The Use of Hormone Antagonists to Inhibit Reproduction in the Lizard, Eublepharis macularis
Dale F. DeNardo DVM, Gabrielle Helmenski. Dept of Biology, Arizona State University, Tempe AZ USA
KEY WORDS: leopard gecko, Eublepharis macularius, tamoxifen, indomethacin, reproduction, inhibition.
While a natural phenomenon, reproductive activity (e.g., vitellogenesis, ovulation, and oviposition) is associated with physiological and behavioral changes that owners would prefer not to experience. Such changes include extended inappetence, weight loss, muscle atrophy, and increased activity (Lloyd, 1990, DeNardo 1996, Stahl, 1997). Furthermore, because the captive environment usually does not duplicate the complexity of the natural nesting environment, gravid females can experience dystocia because of this unwanted and unproductive reproductive activity. While the green iguana is the most popular lizard that experiences "spontaneous" reproductive cycling, such cycling is also exhibited by other reptile taxa (e.g., chameleons, tortoises) and it has resulted in clinical disease and even death in captive individuals of these species also (DeNardo. 1996).
To date, treatment options are limited with owners either enduring this annual process along with the associated potential health risk or having their pet ovariectomized, which is costly and irreversible. However, current knowledge of the endocrine control of the reproductive cycle of reptiles makes it theoretically possible to inhibit hormones critical to vitellogenesis and ovulation and therefore inhibit reproductive activity inexpensively and temporarily, thereby maintaining the potential for reproduction in the future.
A suite of hormones regulates the female reproductive system. Estradiol is the primary stimulus for yolk synthesis in the reptilian liver (Yaron and Widzer, 1978, Ho, et al, 1982), while prostaglandins appear to play a vital role in follicular development and ovulation. In the Italian wall lizard, Podarcis sicula, prostaglandin E (PGE) is important in starting and sustaining oocyte vitellogenic development (via mediation of estradiol) and prostaglandin F2a (PGF2a) induces ovulation (via mediation of progesterone) (Gobetti, et al, 1993). While not the only hormones involved, both estrogens and prostaglandins have substantial roles in regulating female reproductive activity, and, therefore, chemical inhibition of either of these hormones should have a dramatic effect on reproduction.
Tamoxifen is a widely used inhibitor of estrogen binding and has been shown to inhibit estrogen-induced vitellogenesis in birds (Gschwendt, et al, 1982), amphibians (Riegel, et al, 1986), and fish (Le Menn, et al, 1980). It is widely used in studies to ascertain the estrogenic potency of chemicals (Pelissero, et al, 1993). Indomethacin has been shown to inhibit ovarian prostaglandin synthesis in mammals (Espy, et al, 1982), birds (Shimada, et al, 1986), fish (Goetz, 1983), and reptiles (Guillette, et al, 1990). In lizards, indomethacin delays both parturition (Guillette, et al, 1991) and follicle stimulating hormone-induced ovulation (Jones, et al, 1990), but its effect on follicular growth is unknown. Due to their effective inhibition of key reproductive hormones, we hypothesized that slow-release implants of tamoxifen and indomethacin would inhibit reproductive activity in lizards effectively and reversibly, if so, these chemical treatments could provide a clinically valuable alternative to the limited options currently available for assuring reproductive inactivity (e.g., ovariosalpingectomy).
Study animal and
Animals were individually housed at Arizona State University in 51 cm x 20 cm translucent plastic drawer cages (Freedom Breeder, Danville, CA). During the majority of the year, room temperature was 25 ± 1° C (77 ± 1.8° F) and sub-surface supplemental heat was provided using Mylar heat tape (Flex Watt, West Wareham, MA), thereby providing the lizards with a thermogradient of 22 - 36° C (72 - 97° F). Room lights were set to provide a 12:12 photoperiod. Paper towels were used for substrate and a deli cup containing moist vermiculite was provided as a retreat and an ovipositorium. Lizards were fed five to ten crickets dusted with calcium carbonate two to three times weekly. Amount and frequency varied depending on season. Cages were misted twice weekly.
In January, the animal room temperature was dropped to 15° C (59° F) and the supplemental heat source and lights were turned off for approximately one month. This cooling period leads to a stimulation of reproductive activity upon the return to warmer temperatures.
Phase 1: Inhibition of the Onset of Reproductive Activity
Four days after implantation, we began introducing a male into the female cages. Each of the 12 males was haphazardly rotated through the colony, spending 24 to 48 hr with a given female before rotating to another female. Females were exposed to at least one male every week for the duration of the reproductive season. Females were weighed and visually inspected for reproductive activity weekly. Both follicles and eggs are visible and distinguishable through the semi-transparent abdominal skin. When follicles were present, their diameters were measured using calipers. Gravid females were checked daily for the presence of eggs. Upon oviposition, date of oviposition, female mass, egg number, egg mass, and egg quality were recorded. Data was compared between groups using unpaired T-test (StatView, SAS Institute, Cary, NC).
Phase 2: Inhibition of Active Vitellogenesis
Phase 3: Reversibility of Reproductive Inhibition
Beginning 120 days post-implantation, only seven control females were identified with large follicles. Four of these females received a tamoxifen pellet and three received a sham surgery. Tamoxifen was ineffective at arresting follicular development as three of the four tamoxifen treated females and two of the three control females proceeded to ovulate and oviposit eggs resulting from the follicles that were present at the time of surgery (P = 0.846).
One of the ten tamoxifen treated females died during the following cooling period leaving nine tamoxifen treated females for phase three of the experiment. Since this death occurred during the cooling period, the death was not discovered soon enough to permit an effective necropsy. However, because of the timing of the death was more than six months beyond the expected life of the implant, it is unlikely that this death was related to the treatment. Five control females from the first year were randomly selected to remain as controls for phase three. No difference between the groups was detectable, as eight of the nine tamoxifen treated females successfully laid at least one clutch (mean = 3.3 total clutches, 2.6 fertile clutches) during the second season, while all five control females laid at least one clutch (mean = 3.0 total clutches, 2.2 fertile clutches; P = .790 and P = .708 for total clutches and fertile clutches, respectively).
The clinical value of tamoxifen remains in question, but results reported here make it a promising therapeutic option for the temporary inhibition of reproduction. Tamoxifen completely inhibited vitellogenesis during the pellet life span and inhibited successful reproduction for the entire active season.
Furthermore, females effectively treated with tamoxifen one year had normal reproductive effort the following year when they were untreated. Combined, these results suggest that tamoxifen could be used clinically to provide effective but reversible reproductive inhibition.
The failure of tamoxifen to arrest late-stage follicular development is understandable. As a blocker of estrogen binding, tamoxifen should be effective at inhibiting those reproductive processes stimulated by circulating estrogen. These include female receptivity, yolk synthesis by the liver, and yolk deposition into the follicles. It is unknown whether estrogen has any role in follicular maturation and ovulation, and our results suggest that it does not.
Based on our data, tamoxifen would be an effective inhibitor of reproduction only when given prior to or during early follicular growth. In species that respond predictably to seasonal changes (e.g., reduced ambient temperatures), the timing of tamoxifen treatment could be easily determined. However, tamoxifen may be of little value in individuals or species where the onset of reproductive activity is variable.
While, like ovariectomy, tamoxifen implants require anesthesia and entrance into the coelomic cavity, the invasiveness of the procedures are quite different. Ovariectomy requires a surgical plane of anesthesia and extensive manipulation of internal tissues. Contrarily, the implantation procedure can be done using a local anesthetic, a 3 mm incision, simple insertion of the pellet into the coelomic cavity, and a single stitch for wound closure. Total surgical time for pellet implant is approximately two minutes. Therefore, the use of tamoxifen implants potentially provides a less invasive and less time-consuming alternative to ovariectomy. While tamoxifen treatment is not the obvious clinical solution for all cases where the goal is reproductive inhibition, it may be a valuable option in many cases, especially those where only temporary inhibition is desired. Further studies, including trials in more clinically relevant species (e.g., green iguanas, tortoises) are justified.
Arky R. 1999. Physicians Desk Reference. Medical Economics Co, Montvale, NJ.
DeNardo DF. 1996. Dystocias. In Mader DR (ed): Reptile Medicine and Surgery. WB Saunders, Philadelphia, PA: 370-374.
Espy LL, Stein VI, Dumitrescu J. 1982. Survey of antiinflammatory agents and related drugs as inhibitors of ovulation in the rabbit. Fertil Steril, 38:238-247.
Gobetti A, Zerani M, DiFiori MM, Botte V. 1993. Prostaglandins and sex steroids from reptilian (Podarcis sicula sicula) ovarian follicles at different developmental stages. Zool Sci, 10:321-328.
Goetz FW. 1983. Hormonal control of oocyte final maturation and ovulation in fishes. In Hoar WS, Randall DJ, Donaldson EM (eds): Fish Pathology, vol IXB. Academic Press, New York: 117-170.
Gschwendt M, Rincke G, Schuster T. 1982. The estrogen-induced vitellogenin synthesis in chicken liver after estrogen withdrawal or antiestrogen treatment. Mol Cell Endocrinol, 26:231-42.
Guillette U, Gross TS, Matter JH, Palmer BD. 1990. Arginine vasotocin-induced prostaglandin synthesis in vitro by the reproductive tract of the viviparous lizard Sceloporus jarrovi. Prostaglandins, 39:39-51.
Guillette U, Masson GR, DeMarco V. 1991. Effects of prostaglandin-F2-alpha prostaglandin-E2 and arachidonic-acid on the induction of oviposition in vivo and in vitro in oviparous lizards. Prostaglandins, 42:(6)533-540.
Henkel FW, Schmidt W. 1995. Geckoes: Biology, Husbandry, and Reproduction. Krieger Publishing Co. Malabar, FL.
Ho SM, Kleis S, McPherson R, Heisermann GJ, Callard IF. 1982. Regulation of vitellogenesis in reptiles. Herpetologica, 38:40-50.
Jones RE, Orlicky DJ, Austin HB, Rand MS. Lopez KH. 1990. Indomethacin inhibits ovarian PGE secretion and gonadotropin-induced ovulation in a reptile (Anolis carolinensis). J Exp Zool. 255 :57-62.
Le Menn F, Rochefort H, Garcia M. 1980. Effect of androgen mediated by the estrogen receptor of fish liver: vitellogenin accumulation. Steroids, 35:315-28.
Pelissero C, Flouriot G, Foucher JL, Bennetau B, Dunogues J, Le Gac F, Sumpter JP. 1993. Vitellogenin synthesis in cultured hepatocytes: an in vitro test for the estrogenic potency of chemicals. J Steroid Biochem, 44:263-72.
Riegel AT, Jordan VC, Bain RR, Schoenberg DR. 1986. Effects of antiestrogens on the induction of vitellogenin and its mRNA in Xenopus laevis. J Steroid Biochem, 24:1141-9.
Shimada K, Olson DM, Etches RJ. 1986. The effect of indomethacin on ovarian prostaglandin release in hens. Biol Reprod, 35:1147-1153.
Stahl SJ. 1997. Captive management, breeding, and common medical problems of the veiled chameleon (Chamaeleo calpytratus). Proc ARAV, 29-40.
Whittier JM. Crews D. 1986. Ovarian development in red-sided garter snakes. Thamnophis sirtalis parietalis: relationship to mating. Gen Comp Endocr, 61:5-12.
Yaron Z, Widzer L. 1978. The control of vitellogenesis by ovarian hormones in the lizard Xantusia vigi!is. Comp Biochem Physiol, 60:279-284.
You don't need to be a vet to join the Association of Reptilian and Amphibian Veterinarians, nor need to be a member to attend their annual conferences or buy a copy of their conference proceedings. For more information, please visit their website at www.arav.org.
Need to update a veterinary or herp society/rescue listing?
Can't find a vet on my site? Check out these other sites.
|Clean/Disinfect||Green Iguanas & Cyclura||Kids||Prey||Veterinarians|
|Home||About Melissa Kaplan||CND||Lyme Disease||Zoonoses|
|Help Support This Site||Emergency Preparedness|
© 1994-2014 Melissa Kaplan or as otherwise noted by other authors of articles on this site