Katherine L. Rosewell

Estradiol is a protective factor in the adult and aging brain: understanding of mechanisms derived from in vivo and in vitro studies

We have shown that 17beta-estradiol exerts profound protective effects against stroke-like ischemic injury in female rats. These effects are evident using physiological levels of estradiol replacement in ovariectomized rats and require hormone treatment prior to the time of injury. The protective actions of estradiol appear to be most prominent in the cerebral cortex, where cell death is not apparent until at least 4 h after the initiation of ischemic injury and where cell death is thought to be apoptotic in nature. Middle-aged rats remain equally responsive to the protective actions of estradiol. The maintenance of responsiveness of the cerebral cortex to the neuroprotective actions of estradiol was unexpected since responsiveness of the hypothalamus to estradiol decreases dramatically by the time animals are middle-aged. We believe that the protective actions of estradiol require the estrogen receptor-alpha, since estradiol does not protect in estrogen receptor-alpha knockout mice. We have also implemented a method of culturing cerebral cortical explants to assess the protective effects of estradiol in vitro. This model exhibits remarkable parallelisms with our in vivo model of brain injury. We have found that 17beta-estradiol decreases the extent of cell death and that this protective effect requires hormone pretreatment. Finally, 17alpha-estradiol, which does not interact effectively with the estrogen receptor, does not protect; and addition of ICI 182,780, an estrogen receptor antagonist, blocks the protective actions of estradiol. We have begun to explore the molecular and cellular mechanisms of estradiol-mediated protection. In summary, our findings demonstrate that estradiol exerts powerful protective effects both in vivo and in vitro and suggest that these actions are mediated by estrogen receptors.

Age differentially influences estrogen receptor α (ERα) and estrogen receptor β (ERβ) gene expression in specific regions of the rat brain

Abstract Estradiols ability to influence neurochemical events that are critical to female reproductive cyclicity and behavior decreases with age. We tested the hypothesis that decreases in estrogen receptor-α (ERα) and/or ERβ mRNA explain the brains declining responsiveness to estradiol. We assessed ERα and ERβ mRNA levels in intact and ovariectomized estradiol-treated rats. ERβ mRNA was detected in several brain regions and decreased by middle-age in the cerebral cortex and supraoptic nucleus of estradiol-treated rats. ERβ mRNA levels exhibited a diurnal rhythm in the suprachiasmatic nucleus of young and middle-aged rats and this rhythm was blunted in old rats. We examined ERα mRNA in the periventricular preoptic, medial preoptic, ventromedial and arcuate nuclei, and it was decreased only in the periventricular preoptic nucleus of the old rats. In summary, the expression of ERα and ERβ mRNAs is differentially modulated in the aging brain and changes are region specific.

Sex differences in the daily rhythm of vasoactive intestinal polypeptide but not arginine vasopressin messenger ribonucleic acid in the suprachiasmatic nuclei.

The timing of the preovulatory surge of LH in female rodents is tightly coupled to the environmental light/dark cycle. This coupling is mediated by the circadian pacemaker located in the suprachiasmatic nuclei (SCN). Studies indicate that vasoactive intestinal polypeptide (VIP) and arginine vasopressin (AVP), which are synthesized in the SCN, transmit circadian information from the SCN to GnRH neurons, thereby regulating the timing of the LH surge. However, to date, the rhythmic expression of these two peptides in the SCN has only been examined in males. The pattern of VIP expression in males is difficult to reconcile with its role in the LH surge. The purpose of the present study was to assess the rhythm of VIP messenger RNA (mRNA) levels in the SCN of female rats under several endocrine conditions. We compared this rhythm to that in males and to AVP mRNA rhythms in all experimental groups. In all groups of females, VIP mRNA levels were rhythmic, with peak expression occurring during the light phase and ...

Suppression of Vasoactive Intestinal Polypeptide in the Suprachiasmatic Nucleus Leads to Aging-Like Alterations in cAMP Rhythms and Activation of Gonadotropin-Releasing Hormone Neurons

Input from the suprachiasmatic nucleus (SCN) to gonadotropin-releasing hormone (GnRH) neurons is critical to the occurrence of regular cyclic GnRH secretion. It is thought that an essential neuropeptide in the SCN that communicates this cyclic information to GnRH neurons is vasoactive intestinal polypeptide (VIP) and that it may act through cAMP. We tested the hypothesis that (1) aging involves a blunting of cAMP diurnal rhythmicity in the SCN; (2) administration of antisense oligonucleotides (anti-oligos) against VIP, which produces an aging-like pattern in VIP, would lead to an aging-like suppression of cAMP; and (3) this in turn would lead to inhibition of the steroid-induced activation of GnRH neurons. We measured cAMP concentrations in the SCN and rostral preoptic nucleus throughout the day in young and middle-aged rats that were ovariectomized (OVX) or OVX and treated with estradiol. Our results show that cAMP concentrations exhibit a diurnal rhythm in young rats, and that this rhythm is totally abolished by the time rats are middle age. Administration of antisense oligonucleotides against VIP or random oligos suppresses VIP concentrations and abolishes the cAMP rhythm, leading to significantly reduced activation of GnRH neurons. Together, these findings strongly suggest that the SCN conveys diurnal information to GnRH neurons by driving VIP-dependent cAMP rhythms. In addition, aging involves deterioration in this VIP-driven rhythmicity, which impacts the ability of steroids to induce GnRH neuronal activation.

Fos-Induction in Gonadotropin-Releasing Hormone Neurons Receiving Vasoactive Intestinal Polypeptide Innervation Is Reduced in Middle-Aged Female Rats

Abstract A hallmark of reproductive aging in rats is a delay in the initiation and peak, and a decrease in the amplitude, of both proestrous and steroid-induced surges of LH and a decrease in the number of GnRH neurons that express Fos during the surge. The altered timing of the LH surge and the decline in Fos expression in GnRH neurons may be due to changes in the rhythmic expression of vasoactive intestinal polypeptide (VIP), a neuropeptide that carries time-of-day information from the circadian pacemaker, located in the suprachiasmatic nuclei (SCN), to GnRH neurons. The goals of our study were to determine if aging alters 1) the innervation of GnRH neurons by VIP and 2) the ability of VIP to activate GnRH neurons by examining the effects of aging on the number of GnRH neurons apposed by VIP fibers and the number of GnRH neurons that receive VIP input that express Fos. Immunocytochemistry for GnRH and VIP; or GnRH, VIP, and Fos was performed on tissue sections collected from young (2–4 mo), regularly cycling females and middle-aged (10–12 mo) females in constant estrus. The number of GnRH neurons, GnRH neurons apposed by VIP fibers, and GnRH neurons that express Fos and apposed by VIP fibers were counted in both age groups. Our results clearly demonstrate that aging does not alter the number of GnRH neurons that receive VIP innervation. However, the number of GnRH neurons that receive VIP innervation and coexpress Fos decreases significantly. We conclude that the age-related delay in the timing of the LH surge is not due to a change in VIP innervation of GnRH neurons, but instead may result from a decreased sensitivity of GnRH neurons to VIP input.

Aging, estradiol and time of day differentially affect serotonin transporter binding in the central nervous system of female rats

Estrogen-related changes in serotonergic neuronal transmission, including changes in the number of serotonin transporter (SERT) binding sites, have been cited as a possible cause for changes in mood, memory and sleep that occur during the menopausal transition. However, both aging and estradiol regulate SERT binding sites in the brain. The goal of this experiment was to determine how aging and estrogen interact to regulate SERT levels in the forebrain of young and reproductively senescent female Sprague-Dawley rats using [3H]paroxetine. The density of specific [3H]paroxetine binding in various brain regions was compared in young (2-4 months) and reproductively senescent (10-12 months) female rats at three times of day. In most brain regions examined, estrogen and aging independently increased the number of [3H]paroxetine binding sites. The only region that displayed a reduction in [3H]paroxetine binding with age was the suprachiasmatic nucleus (SCN). Time of day influenced [3H]paroxetine binding in the SCN and the paraventricular thalamus (PVT), two regions known to be involved in the regulation of circadian rhythms. Aging and/or estrogen also altered the pattern of binding in these regions. Thus, based on the results of this study, we conclude that aging and estrogen both act to regulate SERT binding sites in the forebrain of female rats, and that this regulation is region specific.

A Period (per)-Like Protein Exhibits Daily Rhythmicity In The Suprachiasmatic Nuclei Of The Rat

The period (per) gene of Drosophila melanogaster is considered an important biological clock gene, since it regulates multiple behavioral rhythms. Per mRNA and protein exhibit circadian rhythms in the fruitfly brain and these rhythms appear to influence each other through a feedback loop. More recently, using the same antibody as was used in the Drosophila studies, PER-like proteins were detected in the suprachiasmatic nuclei (SCN) of male rats. This region of the brain is considered to be a major neural circadian pacemaker in mammals. The purpose of this study was to confirm that PER-like proteins are detectable in the SCN of female rats and to determine whether PER-like proteins exhibit a circadian rhythm. Female rats were killed at several times of day under both light/dark and constant conditions. Using the same anti-PER antibody in Western blots with Enhanced Chemiluminescence (Western-ECL) detection, the levels of the PER-like proteins were quantified in the SCN and cerebral cortex. The antibody identified a doublet band of approximately 170-160 kDa and a single band at 115 kDa. Of the three PER-like proteins only the largest exhibited a daily rhythm in the SCN, which peaked in the middle of the dark and attained its nadir around lights off; levels during the light were intermediate with a tendency towards a second drop around lights on. This rhythm did not persist under constant dim red light.(ABSTRACT TRUNCATED AT 250 WORDS)

Ovarian Expression and Regulation of the Stromelysins During the Periovulatory Period in the Human and the Rat

ABSTRACT The matrix metalloproteinases (MMPs) are postulated to facilitate follicular rupture. In the present study, expression of the stromelysins (MMP3, MMP10, MMP11) was analyzed in the periovulatory human and rat ovary. Human granulosa and theca cells were collected from the dominant follicle at various times after human chorionic gonadotropin (hCG). Intact rat ovaries, granulosa cells, and residual tissue (tissue remaining after granulosa cell collection) were isolated from equine CG (eCG)-hCG-primed animals. Mmp10 mRNA was highly induced in human granulosa and theca cells and intact rat ovaries, granulosa cells, and residual tissue. Localization of MMP10 to granulosa and theca cells in both human and rat ovarian follicles was confirmed by immunohistochemistry. Mmp3 mRNA was unchanged in human cells and rat granulosa cells, but increased in intact rat ovaries and residual tissue. Mmp11 mRNA decreased following hCG treatment in human granulosa and theca cells as well as rat granulosa cells. Regulation of Mmp10 in cultured rat granulosa cells revealed that the EGF inhibitor AG1478 and the progesterone receptor antagonist RU486 suppressed the induction of Mmp10 mRNA, whereas the prostaglandin inhibitor NS398 had no effect. Studies on the Mmp10 promoter demonstrated that forskolin plus PMA stimulated promoter activity, which was dependent upon a proximal AP1 site. In conclusion, there are divergent patterns of stromelysin expression associated with ovulation, with a marked induction of Mmp10 mRNA and a decrease in Mmp11 mRNA, yet a species-dependent pattern on Mmp3 mRNA expression. The induction of Mmp10 expression suggests an important role for this MMP in the follicular changes associated with ovulation and subsequent luteinization.

Ovarian Expression, Localization, and Function of Tissue Inhibitor of Metalloproteinase 3 (TIMP3) During the Periovulatory Period of the Human Menstrual Cycle

ABSTRACT Ovulation involves reorganization of the extracellular matrix of the follicle. This study examines the expression, localization, and potential function of the tissue inhibitor of metalloproteinase 3 (TIMP3) during ovulation in women. The dominant follicle of the menstrual cycle was collected at specified times throughout the ovulatory process: pre-, early, late, and postovulatory. For quantitative studies, the follicle was bisected; granulosa and theca cells were separated and collected. For immunohistochemistry (IHC), the intact follicle was embedded and TIMP3 was localized. Additionally, granulosa cells were collected from women undergoing in vitro fertilization and treated with increasing concentrations of recombinant TIMP3, and cell viability was assessed. Real-time PCR for TIMP3 mRNA revealed an increase in TIMP3 mRNA expression in granulosa cells from the early to the late ovulatory stage. Thecal TIMP3 mRNA expression was constitutive across the periovulatory period. TIMP3 protein was localized by IHC to the granulosa and theca cell layers in pre-, early, and late ovulatory follicles as well as to the vascular bed. The staining was most intense in the granulosa and theca cells in the late ovulatory group. Treatment of human granulosa-lutein cells with exogenous recombinant TIMP3 for 24 h decreased cell viability by 60%. Using human follicles collected throughout the periovulatory period of the menstrual cycle, we have demonstrated that TIMP3 mRNA expression increases and that TIMP3 protein is in the appropriate cellular layers to regulate proteolytic remodeling as the follicle progresses toward ovulation. In addition, we have shown that elevated levels of TIMP3 lead to decreased cell viability.

Induction of proteinases in the human preovulatory follicle of the menstrual cycle by human chorionic gonadotropin

OBJECTIVE To explore the temporal expression in granulosa and theca cells of key members of the MMP and ADAMTS families across the periovulatory period in women to gain insight into their possible roles during ovulation and early luteinization. DESIGN Experimental prospective clinical study and laboratory-based investigation. SETTING University medical center and private IVF center. ANIMAL AND PATIENT(S) Thirty-eight premenopausal women undergoing surgery for tubal ligation and six premenopausal women undergoing assisted reproductive techniques. INTERVENTION(S) Administration of hCG and harvesting of follicles by laparoscopy and collection of granulosa-lutein cells at oocyte retrieval. MAIN OUTCOME MEASURE(S) Expression of mRNA for matrix metalloproteinase (MMPs) and the A disintegrin and metalloproteinase with thrombospondin-like motifs (ADAMTS) in human granulosa cells and theca cells collected across the periovulatory period of the menstrual cycle and in cultured granulosa-lutein cells after hCG. Localization of MMPs and ADAMTSs by immunohistochemistry. RESULT(S) Expression of MMP1 and MMP19 mRNA increased in both granulosa and theca cells after hCG administration. ADAMTS1 and ADAMTS9 mRNA increased in granulosa cells after hCG treatment, however, thecal cell expression for ADAMTS1 was unchanged, while ADAMTS9 expression was decreased. Expression of MMP8 and MMP13 mRNA was unchanged. Immunohistochemistry confirmed the localization of MMP1, MMP19, ADAMTS1, and ADAMTS9 to the granulosa and thecal cell layers. CONCLUSION(S) The collection of the dominant follicle throughout the periovulatory period has allowed the identification of proteolytic remodeling enzymes in the granulosa and theca compartments that may be critically involved in human ovulation. These proteinases may work in concert to regulate breakdown of the follicular wall and release of the oocyte.