Gonadotropin elevation is ootoxic to ovulatory oocytes and inhibits oocyte maturation, and activin decoy receptor ActRIIB:Fc therapeutically restores maturation.

BACKGROUND: Elevated FSH often occurs in women of advanced maternal age (AMA, age ≥ 35) and in infertility patients undergoing controlled ovarian stimulation (COS). There is controversy on whether high endogenous FSH contributes to infertility and whether high exogenous FSH adversely impacts patient pregnancy rates. METHODS: The senescence-accelerated mouse-prone-8 (SAMP8) model of female reproductive aging was employed to assess the separate impacts of age and high FSH activity on the percentages (%) of viable and mature ovulated oocytes recovered after gonadotropin treatment. Young and midlife mice were treated with the FSH analog equine chorionic gonadotropin (eCG) to model both endogenous FSH elevation and exogenous FSH elevation. Previously we showed the activin inhibitor ActRIIB:Fc increases oocyte quality by preventing chromosome and spindle misalignments. Therefore, ActRIIB:Fc treatment was performed in an effort to increase % oocyte viability and % oocyte maturation. RESULTS: The high FSH activity of eCG is ootoxic to ovulatory oocytes, with greater decreases in % viable oocytes in midlife than young mice. High FSH activity of eCG potently inhibits oocyte maturation, decreasing the % of mature oocytes to similar degrees in young and midlife mice. ActRIIB:Fc treatment does not prevent eCG ootoxicity, but it restores most oocyte maturation impeded by eCG. CONCLUSIONS: FSH ootoxicity to ovulatory oocytes and FSH maturation inhibition pose a paradox given the well-known pro-growth and pro-maturation activities of FSH in the earlier stages of oocyte growth. We propose the FOOT Hypothesis ("FSH OoToxicity Hypothesis), that FSH ootoxicity to ovulatory oocytes comprises a new driver of infertility and low pregnancy success rates in DOR women attempting spontaneous pregnancy and in COS/IUI patients, especially AMA women. We speculate that endogenous FSH elevation also contributes to reduced fecundity in these DOR and COS/IUI patients. Restoration of oocyte maturation by ActRIB:Fc suggests that activin suppresses oocyte maturation in vivo. This contrasts with prior studies showing activin A promotes oocyte maturation in vitro. Improved oocyte maturation with agents that decrease endogenous activin activity with high specificity may have therapeutic benefit for COS/IVF patients, COS/IUI patients, and DOR patients attempting spontaneous pregnancies.

Letrozole delays acquisition of water maze task in female BALB/c mice: Possible involvement of anxiety.

Letrozole, an aromatase inhibitor preventing estrogen synthesis from testosterone, is used as an adjuvant therapy in estrogen receptor-positive breast cancer patients. However, like other aromatase inhibitors, it induces many side effects, including impaired cognition. Despite its negative effect in humans, results from animal models are inconsistent and suggest that letrozole can either impair or improve cognition. Here, we studied the effects of chronic letrozole treatment on cognitive behavior of adult female BALB/c mice, a relevant animal model for breast cancer studies, to develop an appropriate animal model aimed at testing therapies to mitigate side effects of letrozole. In Morris water maze, letrozole 0.1 mg/kg impaired reference learning and memory. Interestingly, most of the letrozole 0.1 mg/kg-treated mice were able to learn the new platform position in reversal training and performed similar to control mice in a reversal probe test. Results of the reversal test suggest that letrozole did not completely disrupt spatial navigation, but rather delayed acquisition of spatial information. The delay might be related to increased anxiety as suggested by increased thigmotactic behavior during the reference memory training. The learning impairment was water maze-specific since we did not observe impairment in other spatial tasks such as in Y-maze or object location test. In contrast, the dose of 0.3 mg/kg did not have effect on water maze learning and facilitated locomotor habituation and recognition in novel object recognition test. The current study shows that letrozole dose-dependently modulates behavioral response and that its effects are task-dependent.

Maternal age and gonadotrophin elevation cooperatively decrease viable ovulated oocytes and increase ootoxicity, chromosome-, and spindle-misalignments: '2-Hit' and 'FSH-OoToxicity' mechanisms as new reproductive aging hypotheses.

While there is consensus that advanced maternal age (AMA) reduces oocyte yield and quality, the notion that high FSH reduces oocyte quality and causes aneuploidy remains controversial, perhaps due to difficulties controlling the confounding variables of age and FSH levels. Here, contributions of age and gonadotrophin elevation were separately controlled using a mouse model of human female reproductive aging. Ovulated oocytes were collected from young and midlife mice after 0-, 2.6-, or 17-day treatment with the FSH analog equine chorionic gonadotrophin (eCG), to model both exogenous FSH elevation within a single treatment cycle (as in controlled ovarian stimulation (COS)), and chronic endogenous FSH elevation during multiple cycles (as in diminished ovarian reserve). After 17-day eCG, fewer total oocytes/mouse are ovulated in midlife than young mice, and a precipitous decline in viable oocytes/mouse is observed in midlife but not young mice throughout eCG treatment. eCG is potently ootoxic to ovulatory oocytes and strongly induces chromosome- and spindle-misalignments within 2.6 days of eCG in midlife, but only after 17 days in young mice. These data indicate that AMA increases susceptibility to multiple adverse effects of elevated FSH activity in ovulated oocytes, including declines in total and viable oocytes/mouse, and induction of ootoxicity and aneuploidy. Two hypotheses are proposed for underlying causes of infertility in women. The FSH OOToxicity Hypothesis ('FOOT Hypothesis') posits that high FSH is ootoxic to ovulatory oocytes and that FSH ootoxicity is a root cause of low pregnancy success rates in naturally cycling women with high FSH and IUI patients undergoing COS. The '2-Hit Hypothesis' posits that AMA increases susceptibility to FSH-induced ootoxicity and aneuploidy.

A Forgotten Principle in Immunocytochemistry: Optimal Dilution.

Immunocytochemical (ICC) techniques are frequently used in basic and clinical research. Here, we focus on the importance of using antisera/antibodies at optimal dilutions to achieve specificity and reduce costs. Unfortunately, the basic principle, the necessity to test method specificity of the staining by a series of increasing dilutions of primary antiserum/antibodies, is only occasionally seen in papers using ICC. Many researchers rely on the company's information or others' published data. In this study, we show examples with monoclonal antibodies used in the peroxidase-based ICC technique in mouse and guinea pig brain sections. We show images of ICC staining of phospho-S129 alpha-synuclein in A53T mice and NeuN in guinea pig brains and demonstrate that optimal staining with them can be achieved at least at two to three orders of magnitude higher dilutions than generally used in the literature. We strongly recommend that when antisera/antibodies are used for the first time in any laboratory, independent of what the manufacturer or vendor recommends or are found in the literature, a dilution curve should be set up to identify the optimal dilution. This practice provides not only the highest specificity but is also an economic approach.

Β-endorphin-immunoreactive perikarya appear to receive innervation from NPY-immunoreactive fiber varicosities in the human hypothalamus.

Morphological and pharmacological studies indicate that hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC) neurons communicate with each other in rats and regulate a variety of hypothalamic and extrahypothalamic functions. Indeed, electron microscopic studies revealed NPY-immunoreactive (NPI-IR) synapses on ß-endorphin-IR neurons in the hypothalamus. However, no such connections have been reported in humans. Here, we studied the putative NPY-ß-endorphin associations with high-resolution light microscopic double-label immunocytochemistry in the human hypothalamus. The majority of ß-endorphin-IR perikarya appear to be innervated by abutting NPY-IR fibers in the infundibulum/median eminence, receiving more than 6 contacts (38% of the counted neurons) or three to six contacts (42% of the counted neurons). The rest of the ß-endorphin-IR neurons are lightly innervated by NPY fibers (14%, one-three contacts) or do not receive any detectable NPY-IR axon varicosities (6% of the counted neurons). Since ß-endorphin is cleaved from the proopiomelanocortin (POMC) precursor, the NPY-ß-endorphin connections also provide the foundation for NPY-α-MSH and NPY-ACTH connections and their subsequent physiology. The close anatomical connections between NPY-IR nerve terminals and ß-endorphin-IR neurons reported herein may represent functional synapses and provide the foundation for NPY-stimulated ß-endorphin release. By interacting with ß-endorphin, NPY may have a more widespread regulatory capacity than acting alone on different neurotransmitter systems.

Morphology and distribution of hypothalamic peptidergic systems.

Neuropeptides participate in the regulation of numerous hypothalamic functions that are aimed for sustaining the homeostasis of the organism. These neuropeptides can act in two different levels. They can influence the release of hormones from the adenohypophysis via the portal circulation; in addition, they can act as neurotransmitters/neuromodulators modulating the functioning of numerous hypothalamic neurotransmitter systems. Indeed, most of these peptidergic systems form a complex network in the infundibular and periventricular nuclei of the human hypothalamus, communicating with each other by synaptic connections that may control fundamental physiologic functions. In the present chapter, we provide an overview of the distribution of neuropeptides in the human hypothalamus using immunohistochemistry and high-resolution, three-dimensional mapping.

Substance P-Immunoreactive Fiber Varicosities Appear to Innervate Galaninergic Perikarya in the Human Hypothalamus.

Introduction: Substance P (SP) is a member of the tachykinin family. In the central nervous system, SP participates among others, in the regulation of pain, learning, memory, emotion, and sexual functions. In the periphery, SP affects the gastrointestinal, cardiovascular, and urinary systems. Galanin, similarly to SP, appears to be involved in wide range of physiologic functions, including cognition, waking and sleep, feeding, mood, blood pressure, reproduction, and development, where acts as a trophic factor. The similar distribution of SP-immunoreactive (SP-IR) fibers and galanin-IR perikarya in the human hypothalamus suggests functional interaction between these neuropeptides. Methods: We have utilized double-label immunohistochemistry to reveal these putative juxtapositions. Results: The majority of galanin-IR neurons receive contacting SP-IR fibers that often cover a significant area of the galaninergic perikarya forming multiple en passant type contacts. These SP-galanin juxtapositions are located mainly in the basal part of the infundibulum/median eminence, populating the basal periventricular region as well as the basal perifornical area. Discussion: The density and the morphology of these associations suggest that these contacts are functional synapses and therefore may represent the morphological substrate of the control of SP on multiple functions regulated/modulated by galanin. SP via galanin may modulate anterior pituitary hormone secretion, as contrary to SP, high density of galanin immunoreactivity is present in the median eminence, and by innervating galanin-IR neurons projecting to other parts of the brain, SP can modulate indirectly their activities. Impact statement The present study is the first describing juxtapositions between the substance P (SP)-immunoreactive (IR) and galanin-IR neurons in the human hypothalamus. These juxtapositions may be functional synapses and they may represent the morphological substrate of the control of SP on the galaninergic system. SP via galanin may modulate anterior pituitary hormone secretion, as contrary to SP, high density of galanin immunoreactivity is present in the median eminence. Galanin, released into the hypothalamo-hypophyseal circulation, can reach the anterior pituitary and function as a hypophysiotropic substance and regulates anterior pituitary hormone secretion. SP by innervating galanin-IR neurons, which project to other parts of the brain, can modulate indirectly their activities.

Establishment of a non-human primate model for menopausal hot flushes.

Menopause affects the quality of life of millions of women. With modern lifespan the postmenopausal attenuation of circulating estrogen levels can negatively impact a women's life for 30-40 years. The major hypoestrogenic consequence is hot flushes but decline in cognitive function, sleep disorders, depression/anxiety, cardiovascular disease, and osteoporosis are also characteristic for the menopause. Current treatments of hot flushes include estrogen therapy alone or in combination with progestins, soy products, and serotonin and norepinephrine reuptake inhibitors. However, with the exception of estrogens, none of these have satisfactory efficacy. But estrogens come with the unwanted side effects in the periphery, including stimulation of the uterus and breast leading to elevated cancer risk. Therefore, a tremendous effort has been devoted to developing safer therapies and the research has utilized classic rodent models of hot flush with considerable limitations. As hot flushes are primate-specific symptoms, the development of a non-invasive primate hot flush model would have a tremendous impact on drug development. Therefore, our aim was to develop such a non-human primate (NHP) model a hot flush that both recapitulates flushes women experience and is minimally invasive. We investigated if recent developments in thermal imaging have made it possible to accurately monitor skin temperature via camera imaging. In this study, the skin temperature of an ovariectomized rhesus monkey was measured continuously with an infrared camera in a freely moving animal over long time period. Following mapping skin temperatures of several areas of the neck and face we found that the nose of the monkeys showed that largest changes in skin temperature. In the ovariectomized monkey the temperature of the skin on the nose shows up to 9 °C elevations representing hot flushes. In the untreated monkey, hot flushes occurred more frequently in late afternoon/early evening hours than in the morning and last for several minutes. We observed 58 flushes in the 64 evenings of observation. The average number of hot flushes was 0.51 per evening. Oral administration of biotin (niacin) for seven days exaggerated the number of hot flushes to 2.43 per evening. Oral treatment with estradiol benzoate prevented hot flushes and only 2 flushes were detected in the 12 evenings after treatment, averaging 0.17 per evening. The development of this NHP model of hot flush provides great hope for utilizing it for future drug development and mechanistic studied.

Thyrotropin-releasing hormone axonal varicosities appear to innervate dopaminergic neurons in the human hypothalamus.

Thyrotropin-releasing hormone (TRH) has a critical role in the central regulation of thyroid-stimulating hormone (TSH) from the anterior pituitary, and subsequently, thyroid hormone secretion from the thyroid gland. In addition to its role in the regulation of HPT axis, TRH is a potent regulator of prolactin (PRL) secretion by stimulating PRL secretion either directly from lactotrophs or indirectly via its action on the tuberoinfundibular dopamine (TIDA) neurons. In rodents, the TRH neurons which regulate TSH and thyroid hormone secretion, called hypophysiotropic TRH neurons, are in the medial subdivision of the parvicellular paraventricular nucleus (PVN). In humans, the PVN also contains a large population of TRH neurons, especially in its medial part, but the location of hypophysiotropic TRH neurons is not yet known. In addition to regulating TSH and PRL secretion, TRH also functions as a neurotransmitter/neuromodulator. In rodents and teleosts, TRH axons densely innervate TIDA neurons to inhibit tyrosine hydroxylase (TH) biosynthesis, neuronal firing, and dopamine turnover which may contribute to increasing PRL secretion. No such connections have been reported in humans, although dopaminergic neurons express TRH receptors and TRH also regulates PRL secretion. The objectives of this study were to map TRH-IR and TH-IR structures in the human hypothalamus with single-label light microscopic immunocytochemistry and study their interaction with double-label light microscopic immunocytochemistry. We show that TRH-IR nerve terminals densely surround TH-IR neurons (perikarya and dendrites) in the infundibulum of the human hypothalamus. The micrographs illustrating these juxtapositions were taken by Olympus BX45 microscope equipped with a digital camera and with 100X oil immersion objective. Composite images were created from the consecutive micrographs if the neurons were larger than the frame of the camera, using Adobe Photoshop software. As no gaps between TRH-IR and TH-IR elements were seen, these contacts may be functional synapses by which TRH regulates the activity of dopaminergic neurons and subsequently TSH and PRL secretion.

Brain-Selective Estrogen Therapy Prevents Androgen Deprivation-Associated Hot Flushes in a Rat Model.

Hot flushes are best-known for affecting menopausal women, but men who undergo life-saving castration due to androgen-sensitive prostate cancer also suffer from these vasomotor symptoms. Estrogen deficiency in these patients is a direct consequence of androgen deprivation, because estrogens (notably 17ß-estradiol, E2) are produced from testosterone. Although estrogens alleviate hot flushes in these patients, they also cause adverse systemic side effects. Because only estrogens can provide mitigation of hot flushes on the basis of current clinical practices, there is an unmet need for an effective and safe pharmacotherapeutic intervention that would also greatly enhance patient adherence. To this end, we evaluated treatment of orchidectomized (ORDX) rats with 10ß, 17ß-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective bioprecursor prodrug of E2. A pilot pharmacokinetic study using oral administration of DHED to these animals revealed the formation of E2 in the brain without the appearance of the hormone in the circulation. Therefore, DHED treatment alleviated androgen deprivation-associated hot flushes without peripheral impact in the ORDX rat model. Concomitantly, we showed that DHED-derived E2 induced progesterone receptor gene expression in the hypothalamus without stimulating galanin expression in the anterior pituitary, further indicating the lack of systemic estrogen exposure upon oral treatment with DHED.

Presence of substance P positive terminals on hypothalamic somatostatinergic neurons in humans: the possible morphological substrate of the substance P-modulated growth hormone secretion.

Substance P is an undecapeptide affecting the gastrointestinal, cardiovascular, and urinary systems. In the central nervous system, substance P participates in the regulation of pain, learning, memory, and sexual homeostasis. In addition to these effects, previous papers provided solid evidence that substance P exhibits regulatory effects on growth. Indeed, our previous study revealed that growth hormone-releasing hormone (GHRH) neurons appear to be densely innervated by substance P fibers in humans. Since growth hormone secretion is regulated by the antagonistic actions of both GHRH and somatostatin, in the present paper we have examined the possibility that SP may also affect growth via the somatostatinergic system. Therefore, we have studied the putative presence of juxtapositions between the substance P-immunoreactive (IR) and somatostatinergic systems utilizing double label immunohistochemistry combined with high magnification light microscopy with oil immersion objective. In the present study, we have revealed a dense network of substance P-IR axonal varicosities contacting the majority of somatostatin-IR neurons in the human hypothalamus. Somatostatinergic perikarya are often covered by these fiber varicosities that frequently form basket-like encasements with multiple en passant type contacts, particularly in the infundibular nucleus/median eminence and in the basal periventricular area of the tuberal region. In addition, numerous substance-P-somatostatinergic juxtapositions can be found in the basal perifornical zone of the tuberal area. If these contacts are indeed functional synapses, they may represent the morphological substrate of the control of substance P on growth. Indeed, the frequency and density of these juxtapositions indicate that in addition to the regulatory action of substance P on GHRH secretion, substance P also influences growth by regulating hypothalamic somatostatinergic system via direct synaptic contacts.

Substance P appears to affect growth via growth hormone-releasing hormone (GHRH) neurons in the human hypothalamus.

Substance P is an eleven-amino acid neuropeptide (undecapeptide) with multiple effects on the gastrointestinal, cardiovascular, and urinary systems as well as complex central nervous system functions such as pain, learning, memory, and sexual homeostasis. Previous studies also revealed that substance P exhibits regulatory effects on growth possibly via influencing hypothalamic GHRH release in human. However, the morphological substrate of this phenomenon has not been elucidated yet. In the present study, we examined the putative presence of juxtapositions between the substance P- and GHRH-immunoreactive (IR) systems using double-label immunocytochemistry. High-magnification light microscopy with oil immersion was used to identify putative juxtapositions between these systems. Our studies revealed substance P-IR fiber network abutting on the surface of the majority of GHRH-immunoreactive neurons in the human hypothalamus. These fiber varicosities often cover a significant surface area on the GHRH-IR neurons, forming basket-like encasements with multiple en passant type contacts. The majority of these densely innervated GHRH-IR neurons were found in the infundibular nucleus/median eminence, while substance P-IR fibers often abut on the GHRH-IR neurons in the periventricular zone and basal perifornical area of the tuberal region and in the dorsomedial subdivision of the ventromedial nucleus. The posterior hypothalamus did not contain observable substance P-GHRH associations. The density and the morphology of these intimate associations suggest that substance P influences growth by regulating hypothalamic GHRH release by direct synaptic contacts.

Accessory mammillary bodies formed by the enlarged lateral mammillary nuclei: cytoarchitecture.

Post mortem examination of the hypothalamus of a 79-year-old woman, deceased in cardiac arrest without recorded neurological symptoms, revealed well-defined spherical protrusions located rostro-laterally to the mammillary bodies that appear to be regular size when compared to normal. Cytoarchitectonically, these accessory mammillary bodies are formed by the enlarged lateral mammillary nucleus that is normally a thin shell over the medial. The mammillary nuclei appear to function synergistically in memory formation in rats; however, the functional consequences of the present variation are difficult to interpret due to lack of human data. Most importantly, in addition to the possible functional consequences, lateral mammillary bodies can be falsely identified as various neuropathological processes of the basal diencephalon including gliomas; therefore, it is extremely important to disseminate this unique morphological variant among clinicians.

Dopamine and Stress System Modulation of Sex Differences in Decision Making.

Maladaptive decision making is associated with several neuropsychiatric disorders, including problem gambling and suicidal behavior. The prevalence of these disorders is higher in men vs women, suggesting gender-dependent regulation of their pathophysiology underpinnings. We assessed sex differences in decision making using the rat version of the Iowa gambling task. Female rats identified the most optimal choice from session 1, whereas male rats from session 5. Male, but not female rats, progressively improved their advantageous option responding and surpassed females. Estrus cycle phase did not affect decision making. To test whether pharmacological manipulations targeting the dopaminergic and stress systems affect decision making in a sex-dependent manner, male and female rats received injections of a dopamine D2 receptor (D2R) antagonist (eticlopride), D2R agonist (quinpirole), corticotropin-releasing factor 1 (CRF1) antagonist (antalarmin), and α2-adrenergic receptor antagonist (yohimbine; used as a pharmacological stressor). Alterations in mRNA levels of D2R and CRF1 were also assessed. Eticlopride decreased advantageous responding in male, but not female rats, whereas quinpirole decreased advantageous responding specifically in females. Yohimbine dose-dependently decreased advantageous responding in female rats, whereas decreased advantageous responding was only observed at higher doses in males. Antalarmin increased optimal choice responding only in female rats. Higher Drd2 and Crhr1 expression in the amygdala were observed in female vs male rats. Higher amygdalar Crhr1 expression was negatively correlated with advantageous responding specifically in females. This study demonstrates the relevance of dopaminergic- and stress-dependent sex differences to maladaptive decision making.

Neurotrophic Factor-α1: A Key Wnt-ß-Catenin Dependent Anti-Proliferation Factor and ERK-Sox9 Activated Inducer of Embryonic Neural Stem Cell Differentiation to Astrocytes in Neurodevelopment.

Embryonic neurodevelopment involves inhibition of proliferation of multipotent neural stem cells (NSCs) followed by differentiation into neurons, astrocytes and oligodendrocytes to form the brain. We have identified a new neurotrophic factor, NF-α1, which inhibits proliferation and promotes differentiation of NSC/progenitors derived from E13.5 mouse cortex. Inhibition of proliferation of these cells was mediated through negatively regulating the Wnt pathway and decreasing ß-catenin. NF-α1 induced differentiation of NSCs to astrocytes by enhancing Glial Fibrillary Acidic Protein (GFAP) expression through activating the ERK1/2-Sox9 signaling pathway. Cultured E13.5 cortical stem cells from NF-α1-knockout mice showed decreased astrocyte numbers compared to wild-type mice, which was rescued by treatment with NF-α1. In vivo, immunocytochemistry of brain sections and Western blot analysis of neocortex of mice showed a gradual increase of NF-α1 expression from E14.5 to P1 and a surge of GFAP expression at P1, the time of increase in astrogenesis. Importantly, NF-α1-Knockout mice showed ∼49% fewer GFAP positive astrocytes in the neocortex compared to WT mice at P1. Thus, NF-α1 is critical for regulating antiproliferation and cell fate determination, through differentiating embryonic stem cells to GFAP-positive astrocytes for normal neurodevelopment. Stem Cells 2017;35:557-571.

Corticotropin-releasing hormone (CRH)-immunoreactive (IR) axon varicosities target a subset of growth hormone-releasing hormone (GHRH)-IR neurons in the human hypothalamus.

It is a general consensus that stress is one of the major factors that suppresses growth. Previous studies revealed that the catecholaminergic and neuropeptide Y (NPY) systems, involved in the activation of stress-related neuronal circuits, influence growth hormone (GH)-release via modulating growth hormone-releasing hormone (GHRH) secretion. Indeed, catecholaminergic and NPY-immunoreactive (IR) axon varicosities abut on the surface of the GHRH neurons forming contacts. These juxtapositions appear to be real synapses and may represent the morphological substrate of the impact of stress on growth. In addition to catecholamines and NPY, there is a vast amount of evidence that corticotropin-releasing hormone (CRH), a major stress hormone, also influences GH secretion. Whether this modulatory effect is direct, or indirect, via the hypothalamic GHRH system, has not been elucidated yet. In the present study, we examined the possibility that CRH influences GH secretion via modulating the GHRH release by direct synaptic mechanisms. Since the verification of these synapses by electron microscopy is problematic in human due to the long post mortem time, in order to reveal the putative CRH-GHRH juxtapositions, light microscopic double label immunohistochemistry was utilized. In the infundibular nucleus, a subset (6%) of the GHRH perikarya received abutting CRH fiber varicosities forming multiple contacts while passing by. No gaps appeared between the contacting elements. The morphology of these CRH-GHRH juxtapositions suggests that, among other neurotransmitters/neuromodulators, CRH influences growth by modulating the hypothalamic GHRH secretion via direct synaptic mechanisms.

Treatment with an orally bioavailable prodrug of 17ß-estradiol alleviates hot flushes without hormonal effects in the periphery.

Estrogen deprivation has a profound effect on the female brain. One of the most obvious examples of this condition is hot flushes. Although estrogens relieve these typical climacteric symptoms, many women do not want to take them owing to unwanted side-effects impacting, for example, the uterus, breast and blood. Therefore, there is a need for developing safer estrogen therapies. We show here that treatment with 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), a novel brain-targeting bioprecursor prodrug of the main human estrogen, 17ß-estradiol, alleviates hot flushes in rat models of thermoregulatory dysfunction of the brain. Oral administration of DHED elicits a significant reduction of tail skin temperature (TST) rise representing hot flushes in the morphine-dependent ovariectomized rat model and results in the restoration of estrogen deprivation-induced loss of diurnal rhythm in TST. These beneficial effects occur without detrimental peripheral hormonal exposure; thus, the treatment avoids potentially harmful stimulation of estrogen-sensitive peripheral organs, including the uterus and the anterior pituitary, or the proliferation of MCF-7a breast cancer cell xenografts. Our promising preclinical assessments warrant further considerations of DHED for the development of a brain-selective 17ß-estradiol therapy to relieve hot flushes without undesirable peripheral side-effects.

Activin Decoy Receptor ActRIIB:Fc Lowers FSH and Therapeutically Restores Oocyte Yield, Prevents Oocyte Chromosome Misalignments and Spindle Aberrations, and Increases Fertility in Midlife Female SAMP8 Mice.

Women of advanced maternal age (AMA) (age ≥ 35) have increased rates of infertility, miscarriages, and trisomic pregnancies. Collectively these conditions are called "egg infertility." A root cause of egg infertility is increased rates of oocyte aneuploidy with age. AMA women often have elevated endogenous FSH. Female senescence-accelerated mouse-prone-8 (SAMP8) has increased rates of oocyte spindle aberrations, diminished fertility, and rising endogenous FSH with age. We hypothesize that elevated FSH during the oocyte's FSH-responsive growth period is a cause of abnormalities in the meiotic spindle. We report that eggs from SAMP8 mice treated with equine chorionic gonadotropin (eCG) for the period of oocyte growth have increased chromosome and spindle misalignments. Activin is a molecule that raises FSH, and ActRIIB:Fc is an activin decoy receptor that binds and sequesters activin. We report that ActRIIB:Fc treatment of midlife SAMP8 mice for the duration of oocyte growth lowers FSH, prevents egg chromosome and spindle misalignments, and increases litter sizes. AMA patients can also have poor responsiveness to FSH stimulation. We report that although eCG lowers yields of viable oocytes, ActRIIB:Fc increases yields of viable oocytes. ActRIIB:Fc and eCG cotreatment markedly reduces yields of viable oocytes. These data are consistent with the hypothesis that elevated FSH contributes to egg aneuploidy, declining fertility, and poor ovarian response and that ActRIIB:Fc can prevent egg aneuploidy, increase fertility, and improve ovarian response. Future studies will continue to examine whether ActRIIB:Fc works via FSH and/or other pathways and whether ActRIIB:Fc can prevent aneuploidy, increase fertility, and improve stimulation responsiveness in AMA women.

The prodrug DHED selectively delivers 17ß-estradiol to the brain for treating estrogen-responsive disorders.

Many neurological and psychiatric maladies originate from the deprivation of the human brain from estrogens. However, current hormone therapies cannot be used safely to treat these conditions commonly associated with menopause because of detrimental side effects in the periphery. The latter also prevents the use of the hormone for neuroprotection. We show that a small-molecule bioprecursor prodrug, 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), converts to 17ß-estradiol in the brain after systemic administration but remains inert in the rest of the body. The localized and rapid formation of estrogen from the prodrug was revealed by a series of in vivo bioanalytical assays and through in vivo imaging in rodents. DHED treatment efficiently alleviated symptoms that originated from brain estrogen deficiency in animal models of surgical menopause and provided neuroprotection in a rat stroke model. Concomitantly, we determined that 17ß-estradiol formed in the brain from DHED elicited changes in gene expression and neuronal morphology identical to those obtained after direct 17ß-estradiol treatment. Together, complementary functional and mechanistic data show that our approach is highly relevant therapeutically, because administration of the prodrug selectively produces estrogen in the brain independently from the route of administration and treatment regimen. Therefore, peripheral responses associated with the use of systemic estrogens, such as stimulation of the uterus and estrogen-responsive tumor growth, were absent. Collectively, our brain-selective prodrug approach may safely provide estrogen neuroprotection and medicate neurological and psychiatric symptoms developing from estrogen deficiency, particularly those encountered after surgical menopause, without the adverse side effects of current hormone therapies.

A potentially functional variant in the serotonin transporter gene is associated with premenopausal and perimenopausal hot flashes.

OBJECTIVE: An increase in the use of selective serotonin reuptake inhibitors (SSRIs) and/or serotonin-norepinephrine reuptake inhibitors (SNRIs) to relieve menopausal hot flashes (HFs) has been observed recently. However, response to them has been heterogeneous. We hypothesized that this heterogeneity might be partially attributed to genetic variations in genes encoding the serotonin and/or norepinephrine transporters (SLC6A4 and SLC6A2). As a first step in testing the role of genetics in response to SSRIs/SNRIs, we examined the association between HFs and genetic variants within these two genes. METHODS: We tested 29 haplotype-tagging single nucleotide polymorphisms within SLC6A4 and SLC6A2 for their association with HFs separately for European-American (396 cases and 392 controls) and African-American (125 cases and 81 controls) premenopausal and perimenopausal women. RESULTS: We found that the minor allele of SLC6A4_rs11080121 was associated with protection against HFs (odds ratio, 0.75; 95% CI, 0.60-0.94) only in European-American women. Bioinformatics analyses indicated that rs11080121 is fully correlated with rs1042173 in the 3' untranslated region of SLC6A4. The minor allele of rs1042173 seems to disrupt a conserved binding site for hsa-miR-590-3p microRNA. CONCLUSIONS: Disruption of a microRNA binding site leads to higher expression of SLC6A4, higher expression of SLC6A4 leads to depletion of serotonin in synaptic clefts, and depletion of serotonin triggers the presynaptic autoreceptor feedback mechanism to produce more serotonin, which is protective against HFs. This is the first study to test the association between HFs in both European-American and African-American premenopausal and perimenopausal women and genetic variants in two neurotransmitter transporter genes, SLC6A2 and SLC6A4. This information can be used in tailoring the pharmaceutical use of SSRIs/SNRIs for HF relief.