Male Urinary Chemosignals Differentially Affect Aggressive Behavior in Male Mice

Chemical signals modulate aggressive behavior in mice. For example, male urinary cues enhance aggression against other adults: a resident mouse attacks a male but not a castrated intruder, unless it is anointed with male urine. Our purpose was to understand whether molecules excreted with urine also act as aggression triggers in a different context. Therefore, the effect of urine, or molecules purified from urine, voided by different animals (males or females), was tested on the aggression of male mice against pups. Latency to the first attack, percentage of pups receiving the first attack, and percentage of attacked pups after 5 and 15 min were recorded. At variance with intermale aggression, male urinary chemosignals sprayed on pups reduced infanticide, while female urine did not. Male urine also delayed infanticide when compared to female urine. Pups anointed with low molecular weight dialyzed urine and with the high molecular weight protein fraction were attacked later than controls. Pups anointed with Major Urinary Proteins (MUPs) also were attacked later. The volatiles retained by MUPs act in the same way as adult male urine. MUPs and their ligands did not modify biting of food items. The results show that mice do not perceive male chemosignals as compulsory aggression triggers but rather can consistently and differentially shape their behavior in response to the same molecules according to different contextual events.     

Modulation of exploratory behavior in female mice by protein-borne male urinary molecules

Male pheromones are believed to attract females and repel male mice in open field tests but, when tested in more complex environments, they can attract male mice in usually avoided areas. Females were tested in an apparatus with one dark and one light side, in the absence or presence of male urine or the major urinary proteins (MUPs) bearing the natural ligands. Diestrous females were slower in leaving from the dark area when male urine or MUPs were present in it. Estrogen-primed females showed the opposite behavior, with an increase in the same latency. The light-avoidance behavior of prepubertal females, or females reared without males was not influenced by the presence of male chemosignals. The results show that adult female mice can react to MUP-borne volatiles as to adult male urine and use them as cues of male mice, if they were previously exposed to male cues during infancy. MUP-borne molecules are, thus, the olfactory trace of males in the environment and modulate mice exploratory behavior.     

Estrogen Regulates the Expression and Localization of YAP in the Uterus of Mice

The dynamics of uterine endometrium is important for successful establishment and maintenance of embryonic implantation and development, along with extensive cell differentiation and proliferation. The tissue event is precisely and complicatedly regulated as several signaling pathways are involved including two main hormones, estrogen and progesterone signaling. We previously showed a novel signaling molecule, Serine/threonine protein kinase 3/4 (STK3/4), which is responded to hormone in the mouse uterine epithelium. However, the role and regulation of its target, YES-associated protein (YAP) remains unknown. In this study, we investigated the expression and regulation of YAP in mouse endometrium. We found that YAP was periodically expressed in the endometrium during the estrous cycle. Furthermore, periodic expression of YAP was shown to be related to the pathway under hormone treatment. Interestingly, estrogen was shown to positively modulate YAP via endometrial epithelial receptors. In addition, the knockdown of YAP showed that YAP regulated various target genes in endometrial cells. The knockdown of YAP down-regulated numerous targets including ADAMTS1, AMOT, AMOTL1, ANKRD1, CTNNA1, MCL1. On the other hand, the expressions of AREG and AXL were increased by its knockdown. These findings imply that YAP responds via Hippo signaling under various intrauterine signals and is considered to play a role in the expression of factors important for uterine endometrium dynamic regulation.     

Neural and Hormonal Basis of Opposite-Sex Preference by Chemosensory Signals

In mammalian reproduction, sexually active males seek female conspecifics, while estrous females try to approach males. This sex-specific response tendency is called sexual preference. In small rodents, sexual preference cues are mainly chemosensory signals, including pheromones. In this article, we review the physiological mechanisms involved in sexual preference for opposite-sex chemosensory signals in well-studied laboratory rodents, mice, rats, and hamsters of both sexes, especially an overview of peripheral sensory receptors, and hormonal and central regulation. In the hormonal regulation section, we discuss potential rodent brain bisexuality, as it includes neural substrates controlling both masculine and feminine sexual preferences, i.e., masculine preference for female odors and the opposite. In the central regulation section, we show the substantial circuit regulating sexual preference and also the influence of sexual experience that innate attractants activate in the brain reward system to establish the learned attractant. Finally, we review the regulation of sexual preference by neuropeptides, oxytocin, vasopressin, and kisspeptin. Through this review, we clarified the contradictions and deficiencies in our current knowledge on the neuroendocrine regulation of sexual preference and sought to present problems requiring further study.     

The Same Metabolic Response to FGF21 Administration in Male and Female Obese Mice Is Accompanied by Sex-Specific Changes in Adipose Tissue Gene Expression

The preference for high-calorie foods depends on sex and contributes to obesity development. Fibroblast growth factor 21 (FGF21) beneficially affects taste preferences and obesity, but its action has mainly been studied in males. The aim of this study was to compare the effects of FGF21 on food preferences and glucose and lipid metabolism in C57Bl/6J male and female mice with diet-induced obesity. Mice were injected with FGF21 or vehicle for 7 days. Body weight, choice between standard (SD) and high-fat (HFD) diets, blood parameters, and gene expression in white (WAT) and brown (BAT) adipose tissues, liver, muscles, and the hypothalamus were assessed. Compared to males, females had a greater preference for HFD; less WAT; lower levels of cholesterol, glucose, and insulin; and higher expression of Fgf21, Insr, Ppara, Pgc1, Acca and Accb in the liver and Dio2 in BAT. FGF21 administration decreased adiposity; blood levels of cholesterol, glucose, and insulin; hypothalamic Agrp expression, increased SD intake, decreased HFD intake independently of sex, and increased WAT expression of Pparg, Lpl and Lipe only in females. Thus, FGF21 administration beneficially affected mice of both sexes despite obesity-associated sex differences in metabolic characteristics, and it induced female-specific activation of gene expression in WAT.     

Age-Related Changes in Odor Preferences by House Mice Living in Semi Natural Enclosures

Changes in odor preferences with age were assessed in house mice inhabiting a series of eight 0.1-ha outdoor enclosures. The most recent capture in a live trap was assumed to provide the predominant odor in that trap for one week. Data were analyzed for two separate years for females and males and for three age classes within each sex. Initial analyses confirmed the use of the odor baiting technique; significantly more mice than expected entered traps that had captured another mouse in the previous week relative to traps that had not caught a mouse for at least a week. For female mice two predictions were tested. (1) Young female mice avoid traps with odors from adult males. The findings support this prediction. (2) As they mature, female mice will shift their odor preference and enter significantly more traps odorized by adult males. The findings also support this prediction. Two predictions were tested for male mice. (3) Young male mice will avoid odors from adult males. The data do not support this prediction. (4) As they mature, males will shift their preference and enter more traps odorized by female mice. Some, but not all of the data support this hypothesis.     

Volatile fatty acids in urine and vaginal secretions of cows during reproductive cycle

Levels of volatile fatty acids (VFA) in vaginal secretions (VS) of cows were nearly independent of concentration changes of acids in the gastrointestinal tract, while levels in urine showed marked dependence. During three-week cycles, the VFA concentrations in VS increased shortly before heat and decreased to one third on days 19–22. Some cows showed an immediate increase after heat while in others the increase was slow and gradual. Moderately increased levels at midcycle were probably related to interovulatory follicle growth. Content of VFA in VS of pregnant cows was significantly above the level of the postestrus drop. Some pregnant cows in the first two thirds of gravidity had substantially higher levels of VFA than the other cows. Changes in VFA levels have a potential value for estrus detection and pregnancy diagnosis in cows. The gas chromatographic method used is too slow and tedious for practical application, but a reliable semiquantitative test for rapid proof of VFA or acetic acid in VS would be a very useful device for field use. It would have a substantial impact on the economy of cattle production by minimizing the loss of time due to delayed breeding.The results presented in this communication were presented in part in a dissertation submitted in partial fulfilment of requirements for C.Sc. degree (equivalent of Ph.D.) at the University of Veterinary Medicine, Brno, Czechoslovakia, in 1978.     

Immunogenicity and Cross-Protective Efficacy Induced by Outer Membrane Proteins from Salmonella Typhimurium Mutants with Truncated LPS in Mice

Lipopolysaccharide (LPS) is a major virulence factor present in the outer membrane of Salmonella enterica serovar Typhimurium (S. Typhimurium). Outer membrane proteins (OMPs) from Salmonella show high immunogenicity and provide protection against Salmonella infection, and truncated LPS alters the outer membrane composition of the cell wall. In our previous study, we demonstrated that Salmonella mutants carrying truncated LPS failed to induce strong immune responses and cross-reaction to other enteric bacteria, due to their high attenuation and low colonization in the host. Therefore, we plan to investigate whether outer membrane proteins from Salmonella mutants with truncated LPS resulting from a series of nonpolar mutations, including ∆waaC12, ∆waaF15, ∆waaG42, ∆rfaH49, ∆waaI43, ∆waaJ44, ∆waaL46, ∆wbaP45 and ∆wzy-48, affect immunogenicity and provide protection against diverse Salmonella challenge. In this study, the immunogenicity and cross-protection efficiency of purified OMPs from all mutants were investigated to explore a potential OMP vaccine to protect against homologous or heterologous serotype Salmonella challenge. The results demonstrated that OMPs from three Salmonella mutants (∆waaC12, ∆waaJ44 and ∆waaL46) induced higher immune responses and provided good protection against homologous S. Typhimurium. The OMPs from these three mutants were also selected to determine the cross-protective efficacy against homologous and heterologous serotype Salmonella. Our results indicated that the mutant ∆waaC12 can elicit higher cross-reactivity and can provide good protection against S. Choleraesuis and S. Enteritidis infection and that the cross-reactivity may be ascribed to an antigen of approximately 18.4–30 kDa.     

Xylitol Affects the Intestinal Microbiota and Metabolism of Daidzein in Adult Male Mice

This study examined the effects of xylitol on mouse intestinal microbiota and urinary isoflavonoids. Xylitol is classified as a sugar alcohol and used as a food additive. The intestinal microbiota seems to play an important role in isoflavone metabolism. Xylitol feeding appears to affect the gut microbiota. We hypothesized that dietary xylitol changes intestinal microbiota and, therefore, the metabolism of isoflavonoids in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 5% xylitol diet (XD group) and those fed a 0.05% daidzein-containing control diet (CD group) for 28 days. Plasma total cholesterol concentrations were significantly lower in the XD group than in the CD group (p < 0.05). Urinary amounts of equol were significantly higher in the XD group than in the CD group (p < 0.05). The fecal lipid contents (% dry weight) were significantly greater in the XD group than in the CD group (p < 0.01). The cecal microbiota differed between the two dietary groups. The occupation ratios of Bacteroides were significantly greater in the CD than in the XD group (p < 0.05). This study suggests that xylitol has the potential to affect the metabolism of daidzein by altering the metabolic activity of the intestinal microbiota and/or gut environment. Given that equol affects bone health, dietary xylitol plus isoflavonoids may exert a favorable effect on bone health.     

Possible induction by estrous cows of pheromone production in penmates

The primary attractive odors of estrous cows apparently induced production of secondary attractive odors in penmates. Presence of both odors was reflected by the bull's olfactory behavior. The inducing effect was strongest on the day of estrus and the day before. The information content of the primary and secondary odors was different since a sexually experienced bull was able to distinguish olfactorily between estrous and nonestrous cows. The secondary odors attracted at least transient bull's interest, resulting in sniffing, urine tasting, and flehmen reactions, but not mounting attempts. The induced production of attractive odors and lack of precise smell discrimination in penmates may lead to erroneous detection of estrus in cows and heifers that show false estrous signs without corresponding changes in their reproductive organs. Similar factors may be involved in triggering mounting among feedlot steers (buller steer syndrome).     

Characterization and Comparison of Major Urinary Proteins from the House Mouse, <Emphasis Type="BoldItalic">Mus musculus domesticus</Emphasis>, and the Aboriginal Mouse, <Emphasis Type="BoldItalic">Mus macedonicus</Emphasis>

Urine from the house mouse, Mus musculus domesticus, contains a high concentration of major urinary proteins (MUPs), which convey olfactory information between conspecifics. In wild populations, each individual expresses a different pattern of around 8 to 14 electrophoretically separable MUP isoforms. To examine whether other Mus species express MUPs and exhibit a similar level of individual heterogeneity, we characterized urinary proteins in urine samples from an aboriginal species, Mus macedonicus, captured from different sites in Turkey. Anion exchange chromatography and electrospray ionization mass spectrometry demonstrated that M. macedonicus urine contained a single major peak of mass 18,742 Da, and in contrast to M. m. domesticus, all individuals were the same. The M. macedonicus masses were not predicted from any known MUP gene sequence. Endoproteinase Lys-C (Lys-C) digestion of the purified M. macedonicus urinary protein followed by matrix assisted laser desorption time of flight (MALDI-TOF) mass spectrometry demonstrated that it shared considerable, but not complete, sequence homogeneity with M. m. domesticus MUPs. Three M. macedonicus Lys-C peptides differed in mass from their M. m. domesticus counterparts. These three peptides were further characterized by tandem mass spectrometry. The complete sequences of two were determined, and in conjunction with methyl esterification, the amino acid composition of the third was inferred, and the sequence narrowed down to three permutations. The complete M. macedonicus sequence contained a maximum of seven amino acid substitutions, discernible by tandem mass spectrometry, relative to a reference M. m. domesticus sequence. Six of these were on the surface of the molecule. Molecular modeling of the M. macedonicus sequence demonstrated that the amino acid substitutions had little effect on the tertiary structure. The differences in the level of heterogeneity between the two species are discussed in relation to their environment and behavior. In addition, the differences in protein structure allow speculation into molecular mechanisms of MUP function.     

RFCM-PALM: In-Silico Prediction of S-Palmitoylation Sites in the Synaptic Proteins for Male/Female Mouse Data

S-palmitoylation is a reversible covalent post-translational modification of cysteine thiol side chain by palmitic acid. S-palmitoylation plays a critical role in a variety of biological processes and is engaged in several human diseases. Therefore, identifying specific sites of this modification is crucial for understanding their functional consequences in physiology and pathology. We present a random forest (RF) classifier-based consensus strategy (RFCM-PALM) for predicting the palmitoylated cysteine sites on synaptic proteins from male/female mouse data. To design the prediction model, we have introduced a heuristic strategy for selection of the optimum set of physicochemical features from the AAIndex dataset using (a) K-Best (KB) features, (b) genetic algorithm (GA), and (c) a union (UN) of KB and GA based features. Furthermore, decisions from best-trained models of the KB, GA, and UN-based classifiers are combined by designing a three-star quality consensus strategy to further refine and enhance the scores of the individual models. The experiment is carried out on three categorized synaptic protein datasets of a male mouse, female mouse, and combined (male + female), whereas in each group, weighted data is used as training, and knock-out is used as the hold-out set for performance evaluation and comparison. RFCM-PALM shows ~80% area under curve (AUC) score in all three categories of datasets and achieve 10% average accuracy (male—15%, female—15%, and combined—7%) improvements on the hold-out set compared to the state-of-the-art approaches. To summarize, our method with efficient feature selection and novel consensus strategy shows significant performance gains in the prediction of S-palmitoylation sites in mouse datasets.     

Aberrant Early in Life Stimulation of the Stress-Response System Affects Emotional Contagion and Oxytocin Regulation in Adult Male Mice

Results over the last decades have provided evidence suggesting that HPA axis dysfunction is a major risk factor predisposing to the development of psychopathological behaviour. This susceptibility can be programmed during developmental windows of marked neuroplasticity, allowing early-life adversity to convey vulnerability to mental illness later in life. Besides genetic predisposition, also environmental factors play a pivotal role in this process, through embodiment of the mother’s emotions, or via nutrients and hormones transferred through the placenta and the maternal milk. The aim of the current translational study was to mimic a severe stress condition by exposing female CD-1 mouse dams to abnormal levels of corticosterone (80 µg/mL) in the drinking water either during the last week of pregnancy (PreCORT) or the first one of lactation (PostCORT), compared to an Animal Facility Rearing (AFR) control group. When tested as adults, male mice from PostCORT offspring and somewhat less the PreCORT mice exhibited a markedly increased corticosterone response to acute restraint stress, compared to perinatal AFR controls. Aberrant persistence of adolescence-typical increased interest towards novel social stimuli and somewhat deficient emotional contagion also characterised profiles in both perinatal-CORT groups. Intranasal oxytocin (0 or 20.0 µg/kg) generally managed to reduce the stress response and restore a regular behavioural phenotype. Alterations in density of glucocorticoid and mineralocorticoid receptors, oxytocin and µ- and κ-opioid receptors were found. Changes differed as a function of brain areas and the specific age window of perinatal aberrant stimulation of the HPA axis. Present results provided experimental evidence in a translational mouse model that precocious adversity represents a risk factor predisposing to the development of psychopathological behaviour.     

Molecular Processes in Stress Urinary Incontinence: A Systematic Review of Human and Animal Studies

Stress urinary incontinence (SUI) is a common and burdensome condition. Because of the large knowledge gap around the molecular processes involved in its pathophysiology, the aim of this review was to provide a systematic overview of genetic variants, gene and protein expression changes related to SUI in human and animal studies. On 5 January 2021, a systematic search was performed in Pubmed, Embase, Web of Science, and the Cochrane library. The screening process and quality assessment were performed in duplicate, using predefined inclusion criteria and different quality assessment tools for human and animal studies respectively. The extracted data were grouped in themes per outcome measure, according to their functions in cellular processes, and synthesized in a narrative review. Finally, 107 studies were included, of which 35 used animal models (rats and mice). Resulting from the most examined processes, the evidence suggests that SUI is associated with altered extracellular matrix metabolism, estrogen receptors, oxidative stress, apoptosis, inflammation, neurodegenerative processes, and muscle cell differentiation and contractility. Due to heterogeneity in the studies (e.g., in examined tissues), the precise contribution of the associated genes and proteins in relation to SUI pathophysiology remained unclear. Future research should focus on possible contributors to these alterations.     

Efficient Generation of Hydrazides in Proteins by RadA Split Intein

Protein C-terminal hydrazides are useful for bioconjugation and construction of proteins from multiple fragments through native chemical ligation. To generate C-terminal hydrazides in proteins, an efficient intein-based preparation method has been developed by using thiols and hydrazine to accelerate the formation of the transient thioester intermediate and subsequent hydrazinolysis. This approach not only increases the yield, but also improves biocompatibility. The scope of the method has been expanded by employing Pyrococcus horikoshii RadA split intein, which can accommodate a broad range of extein residues before the site of cleavage. The use of split RadA minimizes premature intein N cleavage in vivo and offers control over the initiation of the intein N cleavage reaction. It is expected that this versatile preparation method will expand the utilization of protein C-terminal hydrazides in protein preparation and modification.     

Patterns of deposition of urine containing chemosignals that affect puberty and reproduction by wild stock male and female house mice (Mus domesticus)

House mice release chemosignals in their urine that influence the timing of puberty and reproductive condition in conspecific females. These experiments tested the possibility that mice depositing urine containing chemosignals that affect puberty and reproduction do so differentially with respect to urine cues from conspecifics. Mice were tested in cages containing samples of urine or water on cotton in wire mesh capsules protruding from the cage floor. Their urine deposition patterns were recorded on squares of filter paper positioned below the cages. Males deposited more urine than females housed in groups, estrous females, diestrous females, or prepubertal females. All groups of mice deposited urine in a nonrandom fashion with regard to urine cues from conspecifics. Male mice deposited more urine near locations previously soiled by females than near water or other males. Grouped females deposited more urine near male urine cues and avoided depositing urine near urine from other group-housed females. Both estrous and diestrous female mice deposited more urine near males than near other urine cues or water, possibly to attract mates. Prepubertal females avoided depositing their urine near male urine and urinated more near urine from grouped females than near other urine types or water. Young females may be avoiding possible male mates until they have attained puberty. This avoidance behavior may enhance the long-term reproductive success of the females that otherwise might mature and mate at too young an age.     

Male and Female Animals Respond Differently to High-Fat Diet and Regular Exercise Training in a Mouse Model of Hyperlipidemia

Inappropriate nutrition and a sedentary lifestyle can lead to obesity, one of the most common risk factors for several chronic diseases. Although regular physical exercise is an efficient approach to improve cardiometabolic health, the exact cellular processes are still not fully understood. We aimed to analyze the morphological, gene expression, and lipidomic patterns in the liver and adipose tissues in response to regular exercise. Healthy (wild type on a normal diet) and hyperlipidemic, high-fat diet-fed (HFD-fed) apolipoprotein B-100 (APOB-100)-overexpressing mice were trained by treadmill running for 7 months. The serum concentrations of triglyceride and tumor necrosis factor α (TNFα), as well as the level of lipid accumulation in the liver, were significantly higher in HFD-fed APOB-100 males compared to females. However, regular exercise almost completely abolished lipid accumulation in the liver of hyperlipidemic animals. The expression level of the thermogenesis marker, uncoupling protein-1 (Ucp1), was significantly higher in the subcutaneous white adipose tissue of healthy females, as well as in the brown adipose tissue of HFD-fed APOB-100 females, compared to males. Lipidomic analyses revealed that hyperlipidemia essentially remodeled the lipidome of brown adipose tissue, affecting both the membrane and storage lipid fractions, which was partially restored by exercise in both sexes. Our results revealed more severe metabolic disturbances in HFD-fed APOB-100 males compared to females. However, exercise efficiently reduced the body weight, serum triglyceride levels, expression of pro-inflammatory factors, and hepatic lipid accumulation in our model.     

Recent Advances in Understanding the Control of Secretory Proteins by the Unfolded Protein Response in Plants

The membrane transport system is built on the proper functioning of the endoplasmic reticulum (ER). The accumulation of unfolded proteins in the ER lumen (ER stress) disrupts ER homeostasis and disturbs the transport system. In response to ER stress, eukaryotic cells activate intracellular signaling (named the unfolded protein response, UPR), which contributes to the quality control of secretory proteins. On the other hand, the deleterious effects of UPR on plant health and growth characteristics have frequently been overlooked, due to limited information on this mechanism. However, recent studies have shed light on the molecular mechanism of plant UPR, and a number of its unique characteristics have been elucidated. This study briefly reviews the progress of understanding what is happening in plants under ER stress conditions.     

Increased scent marking in male Mongolian gerbils by urinary polypeptides of female conspecifics

Male Mongolian gerbils scent mark during a standardized openfield test on an individually different but more-or-less constant level in the absence of females. The presence of females in the same room but without direct contact to the males increases the marking frequencies of the males to an individually different higher level. A similar increase in marking frequency is obtained by application of female urine directly to the nostrils of the males, but not by male gerbil urine or female laboratory mouse urine. This indicates that substances present in female gerbil urine are responsible for the increase in male marking behavior. Using this bioassay, various fractions of female urine were tested for their influence on marking behavior. Our results to date show that the component increasing male scent-marking behavior is hydrophilic and nonvolatile and is contained in the nondialyzable, high-molecular-weight polypeptide fraction of female gerbil urine.     

Metabolic Effects of CCN5/WISP2 Gene Deficiency and Transgenic Overexpression in Mice

CCN5/WISP2 is a matricellular protein, the expression of which is under the regulation of Wnt signaling and IGF-1. Our initial characterization supports the notion that CCN5 might promote the proliferation and survival of pancreatic β-cells and thus improve the metabolic profile of the animals. More recently, the roles of endogenous expression of CCN5 and its ectopic, transgenic overexpression on metabolic regulation have been revealed through two reports. Here, we attempt to compare the experimental findings from those studies, side-by-side, in order to further establish its roles in metabolic regulation. Prominent among the discoveries was that a systemic deficiency of CCN5 gene expression caused adipocyte hypertrophy, increased adipogenesis, and lipid accumulation, resulting in insulin resistance and glucose intolerance, which were further exacerbated upon high-fat diet feeding. On the other hand, the adipocyte-specific and systemic overexpression of CCN5 caused an increase in lean body mass, improved insulin sensitivity, hyperplasia of cardiomyocytes, and increased heart mass, but decreased fasting glucose levels. CCN5 is clearly a regulator of adipocyte proliferation and maturation, affecting lean/fat mass ratio and insulin sensitivity. Not all results from these models are consistent; moreover, several important aspects of CCN5 physiology are yet to be explored.