Induction of ovulation in Xenopuswithout hCG injection: the effect of adding steroids into the aquatic environment

Abstract

BackgroundThe African clawed frog, Xenopus laevis, is widely used in studies of oogenesis, meiotic cell cycle and early embryonic development. However, in order to perform such studies, eggs are normally collected after the injection of hCG into the dorsal lymph sac of fully-grown female frogs following pre-injection of PMSF. Although this protocol is established and used as standard laboratory approach, there are some concerns over whether the injections could cause the transmission of deleterious microorganisms. Moreover, these injection protocols require a competent skilled worker to carry out the procedure efficiently.MethodsRecently, we established a novel method to induce fish ovulation by simply adding the natural maturation-inducing hormone of teleosts, 17 alpha, 20 beta-dihydroxy-4-pregnen-3-one (17,20 beta-DHP), into the surrounding water. In the present study, we demonstrate how we can induce ovulation in frogs using the same methodology.ResultsIn frogs, progesterone was effective in the induction of oocyte maturation in vitro. We then examined the ability of progesterone to induce ovulation in frogs. However treatment of frogs with progesterone alone only occasionally induced ovulation in vivo. The number of oocytes and the frequency of ovulation were significantly lower than that induced by hCG-injection. Thus, conditions were improved by using a combination of progesterone with estradiol and by pre-treating frogs with low concentrations of progesterone or estradiol. Finally, we established an efficient means of inducing ovulation in frogs which involved pre-treatment of frogs with salt solution followed by a mixture of estradiol and progesterone at high concentration. The frequency and numbers of oocytes obtained were identical to those resulting from PMSG-hCG induction. Fertilization rate of eggs ovulated by the new treatment method was comparable to eggs obtained by hCG-injection and juveniles developed normally.ConclusionsTo conclude, we have successfully developed a novel method to induce ovulation in frogs but without the need for a potentially harmful injection strategy.

Reproductive Techniques for Ovarian Monitoring and Control in Amphibians

“…Because the mechanisms controlling reproduction in vertebrates are highly conserved, there are a number of hormones, neuropeptides and commercially available drugs used therapeutically in other taxonomic groups that can also be used reliably in a number of amphibian species ( Table 1). Gonadotropin releasing hormone (GnRH) and Human chorionic gonadotropin (hCG) (or variations thereof, i.e., PMSG and eCG) 17,18 , either individually or in combination, have been used extensively in amphibian captive breeding programs including: the Southern Rocky Mountain boreal (Anaxyrus boreas boreas) 4,19,20 ; the toad, Dusky Gopher frog, Rana sevosa (Langhorne et al, unpublished) 7 ; the Gulf Coast Waterdog, Necturus beyeri 20 ; Wyoming toad, Anaxyrus baxteri 18 ; the bullfrog, Rana catesbiana 21 ; the American toad, Anaxyrus americanus 22 ; the grass frog, Lymnodyaster tasmaniensis 23 ; the Coqui, Eleutherodactylus coqui 24 ; the Xenopus, Xenopus laevis 25 ; the Gunther's toadlet, Pseduophryne guentheri 26 ; the Northern Leopard frog, Lithobates pipiens; the Argentinian Horned-frog, Ceratophrys ornate; the Cranwell's horned-frog, C. cranwelli; the American ground-frog, Odontophrynus americanus 27 ; and the fire salamander (Salamandra) 228 . Steroid hormones, like progesterone (P4), are less commonly reported but have demonstrated good efficacy in eliciting ovulation and oviposition in some species of anurans 16,18,29 .…”

Aqueous exposure to the progestin, levonorgestrel, alters anal fin development and reproductive behavior in the eastern mosquitofish (Gambusia holbrooki)

“…Studies examining the exposure effects of gestagens on reptiles have found evidence of reduced steroid hormone synthesis and yolk development in alligators (Austin, 1991) and altered sex ratios in red-eared slider turtles (Wibbels and Crews, 1995). Changes in gonadal structure, final maturation of eggs, metamorphosis, and reproductive behavior have also been observed in African clawed frogs (Hoffmann and Kloas, 2012;Kvarnryd et al, 2011;Lorenz et al, 2011;Ogawa et al, 2011). In several species of fish, gestagens decrease fecundity, increase ovarian apoptosis, and alter development of secondary sexual characteristics (Paulos et al, 2010;Runnalls et al, 2013;Svensson et al, 2013;Zeilinger et al, 2009;Zucchi et al, 2012).…”

Aromatase expression in Xenopus oocytes: a three cell-type model for the ovarian estradiol synthesis

“…Yolk will provide nutritional reserves necessary for embryo development. More recently, E 2 was also shown to enhance progesterone-induced ovulation, possibly through a stimulatory effect on gonadotropin synthesis (Ogawa et al 2011). In addition to E 2 endocrine effects, E 2 may have autocrine and/or paracrine effects, as E 2 inhibits follicle-enclosed oocyte maturation through inhibition of maturation-inducing steroid (MIS) synthesizing enzymes in vitellogenic species such as Rana pipiens (Schuetz 1972, Spiegel et al 1978, Lin & Schuetz 1983 or Oncorhynchus mykiss ( Jalabert et al 1984).…”