Female Knockout Research Articles

An Evolutionarily Conserved Olfactory Receptor Is Required for Sex Differences in Blood Pressure

Olfactory receptor 558 (Olfr558) is expressed in the kidney where its functional role is unknown. Using RNAScope, we find that Olfr558 localizes to vascular smooth muscle, including the renal afferent arteriole. Thus, we measured blood pressure (BP) in Olfr558 wild-type (WT) and knockout (KO) mice. Systolic (SBP), diastolic (DBP), and mean arterial pressure (MAP) were measured in whole-body Olfr558 wild-type (WT) and knockout (KO) via telemetry. It is known that males have a higher BP than premenopausal females in both humans and mice; indeed, we find Olfr558WT males (M) have higher BP than females (F, n=7-9 per group), including MAP (M:101.0±1.6 vs. F:89.0±0.9 mmHg, p<0.0001), SBP (M:113±2 vs. F:102±1mmHg, p=0.0025), and DBP (M: 89±2 vs. F:75±01mmHg, p<0.0001). However, sex differences in BP are entirely absent in Olfr558 KO (MAP M: 96±2 vs. F: 95±1mmHg; SBP M: 110±2 vs. F: 108±2mmHg; DBP M: 81±2 vs. F: 82±2mmHg). Of note, other sex differences (body weight) are intact in KO, and KO males and females are fertile. To characterize this phenotype, renin expression was analyzed by qPCR, plasma renin activity (PRA) was measured by ELISA, and vasoreactivity was examined ex vivo by wire myography (responses to potassium chloride (KCl), phenylephrine (PE), acetylcholine (Ach), and sodium nitroprusside (SNP) in aortic and mesenteric rings). In male KOs (n=10-12), there was a decrease in kidney renin mRNA (0.4±0.1 vs WT: 1±0.1 p=0.001) and PRA (201±21 vs WT: 402±15, p=0.001); these parameters were unchanged in female KOs. Male KO aortic rings exhibit less constriction to PE, male KO mesenterics exhibit more relaxation to SNP, and female KO aortic rings exhibit more constriction to KCl. In females, pulse wave velocity (PWV) and tensile testing revealed that KO females have increased PWV (KO: 4.6±0.4 vs WT: 3.2±0.2 m/s, p=0.0069, n=8-11), but no changes in tensile testing. The human ortholog of Olfr558, OR51E1, was previously identified as a locus associated with DBP. Using UK Biobank data (N~450k), we find that a rare OR51E1 missense variant has a statistically significant sex interaction effect with DBP ( p=0.015), increasing DBP (+6.28mmHg) in women but decreasing it (-4.77mmHg) in men (similar to the phenotype in M and F Olfr558 KO mice). Finally, we express OR51E1 variants in HEK 293T cells and characterize how two different clinically relevant OR51E1 variants influence OR51E1 signaling. In sum, our findings demonstrate that an evolutionarily conserved olfactory receptor is required for sex differences in BP. Olfr558 regulates BP in males via renin and vascular reactivity, but via arterial stiffness in females. Funding: R56DK107726 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of Tyro3 causes anxiety-relevant behavioural changes in female mice.

White-matter brain abnormalities have been found across a variety of psychiatric disorders. The extent of white matter pathology is proposed to be predictive of the severity of anxiety disorders. However, it is still unknown whether disruptions of white matter integrity precede, and are sufficient to give rise to, the behavioural symptoms. Interestingly, mood disturbances feature prominently in central demyelinating diseases such as multiple sclerosis. It is unclear whether the greater frequency of neuropsychiatric symptoms is linked to underlying neuropathology. In this study, we characterised male and female Tyro3 knockout (KO) mice using a variety of behavioural paradigms. Anxiety-related behaviours were assessed with the elevated-plus maze and light-dark box. Fear memory processing was assessed using fear conditioning and extinction paradigms. Finally, we assessed immobility time in the Porsolt swim test as a measure of depression-related behavioural despair. Surprisingly, loss of Tyro3 did not lead to manifestation of major shifts in baseline behaviour. We noted significant differences in habituation to novel environments and post-conditioning freezing levels of female Tyro3 KO mice, which are consistent with the female bias in anxiety disorders and could be indicative of maladaptive stress-responses. This study has demonstrated that white matter pathology related to a loss of Tyro3 is associated with pro-anxiety behavioural responses of female mice. Future studies could probe their contribution to increased risk for neuropsychiatric disorders when combined with stressful triggering events.

Sarcolipin ablation decreases whole body VO2 in young female nice

The sarco(endo)plasmic reticulum calcium (Ca 2+ ) ATPase (SERCA) pump is a major regulator of cytosolic Ca 2+ in striated muscle. Sarcolipin (SLN) reduces SERCA-mediated Ca 2+ pumping efficiency and is a key regulator of muscle metabolism and contractile function. With SERCA activity comprising approximately 12% of whole-body metabolic rate, the inefficient SERCA Ca 2+ cycling promoted by SLN would be predicted to increase metabolic rate. Although SLN is involved in adaptive diet-induced thermogenesis and overexpression of SLN increases metabolic rate and protects against diet-induced obesity, we found previously that SLN ablation had no effect on whole body metabolic rate or body composition in male mice that were fed a standard chow diet. However, unpublished work from our lab indicates that SLN expression in mouse soleus is significantly higher in females compared with males. Therefore, the purpose of this study was to investigate the effects of SLN ablation on body composition and metabolic rate of female mice. Adult (4-6mo) female wild type (WT) and SLN knockout (KO) mice (n=4 per group) were used to collect anthropometric measures (adiposity and body weight) and were housed in a comprehensive lab animal monitoring system (CLAMS) over a 48-hour period to assess daily whole body VO2 and total cage activity. With SLN ablation, total whole body VO 2 (ml/kg/hr) was lower (p<0.05) in the KO females (3534 ± 95.1) compared to WT females (5080 ± 98.8), with no differences (p>0.05) found for total cage activity (counts) (WT, 14935.42 ± 1799.6 vs. KO, 11860.83 ± 679.48), body weight (grams) (WT, 20.97 ± 0.76 vs. KO, 22.18 ± 0.80), or adiposity (arbitrary units) (WT, 5.18 ± 1.71 vs. KO, 2.12 ± 0.36). These results showing that high levels of endogenous muscle SLN expression in female mice influence whole-body metabolism even under chow-fed conditions, demonstrate a potential important sex difference in whole body physiology and metabolism. Natural Sciences and Engineering Research Council of Canada (NSERC) [grant number 311922-05 (to A.R.T.)] This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Mustn1 ablation in skeletal muscle results in increased glucose tolerance concomitant with upregulated GLUT expression in male mice.

Glucose homeostasis is closely regulated to maintain energy requirements of vital organs and skeletal muscle plays a crucial role in this process. Mustn1 is expressed during embryonic and postnatal skeletal muscle development and its function has been implicated in myogenic differentiation and myofusion. Whether Mustn1 plays a role in glucose homeostasis in anyway remains largely unknown. As such, we deleted Mustn1 in skeletal muscle using a conditional knockout (KO) mouse approach. KO mice did not reveal any specific gross phenotypic alterations in skeletal muscle. However, intraperitoneal glucose tolerance testing (IPGTT) revealed that 2-month-old male KO mice had significantly lower glycemia than their littermate wild type (WT) controls. These findings coincided with mRNA changes in genes known to be involved in glucose metabolism, tolerance, and insulin sensitivity; 2-month-old male KO mice had significantly higher expression of GLUT1 and GLUT10 transporters, MUP-1 while OSTN expression was lower. These differences in glycemia and gene expression were statistically insignificant after 4 months. Identical experiments in female KO and WT control mice did not indicate any differences at any age. Our results suggest a link between Mustn1 expression and glucose homeostasis during a restricted period of skeletal muscle development/maturation. While this is an observational study, Mustn1's relationship to glucose homeostasis appears to be more complex with a possible connection to other key proteins such as GLUTs, MUP-1, and OSTN. Additionally, our data indicate temporal and sex differences. Lastly, our findings strengthen the notion that Mustn1 plays a role in the metabolic capacity of skeletal muscle.

Abstract 528: Endothelial Estrogen Receptor Alpha Deletion Promotes Abdominal Aortic Aneurysm In Male Mice

Introduction: Although men have a greater incidence of abdominal aortic aneurysm (AAA) compared with women, the role of sex-hormones in mediating these differences is not well understood. Given the known effect of estrogen receptor alpha (ERα) signaling in maintaining vascular wall homeostasis, the present study examines its role in the pathogenesis of AAA. Specifically, we scrutinize whether endothelial cell (EC) ERα protects against AAA via increased lysyl oxidase (LOX) activation and regulation of vascular smooth muscle cell (VSMC) phenotype. Methods: AAA was induced in male and female ECERα knockout (ECERαKO) mice and their littermates (n=6-11) by administering β-Aminopropionitrile (BAPN) via drinking water (1 mg/ml; 0.15 g/kg/d) from 3 days prior to angiotensin II (Ang II; 1 μg/kg/day) infusion for 2 weeks. The development of AAA was assessed by ultrasound measurement of aortic luminal dilation and histology. Relevant molecular mechanisms were examined in abdominal aorta and in human aortic endothelial cells (HAECs). Results: BAPN Ang II resulted in an increased incidence of AAA in male ECERαKO mice (72%), whereas female ECERαKO mice did not develop aneurysms. No difference in AAA incidence was observed between male and female littermate control mice (12.5%). Elastin fragmentation was significantly higher only in male ECERαKO mice compared to littermates, while female ECERαKO and their littermates had comparable elastin fragmentation. Ang II receptor type 1a mRNA expression was higher in male ECERαKO, whereas female ECERαKO mice had higher Ang II receptor type 1b mRNA expression. Gene expression of smooth muscle alpha-actin and smooth muscle 22 (contractile VSMC markers) was significantly higher in female aortas compared to males irrespective of genotype, whereas, osteopontin, synthetic VSMC marker was only upregulated in male ECERαKO. HAECs treated with estradiol (10 nM, 24 h) significantly increased LOX activity. Conclusions: Our findings identify a critical role of endothelial ERα in protection against AAA in male mice likely through increased LOX activity and maintenance of VSMC phenotype. Further studies are needed to delineate the crosstalk between endothelial ERα and VSMCs, and their impact in the pathogenesis of AAA.

Melanocortin 4 receptor signaling in Sim-1 neurons permits sexual receptivity in female mice

Female sexual dysfunction affects approximately 40% of women in the United States, yet few therapeutic options exist for these patients. The melanocortin system is a new treatment target for Hypoactive Sexual Desire Disorder (HSDD), but the neuronal pathways involved are unclear. In this study, female MC4R knockout mice lacking melanocortin 4 receptors (MC4Rs) paired with males were found to approach males less and have reduced receptivity to copulation, as indicated by a low lordosis quotient. The mice were then bred to express MC4Rs exclusively on Sim1 neurons (tbMC4RSim1 mice) or on oxytocin neurons (tbMC4ROxt mice). Lordosis behavior was normalized in tbMC4RSim1 mice and improved in tbMC4ROxt mice. In contrast, approach behavior was unchanged in tbMC4RSim1 mice but greatly increased in tbMC4ROxt animals. The changes were independent of melanocortin-driven metabolic effects. These results implicate MC4R signaling in Oxt neurons in appetitive behaviors and MC4R signaling in Sim1 neurons in female sexual receptivity, while suggesting melanocortin-driven sexual function does not rely on metabolic neural circuits. R01HD081792 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Liver Androgen Receptor Knockout Delayed the Onset of High Fructose Diet-Induced Dysglycemia in Female Mice

Introduction: Previous research has indicated that a lipid-independent pathogenic mechanism plays a role in androgen-induced hepatic insulin resistance where liver androgen receptors may play a role in modulating disease. Consequently, this study aims to investigate the pathophysiology of high fructose diet (HFrD) induced dysglycemia in male and female liver androgen receptor knockout (LivARKO) mice. Andrisse et al 2021 showed that deleting the liver androgen receptor prevented female mice from developing hyperandrogenemia (HA)-induced insulin resistance. From this, we asked the question, does LivARKO prevent HFrD induced dysglycemia? Hypothesis: It was hypothesized that HFrD LivARKO female mice would display impaired whole body glucose metabolism in comparison to the Control diet fed LivARKO female mice, suggesting that AR does not play a significant role in regulating HFrD-induced dysglycemia.Methods: We performed insulin tolerance tests (ITT), glucose tolerance tests (GTT), pyruvate tolerance tests (PTT), and glucose stimulated insulin secretion (GSIS) tests on LivARKO female mice fed a high fat diet (HFD) or a control diet (CD) (from Research Diets Inc) during months 1 or 2 after starting the diet. Results: After 1-month, LivARKO female mice fed a Control diet had impaired glucose tolerance compared to LivARKO-HFrD female mice; however, after 2-months, the LivARKO-HFrD female mice displayed impaired glucose tolerance above that of the Control diet LivARKO female mice. LivARKO female mice fed a high fructose diet (HFrD) did not display increased fasting insulin levels compared to LivARKO female mice on a control diet. HFrD LivARKO mice did not display any increase in GSIS at 15-min or 30-min. Conclusion: These findings suggest that LivARKO delayed the onset of HFrD-Induced dysglycemia in female mice. Further research may consist of molecular data to elucidate the mechanistic pathways involved in these whole body phenotypes observed. The cellular molecular regulation of differing mechanisms of insulin resistance. 1R01DK126892-01A1 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Loss of A Metalloproteinase and Disintegrin 15 leads to exacerbated myocardial hypertrophy and dilation through activation of the calcineurin pathway in male but not female micev

Cardiac dysfunction associated with cardiomyopathies is characterized by myocardial hypertrophy and dilation involving many cellular and molecular signalling events. A disintegrin and metalloproteinases (ADAMs) are membrane-bound proteinases with diverse functions, whose role in heart disease remains underexplored. ADAM15 is expressed in the heart and is downregulated in the failing human heart. In left ventricular (LV) specimens, ADAM15 levels did not change in patients with concentric hypertrophy (preserved ejection fraction) but was markedly decreased in hypertrophic and dilated failing cardiomyopathy. We investigated the role of ADAM15 in pressure overloaded cardiomyopathy in both male and female C57BL/6 mice. Loss of ADAM15 alone did not cause cardiomyopathy in both male and female mice up to 1 year of age. Male and female wildtype (WT) and ADAM15 knockout ( Adam15 -/- ) mice were subjected to cardiac pressure overload by transverse aortic constriction (TAC) at 8 weeks of age. Adam15 -/- female mice had comparable cardiac function and hypertrophy to their WT controls while, Adam15 -/- male mice exhibited worsened decompensated hypertrophy and dilation with increased activation of mitogen activated protein kinases (pJNK, pERK1/2) compared to WT. Expression of integrin-α7 (but not integrin β1) increased significantly more in male Adam15 -/- -TAC hearts, while the interaction of integrins with basement membrane (laminin) decreased consistent with increased LV dilation. Activity of calcineurin was higher in male Adam15 -/- -TAC hearts along with greater de-phosphorylation of its target transcription factor, NFAT1. Calcineurin inhibition (cyclosporin A) blocked the excess hypertrophy and dilation in male Adam15 -/- mice post-TAC. Calcineurin signalling has been shown to be inhibited by 17β-estradiol (E2), the circulating form of estrogen. Thus, the sex-dependent phenotypic differences in male and female Adam15 -/- mice following pressure overload could be attributed to the inherent increased inhibition of calcineurin-mediated signalling due to the increased expression of estrogen in female mice. Research supported by Canadian Institutes of Health Research (CIHR) grant This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Sex-specific effect of AQP9 deficiency on hepatic triglyceride metabolism in mice with diet-induced obesity.

Studies in obese rats and human cell models of non-alcoholic fatty liver disease have indicated that knockdown of the hepatic glycerol channel aquaporin 9 (AQP9) leads to decreased hepatic steatosis. However, a study in leptin receptor-deficient mice did not find that knockout (KO) of AQP9 alleviated hepatic steatosis. The aim of this study was to investigate the effect of high-fat diet (HFD) on hepatic glycerol and triglyceride metabolism in male and female AQP9 KO mice. Male and female AQP9 KO mice and wild-type (WT) littermates were fed a HFD for 12weeks. Weight, food intake and blood glucose were monitored throughout the study and tissue analysis included determination of hepatic triglyceride content and triglyceride secretion. The expression of key molecules for hepatic glycerol and triglyceride metabolism was evaluated using qPCR and western blotting. AQP9 KO and WT mice demonstrated a similar weight gain throughout the study period, and we found no evidence for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. Instead, we show that the effect of AQP9 deficiency on hepatic lipid metabolism is sex-specific, with only male AQP9 KO mice having a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α. Male AQP9 KO mice had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels. Thus, we found no evidence for AQP9 inhibition being a target for alleviating the development of hepatic steatosis in mice with diet-induced obesity. KEY POINTS: This study investigates the effect of AQP9 deficiency on hepatic triglyceride metabolism in both male and female mice fed a high-fat diet (HFD) for 12weeks. No evidence was found for AQP9 deficiency being associated with a reduced hepatic accumulation of triglyceride or a reduced blood glucose level. The effect of AQP9 deficiency on hepatic triglyceride metabolism is sex-specific. Male AQP9 KO mice had a reduced hepatic secretion of triglycerides and an elevated expression of peroxisome proliferator-activated receptor α, which likely promotes an increased hepatic fatty acid oxidation. Male AQP9 KO had an elevated blood glucose level after 12weeks of HFD when compared to baseline levels.

Structural Analysis Implicates CASK-Liprin-α2 Interaction in Cerebellar Granular Cell Death in MICPCH Syndrome.

Microcephaly with pontine and cerebellar hypoplasia (MICPCH) syndrome is a neurodevelopmental disorder caused by the deficiency of the X-chromosomal gene CASK. However, the molecular mechanisms by which CASK deficiency causes cerebellar hypoplasia in this syndrome remain elusive. In this study, we used CASK knockout (KO) mice as models for MICPCH syndrome and investigated the effect of CASK mutants. Female CASK heterozygote KO mice replicate the progressive cerebellar hypoplasia observed in MICPCH syndrome. CASK KO cultured cerebellar granule (CG) cells show progressive cell death that can be rescued by co-infection with lentivirus expressing wild-type CASK. Rescue experiments with CASK deletion mutants identify that the CaMK, PDZ, and SH3, but not L27 and guanylate kinase domains of CASK are required for the survival of CG cells. We identify missense mutations in the CaMK domain of CASK derived from human patients that fail to rescue the cell death of cultured CASK KO CG cells. Machine learning-based structural analysis using AlphaFold 2.2 predicts that these mutations disrupt the structure of the binding interface with Liprin-α2. These results suggest that the interaction with Liprin-α2 via the CaMK domain of CASK may be involved in the pathophysiology of cerebellar hypoplasia in MICPCH syndrome.

Aerobic Exercise Modulates Visceral Adipose Tissue of Estrogen Deprived Rats in an Experimental Model of Dyslipidemia

Abstract Introduction Menopausal women have an increase deposition of body fat and changes in the lipid profile, being especially susceptible to cardiovascular diseases, and type 2 diabetes. However, physical activity can mitigate this situation. Thus, the aim of the present study is to evaluate the effects of moderate aerobic exercise on visceral adipose tissue (VAT) of female LDL-receptor knockout ovariectomized mice. Methods We used 48 animals, divided into six groups (n = 8/per group): sedentary control (SC), sedentary ovariectomized control (SCO), trained ovariectomized control (TCO), sedentary non-ovariectomized LDL-receptor knockout (KS), sedentary ovariectomized LDL-receptor knockout (KOS), and trained LDL-receptor knockout ovariectomized (KOT). We analyzed the VAT through morphometric and stereological parameters in hematoxylin and eosin stained sections. Additionally, we evaluated biochemical parameters as glucose, triglycerides, and total cholesterol. Finally, immunohistochemical techniques for matrix remodeling, inflammation, apoptosis, and oxidative stress were evaluated. Results We observed that menopause is related to increased visceral adiposity, inflammation, oxidative stress, macrophages activity, serum levels of glucose, triglycerides, and total cholesterol. However, exercise was effective in reducing these parameters, as well as being associated with increased vascularization of VAT and interstitial volume density. Conclusions Moderate exercise is a key factor in mitigating the effects of dyslipidemia in estrogen deprivation. However, further studies are needed to corroborate with our findings.

β-Arrestin 2 knockout prevents bone loss in response to continuous parathyroid hormone stimulation in male and female mice

ABSTRACT Background β-Arrestin 2 (β-arr2) binds activated parathyroid hormone (PTH) receptors stimulating internalization. PTH stimulates both anabolic and catabolic effect on bone depending on the way it is administered. Intermittent PTH stimulation increases trabecular bone formation in mice, but this is decreased in mice lacking β-arr 2, suggesting a role for β-arr 2 in the anabolic effects of PTH. The role of β-arr 2 in the catabolic effects of continuous PTH (cPTH) treatment is not known. Objective To assess the effects of cPTH administration on bone in mice lacking β-arr 2 compared to wild-type (WT). Methods Groups of male and female WT or β-arr2 knockout (KO) mice were administered either PTH or phosphate-buffered saline by osmotic pumps for 2 weeks. Following treatment, serum calcium and phosphate levels were measured, bone structure and mineral density were measured by microcomputed tomography, and bone cells measured by static and dynamic histomorphometry. Results β-arr2 KO had no effects on skeletal development in mice of either sex. PTH treatment caused hypercalcemia and hypophosphatemia and decreased trabecular and cortical bone only in male WT mice. β-arr2 KO in male mice completely abrogated the effects of PTH on bone, while in female β-arr2 KO mice, PTH treatment increased trabecular bone with no effects on cortical bone. Conclusions These results demonstrate a profound sex effect on skeletal responses to cPTH treatment, suggesting a protective effect of estrogen on bone loss. β-arr2 plays a role in restraining the anabolic effects of PTH in both male and female mice.

Abstract 6030: Asparagine metabolism is linked to sex differences in colorectal cancer growth

Abstract Colorectal cancer (CRC) is the third leading cause of cancer-related deaths in men and women in the United States. We have shown that tumors from female CRC patients have increased asparagine (Asn) which is positively associated with poorer overall survival for female patients only (n=196, stage I-III). Furthermore, analysis of asparagine synthetase (ASNS) expression shows that ASNS is similarly associated with poorer survival for females with CRC only. Currently it is not understood why there are sex-specific differences in tumor metabolism of Asn, or the roles of ASNS in CRC progression. Therefore, we hypothesize that limiting ASNS-driven Asn production is a viable target to suppress CRC development in a sex-specific manner. Therefore, we used a combination of in vitro and in vivo models with human CRC ASNS knock out (KO) cells to generate additional hypotheses that may reveal mechanisms that underlie this finding. Using a 3D spheroid model, we cultured HCT116 ASNS KO and isogenic HCT116 ASNS WT cell lines and modulated Asn supply. During 7 days of growth, depletion of Asn slowed the growth of ASNS KO cells compared to WT, whereas Asn supplementation (400 μM) recovered growth in both cell lines; however, spheroid growth was less in the KO cells. Subcutaneous xenograft of these cell lines in male and female mice (n=10/genotype/sex) showed that loss of ASNS reduced tumor burden in both male and female mice, and improved survival in female mice alone. Pathological assessment of tumors indicated poorly differentiated carcinoma and presence of viable tumor mass in both groups. However, the mitotic activity index, and cellular proliferation (measured by Ki67 staining) was significantly reduced in female ASNS KO mice compared to ASNS WT, showing that the tumor cells with ASNS KO are growing slower than those of ASNS WT counterparts. A multi-omics approach was then used to investigate metabolic pathways and signaling networks altered between the tumor genotypes. Tumor tissue metabolomics revealed significant decreases in nucleotides, amino acids, fatty acids within the ASNS KO compared to WT tumors. Using RNA-Seq, gene set enrichment and Ingenuity Pathway Analysis (IPA), data were integrated revealing disruption to Wnt/B-catenin signaling and immune responses associated with ASNS KO in female mice. Serum metabolomics also revealed a higher ratio of circulating glutamine to glutamate in the female mice with ASNS KO xenografts compared to WT, and a decreased expression of the tumor aspartate-glutamate transporter SLC1A3, therefore tumors with ASNS KO could have decreased ability to transport metabolites needed for Asn generation. In summary we identified that lack of ASNS suppresses tumor progression in a pre-clinical CRC model, and significantly improves survival in female mice only. Validation and further mechanistic experiments will reveal the robustness of this finding. Citation Format: Caroline Johnson, Oladimeji Aladelokun, Sajid Khan. Asparagine metabolism is linked to sex differences in colorectal cancer growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6030.

Sex Difference in Cardioprotection against Acute Myocardial Infarction in MAO-B Knockout Mice In Vivo.

To induce the deletion of MAO-B, MAO-B KO mice (Myh6 Cre+/MAO-Bfl/fl) and wild-type (WT, Cre-negative MAO-Bfl/fl littermates) were fed with tamoxifen for 2 weeks followed by 10 weeks of normal mice chow. Myocardial infarction (assessed by TTC staining and expressed as a percentage of the area at risk as determined by Evans blue staining)) was induced by 45 min coronary occlusion followed by 120 min of reperfusion. The mortality following I/R was higher in male compared to female mice, with the lowest mortality found in MAO-B KO female mice. IS was significantly higher in male WT mice compared to female WT mice. MAO-B KO reduced IS in male mice but had no further impact on IS in female MAO-B KO mice. Interestingly, there was no difference in the plasma estradiol levels among the groups. The cardiomyocyte-specific knockout of MAO-B protects male mice against acute myocardial infarction but had no effect on the infarct size in female mice.

RAGE is a critical factor of sex-based differences in age-induced kidney damage.

Introduction: Advanced glycation end products (AGEs) are a heterogeneous group of molecules with potential pathophysiological effects on the kidneys. Fibrosis together with the accumulation of AGEs has been investigated for its contribution to age-related decline in renal function. AGEs mediate their effects in large parts through their interactions with the receptor for AGEs (RAGE). RAGE is a transmembrane protein that belongs to the immunoglobulin superfamily and has the ability to interact with multiple pro-inflammatory/pro-oxidative ligands. The role of RAGE in aging kidneys has not been fully characterized, especially for sex-based differences. Methods: Therefore, we analyzed constitutive RAGE knockout (KO) mice in an age- and sex-dependent manner. Paraffin-embedded kidney sections were used for histological analysis and protein expression of fibrosis and damage markers. RNA expression analysis from the kidney cortex was done by qPCR for AGE receptors, kidney damage, and early inflammation/fibrosis factors. FACS analysis was used for immune cell profiling of the kidneys. Results: Histological analysis revealed enhanced infiltration of immune cells (positive for B220) in aged (>70weeks old) KO mice in both sexes. FACS analysis revealed a similar pattern of enhanced B-1a cells in aged KO mice. There was an age-based increase in pro-fibrotic and pro-inflammatory markers (IL-6, TNF, TGF-β1, and SNAIL1) in KO male mice that presumably contributed to renal fibrosis and renal damage (glomerular and tubular). In fact, in KO mice, there was an age-dependent increase in renal damage (assessed by NGAL and KIM1) that was accompanied by increased fibrosis (assessed by CTGF). This effect was more pronounced in male KO mice than in the female KO mice. In contrast to the KO animals, no significant increase in damage markers was detectable in wild-type animals at the age examined (>70weeks old). Moreover, there is an age-based increase in AGEs and scavenger receptor MSR-A2 in the kidneys. Discussion: Our data suggest that the loss of the clearance receptor RAGE in male animals further accelerates age-dependent renal damage; this could be in part due to an increase in AGEs load during aging and the absence of protective female hormones. By contrast, in females, RAGE expression seems to play only a minor role when compared to tissue pathology.

Melanocortin 4 receptor signaling in Sim1 neurons permits sexual receptivity in female mice.

Female sexual dysfunction affects approximately 40% of women in the United States, yet few therapeutic options exist for these patients. The melanocortin system is a new treatment target for hypoactive sexual desire disorder (HSDD), but the neuronal pathways involved are unclear. In this study, the sexual behavior of female MC4R knockout mice lacking melanocortin 4 receptors (MC4Rs) was examined. The mice were then bred to express MC4Rs exclusively on Sim1 neurons (tbMC4RSim1 mice) or on oxytocin neurons (tbMC4ROxt mice) to examine the effect on sexual responsiveness. MC4R knockout mice were found to approach males less and have reduced receptivity to copulation, as indicated by a low lordosis quotient. These changes were independent of body weight. Lordosis behavior was normalized in tbMC4RSim1 mice and improved in tbMC4ROxt mice. In contrast, approach behavior was unchanged in tbMC4RSim1 mice but greatly increased in tbMC4ROxt animals. The changes were independent of melanocortin-driven metabolic effects. These results implicate MC4R signaling in Oxt neurons in appetitive behaviors and MC4R signaling in Sim1 neurons in female sexual receptivity, while suggesting melanocortin-driven sexual function does not rely on metabolic neural circuits.

Vesicular Zinc Modulates Cell Proliferation and Survival in the Developing Hippocampus.

In the brain, vesicular zinc, which refers to a subset of zinc that is sequestered into synaptic vesicles by zinc transporter 3 (ZnT3), has extensive effects on neuronal signalling and modulation. Vesicular zinc-focused research has mainly been directed to its role in the hippocampus, particularly in adult neurogenesis. However, whether vesicular zinc is involved in modulating neurogenesis during the early postnatal period has been less studied. As a first step to understanding this, we used ZnT3 knockout (KO) mice, which lack ZnT3 and, thus, vesicular zinc, to evaluate cell proliferation at three different age points spanning postnatal development (P6, P14, and P28). The survival and the neuronal phenotype of these cells was also assessed in adulthood. We found that male ZnT3 KO mice exhibited lower rates of cell proliferation at P14, but a greater number of these cells survived to adulthood. Additionally, significantly more cells labelled on P6 survived to adulthood in male and female ZnT3 KO mice. We also found sex-dependent differences, whereby male mice showed higher levels of cell proliferation at P28, as well as higher levels of cell survival for P14-labelled cells, compared to female mice. However, female mice showed greater percentages of neuronal differentiation for P14-labelled cells. Finally, we found significant effects of age of BrdU injections on cell proliferation, survival, and neuronal differentiation. Collectively, our results suggest that the loss of vesicular zinc affects normal proliferation and survival of cells born at different age points during postnatal development and highlight prominent sex- and age-dependent differences. Our findings provide the foundation for future studies to further probe the role of vesicular zinc in the modulation of developmental neurogenesis.

Egg envelope formation of medaka Oryzias latipes requires ZP proteins originating from both the liver and ovary

Teleost oocytes are surrounded by a structure called chorion or egg envelopes, which is composed of zona pellucida (ZP) proteins. As a result of the gene duplication in teleost, the expression site of the zp genes, coding the major component protein of egg envelopes, changed from the ovary to the maternal liver. In Euteleostei, there are three liver-expressed zp genes, named choriogenin (chg) h, chg hm, and chg l, and the composition of the egg envelope is mostly made up of these Chgs. In addition, ovary-expressed zp genes are also conserved in the medaka genomes, and their proteins have also been found to be minor components of the egg envelopes. However, the specific role of liver-expressed versus ovary-expressed zp genes was unclear. In the present study, we showed that ovary-synthesized ZP proteins first form the base layer of the egg envelope and then Chgs polymerize inwardly to thicken the egg envelope. To analyze the effects of dysfunction of the chg gene, we generated some chg knockout medaka. All knockout females failed to produce normally fertilized eggs by the natural spawning. The egg envelopes lacking Chgs were significantly thinner, but layers formed by ZP proteins synthesized in the ovary were found in the thin egg envelope of knockout as well as wildtype eggs. These results suggest that the ovary-expressed zp gene is well conserved in all teleosts, including those species in which liver-derived ZP proteins are the major component, because it is essential for the initiation of egg envelope formation.

Skeletal phenotypes in secreted frizzled-related protein 4 gene knockout mice mimic skeletal architectural abnormalities in subjects with Pyle’s disease from SFRP4 mutations

Mutations in SFRP4 cause Pyle’s bone disease with wide metaphyses and increased skeletal fragility. The WNT signaling pathway plays important roles in determining skeletal architecture and SFRP4 is a secreted Frizzled decoy receptor that inhibits WNT signaling. Seven cohorts of male and female Sfrp4 gene knockout mice, examined through 2 years of age, had a normal lifespan but showed cortical and trabecular bone phenotypes. Mimicking human Erlenmeyer flask deformities, bone cross-sectional areas were elevated 2-fold in the distal femur and proximal tibia but only 30% in femur and tibia shafts. Reduced cortical bone thickness was observed in the vertebral body, midshaft femur and distal tibia. Elevated trabecular bone mass and numbers were observed in the vertebral body, distal femur metaphysis and proximal tibia metaphysis. Midshaft femurs retained extensive trabecular bone through 2 years of age. Vertebral bodies had increased compressive strength, but femur shafts had reduced bending strength. Trabecular, but not cortical, bone parameters in heterozygous Sfrp4 mice were modestly affected. Ovariectomy resulted in similar declines in both cortical and trabecular bone mass in wild-type and Sfrp4 KO mice. SFRP4 is critical for metaphyseal bone modeling involved in determining bone width. Sfrp4 KO mice show similar skeletal architecture and bone fragility deficits observed in patients with Pyle’s disease with SFRP4 mutations.

Oral Administration of Vitamin D3 Prevents Corneal Damage in a Knock-Out Mouse Model of Sjögren's Syndrome.

Vitamin D deficiency has been associated with dry eye development during Sjögren's syndrome (SS). Here, we investigated whether repeated oral vitamin D3 supplementation could prevent the corneal epithelium damage in an SS mouse model. 30 female mouse knock-out for the thrombospondin 1 gene were randomized (six per group) in untreated mice euthanized at 6 weeks as negative control (C-) or at 12 weeks as the positive control for dry eye (C+). Other mice were sacrificed after 6 weeks of oral vitamin D3 supplementation in the drinking water (1000, 8000, and 20,000 IU/kg/week, respectively). The C+ mice showed alterations in their corneal epithelial morphologies and thicknesses (p < 0.01 vs. C-), while the mice receiving 8000 (M) and 20,000 (H) IU/kg/week of vitamin D3 showed preservation of the corneal epithelium morphology and thickness (p < 0.01 vs. C+). Moreover, while the C+ mice exhibited high levels and activity of corneal tumor necrosis factor alpha converting enzyme (TACE), neovascularization and fibrosis markers; these were all reduced in the M and H mice. Oral vitamin D3 supplementation appeared to counteract the negative effect of TACE on corneal epithelium in a mouse model of SS-associated dry eye.