiating liver to be solely dependent on the feminine GH profile for expression in the adult female. In contrast, adult expression of CYP2C6 and CYP2E1 in the female rat appears to require no GH imprinting.

Keywords: Cytochromes P450; Development; Feminization; Growth hormone; Imprinting; Sexual dimorphism.

Figure 1.

Plasma GH of female rat pups treated solely or with a combination of MSG, GH, GRF or diluents. Commencing on the day of birth (D. 1), female neonates were injected, s.c., with either MSG, GH, MSG + GH, GRF, MSG + GRF or diluents (CONTROL) for a treatment period and at a dose described under Materials and Methods. Results are presented as the mean ± SD of at least 6 pups/group. * P < 0.01 vs CONTROL; ^† P < 0.01 vs. MSG treatment alone.

Figure 2.

Circulating GH levels obtained from individual, undisturbed catheterized adult (~30 weeks of age) female rats neontally treated with either MSG, GH, MSG + GH, GRF, MSG + GRF or diluents (CONTROL) for 8 continuous hours at 15 minute intervals. Similar results were obtained from 4 to 5 additional animals in each treatment group. See Materials and Methods for details of neonatal treatments.

Figure 3.

Neonatal GH effects on maturational body weight gains (top) and Lee indices (bottom). Results are presented as the means ± SD of at least 15 rats/group. See Materials and Methods for details of neonatal treatments. Body weights: *P < 0.01 vs. CONTROL; ^† P < 0.01 vs. MSG treatment alone. Lee Indices: see Results for statistical significances.

Figure 4.

Plasma levels of circulating GH in adult (~30 weeks of age) female rats neonatally treated with MSG, MSG + GH, MSG + GRF or diluents (CONTROL) implanted with either rat GH or diluent containing osmotic mini pumps. Plasma levels of circulating GH were obtained from individual, undisturbed catheterized adult females for 7 continuous hours at 15 min intervals. Peritoneally implanted osmotic mini pumps were set to continuously deliver 15 μg of rat GH/kg bd wt / hr or just GH diluent in CONTROL rats. Similar results were obtained from 3 to 4 additional animals in each treatment group. See Materials and Methods for all treatment details. (Note different scales on the y-axes.)

Figure 5.

Neonatal GH effects on adult hepatic expression of female specific CYP2C12 (top) and female-predominant CYP2C7 (bottom). Hepatic CYP isoform levels (mRNA and protein) were determined in adult female rats neonatally treated solely or with a combination of vehicles (e.g. CONTROL), MSG, GH or GRF (upper case labels on x-axis). Half of the animals in groups neonatally exposed to MSG (i.e., MSG, MSG + GH and MSG + GRF) were implanted with rat GH releasing osmotic mini pumps to restore the continuous feminine GH profile (Fig. 4). All other animals received GH-vehicle releasing osmotic mini pumps (lower case labels on x-axis). Results are presented as a percentage of mRNA or protein in CONTROL rats arbitrarily designated as 100%. Values are means ± SD of at least 6 rats / data point. *P < 0.05, **P < 0.01 vs. CONTROL; ^† P < 0.05, ^††P < 0.01 compares adult vehicle treatment of neonatal MSG + GH and MSG + GRF with vehicle treated MSG alone; ^##P < 0.01 compares adult GH treatment with vehicle treatment of rats exposed to the same neonatal treatment. See Materials and Methods for all treatment details.

Figure 6.

Neonatal GH effects on adult hepatic expression of female-predominant CYP2C6 (top) and CYP2E1 (bottom). Hepatic CYP isoform levels (mRNA and protein) were determined in adult female rats neonatally treated solely or with a combination of vehicles (e.g. CONTROL), MSG, GH or GRF (upper case labels on x-axis). Half of the animals in groups neonatally exposed to MSG (i.e., MSG, MSG + GH and MSG + GRF) were implanted with rat GH releasing osmotic mini pumps to restore the continuous feminine GH profile (Fig. 4). All other animals received GH-vehicle releasing mini pumps (lower case labels on x-axis). Results are present as a percentage of mRNA or protein in CONTROL rats arbitrarily designated as 100%. Values are means ± SD of at least 6 rats / data point. *P < 0.05, **P < 0.01 vs. CONTROL; ^#P < 0.05, ^##P < 0.01 compares adult GH treatment with vehicle treatment of rats exposed to the same neonatal treatment. See Materials and Methods for all treatment details.

Figure 7.

Neonatal GH effects on adult hepatic expression of male-specific CYP2C11 (top) and sex-independent albumin (bottom). Hepatic mRNA levels of CYP2C11 and protein levels of albumin were determined in adult female rats neonatally treated with various regimens of vehicles (e.g. CONTROL), MSG, GH or GRF (upper case labels on x-axis). Half of the animals in groups neonatally exposed to MSG were implanted with rat GH releasing osmotic mini pumps to restore the continuous feminine GH profile (Fig. 4). All other animals received GH-vehicle releasing mini pumps (lower case labels on x-axis). Results are presented as a percentage of mRNA or protein in CONTROL rats artbitrarily designated as 100%. Values are means ± SD of at least 6 rats / data point. *P < 0.01 vs. CONTROL; ^#P < 0.01 compares adult GH treatment with vehicle treatment of rats exposed to the same neonatal treatment. See Materials and Methods for all treatment details. (CYP2C11 mRNA was not determined in GRF-alone and MSG + GRF treated rats.)

Figure 8.

Perinatal GH imprinting of sex-dependent differences in adult hepatic CYP isoforms. In half of the newborns, pituitary GH secretion is blocked by MSG administration. The remaining pups are untreated (i.e., MSG-vehicle) allowing GH imprinting of the developing liver (depicted as darker and surrounded by rays). Expression of the principle CYP isoforms, female- specific CYP2C12 and CYP2C7 as well as male-specific CYP2C11 are solely regulated by the adult sex-dependent GH profiles, and consequently are only expressed during adulthood. Accordingly, these hepatic drug metabolizing enzymes are first measured in adult rats, and for experimental purposes, devoid of pituitary GH secretion by either hypophysectomy of the GH- imprinted rats or by the permanent inhibition of somatotroph section by the perinatally administered MSG. Whereas the GH-imprinted females are incapable of expressing CYP2C12 and CYP2C7 in the absence of adult GH, considerable GH-dependent expression levels of these isoforms are observed in neonatally MSG-treated adult females. Expression of hepatic CYP2C11 in adult males, either GH-imprinted, or not, is totally suppressed in the absence of adult GH secretion. Restoration of the circulating sex-dependent GH profiles discloses an inherent sexual dimorphism. Renaturalization of the feminine continuous GH profile restores normal-like (100%) expression levels of CYP2C12 and CYP2C7 to all females; GH imprinted and imprint-blocked. In contrast, replacement of the masculine episodic GH profile restores normal-like (100%) levels of CYP2C11 to the GH imprinted males, but is completely ineffective inducing the enzyme in males not imprinted by GH. The results demonstrate that the ability of the adult male to express the principle male-specific CYP2C11 isoform is totally dependent on perinatal GH imprinting without which the isoform is permanently incapable of being expressed. In the case of the female, perinatal GH imprints the developing liver so that in adulthood, the expression of the major female isoforms, CYP2C12 and CYP2C7, are now solely dependent upon the continuous feminine GH profile. In the absence of GH imprinting, the female- dependent isoforms are unrestrictively inducible by the feminine as well as masculine GH profile and even in the absence of GH. Thus, perinatal GH imprinting assures that the expression of female CYPs are solely dependent on the feminine continuous GH profile, while expression of the male isoform is dependent on the masculine episodic GH profile.