William C. Agosta

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)

Fission fragment ionization (252Cf) mass spectrometry. Positive and negative spectra and decomposition mechanisms for seven compounds

The 252Cf fission fragment ionization mass spectra (both positive and negative ions) have been determined for alanine, arginine, sucrose, guanosine, 5′-adenosine monophosphate alanylalanylalanine, and lysyltyrosylthreonine. The lower m/z limit is 18 for the positive and 26 for the negative spectra tabulated. Mass to ion charge values are given with millimass precision, which permits deduction of atomic compositions for many of the observed ions. Reasonable mechanisms for the production of some of these ions are postulated. The significant findings of the study are: 1. (1) The amount of fragmentation occurring is large both in that quasi-molecular ion intensities are low and that many small ions are produced with large intensities. For example, only for alanine is the intensity of the quasi-molecular ion greater than 10% of the total ionization, and for arginine the CN− ion comprises 56% of the total negative ionization. 2. (2) The spectra observed have characteristically a quasi-molecular ion, a large gap without ions, and many ions at low mass. 3. (3) Detectable amounts of quasi-molecular ions are present in all the spectra. 4. (4) Reactions invoking known chemistry of gaseous positive and negative ions can be written for the formation of many of the fragment ions. 5. (5) The results indicate that the ionization process occurring is primarily a relatively high-energy one since it produces much fragmentation, but it has a low-energy component which is involved in the formation of the quasi-molecular ions observed here and in previosly published work. An ancillary finding is that the fission fragment ionization method seems to have a high sensitivity for producing anions from certain inorganic trace impurities.

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.

Responses of the male golden hamster to mixtures of odorants identified from vaginal discharge.

A series of aliphatic acids and alcohols was isolated and identified from hamster vaginal discharge. These materials along with a previously identified hamster attractant pheromone, dimethyl disulfide, were assayed in amounts characteristic of a single female collection for their ability to attract normal males and to promote copulatory behavior toward female surrogates. Estrous hamster vaginal discharge attracted normal males and stimulated copulatory attempts toward scented surrogates. Male copulatory behavior was unmodified by: dimethyl disulfide, the aliphatic acids, the aliphatic alcohols or a grand mixture of all these identified components of vaginal discharge. Therefore there must be some, as yet unidentified, material in vaginal discharge which induces normal males to engage in copulatory behavior. Neither the acids nor the alcohols when presented as mixtures had significant effects on male attraction. The attractiveness of a grand mixture of dimethyl disulfide and the series of aliphatic acids and alcohols was comparable to the attractiveness of dimethyl disulfide alone. Thus, the presence of small amounts of dimethyl disulfide can account for much of the ability of whole vaginal discharge to attract males to the odor source even when this compound is assayed in the presence of large amounts of inactive biological odorants.

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.

PHEROMONES OF HAMSTER VAGINAL DISCHARGE Attraction to Femtogram Amounts of Dimethyl Disulfide and to Mixtures of Volatile Components

The responses of normal male hamsters to the odors of several components of estrous female hamster vaginal discharge (FHVD) were quantified with a two-bottle preference test in order to clarify the role of dimethyl disulfide (DMDS) and dimethyl trisulfide (DMTS) in the attraction of males to females. These two compounds were originally isolated and identified in two separate, behaviorally active fractions of FHVD, one containing DMDS and the second containing DMTS. However, only authentic DMDS proved to be attractive to males; DMTS was inactive in the amounts tested (50 ng to 10 μg). When smaller amounts of highly purified samples of DMTS (0.03–3.32 ng) are assayed, measurable amounts of activity are obtained. This activity of authentic DMTS is not due to any intrinsic activity of its own but rather can be ascribed to the activity of the residual DMDS (0.07%) that it contains. Highly purified samples of authentic DMDS (containing 0.03% DMTS) were again found to be attractive to males. Doses as small as 500 fg were significantly active when compared to controls. Arguments are presented suggesting that the behaviorally effective dose of DMDS is several orders of magnitude less than 500 fg. Deliberate mixtures of DMDS and DMTS failed to show any synergistic effects. In fact, at the two lowest doses examined (0.1 and 0.46 ng total sulfides), the response obtained with the mixture was smaller than that expected if DMDS were the only odorant in the solution. Therefore, DMTS seems to suppress the activity of DMDS.

Chemical Studies of Hamster Reproductive Pheromones

Male golden hamsters respond to the vaginal discharge of females in a number of ways. The vaginal discharge attracts males, stimulates their sexual behavior, decreases their aggressive behavior, reduces their tendency to scent mark with the flank gland, and increases their plasma testosterone levels (cf, Johnston, 1977), It is not clear to what extent these are responses to different compounds in the discharge and to what extent they are different aspects of the response to a single compound or mixture. This is one of the questions which we are attempting to answer as we study the chemistry of the discharge. This paper is primarily concerned with our recent work on the substances in vaginal discharge which stimulate sexual behavior. However, since attraction is an important component of the sexual response, we first will review the results of earlier work on the attractant response.

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)

Cyclic variations in the concentration of sex attractant pheromone in hamster vaginal discharge

The concentration of dimethyl disulfide (DMDS), a sex-attractant pheromone of Syrian golden hamsters (Mesocricetus auratus), changes during the 4-day estrous cycle of the female. Pheromone concentration in hamster vaginal discharge (HVD) is maximum on Day 1, at the time of ovulation (320 ng DMDS/g of HVD), falls on Day 2 (227 ng/g), is minimal on Day 3 (78 ng/g), and then begins to rise on Day 4 (147 ng/g). The largest total amount of DMDS (5.6 μg) is present in the vagina on Day 2, the day on which the maximum HVD collection is obtained. However the peak in the cycle of DMDS concentration leads the peak in the amount of HVD collected by 1 and sometimes by 2 cycle days. There is sufficient DMDS present each day in the females vagina to account for her persistant attractiveness to males in the typical laboratory bioassay.

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.