Age-dependent onset of liver-specific IGF-I gene deficiency and its persistence in old age: implications for postnatal growth and insulin resistance in LID mice

To explore the limitations of the liver-specific IGF-I gene-deficient (LID) model and to further evaluate the role of endocrine IGF-I in early postnatal life and old age, we have studied these mice during the prepubertal period (from birth to 3 wk of age) and when they are 2 yr old. During the first 2 wk of life, IGF-I gene deficiency and the resulting reduction in serum IGF-I levels in LID mice did not reach sufficiently low levels when mice experience the most rapid and growth hormone (GH)-independent growth. It suggests that the role of liver-derived IGF-I in prepubertal, GH-independent postnatal growth cannot be established. From our previous studies, liver IGF-I mRNA level was abolished in adult LID mice, which causes elevated GH level, insulin resistance, pancreatic islet enlargement, and hyperinsulinemia. Interestingly in 2-yr-old LID mice, although liver IGF-I mRNA and serum IGF-I levels were still suppressed, serum insulin and GH levels had returned to normal. Compared with same-sex control littermates, aged male LID mice had significantly reduced body weight and fat mass and exhibited normal insulin sensitivity. On the other hand, aged female LID mice exhibited normal weight and marginal resistance to insulin actions. The pancreatic islet percentage (reflecting islet cell mass) was also restored to normal levels in aged LID mice. Thus, although the IGF-I gene deficiency is well maintained into old age, the insulin sensitivity, islet enlargement, and hyperinsulinemia that occurred in young adult mice have been mostly restored to normal levels, further supporting the age-dependent and sexual dimorphic features of the LID mice.     

Characterisation and expression of Sox9 in the Leopard gecko, Eublepharis macularius

Since the discovery of the sex-determining gene, Sry, a number of genes have been identified which are involved in sex determination and gonadogenesis in mammals. Although Sry is known to be the testis-determining factor in mammals, this is not the case in non-mammalian vertebrates. Sox9 is another gene that has been shown to have a male-specific role in sex determination, but, unlike Sry, Sox9 has been shown to be involved in sex determination in mammals, birds, and reptiles. This is the first gene to be described that has a conserved role in sex determination in species with either chromosomal or environmental sex-determining mechanisms. Many reptiles do not have sex chromosomes but exhibit temperature-dependent sex determination (TSD). Sox9 has been shown to be expressed in both turtle and alligator during gonadogenesis. To determine if Sox9 also has a role in a gecko species with TSD, we studied gonadal expression of Sox9 during embryonic development of the Leopard gecko (Eublepharis macularius). Gecko Sox9 was found to be highly conserved at the nucleotide level when compared to other vertebrate species including human, chick, alligator, and turtle. Sox9 was found to be expressed in embryos incubated at the male-producing temperature (32.5 degrees C) as well as in embryos incubated at the female-producing temperatures (26 and 34 degrees C), Northern blot analysis showed that Sox9 was expressed at both temperatures from morphological stages 31 to 37. mRNA in situ hybridisation on isolated urogenital systems showed expression at both female- and male-producing temperatures up to stage 36. After this stage, no expression was seen in the female gonads but expression remained in the male. These data provide further evidence that Sox9 is an essential component of a testis-determining pathway that is conserved in species with differing sex-determining mechanisms.     

Effects of growth hormone on hypothalamic catalase and Cu/Zn superoxide dismutase

Age-associated changes in hypothalamic catalase activity and level, and Cu/Zn superoxide dismutase (Cu/Zn SOD) activity were examined in Ames dwarf mice with growth hormone (GH) deficiency and prolonged lifespan, in PEPCK-hGH transgenic mice with overexpression of GH and reduced lifespan, and compared to values measured in normal controls. Hypothalami from young (3-4 months), middle-aged (9-10 months), and old (19-23 months) male mice were examined using spectrophotometric assay and Western blot. In dwarf mice, Cu/Zn SOD and catalase activities declined with age, and were higher than the corresponding normal values in young and middle-aged groups. Catalase levels also declined with age, but were similar to values in normal controls. In GH transgenic mice, age-associated decline of both catalase and Cu/Zn SOD occurred earlier than in normal animals. Catalase levels and activities in transgenic animals were similar to controls, whereas Cu/Zn SOD activity was higher in transgenics than in normal mice. The present results suggest that dwarf mice, during early life, have enhanced hypothalamic free radical defenses, which may contribute to their extended lifespan. However, from the present results in GH transgenic mice, it is impossible to conclude whether early decline of hypothalamic catalase and Cu/Zn SOD in these animals represents a correlate of accelerated aging, or contributes to their reduced lifespan.     

Pulsatile growth hormone secretion persists in genetic growth hormone-releasing hormone resistance

Growth hormone (GH) secretion is regulated by GH-releasing hormone (GHRH), somatostatin, and possibly ghrelin, but uncertainty remains about the relative contributions of these hypophysiotropic factors to GH pulsatility. Patients with genetic GHRH receptor (GHRH-R) deficiency present an opportunity to examine GH secretory dynamics in the selective absence of GHRH input. We studied circadian GH profiles in four young men homozygous for a null mutation in the GHRH-R gene by use of an ultrasensitive GH assay. Residual GH secretion was pulsatile, with normal pulse frequency, but severely reduced amplitude (<1% normal) and greater than normal process disorder (as assessed by approximate entropy). Nocturnal GH secretion, both basal and pulsatile, was enhanced compared with daytime. We conclude that rhythmic GH secretion persists in an amplitude-miniaturized version in the absence of a GHRH-R signal. The nocturnal enhancement of GH secretion is likely mediated by decreased somatostatin tone. Pulsatility of residual GH secretion may be caused by oscillations in somatostatin and/or ghrelin; it may also reflect intrinsic oscillations in somatotropes.     

The consequences of altered somatotropic system on reproduction

Although the primary control of gonadotropin secretion is by the hypothalamic GnRH and the gonadal function is controlled by the pituitary gonadotropins and prolactin, the emerging evidence suggests a vital role of the somatotropic axis, growth hormone (GH), and insulin-like growth factor-I (IGF-I) in the control of the pituitary and gonadal functions. It has been shown that GH deficiency, GH resistance, and experimental alterations in IGF-I secretion modify folliculogenesis, ovarian maturation, ovulation, and pregnancy, and in the male, GH/IGF-I plays an important role in spermatogenesis and the Leydig cell function. The primary focus of this review is to examine the role of GH/ IGF-I on the onset of puberty, fertility, pituitary, and gonadal endocrine functions. A number of studies have revealed that fertility is affected in GH-deficient dwarf and in IGF-I gene-ablated mice, possibly due to subnormal function of either the pituitary gland or the gonads. In the female GH receptor gene knockout (GHR-KO) mice, there was impairment in follicular development, ovulation rate, sexual maturation, production of and responsiveness to pheromonal signals, and the corpus luteum function. In IGF-I-deficient male GHR-KO mice, puberty is delayed, spermatogenesis is affected, and neuroendocrine-gonadal function is attenuated. Similarly, in some of the human Laron syndrome patients, puberty is delayed due to GH resistance. These data suggest that, in addition to GnRH and gonadotropins, GH/IGF-I influences the pituitary and gonadal functions in animals and humans.     

Loss of Fgfr2 leads to partial XY sex reversal

In mammals, sex is determined in the bipotential embryonic gonad by a balanced network of gene actions which when altered causes disorders of sexual development (DSD, formerly known as intersex). In the XY gonad, presumptive Sertoli cells begin to differentiate when SRY up-regulates SOX9, which in turn activates FGF9 and PGDS to maintain its own expression. This study identifies a new and essential component of FGF signaling in sex determination. Fgfr2 mutant XY mice on a mixed 129/C57BL6 genetic background had either normal testes, or developed ovotestes, with predominantly testicular tissue. However, backcrossing to C57BL6 mice resulted in a wide range of gonadal phenotypes, from hypoplastic testes to ovotestes with predominantly ovarian tissue, similar to Fgf9 knockout mice. Since typical male-specific FGF9-binding to the coelomic epithelium was abolished in Fgfr2 mutant XY gonads, these results suggest that FGFR2 acts as the receptor for FGF9. Pgds and SOX9 remained expressed within the testicular portions of Fgfr2 mutant ovotestes, suggesting that the Prostaglandin pathway acts independently of FGFR2 to maintain SOX9 expression. We could further demonstrate that double-heterozygous Fgfr2/Sox9 knockout mice developed ovotestes, demonstrating that both Fgfr2 and Sox9 can act as modifier intersex genes in the heterozygous state. In summary, we provide evidence that FGFR2 is important for male sex determination in mice, thereby rendering human FGFR2 a candidate gene for unsolved DSD cases such as 10q26 deletions.     

Growth hormone receptor gene deficiency causes delayed insulin responsiveness in skeletal muscles without affecting compensatory islet cell overgrowth in obese mice

Growth hormone (GH), acting through its receptor (GHR), is essential for somatic growth and development and maintaining metabolic homeostasis. GHR gene-deficient (GHR(-/-)) mice exhibit drastically diminished insulin-like growth factor-I (IGF-I) levels, proportional growth retardation, elevated insulin sensitivity, and reduced islet beta-cell mass. Unlike the liver, which is mostly unaffected by changes in IGF-I level, skeletal muscles express high levels of IGF-I receptor (IGF-IR). The net result of a concurrent deficiency in the actions of both GH and IGF-I, which exert opposite influences on insulin responsiveness, has not been evaluated. We studied insulin-stimulated early responses in the insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and p85 subunit of phosphatidylinositol 3-kinase. Upon in vivo insulin stimulation, skeletal muscles of GHR(-/-) mice exhibit transient delayed responses in IR and IRS-1 phosphorylation but normal levels of p85 association with IRS-1. This is in contrast to normal/elevated insulin responses in hepatocytes and indicates tissue-specific effects of GHR gene deficiency. In addition to stimulating normal islet cell growth, GH may participate in islet cell overgrowth, which compensates for insulin resistance induced by obesity. To determine whether the islet cell overgrowth is dependent on GH signaling, we studied the response of male GHR(-/-) mice to high-fat diet (HFD)-induced obesity. After 17 wk on a HFD, GHR(-/-) mice became more significantly obese than wild-type mice and exhibited increased beta-cell mass to a slightly higher extent. These data demonstrate that GH signaling is not required for compensatory islet growth. Thus, in both muscle insulin responsiveness and islet growth compensation, normal levels of GH signals do not seem to play a dominant role.     

Increased P85alpha is a potent negative regulator of skeletal muscle insulin signaling and induces in vivo insulin resistance associated with growth hormone excess

Insulin resistance is a cardinal feature of normal pregnancy and excess growth hormone (GH) states, but its underlying mechanism remains enigmatic. We previously found a significant increase in the p85 regulatory subunit of phosphatidylinositol kinase (PI 3-kinase) and striking decrease in IRS-1-associated PI 3-kinase activity in the skeletal muscle of transgenic animals overexpressing human placental growth hormone. Herein, using transgenic mice bearing deletions in p85alpha, p85beta, or insulin-like growth factor-1, we provide novel evidence suggesting that overexpression of p85alpha is a primary mechanism for skeletal muscle insulin resistance in response to GH. We found that the excess in total p85 was entirely accounted for by an increase in the free p85alpha-specific isoform. In mice with a liver-specific deletion in insulin-like growth factor-1, excess GH caused insulin resistance and an increase in skeletal muscle p85alpha, which was completely reversible using a GH-releasing hormone antagonist. To understand the role of p85alpha in GH-induced insulin resistance, we used mice bearing deletions of the genes coding for p85alpha or p85beta, respectively (p85alpha (+/-) and p85beta(-/-)). Wild type and p85beta(-/-) mice developed in vivo insulin resistance and demonstrated overexpression of p85alpha and reduced insulin-stimulated PI 3-kinase activity in skeletal muscle in response to GH. In contrast, p85alpha(+/-)mice retained global insulin sensitivity and PI 3-kinase activity associated with reduced p85alpha expression. These findings demonstrated the importance of increased p85alpha in mediating skeletal muscle insulin resistance in response to GH and suggested a potential role for reducing p85alpha as a therapeutic strategy for enhancing insulin sensitivity in skeletal muscle.     

The relationship between age and genotype and circulating concentrations of triiodothyronine (T3), thyroxine (T4), and growth hormone in commercial meat strain chickens

The presence of the sex-linked dwarf gene (dw) in homozygous male (dw/dw) and female (dw/-) meat strain chickens is associated with a significant reduction in circulating levels of triiodothyronine (T3). Heterozygous (Dw/dw) male broiler strain chickens have T3 concentrations similar to those in homozygous (Dw/Dw) male broilers. Genetically normal (Dw/Dw) but significantly slower growing roaster strain male meat chickens had consistently higher T3 than the faster growing broilers at all ages in one experiment but only at 8 weeks in a second experiment. Age and not growth rate appears to have a greater influence on serum T3 concentrations in the slow- and fast-growing normal strains. Growth hormone levels were significantly higher in the dwarf chickens at all ages and in all three experiments. The heterozygous and homozygous broilers had similar GH levels and the slow-growing, genetically normal roasters had intermediate concentrations between the broiler and dwarf lines. GH was influenced to a greater extent by the rate of body weight gain than by increasing age in the genetically normal fast and slow growing strains.     

A phenotypic spectrum of sexual development in Dax1 (Nr0b1)-deficient mice: consequence of the C57BL/6J strain on sex determination

Nuclear receptor subfamily 0, group B, member 1 (Nr0b1; hereafter referred to as Dax1) is an orphan nuclear receptor that regulates adrenal and gonadal development. Dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (Dax1) mutations in the mouse are sensitive to genetic background. In this report, a spectrum of impaired gonadal differentiation was observed as a result of crossing the Dax1 knockout on the 129SvIm/J strain onto the C57BL/6J strain over two generations of breeding. Dax1-mutant XY mice of a mixed genetic background (129;B6Dax1(-/Y) [101 total]) developed gonads that were predominantly testislike (n = 61), ovarianlike (n = 27), or as intersex (n = 13). During embryonic development, Sox9 expression in the gonads of 129;B6Dax1(-/Y) mutants was distributed across a wide quantitative range, and a threshold level of Sox9 (>0.4-fold of wild-type) was associated with testis development. Germ cell fate also varied widely, with meiotic germ cells being more prevalent in the ovarianlike regions of embryonic gonads, but also observed within testicular tissue. Ptgds, a gene associated with Sox9 expression and Sertoli cell development, was markedly downregulated in Dax1(-/Y) mice. Stra8, a gene associated with germ cell meiosis, was upregulated in Dax1(-/Y) mice. In both cases, the changes in gene expression also occurred in pure 129 mice but were amplified in the B6 genetic background. Sertoli cell apoptosis was prevalent in 129;B6Dax1(-/Y) gonads. In summary, Dax1 deficiency on a partial B6 genetic background results in further modulation of gene expression changes that affect both Sertoli cell and germ cell fate, leading to a phenotypic spectrum of gonadal differentiation.     

Testicular type Sox9 is not involved in sex determination but might be in the development of testicular structures in the medaka, Oryzias latipes

Testicular type Sox9 is the most upstream conserved gene in the sex determining cascade among vertebrate. However, in medaka, only one Sox9 gene was identified as expressed in the ovary; no other Sox9 gene was reported expressed in the testis. We explored the medaka genome and cloned a novel testicular type Sox9 cDNA. Phylogenetic analysis revealed that both our isolated Sox9 and the already reportedly cloned medaka Sox9 belongs zebrafish Sox9a branch. Therefore, we named our gene Sox9a2. Unexpectedly, Sox9a2 mRNA was expressed in somatic cells surrounding germ cells at similar high levels in both sexes during early gonadal sex differentiation. However, at the initial stage of testicular tubules development, the expression of Sox9a2 was maintained only in XY gonads, and was remarkably reduced in XX gonads. These results suggest that Sox9a2 is not involved in early sex determination and differentiation, but is involved in the later development of testicular tubules in medaka.     

Fertility of transgenic female mice expressing bovine growth hormone or human growth hormone variant genes

Although growth hormone (GH) exerts various direct and indirect stimulatory effects on gonadal development and function, excessive levels of GH in acromegalic patients and in transgenic animals are often associated with reproductive disorders. We have examined reproductive performance of transgenic female mice expressing the following hybrid genes: mouse metallothionein-1 (MT)/human placental GH variant (hGH.V), MT/bovine GH(bGH), and phosphoenolpyruvate carboxykinase (PEPCK)/bGH. This allowed us to evaluate the effects of chronic GH excess in three animal models and to obtain some information on the significance of the lactogenic activity of the foreign GH (hGH.V vs. bGH) and on the developmental stage of transgene expression (MT vs. PEPCK). Transgenic animals from each line had elevated plasma insulin-like growth factor-I levels and greatly increased adult body weight. Plasma bGH levels were significantly higher in PEPCK/bGH than in MT/bGH transgenic mice. Approximately 20% of transgenic MT/hGH.V and MT/bGH females and over 60% of transgenic PEPCK/bGH females were infertile. Transgenic females that did reproduce ovulated either a normal or increased number of eggs but exhibited a variety of reproductive disorders including increased interval between pairing with a male and conception, increased interval between litters, reduced number of litters, reduced fetal growth, increased pre- and postnatal mortality, and alterations in sex ratio. Among adult offspring of these females, the proportion of transgenic animals was significantly less than the expected 50%. While some characteristics (e.g., fetal crown-rump length and weight on Day 14 of pregnancy) were affected to a comparable extent in transgenic females from all three lines, MT/hGH.V and PEPCK/bGH females were, in general, more severely affected than the MT/bGH animals. Sterility of PEPCK/bGH females appeared to be due to luteal failure since treatment with progesterone led to pregnancy. Greatly increased intervals between successive litters appeared to be due to failure to mate during postpartum estrus and to sterile matings during this period. Reduced fetal size and weight may have been due to chronic glucocorticoid excess because comparable changes could be induced in normal females by injections of dexamethasone during pregnancy, and plasma corticosterone levels were previously shown to be elevated in transgenic mice from each of these lines. Comparison of these results with data obtained from matings of normal female mice to transgenic males from the same lines suggests that reduced fetal growth is due primarily to maternal genotype, while reduced "transmission" of the hybrid genes is not, and presumably reflects increased mortality of transgenic progeny at various stages of development.(ABSTRACT TRUNCATED AT 400 WORDS)     

Direct effects of triiodothyronine on production of anterior pituitary hormones and gonadal steroids in goldfish

The present study investigated the effects of triiodothyronine (T3) on pituitary gonadotropin (GTH) subunits, thyroid stimulating hormone (TSH) β subunit, and growth hormone (GH) mRNA levels, as well as gonadal steroid secretion during different stages of reproduction in goldfish. Goldfish pituitary cells cultured with T3 exhibited lower tshβ mRNA levels in all reproductive stages and lower luteinising hormone β (lhβ) mRNA levels in early recrudescence, whereas gh and fshβ mRNA levels were not altered. T3 injections significantly reduced circulating oestrogen (OE2) concentrations in early and mid recrudescent male goldfish, but were without effect on the circulating level of OE2 in female fish. T3 injections also reduced circulating levels of testosterone in both male and female goldfish during the mid stage of gonadal recrudescence. In vitro culture of goldfish ovarian follicles at the late stage of gonadal recrudescence, in the presence of T3, resulted in reduced OE2 secretion; no consistent effect of T3 on testosterone secretion was observed in cultured goldfish ovarian follicles and testis. These findings support the hypothesis that T3 impairs reproduction by inhibiting production of gonadal steroids and pituitary luteinising hormone production in goldfish. Mol. Reprod. Dev. 79: 592–602, 2012. © 2012 Wiley Periodicals, Inc.