A Call to Action for Improving LGBTQIA2S+ Inclusive Policies and Practices in Educating Science and Medical Professionals.

Based on Nature's novel sex and gender guidelines, we share a call to action to enact policy changes in medical and scientific education. We conducted a literature search of current policies and practices affecting sex and gender minorities. Our work indicated a scarcity of guidelines and curricula dedicated to standardizing LGBTQIA2S+ topics. Educational policies must be enacted to ensure that sex and gender guidelines are implemented across all institutions as it impacts the future of healthcare and science. It is essential that sex and gender considerations be mandated topics in both medical and scientific education.

A novel variant of the cleidocervicalis muscle with clinical implications for nerve compression/entrapment.

PURPOSE: The cleidocervicalis muscle occurs in approximately 1-3 % of the population that may be confused with pathological neck masses. We describe a novel variant of the muscle and its clinical implications. METHODS: This is a case report of a cleidocervicalis muscle variant identified during routine cadaveric dissection. RESULTS: The muscle identified originated on the C5 vertebra and inserted on the clavicle medial to the trapezius muscle. Innervation was provided by a C6 spinal nerve branch. Notably, a branch of the supraclavicular nerve was closely associated with the muscle, raising the possibility of compression of this nerve. CONCLUSIONS: Presence of a cleidocervicalis muscle should be considered in cases of shoulder pain consistent with supraclavicular nerve entrapment or compression.

KNDy (kisspeptin/neurokinin B/dynorphin) neurons are activated during both pulsatile and surge secretion of LH in the ewe.

KNDy (kisspeptin/neurokinin B/dynorphin) neurons of the arcuate nucleus (ARC) appear to mediate the negative feedback actions of estradiol and are thought to be key regulators of pulsatile LH secretion. In the ewe, KNDy neurons may also be involved with the positive feedback actions of estradiol (E(2)) to induce the LH surge, but the role of kisspeptin neurons in the preoptic area (POA) remains unclear. The goal of this study was to identify which population(s) of kisspeptin neurons is (are) activated during the LH surge and in response to the removal of E(2)-negative feedback, using Fos as an index of neuronal activation. Dual-label immunocytochemistry for kisspeptin and Fos was performed on sections containing the ARC and POA from ewes during the luteal phase of the estrous cycle, or before or after the onset of the LH surge (experiment 1), and from ovary-intact, short-term (24 h) and long-term (>30 d) ovariectomized (OVX) ewes in anestrus (experiment 2). The percentage of kisspeptin neurons expressing Fos in both the ARC and POA was significantly higher during the LH surge. In contrast, the percentage of kisspeptin/Fos colocalization was significantly increased in the ARC, but not POA, after both short- and long-term E(2) withdrawal. Thus, POA kisspeptin neurons in the sheep are activated during, and appear to contribute to, E(2)-positive feedback, whereas ARC kisspeptin (KNDy) neurons are activated during both surge and pulsatile modes of secretion and likely play a role in mediating both positive and negative feedback actions of E(2) on GnRH secretion in the ewe.

Evidence that the arcuate nucleus is an important site of progesterone negative feedback in the ewe.

There is now considerable evidence that dynorphin neurons mediate the negative feedback actions of progesterone to inhibit GnRH and LH pulse frequency, but the specific neurons have yet to be identified. In ewes, dynorphin neurons in the arcuate nucleus (ARC) and preoptic area (POA) are likely candidates based on colocalization with progesterone receptors. These studies tested the hypothesis that progesterone negative feedback occurs in either the ARC or POA by determining whether microimplants of progesterone into either site would inhibit LH pulse frequency (study 1) and whether microimplants of the progesterone receptor antagonist, RU486, would disrupt the inhibitory effects of peripheral progesterone (study 2). Both studies were done in ovariectomized (OVX) and estradiol-treated OVX ewes. In study 1, no inhibitory effects of progesterone were observed during treatment in either area. In study 2, microimplants of RU486 into the ARC disrupted the negative-feedback actions of peripheral progesterone treatments on LH pulse frequency in both OVX and OVX+estradiol ewes. In contrast, microimplants of RU486 into the POA had no effect on the ability of systemic progesterone to inhibit LH pulse frequency. We thus conclude that the ARC is one important site of progesterone-negative feedback in the ewe. These data, which are the first evidence on the neural sites in which progesterone inhibits GnRH pulse frequency in any species, are consistent with the hypothesis that ARC dynorphin neurons mediate this action of progesterone.

Neurokinin B acts via the neurokinin-3 receptor in the retrochiasmatic area to stimulate luteinizing hormone secretion in sheep.

Recent data have demonstrated that mutations in the receptor for neurokinin B (NKB), the NK-3 receptor (NK3R), produce hypogonadotropic hypogonadism in humans. These data, together with reports that NKB expression increases after ovariectomy and in postmenopausal women, have led to the hypothesis that this tachykinin is an important stimulator of GnRH secretion. However, the NK3R agonist, senktide, inhibited LH secretion in rats and mice. In this study, we report that senktide stimulates LH secretion in ewes. A dramatic increase in LH concentrations to levels close to those observed during the preovulatory LH surge was observed after injection of 1 nmol senktide into the third ventricle during the follicular, but not in the luteal, phase. Similar increases in LH secretion occurred after insertion of microimplants containing this agonist into the retrochiasmatic area (RCh) in anestrous or follicular phase ewes. A low-dose microinjection (3 pmol) of senktide into the RCh produced a smaller but significant increase in LH concentrations in anestrous ewes. Moreover, NK3R immunoreactivity was clearly evident in the RCh, although it was not found in A15 dopaminergic cell bodies in this region. These data provide evidence that NKB stimulates LH (and presumably GnRH) secretion in ewes and point to the RCh as one important site of action. Based on these data, and the effects of NK3R mutations in humans, we hypothesize that NKB plays an important stimulatory role in the control of GnRH and LH secretion in nonrodent species.

Evidence that gamma-aminobutyric acid is part of the neural circuit mediating estradiol negative feedback in anestrous ewes.

Seasonal anestrus in ewes is driven by an increase in response to estradiol (E2) negative feedback. Compelling evidence indicates that inhibitory A15 dopaminergic (DA) neurons mediate the increased inhibitory actions of E2 in anestrus, but these neurons do not contain estrogen receptors. Therefore, we have proposed that estrogen-responsive afferents to A15 neurons are part of the neural circuit mediating E2 negative feedback in anestrus. This study examined the possible role of afferents containing gamma-aminobutyric acid (GABA) and nitric oxide (NO) in modulating the activity of A15 neurons. Local administration of NO synthase inhibitors to the A15 had no effect on LH, but GABA receptor ligands produced dramatic changes. Administration of either a GABA A or GABA B receptor agonist to the A15 increased LH secretion in ovary-intact ewes, suggesting that GABA inhibits A15 neural activity. In ovariectomized anestrous ewes, the same doses of GABA receptor agonist had no effect, but combined administration of a GABA A and GABA B receptor antagonist to the A15 inhibited LH secretion. These data are consistent with the hypothesis that endogenous GABA release within the A15 is low in ovary-intact anestrous ewes and elevated after ovariectomy. Using dual immunocytochemistry, we observed that GABAergic varicosities make close contacts on to A15 neurons and that A15 neurons contain both the GABA A-alpha1 and the GABA B-R1 receptor subunits. Based on these data, we propose that in anestrous ewes, E2 inhibits release of GABA from afferents to A15 DA neurons, increasing the activity of these DA neurons and thus suppressing episodic secretion of GnRH and LH.

Endocrine basis for disruptive effects of cortisol on preovulatory events.

Stress activates the hypothalamo-pituitary-adrenal axis leading to enhanced glucocorticoid secretion and concurrently inhibits gonadotropin secretion and disrupts ovarian cyclicity. Here we tested the hypothesis that stress-like concentrations of cortisol interfere with follicular phase endocrine events of the ewe by suppressing pulsatile LH secretion, which is essential for subsequent steps in the preovulatory sequence. Cortisol was infused during the early to midfollicular phase, elevating plasma cortisol concentrations to one third, one half, or the maximal value induced by isolation, a commonly used model of psychosocial stress. All cortisol treatments compromised at least some aspect of reproductive hormone secretion in follicular phase ewes. First, cortisol significantly suppressed LH pulse frequency by as much as 35%, thus attenuating the high frequency LH pulses typical of the preovulatory period. Second, cortisol interfered with timely generation of the follicular phase estradiol rise, either preventing it or delaying the estradiol peak by as much as 20 h. Third, cortisol delayed or blocked the preovulatory LH and FSH surges. Collectively, our findings support the hypothesis that stress-like increments in plasma cortisol interfere with the follicular phase by suppressing the development of high frequency LH pulses, which compromises timely expression of the preovulatory estradiol rise and LH and FSH surges. Moreover, the suppression of LH pulse frequency provides indirect evidence that cortisol acts centrally to suppress pulsatile GnRH secretion in follicular-phase ewes.

The premammillary hypothalamic area of the ewe: anatomical characterization of a melatonin target area mediating seasonal reproduction.

Recent evidence suggests that the ovine premammillary hypothalamic area (PMH) is an important target for the pineal hormone, melatonin, and its role in seasonal reproduction. In rodents, the PMH is a complex region consisting of several cell groups with differing neurochemical content and anatomical connections. Therefore, to obtain a better understanding of the potential neural targets for melatonin in this area of the sheep brain, we have undertaken a detailed anatomical characterization of the PMH, including its nuclear divisions and the location of neuropeptide/neurotransmitter cells within them. By combining immunocytochemistry for NeuN, a neuronal marker, with Nissl staining in anestrous, ovariectomized, estradiol-treated ewes, we identified three nuclei within the PMH: a caudal continuation of the hypothalamic arcuate nucleus (cARC), the ventral division of the premammillary nucleus (PMv), and the ventral tuberomammillary nucleus (TMv). The cARC contained neurons that were immunoreactive for tyrosine hydroxylase, dynorphin, estrogen receptor alpha, cocaine- and amphetamine-regulated transcript peptide (CART), and nitric oxide synthase (NOS). The PMv was also characterized by the presence of cells that contained NOS and CART, although the size of these cells was larger than that of their corresponding phenotype in the cARC. By contrast, in the TMv, of the markers examined in the present study, only fibers immunoreactive for orexin were seen. Thus, the ovine PMH is a heterogeneous region comprised of three subdivisions, each with distinct morphological and neurochemical characteristics. This anatomical map of the PMH provides a basis for future studies to determine the functional contribution of each component to the influence of melatonin on seasonal reproduction.

Endotoxin inhibits the surge secretion of gonadotropin-releasing hormone via a prostaglandin-independent pathway.

Immune/inflammatory challenges, such as bacterial endotoxin, disrupt gonadotropin secretion and ovarian cyclicity. We previously determined that endotoxin can block the estradiol-induced LH surge in the ewe. Here, we investigated mechanisms underlying this suppression. First, we tested the hypothesis that endotoxin blocks the estradiol-induced LH surge centrally, by preventing the GnRH surge. Artificial follicular phases were created in ovariectomized ewes, and either endotoxin or vehicle was administered together with a surge-inducing estradiol stimulus. In each ewe in which endotoxin blocked the LH surge, the GnRH surge was also blocked. Given this evidence that endotoxin blocks the estradiol-induced LH surge at the hypothalamic level, we began to assess underlying central mechanisms. Specifically, in view of the prior demonstration that prostaglandins mediate endotoxin-induced suppression of pulsatile GnRH secretion in ewes, we tested the hypothesis that prostaglandins also mediate endotoxin-induced blockade of the surge. The prostaglandin synthesis inhibitor flurbiprofen was delivered together with endotoxin and the estradiol stimulus. Although flurbiprofen abolished endotoxin-induced fever, which is a centrally generated, prostaglandin-mediated response, it failed to reverse blockade of the LH surge. Collectively, these results indicate endotoxin blocks the LH surge centrally, suppressing GnRH secretion via a mechanism not requiring prostaglandins. This contrasts with the suppressive effect of endotoxin on GnRH pulses, which requires prostaglandins as intermediates.

The suprachiasmatic nucleus: a clock of multiple components.

Although impressive progress has been made in understanding the molecular basis of pacemaker function in the suprachiasmatic nucleus (SCN), fundamental questions about cellular and regional heterogeneity within the SCN, and how this heterogeneity might contribute to SCN pacemaker function at a tissue level, have remained unresolved. To reexamine cellular and regional heterogeneity within the SCN, the authors have focused on two key questions: which SCN cells are endogenously rhythmic and/or directly light responsive? Observations of endogenous rhythms of electrical activity, gene/protein expression, and protein phosphorylation suggest that the SCN in mammals examined to date is composed of anatomically distinct rhythmic and nonrhythmic components. Endogenously rhythmic neurons are primarily found in rostral, dorsomedial, and ventromedial portions of the nucleus; at mid and caudal levels, the distribution of endogenously rhythmic cells in the SCN has the appearance of a "shell." The majority of nonrhythmic cells, by contrast, are located in a central "core" region of the SCN, which is complementary to the shell. The location of light-responsive cells, defined by direct retinohypothalamic input and light-induced gene expression, largely overlaps the location of nonrhythmic cells in the SCN core, although, in hamsters and mice light-responsive cells are also present in the ventral portion of the rhythmic shell. While the relative positions of rhythmic and light-responsive components of the SCN are similar between species, the precise boundaries of these components, and neurochemical phenotype of cells within them, are variable. Intercellular communication between these components may be a key feature responsible for the unique pacemaker properties of the SCN observed at a tissue and whole animal level.

Evidence that thyroid hormones act in the ventromedial preoptic area and the premammillary region of the brain to allow the termination of the breeding season in the ewe.

Thyroid hormones are permissive for various species to enter seasonal anestrus. In the ewe they act centrally to permit the onset of potent estradiol-negative feedback responsible for anestrus, but the specific sites of action are unknown. Therefore, we tested whether T(4) replacement via chronic microimplants in any of five brain areas could reverse the reproductive effects of thyroidectomy. Diffusion of (125)I-T(4) from the microimplant was largely (>98%) limited to a 1.2-mm radius. A marked decline in LH concentration in ovariectomized, estradiol-treated ewes was used as an index for anestrus. In experiment 1, all thyroidectomized (THX) ewes with microimplants in the medial preoptic area, A15 area, and medial basal hypothalamus failed to enter anestrus; instead, LH levels remained elevated, similar to those in untreated THX controls. In ventromedial preoptic area (vmPOA)-microimplanted ewes, only the two animals with the most caudal microimplants entered anestrus, as did thyroid-intact controls and THX ewes receiving icv or sc T(4) replacement. In experiment 2, all vmPOA-treated ewes with similar placements to those effective in experiment 1 along with all ewes microimplanted in the premammillary region entered neuroendocrine anestrus. Thus, the premammillary region and vmPOA are brain sites in which thyroid hormones act to permit the onset of seasonal anestrus.

Does cortisol mediate endotoxin-induced inhibition of pulsatile luteinizing hormone and gonadotropin-releasing hormone secretion?

Bacterial endotoxin (lipopolysaccharide), a commonly used model of immune/inflammatory stress, inhibits reproductive neuroendocrine activity and concurrently induces a profound stimulation of the hypothalamo-pituitary-adrenal axis. We employed two approaches to test the hypothesis that enhanced secretion of cortisol mediates endotoxin-induced suppression of pulsatile GnRH and LH secretion in the ovariectomized ewe. First, we mimicked the endotoxin-induced increase in circulating cortisol by delivering the glucocorticoid in the absence of the endotoxin challenge. Within 1-2 h, experimentally produced increments in circulating cortisol suppressed pulsatile LH secretion in a dose-dependent fashion. Second, we blocked the endotoxin-induced stimulation of cortisol secretion using the drug metyrapone, which inhibits the 11-beta hydroxylase enzyme necessary for cortisol biosynthesis. In the absence of a marked stimulation of cortisol secretion, endotoxin still profoundly inhibited pulsatile GnRH and LH secretion. We conclude that, although enhanced cortisol secretion may contribute to endotoxin-induced suppression of reproductive neuroendocrine activity, the marked stimulation of the glucocorticoid is not necessary for this response. Our findings are consistent with the hypothesis that immune/inflammatory stress inhibits reproductive neuroendocrine activity via more than one inhibitory pathway, one involving enhanced secretion of cortisol and the other(s) being independent of this glucocorticoid.

Temporal requirements of thyroid hormones for seasonal changes in LH secretion.

The transition between breeding and anestrous seasons in ewes is driven by an endogenous rhythm in responsiveness to estradiol negative feedback. One stage of this rhythm, the transition to anestrus, requires the presence of thyroid hormone during a window of responsiveness that opens in the late breeding season. The primary goal of this study was to assess when ewes lose responsiveness to thyroid hormone (i.e. when the window closes). In addition, we investigated whether thyroid hormone influences aspects of seasonality other than the transition to anestrus. Ovariectomized ewes maintained in a simulated natural photoperiod were implanted with estradiol, thyroidectomized, and treated with T(4) for 100 d beginning at progressively later dates during the anestrous season. Onset of neuroendocrine anestrus (decrease in LH), latency to anestrus, and time of onset of the subsequent neuroendocrine breeding season (rise in LH) were determined. Ewes gradually lost responsiveness to T(4) during the latter half of the anestrous season, as judged by increasing latency to the decrease in LH and, eventually, failure to exhibit a decrease in LH. Progressively later T(4) replacements also caused progressive delays in the subsequent breeding season. In contrast, the annual PRL cycle was not significantly affected by thyroidectomy or T(4) replacement. These findings indicate that 1) responsiveness to T(4) is lost gradually during the mid to late anestrous season; 2) thyroid hormones can influence the timing of the breeding season and thus may be required for the maintenance or entrainment of the endogenous reproductive rhythm; 3) thyroid hormones are not required for all seasonal neuroendocrine cycles.