Sex-specific changes in preoptic regulatory factor-1 and preoptic regulatory factor-2 mRNA expression in the rat brain during development

Preoptic regulatory factor-1 (Porf-1) and Preoptic regulatory factor-2 (Porf-2) are two novel peptide genes which are expressed in the central nervous system. Expression is modified by age and by hormones of the reproductive system. In this study nuclease protection assays were employed to investigate Porf-1 and Porf-2 mRNA expression in the cerebral cortex (CC), hippocampus (HIPP), preoptic area (POA) and medial basal hypothalamus (MBH) of male and female rats, aged 15, 30 and 60 days. Porf-1 and Porf-2 mRNA expression tended to decrease from 15 to 60 days, with two exceptions. Porf-2 in the hippocampus of female rats, and Porf-1 in the MBH of the male rats, were found instead to increase with age. There were distinctive sex differences inporf-1 andporf-2 gene expression. Consistently higher mRNA levels were measured for both genes in the POA of female rats at all ages examined, and this difference reached statistical significance for Porf-1 at the age of 60 days. In contrast, levels of Porf-2 mRNA were higher in MBH of male than female rat MBH at 15 days, and Porf-1 mRNA was substantially more abundant in male than in female rat MBH at 30 and 60 days of age. These results indicate that there is sexual dimorphism and regional specificity in the developmental expression of these genes.     

Expression of the preoptic regulatory factor-1 and ?2 genes in rat testis

Hormone-responsive peptides play a vital role in development and regulation of testicular function. The preoptic regulatory factors, porf-1 and porf-2, were originally discovered in the rat brain, but are also expressed in the rat and human testis. In the brain expression is age-related, hormone-responsive, region-specific, and gender-related, suggesting that porf-1 and porf-2 are involved in gender-specific brain development and function. Tissue-specific porf-1 and porf-2 mRNAs are also found in the testis and hypophysectomy may alter testicular porf-2 expression. It was thus of interest to further examine porf-1 and porf-2 expression in the testis to evaluate their potential as hormone-responsive peptides that regulate testicular development and function. Testicular expression of both porf-1 and −2 was analyzed as a function of maturational stage, aging and hypophysectomy by the solution hybridization/nuclease protection assay, and cellular location determined byin situ hybridization histochemistry. Expression was quantitatively compared in normal male rats at 15,30, and 60 d (n=4) and at 2, 6, 12, and 24 mo of age (n=5). During development porf-1 is expressed at a constant level at 15, 30, and 60 d, then declines significantly with advancing age; levels at 24 mo are only 20% of those seen at 2 mo (p<0.05). In contrast, porf-2 expression is highest at 15 d of age and steadily declines at 30 and 60 d, plateaus in the mature adult (6 and 12 mo), then exhibits an additional significant decline in the aged 24 mo animals (6 vs 24 mo,p<0.05). Hypophysectomy of young adult rats at day 42 results in increased testicular expression 12 d later of both porf-1 (p<0.05) and porf-2 (p<0.005) compared to intact 54-d-old rats (n=5).In situ hybridization histochemistry confirms that both porf-1 and porf-2 are expressed in the mature testis at 60 d of age. Porf-2 mRNA is localized to immature germ cells including spermatogonia and primary spermatocytes. Porf-1 mRNA is associated with mature sperm and at low levels in the Sertoli cell cytoplasm surrounding spermatocytes. These data suggest that porf-2 is a pituitary hormone-responsive factor in the developing testis and that both porf-1 and porf-2 have cell-type specific functions in the germ cell compartment of the mature testis.     

Relation of gonadal hormones to differential LH response to naloxone in prepubertal male and female rats

Naloxone (NAL) has been shown to induce LH release in female but not in male rats 10-25 days of age. The purpose of this study was to examine the role of neonatal gonadal hormones on NAL-induced LH release in male and female rats 15, 25, and 35 days of age. On each of these days rats received a s.c. injection of either NAL (5 mg/kg) or physiological saline, and blood was collected 30 min later by decapitation. At 15 days of age, NAL induced LH release in intact and ovariectomized (OVX) female rats, and in male rats castrated (CAST) on the 1st day of life (neonate CAST males). Injection of 10 micrograms estradiol benzoate (EB) 24 h prior to NAL administration blocked NAL-induced LH release in these rats. NAL had no effect on LH release in 15- or 25-day-old intact and CAST male rats or in female rats given 2 mg testosterone propionate at 3 days of age (androgenized female rats). At 35 days of age, NAL induced LH release in intact, OVX, and OVX-EB treated female rats, and in neonate CAST and neonate CAST-EB treated male rats. NAL had no effect on serum LH levels in androgenized female rats. NAL induced LH release in intact and CAST 35-day-old male rats, but pretreatment with estrogen prevented NAL from eliciting LH release. These results indicate that neonatal exposure to androgen is responsible for the sex difference in the LH response to NAL observed in prepubertal male and female rats before 30 days of age.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential gene expression in response to ventricular unloading in rat and human myocardium

Left ventricular unloading by mechanical assist devices induces myocardial atrophy. We aimed to systematically identify differentially expressed genes in a model of physiological atrophy (unloading of healthy rat myocardium) and compare these changes to those in a unloaded, failing human heart. METHODS: Atrophy in rat hearts was induced by heterotopic transplantation of a donor heart into the abdomen of an isogenic recipient. After one week, donor and recipient RNA was isolated. Differential gene expression was assessed by subtractive hybridization. Two screens with radioactive probes were performed to verify differentially expressed clones. Positive clones were sequenced and cDNA of genes of known homology were used as probes for hybridization with RNA from separate atrophied rat hearts and human tissue from a normal, failing or failing and unloaded left ventricle. RESULTS: We picked 1880 clones from the subtractive hybridization procedure (940/940: forward/reverse runs assessing up- or down-regulation, respectively). The first screen verified 465/140 and the second screen verified 67/30 clones. 24/23 clones were sequenced and 14/10 homologies to known genes were found. In the atrophied heart, respiratory chain and metabolic genes were down-regulated (NADH-DH, cytochrome c oxidase, acetyl-CoA synthetase, myoglobin) and cellular recognition and stress genes were up-regulated (MHC1 and 2, HSP70). In the human heart, cytochrome c oxidase, acetyl-CoA synthetase, and myoglobin expression was increased in the failing heart and returned to normal with unloading. Unloading also resulted in up-regulation of HSP70. CONCLUSIONS: The genetic responses of failing human and healthy rat myocardium to mechanical unloading show similarities that appear to be independent of species differences and/or underlying disease. Thus, heterotopic heart transplantation is a relevant model for investigating the mechanisms of mechanical unloading.     

Sexually dimorphic expression of the growth hormone-releasing hormone gene is not mediated by circulating gonadal hormones in the adult rat

The sexual dimorphism characterizing GH secretion in the rat is thought to be related to differences in the hypothalamic synthesis and release of the GH-regulating peptides, GH-releasing hormone (GHRH), and somatostatin. Therefore, the influence of gender and sex steroid hormones on hypothalamic expression of the GHRH gene in adult rats were examined. GHRH messenger RNA (mRNA) levels were measured in individual rat hypothalami by Northern hybridization analysis using a 32P-labeled complementary DNA encoding rat GHRH. Destruction of hypothalamic GHRH neurons by neonatal treatment with monosodium glutamate caused similar 3-fold reductions in the levels of GHRH mRNA in adult male and female animals. In three separate experiments, hypothalamic GHRH mRNA concentrations in male rats were 2- to 3-fold greater than in randomly cycling females (four or five rats per group; P less than 0.01). In spite of the greater abundance of GHRH mRNA abundance in the male rat hypothalamus, circulating gonadal steroids lacked the ability to modulate GHRH gene expression in adult animals, since neither gonadectomy nor pharmacological sex steroid replacement changed GHRH mRNA levels in the hypothalamus of male and female adult rats. Furthermore, GHRH mRNA concentrations in female rats were similar during the proestrus, estrus, and diestrus phase of the estrous cycle. Also, GH inhibited hypothalamic GHRH gene expression in a sex-specific manner. Exposure to high levels of GH secreted by the MtTW15 tumor for 4 weeks reduced GHRH mRNA concentrations 7-fold in male rats (P less than 0.001) but only 2-fold in females (P less than 0.05). These studies demonstrate that GHRH gene expression in the rat hypothalamus is sexually dimorphic. Basal mRNA levels are greater in male rats, and expression in male hypothalami is more sensitive to feedback inhibition by GH than expression in females. There is no evidence for regulation of GHRH mRNA levels by either testosterone or estrogen in adult rats. These gender differences in GHRH gene expression likely contribute to the generation of a sex-specific pattern of GH secretion.     

Differential regulation of female rat olfactory preference and copulatory pacing by the lateral septum and medial preoptic area

We examined the effect of bilateral radiofrequency lesions in the lateral septum (LS) or medial preoptic area (MPOA) on olfactory preference (time spent sniffing odors derived from either a stud or castrated male) and pacing behavior (paracopulatory behavior in a cage with a compartment inaccessible to males) in ovariectomized rats following different hormonal regimes. Sham-operated and LS-lesioned females, but not MPOA-lesioned females, preferred odors from the stud males to the castrated males. MPOA lesions significantly decreased total nose-poking time, compared to that of sham-operated females. When mounted by stud males, both LS- and MPOA-lesioned females showed significantly higher lordosis quotients than sham-operated females. On the other hand, LS-lesioned females spent a significantly longer time in the male compartment, and stayed with the males even after they were mounted. These results suggest that the LS and MPOA play different roles in the sociosexual activity in female rats, and that the two regions exert an inhibitory influence on lordosis.     

Regulation of Kiss1 gene expression in the brain of the female mouse

The Kiss1 gene encodes a family of neuropeptides called kisspeptins, which activate the receptor G protein-coupled receptor-54 and play a role in the neuroendocrine regulation of GnRH secretion. We examined whether estradiol (E2) regulates KiSS-1 in the forebrain of the female mouse by comparing KiSS-1 mRNA expression among groups of ovary-intact (diestrus), ovariectomized (OVX), and OVX plus E2-treated mice. In the arcuate nucleus (Arc), KiSS-1 expression increased after ovariectomy and decreased with E2 treatment. Conversely, in the anteroventral periventricular nucleus (AVPV), KiSS-1 expression was reduced after ovariectomy and increased with E2 treatment. To determine whether the effects of E2 on KiSS-1 are mediated through estrogen receptor (ER)alpha or ERbeta, we evaluated the effects of E2 in OVX mice that lacked functional ERalpha or ERbeta. In OVX mice that lacked functional ERalpha, KiSS-1 mRNA did not respond to E2 in either the Arc or AVPV, suggesting that ERalpha is essential for mediating the inhibitory and stimulatory effects of E2. In contrast, KiSS-1 mRNA in OVX mice that lacked functional ERbeta responded to E2 exactly as wild-type animals. Double-label in situ hybridization revealed that virtually all KiSS-1-expressing neurons in the Arc and AVPV coexpress ERalpha, suggesting that the effects of E2 are mediated directly through KiSS-1 neurons. We conclude that KiSS-1 neurons in the Arc, which are inhibited by E2, may play a role in the negative feedback regulation of GnRH secretion, whereas KiSS-1 neurons in the AVPV, which are stimulated by E2, may participate in the positive feedback regulation of GnRH secretion.     

Proteins regulated by gonadal steroids in the medial preoptic and ventromedial hypothalamic nucleic of male and female rats

Protein profiles of brain areas mediating effects of steroid hormones on copulation were compared between animals in gonadal steroid states predictive of either the presence or absence of copulatory activity. A broad range of proteins present in micropunches of tissue from the medial preoptic area (MPO) and from the ventromedial hypothalamus (VMH) were compared between male and female rats with gonadal steroids present or absent. Half of the animals of each gender were gonadectomized 1 month prior to sacrifice. The remaining males were left intact, while the remaining females were gonadectomized, implanted with estrogen capsules, and injected with progesterone prior to sacrifice. These females were screened for sexual receptivity immediately prior to sacrifice. Proteins from the MPO and VMH of each animal were separated by two-dimensional gel electrophoresis, silver stained, and quantified by computerized optical densitometry. Several proteins differed in density between gels of high-steroid males and and females and between high-steroid and absent-steroid animals of one or both genders. Two previously reported sex differences were replicated and found to depend on activational effects of gonadal steroids. Several interesting reversal patterns were noted between MPO and VMH, including three proteins that were affected by gonadectomy in the MPO of males, but not females, and in the VMH of females, but not males, thus correlating with sexual function. These included serum albumin (a possible index of local area blood flow) and neuron-specific enolase, a glycolytic enzyme of anaerobic metabolism. A probable genetic polymorphism was discovered at a locus whose expression appears to be regulated by gonadal steroids.(ABSTRACT TRUNCATED AT 250 WORDS)     

Postnatal development of gonadotropin-releasing hormone and cyclophilin gene expression in the female and male rat brain

The rat preoptic area-anterior hypothalamic continuum (POA-AH) contains about 400-800 neurons that express the decapeptide GnRH and the 56-amino-acid GnRH-associated peptide. Originating from the olfactory placode, these neurons migrate and establish their final distribution and connections in the POA-AH several days before birth. The aim of the present study was to examine whether the biosynthesis of the mRNA encoding the precursor (proGnRH) common to GnRH and GnRH-associated peptide undergoes postnatal changes corresponding to the development of sexual maturation. The POA-AH content of proGnRH messenger RNA (mRNA) was followed from postnatal day 1 to day 90 in female and male Sprague-Dawley rats killed by decapitation between 1000-1200 h. Cytoplasmic RNA fractionated from individual POA-AH homogenates was purified using proteinase K digestion. Cytoplasmic proGnRH mRNA was quantitated simultaneously with cyclophilin mRNA (an internal standard control) using solution hybridization-RNase protection assay, with the protected fragments separated through polyacrylamide gel electrophoresis. In the POA-AH, the concentrations of proGnRH mRNA (femtograms mRNA per microgram total RNA) increased significantly with age in both sexes (P less than 0.001). In males, proGnRH mRNA levels increased by day 30 some 2-fold over the values of days 5 and 10, and the levels established on day 30 were maintained through adulthood. In females, the first rise in proGnRH mRNA levels occurred on day 30, followed by an additional increase on day 45 to levels seen in adulthood. Levels of proGnRH mRNA established in adulthood were significantly higher in females than in males (P less than 0.03). The concentrations of cyclophilin mRNA (picograms mRNA per microgram total RNA) remained essentially unchanged in both sexes during the same period of time when proGnRH mRNA levels were increasing. These results provide evidence for postnatal sex-related increases in the levels of proGnRH mRNA in the rat POA-AH, which are likely to reflect differential regulation by gonadal steroids.     

Sexual differentiation of Kiss1 gene expression in the brain of the rat

The Kiss1 gene codes for kisspeptins, which have been implicated in the neuroendocrine regulation of reproduction. In the brain, Kiss1 mRNA-expressing neurons are located in the arcuate (ARC) and anteroventral periventricular (AVPV) nuclei. Kiss1 neurons in the AVPV appear to play a role in generating the preovulatory GnRH/LH surge, which occurs only in females and is organized perinatally by gonadal steroids. Because Kiss1 is involved in the sexually dimorphic GnRH/LH surge, we hypothesized that Kiss1 expression is sexually differentiated, with females having more Kiss1 neurons than either males or neonatally androgenized females. To test this, male and female rats were neonatally treated with androgen or vehicle; then, as adults, they were left intact or gonadectomized and implanted with capsules containing sex steroids or nothing. Kiss1 mRNA levels in the AVPV and ARC were determined by in situ hybridization. Normal females expressed significantly more Kiss1 mRNA in the AVPV than normal males, even under identical adult hormonal conditions. This Kiss1 sex difference was organized perinatally, as demonstrated by the observation that neonatally androgenized females displayed a male-like pattern of adulthood Kiss1 expression in the AVPV. In contrast, there was neither a sex difference nor an influence of neonatal treatment on Kiss1 expression in the ARC. Using double-labeling techniques, we determined that the sexually differentiated Kiss1 neurons in the AVPV are distinct from the sexually differentiated population of tyrosine hydroxylase (dopaminergic) neurons in this region. Our findings suggest that sex differences in kisspeptin signaling from the AVPV subserve the cellular mechanisms controlling the sexually differentiated GnRH/LH surge.     

Differential regulation of anterior pituitary prodynorphin and gonadotropin-subunit gene expression by steroid hormones.

Prodynorphin is expressed by neurons of the hypothalamus and gonadotrophs of the anterior pituitary gland (AP) and plays a role in the negative feedback regulation of the reproductive neuroendocrine axis. The present study examined whether gonadal steroid hormones are capable of modulating pituitary prodynorphin expression in immature, female rats. Steroids were administered via subcutaneous Silastic implants and rats were killed at 29 days of age. Northern blot analysis was used to measure AP prodynorphin, luteinizing hormone-beta (LH beta), follicle-stimulating hormone-beta (FSH beta), and common alpha-subunit mRNA levels (normalized to 18S ribosomal RNA). Treatment groups (n = 5-6) consisted of control (CNT; empty implants), estradiol (E2; 4 days), E2 + progesterone (E2 + P4; 8 days and 4 days, respectively), and dihydrotestosterone (DHT; 4 days). Pituitary prodynorphin mRNA was significantly suppressed in only the DHT-treated animals (26 +/- 10% of CNT, p < 0.01). LH beta mRNA was suppressed by all steroid treatments (p < 0.01), FSH beta was lower in only the E2 group, and alpha-subunit was reduced in both the E2 + P4 and DHT groups (p < 0.01). Serum LH was suppressed by all steroid treatments but FSH was reduced in only the E2 and E2 + P4 groups (p < 0.01). Treatment of prepubescent rats with continuous high levels of gonadal steroids is known to severely reduce endogenous hypothalamic gonadotropin releasing hormone (GnRH) release and this is supported by our observation of reduced gonadotropin-subunit gene expression.(ABSTRACT TRUNCATED AT 250 WORDS)     

Differential cytokine gene expression in the diaphragm in response to strenuous resistive breathing

Strenuous resistive breathing induces plasma cytokines that do not originate from circulating monocytes. We hypothesized that cytokine production is induced inside the diaphragm in response to resistive loading. Anesthetized, tracheostomized, spontaneously breathing Sprague-Dawley rats were subjected to 1, 3, or 6 hours of inspiratory resistive loading, corresponding to 45-50% of the maximum inspiratory pressure. Unloaded sham-operated rats breathing spontaneously served as control animals. The diaphragm and the gastrocnemius muscles were excised at the end of the loading period, and messenger ribonucleic acid expression of tumor necrosis factor-alpha, tumor necrosis factor-beta, interleukin (IL)-1alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, IFN-gamma, and two housekeeping genes was analyzed using multiprobe RNase protection assay. IL-6, IL-1beta, and, to lesser extents, tumor necrosis factor-alpha, IL-10, IFN-gamma, and IL-4 were significantly increased in a time-dependent fashion in the diaphragms but not the gastrocnemius of loaded animals or in the diaphragm of control animals. Elevation of protein levels of IL-6 and IL-1beta in the diaphragm of loaded animals was confirmed with immunoblotting. Immunostaining revealed IL-6 protein localization inside diaphragmatic muscle fibers. We conclude that increased ventilatory muscle activity during resistive loading induces differential elevation of proinflammatory and antiinflammatory cytokine gene expression in the ventilatory muscles.     

高脂膳食对小鼠脑内铁调节蛋白-2基因表达的影响

目的 探讨高脂膳食对脑老化及脑铁代谢的影响,为合理膳食预防脑老化提供科学依据.方法 24只ICR小鼠分4组:脑老化模型组、高脂膳食组、脑老化+高脂膳食组和对照组.连续造模10 w后,以RT-PCR法检测各组脑内铁调节蛋白-2(IRP-2)表达水平.结果 脑老化组和高脂膳食组小鼠IRP-2表达水平均低于对照组且差异显著(P＜0.05);高脂膳食组与脑老化模型组、脑老化+高脂膳食组间IRP-2表达无显著差异(P＞0.05).结论 高脂膳食可降低小鼠脑内IRP-2表达,这可能是高脂膳食诱发脑老化及神经退行性变的机制之一.     

Development of gonadal feedback regulation of gonadotropin gene expression and secretion in female rats.

The postnatal development of the gonadal negative feedback control of gonadotropins was studied in female rats. Neonatal (5-day-old) and randomly cycling young (60-day-old) and more mature (180-day-old) adult rats were ovariectomized, and half of them received Silastic implants containing the synthetic estrogen, diethylstilbestrol. The neonatal rats were killed 5, 10 or 15 days, and the adult rats 7 days after the operation. Age-matched and sham-operated animals served as controls. There were no statistically significant responses of serum LH or FSH concentrations or of the pituitary gonadotropin subunit mRNA levels to ovariectomy at any of the neonatal ages. A marked increase (p < 0.01) after ovariectomy was seen in serum gonadotropins and in the cognate mRNA levels at both adult ages. In spite of the weak feedback response of the neonatal rats to ovariectomy, diethylstilbestrol suppressed the basal pituitary gonadotropin concentrations and the specific LH and FSH beta-chain mRNAs (p < 0.01-0.05). These results demonstrate that the gonadal negative feedback regulation of gonadotropin synthesis and secretion is not fully developed in neonatal and prepubertal female rats before 20 days of age. This is probably due to the steroidogenic quiescence of the ovaries in early life. However, the capability of the pituitary to respond to negative estrogen feedback has developed in the neonatal female, as demonstrated by the suppressive effects of diethylstilbestrol treatment on gonadotropin secretion.     

Pituitary and gonadal response to LH releasing hormone administration in the female and male cheetah

Luteinizing hormone releasing hormone (LHRH, 50 micrograms) or saline was administered (i.m.) to adult female and male cheetahs under anaesthesia to evaluate pituitary and gonadal response. Serum LH levels did not fluctuate over a 120-min sampling period in saline-treated animals. Serum LH concentrations were raised (P less than 0.05) in both female and male cheetahs after LHRH injection, the temporal response being similar to previously reported results in unanaesthetized, domestic carnivores. The magnitude of the LHRH-induced LH response was sex-dependent. Over a 120-min post-injection period both saline control and LHRH-induced LH levels were about twofold greater in males than females. Although LHRH had no acute influence on ovarian oestradiol-17 beta production in the female, serum testosterone levels were raised (P less than 0.05) in male cheetahs by 60 min after treatment. This study (1) provides introductory endocrine information on the cheetah, an endangered species, and (2) indicates that exogenous LHRH is effective in acutely altering pituitary (female) and pituitary/gonadal (male) function in an anaesthetized, non-domestic felid.     

Effects of thyroid hormones on angiotensinogen gene expression in rat liver, brain, and cultured cells.

We examined the effect of chronic hypo- and hyper- thyroidism on angiotensinogen (AOG) gene expression in rat liver and brain. Chronic hypothyroidism resulted in approximately a 50% decrease in plasma AOG and AOG messenger RNA (mRNA) concentrations in liver, diencephalon, and brain stem. In contrast, plasma AOG and liver AOG mRNA concentrations were elevated by about 75% during hyperthyroidism, but no change was seen in diencephalon and brain stem. In vitro, the effect of T3 on AOG secretion by rat hepatoma cell lines H35 and H4IIEC-3 depended on the type of cell line used and on the growth status of the cells. At confluency, H35 cells were more responsive to T3 than H4IIEC-3 cells. In addition, subconfluent H35 cells were less responsive to T3 than confluent ones, although no difference was observed in the number of nuclear T3 binding sites or in the responsiveness to dexamethasone. T3 also increased AOG mRNA concentration in confluent H35 cells. Finally, AOG secretion by primary cultures of rat astrocytes increased approximately 1.8-fold following exposure to T3. The fact that T3 increased the production of AOG by these various types of cell culture in vitro suggests that it acted directly upon these cells, and that the effect of thyroid hormone was not dependent on the prior stimulation of another hormone. However, the difference in responsiveness between confluent and subconfluent H35 cells indicates that the action of thyroid hormones may be dependent on the induction of secondary genes within these cells.