Prenatal exposure to phenobarbital permanently decreases testosterone and causes reproductive dysfunction.

Exposure of rats to phenobarbital during late prenatal development decreased the concentration of testosterone in plasma and the brain during the late fetal, early postnatal, pubertal, and adult periods, By decreasing the production of testosterone in the brain during the period of sexual differentiation, phenobarbital may lead to sexual dysfunction in later life.

The testicular feminized rat: a naturally occurring model of androgen independent brain masculinization.

Although genotypically male (XY), the testicular feminized rat develops as an anatomic female because of an inherited deficiency in intracellular androgen receptors that prevents androgen imprinting of sexual primordia. However, the ability of testicular feminized rats to exhibit male-like sexual behavior and little feminine sexual behavior suggests that the brain can be masculinized without androgens.

Phenobarbital exposure in utero: alterations in female reproductive function in rats.

Phenobarbital administration to pregnant rats from day 12 to day 19 of gestation suppressed body weight gain and produced significant effects on reproductive function in their offspring. These effects included delays in the onset of puberty, disorders in the estrous cycle, and infertility. Moreover, the animals exposed to phenobarbital in utero showed altered concentrations of sex steroids, gonadotrophic hormones, and estrogen receptors. These findings suggest that phenobarbital treatment during prenatal development can produce permanent alterations in sexual maturation.

Gender differences in drug metabolism regulated by growth hormone.

Gender differences in drug metabolism in rats have been known for more than 60 years when it was first reported that the much shorter duration of drug action in the male was due to the effects of testicular androgens. More recent studies have demonstrated that this sexual dimorphism in rat drug metabolism results from the differential expression of a possible dozen, or so sex-dependent hepatic forms of cytochrome P450. Moreover, it is the sexually dimorphic plasma profiles of growth hormone, and not androgens, that directly regulate the expression of these individual forms of hepatic cytochrome P450. Male rats secrete growth hormone in an "on-off" episodic rhythm in which interpulse periods contain no detectable levels of the hormone. Growth hormone secretion in the female rat is also pulsatile, but can be characterized as "continuous" since hormone levels are always present in the circulation. It would appear that the duration of the interpulse period is at least one "signal" in the growth hormone profile regulating hepatic expression of the sex-dependent forms of cytochrome P450, and thus establishing the gender differences in drug metabolism. The exaggerated gender differences in rat drug metabolism (i.e. 300-500%) have made it the standard, and understandably an ideal model in which to investigate the mechanisms regulating these dimorphisms. However, it is also possible that these studies have limited value when extrapolated to other species, such as humans, in which the magnitude of the sexual differences are much smaller, and the dimorphism may be reversed (F > M). In this regard, the mouse model, in which the sexual differences (F > M) in drug metabolizing enzyme activities vary by only 40-100%, are also regulated by sex-dependent plasma growth hormone profiles, and may be more representative of the vast majority of outbred species in which only subtle gender differences occur in drug metabolism.

Gender differences in the responsiveness of the sex-dependent isoforms of hepatic P450 to the feminine plasma growth hormone profile.

Most of the constitutive hepatic P450 isoforms expressed in the rat exhibit dramatic gender differences. Whereas only male hepatocytes contain CYP2A2, 2C11, and 3A2, only female hepatocytes express CYP2C12 and 3- to 4-fold greater levels of CYP2C7. This sexually dimorphic expression of hepatic P450 isoforms is regulated by the gender-dependent secretory GH profiles, i.e. episodic in males and continuous in females. In the case of the feminine GH profile, the continuous presence of the hormone in the circulation completely suppresses male-specific CYP2A2, 2C11, and 3A2, while stimulating full expression of female-dependent CYP2A1, 2C7, 2C12, and non-P450 testosterone 5alpha-reductase (type 1). The gender-dependent expression of the P450s can be reversed by exposing male rats to the continuous feminine plasma GH profile and females to the episodic masculine GH profile. Under these conditions, females will now express the male-specific isoforms and suppress the female-dependent forms, whereas the opposite will occur in the males. Nevertheless, it is not clear whether the levels of expression or suppression are comparable in male and female rats exposed to the same sex-dependent GH profiles. In the present study, we have renaturalized the circulating feminine GH profile in euthyroid-maintained, hypophysectomized female and male rats at six concentrations ranging from 3-100% of normal. Continuous monitoring of GH levels revealed indistinguishable plasma profiles in females and males at each dosage administered. In the case of females, restoration of the feminine-like plasma GH profile at a concentration that was 3% of the normal level restored expression levels (i.e. mRNA, protein, and/or catalytic activity) of female-dependent CYP2C12, 2A1, and 5alpha-reductase to 50% or greater of normal and fully suppressed expression of male-specific CYP2A2, 2C11, and 3A2. Twice the dosage of the hormone (6% of normal) was required to restore female-predominant CYP2C7 to 50% of normal in hypophysectomized female rats. In contrast, we found that all of the measured isoforms were significantly less responsive to the inductive and suppressive effects of the feminine-like GH profile when administered to male rats. While suppression of the male-specific isoforms (i.e. CYP2A2, 2C11, and 3A2) in male rats required concentrations of GH in the feminine profile 2-3 times greater than were effective in female rats, no dosage of the hormone was as effective in inducing female-dependent P450s (i.e. CYP2A1, 2C7, and 2C12) in males as in females. Clearly, the continuous feminine GH profile was more effective at inducing and suppressing gender-dependent isoforms of hepatic P450 when restored to female rats, where it is normally secreted, than in males. As GH profiles appear to be the sole factor responsible for regulating the sexually dimorphic expression of hepatic P450 isoforms in adult rats, the differential responsiveness of male and female rats to the feminine GH profile are likely to be inherently induced by irreversible imprinting during a critical developmental period.

Nominal growth hormone pulses in otherwise normal masculine plasma profiles induce intron retention of overexpressed hepatic CYP2C11 with associated nuclear splicing deficiency.

Restoration of circulating masculine GH profiles at minipulse amplitudes (i.e. approximately 10% of normal) to hypophysectomized male rats and neonatal administration of monosodium glutamate (MSG), producing a similar plasma GH profile, both result in an overexpression (approximately 200-300%) of CYP2C11 messenger RNA (mRNA), the predominant hepatic cytochrome P450 (CYP) drug-metabolizing enzyme in adult male rats. Coincident with the severalfold elevation in transcript level is a modest 10-30% overexpression of CYP2C11 protein and its catalytic activities. Using hepatic tissue from adult, neonatally MSG-treated rats, we have cloned a variant species of CYP2C11 mRNA containing all of the essential elements of a full-length complementary DNA, including initiating codon, termination codon, and polyadenylase tail. In addition, the transcript contains a 742-bp intervening sequence (identical to the complete terminal intron) between the last and penultimate exons, and an intron-specific oligo probe for Northern blotting demonstrates the presence of the variant transcript in liver of MSG-treated rats. Associated with the overexpression and intron retention of the transcript is a 50% reduction in the nuclear splicing capacity of the liver for model precursor CYP2C11 mRNA. It is proposed that this splicing defect may be a consequence of the mini-GH pulses (secreted in otherwise normal masculine plasma profiles) signaling abnormal processing of precursor CYP2C11 mRNA to produce a substantial portion of intron retained, nontranslatable transcript.

Testicular maintenance and masculinized development of the vagina of the androgen-insensitive male rat pseudohermaphrodite.

The vaginae of about one-third of androgen-insensitive male rat pseudohermaphrodites never open nor can they be opened by the administration of estrogen, progesterone or andorgens. The vaginae of the remaining pseudohermaphrodites open significantly later than normal females, and, once open, require the presence of the testes or exogenous estrogen to maintain their patency. These defects in the vaginal development of the pseudohermaphrodite may be due to some perinatal masculinization of the perineum, unlike most of the phenotypically female development exhibited by this animal.

Masculine-liky hypothalamic-pituitary axis in the androgen-insensitive genetically male rat pseudohermaphrodite.

The hypothalamic-pituitary sex of the androgen insensitive, genetically male rat pseudohermaphrodite was studied by examining its vaginal cytology, response to ovarian transplants and urinary steroidal excretion patterns. More than half the pseudohermaphrodites studied were in constant vaginal estrus, while the remaining rats displayed either persistent diestrus or irregular cyclicity tending towards lengthened estrus. Following gonadectomy and ovarian transplantation, normal females displayed regular 14-day cycles while pseudohermaphrodites remained in constant vaginal estrus. In pseudohermaphrodites with ovarian transplants, only C19 steroids were detected in the urine while females excreted both C21 and C19 steroids. Indicative of the urinary findings, transplants in females had corpora lutea and maturing follicles while grafts from pseudohermaphrodites and males contained follicular cysts and luteinized theca. In addition, distribution and activity of histochemical 3beta-hydroxy-delta5 steroid oxidoreductase were similar in the grafts from pseudohermaphrodites and males, but unlike the females. Although previous reports have shown that much of the sex-dependent differentiation of the genetic male rat pseudohermaphrodite is phenotypically female, our results suggest that the phenotype of the hypothalamic-pituitary axis of this animal is, at least in certain respects, male.

Depletion of serum growth hormone in adult female rats by neonatal monosodium glutamate treatment without loss of female-specific hepatic enzymes P450 2d (IIC12) and steroid 5 alpha-reductase.

The sexually dimorphic profiles of pituitary GH secretion play a key role in regulating the expression of several sex-dependent and developmentally controlled P-450 enzymes in rat liver. Current models for P-450 regulation by GH, however, are primarily based on hypophysectomy and GH replacement experiments. The present study examines the effects on hepatic P-450 expression of neonatal injections of monosodium glutamate (MSG), which allows for the nonsurgical suppression of adult GH levels. Furthermore, the levels of other pituitary-dependent hormones, such as testosterone and estradiol, are largely unchanged in the MSG-treated rats. Although hypophysectomy and GH replacement experiments have previously demonstrated that expression of the female-specific hepatic enzymes P-450 2d (gene product IIC12) and steroid 5 alpha-reductase is strikingly dependent on continuous GH exposure, neither enzyme was decreased in adult female rat liver after the elimination of plasma GH (less than 2 ng/ml) by neonatal MSG treatment. Moreover, although the loss of circulating GH appears to be largely responsible for the more than 10- to 20-fold elevation of the male-specific hepatic P-450 forms 2a (gene product IIIA2) and RLM2 (gene product IIA2) in hypophysectomized female rats, no such elevation of the male-specific P-450s was observed in the GH-deficient MSG-treated female rats. In contrast, the female-predominant forms P-450j (gene product IIE1) and 3 (gene product IIA1) were both elevated in adult rat liver after neonatal MSG treatment, in agreement with earlier hypophysectomy studies and demonstrating the suppressive effects that GH can exert on expression of these P-450 forms. Thus, although MSG and hypophysectomy both produce GH depletion, the responsiveness of the hepatic P-450s to these endocrine manipulations differs, allowing for an expanded understanding of the role of GH in P-450 expression.

Signalling elements in the ultradian rhythm of circulating growth hormone regulating expression of sex-dependent forms of hepatic cytochrome P450.

Neonatal male rats were treated with monosodium glutamate (MSG) at either 2 or 4 mg/g BW on alternate days during the first 10 days of life. As adults, the 4 mg MSG-treated rats displayed the obesity, growth retardation, and reduced pituitary weights that typify this syndrome. These animals had no detectable plasma GH as determined from serial blood samples taken every 20 min for 8 consecutive h. Associated with this loss of circulating GH was an induction of the female-specific hepatic cytochrome P450 2d (gene IIC12) and the disappearance of the male-specific form of cytochrome P450 2c (gene IIC11). The catalytic activities of cytochrome P450 2c (i.e. androgen 16 alpha- and 2 alpha-hydroxylase), sex-dependent hexobarbital hydroxylase and total cytochrome P450 were similarly feminized. Rats exposed to the 2-mg dose of MSG were also obese, but their growth rates and pituitary sizes were not as severely affected as in the 4 mg MSG-treated rats. Circulating GH in these lower dosed males was secreted in a pulsatile pattern similar to that in normal males, except that the pulse amplitude was reduced as much as 90%. In spite of this profound decline in GH peak heights in the 2 mg MSG-treated males, liver metabolism was characteristically masculine. That is, female-specific cytochrome P450 2d remained undetectable, while the male forms of cytochrome P450 2c, 2a (gene IIIA2), and RLM2 (gene IIA2), their respective catalytic steroid hydroxylase activities, and associated sex-dependent drug-metabolizing enzymes were expressed at the level of or greater than that in normal males. Thus, while an ultradian pulse of circulating GH is necessary for the characteristically masculine profile of sex-specific forms of hepatic cytochrome P450, neither the amplitude of the secretory peaks nor their total GH content is critical, and these can be greatly reduced from the normal male levels.

Aberration of heme and hemoprotein in aged female rats.

The magnitude and duration of drug action is determined partially by the activity of the drug metabolizing enzyme systems in the liver. The pharmacological effectiveness of many drugs is altered during the aging process. In this study, the regulation of heme metabolism and hemoprotein content was examined in livers of aged female rats. The activities of hexobarbital hydroxylase and aniline hydroxylase, indicators of mono-oxygenase function, were decreased in aged rats by 31% and 24%, respectively, as compared to values in young rats. This was accompanied by a proportional decrease in the level of cytochrome P-450 (26%). Additionally, the activity of delta-aminolevulinic acid synthetase (ALA-S), the rate-limiting enzyme in heme synthesis, and the microsomal concentration of heme were also decreased by 33% and 26%, respectively, in these animals. In contrast, the basal activity of microsomal heme oxygenase (MHO), the rate-limiting enzyme in heme degradation, and the percent heme saturation of tryptophan pyrrolase (TPO), a sensitive indicator of changes in the availability of heme in the "regulatory" heme pool, were increased by (87%) and (31%), respectively, in the aged rats. The serum concentration of bilirubin, an indicator of erythrocyte breakdown and/or liver function was likewise increased in these animals. In view of these findings, we suggest that the high activity of MHO and the low level of ALA-S may be a significant causative factor for the decreased microsomal concentration of heme, cytochrome-P-450 and its dependent monooxygenase activities in senescent female rats.

Androgenic repression of hexobarbitone metabolism and action in Crl:CD-1 (ICR)BR mice.

Mice of the Crl:CD-1 (ICR)BR strain exhibit a sexual dimorphism in hexobarbitone metabolism and action. Compared to females, males have a lower Vmax and a higher Km for hepatic microsomal hexobarbitone hydroxylase. In agreement with the enzyme studies, hexobarbitone-induced sleeping times were greater for males than for females. Results from experiments measuring hexobarbitone metabolism and action in castrate, testosterone and gonadotropin-treated mice indicate that the sexual differences in drug metabolism and action found in Crl:CD-1 (ICR)BR mice are due to the normally repressive effects of testicular androgens on the activities of the hepatic mono-oxygenases. These findings are in dramatic contrast to studies with rats where it has been shown that androgens induce mono-oxygenases. Furthermore, in the case of the mouse, changes in the activity of hexobarbitone hydroxylase in response to alterations in androgen levels require weeks, while in the rat, androgenic-induced changes are apparent within a matter of days.

Sexual dimorphism and the effects of the X-linked Tfm locus on hexobarbitone metabolism and action in mice.

1 Normal males of the testicular feminized strain of mice (Tfm) had longer hexobarbitone-induced sleeping times than females, and hepatic hexobarbitone hydroxylase activity different in that the Km was higher and the Vmax lower in the male. 2 Castration and androgen replacement studies indicated that testicular androgens were responsible for the sexual differences in drug metabolism found in this mouse strain. 3 Hepatic hexobarbitone metabolism and action were feminized in the intact, androgen-insensitive, genetically male Tfm mouse. Furthermore, hexobarbitone hydroxylase activities were less responsive to large doses of testosterone in Tfm mice than in normal males. 4 The Tfm mouse with a deficiency in androgen receptors responded to the enzyme-inductive effects of phenobarbitone and softwood bedding, indicating that these inducers do not act through the androgen receptors.

Sex differences in the ultradian pattern of plasma growth hormone concentrations in mice.

Ultradian patterns of plasma GH concentration were determined in adult male and female mice. Serial blood samples were collected every 15 min over 8 h through surgically placed chronic indwelling right atrial catheters and assayed for GH content by an homologous radioimmunoassay. In both sexes, GH concentrations fluctuated episodically from baseline values that were often in the range of 2 micrograms/l which approached the limit of assay sensitivity, to peak values sometimes reaching 100 micrograms/l. Male mice, however, demonstrated a regular periodicity of GH peaks approximately every 2.5 h with interposed stable baseline concentrations that were significantly longer in duration than in females. The absence of extended baseline concentrations in females reduced cycle length to an average of 1.4 h and significantly increased the overall mean GH concentration. The duration, height and area of GH peaks and the average concentration between peaks were comparable in males and females. Sexual dimorphism in the ultradian patterns of serum GH concentrations have been shown in both mice and rats to regulate the expression of several sex-specific phenotypes. Comparing endogenous GH patterns in mice and rats demonstrates that males of both species have less frequent peaks than females over the same interval of time. This separation of GH peaks appears to be an essential element for the expression of masculine GH-dependent traits.

Pituitary hormone secretion in the genectically male rat pseudohermaphrodite.

Pituitary content or concentration of follicle-stimulating hormone (FSH), prolactin and growth hormone in the genetically androgen insensitive male rat pseudohermaphrodite is intermediate between normal male and female rats, while pituitary luteinizing hormone (LH) concentration and serum FSH levels are the same as in the normal male. The concentration of serum LH, prolactin and growth hormone indicated no sexual dimorphism. Although the pseudohermaphrodite is genetically male with a female phenotype, our results suggest some degree of masculinization of the hypothalamic-pituitary system.

Effects of new multi-site hormone blockers on the fertility of male rats.

The effects on the fertility of adult male rats of six new synthetic steroids: I, 3-cyano-5alpha-androst-1-en-17-one; II, the 17beta-acetate form of I; III, 17beta-hydroxy-5beta-cyano-androstan-3-one; IV, 6-methylpregnenolone; V, 17beta-hydroxy-17alpha-ethynyl-5beta-cyano-19-norandrostan-3-one; and VI, 19-norspiroxenone (oestr-4-en-3-one-spiro-17alpha-2'-[tetrahydrofuran]) have been tested. After 6 weeks of treatment with daily doses of 5 mg (I, II, III), 15 mg (IV) or 10 mg (V, VI) only steroid VI blocked the completion of spermatogenesis and reduced the number of foetuses sired in at least five females/male. Steroid VI also diminished seminal vesicular, prostatic, testicular and epididymal weights. It inhibited the testicular enzymes, 3beta-hydroxysteroid dehydrogenase-delta4-5-3-oxosteroid isomerase system, 17alpha-hydroxylase, and C17-20 lyase markedly, but did not affect the adrenal dehydrogenase-isomerase system. It depressed, strikingly, testicular and serum levels of testosterone and 5alpha-dihydrotestosterone and reduced pituitary and serum levels of FSH and LH. Although marked depression of target organ weights also occurred with steroids II, IV and V, and reduction of androgen levels and LH in the circulation with III, IV and V, only VI was a potent blocker of male fertility with the exception of a slight block of the siring of viable foetuses by steroids IV and V. The major difference in site of action of steroid VI from the others was the depression of pituitary and serum levels of FSH along with a marked diminution of testicular content of both testosterone and 5alpha-dihydrotestosterone. 19-Norspiroxenone in the rat is a potent anti-oestrogen without inherent oestrogenicity and is anti-uterotrophic. Thus, VI may affect male fertility by virtue of its potent anti-oestrogenic action in the hypothalamus or testis.

Selective androgen insensitivity of hepatic drug-metabolizing enzymes in senescent mice.

The normal sexual dimorphism in murine hepatic hexobarbital metabolism (i.e. females greater than males) was found to be absent in senescent animals. Hexobarbital metabolism, expressed as microsomal activity of hexobarbital hydroxylase and hexobarbital-induced sleep time, in senescent male mice was similar to that in females, but significantly greater than that found in young adult males. No age-related changes in hexobarbital metabolism were observed in intact females. In addition, experiments involving gonadectomies and testosterone administration indicated that both male and female senescent mice were insensitive to the normally repressive effects of androgens on hexobarbital hydroxylase. In contrast, the sexual dimorphism in the activity of p-nitrophenol UDP-glucuronosyltransferase was maintained in the senescent mice as well as the usual responsiveness to testosterone regulation. Furthermore, the growth-promoting effects of androgen on the kidneys and seminal vesicles were similarly expressed in young and old mice. Thus, our results suggest the development of an age-dependent and selective insensitivity of hexobarbital hydroxylase to androgenic regulation in the aging mouse.

Subnormal concentrations in the feminine profile of circulating growth hormone enhance expression of female-specific CYP2C12.

When newborn female rats were treated with monosodium glutamate, 4 mg/g body weight, on days 1 and 3 of life, circulating growth hormone concentrations were permanently reduced 75-85% in adulthood, whereas the feminine secretory profile characterized by frequent growth hormone pulses, separated by short-lived, measurable troughs, persisted. Associated with this reduction in growth hormone secretion was a mild obesity and a slight depression in peripubertal body weight. In contrast, expression of growth hormone-dependent, female-specific CYP2C12 was increased by almost 100% when measured at both its protein and mRNA levels. In agreement, this supraphysiological expression of CYP2C12 was reflected at a pharmacologic level by a simultaneous elevation in in vitro and in vivo hexobarbital metabolism. When growth hormone secretion was pulsatile (i.e. masculine) or was eliminated from the circulation (i.e. hypophysectomy), hepatic CYP2C12 protein and mRNA were undetectable. The present findings suggest that the normal levels of plasma growth hormone found in female rats are not necessarily optimum for the expression of female-specific CYP2C12.

Growth hormone regulation of hepatic drug-metabolizing enzymes in the mouse.

Hepatic microsomal hexobarbital hydroxylase and aminopyrine N-demethylase activities increased in adult male mice following hypophysectomy to female-like levels, eliminating the normal sexually dimorphic pattern of these enzymes. Exogenous growth hormone replacement (0.08 I.U./100 g body weight/day) re-established the lower masculine activities only when administered subcutaneously once every 12 hr. Enzyme activities remained elevated at female-like levels when the same total dose of growth hormone was infused continuously using osmotic pumps or was injected once every 6 hr. These data suggest that, despite the reversed orientation of sex differences in hepatic drug-metabolizing enzymes between rats and mice (i.e. higher enzyme activities in female mice and male rats), the basic hormonal regulatory axis is similar in the two species. Cyclic fluctuations of systemic growth hormone concentrations masculinize kinetic parameters of hepatic hexobarbital hydroxylase and aminopyrine N-demethylase in both species. Rats and mice differ in that these similar hormonal signals lower the apparent Vmax in male mice, while markedly increasing the enzyme activities in male rats. It appears more likely, therefore, that species- and sex-specific differences in the total hepatic cytochrome P-450 isoenzyme populations produce the reversed sex-dependent pattern of hexobarbital hydroxylase and aminopyrine N-demethylase.

Effects of neonatally administered monosodium glutamate on the sexually dimorphic profiles of circulating growth hormone regulating murine hepatic monooxygenases.

Neonatal male and female mice were treated with monosodium glutamate (MSG) at either 2.0 or 4.0 mg/g body weight on alternate days during the first 9 days of life. As adults, mice were catheterized to obtain unstressed, serial blood samples for the determination of ultradian profiles of circulating growth hormone. In addition, monooxygenase levels (i.e. steroid hydroxylases and drug-metabolizing enzymes) were measured in hepatic microsomes. Generally, both doses of MSG produced the same developmental defects. Mice neonatally exposed to the amino acid developed a syndrome characterized by retarded growth, obesity and reduced organ weights. While vehicle-treated mice secreted growth hormone in sexually dimorphic patterns defined by pulse frequency (i.e. F > M), hormone concentrations in plasma samples obtained during 8 continuous hr of serial blood collections from both male and female MSG-treated mice were barely detectable at best, and exhibited no pulsatility. Approximately 15% of the measured monooxygenases were male-predominant, 35% were female-predominant and 50% had no sex differences. The enhanced expression of the hepatic monooxygenases in response to MSG-induced depletion of plasma growth hormone indicates that the hormone basically functions as a suppressor of the murine enzyme system.