Effects of gossypol on sperm viability and plasma sexsteroid hormones in male sea lamprey, Petromyzon marinus
Jacques Rinchard a, Andrzej Ciereszko a,b, Konrad Dabrowski a,*,Joseph Ottobre c
a School of Natural Resources, The Ohio State Uni6ersity, 2021 Coffey Road, Columbus, OH 43210, USAb Polish Academy of Sciences, 10-718 Olsztyn, Poland
c Department of Animal Sciences, The Ohio State Uni6ersity, Columbus, OH 43210, USA
Received 17 May 1999; received in revised form 4 August 1999; accepted 5 August 1999
Abstract
Male sea lampreys Petromyzon marinus were injected with different doses of gossypol acetic acid in an attempt tosterilize them for use in a program for controlling the sea lampreys through the release of sterile males. Two lots ofsea lamprey were used in these experiments. The first lot was divided into three groups and fish were injectedintraperitoneally (i.p.) with 0.2 ml 50% ethanol as a control group or with gossypol suspended in ethanol at 100 and200 mg/kg. The second lot was also divided into three groups and fish were either injected i.p. with vehicle as controlsor gossypol at 25 and 50 mg/kg. Sperm weight, concentrations and motility were recorded after 31, 36 and 40 daysor 24, 28 and 33 days in lots 1 and 2, respectively. Blood was collected from the caudal vessel prior to injections withgossypol and after 40 or 33 days in lots 1 and 2, respectively. Plasma levels of estradiol-17b (E2), testosterone (T),progesterone (P) and 17,20b-dihydroxy-4-pregnen-3-one (17,20bP) were measured by radioimmunoassay. At the endof the experiment, the testis were removed and fixed in Bouin’s solution for histological examination. High mortalitywas observed at the day of injection in the group treated with 200 mg/kg (84.6%), 100 mg/kg (41.7%), and 50 mg/kg(25%). Sperm concentrations were higher in control fish in comparison to most of the treated groups during the firstsperm sampling (day 31 or 24), but then differences disappeared. At each sampling, sperm motility was higher incontrol groups than in treated groups and significant differences were observed (e.g. between control and 50 mggossypol/kg). Fertility, evaluated at optimized sperm/egg ratio (5×104 sperm/egg) did not differ among treatmentsand controls. Changes in mean plasma sex steroid levels in the various treated groups were not significant, but a trendof decreasing plasma E2 was observed with increasing dose of gossypol. The structure of the testis was examined atthe end of the experiment using light microscopy and appeared to be unchanged in gossypol-treated lampreys ascompared to that of control animals. These findings suggest that gossypol injection into lamprey interferes with someof the reproductive parameters. Although high mortality rate precludes a single i.p. injection of gossypol as afavorable mode of chemosterilization in the lamprey, it would be useful to reassess the means of transfer of gossypolto reproductive tissues. This notion is further supported by the results of an in vitro sperm toxicity assay which
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* Tel.: +1-614-2924555; fax: +1-614-2927432.E-mail address: [email protected] (K. Dabrowski)
The invasion of the sea lamprey, Petromyzonmarinus, through the Weeland canal, designed toconnect Lakes Ontario and Erie at the turn of thecentury, is commonly recognized as having a greatecological impact on the Great Lakes. The ab-sence of natural predators combined with thepresence of ample food and suitable habitat al-lowed sea lamprey populations in the Great Lakesto grow at an explosive rate. This resulted insevere impact on native fish populations such aslake trout, Sal6elinus namaycush, and lakewhitefish, Coregonus clupeaformis (Ferreri et al.,1995). Different control methods, including me-chanical and electrical barriers, chemical lampri-cides (such as 3-trifluoromethyl-4-nitrophenol or5,2%-dichloro-4%-nitrosalicylanilide), trapping, sex-ratio manipulation (male sterilization usingbisazir, hormonal treatment or radiation), attrac-tants and repellants (pheromones) have been de-veloped by the Great Lakes Fishery Commissionto eradicate or minimize sea lamprey population(Holmes, 1995). However, total reliance on a sin-gle control method is considered unwise (Lamsaet al., 1980). Therefore, a search for alternativelamprey control methods is necessary to create anintegrated control program.
Gossypol [1,1%,6,6%,7,7% - hexahydroxy - 5,5% - di-isopropyl-3,3%-dimethyl-(2,2%-binaphthalene)-8,8%-dicarboxaldehyde], a yellow pigment extractedfrom the seeds, leaves, stems and roots of thecotton plants of the genus Gossypium sp. (familyMalvaceae), has been reported to be a potentmale non-steroid contraceptive in numerous spe-cies, including invertebrates, sea urchin (Burgos etal., 1980), birds, Japanese quail (Lin et al., 1988),domestic cock (Mohan et al., 1989), and mam-mals, rat (Hadley et al., 1981; Baccetti et al.,1986), hamster (Chang et al., 1980; Waller et al.,1981), boar (Tso and Lee, 1981), monkey (Shandi-lya et al., 1982; Stephens, et al., 1983) and man(Coutinho et al., 1985; Waites et al., 1998). How-
ever, there were species differences between ani-mals in terms of doses required for an antifertilityeffect. For examples, oral administration of gossy-pol induced sterility in rats and hamsters whengiven at a dose of 5 or 10 mg/kg daily for 12weeks (Chang et al., 1980). In domestic cock, adaily dose of 40 mg/kg for 18 days caused adecrease of sperm concentration and a drop tozero fertility (Mohan et al., 1989), whereas incynomolgus monkeys, Macaca fascicularis, a dailydose of 10 mg/kg for 6 months was required tosuppress sperm production and motility (Shandi-lya et al., 1982). Evidence exists that gossypol, isdetrimental to spermatocytes, spermatids andspermatozoa (Xue, 1985), and may decrease con-centration or inhibit sperm motility. In addition,gossypol increases sperm abnormalities and in-hibits testicular steroidogenesis. Thus, this antifer-tility agent could be used as an alternativesterilant to eradicate or minimize sea lampreypopulations in the Great Lakes since the steriliza-tion of a portion of the natural population willproduce both a decrease of the reproductive po-tential of treated males and a decrease in thereproductive capacity of the remaining normalindividuals (females) in the population (Knipling,1968). One practical way of treating sea lampreyfor sterilization would involve a single injection ofgossypol. This mode of administration (i.e. a sin-gle injection) is currently used with anotherchemosterilant, bisazir (p,p-bis(1-aziridinyl)-N-methylphosphinothioic amide) (Hanson and Man-ion, 1980). Hanson and Manion (1980) reportedthat a dose of 100 mg bisazir/kg injected in-traperitoneally sterilized lampreys and thus re-duced production of viable larvae. However, theuse of a single injection necessitates the use ofhigh concentrations of the chemosterilant, whichcould also affect non-reproductive aspects of sealamprey physiology. In mice, a single i.p. injectionof gossypol at doses B20 mg/kg resulted in 100%survival, whereas all mice died at doses over 40mg of gossypol/kg (Rao et al., 1985).
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The objectives of this study were to investigatethe effects of a single injection of gossypol on thesperm characteristics (quantity, motility, concen-tration, fertilization rate) of sea lamprey and onthe levels of blood plasma sex steroids (testos-terone, progesterone, estradiol-17b and 17,20b-di-hydroxy-4-pregnen-3-one) in this species.Additionally, in vitro effects of gossypol on fertil-ization rate of spermatozoa were examined.
2. Material and methods
2.1. Chemicals
Gossypol acetic acid was purchased fromSigma Chemical Company (St. Louis, MO,USA). [1,2,6,7-3H]testosterone (96.5 Ci/mmol),[2,4,6,7,16,17-3H]estradiol (141 Ci/mmol) and[1,2,6,7,16,17-3H]progesterone (100-160 Ci/mmol)were purchased from NEN Life Science Products(Boston, MA, USA). [3H]17,20b-dihydroxy-4-pregnen-3-one was a gift from Dr A. Fostier(INRA, Rennes, France). Unlabelled steroidswere purchased from ICN Pharmaceuticals (CostaMesa, CA, USA), Sigma Chemical Company andSteraloids (Wilton, NH, USA). The testosteroneantiserum was provided by Dr R.E. Ciereszko(Institute of Animal Physiology, University ofAgriculture and Technology, Olsztyn, Poland),the estradiol-17b antiserum by Dr R.L. Butcher(West Virginia University, WV, USA), the proges-terone antiserum by Dr G.D. Niswender (Colo-rado State University, CO, USA) and the17,20b-dihydroxy-4-pregnen-3-one antiserum byDr A. Fostier. The characteristics of these antis-era have been reported previously (Butcher et al.,1974; Fostier and Jalabert, 1986; Johnson et al.,1988; Dabrowski et al., 1995, respectively). Otherchemicals were obtained from Sigma ChemicalCompany, Packard (Downers Grove, IL, USA),Pharmacia Fine Chemicals AB (Uppsala, Sweden)and Matheson and Bell (Norwood, OH, USA).
2.2. Animals and experimental procedure
Sea lampreys were sent by air from the LakeHuron Biological Station, Michigan, to Colum-
bus, OH on May 28 (n=47) and June 5 (n=46),1997. These animals had been captured in theCheboygan River, Michigan, on their anadro-mous migration after their parasitic phase in LakeHuron and averaged 227.8921.3 g in bodyweight. At this period, sea lamprey males are inthe final stage of their spermatogenesis and testic-ular ampullae become filled with matured sperma-tozoa (Sower and Gorbman, 1999). Sea lampreyspawn only once in their lifetime, after which theydied. At spawning, all spermatozoa that wereproduced are released.
The males of the first lot (May 28) were dividedinto three groups. Lampreys in the control groupwere injected intraperitoneally with 0.2 ml/kg of50% ethanol (average weight: 237.4943.1 g, n=12). Lampreys in the other groups were injectedwith gossypol diluted in ethanol at doses of 100and 200 mg/kg (average weights: 255.2946.7 g,n=12 and 243.0941.4 g, n=13, respectively).The males of the second lot (June 5) were alsodivided into three groups. Again, controls wereinjected with 0.2 ml/kg of 50% ethanol (averageweight: 197.1963.7 g, n=12). The other groupswere injected with gossypol at doses of 25 and 50mg/kg (average weights: 214.2947.7 g, n=12and 220.0956.5 g, n=12, respectively). Eachgroup was kept separately in darkness in circulartanks supplied with water at ambient temperature(12–16°C). Females from both lots (n=20) weremaintained together separated from males in atank with the same conditions. Before sampling,fish were anaesthetized in a solution of 0.01%tricaine (MS-222, Argent, Redmond, WA, USA)with 0.3% sodium bicarbonate.
2.3. E6aluation of the quality of the semen
Semen specimens were collected by stripping.This was done by applying abdominal massagetoward a genital papilla. Specimens were collectedinto 1.5 ml Eppendorf tubes. This procedure wasrepeated three times. Collection followed the sin-gle injection of gossypol by 31, 36 and 40 days,and 24, 28 and 33 days in lots 1 and 2, respec-tively. Special care was taken to avoid urine con-tamination. The semen was examined for weight,sperm concentration and motility. Sperm concen-
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tration was estimated microscopically using Dou-ble Neubauer Counting Chamber (Ciereszko andDabrowski, 1993). The sperm motility was evalu-ated at room temperature after dilution 100 timesin 10% modified ‘lamprey Ringer’ solution (137mM NaCl, 12 mM KCl, 1 mM CaCl2, 10 mMHEPES, pH 7.4 adjusted with NaOH) (Kobayashiand Yamamoto, 1994) by the same observer andrecorded as percent motile sperm.
2.4. Fertilization
Gametes (sperm and ovas) were obtained afterspontaneous spermiation or ovulation by strip-ping. Fertilization trials were performed two timesfollowing gossypol injection (days 36 and 40 anddays 28 and 33 in lots 1 and 2, respectively). Petridishes were used with 200 eggs and 50 000 sper-matozoa per egg. Gametes were activated with 10ml of water and incubated at room temperaturefor 5 h. After reaching the two-cell embryos stage(Piavis, 1961), eggs were transferred to California-type hatching trays. Survival rate was estimatedby calculation of % of fertilization at two-cellembryos stage (5 h after fertilization) and athatching.
2.5. Blood samples and steroidradioimmunoassays
Blood was collected in male sea lampreys fromthe caudal vessel into a heparinized syringe imme-diately prior to and after (40 and 33 days in lots1 and 2, respectively) the injection of gossypol.After centrifugation (1500×g for 10 min at 4°C),plasma from individual blood samples was frozen
and stored at−20°C until assay. All plasma sexsteroids (testosterone, T; estradiol-17b, E2;progesterone, P; and 17,20b-dihydroxy-4-pregnen-3-one, 17,20bP) were measured by radioim-munoassays (methods similar to Ottobre et al.,1989) following an extraction with ethyl ether (E2,T and 17,20bP) or petroleum ether (P). All assayswere validated for use with sea lamprey (Table 1).
2.6. Gonad histology
At the end of the experiment, lampreys weredecapitated. The testes were removed from two tothree animals per treatment, immersed in Bouin’sfixative for 24 h and then preserved in 70% ethylalcohol until use. Testes, dehydrated and embed-ded in paraffin, were cut at 7 mm and stained withMayer’s hematoxylin and eosin.
2.7. In 6itro effect of gossypol on fertilizationrate of spermatozoa
Gossypol acetate was freshly prepared by dis-solving it in 100% ethyl alcohol to a concentrationof 10 mM before dilution to yield the desiredconcentration. Fertilization trials in duplicate us-ing semen from four individual males were per-formed as described above (200 eggs and 50 000spermatozoa per egg). Semen from several maleswere mixed and preincubated during 30 min with1% ethanol (control 1) dissolved in immobilizingsolution of ‘lamprey Ringer’ or 100 mM gossypolacetate dissolved in 1% ethanol. A second control(control 2) was carried out to check if the gossy-pol introduced at the time of fertilization wouldhave an effect. In this case, gossypol was added
Table 1Radioimmunoassay characteristics of steroid hormones in sea lamprey
0.9970.999Accuracy (coefficient of determination) 0.995 0.9981 2Sensitivity (pg/ml) 1 1
73.58280Recovery of extraction (%) 82Parallelism Serial dilutions of plasma samples showed parallelism with the standard
curve between 25 and 200 ml
J. Rinchard et al. / Toxicology Letters 111 (2000) 189–198 193
Fig. 1. Relationship between the concentration of gossypolinjected to sea lamprey and the rate of mortality (y=0.42x+0.57, r=0.98, PB0.01).
tality rate of sea lamprey (Fig. 1). Additionally,at the first sampling, semen collected from allgossypol-treated males was yellow and containedyellow sediment. The weight of the yellow sedi-ment was less than 1% of the weight of thesemen.
3.1. Effects of gossypol on semen quality
A single intraperitoneal injection of gossypol tomale sea lamprey at a dose of 25–200 mg/kg hadvarious effects with respect to sperm qualityparameters. Throughout the experiment theweight of semen released by individuals afterstripping ranged from 0.1 to 2.6 g and from 0.2to 3.3 g in control and gossypol-treated groups,respectively. Means are reported in Fig. 2A. In allgossypol-treated groups, the semen weightshowed a trend to decrease gradually over time,although the changes were not significant (P\0.05). Some semen collected from the gossypol-treated lamprey did not contain anyspermatozoa. Sperm concentration was lowerthan controls in all gossypol-treated groups (con-centrations in individual samples ranged from0.01 to 2.12×109 cells/ml). However, no signifi-cant differences (PB0.05) were found betweengossypol-treated and control groups (Fig. 2B),except between the control group and the groupstreated with 100 and 200 mg/kg of gossypol dur-ing the first sampling. The sperm concentrationstarted to return to the normal value during thetwo following samplings. At each sampling date,sperm motility was higher in control groups thanin treated groups. At the first sampling, thesperm motility was significantly (PB0.05) higherin the control and in the group treated with 100mg/kg of gossypol than in the group treated with200 mg/kg. The sperm motility was also signifi-cantly (P\0.05) lower in the group treated with100 mg/kg compared to control at the end of theexperiment (Fig. 2C). In the second lot of sealamprey, the motility of the spermatozoa wassignificantly (PB0.05) higher during the secondsampling in the control group compared to thegroup injected with 25 or 50 mg/kg of gossypoland during the third sampling between the con-trol and the group treated with 50 mg/kg gossy-
into the fertilization medium just prior to mixinggametes. The final concentration of gossypol incontrol 2 was 100 mM after mixing gametes.
2.8. Statistical analysis
The data are presented as the mean9SEM.Each parameter was first analyzed statistically bya one way analysis of variance (ANOVA) forchanges that might have occurred during the ex-perimental period. Then each parameter wascompared simultaneously among all treatmentsusing two way ANOVA to test for treatmenteffects. Analysis of variance was followed byFisher least significant differences test. Data ex-pressed as percentages were arcsine transformedbefore analysis. A Bartlett’s test was used toverify homogeneity of variance (Dagnelie, 1975).The relationship between particular parameterswas estimated by calculation of the correlationcoefficient (r) and its statistical significance wastested (Dagnelie, 1975).
3. Results
High mortality was observed on the day ofgossypol intraperitoneal injection in the groupsreceiving 50 mg/kg, 100 mg/kg, and 200 mg/kg.There was a significant linear relationship be-tween the dose of gossypol injected and the mor-
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Fig. 2. Effects of gossypol on semen characteristics of male sea lamprey (n ranged from 2 to 7). (A) Semen weight; (B) spermconcentration; (C) sperm motility; (D) fertilization rate. Data are expressed as mean9SEM. Error bars were not indicated when toosmall. At each sampling, means with different letter are significantly (PB0.01) different from each other.
pol (Fig. 2C). Sperm from treated with 25–100mg/kg had lower fertilizing abilities than controls,but these differences were not significant (P\0.05, Fig. 2D). Fertilization rates were signifi-cantly correlated with the sperm motility(r=0.79, n=47, PB0.001) (Fig. 3).
3.2. Effects of gossypol on plasma sex steroids
There were some trends among groups, asnoted below, in the changes of plasma levels ofthe four examined sex steroids, but no significantdifferences were observed (Fig. 4). There was aslight decrease in plasma E2 concentrations asso-ciated with the increase of the dose of gossypol(Fig. 4A). The low doses of gossypol (25 and 50mg/kg) induced an increase of the P levels,whereas the high doses (100 and 200 mg/kg)decreased those levels (Fig. 4B). Plasma levels ofT and 17,20bP were low compared to plasma E2and P in all groups of sea lamprey (Fig. 4C,D).
3.3. Effects of gossypol on the morphology of thetestis
At the end of the experiment, the morphologyof the testis in control and gossypol treated
Fig. 3. Relationship between the fertilization rate and thesperm motility in sea lamprey (y=36.3 log(x)+17.4; r=0.79,PB0.01).
J. Rinchard et al. / Toxicology Letters 111 (2000) 189–198 195
Fig. 4. Effects of gossypol on plasma sex steroids of male sea lamprey (n=3). (A) Estradiol-17b; (B) progesterone; (C) testosterone;(D) 17,20b-dihydroxy-4-pregnen-3-one. Data are expressed as mean9SEM.
groups was similar. In both groups, the testislobules (analogous to seminiferous tubules inmammals) contained large amounts of spermato-zoa. Moreover the structure of the spermatogenicepithelium from the gossypol-treated groups ap-peared to be unchanged compared to controlgroups.
3.4. In 6itro effect of gossypol on fertilizationrate of spermatozoa
The fertilization rate in control sperm sampleswithout gossypol (control 1) and with gossypolintroduced in vitro with sperm (control 2) reached56.5921.2% and 57.6922.0%, respectively. Thepreincubation of sperm with 100 mM gossypol for30 min produced a significant (PB0.01) decreaseof fertilization rate, 28.4910.7% (Fig. 5).
Fig. 5. In vitro effect of gossypol exposure on sea lampreyfertilization rate (n=3). Data are expressed as mean9SEM.Means with the same letter are not significantly (P\0.05)different from each other.
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4. Discussion
It has been previously reported that oral ad-ministration of gossypol at a dose of 12–40 mg/kg/day for 5–12 weeks induced infertility in therat, hamster, guinea pig, monkey and men (Shi etal., 1987). The degree of damage caused by gossy-pol on spermatogenesis was described to consis-tently correlate with the dose and duration ofadministration. In our study, sea lamprey receiveda single i.p. injection of gossypol acetic acid.Although this method is not commonly used inhigher vertebrates, oral administration is not pos-sible because during their spawning-run sea lam-preys do not feed. However, this sterilizationprocedure (single injection of chemosterilant) mayrequire the use of high concentrations of gossypol(100 and 200 mg/kg), which resulted in high mor-tality (41.7 and 84.6%, respectively).
Gossypol has been proven as a potent spermici-dal agent in mammals and invertebrates. Its ac-tion on sperm cells includes reduction in motility,and after prolonged exposure it causes distortionand lysis of the acrosome. In addition, gossypolinduced a flagellar lesion described as segmentalaplasia of the mitochondrial sheath (Porat, 1990;Randel et al., 1992). Gossypol can also induceapoptosis of germ cells (Teng, 1995). The mecha-nisms of sperm motility inhibition by gossypol inmammals are complex and include a peroxidebreakdown of membrane phospholipids (Forneset al., 1993), inhibition of sperm-specific lacticacid dehydrogenase (Stephens et al., 1983), and adecrease in sperm cAMP (Cowart et al., 1994).The results of our in vitro experiment indicatedthat gossypol is spermicidal toward sea lampreyspermatozoa (Fig. 5), similar to what has beenobserved in mammals (10 to 50 mg/ml; Lin et al.,1994; Zavos and Zarmakoupis-Zavos, 1996) andinvertebrates (50 mM; Burgos et al., 1997). How-ever, its effect in vivo was less evident under theconditions of our experiment. It is possible thatthe spermicidal effect of gossypol would be en-hanced by prolonged exposure of sperm to thiscompound, particularly during final stages ofspermatogenesis, i.e. at 2–3 months prior tospawning. However, we found that higher dosesof gossypol are lethal to sea lampreys. For this
reason, the usefulness of gossypol in a program ofsterilization of male lampreys needs to be recon-sidered, particularly in the light of different cyto-toxicity of different enantiomers, (+ ) and (− )isomers, and derivatives (Lin and Rikihisa, 1987;Wang et al., 1992). Some fish species may be lesssensitive to gossypol than others. For instance,Robinson and Tiersch (1995) found that long-term feeding of cottonseed meal resulted in signifi-cant accumulation of gossypol in the liver ofchannel catfish, Ictalurus punctatus. In addition,this diet increased sperm motility. It needs to beestablished if the fish reproductive system is moreresistant to gossypol toxicity than is themammalian.
The function and pattern of secretion ofsteroids in lampreys differ from those of teleostsand higher vertebrates (Sower et al., 1985). Thus,in male sea lamprey, the level of E2 increasedsignificantly during spermiation whereas the levelsof T remained extremely low. Linville et al. (1987)reported that the levels of P were significantlyhigher in lamprey males compared to femalesduring final maturational stages. However, therole of this steroid in male reproductive physiol-ogy is yet unknown.
In the present experiment, the levels of plasmasex steroids (P, E2 and T) in the control groupswere similar to those reported in male sea lampreyduring their reproductive season (Sower et al.,1985 and Linville et al., 1987). Although notsignificant, a slight decrease of plasma E2 with theincrease of the dose of gossypol injected wasobserved in male sea lampreys. This finding is inconcert with the observed antiandrogenic effect ofhigh dose of gossypol in rats. Thus, a significantdecrease of plasma T, the major steroid in malemammals associated with reproduction, has beenreported in rats fed gossypol acetic acid (Oko andHrudka, 1984; Lin et al., 1980) (30 mg/kg/day for30 days and 5 weeks, respectively) or implantedwith gossypol acetate pellets (Ahluwalia et al.,1986) (10–20 mg for 7 weeks). However, generalcytotoxic effects found in mammalian gonadswere not evident in poikilothermic lamprey. Incontrast, no changes in plasma T concentrationsamong control and gossypol-treated malecynomolgus monkeys (Macaca fascicularis) at a
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dose of 5 or 10 mg/kg/day for 6 months, weredetected by Shandilya et al. (1982). These resultssuggested that only high concentrations of gossy-pol can induce a decrease of the major steroids(E2 in sea lamprey or T in mammals) associatedwith spermiation. Demonstration of an inhibitoryeffect of gossypol on plasma sex steroids mayhave been difficult in the current study since onlya single injection of gossypol was used. The de-crease of the E2 levels observed in sea lampreycould be due to an action of gossypol on thehypothalamo–hypophysial axis as reported in ratby Ahluwalia et al. (1986). If the gossypol acts onthis axis, we should also probably observe somechanges in the concentration of other steroids.However, in our experiment, the levels of P, Tand 17,20bP in plasma of sea lamprey showed nodistinct response over the dose range of gossypolinjected.
In conclusion, a single injection of gossypol inmale sea lamprey interferes with some of thereproductive parameters examined in the currentstudy. An in vitro sperm toxicity assay confirmedantifertility action of gossypol in this species. Al-though high mortality rate precludes a single i.p.injection of gossypol as a favorable mode ofchemosterilization in the lamprey, the observa-tions made in the current study suggest that itmay be worthwhile to reassess the means of trans-fer of gossypol to reproductive tissues in the sealamprey.
Acknowledgements
This study was supported by the Great LakesFishery Commission, Ann Arbor, MI (projectNo. 20021327) and Ohio Sea Grant College Pro-gram, Columbus, OH (project No.NA46RG0482). We are indebted to Drs R.J.Butcher, R.E. Ciereszko, A. Fostier, G.D.Niswender and C.B. Schreck for their donation ofantiserum and radioactive hormones, to D.Gloeckner for his technical assistance. Salarieswere partly provided by State and Federal Fundsawarded to the Ohio Agriculture Research andDevelopment Center, Wooster, Ohio.
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