The primary structure of aphrodisin.

Aphrodisin is a protein which is secreted in hamster vaginal discharge and acts via the vomeronasal organ of the accessory olfactory system to elicit copulatory behavior in male hamsters. The complete primary structure of aphrodisin was determined by sequence analysis of intact aphrodisin after unblocking the amino terminus with pyroglutamate aminopeptidase and from peptides generated by trypsin and Lys-C digests. Alignment of the peptides was obtained from sequence analysis of peptides from cyanogen bromide and hydroxylamine cleavages. The protein consists of 151 residues of Mr = 17,000. It has disulfide bonds linking cysteine residues at positions 38 and 42 and at 57 and 149. N-acetylglucosamine residues are linked to asparagines at positions 41 and 69. Based on its similarity to the major urinary proteins in rats and mice, aphrodisin is a putative member of the alpha 2u-globulin superfamily of extracellular proteins.

Experiential and endocrine dependence of gonadotropin responses in male mice to conspecific urine.

Previous research has shown that a urinary pheromone of female mice acts via the vomeronasal organ of the accessory olfactory system to elicit rapid release of luteinizing hormone (LH) in conspecific males. Several experiments were conducted to examine the importance of sexual experience for gonadotropin responses in male mice to female urine, male urine, saline, or mixtures of these stimuli. Both sexually naive and sexually experienced male mice had significantly higher plasma LH levels after presentations of female urine than after presentations of male urine. However, sexual experience appeared to increase the reliability of the short-latency gonadotropin response to female urine relative to a sexually neutral component of urine such as sodium chloride, and male urine appeared to suppress spontaneous LH secretion episodes in both naive and sexually experienced males. Subsequent experiments with sexually experienced subjects demonstrated that male mouse urine is a powerful suppressant of LH release in other males. Specifically, female mouse urine mixed with male urine failed to elicit LH responses in male subjects, whereas female urine mixed with saline was highly effective. Urine obtained from castrated male donors was as potent as urine from intact males in suppressing the gonadotropin response to female urine. The suppressive activity in male mouse urine thus does not appear to be critically dependent on gonadal hormones. The existence of a potent stimulatory pheromone in female urine and a potent suppressive pheromone in male urine makes male mice an excellent model system for studying the neural regulation of LH secretion.

Chemical properties of a female mouse pheromone that stimulates gonadotropin secretion in males.

Female mouse urine contains a pheromone that acts via the vomeronasal organ of conspecific males to stimulate a rapid increase in circulating levels of luteinizing hormone. A bioassay based on this male response was used to test biochemical preparations of female urine. Retention of significant biological activity by the urine after dialysis indicated that the activity is associated with urinary protein. Complete loss of activity from the urine after adsorption chromatography on a neutral polystyrene column suggested that the protein functions as a pheromone carrier. Assay of gel permeation chromatography fractions, before and after degradation of the urinary proteins with proteolytic enzymes, demonstrated that the protein is not necessary for the male response in the bioassay. Its resistance to vigorous proteolytic enzyme treatment further indicates that the pheromone is not a peptide. High biological activity, indistinguishable from that of the unfractionated urine, was isolated in a protein-depleted, presumably low molecular weight fraction containing compounds that are retarded by adsorption on Sephadex. The chemical properties of this female mouse pheromone are markedly different from those of a recently purified female hamster pheromone that also acts via the vomeronasal organ.

Purification and analysis of a proteinaceous aphrodisiac pheromone from hamster vaginal discharge.

Hormonally regulated proteinaceous material secreted in hamster vaginal discharge is detected via the vomeronasal organ and elicits copulatory behavior in males. The major soluble protein in estrous vaginal discharge has been isolated, characterized by molecular weight and amino acid content, and shown to have substantial aphrodisiac activity. The aphrodisiac activity of the purified protein is abolished by heating or proteolysis, and the native protein retains the activity after procedures for removing possible ligands such as volatile odorants, steroids, and peptides. This evidence that the protein is a reproductive pheromone indicates that the mammalian vomeronasal organ can mediate sensory detection of behaviorally relevant macromolecules.

Male hamster investigatory and copulatory responses to vaginal discharge: an attempt to impart sexual significance to an arbitrary chemosensory stimulus.

Hamster vaginal discharge elicits intense genital investigation and facilitates overt copulatory behavior toward anesthetized males (female surrogates) whose hindquarters have been scented with this material. The ability of an arbitrary chemosensory stimulus to acquire behavioral activity like that of vaginal discharge through association with maternal stimuli and/or adult sexual experience was examined in male hamsters. Vanillin was used as the arbitrary stimulus because it is attractive to hamsters, is not likely to be a natural constituent of hamster scents, is not known to exert any adverse physiological effects, and is a subliming solid with an extremely long persistence when used as an artificial scent. The males were reared by vanillin-scented or control solvent (water)-scented foster mothers, and in adulthood were paired repeatedly with vanillin- or solvent-scented receptive females. Behavioral testing with scented surrogates was performed one week preceding, and again following, the sexual pairings. Rearing by vanillin-scented mothers modestly but significantly increased the amount of time sexually naive males spent investigating the hindquarters as compared to other body regions of vanillin-scented surrogates. However, neither neonatal nor adult interactions with vanillin-scented females imparted to this stimulus the capacity to facilitate overt copulatory behavior. Also, regardless of the males' exposure history, only vaginal discharge caused the males to direct their investigatory behavior predominantly toward the hindquarters. The characteristic investigatory and copulatory responses exhibited by male hamsters toward vaginal discharge thus do not appear to be readily developed toward arbitrary chemosensory stimuli associated with particular females to which the males have been exposed.

Male hamster investigatory and copulatory responses to vaginal discharge: relationship to the endocrine status of females.

The ability of vaginal discharge from ovariectomized (OVEX) or hypophysectomized (HYPOX) female hamsters to elicit intense genital investigation and to facilitate overt copulatory behavior in males was compared with that of estrous vaginal discharge. The discharges were collected by vaginal lavage with water. In order to avoid exposure of experimental males to female stimuli other than vaginal discharge, the behavioral tests employed anesthetized males (female surrogates) whose hindquarters were scented with the collected vaginal material or with control solvent (water). Both the OVEX and HYPOX discharges elicited intense genital investigation and significantly increased the incidence of intromission attempts toward the scented surrogates. However, both types of discharge had significantly less behavioral activity than estrous vaginal discharge. In a subsequent experiment, a water dilution series of estrous vaginal discharge was tested for the ability to promote genital investigation and copulatory behavior toward scented surrogates. The amount of discharge typically extruded by one estrous female in response to tactile genital stimulation can be diluted one hundred-fold without appreciable reductions in its behavioral activity. These results demonstrate that the behavioral activity of hamster vaginal discharge is related to the endocrine status of females, and suggest that previous failures to demonstrate clear dependence on ovarian function might have been due to ceiling effects in laboratory tests.

Chemical studies of hamster vaginal discharge: male behavioral responses to a high molecular weight fraction require physical contact.

This report describes the isolation and behavioral testing in normal male hamsters of a high molecular weight fraction (HMF) of vaginal discharge which accounts for much of the aphrodisiac activity in the discharge. The HMF encompasses a group of proteins which elute as a relatively narrow, major peak upon agarose gel filtration of estrous vaginal discharge. The crude fraction from gel filtration retains a variety of volatiles including sulfur-containing compounds which we have previously found to account for much of the initial attraction of males to the female but which do not, themselves, facilitate overt copulatory behavior. Procedures for markedly reducing the presence of such volatiles to yield the HMF are described. In behavioral assays using anesthetized males as surrogate females, scenting the hindquarters of the surrogates with the HMF elicits intense genital investigation by experimental males, although this effect on investigatory behavior is not as dramatic as that of the unfractionated vaginal discharge. Like the unfractionated discharge, the HMF significantly increases the incidence of intromission attempts toward scented surrogates. To assess whether physical contact with the HMF is required for behavioral activity, as would be expected if the active material is proteinaceous, series of preference tests were performed using vanillin as a competing stimulus under conditions in which physical contact with the stimuli either was possible or was prevented. The unfractionated discharge was preferred in both conditions, whereas significant preferences for the HMF were exhibited only if it could be contacted by the snout of experimental males. The active material in the HMF thus appears to be of extremely low volatility, at least prior to physical contact with it by the male.

Male hamster copulatory responses to a high molecular weight fraction of vaginal discharge: effects of vomeronasal organ removal.

The importance of the vomeronasal (accessory olfactory) system for the copulatory responses of male hamsters to a high molecular weight fraction (HMF) of vaginal discharge was assessed in animals that had their vomeronasal organs (VNO) removed. These organs were extirpated bilaterally using an oral approach through the palate so as to eliminate the peripheral afferents to the accessory olfactory bulb (AOB) with minimal or no damage to the main olfactory system. The selective peripheral deafferentation procedure was verified by applying horseradish peroxidase intranasally following intraperitoneal injections of epinephrine to facilitate the vomeronasal pumping mechanism that draws fluids into the VNO. Heavy, bilateral anterograde labeling was evident in the olfactory nerve afferents within the main olfactory bulb of males that had their VNO removed and of animals that received sham surgery. Sham-operated males also had heavy, bilateral labeling in the vomeronasal nerve afferents within the AOB, whereas no such labeling occurred among animals with bilateral removal of the VNO. In sham-operated animals, both the HMF and the unfractionated discharge significantly increased the incidence of intromission attempts toward anesthetized males (surrogate females) whose hindquarters were scented with these stimuli. The unfractionated discharge also produced a significant elevation of overt copulatory behavior in males with selective peripheral deafferentation of the vomeronasal system, whereas the HMF did not facilitate copulatory behavior in these animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Chemical studies of hamster vaginal discharge: effects of endocrine ablation and protein digestion on behaviorally active macromolecular fractions.

Our previous studies have demonstrated that macromolecular fractions of hamster vaginal discharge elicit intense genital investigation and facilitate copulatory behavior toward anesthetized males (female surrogates) whose hindquarters have been scented with these fractions, and that the aphrodisiac activity is significantly reduced in the vaginal discharge of ovariectomized (OVEX) or hypophysectomized (HYPOX) females. The present series of studies compared the protein compositions of estrous, diestrous, OVEX, and HYPOX vaginal discharge, and assessed whether protein digestion of estrous vaginal discharge affects its aphrodisiac activity. Polyacrylamide gel electrophoresis of vaginal lavages showed that the endocrine status of females has a profound effect on the protein composition of the vaginal discharge. The concentration of the major proteins dropped by about ten-fold in going from the estrous to the diestrous condition. The concentration of these same proteins appear to be at least another order of magnitude lower in the vaginal discharges of OVEX and HYPOX females. These major proteins had molecular weights greater than 10,000 Daltons. A macromolecular fraction of estrous vaginal discharge isolated by gel filtration at elevated temperature (conditions known to separate some protein-bound steroids) and containing proteins of molecular weight greater than 10,000 Daltons was found to be modestly but significantly less active than estrous discharge in its ability to elicit intense genital investigation, and not significantly different from estrous discharge in its ability to facilitate overt copulatory behavior toward scented surrogates. Pronase digestion of the proteins in this fraction did not alter its effects on investigatory behavior toward scented surrogates but significantly reduced its ability to facilitate copulatory behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Methyl thiolbutyrate: a reliable correlate of estrus in the golden hamster.

A volatile sulfur-containing compound that is characteristic of estrous (Day 1) hamster vaginal discharge has been identified as methyl thiolbutyrate (MTB). From numerous measurements of MTB concentration on each day of the estrous cycle, it is clear that this compound could well serve as a natural chemical signal of the estrous state of the female. Both the ability of MTB to attract male hamsters and its ability to facilitate copulatory behavior toward female surrogates were evaluated in behavioral experiments. The responses to MTB were compared to those elicited by dimethyl disulfide (DMDS), by a mixture of MTB and DMDS, by a grand mixture of MTB and DMDS with thirteen other known volatile constituents of the vaginal discharge, and by fresh vaginal discharge. In contrast to fresh vaginal discharge, none of the identified compounds or mixtures facilitated male copulatory behavior. Most of the attraction observed in the behavioral experiments could be attributed to the DMDS in the test samples. The MTB is readily converted to DMDS and may serve as a precursor of DMDS in the vaginal discharge.