Hormonal and developmental regulation of expression of the hepatic microsomal steroid 16 alpha-hydroxylase cytochrome P-450 apoprotein in the rat

The hormonal regulation of the sexually differentiated cytochrome P-450 isozyme which catalyzes 16 alpha-hydroxylation of testosterone and 4-androstene-3,17-dione in male rat liver (P-450(16) alpha) was investigated. Estradiol valerate injection of male rats caused a decrease in P-450(16) alpha levels to almost the female level, while methyltrienolone injection had the reverse effect in female animals. Hypophysectomy abolished the sex difference in P-450(16) alpha levels. Human growth hormone infusion into male rats, mimicking the female pattern of growth hormone secretion, caused a feminization of P-450(16) alpha levels. The same effect was also seen in hypophysectomized rats of both sexes. In contrast, a different administration schedule involving 12 h injections of human growth hormone, mimicking the male pattern of growth hormone secretion, caused a masculinization of P-450(16) alpha levels in hypophysectomized rats, at a daily dose which causes feminization when given by infusion. Thus, the level of expression of P-450(16) alpha in the liver is dependent on the temporal pattern of blood growth hormone levels. While infusion of rat growth hormone into male rats also feminized the P-450(16) alpha levels, infusion of ovine prolactin had no effect. Ontogenic studies showed that the developmental pattern of P-450(16) alpha expression in the liver coincided with the known pattern of development of the sexual differentiation of hepatic steroid 16 alpha-hydroxylase activity and of the diurnal pattern of growth hormone secretion.     

Loss of growth hormone-dependent characteristics of rat hepatocytes in culture

Summary The liver of rodents is sexually differentiated, i.e. the female liver differs from the male liver. This differentiation is largely controlled by the pattern of growth hormone (GH) secretion. We have attempted to maintain GH-dependent differentiation of cultured rat hepatocytes. We examined the level of alcohol dehydrogenase (ADH) activity, which responds to GH and is higher in female than in male liver, and the estrogen receptor, which is dependent on GH but is present in equal amounts in males and females. ADH activity was maintained in cells from male rats, but fell by 40% in cells from females in medium supplemented with insulin and dexamethasone. The estrogen receptor content of female cells fell dramatically to undetectable levels within 2 d of culture. Extensive supplementation of the medium failed to prevent the decrease in ADH activity in female cells; similarly, the addition of female sex steroids; rat serum; pituitary extracts; rat, human, or bovine GH; or ovine prolactin failed to maintain the enzyme activity. Insulin, dexamethasone, thyroid hormone plus GH or prolactin, or the combination of all five hormones also failed to prevent the loss of estrogen receptors. Short-term cultures of rat hepatocytes, although retaining the liver-specific expression of ADH at the male level, lose GH-dependent expression of the estrogen receptor and stimulation of ADH activity. Supported by grants AA 00081 and AA 06434 from the National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD.     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

On the regulation of sex-hormone-binding globulin--a challenge of an old dogma and outlines of an alternative mechanism

In this review, the different factors known to affect SHBG levels are discussed with respect to their possible significance in the physiological regulation of this protein: Sex steroids, puberty, nutritional status, thyroid hormones and liver disease. It is concluded that the serum levels of SHBG are related to general metabolic factors, nutritional status, growth and ageing than to the estrogen/androgen balance. The authors suggest that SHBG is regulated primarily by growth hormone, somatomedin-C and possibly other growth factors. Growth hormone may promote SHBG synthesis in the liver while somatomedin-C may stimulate its extravasation and uptake in target tissues. It is suggested that sex steroids merely have an indirect, modulating influence.     

Differentiation of rabbit adipocyte precursor cells in a serum-free medium

Summary A serum-free, hormone-supplemented medium containing insulin, transferrin, and triiodothyronine (ITT medium), able to support differentiation of rat adipose precursor cells, has been used to study the regulation of the development of adipocytes in the rabbit. Adipose conversion was assessed by the appearance of glycerol-3-phosphate dehydrogenase activity. Stromal-vascular cells from rabbit perirenal adipose tissue differentiated to a very low extent or not at all in ITT medium. Supplementation of ITT medium with growth hormone or fibroblast growth factor did not increase the proportion of differentiated cells. In contrast, rabbit stromal-vascular cells were able to differentiate in ITT medium supplemented with glucocorticoids (dexamethasone, corticosterone) whereas sex steroids (β-estradiol, testosterone, progesterone) did not affect the differentiation process. In the presence of both dexamethasone and insulin, 20 to 50% of rabbit stromal-vascular cells differentiated into adipocytes within 2 wk of culture. The stimulatory actions of dexamethasone or insulin were dose-dependent. Insulin-like growth Factor-I (IGF-I), did not replace insulin under our culture conditions and had only a slight effect when added along with dexamethasone (100 nM) and insulin (1.7 nM). The results suggest that glucocorticoids, in association with insulin, may play an important role in the development of adipocytes from rabbit precursor cells. This work was supported by grant 4388 from the Institut National de la Recherche Agronomique, France.     

Hes1 regulates formations of the hypophyseal pars tuberalis and the hypothalamus

The hypophyseal pars tuberalis surrounds the median eminence and infundibular stalk of the hypothalamus as thin layers of cells. The pars tuberalis expresses MT1 melatonin receptor and participates in mediating the photoperiodic secretion of pituitary hormones. Both the rostral tip of Rathke’s pouch (pars tuberalis primordium) and the pars tuberalis expressed αGSU mRNA, and were immunoreactive for LH, chromogranin A, and TSHβ in mice. Hes genes control progenitor cell differentiation in many embryonic tissues and play a crucial role for neurulation in the central nervous system. We investigated the Hes1 function in outgrowth and differentiation of the pars tuberalis by using the markers for the pars tuberalis. In homozygous Hes1 null mutant embryos, the rostral tip was formed in the basal-ventral part of Rathke’s pouch at embryonic day (E)11.5 as well as in wild-type embryos. In contrast to the wild-type, the rostral tip of null mutants could not extend rostrally with age; it remained in the low extremity of Rathke’s pouch during E12.5–E13.5 and disappeared at E14.5, resulting in lack of the pars tuberalis. Development of the ventral diencephalon was impaired in the null mutants at early stages. Rathke’s pouch, therefore, could not link with the nervous tissue and failed to receive inductive signals from the diencephalon. In a very few mutant mice in which the ventral diencephalon was partially sustained, some pars tuberalis cells were distributed around the hypoplastic infundibulum. Thus, Hes1 is required for development of the pars tuberalis and its growth is dependent on the ventral diencephalon.     

Sexual dimorphic expression of ADH in rat liver: importance of the hypothalamic-pituitary-liver axis

Hepatic alcohol dehydrogenase (ADH) activity is higher in female than in male rats. Although sex steroids, thyroid, and growth hormone (GH) have been shown to regulate hepatic ADH, the mechanism(s) for sexual dimorphic expression is unclear. We tested the possibility that the GH secretory pattern determined differential expression of ADH. Gonadectomized and hypophysectomized male and female rats were examined. Hepatic ADH activity was 2.1-fold greater in females. Because protein and mRNA content were also 1.7- and 2.4-fold greater, results indicated that activity differences were due to pretranslational mechanisms. Estradiol increased ADH selectively in males, and testosterone selectively decreased activity and mRNA levels in females. Effect of sex steroids on ADH was lost after hypophysectomy; infusion of GH in males increased ADH to basal female levels, supporting a role of the pituitary-liver axis. However, GH and L-thyroxine (T4) replacements alone in hypophysectomized rats did not restore dimorphic differences for either ADH activity or mRNA levels. On the other hand, T4 in combination with intermittent administration of GH reduced ADH activity and mRNA to basal male values, whereas T4 plus GH infusion replicated female levels. These results indicate that the intermittent male pattern of GH secretion combined with T4 is the principal determinant of low ADH activity in male liver.     

Steroids as Potential Modulators of Thyroid Hormone Activity in Anuran Metamorphosis

SYNOPSIS. Anuran metamorphosis is controlled by the complexinteraction of several hormones. Although thyroid hormone isthe major stimulatory hormone in metamorphosis and likely regulatesall of the metamorphic genes directly, other hormones are involvedin regulating thyroid hormone secretion and activity. Corticoids(in particular corticosterone) and the sex steroids (especially17ß estradiol) all potentially regulate thyroid hormoneactivity both by affecting hypothalamic and pituitary controlof thyroid hormone secretion and by interacting with thyroidhormones peripherally. Although there is likely a role for endogenouscorticoids in anuran metamorphosis, the role for endogenousestrogens remains to be shown. The role of these "modulators"must be fully understood, if our understanding of hormonal controlof metamorphosis is to be complete.     

Mycoplasma provides for the spreading of opossum kidney cells in a serum-free,defined medium

Summary During the course of investigating the growth and differentiation of opossum kidney cells in serum-free medium, it was observed that a mycoplasma contamination (M. hyorhinis) contributed to the spreading of cells. Contaminated cells, seeded on collagen-coated plates, spread out and grew to confluency in Dulbecco’s modified eagle’s medium/Ham’s F12 nutrient mixture containing insulin, transferrin, sodium selenite, and bovine serum albumin fraction V. In addition, differentiated characteristics, including parathyroid hormone-inhibitable, sodium-dependent phosphate transport, were expressed by these cells grown in this medium. After the infection was eradicated, the contamination-free cells would not spread out and proliferate in the same serum-free medium as they had done in the presence of mycoplasma. Normal cellular development, however, was attained after cells were plated in serum-free medium that included fetuin. Cells once again spread out, grew to confluency, and were able to express their differentiated characteristics.     

Gibberellin A1 dwarfism and shoot elongation in higher plants

Evidence is presented to support the generalization that gibberellin A₁ is the main ‘gibberellin hormone’ that is activeper se in the control of elongation growth in higher plants. The evidence is based on a combination of chemical and genetic studies using single gene mutants of maize, pea and rice.     

Characterization of a rat anterior pituitary cell bioassay

Summary We have described the protocols and characterization of a pituicyte culture, which became established as a reliable and reproducible bioassay for the secretion of follicle-stimulating hormone (FSH) and luteinizing hormone (LH). The bioassay was used to measure the bioactivity of factors that inhibit and stimulate gonadotrophin secretion. The protocol that was used involved the culling of female Wistar rats (200 to 250 g weight), at random stages of their cycle, and dispersal of their pituicytes in a concentration of 0.4 × 10⁶ cells · ml⁻¹ · well⁻¹ in serum-free medium (Dulbecco’s modified Eagle’s medium/Ham’s F12 mixture, supplemented with insulin and transferrin) in Falcon 3047 24-well culture plates. After 24 h of pre-culture, the medium was changed and the cells cultured for a further 48 h. The supernatant was removed and assayed for basal secretion of FSH and LH. The cells were then stimulated with 10⁻⁸ M GnRH for 4 h and the supernatant assayed for gonadotrophin-releasing hormone (GnRH)-stimulated FSH and LH secretion. All samples were assayed as pairs of duplicates (i.e. quadruplicate samples) which were randomly added to the plates to minimize plate effects. Random number tables were used to achieve this randomization.     

Sex Steroids and the Differentiation of Avian Reproductive Behavior

Experiments in which avian embryos are treated with sex steroidsor steroid antagonists suggest that sexual differentiation ofreproductive behavior (and thus differentiation of the brainmechanisms for such behavior) is controlled by steroids producedby the embryonic gonads. In chickens and Japanese quail, maleshatched from eggs treated with estradiol or testosterone duringincubation are feminized (demasculinized); they fail to exhibitmasculine sexual behavior as adults, and no longer are behaviorallydistinguishable from females. Some evidence suggests that testosteronemay mimic the feminizing action of estradiol by being convertedto an estrogen in the embryonic brain. Genetic female quailexposed to an antiestrogen during embryonic development aremasculinized; they exhibit an increased ability to display themasculine copulatory pattern. Thus the behavior of these speciesis feminized by embryonic exposure to sex steroids, the anhormonal(neutral) sex for behavioral differentiation appears to be themale, and females appear to result from estrogen produced bythe embryonic ovaries. In contrast, sex steroid treatment ofmammals early in development masculinizes behavior, the femaleis the neutral sex, and males result from fetal androgen secretion.These opposite patterns of psychosexual differentiation in birdsand mammals are correlated with a major difference between theavian and mammalian sex-determining mechanism. Implicationsfor other vertebrates are discussed.     

Mechanism of puberty

The hypothalamo-pituitary-gonadal axis in children is fully functional in fetal life and immediately after birth. The reason why it declines with advancing years of childhood is not clear but gonadotropin pulsatility is at a nadir at 6 years of age. From that time pulsatile gonadotropin starts to reappear but, again, the reason why this happens is completely unknown. All of the events of puberty can be ascribed to pulsatile gonadotropin-releasing hormone stimulation causing pulsatile gonadotropin stimulation of sex steroids. The sex steroids explain the development of the pubertal characteristics; the fact that girls have an earlier growth spurt than boys is explained by the differential effect of oestradiol and testosterone on hypothalamic control of pituitary growth hormone secretion.     

Sex steroid effects on the development and functioning of the growth hormone axis

Summary 1. The secretory pattern of growth hormone (GH) is sexually dimorphic in the adult rat. However, this difference between the sexes does not become apparent until after the onset of puberty, suggesting that pubertal sex steroids play an important role in the manifestation of this phenomenon.2. We have addressed the question as to whether there exists a sexual dimorphism in the hypothalamic neuropeptides that regulate GH release from the anterior pituitary,i.e., somatostatin (SS) and growth hormone-releasing hormone (GHRH). In addition, we have investigated whether the developmental changes in the GH secretory pattern are correlated with changes in these neuropeptides. The effect of testosterone treatment on SS and GHRH neurons during both the neonatal period and adulthood have also been studied.3. We have found that the synthetic capacity, as reflected in relative messenger RNA (mRNA) levels, of both SS and GHRH neurons changes throughout development in both male and female rats. These mRNA levels are sexually dimorphic at certain times during maturation and can be modulated by changes in testosterone levels, suggesting that sex steroid modulation of these two neuropeptide systems could at least partially account for the sexual dimorphism seen in the adult GH secretory pattern.4. The neonatal steroid environment has also been suggested to be involved in the generation of the final adult GH secretory pattern, although the mechanisms underlying this effect are even less well understood. In support of the hypothesis that the neonatal steroid environment plays an important role in organizing the GH axis, we have found that the number of GHRH neurons in the adult brain, as well as their sensitivity to adult steroids, is modulated by neotatal testosterone treatment. The number of SS neurons in the periventricular and paraventricular nuclei were not modulated by neonatal steroids; however, the synthetic capacity of these neurons does appear to be influenced by the neonatal steroid environment.5. These studies suggest that both the neonatal and adult sex steroid environments influence the adult GH secretory pattern by modulating GHRH and SS neurons.     

Dmrt1 polymorphism covaries with sex‐determination patterns in Rana temporaria

Patterns of sex‐chromosome differentiation and gonadal development have been shown to vary among populations of Rana temporaria along a latitudinal transect in Sweden. Frogs from the northern‐boreal population of Ammarnäs displayed well‐differentiated X and Y haplotypes, early gonadal differentiation, and a perfect match between phenotypic and genotypic sex. In contrast, no differentiated Y haplotypes could be detected in the southern population of Tvedöra, where juveniles furthermore showed delayed gonadal differentiation. Here, we show that Dmrt1, a gene that plays a key role in sex determination and sexual development across all metazoans, displays significant sex differentiation in Tvedöra, with a Y‐specific haplotype distinct from Ammarnäs. The differential segment is not only much shorter in Tvedöra than in Ammarnäs, it is also less differentiated and associates with both delayed gonadal differentiation and imperfect match between phenotypic and genotypic sex. Whereas Tvedöra juveniles with a local Y haplotype tend to ultimately develop as males, those without it may nevertheless become functional XX males, but with strongly female‐biased progeny. Our findings suggest that the variance in patterns of sex determination documented in common frogs might result from a genetic polymorphism within a small genomic region that contains Dmrt1. They also substantiate the view that recurrent convergences of sex determination toward a limited set of chromosome pairs may result from the co‐option of small genomic regions that harbor key genes from the sex‐determination pathway.