Glucocorticoids Regulate Kisspeptin Neurons during Stress and Contribute to Infertility and Obesity in Leptin-Deficient Mice

Stressors generate adaptive responses, including transient suppression of reproductive function. Natural selection depends on successful reproduction, but inhibition of reproduction to survive famine or escape predation allows animals to survive to reproduce at a later time. The cellular locations and mechanisms responsible for inhibiting and reactivating the reproductive axis during and after stress, respectively, are not well understood. We demonstrated that stress-induced elevation in glucocorticoids affects hypothalamic neurons that secrete kisspeptin (KISS1), an important reproductive hormone. Stressors that stimulated glucocorticoid secretion, as well as glucocorticoid administration itself, inhibited Kiss1 mRNA expression, while conditions that did not change glucocorticoid secretion did not alter Kiss1 mRNA expression. In mice lacking glucocorticoid receptor specifically in kisspeptin-containing neurons, Kiss1 mRNA expression was no longer inhibited during restraint stress despite a rise in corticosterone, and both testosterone and copulatory behaviors showed accelerated recovery in the post-traumatic period. We also demonstrated that increased glucocorticoid secretion contributed to infertility and obesity in leptin-deficient mice. Leptin deficiency creates a chronic state of perceived starvation, and leptin-deficient mice exhibit elevated plasma glucocorticoid concentrations, morbid obesity, and infertility. Leptin-deficient, glucocorticoid-deficient mice exhibited decreased body weight and fat composition, decreased hyperphagia, and normal fertility. When supplemented with glucocorticoids back to the initial levels present in leptin deficiency, these mice gained weight and became infertile. Thus, leptin is not required for fertility as previously believed, and glucocorticoids can contribute to obesity and suppress fertility independently of leptin signaling. Together, these findings implicate glucocorticoids in the regulation of obesity and reproductive inhibition during stress, including perceived starvation caused by leptin deficiency. These studies may provide novel mechanisms and molecular targets in the reproductive and metabolic aspects of disorders characterized by glucocorticoid dysregulation, including post-traumatic stress disorder, anorexia nervosa, and mood disorders

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty

Reproductive Hormone-Dependent and -Independent Contributions to Developmental Changes in Kisspeptin in GnRH-Deficient Hypogonadal Mice

Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty

Effects of estradiol (E2) on <i>hpg</i> AVPV and ARC kisspeptin immunostaining patterns.

<p><b>A</b>) Representative images of AVPV kisspeptin immunostaining patterns from gonad intact or ovariectomized (OVX) 60 day old WT and <i>hpg</i> females treated with sham or E2-containing Silastic capsules for 7 days. Scale bar  = 100 µm. <b>B</b>) AVPV kisspeptin-immunoreactive neurons from each group in (A) were counted and compared. Values are mean ± SEM. Different letters denote significant differences (ANOVA, Neuman-Keuls post hoc), P<0.05. <b>C)</b> Representative images of kisspeptin immunostaining patterns in the ARC of intact diestrus or OVX female WT or <i>hpg</i> mice, treated with sham or E2-containing Silastic capsules for 7 days. Scale bar  = 200 µm.</p

Altered distribution pattern of kisspeptin immunostaining in the ARC of <i>hpg</i> mice.

<p>Kisspeptin ICC across postnatal development (age 10, 30, 45 and 60 days) in WT and <i>hpg</i> females (<b>A</b>) and males (<b>B</b>). Kisspeptin ICC staining pattern in the ARC of both female and male WT mice (<i>left of dashed line</i>) is dominated by densely stained fibers that obscure kisspeptin-positive cell bodies (<i>white arrowheads</i>). In <i>hpg</i> mice (<i>right of dashed line</i>), ARC kisspeptin ICC shows reduced fiber staining and increased clusters of large, darkly stained cell bodies (<i>black arrowheads</i>). Scale bar  = 200 µm.</p

Developmental expression of <i>Kiss1</i> in the ARC of male and female <i>hpg</i> and WT mice.

<p>Quantitative analysis of Kiss1 mRNA levels in the ARC measured by qRT-PCR of WT and <i>hpg</i> females (<b>A</b>) and males (<b>B</b>) across postnatal development. Values are mean ± SEM. WT, <i>white bars</i>; <i>hpg</i>, <i>black bars.</i> Different letters denote significant differences (ANOVA, Neuman-Keuls post hoc). *, denotes significant differences between genotypes, P<0.05.</p

Immunocytochemistry (ICC) of kisspeptin neurons in the AVPV during postnatal development in wild-type (WT) and <i>hpg</i> mice.

<p><b>A</b>) Representative images of kisspeptin immunostaining from AVPV regions along the third ventricle (<i>dashed line</i>) of female and male WT and <i>hpg</i> mice at postnatal ages 10, 30, 45 and 60 days. <b>B, C</b>) Quantitation of the number of immunoreactive kisspeptin neurons/section in the AVPV of WT and <i>hpg</i> female (<b>B</b>) and male (<b>C</b>) mice. Values are mean ± SEM. WT, <i>white bars</i>; <i>hpg</i>, <i>black bars</i>. Significant differences between ages are denoted by different letters (WT, <i>lower case</i>, <i>hpg</i>, <i>upper case</i>; ANOVA, Neuman-Keuls post hoc); *, denotes significant difference between genotypes. <i>Solid horizontal line</i> across B and C indicates the mean of the number of immunoreactive neurons in the AVPV of <i>hpg</i> females and males from P30 onward, and the <i>dotted lines</i> indicate ±2 standard deviations from the mean. P<0.05. Scale bar  = 100 µm.</p

<i>Kiss1</i> expression is increased in the ARC of female and male <i>hpg</i> mice.

<p><b>A</b>) Representative dark-field images of silver grain Kiss1 mRNA signal as measured by ISH in the ARC of pubertal-aged WT and <i>hpg</i> females (30 days) and males (45 days). Scale bar  = 200 µm. <b>B</b>) Male and female Kiss1 positive neurons/section in the <i>hpg</i> ARC compared to WT. <b>C</b>) Mean of representative Kiss1 mRNA silver grain signal per neuron in the male and female <i>hpg</i> ARC sections compared to WT (mean signal intensity). Values are mean ± SEM. WT, <i>white bars</i>; <i>hpg, black bars</i>. Different letters denote significant differences, P<0.05.</p

<i>Kiss1</i> expression in the AVPV of WT and <i>hpg</i> females and males across postnatal development.

<p>Quantitative analysis of Kiss1 mRNA levels in the AVPV of (<b>A</b>) females and (<b>B</b>) males as measured by qRT-PCR. Values are mean ± SEM. WT, <i>white bars</i>; <i>hpg, black bars</i>. Different letters denote significant differences (ANOVA, Neuman-Keuls post hoc); *, denotes significant difference between genotypes, P<0.05.</p

Effects of cotrimoxazole prophylaxis on Talaromyces marneffei

The dimorphic fungus Talaromyces marneffei (TM) is a common cause of HIV-associated opportunistic infections in Southeast Asia. Cotrimoxazole (CTX) inhibits folic acid synthesis which is important for the survival of many bacteria, protozoa, and fungi and has been used to prevent several opportunistic infections among HIV/AIDS patients. We question whether CTX is effective in preventing TM infection. To investigate this question, we conducted an 11-year (2005–2016) retrospective observational cohort study of all patients on the Chinese national antiretroviral therapy (ART) programme in Guangxi, a province with high HIV and TM burden in China. Survival analysis was conducted to investigate TM cumulative incidence, and Cox regression and propensity score matching (PSM) were used to evaluate the effect of CTX on TM incidence. Of the 3359 eligible individuals contributing 10,504.66 person-years of follow-up, 81.81% received CTX within 6 months after ART initiation, and 4.73% developed TM infection, contributing 15.14/1,000 person-year TM incidence rate. CTX patients had a significantly lower incidence of TM infection than non-CTX patients (4.11% vs. 7.53%; adjusted hazard ratio (aHR) = 0.50, 95% CI 0.35–0.73). CTX reduced TM incidence in all CD4+ cell subgroups (+ cell count <50 cells/μL in both Cox regression and the PSM analyses. In conclusion, in addition to preventing other HIV-associated opportunistic infections, CTX prophylaxis has the potential to prevent TM infection in HIV/AIDS patients receiving ART.</p