Research_Sample

Effects of Inhibitors to Prostaglandin’s Effects on Female Garter Snakes Pg. 1

Portland State UniversityP.O. Box 751, Portland, Oregon 97207-0751

Effects of Inhibitors to Prostaglandin’s Impact on the Behavior and Estrogen Release in Female Red-Sided Garter Snakes

Prepared byRadford C. Bean

Student

For Dr. Joan WhittierAssociate Professor of Biology

Portland State University

Pg. 2 American Journal of Animal Physiology

PGF2 is found to be a uterine luteolytichormone in guinea pigs and sheep, therebyterminating corpus luteum (reviewed byMoore 1985). It is also a uterine vaso-constrictor.

Prostaglandins stimulate FSH and LHrelease from the pituitary gland. Prostaglandinalso may be responsible for the estradiolfeedback on GRH.

In sheep, systemic administration of PGF2caused plasma LH to increase, while admin-istration of indomethacin caused a decrease inplasma LH. Aspirin given to cows caused adecrease in LH. Prostaglandins have manycharacteristics of inflammatory mediators.

Increased levels of prostaglandin in theplasma of female red-sided garter snakes hasbeen found to increase the latency of femalesto mate (PGF2 – 0.05ug/g BW). A higherdosage of PGF2 (5.0ug/g BW) has renderedfemales unattractive to males within 24 hoursof treatment (Whittier and Crews 1985). Thisexperiment attempts to establish the effectsinhibiting prostaglandin will have on male andfemale courtship behavior, and plasma levelsof estrogen in the females.

When a sexually active female isintroduced into an aquarium of adult males,the males form a mating ball around thefemale. Male courtship in garter snakes, aswith other species of snakes, involves the

stroking of the female’s back by the male’schin. This is a result of a pheromone releasedby the female (Whittier and Crews 1986;Crews and Garstka 1982). This chin strokingbehavior provides a means of counting thenumber of males courting, determining if thecertain prostaglandin inhibitors have an effecton male and female behavior.

Methods

Animals and animal maintenance

Adult, sexually mature male and femalered-sided garter snakes were collected inManitoba, Canada. All snakes were marked byclippings to various ventral scales. Thefemales were kept in hibernation for a periodof 6 months at 60 C, 0L, and 24D. All thefemales used for the experiments had snout tovent lengths > 45cm and weights > 32.5g.

Six, 10-gallon aquariums housed sevenmales each, except for one aquarium thathoused on six. A Vitalite provided illuminationfor each aquarium. The substrate in eachaquarium consisted of hardwood chips.

All the males and females were fed smeltand provided with water.

Abstract: Various prostaglandin inhibitors were administered to female red-sided garter snakes(Thamnophis sirtalis parietalis) to measure the effects on male attractiveness to female gartersnakes and the impact on plasma levels of estrogen in females. A 1% solution of sodiumbicarbonate was used as the control. Inhibitors consisting of 8mg/ml indomethacin and 80mg/mlacetaminophen were injected into the females. After a period of incubation, the females wereintroduced to males and their receptivity monitored. If prostaglandin was inhibited by the varioustreatments, it would be reasonable to expect that after the females mated they would still bereceptive and attractive to male garter snakes. Females observed during the mating andimmediately on completion showed that the mated females were unreceptive to other males, andthat the unmated males lost interest in the mated females.

Twenty-four hours after completion of the experiments, the females were bled. Inhibition ofprostaglandin should result in low levels of estrogen 24 hours later.


Treatments and administration

Three various treatments were made upconsisting of 1% NaHCO3, 8mg/mlindomethacin (Sigma Chem. CO., Lot Date129C-0052) and 80mg/ml acetaminophen(Sigma Chem. Co., Lot Date 71F-0160).Sodium bicarbonate was used as the controlwas used as the control. These treatments werekept refrigerated when not being used.

Three groups of twelve females receivedone of the three treatments. Their body weightx .001ml determined the dosage administeredto them. The dosage was injected into theirbody cavity, and the females placed in aholding aquarium for a period of 20 minutesfrom the time of injection. The treatmentsadministered to each female were recordedalong with each female’s individual number(determined by the scale clippings).

Behavioral measurements

The experiment was run six times during aperiod of 3 weeks. One female each wasintroduced into one of the five aquariumshousing seven males, or into the sixthaquarium housing six males. Every 5 minutesthe number of males courting each female wasrecorded. This recording continued over aperiod of 1 hour. It also was recorded when afemale mated and the effect it had on theunmated males.

Extraction, chromatography and RIA of theblood samples

All mated and unmated females were bledusing the method of withdrawing blood fromthe ventricle. The collected samples were sentto the Oregon Regional Primate ResearchCenter for radio immunoassaying.

Statistical methods

The statistical significance of the results ofthe inhibitors with regards to the control wasfigured using the Chi square formula and the2 X 2 contingency tables. P < 0.05 wasconsidered significant.

X2 =df=1

H0: Mating unrelated to treatment.H1: Treatment influences mating.

If x2 value exceeds 3.84 (from table) reject H0.

The Kruskal-Wallis test was used todetermine the ranking of the three treatmentsto E2 levels in the female’s blood plasma. P <0.05 was considered significant. The one-tailed test was used to determine thesignifiacnace among 15 comparisons.

z = 2.713

x =

Results

One of three treatments was administeredto thirty-six females removed fromhibernation. Each run consisted of two femalesfrom each treatment. A minimum of 10minutes elapsed before observing the firstmating. The average number of courting malesfor each treatment, whether mating occurred ornot, is listed in Table 1.

x2 N AD BC∠ N 2 )2⁄∠(A B ) C D ) A C ) B D )+(+(+(+(

--------------------------------------------------------------------------------=

)/1/1)(12/)131(31( vu nnz ++

Pg. 4 American Journal of Animal Physiology

The percentage of males courting femalesin which mating occurred was similar betweenthe control and the two treatments (Figure 1).The same case held true for the males courtingwith no resulting copulation (Figure 2).

Courtship Bouts Where Mating Occurred

Figure 1. Percentage of males courtingfemales for each treatment in which copulationoccurred.

Upon intromission, all other males in theaquarium lost their attraction for the femaleand no further stroking of the female’s back bythe males was observed. This was the case forall the females that mated, regardless of thetreatment. All the females that mated haddeposited in the cloaca a copulatory plug (Rossand Crews 1977). It is not certain whether ornot a pheromone contained in the copulatoryplug interferes with further femaleattractiveness to males (Whittier, Mason andCrews 1985; Ross and Crews 1977). Thefemales administered the control substance

had a higher number of successful matings;half of those injected with NaHCO2 mated.Those females injected with the two inhibitorshad lower rates of mating success compared tothe control value (Figure 3). The difference insuccess was not significant (x2 = 7.37 X 10-1

H0 was not rejected: x2 < 3.84).

Courtship Bouts Without Mating

Figure 2. Percentage of observed malescourting females for each treatment in whichno mating took place.

Treatments to Female Garter Snakes

Figure 3. Percentage of females that matedbased on the type of treatment each received.

Test results show that all the matedfemales, regardless of treatment, had higherlevels of E2 in their blood plasma with the

Table 1: Average Number of Males Courting

NaHCO3 Acetamino-phen

Indometh-acin

Mated 4 4 3

Unmated 2 2 2

0

1

2

3

4

5

6

7

N aHC O 2 A cetam inophen Indom ethacin

T reatm ent

0

2

4

6

8

10

12

14

16

18


T reatm ent

0

5

10

15

20

25

30

35


T reatm ent


exception of one case. Female #94 had an E2level of 2.320ng/ml. Since this is notconsistent with the results for the other matedfemales, I chose to exclude that E2 value fromthe data. Two out of three mated femalestreated with indomethacin had E2 levelsconsiderably lower than those mated femalesthat received the other two types of treatments.The difference in E2 levels in femalesadministered indomethacin was significant,compared to the E2 levels in those femalestreated with the control or acetaminophen(Table 2).

Discussion

From the results obtained during theexperiment, the inhibitors had no effect onprostaglandin’s impact on male and femalemating behavior. Inhibiting prostaglandinshould have resulted in the females still beingreceptive to further male courtshipimmediately following copulation; this did notoccur. Furthermore, all unsuccessful malesshould have shown continued interest in thefemale, even though she had already mated.Prior research showed that prostaglandinsincrease latency and inhibit receptivity in thefemales (Whittier and Crews 1986). Based onFigures 1 and 2, acetaminophen andindomethacin had no effect on male courtship.In both cases, mated and unmated, thepercentages are similar to the control in eachgraph.

Of the three females treated withindomethacin that mated, the two females withthe lowest E2 levels are causing the

insignificance between those females and thefemales administered with the othertreatments. Indomethacin may, therefore, haveimpacted estrogen by suppressing the E2 levelsin those two females. A larger sampling offemales administered with indomethacin isneeded to see if the above results hold true inmost cases.

References

1) Moore P. K. Prostanoids: Pharmacological,Physiological and Clinical Relevance.England. Cambridge University Press,1985.

2) Whittier J. M, Crews D. “Effects ofProstaglandin F on Sexual Behavior andOvarian Function in Female Garter Snakes(Thamnophis sirtalis parietalis).”Endocrinology 119 (1986): 787-792.

3) Whittier J.M, Crews D. “OvarianDevelopment in Red-sided Garter Snakes,Thamnophis sirtalis parietalis,Relationship to Mating.” General andComparative Endocrinology 61 (1982): 5-12.

4) Crews D., Garstka W. R. “The EcologicalPhysiology of a Garter Snake.” ScientificAmerican 247 (1986): 158-168.

5) Siegel S., Castellan N. J., Jr. “Table C.”Non-parametric Statistics for theBehavioral Sciences. McGraw-Hill, 1988.

6) Whittier J. M., Mason R. T., Crews D.“Mating in the Red-sided Garter Snake,Thamnophis sirtalis parietalis:Differential Effects on Male and FemaleSexual Behavior.” Behavioral EcologicalSociobiology 16 (1985): 257-261.

7) Ross P., Jr., Crews D. “Influence of theSeminal Plug on Mating Behavior in theGarter Snake.” Nature 267 (1977: 344-345.

Table 2. Average Sum of Females

NaHCO3Acetamino-

phenIndometh-

acin

Mated 25.98a 26.63a 22.7b

Unmated 8.5b 10.7b 9.13b

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