Adult Male Urine Research Articles

Three-dimensional printed polylactic acid and hydroxyapatite composite scaffold with urine-derived stem cells as a treatment for bone defects

Bone defects still pose various challenges in osteology. As one treatment method for bone defects, tissue engineering requires biomaterials with good biocompatibility and stem cells with good differentiation. This study aimed to fabricate a 3D-printed polylactic acid and hydroxyapatite (PLA/HA) composite scaffold with urine-derived stem cells (USCs) to study its therapeutic effect in a rat model of skull defects. USCs were isolated and extracted from the urine of healthy adult males and inoculated onto PLA/HA and PLA scaffolds fabricated by 3D printing technology. A total of 36 skull defect models in eighteen Sprague–Dawley rats were randomly divided into a control group (no treatment of the defects), PLA group (treated with PLA scaffolds with USCs), and PLA/HA group (treated with PLA/HA scaffolds with USCs). The therapeutic efficacy was evaluated by real-time PCR, microcomputed tomography (micro-CT), and immunohistochemistry at 4, 8, and 12 weeks. We found that the PLA/HA scaffold loaded with USCs effectively promoted new bone regeneration in the defect area. CT images showed that in the PLA/HA group, the defect area was almost entirely covered by newly formed bone (coverage of 96.7 ± 1.6%), and the coverage was greater than that in the PLA group (coverage of 74.6 ± 1.9%) at 12 weeks. Histology and immunohistochemical staining showed the highest new bone formation on the PLA/HA scaffolds containing USCs in the defect site at 12 weeks. These findings demonstrate the broad application prospects of PLA/HA scaffolds with USCs in bone tissue engineering.Graphical abstract

Differentiation of protein species of alpha-2u-globulin according to database entries: A half-theoretical approach

The alpha-2u-globulin protein, the main subject of this study, is the most abundant protein in adult male rat urine. In this investigation there are 19 spots identified as alpha-2u-globulin by 2-DE and MALDI-TOF/TOF-MS. All of them are in the low molecular weight region, at approximately 15–25kDa. Searches within both, SwissProt and NCBI databases gave different from each other, but on the majority the same database entry as the highest ranked result for all spots. For this half-theoretical approach all entries from the NCBI database for the protein alpha-2u-globulin were considered in more detail. The sequences and the masses of the theoretically resulting tryptic peptides were compared with the PMF spectra of the spots identified as alpha-2u-globulin. This study presents a trial to distinguish between different protein species of that protein, only on the amino acid level. Other modifications like phosphorylation, glycosylation or any other group modification could not be considered here. Different protein species can be predicted for groups of closer positioned spots. Statements about which spot contains which peptide variant can be done, too. But it was found that several spots contain more than one protein species. Biological significanceIn this investigation a half-theoretically approach was shown to differentiate between protein species of different spots identified as the same protein. Different positions of spots in 2-DE gels mean different contents of protein species. Here, peptide masses from sequences of protein database entries were searched in the PMF spectra of alpha-2u-globulin. Different peptide variants could be assigned to different spots. It was also found that several spots contain more than one peptide variant and therefore more than one protein species. In addition, the insufficiency of the database has been demonstrated.

Airborne molecules released from male mouse urine affect female exploratory behavior

Male mouse urine delivers a wide range of molecules, which may be involved in intraspecific chemical communication. These include the Major Urinary Proteins (MUP), which bind volatile odorant molecules and slowly release them from urine marks. The aim of this work is to evaluate the role of volatile molecules in eliciting exploratory behavior, in comparison to MUP. Female mice were exposed to male mouse urine, either diluted or not, or to MUP stripped of ligands. Gas chromatography and mass spectrometry of the stimuli were performed to verify the presence and identify the odorant molecules in urine and to assess the absence of MUP ligands. The exploratory behavior of adult female mice was analyzed in a cage, in the presence of two stimuli on opposite sides, but preventing direct contact with them. Four stimuli were presented in pairs: adult male mouse urine, MUP stripped of ligands, urine diluted 100 times and water as control. The results show that adult female mice explore urine, as little as 150 nl, but do not explore MUP stripped of ligands. These data show that male urine airborne molecules, effective at very low doses, mediate initial stimulus exploration by female mice.

Why do capuchin monkeys urine wash? An experimental test of the sexual communication hypothesis using fMRI

Urine washing (UW) consists of depositing urine on the hands and vigorously rubbing the body. As urine contains chemical and pheromonal cues, UW may convey socially relevant information. Although ritualized UW is observed in many New World primates, including capuchin monkeys, the functional significance of UW remains unclear. In this experiment, we investigated the social signaling hypothesis of UW. Specifically, we hypothesized that UW by males conveys socially relevant signals that females can detect. We used functional magnetic resonance imaging (fMRI) to test whether adult female capuchins show differential brain activation in response to adult male and juvenile male capuchin urine. We expected to see changes in activation of structures involved in olfactory processing, including the piriform cortex, medial preoptic and anterior hypothesis, orbitofrontal cortex, hippocampus, and cerebellum. Data were acquired from four adult female capuchin monkeys. Presentations of odor stimuli (obtained from unfamiliar males) were made during fMRI acquisition using a standard ON-OFF design. All fMRI data were spatially normalized to a template and analyzed using the FMRI Expert Analysis Tool Version 5.98, part of the FMRIB's Software Library (www.fmrib.ox.ac.uk/fsl). Whole brain analyses revealed significant activations in the inferior temporal cortex, parahippocampal gyrus, precuneus, hippocampus, pulvinar, and cerebellum when females were presented with the adult male urine. Notably, significantly greater signal activation was observed in several regions associated with olfactory processing, when subjects were presented with adult male urine as compared with urine from juvenile males. Our results indicate that UW serves a social communicative function in capuchins, providing support for the sexual signaling hypothesis.

Profiles of Volatiles in Male Rat Urine: The Effect of Puberty on the Female Attraction

Rat urine contains many volatile constituents that may be used for chemical communication. The levels of certain urinary volatiles are strongly dependent on the sex and endocrine status (e.g., puberty). We performed chemical and behavioral studies to identify the volatiles in adult male rat urine that attract mature females. Our results demonstrated that adult male rats have higher levels of 2-heptanone (2-HP), 4-methylphenol (4-MP), and 4-ethylphenol (4-EP) than prepubescent male rats; furthermore, female rats are more attracted to the odor of adult male rat urine than that of prepubescent males. When prepubescent rat urine was supplemented with 2-HP, 4-MP, and 4-EP to the levels found in adult male urine, the attractiveness of the urine to females was markedly enhanced. Our results suggested that this attraction is due to an increased level of chemosignaling.

Attenuation of the Production of Inositol 1,4,5-Trisphosphate in the Mouse Vomeronasal Organ by Antibodies Against the q/11 Subfamily of G-Proteins

The social and reproductive behaviors of most mammals are modulated by pheromones, which are perceived by the vomeronasal organ (VNO). Vomeronasal transduction in vertebrates is activated through G-protein-coupled receptors, which in turn leads to the generation of inositol 1,4,5-trisphosphate (IP(3)) and diacylglycerol (DAG) by the activity of phospholipase C. DAG has been shown to gate the transient receptor potential channel 2, whereas IP(3) may play a role in stimulating the release of calcium from the endoplasmic reticulum store. To investigate the role of the alpha subunits of G(q/11) in the transduction process, microvillar membranes from female mice VNO were preincubated with a selective C-terminal peptide antibody against Galpha(q/11) and then stimulated with adult male urine. Incubation of VNO membranes with antibodies against Galpha(q/11) blocked the production of IP(3) in a dose-dependent manner. We were also able to impair the production of IP(3) when we stimulated with 2-heptanone or 2,5-dimethylpyrazine in the presence of antibodies against the alpha subunit of G(q/11). 2-Heptanone is a known pheromone that has been linked to VIR receptors. Thus, our observations indicate that the alpha subunits of G(q/11) play a role in pheromonal signaling in the VNO.

Characteristics of the carbohydrate moiety and the intermolecular associations of the high-molecular-weight forms of alpha 2u-globulin.

We have investigated some of the biochemical characteristics of the glycosylated forms of α2u-globulin, the major protein of adult male rat urine. Labeled α2u-globulin that was secreted into the culture medium of primary monolayer cultures of hepatocytes was immunoprecipitated from the medium with antibody specific for urinary α2u-globulin and the complex used for further study. α2u-Globulin existed in the medium as a high-molecular-weight aggregate that could be dissociated with reducing agents and detergents into two glycosylated molecules of Mr 75000 and 50000 and one nonglycosylated molecule of Mr 20000. The glycosylated forms of α2u-globulin were shown by kinetic studies not to be precursors of the molecule of Mr 20000. When the glycoproteins were synthesized in the presence of radioactive sugar and amino acid precursors, the ratio of sugar to amino acid suggested that the highest-molecular-weight glycoprotein had 1.5–2 times the carbohydrate content of the faster-migrating glycoprotein. The oligosaccharide moiety, isolated from an exhaustive pronase digest of the combined glycoprotein molecules, was of Mr 5000 as determined by gel nitration methods. Sequential glycosidase digestion with neuraminidase, β-galactosidase, and β-N-acetylglucosaminidase produced a core oligosaccharide of Mr 1200. Anion-exchange chromatography demonstrated that the charge microheterogeneity of the α2u-globulin glycopeptides could be abolished by treatment with neuraminidase. Our results indicate that the oligosaccharide moiety of α2u-globulin is similar to those oligosaccharides found on other serum proteins, and suggest that formation of the species of α2u-globulin of Mr 75000 and 50000 requires either the attachment of many oligosaccharide chains to a single polypeptide of Mr 20000 or the covalent linkage of poly-peptides containing one or more oligosaccharide chains to form dimers and trimers.

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.

Involvement of G(q/11) in signal transduction in the mammalian vomeronasal organ.

Social behaviors of most mammals are profoundly affected by pheromones. Pheromones are detected by G-protein coupled receptors in the vomeronasal organ (VNO). To investigate the role of G alpha(q/11) in vomeronasal signal transduction pathways, microvillar membranes from murine VNO were prepared. Incubation of such membranes from prepubertal females with adult male urine results in an increase in production of inositol-(1,4,5)-trisphosphate (IP(3)). This stimulation is mimicked by GTP gamma S, blocked by GDP beta S and is tissue specific. Furthermore, use of bacterial toxins such as pertussis that lead to ADP-ribosylation of the G-protein alpha subunits of G(o) and G(i2) do not block the increase in IP(3) levels but U-73122, a PLC inhibitor, blocks the production of IP(3). Studies with monospecific antibodies revealed the presence of three G-proteins, G alpha(o), G alpha(i2) and G alpha(q/11)-related protein, in vomeronasal neurons, concentrated on their microvilli. Our observations indicate that pheromones in male urine act on vomeronasal neurons in the female VNO via a receptor-mediated, G alpha(q/11)-protein-dependent increase in IP(3) levels.

Use of the “SMITEST” PSA card to identify the presence of prostate-specific antigen in semen and male urine

To determine whether the prostate-specific antigen (PSA) could be identified in semen using the “SMITEST” PSA immunochromatographic membrane test card, we examined semen and other body fluids, including urine. Although PSA activity was detected in semen with high sensitivity using the “SMITEST” PSA card, it was also detected in adult male urine. However, the lower detectable limit in the urine was 1000-fold lower than that in semen. The concentration of PSA in adult male urine was found to be 800ng/ml using the card. PSA activity usually can be detected in urine of individuals over 14 years old and it has been detected in urine from children as young as 11 years old. Therefore, the appearance of PSA in urine may occur anytime between the age of 12 and 14 years. To determine the stability of PSA activity in urine, dried samples of urine on filter paper were kept at room temperature for up to 3 years. Although the immunoreactive line showing PSA activity became weak after storage, it was still detectable, but faint, after 3 years. In addition, PSA activity was not detected in male serum or saliva and in the urine from human females, male cats or male dogs using the PSA card. We conclude that the PSA card is useful for identification of PSA in both semen and adult male urine.

The highs and lows of chemical communication in giant pandas (Ailuropoda melanoleuca): effect of scent deposition height on signal discrimination

The giant panda's (Ailuropoda melanoleuca) social system is not well known, but it is clear that competition and avoidance characterizes social interaction for this solitary species throughout most of the year. Chemical communication appears to play a major role in structuring these interactions. Pandas adopt four distinct marking postures that deposit anogenital gland secretions or urine at varying heights: squat, reverse, legcock and handstand. In this experiment we tested the behavioral responses of 28 captive pandas belonging to all age-sex groups to conspecific odors placed to mimic the height of these postures. We tested three odor stimuli: male urine, and male and female glandular secretions. In all cases, pandas spent more time investigating higher odors than lower odors, suggesting that pandas place more importance on gathering information about individuals that adopt elevated postures. Pandas, particularly subadult males, also showed evidence of avoiding areas where adult male urine was deposited to mimic the handstand posture. Based on these and other findings, we propose that elevated postures in pandas may function to communicate competitive ability and possibly aggressive intent. Height of odor deposition, especially in the handstand posture, may be associated with body size, a major determinant of competitive ability. Selection may favor signalers that capitalize on the assessment activities of assessors by depositing scents to emphasize height cues. If such signals of competitive ability are used to mediate aggressive interactions, subsequent selection may favor their use only when the signaler is prepared to escalate in a contest.

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.

Urinary chemical cues affect light avoidance behaviour in male laboratory mice,Mus musculus

Chemical signals from conspecifics can influence the behaviour and neuroendocrine axis of mice. Several different molecules are excreted with urine, depending on hormonal level, and can indicate the sex of the emitter. In male mice, these chemicals are the major urinary proteins (MUPs) and some small volatile odorant molecues that are found bound to them. We tested adult males for light avoidance behaviour in a two-chamber apparatus, with one light and one dark side, in the presence or absence of male urinary substances. The presence of chemical cues on either side of the cage was expected to modify light avoidance behaviour. The volatiles released from purified MUPs had the same effect as whole adult male urine, in that they induced a faster onset of exploration of the light compartment. The results show that mice can use the information carried by the odorant molecules released by MUPs to recognize the urine of male mice, and respond appropriately.

CHEMICAL SIGNALS IN MALE HOUSE MICE URINE: PROTEIN-BOUND MOLECULES MODULATE INTERACTIONS BETWEEN SEXES

AbstractThe role of urinary chemosignals in sexual interactions was investigated in pairs of adult mice. Based on previous findings, volatile molecules bound by the Major Urinary Proteins (MUPs) from adult male urine were thought to be sufficient to carry information about the sex of the emitter, and thus sufficient to modify the behaviour of conspecifics. In the first experiment, virgin and stud adult males were exposed to receptive females painted or not with MUPs-borne molecules. Both virgin and stud males showed an increased latency to the first anogenital sniff and a reduced number of sniffings towards MUPs-treated females. This suggests that adult mice are repelled by MUPs-borne volatile molecules, even in the presence of female stimuli conveyed by receptive mates. In the second experiment only stud males were tested, with ovariectomized or estrogen-primed females. These latter were either untreated, painted with MUPs-borne molecules or MUPs without volatile ligands. Ovariectomized females and those treated with MUPs without ligands received less sniffs than the other two groups. Estrogen-primed females were mounted more times, with a shorter latency. Ovariectomized females and females treated with MUPs-borne ligands were attacked earlier by males. The presence of chemical cues from male urine is thus sufficient to modify the behaviour of stud males towards receptive females. In particular, MUPs-borne volatiles are sufficient to act as male cues and to induce aggression towards receptive females. It can be speculated that in nature adult male mice rely on olfactory cues like MUPs-borne odorants to firstly identify a male conspecific and possibly use similar chemical cues from their own urine to signal their presence.

Protein-bound male urinary pheromones: differential responses according to age and gender.

The attractive properties of male urinary pheromones were tested on adult or prepubertal male and female mice. An androgen-dependent protein is present in adult male urine (major urinary protein, MUP) which has been suggested to be a pheromone-binding protein. We tested the pheromonal properties of the protein-bound volatiles in a test of attractiveness. These molecules, that co-purify with MUP, attract females and repel adult males. In prepubertal animals, females are repelled and males are attracted by the same stimuli. These results are similar to those obtained by others with adult male whole urine. Therefore MUP binds molecules with a pheromonal activity, and these molecules are sufficient to act as male signals.

Chemosignals and activity of wild stock house mice, with a note on the use of running wheels to assess activity in rodents

Two control tests and an experimental test were used to explore the effects of urinary chemosignals on activity in house mice ( Mus domesticus). Control tests revealed that 1. (a) house mice of both sexes and different ages cling to the wheel for significant and variable proportions of their revolutions and thus correction factors must be employed to obtain useable results, and 2. (b) there is no social facilitation or inhibition of wheel running when mice are permitted to run in wheels housed near one another in the same room as opposed to being in separate rooms. Using running wheels for measurement periods of up to a week, we tested whether any of eight urinary odours or water (control) influenced activity in male and female mice that were prepubertal, pubertal, or adult. 1. (1) Prepubertal females were more active when treated with adult male urine than when treated with prepubertal male urine or water. 2. (2) Females of all three ages were generally more active when treated with urine from pregnant or lactating females when compared with the control treatment. 3. (3) Prepubertal and pubertal males were more active when treated with urine from adult males than when treated with water. 4. (4) Pubertal and adult males exhibited heightened running wheel activity when exposed to cues from oestrous, dioestrous, grouped females or prepubertal females. 5. (5) Pubertal and adult females exhibited greater activity when exposed to urine from adult males. These findings can be interpreted with regard to the physiology and behavioural biology of the mice.

Acceleration of puberty onset in female mice by male urinary proteins.

1. Puberty onset in female mice is accelerated by exposure to conspecific adult male urine, which acts through the vomeronasal organ and the accessory olfactory system. A distinctive component of adult male mouse urine is the major urinary protein complex (MUP), which is a lipocalin; it has a hydrophobic pocket that binds small endogenous volatile molecules. The MUP gene family also codes for a hexapeptide, which has four residues in common with the N-terminal region of MUP. 2. MUP, the volatiles bound to MUP and the MUP-related hexapeptide have been tested for the induction of puberty acceleration by measuring the increase in uterus weight related to the first pro-oestrus phase. MUP, together with its bound volatiles, induces puberty acceleration. Its activity is retained even when the volatiles have been removed either by organic extraction or competition displacement with a high-affinity ligand. 3. MUP-related hexapeptide also induces puberty acceleration in female mice. In contrast, the odorants bound to MUP do not exert this effect. 4. It is proposed that the vomeronasal organ contains receptors that recognize the short N-terminal consensus sequence, N-Glu-Glu-Ala-X-Ser (where X is a polar residue), common to both MUP and the hexapeptide.

Pheromonally accelerated puberty is enhanced by previous experience of the same stimulus

The influence of rearing conditions on pheromone-induced puberty acceleration was tested on Swiss mice. Litters were reared in one of three conditions: with either both parents, or with two females, or finally with two females in the presence of urinary pheromonal cues from adult males, which are known to induce puberty acceleration. Nine days after weaning the females were exposed to either prepubertal or adult male urine. In the groups reared with either both parents or with two females and the pheromonal cues from stranger males, females treated with adult male urine presented heavier uteri and more cornified vaginal smears than females reared in the same conditions but subsequently treated with prepubertal males urine. In the group reared simply with two females, the differences in both uterus weight and vaginal smears did not reach statistical significance. The data support the notion that early experience of pheromonal cues may influence the response to pheromones in a later period, even if the preweaning exposure to males had no direct influence on early signs of puberty onset.

Inhibition of sexual maturation in juvenile female and male mice by a chemosignal of female origin

Maturation of juvenile house mice ( Mus domesticus) exposed to synthetic 2,5-dimethylpyrazine was compared to the maturation of juvenile male and female mice exposed to urine from adult males and grouped females. Exposure of juvenile male mice to the synthetic compound significantly inhibited the growth of their testes and sex-accessory glands in a fashion similar to the urine of adult males and grouped females. The uterine weight and the presence of ova in the oviducts in females was strongly affected by synthetic stimuli. The majority of females (76–84%) exposed either to grouped females' urine or to the synthetic compound did not exhibit significant uterine weight increase and did not achieve pubertal ovulation before the age of 30 days as it has been reported for control mice. It is suggested that 2,5-dimethylpyrazine delays puberty of juvenile conspecifics in a population and may function whenever the living conditions of growing animals are below the level required for successful reproduction.

Analysis of rat urine proteins and allergens by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting

Background: In rats, urine has been identified as a major source of the allergens that cause laboratory animal allergy an important occupational health problem. Methods and Results: Urinary proteins and allergens of Wistar rats were studied by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting. Proteins excreted by male and female rats during puberty were similar and of low molecular weight. In adulthood, moderate increases in diffuse staining of 26 and 21 kd occurred in female urine. In males the 17 kd protein increased dramatically and the 23 and 21 kd proteins moderately. The urine excretion of high-molecular-weight proteins (75 to 63 kd) increased with age in males (females not studied). Immunoblot studies with six sera showed allergens in urine of male and female rats of all ages, three of which were present in all urine (75, 68, and 21 kd). Three allergens (17, 16, and 15 kd) in female urine may be allergenically similar to the 17 kd allergen in adult male urine. Adult male urine allergens were studied further with sera from 83 rat-hypersensitive subjects. Major allergens were identified at 23, 21, and 17 kd, and all sera had IgE to one or more of these proteins. Twenty-seven percent had IgE to 68 and 63 kd allergens. Minor allergens were identified at 75, 51, and 44 kd. Conclusions: Rat urine is an important source of the major allergens associated with rat hypersensitivity. Age and sex markedly influence the protein and allergenic constituents of rat urine.