Young Adult Female Rats Research Articles

Correlation of estrogen β-receptor messenger RNA with endogenous levels of plasma estradiol and progesterone in the female rat hypothalamus, the bed nucleus of stria terminalis and the medial amygdala

Estrogen receptor β (ERβ) has been previously mapped in the rat central nervous system. This study aims to explore the regulation of ERβ mRNA as it is expressed in the intact and cycling female rat brain. Young adult female rats (90+ day, N=20) were screened for estrous phases via vaginal cytology and sacrificed. Brains and blood were collected and processed for in situ hybridization and estradiol (E2) and progesterone (P4) hormone assays, respectively. ERβ mRNA levels exhibited significant correlations with ovarian steroid ratios (E2/P4) in various brain regions, including the bed nucleus of stria terminalis, the medial nucleus of amygdala, and the anteroventral periventricular nuclei but not the paraventricular and the supraoptic nuclei or the preoptic area of the hypothalamus. No regulatory changes were detected in the cortex. Specifically, in the affected regions, higher P4 levels were significantly correlated with higher ERβ mRNA expression. In contrast, there was a tendency for higher E2 levels to be correlated with lower ERβ mRNA expression, but this tendency reached significance only in the bed nucleus of stria terminalis. These results suggest that ERβ mRNA is regulated in the intact and cycling female rat hypothalamic as well as extrahypothalamic brain regions, and the circulating ovarian hormones play a critical role.

Effect of Zn deficiency and subsequent Zn repletion on bone mineral composition and markers of bone tissue metabolism in 65Zn-labelled, young-adult rats.

The objective of the present study was to investigate the effect of changing skeletal Zn load (mobilization/restoring) on bone mineral composition and bone tissue metabolism. For this purpose, 36 65Zn-labelled, young-adult female rats were fed with either a purified diet with sufficient Zn (21 microg/g, control) for 26 days, or deficient Zn (1.4 microg/g) for 12 days followed by 14 days repletion with the control diet. The animals were killed at the onset of the study (reference: n=4), at the end of the Zn deficiency episode (control: n=4; Zn deficiency: n=4), subgroups (n=4) of Zn repleted animals at repletion days 2, 4, 7, 10 and 14, and at day 14 the remaining controls also (n=4). Zn deficiency reduced skeletal Zn concentration from 198 to 155 microg/g of bone dry matter. About half of mobilized skeletal Zn was refilled within 2 days of repletion and was completely restored until the end of the study. Concentrations of bone ash, Ca, P and Mg remained constant (means in bone dry matter: 51% bone ash, 191 mg Ca/g, 95 mg P/g, 4.4 mg Mg/g). Blood plasma concentrations of osteocalcin and daily urinary excretions of pyridinoline PYD and dexoxypyridinoline DPD were unaffected by treatment (mean: 57 ng/ml, 222 nmol/day, 137 nmol/day). Also daily urinary excretions of Ca, P and Mg remained fairly constant (means: 0.26 mg/day, 16 mg/day, 1.5 mg/day). 65Zn autoradiography of femur sections revealed a pronounced Zn exchange in the area of the metaphysis and epiphysis. We conclude that transient mobilization and restoration of skeletal Zn occurs mainly in trabecular bone, and does not involve major changes in bone mass, macro mineral content, or bone tissue turnover in young-adult rats.

Functional Integrity of ErbB-4/-2 Tyrosine Kinase Receptor Complex in the Hypothalamus Is Required for Maintaining Normal Reproduction in Young Adult Female Rats

Functional Integrity of ErbB-4/-2 Tyrosine Kinase Receptor Complex in the Hypothalamus Is Required for Maintaining Normal Reproduction in Young Adult Female Rats

Functional integrity of ErbB-4/-2 tyrosine kinase receptor complex in the hypothalamus is required for maintaining normal reproduction in young adult female rats.

ErbB-1 tyrosine kinase receptors are necessary for maintaining female reproduction by modulating the release of LH-releasing hormone (LHRH). Changes in ErbB-1 signaling capacity in aging rats are linked to compromised reproduction. The interactive and synergistic nature of different members of ErbB receptors in mediating signal transduction exists in many cellular systems. Particularly, the interactions among ErbB-1 and ErbB-2 or ErbB-4 and ErbB-2 are known to be involved in the stimulation of LHRH secretion during sexual maturation. Thus, ErbB-4/-2 receptors may also play a role in maintaining reproduction during adulthood, and consequently, alteration in ErbB-4/-2 signaling capacity may contribute to compromised reproductive competence during aging. By in situ hybridization histochemistry, ErbB-4/-2 mRNAs were detected in the preoptic area (POA) and arcuate nucleus, which are important areas involved in the control of LHRH neuronal activity. RT-PCR analyses showed that levels of ErbB-4/-2 mRNA increased to a maximal value in the POA of young adult animals before the LH surge. However, no such increase was found in middle-aged female rats. The timing of the decrease in ErbB-4 mRNA in the median eminence-arcuate nucleus of middle-aged rats was delayed compared with that in young adult animals. Disruption of functional ErbB-4/-2 receptor complex by blocking ErbB-2 receptor synthesis in the hypothalamus via an infusion of ErbB-2 antisense oligodeoxynucleotide resulted in an estrous acyclicity in young adult rats. These results indicate that changes in ErbB-4/-2 gene expression and functional integrity of this ErbB-4/-2 receptor complex in the hypothalamus of middle-aged female animals may lead to an altered preovulatory LH release. Thus, the ErbB-4/-2 receptor complex is a physiological component necessary for maintaining female reproduction.

Altered gene activity of epidermal growth factor receptor (ErbB-1) in the hypothalamus of aging female rat is linked to abnormal estrous cycles.

Activation of the ErbB-1 receptor is necessary for initiating mammalian female puberty by stimulating the release of LH-releasing hormone. It remains unclear whether ErbB-1 is also required in governing reproduction during adulthood and whether altered ErbB-1 signaling is linked to changes in gonadotropin secretion in aging females. The present study examined these issues. RT-PCR was employed to determine changes in ErbB-1 mRNA levels during proestrus in both young adult (YA) and middle-aged (MA) female rats. Before the LH surge, expression levels in the preoptic area of YA rats increased to a maximal value. No such increase in ErbB-1 mRNA was found in MA rats. This difference was confirmed by the analysis of in situ hybridization histochemistry, where a stronger mRNA signal was observed in the preoptic area of YA rats compared with MA females. ErbB-1 protein levels measured by Western blot reflected this difference. A peak level of ErbB-1 mRNA in the median eminence-arcuate nucleus was detected at 0800 h in YA rats, but it was delayed in MA animals. There were intense ErbB-1 mRNA-positive cells in the arcuate nucleus. Pharmacological blockade of ErbB-1 receptor-mediated signal transduction resulted in the disruption of estrous cyclicity in YA rats. These results indicate that ErbB-1 receptors are necessary for maintaining normal estrous cycles. Consequently, age-related alterations in hypothalamic ErbB-1 gene activity may contribute to a delayed preovulatory LH secretion in aging females. Thus, the ErbB-1 signaling system plays an important role in the control of female reproduction during adulthood.

Estrogen-induced changes in place and response learning in young adult female rats.

Many findings suggest that changes in circulating estrogen levels influence cognition, in some cases impairing performance and in others enhancing performance. One interpretation of these mixed effects is that estrogen biases the strategy used to solve a task. To test this idea, young adult female rats, ovariectomized for 21 days, were trained after acute hormone or control treatment in 2 very similar tasks with different cognitive requirements. One task required place learning and the other response learning. Rats given two 10-microg injections of estradiol 48 and 24 hr before training learned the place task significantly faster than did rats without estradiol. Conversely, rats without estradiol performed better on the response task than did rats with replacement. These data suggest that the cognitive actions of estrogen may be task-specific by modulating the relative contribution of different learning and memory systems.

Selected contribution: estrogen receptor-alpha gene transfer inhibits proliferation and NF-kappaB activation in VSM cells from female rats.

Epidemiological studies have demonstrated that hormone replacement therapy with estrogen (E2) or E2 plus progesterone in postmenopausal women decreases the age-associated risk of cardiovascular disease by 30-50%. Treatment of vascular smooth muscle (VSM) cells with physiological concentrations of E2 has been shown to inhibit growth factor-stimulated cell proliferation. In this study, we tested the hypothesis that E2 inhibits the age-associated increase in VSM cell proliferation by inhibiting nuclear factor (NF)-kappaB pathway. We investigated the effects of E2 treatment and adenovirus-mediated estrogen receptor (ER)-alpha gene transfer on cell proliferation and NF-kappaB activation using VSM cells cultured from 3-mo-old and 24-mo-old Fischer 344 female rats. Our results demonstrate that VSM cell proliferation was significantly increased (P < 0.05) in aged compared with young adult female rats. Treatment of VSM cells with physiological concentrations of E2 inhibited VSM cell proliferation, and this inhibition was significantly greater (P < 0.05) in cells from aged female rats compared with young adults. The inhibitory effects of E(2) on cell proliferation in aged female rats were significantly potentiated by overexpression of the human ER-alpha gene into VSM cells. Constitutive and interleukin (IL)-1beta-stimulated NF-kappaB activation was significantly greater (P < 0.05) in VSM cells from aged compared with young female rats. E2 treatment of VSM cells from aged female rats inhibited both constitutive and IL-1beta-stimulated NF-kappaB activation. ER-alpha gene transfer into VSM cells from aged female rats further augmented the inhibitory effects of E2. In conclusion, our data demonstrate that constitutive and IL-1beta-stimulated NF-kappaB activation is increased in VSM cells from aged female rats due to loss of E2 and this can be restored back to normal levels by ER-alpha gene transfer and E2 treatment. In addition, increased NF-kappaB signaling may be responsible for increased incidence of cardiovascular disease in postmenopausal females.

Immunohistochemical localization of beta 1-adrenergic receptors in the liver of male and female F344 rat.

The distribution of beta(1)-adrenergic receptors in the liver of Fischer 344 (F344) rat has been examined by an immunohistochemical method. The study was carried out on formalin-fixed and paraffin-embedded livers from young adult, middle-aged, and old female and male F344 rats. An antibody specific for the beta(1)-adrenoreceptor subtype was used. A positive reaction was found in the liver parenchyma of female and male rats from all age groups. Within the liver lobule, a clear zonation is observed, with the beta(1)-adrenoreceptor positivity most evident in pericentral zone hepatocytes and a gradual fading of the immunostaining from pericentral to periportal zone hepatocytes, which may be completely negative. Immunoreactivity is localized on the cell membrane and on the membrane of peripheral cytoplasmic vesicles, and is mostly confined to the cell side facing vascular space. The intensity of immunostaining seems to be slightly higher in the 6- and 10-month-old female rats as compared to the matched male rats and to the senescent female rats. No age-related changes in the intensity of immunostaining are appreciable in male rats. However, no definite conclusion could be drawn about the existence of gender-related differences or age-related changes in the density of beta(1)-adrenoreceptors. A low density of beta1-adrenoreceptor was observed in the spontaneous preneoplastic lesions of the livers from senescent rats.

Macrophage depletion impairs oligodendrocyte remyelination following lysolecithin-induced demyelination.

An association between macrophages and remyelination efficiency has been observed in a variety of different models of CNS demyelination. In order to test whether this association is causal or coincidental, we have examined the effects of macrophage depletion on the rate of remyelination of lysolecithin-induced demyelination in the spinal cord of young adult female rats. Macrophage depletion was achieved by reducing the monocyte contribution to the macrophages within the lesion using the clodronate-liposome technique. This technique not only resulted in a decrease in Ox-42-positive cells in the spleen of treated animals but also in the levels of macrophage scavenger receptor type B mRNA expression within the demyelinating lesion. In animals treated with clodronate-liposomes throughout the remyelination process, there was a significant decrease in the extent of oligodendrocyte remyelination at 3 weeks after lesion induction, but no effect on Schwann cell remyelination. If macrophage depletion was delayed until the second half of the remyelination phase, then there was no effect on the repair outcome, implying that macrophages are required for the early stages of CNS remyelination. The results of this study indicate that the macrophage response is an important component of successful CNS remyelination and that approaches to the treatment of demyelinating disease based on inhibition of the inflammatory response may also impair regenerative events that follow demyelination.

Enhanced anticonvulsant activity of neuroactive steroids in a rat model of catamenial epilepsy.

Perimenstrual catamenial epilepsy may in part be due to withdrawal of the endogenous progesterone-derived neurosteroid allopregnanolone that potentiates gamma-aminobutyric acidA (GABA(A)) receptor-mediated inhibition. Here we sought to determine whether the anticonvulsant potencies of neuroactive steroids, benzodiazepines, phenobarbital (PB), and valproate (VPA) are altered during the heightened seizure susceptibility accompanying neurosteroid withdrawal in a rat model of perimenstrual catamenial epilepsy. Test drugs were evaluated for their ability to alter the convulsant activity of pentylenetetrazol (PTZ) in young adult female rats, in pseudopregnant rats with prolonged exposure to high levels of progesterone (and its neurosteroid metabolites), and in pseudopregnant rats 24 h after acute withdrawal of neurosteroids by treatment with the 5alpha-reductase inhibitor finasteride. Test drugs were administered at doses equivalent to twice their ED50 values for protection against PTZ-induced clonic seizures in naive young adult female rats. The anticonvulsant activity of allopregnanolone (5 mg/kg, s.c.), pregnanolone (5 mg/kg, s.c.), allotetrahydrodeoxycorticosterone (15 mg/kg, s.c.), and tetrahydrodeoxycorticosterone (10 mg/kg, s.c.) were enhanced by 34-127% after neurosteroid withdrawal. The anticonvulsant activity of PB (65 mg/kg, i.p.) was also enhanced by 24% in neurosteroid-withdrawn animals. In contrast, the anticonvulsant activity of diazepam (4 mg/kg, i.p.), bretazenil (0.106 mg/kg, i.p.), and VPA (560 mg/kg, i.p.) were reduced or unchanged in neurosteroid-withdrawn animals. The anticonvulsant activity of neuroactive steroids is potentiated after neurosteroid withdrawal, supporting the use of such agents in the treatment of perimenstrual catamenial epilepsy.

Aging and gender affect the response of thyrotropin (TSH) to gastrin releasing peptide (GRP) in rats

We had previously shown that GRP acts directly at the pituitary gland inhibiting basal and TRH-stimulated TSH secretion in adult male rats. In this study we showed a gender dimorphism in this response of old animals pituitaries to GRP. In both female and male young adult animals, GRP-incubated pituitaries showed approximately 50% less basal and TRH-stimulated TSH secretion to the medium, without affecting the pituitary content of TSH. However, GRP did not have any significant effect upon TSH secretion in old male rats, but the old female showed the same degree of response to GRP as the young adult female rat, regarding basal and TRH-stimulated TSH secretion, while the TSH pituitary content after GRP incubation was higher than that of the young female group. Our data suggest a loss of thyrotrope responsiveness to GRP in aged male rats that could contribute to the decrease in TSH pituitary stores leading to lower basal and TRH-stimulated TSH secretion. Meanwhile, the preservation of GRP responsiveness could help in the relative maintenance of these parameters in the old female rat.

Memory retention is modulated by acute estradiol and progesterone replacement.

Ovarian hormones alter spine density of hippocampal granule and pyramidal cells in young adult and aging female rats (P. Miranda, C. L. Williams, & G. Einstein, 1999; C. S. Woolley, 1998). The present study used a delayed matching-to-place version of the water maze to investigate a behavioral correlate of these hormone-induced changes in hippocampal connectivity in 3- and 8-month-old female rats. When primed with 10-microg injections of estradiol 72 and 48 hr before testing, the memory retention of ovariectomized rats was improved compared with retention after priming with oil. A single injection of progesterone maintained this enhancement if testing occurred within 8 hr of the progesterone injection but not if testing occurred more than 24 hr after the progesterone injection. These findings indicate that estradiol and progesterone alter memory retention and suggest that these changes may be the result of hormone-induced increases in hippocampal connectivity.

Age-related differences in the toxicity of ochratoxin A in female rats

Ochratoxin A (OTA) is a mycotoxin found in food and feedstuffs of plant and animal origin. OTA exposure is related to nephropathy in humans. Age-related differences, especially in nephro- and immunotoxicity of OTA, were investigated in young adult (aged 12 weeks) and old (aged 27–30 months) female SPF Wag rats, treated by gavage with 0, 0.07, 0.34 or 1.68 mg OTA/kg body weight for 4 weeks. In both age groups, survival was significantly decreased in the highest dose group. Clinical condition, body weight, clinical chemistry parameters (ALAT, ASAT, creatinin and urea) and target organs (as identified by weight and pathology — kidney, liver, adrenals, forestomach and brain) were affected by age and dose, but often more severely in old than in young rats. OTA induced primarily nephropathy. Old rats were more sensitive to induction of tubular karyomegaly and vacuolation/necrosis. In young rats, OTA induced a dose-related thickening of the basement membrane and reduction in splenic T-cell fraction. Decreased IgG levels were seen at 0.34 mg/kg OTA (young and old rats) and 1.68 mg/kg OTA (young rats). Vacuolation of the white brain matter (cerebellar medulla and ventral parts of the brain stem) was significantly increased in young rats at 0.34 and 1.68 mg/kg OTA and in old rats at 0.07 and 0.34 mg/kg OTA. It was concluded that: (1) the profiles of OTA toxicity for both age groups are similar, with the kidney and possibly the brain being primary target organs; (2) based on clinical and pathological data old rats are more sensitive to OTA than young rats; and (3) the immune system is probably not the primary target of OTA toxicity.

Variations of Lacrimal Fluid Peroxidase Activity in Female and Male Rats

Previous research from this laboratory showed that human lacrimal fluid peroxidase has cyclic variations during the menstrual cycle, correlated with plasma levels of 17β-oestradiol. In the present investigation, variations of enzyme activity and total protein content during the oestrous cycle of young adult female rats are analysed. Effects from circadian rhythm and a gender-related influence are also examined. In female rats, as in women, lacrimal fluid peroxidase activity shows cyclic variations; in fact, it significantly (p < 0.05) changes during the different phases of the oestrous cycle. In contrast, in males such variations do not occur. Thus, we suggest that gender seems to exert a significant influence on the secretion of this specific tear protein, probably by a direct effect of oestrogens.

Bismuth biokinetics and kidney histopathology after bismuth overdose in rats.

Bismuth induced nephrotoxicity has been reported to occur after acute overdoses of Bi-containing therapeutic drugs. We studied the development of bismuth induced nephropathy and bismuth biokinetics in rats. Bismuth nephropathy was induced in 33 young adult female Wistar rats weighing ca. 175 g by feeding them a single overdose of colloidal bismuth subcitrate containing 3.0 mmol Bi/kg at (t = 0). Control animals (n = 7) were fed the vehicle only. The animals were sacrificed after 1-48 h. Plasma creatinine increased from 51 +/- 6 micromol/l at t = 0 to 550 +/- 250 micromol/l after 48 h in the experimental group. The S3 segment of the proximal tubule showed epithelial cell vacuolation after 1 h and necrosis after 3 h. Cells of the S1/S2 segment demonstrated vacuolation after 6 h and necrosis after 12 h. Biokinetics of bismuth in blood could best be described with a one-compartment model characterized by an absorption half-life of 0.32 h and an elimination halflife of 16 h. The peak concentration of about 7.0 mg Bi/l was reached after 2 h. In conclusion, cells of the S3 segment of the proximal tubule necrotized first after an oral colloidal bismuth subcitrate overdose and biokinetics of Bi in blood was best described by a one-compartment model.

Endocrine regulation and extended follow up of longitudinal growth in intrauterine growth-retarded rats.

Bilateral uterine artery ligation in late gestation was performed in pregnant dams in order to determine the effects of intrauterine growth retardation (IUGR) on long-term postnatal somatic growth and the GH neuroendocrine axis in the adult female and male rat. Body weight (BW), nose-anus length (NAL) and tail length (TL) were recorded at regular intervals in both the IUGR and control (CON) offspring until the age of 93 days. Spontaneous 6-h GH secretory profiles and serum IGF-I were determined around the age of 100 days in both the IUGR and the CON group. No catch-up growth in BW, NAL or TL was observed in young adult male IUGR rats. Female IUGR rats did catch up in NAL beyond the age of 57 days and in TL before weaning, but did not catch up at any time in BW. Spontaneous 6-h GH secretory profiles in female and male IUGR rats at a mean age of 100+/-4 days were similar to their controls at a mean age of 101+/-4 days. Overall median 6-h rat GH plasma concentrations, rat GH peak amplitude, number of rat GH peaks and sum of peak area were not significantly different. Median serum IGF-I levels in young adult female and male IUGR rats showed no difference when compared with their respective controls. These results demonstrate that IUGR, after bilateral uterine artery ligation in late gestation, leads to incomplete BW catch-up growth in young adult rats of both sexes with physiological GH/IGF-I secretion, suggesting intrauterine modulation of tissue responsiveness to GH and IGF-I. Female IUGR rats do catch up in NAL and TL, developing disturbed body proportions.

The GH secretagogues ipamorelin and GH-releasing peptide-6 increase bone mineral content in adult female rats.

Growth hormone (GH) is of importance for normal bone remodelling. A recent clinical study demonstrated that MK-677, a member of a class of GH secretagogues (GHSs), increases serum concentrations of biochemical markers of bone formation and bone resorption. The aim of the present study was to investigate whether the GHSs, ipamorelin (IPA) and GH-releasing peptide-6 (GHRP-6), increase bone mineral content (BMC) in young adult female rats. Thirteen-week-old female Sprague-Dawley rats were given IPA (0.5 mg/kg per day; n=7), GHRP-6 (0.5 mg/kg per day; n=8), GH (3.5 mg/kg per day; n=7), or vehicle administered continuously s.c. via osmotic minipumps for 12 weeks. The animals were followed in vivo by dual X-ray absorptiometry (DXA) measurements every 4th week. After the animals were killed, femurs were analysed in vitro by mid-diaphyseal peripheral quantitative computed tomography (pQCT) scans. After this, excised femurs and vertebrae L6 were analysed by the use of Archimedes' principle and by determinations of ash weights. All treatments increased body weight and total tibial and vertebral BMC measured by DXA in vivo compared with vehicle-treated controls. However, total BMC corrected for the increase in body weight (total BMC:body weight ratio) was unaffected. Tibial area bone mineral density (BMD, BMC/area) was increased, but total and vertebral area BMDs were unchanged. The pQCT measurements in vitro revealed that the increase in the cortical BMC was due to an increased cross-sectional bone area, whereas the cortical volumetric BMD was unchanged. Femur and vertebra L6 volumes were increased but no effect was seen on the volumetric BMDs as measured by Archimedes' principle. Ash weight was increased by all treatments, but the mineral concentration was unchanged. We conclude that treatment of adult female rats with the GHSs ipamorelin and GHRP-6 increases BMC as measured by DXA in vivo. The results of in vitro measurements using pQCT and Archimedes' principle, in addition to ash weight determinations, show that the increases in cortical and total BMC were due to an increased growth of the bones with increased bone dimensions, whereas the volumetric BMD was unchanged.

Preserved olfactory short-term memory after combined cholinergic and serotonergic lesions using 192 IgG-saporin and 5,7-dihydroxytryptamine in rats.

Young adult Long-Evans female rats were subjected to intracerebroventricular injections of 150 microg 5,7-dihydroxytryptamine (5,7-DHT), 2 microg 192 IgG-saporin, or a combination of both neurotoxins. All rats were tested for olfactory recognition (short-term memory) using a task based on spontaneous exploration of odor sources. Compared with animals undergoing sham operations, 5,7-DHT reduced the concentration of serotonin by 60-80% in the frontoparietal cortex, hippocampus, striatum and the olfactory bulbs. After 192 IgG-saporin treatment, acetylcholine concentrations were reduced by approximately 40% in all these structures, except the striatum. Neither lesion induced a significant deficit in olfactory recognition. These data suggest that combined lesions of cholinergic and serotonergic neurons in the rat brain do not alter olfactory perception or olfactory short-term memory.

Neuroprotective effects of female gonadal steroids in reproductively senescent female rats.

Young adult female rats sustain smaller infarcts after experimental stroke than age-matched males. This sex difference in ischemic brain injury in young animals disappears after surgical ovariectomy and can be restored by estrogen replacement. We sought to determine whether ischemic brain injury continues to be smaller in middle-aged, reproductively senescent female rats compared with age-matched males and to test the effect of ovarian steroids on brain injury after experimental stroke in females. Four groups of 16-month old Wistar rats (males [n=9], untreated females [n=9], and females pretreated with 17beta-estradiol [25-microgram pellets administered subcutaneously for 7 days; n=9] or progesterone [10-mg pellets administered subcutaneously for 7 days; n=9] were subjected to 2 hours of middle cerebral artery occlusion with the intraluminal filament technique, followed by 22 hours of reperfusion. Physiological variables and laser-Doppler cerebral cortical perfusion were monitored throughout ischemia and early reperfusion. In a separate cohort of males (n=3), untreated females (n=3), females pretreated with 17beta-estradiol (n=3), and females pretreated with progesterone (n=3), end-ischemic regional cerebral blood flow was measured by [(14)C]iodoantipyrine autoradiography. As predicted, infarct size was not different between middle-aged male and female rats. Cortical infarcts were 21+/-5% and 31+/-6% of ipsilateral cerebral cortex, and striatal infarcts were 44+/-7% and 43+/-5% of ipsilateral striatum in males and females, respectively. Both estrogen and progesterone reduced cortical infarct in reproductively senescent females (5+/-2% and 16+/-4% in estrogen- and progesterone-treated groups, respectively, compared with 31+/-6% in untreated group). Striatal infarct was smaller in the estrogen- but not in the progesterone-treated group. Relative change in laser-Doppler cerebral cortical perfusion from preischemic baseline and absolute end-ischemic regional cerebral blood flow were not affected by hormonal treatments. We conclude that the protection against ischemic brain injury found in young adult female rats disappears after reproductive senescence in middle-aged females and that ovarian hormones alleviate stroke injury in reproductively senescent female rats by a blood flow-independent mechanism. These findings support a role for hormone replacement therapy in stroke injury prevention in postmenopausal women.

Effects of hyperstimulation with gonadotrophins and age of females on oocytes and their metaphase II status in rats.

The present study was undertaken to evaluate the effects of hyperstimulation and aging on the number and proportion of oocytes in the metaphase II stage in female Wistar rats. It explored the validity of the hypothesis that a combination of hyperstimulation with pregnant mare serum gonadotrophins (PMSG) and age could compromise, to a greater extent, the oocyte quality as indicated by the proportion of ovulated oocytes in the metaphase II stage. Female Wistar rats were stimulated with varying doses of PMSG and human chorionic gonadotrophins (hCG) and the number and proportion of ovulated oocytes in the metaphase II stage were examined and compared between different groups of young adult (8-10 weeks old) and aging (30-32 weeks old) female rats. While spontaneous ovulation occurred in all young adult rats, only 50% of the aging rats did. The ovulation rate in aging rats was increased from 50 to 93% when non-PMSG-stimulated rats were given a dose of 10 IU of hCG at proestrus. The lower number of ovulated oocytes noted, even in those hyperstimulated with high doses of PMSG/hCG, also indicated a reduction in fertility in aging rats. Under the influence of high doses of PMSG, all aging rats ovulated, but as with the young adult rats, a higher dose of hCG was needed to achieve the maximum number of ovulated oocytes from the PMSG-induced expanded pool of preovulatory follicles. However, the average number of ovulated oocytes in aging rats was, nevertheless, still significantly lower than in young adult rats even when approximation of weight was considered. No consistent significant difference in proportion of normal oocytes was noted within groups and between young adult and aging rats. A lower proportion of ovulated oocytes was arrested at the metaphase II stages when rats, whether they were young adult or aging, were hyperstimulated with 40 IU of PMSG. However, this proportion was restored to normal (about 100%) when a higher dose of hCG, which is a signal responsible for initiating oocyte maturation, was used. Results of the present study showed that there appears to be an age-related reduction of sensitivity of the preovulatory follicles to the ovulation induction signal of hCG and thus higher doses of hCG were needed to ovulate the PMSG-induced expanded pool of dominant follicles. In older rats, apart from the obvious depletion of the pool of follicles, the evidence from the present study suggests that some of these older rats do have follicles, but that these were unable to develop to preovulatory follicles, probably because of the absence of sufficiently high levels of gonadotrophins essential for the initiation of folliculogenesis. PMSG-hyperstimulation can affect nuclear maturation; the proportion of ovulated oocytes not arrested at the metaphase II stage was higher. However, the proportion of ovulated oocytes at the metaphase II was restored to normal by increasing the dose of hCG use. Hence, meiotic aberration in rats is not age-dependent but rather dependent on the amplitude of the luteinizing hormone (LH)/hCG surge present. The results from this study nullified the hypothesis that hyperstimulation in combination with aging would lead to a higher proportion of abnormality in ovulated oocytes with respect to their being at inappropriate meiotic stages.