Direct effect of estradiol on the number of dopamine receptors in the anterior pituitary of ovariectomized rats

The effect of 17 beta-estradiol (17 beta E2) on anterior pituitary dopaminergic receptor (D2) content was studied in vitro in relation to PRL secretion. Anterior pituitaries from ovariectomized rats were incubated for short periods in medium 199, with or without the steroid. Dopamine (DA) receptors in partially purified pituitary membranes were quantified by equilibrium binding using [3H]spiperone; the PRL released into the incubation medium was analyzed by RIA. Addition of 10(-10) to 10(-6) M 17 beta E2 to the incubation medium of anterior pituitaries rapidly and reversibly decreased the number of DA receptors (P less than 0.01 to 0.001), while increasing PRL release, in a dose-related fashion. The maximal effect on both receptor numbers and PRL secretion was obtained with 10(-8) M 17 beta E2. This effect involved no change in receptor affinity (Kd = 0.11 +/- 0.01 nM in presence or in absence of 17 beta E2). This estrogen-induced decrease in DA-binding capacity was apparently not the result of the occupation of spiperone binding sites by the steroid, since after a 30-min incubation with 10(-8) M [3H]17 beta E2, no radioactivity was detectable on the partially purified membranes. Moreover, the presence of 17 beta E2 at the same time as the labeled D2 ligand did not modify the kinetics of association or dissociation of spiperone with pituitary membranes. This decrease in anterior pituitary DA receptor content and the increase in PRL release were already significant after a 7-min incubation in the presence of 10(-8) M 17 beta E2. Finally, these effects of 17 beta E2 were not mimicked by its 17 alpha-stereoisomer, nor by progesterone, or testosterone. These results suggest that the stimulatory effect of 17 beta E2 on PRL secretion may be due, at least in part, to the desensitization of anterior pituitary cells to DA. The steroid may produce this desensitization directly by decreasing the number of D2. The short latency of this effect likely discards the possibility of a genomic action of 17 beta E2.     

A comparison of the disappearance rates of luteinizing hormone from intact and ovariectomized rats

Hypophysectomy was performed on intact female rats during the proestrous LH surge and on ovariectomized rats, and plasma concentrations of LH were measured every 5 min for approximately the next hour. The disappearance rate of LH from ovariectomized rats corresponded to an initial half-life of 23.1 +/- 2.9 min, which is within the range previously reported for this species. The disappearance of LH from intact rats, however, corresponded to an initial half-life of 13.7 +/- 0.7 min. In a second experiment, blood was taken from ovariectomized rats and from intact female rats during the LH surge. The sera were injected iv into hypophysectomized female rats (with ovaries intact) bearing chronic venous catheters, and plasma concentrations of LH were measured every 5 min for the next hour. LH from ovariectomized rats disappeared at a rate corresponding to an initial half-life of 22.7 +/- 2.2 min, while the half-life for LH from proestrous intact rats was 13.4 +/- 1.2 min. These results suggest a change in the circulating form of LH following ovariectomy, in that LH in the ovariectomized rat is cleared more slowly. Further, it appears that LH is removed from the circulation of the intact rat at a higher rate than has previously been reported.     

雌激素对去卵巢大鼠胰岛β细胞数量和功能的影响

目的 观察大鼠在去卵巢和雌激素替代治疗状态下,经小剂量链脲佐菌素(STZ)干预后胰岛β细胞数量和功能的变化. 方法 30只雌性SD大鼠随机分为5组,正常对照组、STZ组、去卵巢组、去卵巢+STZ组和雌激素组.正常对照组和STZ组行假手术,其余均行去卵巢手术.术后雌激素组给予雌激素治疗,3周后STZ组、去卵巢+STZ组、雌激素组给予STZ(40 mg/kg)干预,检测各组大鼠血糖、血胰岛素水平,平均β细胞面积和相对β细胞数量,β细胞凋亡数量,β细胞内的促凋亡基因Bax、抗凋亡基因Bcl-2及增殖细胞核抗原(PCNA)蛋白表达水平. 结果 在STZ作用下,去卵巢大鼠口服葡萄糖耐量(OGTT)各时点血糖均较未去卵巢大鼠明显升高(P<0.05),且各时点胰岛素分泌水平均减少(P<0.05);胰岛素生成指数(△I30/△G30)及修正的β细胞胰岛素分泌指数MBCI下降,β细胞内胰岛素蛋白表达水平、相对β细胞数量和平均β细胞面积均明显下降(P<0.05),Bcl-2/Bax比值下降(0.41±0.03对0.76±0.05,P<0.05),β细胞的凋亡指数升高(t=2.957,P<0.05).去卵巢后给予雌激素替代治疗则对上述变化有明显的改善作用,OGTT各时点血糖水平显著下降(P<0.05),β细胞内胰岛素蛋白表达水平及血胰岛素水平的升高,Bcl-2/Bax比值上升(0.71±0.05对0.41±0.03),β细胞的凋亡指数下降(t=2.782,P<0.05). 结论 去卵巢大鼠对外来刺激明显易感,小剂量STZ即导致胰岛β细胞的凋亡增加,数量减少,胰岛素分泌水平的下降,血糖明显升高.补充雌激素能够对抗去卵巢带来的对胰岛β细胞的不利影响,改善β细胞的凋亡,具有保护作用.     

Age-related alterations of proteasome structure and function in aging epidermis

Recent studies on the effect of aging in epidermal cells have evidenced a decrease of proteasome activity and content, suggesting that proteasome is down-regulated in aged cells. The 20S proteasome is the major proteolytic system that has been implicated in removal of abnormal and oxidatively damaged proteins. Therefore, a decreased proteasome content may explain, at least in part, the well-documented age-related accumulation of oxidized proteins. To gain further insight in other mechanisms that may be implicated in a decreased activity of the proteasome with age, 20S proteasome has been purified from the epidermis from donors of different ages: young, middle-aged and old. The patterns of proteasome subunits have been analyzed by 2D gel electrophoresis to determine whether its structure is also affected with age. The 2D gel pattern of proteasome subunits was found to be modified for four subunits, indicating that the observed decline in proteasome activity with age may also be related to alterations of its subunits. These subunit alterations are likely to be involved in the age-related decrease of proteasome activity since the specific peptidase activities of the purified proteasome were found to be decreased with age.     

Age-related differences in the release of luteinizing hormone and gonadotropin-releasing hormone in ovariectomized rats

The effect of aging on the release of gonadotropin-releasing hormone (GnRH) in vitro and of luteinizing hormone (LH) both in vivo and in vitro in ovariectomized (Ovx) rats was studied. Old (21-24 months) and young (3-4 months) rats were Ovx before use. They were injected subcutaneously with estradiol benzoate (25 micrograms/kg) or sesame oil for 3 days and then challenged with GnRH (0.5, 2 or 10 micrograms/kg) via a jugular catheter. Blood samples were collected immediately before and at 5, 10, 20, 40 and 60 min following GnRH injection. For in vitro study, Ovx rats were decapitated. The anterior pituitary glands (APs) were incubated with GnRH (0.1 or 10 nM) and estradiol (0, 0.1, 1 or 10 nM) at 37 degrees C for 30 min. The mediobasal hypothalamus was superfused with Locke's solution at 37 degrees C for 210 min, and stimulated with 60 mM KCl at 90 and 150 min. The medium samples were collected at 10-min intervals. Concentrations of GnRH and LH in plasma and medium samples were measured by radioimmunoassay. In all rats, the basal and GnRH-stimulated levels of plasma LH were lower in old than in young rats. The spontaneous release of LH in vitro from APs of Ovx rats was increased by aging, whereas GnRH-stimulated release of LH in vitro was lower in old than in young animals. The potassium-stimulated, but not spontaneous, release of GnRH was lower in old than in young Ovx rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Age-related changes in pituitary/thyroid function in chickens

Pituitary cyclic AMP-dependent protein kinase (cAMP-PK) activity ratios are not increased in response to thyrotropin-releasing hormone (TRH) in old chickens (retired White Leghorn breeders). This reduced responsiveness may be due to reduced hypothalamic function, reduced thyrotrope function, or to a reduction in TRH membrane receptors. The thyroid cAMP-PK activity ratio of old males does not respond to TRH treatment but the thyroid of old females does have an increased activity ratio ater TRH injection. Both males and females have a much higher basal cAMP-PK activity ratio than young birds. This higher basal level is thought to be due to an increased thyroid-stimulating hormone (TSH) stimulation, and the failure of old males to increase activity after TRH injections may be due to a loss of the thyroid's ability to respond to direct TRH stimulation.     

Pituitary responsiveness to LH-RH in intact and ovariectomized androgen-sterilized rats.

Serum LH changes in response to LH-RH injection were measured in intact and ovariectomized, steroid-treated female rats which were androgenized neonatally with 1,250 microgram testosterone propionate (TP) on day 5. At a dose of 20 ng LH-RH/100 g b.w., serum LH levels in intact rats increased over pre-injection levels, and at a dose of 100 ng LH-RH/100 g b.w., LH concentrations 15 min after injection were higher in nembutal-blocked proestrous rats than in androgen-sterilized rats. However, the ovulation response was not different between the groups. In ovariectomized estradiol benzoate (EB)-treated, androgen-sterilized rats, serum LH concentrations 15 and 60 min after LH-RH injection were lower than in similarly treated control rats. This effect was not secondary to the anovulatory state of the animal, since it also occurred in ovariectomized EB-treated prepuberal rats and in rats ovariectomized prepuberally and treated with EB in adulthood. Also, after treatment with 5alpha-dihydrotestosterone propionate (5alpha-DHTP), pituitary responsiveness to LH-RH in androgen-sterilized rats was lower than in control rats, which suggests that the subnormal response in the estrogen-treated rats was not due to a relative insensitivity to estrogen in the androgen-sterilized rats. The relatively high pituitary responsiveness to LH-RH in intact androgen-sterilized rats is probably due to the high circulating estrogen levels. The subnormal pituitary responsiveness to LH-RH after ovariectomy and estradiol treatment suggests, in addition to an effect on the hypothalamus, also a direct effect of neonatal androgen administration on the pituitary.     

Age-related alterations in the strength and collagen content of left colon in rats

The biomechanical properties and intestinal wall composition of left colon were studied in 4-month-old, 14-month-old, and 27-month-old male rats. The hydroxyproline content and hydroxyproline concentration in old rats were increased by 36% and 26%, respectively, compared with young rats and by 20% and 17%, respectively, compared with middle-aged rats. In middle-aged rats the maximum load increased by 21%, compared with young rats. In old rats, however, the maximum load decreased by 13%, compared with middle-aged rats. Histological examination showed that the mean crypt height was 9% higher in middle-aged rats and 12% higher in old rats than in young rats. In conclusion, an accumulation of collagenous proteins was found in old rats compared with middle-aged rats and this was accompanied by a decrease in the strength, which may deteriorate the fuctional integrity of the left colonic wall with age.

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Résumé Les propriétés bio-mécaniques et la composition de la paroi intestinale du colon gauche ont été étudiées chez des rats males âgés de 4 mois, 14 mois et 27 mois. La teneur en hydroxyproline et la concentration d'hydroxyproline chez les rats âgés était augmentée de 36 et 26% respectivement en comparaison avec les jeunes rats et de 20 et 17% respectivement comparé aux rats d'âge moyen. Chez ces derniers la charge maximale augmentait de 21% par rapport aux rats jeunes. Toutefois les rats âgés avaient une charge moyenne diminuée de 13% par rapport aux rats d'âge moyen. L'examen histologique montrait que la hauteur moyenne des cryptes était de 9% plus élévée chez les rats d'âge moyen et de 10% plus élevée chez les rats âgés que chez les jeunes rats. En conclusion une accumulation de protéïne collagénique a été trouvée chez les rats âgés par comparaison aux rats d'âge moyen et ceci était accompagné par une diminution de la force qui pourrait détériorer l'intégrité fonctionnelle de la paroi du colon gauche avec l'âge.

     

Age-related alterations in pituitary and testicular functions in long-lived growth hormone receptor gene-disrupted mice

The somatotropic axis, GH, and IGF-I interact with the hypothalamic-pituitary-gonadal axis in health and disease. GH-resistant GH receptor-disrupted knockout (GHRKO) male mice are fertile but exhibit delayed puberty and decreases in plasma FSH levels, testicular content of LH, and prolactin (PRL) receptors, whereas PRL levels are elevated. Because the lifespan of GHRKO mice is much greater than the lifespan of their normal siblings, it was of interest to compare age-related changes in the hypothalamic-pituitary-gonadal axis in GHRKO and normal animals. Plasma IGF-I, insulin, PRL, LH, FSH, androstenedione and testosterone levels, and acute responses to GnRH and LH were measured in young (2-4 and 5-6 months of age) and old (18-19 and 23-26 months of age) male GHRKO mice and their normal siblings. Plasma IGF-I was not detectable in GHRKO mice. Plasma PRL levels increased with age in normal mice but declined in GHRKO males, and did not differ in old GHRKO and normal animals. Plasma LH responses to acute GnRH stimulation were attenuated in GHRKO mice but increased with age only in normal mice. Plasma FSH levels were decreased in GHRKO mice regardless of age. Plasma testosterone responses to LH stimulation were attenuated in old mice regardless of genotype, whereas plasma androstenedione responses were reduced with age only in GHRKO mice. Testicular IGF-I mRNA levels were normal in young and increased in old GHRKO mice, whereas testicular concentrations and total IGF-I levels were decreased in these animals. These findings indicate that GH resistance due to targeted disruption of the GH receptor gene in mice leads to suppression of testicular IGF-I levels, and modifies the effects of aging on plasma PRL levels and responses of the pituitary and testes to GnRH and LH stimulation. Plasma testosterone levels declined during aging in normal but not in GHRKO mice, and the age-related increase in the LH responses to exogenous GnRH was absent in GHRKO mice, perhaps reflecting a delay of aging in these remarkably long-lived animals.     

Age-related alterations in liver mitochondrial bioenergetics of diabetic Goto-Kakizaki rats

Type 2 diabetes mellitus is one of the most common chronic metabolic diseases in man. Due to long-term complications of the disease, severely decreasing the quality of life of diabetic patients, early interventions to obviate the risk of complications are of major importance. Therefore, diabetic animal models are of major importance in research for interventional treatment of type 2 diabetes. In this work we investigated the possible alterations in mitochondrial energetic metabolism of Goto-Kakizaki (GK) rats during the progression of the disease, since glucose metabolism is closely related to intracellular ATP content. For that reason, respiratory indexes (state 4, state 3, RCR and ADP/O) were evaluated either in the presence of NAD- or FAD-linked substrates (glutamate + malate and succinate, respectively) in mitochondrial preparations of GK and control rats with 8, 12, 26 and 52 weeks of age. Until the age of 1 year (52 weeks) we found no impairment of mitochondrial respiratory indexes both in the presence of glutamate + malate and succinate. In conclusion, this study indicates that GK rat is a good model for studying the initial events of diabetes, since it presents no impairment of liver mitochondrial functions during the first year of life, contrasting clearly with pharmacological induced diabetes. Received: 7 May 1999 / Accepted in revised form: 17 December 1999     

Effects of chronic prednisolone treatment on bone resorption and bone composition in intact and ovariectomized rats and in ovariectomized rats receiving beta-estradiol

To examine the interactions between estrogen deficiency and glucocorticoid excess on bone metabolism the osteopenic effects of a standard dose of prednisolone (2 mg/kg BW.day) were studied in sham-ovariectomized (Sham-OVX), ovariectomized (OVX), and OVX rats given replacement beta-estradiol (OVX + E2). For 12 weeks six groups of female albino rats aged 4 months which had their skeletons labeled with 45Ca were fed matched amounts of low-calcium (0.1% Ca) hydroxyproline-free diet. The six treatment groups were: group 1, Sham-OVX; group 2, Sham-OVX + prednisolone; group 3, OVX; group 4, OVX + prednisolone; group 5, OVX + E2; group 6, OVX + E2 + prednisolone. Bone resorption was estimated by studying the urinary excretion of hydroxyproline and 45Ca. Parathyroid function was assessed indirectly from urinary cAMP excretion. Treatments did not influence parathyroid activity or serum levels of calcium or 1,25-dihydroxyvitamin D. However, ovariectomy increased bone resorption and induced osteopenia whereas prednisolone decreased bone resorption and formation and caused osteopenia. Ovariectomy increased the rate of bone resorption in prednisolone-treated rats; prednisolone lowered the rates of bone resorption and formation in OVX rats. The osteopenic effects of prednisolone and ovariectomy were additive and independent. E2 protected bone from the osteopenic effects of ovariectomy but did not affect bone loss induced by prednisolone. These results suggest prophylactic estrogen should help to avoid bone loss from estrogen deficiency in patients requiring chronic high dose glucocorticoid treatment.     

Differential stress reactivity in intact and ovariectomized prepubertal and adult female rats

The pubertal development of the hypothalamic-pituitary-adrenal (HPA) axis has received relatively little experimental attention. As puberty is marked by an increase in the susceptibility to various psychiatric disorders that may be related to HPA dysfunction, it is imperative to elucidate the pubertal development of this neuroendocrine axis. To date, the limited research in this area has been conducted primarily on males. Presently, we investigated stress responsiveness, as measured by both stress hormones (e.g., corticotropin (ACTH) and corticosterone) and gonadal steroids, in intact and ovariectomized prepubertal and adult female rats before and after a 30-min session of restraint stress. We report here that intact prepubertal females exhibit an extended corticosterone stress response (30-45 min longer) compared to intact adults. Moreover, ovariectomized prepubertal females continue to exhibit a prolonged stress-induced corticosterone and progesterone response compared to ovariectomized adults, indicating these protracted responses prior to puberty are independent of ovarian hormones. ACTH levels were not significantly different between intact and ovariectomized prepubertal and adult animals at all the post-stress time points measured, suggesting that the prolonged corticosterone response in prepubertal females is due to an enhanced sensitivity to ACTH at the level of the adrenal cortex. Taken together, these data indicate that stress reactivity changes dramatically during puberty in females. Furthermore, these data demonstrate additional development of the HPA axis during pubertal maturation, resulting in a more quickly terminated stress response in adulthood.     

Age-related changes in the mechanisms of LHRH-stimulated LH release from pituitary cells in vitro.

In vitro release of LH in response to LHRH, phorbol myristate acetate (PMA), the ionophore A23187, and nifedipine was evaluated in primary cultures of anterior pituitary cells from intact mature (6 to 7 month) and old (23 to 24 month) male Wistar rats. LH release from pituitary cells is reduced approximately 30% and 60% after 4 and 48 h of 10(-7) M LHRH stimulation in cells of old rats, respectively. This impairment may be secondary to a loss of LHRH receptors. LHRH-stimulated LH release from cells of mature rats was inhibited 70% by the voltage-gated calcium channel blocker, nifedipine (10(-6) M), whereas LHRH-stimulated LH release from cells of old rats was too low to detect the effects of this drug. Age changes can be partially reversed by A23187 and PMA during 4 h, but not 48 hrs of stimulation. It therefore appears that short- and long-term (4 h and 48 h, respectively) stimulation of LH release may proceed through separate mechanisms that are differentially affected by aging.     

Quantification of vasoactive intestinal peptide immunoreactivity in the anterior pituitary glands of intact male and female, ovariectomized, and estradiol benzoate-treated rats.

There are considerable data suggesting that vasoactive intestinal peptide (VIP) is involved in the regulation of PRL secretion; however, the role and cell of origin of anterior pituitary VIP remain to be determined. Immunocytochemical (ICC) studies have generally failed to detect VIP-immunoreactive (IR) cells in the pituitary of the untreated rat, although VIP-IR cells have been observed in the pituitaries of hypothyroid or estrogen-treated rats. This study was designed to examine the cellular distribution and tissue content of VIP in the anterior pituitary gland of rats under selected endocrine conditions known to alter the rates of PRL and VIP synthesis and secretion. To this end, anterior pituitary VIP and PRL content (ICC and RIA) and serum PRL levels were determined in ovariectomized (OVX) and OVX rats 3 days after treatment with 7 or 70 micrograms estradiol benzoate (EB). For comparison, pituitary VIP and PRL content (ICC and RIA) and serum PRL levels in untreated male and diestrous female rats were determined. Immunostaining for VIP was accomplished using a newly developed primary antiserum. Significant numbers of VIP-IR cells per 5-microns section were found in the anterior pituitary glands of all animals examined (275 +/- 33 in diestrous to 481 +/- 103 cells in male rats). VIP was not colocalized with PRL in any of the pituitaries regardless of steroid treatment or sex. Furthermore, the number of VIP-IR cells per pituitary gland was not significantly correlated with sex or EB treatment. Treatment with 70 micrograms, but not 7 micrograms, EB significantly increased the pituitary content of VIP and serum PRL levels compared to those after ovariectomy. However, both EB treatments resulted in a significant increase in pituitary PRL content compared to that in untreated OVX rats. Pituitaries from male rats had several-fold more VIP and less PRL content than pituitaries from diestrous rats. These data show that 1) in contrast to previous ICC studies, VIP-IR cells are readily detected in the anterior pituitary of intact male and female and OVX as well as EB-treated rats; 2) VIP is localized to cells other than lactotrophs, regardless of the steroid background; and 3) marked changes in anterior pituitary VIP content are not accompanied by changes in VIP-IR cell number.     

Age-related differences in the spontaneous and thyrotropin-releasing hormone-stimulated release of prolactin and thyrotropin in ovariectomized rats

Effect of estradiol on the spontaneous and thyrotropin-releasing hormone (TRH)-stimulated release of prolactin (PRL) and thyrotropin (TSH) in young and aged ovariectomized (Ovx) rats was investigated. Old (22-26 months) and young (3 months) female rats were Ovx 3 weeks before use. They were injected subcutaneously with estradiol benzoate (EB, 25 micrograms/kg) or sesame oil for 3 days and were catheterized via the right jugular vein. Twenty hours after the last administration of EB, rats were injected with TRH (10 micrograms/kg) through the catheter. Blood samples were collected before and 5, 10, 20, 40 and 60 min after TRH injection. On the day following blood sampling, all rats were decapitated. The anterior pituitary glands (APs) were excised, and incubated with or without TRH (10 ng/ml) at 37 degrees C for 30 min. The basal level of PRL concentration in plasma samples was 5-fold higher in old Ovx rats than in young Ovx rats. Five min after TRH injection, the increase in plasma PRL was greater in old animals than in young animals. Plasma PRL remained higher in old animals than in young animals at 10, 20, 40 and 60 min following TRH challenge. Administration of EB to old and to young Ovx rats produced increases in both basal and TRH-stimulated secretions of PRL, but did not affect the difference in plasma PRL patterns between old and young animals. The release of PRL from APs was increased significantly in all rats after a 30-min incubation with TRH. In Ovx rats injected with oil, the basal release of PRL in vitro was increased with age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of met enkephalin, leu enkephalin and their retro analogs on the lactotropic function of the pituitary gland

When administered subcutaneously to male rats, the novel peptides retromet-enkephalin and retro-leu-enkephalin stimulated prolactin (PRL) release less powerfully than morphine but somewhat greater than met-enkephalin. Leu-enkephalin did not affect PRL secretion in vivo. Neither morphine nor any of opioid peptides under study were able to stimulate basal or the dopamine-inhibited PRL release from cultured adenohypophyseal cells of male rats. It is concluded that stimulatory effects of opiates and opioid peptides on PRL secretion in rats are mediated via the hypothalamus or some other neural structures.