Neonatal androgenization of hypogonadal (hpg) male mice does not abolish estradiol-induced FSH production and spermatogenesis

BACKGROUND: Testicular development is arrested in the hypogonadal (hpg) mouse due to a congenital deficiency in hypothalamic gonadotropin-releasing hormone (GnRH) synthesis. Chronic treatment of male hpg mice with estradiol induces FSH synthesis and secretion, and causes testicular maturation and qualitatively normal spermatogenesis. As estradiol negative feedback normally inhibits FSH production in the male, this study tested whether this paradoxical response to estradiol in the male hpg mouse might be due to inadequate masculinisation or incomplete defeminization in the neonatal period. Previous studies have demonstrated that treatment of hpg mice with testosterone propionate in the immediate neonatal period is necessary to allow full reproductive behaviors to be expressed following suitable endocrine stimulation at adult ages. METHODS: Hpg mice were treated with 100 μg testosterone propionate or vehicle on postnatal day 2. At 35 days of age, subgroups of these mice were treated with silastic implants containing estradiol or cholesterol. Reproductive behavior was scored in tests with steroid-primed female mice, then testicular development was assessed histologically, and measures of pituitary FSH content made at 85 days of age. RESULTS: The neonatal testosterone propionate treatment successfully defeminized female litter mates, as revealed by impaired vaginal opening and deficiencies in lordosis behavior, and it allowed appropriate male reproductive behavior to be expressed in a proportion of the hpg males when tested at an adult age. However, neonatal androgen supplementation did not block or even reduce the subsequent actions of estradiol in increasing pituitary FSH content, nor did it affect the ability of estradiol to induce qualitatively normal spermatogenesis. CONCLUSION: The ability of the hpg male to show a "female" neuroendocrine response to estradiol is not a result of inadequate androgenization during neonatal development, and thus the actions of estradiol revealed in this rodent model are not an artefact of incomplete sexual differentiation, but reflect a physiological role of estradiol occurring during a specific early temporal window of male reproductive development

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse

<p>Abstract</p> <p>Background</p> <p>The hypogonadal (hpg) mouse is widely used as an animal model with which to investigate the endocrine regulation of spermatogenesis. Chronic treatment of these GnRH-deficient mice with estradiol is known to induce testicular maturation and restore qualitatively normal spermatogenesis. The aim of the current studies was to investigate whether these effects of estradiol are direct effects in the testis, or indirect actions via paradoxical stimulation of FSH secretion from the pituitary gland.</p> <p>Methods</p> <p>Initially, Western blot and immunohistochemistry were used to analyse tissues from hpg mice to identify potential sites of action of estradiol. In the main study, hpg mice were treated for 50 days with either an estradiol implant or daily injections of recombinant human FSH, or a combination of both, to determine whether estradiol would have an additive or synergistic effect with FSH on testis development, as assessed by histological analysis and stereological quantification of Leydig, Sertoli and germ cell proliferation.</p> <p>Results</p> <p>Western blot analysis revealed ERα immunoreactive bands of appropriate molecular weight in extracts of testis and pituitary glands from hpg mice, and immunohistochemical studies confirmed ERα in nuclei of anterior pituitary cells and Leydig and peritubular cells in hpg mice. Histological and morphometric analyses revealed that estradiol treatment alone was as effective as FSH in promoting Sertoli cell production and proliferation of the seminiferous epithelium, resulting in the production of elongating spermatids. Combined estradiol and FSH treatment did not produce a greater effect than either treatment alone, though an increased dose of FSH significantly increased seminiferous tubule volume and testis weight and increase Sertoli cell numbers further within the same time frame. In contrast, estradiol caused substantial increases in the wet weight of the seminal vesicles, whereas FSH was without effect on this tissue, and did not augment the actions of estradiol.</p> <p>Conclusion</p> <p>As ERalpha receptor is abundantly expressed in the pituitary gland of hpg mice, and estradiol did not exert effects on testis development over and above those of FSH, we conclude that the action of estradiol on testis development in <it>hpg </it>mice is predominantly via the stimulation of pituitary FSH release.</p

Whole-body and adipose tissue-specific mechanisms underlying the metabolic effects of fibroblast growth factor 21 in the Siberian hamster.

OBJECTIVE: Fibroblast growth factor 21 (FGF21) has been shown to rapidly lower body weight in the Siberian hamster, a preclinical model of adiposity. This induced negative energy balance mediated by FGF21 is associated with both lowered caloric intake and increased energy expenditure. Previous research demonstrated that adipose tissue (AT) is one of the primary sites of FGF21 action and may be responsible for its ability to increase the whole-body metabolic rate. The present study sought to determine the relative importance of white (subcutaneous AT [sWAT] and visceral AT [vWAT]), and brown (interscapular brown AT [iBAT]) in governing FGF21-mediated metabolic improvements using the tissue-specific uptake of glucose and lipids as a proxy for metabolic activity. METHODS: We used positron emission tomography-computed tomography (PET-CT) imaging in combination with both glucose (18F-fluorodeoxyglucose) and lipid (18F-4-thiapalmitate) tracers to assess the effect of FGF21 on the tissue-specific uptake of these metabolites and compared responses to a control group pair-fed to match the food intake of the FGF21-treated group. In vivo imaging was combined with ex vivo tissue-specific functional, biochemical, and molecular analyses of the nutrient uptake and signaling pathways. RESULTS: Consistent with previous findings, FGF21 reduced body weight via reduced caloric intake and increased energy expenditure in the Siberian hamster. PET-CT studies demonstrated that FGF21 increased the uptake of glucose in BAT and WAT independently of reduced food intake and body weight as demonstrated by imaging of the pair-fed group. Furthermore, FGF21 increased glucose uptake in the primary adipocytes, confirming that these in vivo effects may be due to a direct action of FGF21 at the level of the adipocytes. Mechanistically, the effects of FGF21 are associated with activation of the ERK signaling pathway and upregulation of GLUT4 protein content in all fat depots. In response to treatment with FGF21, we observed an increase in the markers of lipolysis and lipogenesis in both the subcutaneous and visceral WAT depots. In contrast, FGF21 was only able to directly increase the uptake of lipid into BAT. CONCLUSIONS: These data identify brown and white fat depots as primary peripheral sites of action of FGF21 in promoting glucose uptake and also indicate that FGF21 selectively stimulates lipid uptake in brown fat, which may fuel thermogenesis

Cold Exposure Drives Weight Gain and Adiposity following Chronic Suppression of Brown Adipose Tissue.

Therapeutic activation of thermogenic brown adipose tissue (BAT) may be feasible to prevent, or treat, cardiometabolic disease. However, rodents are commonly housed below thermoneutrality (~20 °C) which can modulate their metabolism and physiology including the hyperactivation of brown (BAT) and beige white adipose tissue. We housed animals at thermoneutrality from weaning to chronically supress BAT, mimic human physiology and explore the efficacy of chronic, mild cold exposure (20 °C) and β3-adrenoreceptor agonism (YM-178) under these conditions. Using metabolic phenotyping and exploratory proteomics we show that transfer from 28 °C to 20 °C drives weight gain and a 125% increase in subcutaneous fat mass, an effect not seen with YM-178 administration, thus suggesting a direct effect of a cool ambient temperature in promoting weight gain and further adiposity in obese rats. Following chronic suppression of BAT, uncoupling protein 1 mRNA was undetectable in the subcutaneous inguinal white adipose tissue (IWAT) in all groups. Using exploratory adipose tissue proteomics, we reveal novel gene ontology terms associated with cold-induced weight gain in BAT and IWAT whilst Reactome pathway analysis highlights the regulation of mitotic (i.e., G2/M transition) and metabolism of amino acids and derivatives pathways. Conversely, YM-178 had minimal metabolic-related effects but modified pathways involved in proteolysis (i.e., eukaryotic translation initiation) and RNA surveillance across both tissues. Taken together these findings are indicative of a novel mechanism whereby animals increase body weight and fat mass following chronic suppression of adaptive thermogenesis from weaning. In addition, treatment with a B3-adrenoreceptor agonist did not improve metabolic health in obese animals raised at thermoneutrality

Conserved Expression of the Glutamate NMDA Receptor 1 Subunit Splice Variants during the Development of the Siberian Hamster Suprachiasmatic Nucleus

Glutamate neurotransmission and the N-methyl-D-aspartate receptor (NMDAR) are central to photic signaling to the master circadian pacemaker located in the hypothalamic suprachiasmatic nucleus (SCN). NMDARs also play important roles in brain development including visual input circuits. The functional NMDAR is comprised of multiple subunits, but each requiring the NR1 subunit for normal activity. The NR1 can be alternatively spliced to produce isoforms that confer different functional properties on the NMDAR. The SCN undergoes extensive developmental changes during postnatal life, including synaptogenesis and acquisition of photic signaling. These changes are especially important in the highly photoperiodic Siberian hamster, in which development of sensitivity to photic cues within the SCN could impact early physiological programming. In this study we examined the expression of NR1 isoforms in the hamster at different developmental ages. Gene expression in the forebrain was quantified by in situ hybridization using oligonucleotide probes specific to alternatively spliced regions of the NR1 heteronuclear mRNA, including examination of anterior hypothalamus, piriform cortex, caudate-putamen, thalamus and hippocampus. Gene expression analysis within the SCN revealed the absence of the N1 cassette, the presence of the C2 cassette alone and the combined absence of C1 and C2 cassettes, indicating that the dominant splice variants are NR1-2a and NR1-4a. Whilst we observe changes at different developmental ages in levels of NR1 isoform probe hybridization in various forebrain structures, we find no significant changes within the SCN. This suggests that a switch in NR1 isoform does not underlie or is not produced by developmental changes within the hamster SCN. Consistency of the NR1 isoforms would ensure that the response of the SCN cells to photic signals remains stable throughout life, an important aspect of the function of the SCN as a responder to environmental changes in quality/quantity of light over the circadian day and annual cycle

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-4

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>s containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™) for 50 days. Scale bars represent 50 μm

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-1

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>s) or testis of hypogonadal mice (, left panels) or age-matched wild-type mice (+/+, right panels). Note that many anterior pituitary cells express nuclear ERα immunoreactivity in both and +/+ mice. ERα-ir is abundant in the Leydig cells (lc) and peritubular myoid cells (pt) in the testis of mice, and is also present in some Sertoli cells (sc) in wild-type mice. Bottom left panel shows a control study in which the primary antiserum was omitted, the section is from a testis. All photomicrographs were taken at the same magnification so the 20 μm scale bar in top left panel applies to all panel

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-6

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>tom) from hypogonadal () and age-matched wild-type (+/+) mice. Membranes were stained with MC-20 ERα antiserum (Santa Cruz)

Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse-5

<p><b>Copyright information:</b></p><p>Taken from "Effects of estradiol and FSH on maturation of the testis in the hypogonadal (hpg) mouse"</p><p>Reproductive biology and endocrinology : RB&E 2008;6():4-4.</p><p>Published online 29 Jan 2008</p><p>PMCID:PMC2254628.</p><p></p>that had been treated for 50 days with subcutaneous implants containing either cholesterol (chol) or 2% estradiol (E), and also daily treatment with vehicle, 1 or 5 U of recombinant human FSH (Gonal-F™). Values are group mean ± SEM, group sizes as indicated. *P < 0.05 and ***P < 0.001 vs group treated with cholesterol implants and vehicle