Ogawa A et al. (2011), Induction of ovulation in Xenopus without hCG i...

XB-ART-42721

Reprod Biol Endocrinol 2011 Jan 21;9:11. doi: 10.1186/1477-7827-9-11.

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Induction of ovulation in Xenopus without hCG injection: the effect of adding steroids into the aquatic environment.

Ogawa A , Dake J , Iwashina YK , Tokumoto T .

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The 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. Recently, 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. In 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. To conclude, we have successfully developed a novel method to induce ovulation in frogs but without the need for a potentially harmful injection strategy.

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Species referenced: Xenopus laevis
Genes referenced: dpys il12b rpsa tbx2 tes

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	Figure 1. The effect of various steroid hormones on the in vivo induction of Xenopus ovulation. The left column represents the results of induction by hCG injection. Annotations underneath each column indicate substance and concentration for the induction of ovulation as following: Et0.01, ethanol 0.01%; DHP, 17,20Î²-DHP; P, progesterone; Tes, testosterone; And, androstenedion; E, 17Î²-estradiol. Each compound was added to 0.1 M NaCl at a final concentration of 1 Î¼M except progesterone (additional high concentrations were examined at 10 Î¼M; P10, 20 Î¼M; P20, 40 Î¼M; P40). After 24 to 30 hr incubation, the numbers of ovulated eggs were counted as described in the Materials and Methods. Each value represents the mean of data from more than three different females. The number of ovulated frogs per number treated is indicated over each column. Vertical lines indicate standard deviation.
	Figure 2. The effect of steroid pre-treatment upon progesterone-induced ovulation. Frogs were pre-treated with ethanol (Et), estradiol (E) or progesterone (P) at the indicated concentration for a period of one day before induction by progesterone. The left column represents induction results in response to PMSG-hCG injection. After 24 to 30 hr incubation, the number of ovulated eggs was counted as described in the Materials and Methods. Annotations underneath each column indicate substance and concentration for both pre-treatment and the induction of ovulation. For example, Et0-P20 represents pre-treatment with 0% ethanol for one day followed by induction by a mixture of 20 Î¼M progesterone the following day. Each value represents the mean of data from more than three different females. The number of ovulated frogs per number treated is indicated over each column. Vertical lines indicate standard deviation.
	Figure 3. Induction of ovulation by a mixture of steroids. Frogs were treated with a mixture of estradiol (E) and progesterone (P) at the indicated concentration. The left column represents induction results in response to PMSG-hCG injection. After 24 to 30 hr incubation, the numbers of ovulated eggs were counted as described in the Materials and Methods. Each value represents the mean of data from more than three different females. The number of ovulated frogs per number treated is indicated over each column. Vertical lines indicate standard deviation.
	Figure 4. Synergistic effects of pre-treatment and a cocktail of steroids upon induction of ovulation. Frogs were pre-treated with or without (Ethanol alone) steroids, or at the indicated concentrations, for one day before ovulation induction by a mixture of steroids. Frogs were then treated with a mixture of estradiol and progesterone at the indicated concentrations. After 24 to 30 hr incubation, the numbers of ovulated eggs were counted as described in the Materials and Methods. Annotations underneath each column indicate substance and concentration for both pre-treatment and the induction of ovulation. For example, Et0-P20E10 represents pre-treatment with 0% ethanol for one day followed by induction by a mixture of 20 Î¼M progesterone and 10 Î¼M estradiol the following day. The left column represents the induction results in response to PMSG-hCG injection. Each value represents the mean of data from more than three different females. The number of ovulated frogs per number treated is indicated over each column. Vertical lines indicate standard deviation.
	Figure 5. Externally applied steroids induced natural spawning. (A) Artificial insemination was conducted for eggs squeezed during spawning induced by a mixture of steroids (Steroid). As a control, the fertilization rate of eggs ovulated by PMSG-hCG injection was examined (hCG). Fertilisation rate (in %) was assessed by counting the number of eggs dividing to the 4 cell stage following insemination. Each value represents the mean of data from three different females. Vertical lines indicate standard deviation. (B) A photograph of frogs developed from eggs ovulated by steroids.

References [+] :

Barr, Method for estimating the number of eggs laid by Xenopus laevis in response to the the injection of gonadotropin. 1967, Pubmed, Xenbase