Proteinaceous pheromone affecting female receptivity in a terrestrial salamander

Science

Volumn 285, Issue 5435, 1999, Pages 1907-1909

  Rollmann, Stephanie M ^a   Houck, Lynne D ^b   Feldhoff, Richard C ^c  

^a UNIVERSITY OF CHICAGO   (United States)

^b OREGON STATE UNIVERSITY   (United States)

^c University of Louisville School of Medicine   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

PHEROMONE; PROTEIN;

AMINO ACID SEQUENCE; ANIMAL TISSUE; ARTICLE; COURTSHIP; HORMONE ACTION; MALE; MOLECULAR CLONING; NONHUMAN; NUCLEOTIDE SEQUENCE; OVULATION; PRIORITY JOURNAL; PROTEIN ISOLATION; RNA TRANSCRIPTION; SALAMANDER; SEQUENCE ANALYSIS; SEQUENCE HOMOLOGY; SEXUAL BEHAVIOR; SUBMANDIBULAR GLAND;

AMINO ACID SEQUENCE; ANIMALS; FEMALE; HUMANS; MALE; MOLECULAR SEQUENCE DATA; SEQUENCE HOMOLOGY, AMINO ACID; SEX ATTRACTANTS; SEXUAL BEHAVIOR, ANIMAL; URODELA;

AMPHIUMA MEANS; ANIMALIA; CAUDATA; PLETHODON JORDANI; PLETHODONTIDAE;

EID: 0033578813     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.285.5435.1907     Document Type: Article

References (20)

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12. Each male was anesthetized in a mixture of 4% ether in water. The mental gland was excised and placed into a 0.8 mM acetylcholine chloride solution for 30 min. Extracted pheromone components were then centrifuged (10 min; 14,000g) and the supernatant frozen at -80°C. Pooled gland extracts were later thawed and filtered through a 0.2-μm filter (non-protein binding) and applied to a Mono-Q column (FPLC HR 5/5; Pharmacia, Piscataway, NJ) at 50 mM tris-HCl, pH 8.0. The column was eluted with the same buffer, at 1 ml/min, with a NaCl gradient (5.0 mM NaCl/min). To further purify PRF, we ran enriched fractions of PRF isoforms separately on the Mono-Q column again and eluted the column with a NaCl gradient of 3.3 mM NaCl/min. The PRF fractions were further purified on a Sephadex Superfine G-75 column (1.6 cm by 15.5 cm; Pharmacia, Piscataway, NJ) previously equilibrated with one-half strength Dulbecco's phosphate-buffered saline at a flow rate of 7.9 ml/hour. A pool of the highly purified PRF isoforms was adjusted to 0.7 mg/ml, the approximate concentration of PRF present in the whole extract used in previous experiments (3), and analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on 15% tris-tricine gels. No contaminants were detected on SDS-PAGE gels when 5 μg was stained with Coomassie Brilliant Blue R-250, suggesting a purity of ≥ 98%. Reversed-phase HPLC analysis of similarly prepared PRF isoforms suggests that typical purity is ∼99%. We did not use isoforms prepared from cloned genes in this bioassay system because it is unknown if the activity of individual isoforms will depend only on the primary amino acid sequence or will include posttranslational modifications.
13. About 400 adult males and 400 gravid females were collected from a single population in North Carolina (83°33′38″N longitude and 35°10′49″W latitude). The appropriate collecting permits (North Carolina permit number 0120 to S.M.R.) and animal care protocols (University of Chicago animal care and use protocol number 70511; Oregon State University number 1997) were obtained. After collection, courtship encounters were staged between male-female pairs to assess individual tendency to mate under laboratory conditions. Every salamander received multiple courtship opportunities. Males and females were then ranked according to courtship propensity. For the experiments, we selected males and females that had the highest courtship propensities, in order to maximize the probability of obtaining animals willing to court under experimental conditions. We are aware that such prescreening may bias the results by increasing overall courtship rates. Any bias, however, would tend to minimize the probability of demonstrating courtship pheromone effects and thus render significant results more reliable. The experiment comprised eight trial nights, with three "days off" between each trial night. Courtship encounters were staged by placing male-female pairs into individual courtship boxes at dusk. Delivery of male courtship pheromones was controlled experimentally because each male lacked a mental gland. The time a pair spent in tail-straddling walk was recorded. An increase in female receptivity was operationally defined as a decrease in the time a pair spent in tail-straddling walk. A female that engaged in tail-straddling walk became a candidate for treatment delivery when the courting male turned back and physically contacted the female's snout with his chin. At this time, the first of three 5-μl drops of the appropriate treatment solution was delivered to the nares of the female. Typically, the male delivers courtship pheromones multiple times during tail-straddling walk. The experimental delivery of three drops (10 min apart) mimics natural male delivery. Females received either a pheromone (purified PRF pool from the G-75 column) solution or saline (control) solution. If a female received a treatment solution on the first trial night, the female received the opposite treatment on the following trial night. The saline (control) solution was the same vehicle used in the purification of PRF.
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20. We thank S. J. Arnold, E. Adams, P. Andolfatto, P. Feldhoff, M. Kreitman, T. Morton, C. Palmer, E. Stahl, R. Steinberg, and J.-B. Warne-Reese for technical advice, help in the field, or manuscript review. We also wish to thank the Highlands Biological Station (HBS) and R. Bruce for the use of their facilities. Supported by NSF grants IBM-9709355 (R.C.F.) and IBM-9615473 (S.J.A. and L.D.H.), NSF dissertation improvement grant 9801210 (S.M.R.), Hinds Fund University of Chicago (S.M.R.), and HBS Grants in Aid (L.D.H. and S.M.R.).