Zygote 13 (August), pp. 265268. C 2005 Cambridge University Press doi:10.

1017/S0967199405003345 Printed in the United Kingdom

Role of prolactin in nuclear maturation and ovulation in amphibian oocytes

In es Ramos, Susana Cisint, Marcela F. Medina, Claudia A. Crespo and Silvia N. Fern andez
Instituto Superior de Investigaciones Biologicas (INSIBIO), Departamento de Biolog a del Desarrollo, Universidad Nacional de Tucum an, Argentina Date submitted: 24.01.05. Date accepted: 29.04.05

Summary
The present study was undertaken to determine the effect of prolactin (Prl) on Bufo arenarum oocyte maturation and ovulation, two characteristic events of the breeding period, the stage of the sexual cycle in which gamete growth is complete. We observed that Prl, at the doses assayed, did not affect nuclear maturation per se. In addition, when follicles were pretreated with Prl and progesterone was later added to the medium as a physiological nuclear maturation inducer, the percentage of germinal vesicle breakdown obtained with the steroid was unaffected by Prl. The analysis of the in vitro ovulation process demonstrated that pituitary homologous homogenate (PHH) produced a dose- and month-dependent stimulating effect. The maximum percentage of ovulated oocytes was obtained from the end of July to October, the period in which oviposition naturally occurs. Prl by itself did not affect the ovulation process, but when both the hormone and PHH were present in the incubation medium, a signicant increase in ovulated oocytes was observed. The results suggest that Prl does not participate in oocyte maturation; however, its presence in the incubation medium would increase oocyte sensitivity to the action of the physiological ovulation inducers. Keywords: Amphibian, Nuclear maturation, Ovulation, Prl

Introduction
During the sexual cycle the gonad of Bufo arenarum females shows three clearly identiable stages: a postovulatory or gonadal recovery period, a quiescent or hibernation period and a breeding or reproductive 1980). During the rst period (Valdez Toledo & Pisano, two stages, the growth and development of the oocytes occur and the gametes, in the ovary, remain arrested at the prophase of the rst meiotic division (Masui, 1985). During the breeding period the ovary exhibits a maximum degree of development and is ready for ovulation, which frequently occurs in early spring. Under physiological conditions, ovulation is restricted to those fully grown oocytes that have undergone the maturation process.
All correspondence to: Dra. In es Ramos. Chacabuco 461, 4000 S. M. de Tucum an, Argentina. Fax: + 54 381 4247752560. e-mail: inramos@fbqf.unt.edu.ar Instituto Superior de Investigaciones Biologicas (INSIBIO), Departamento de Biolog a del Desarrollo, Universidad Nacional de Tucum an, Chacabuco 461, 4000 Tucum an, Argentina.

Oocyte maturation is associated with the events leading up to meiotic resumption and to the progression of the cycle from prophase I to metaphase II 1993). This process (Sadler & Maller, 1981; Liu & Patino, is morphologically characterized by the migration of the nuclear or germinal vesicle and the later breakdown of the nuclear envelope (Ramos et al., 1998). Both maturation and ovulation, temporally coupled, are known to be under the control of the hypothalamuspituitary axis (Whittier & Crews, 1987). Previous results from our laboratory suggest that, among the pituitary hormones, not only gonadotropins but also prolactin (Prl) are involved in the control of amphibian gonadal activity. The aim of this work was to analyse the inuence of Prl on amphibian oocyte maturation and ovulation.

Materials and methods

Animals Sexually mature Bufo arenarum females were freshly collected in the surroundings of San Miguel de

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Table 1 Effect of prolactin (Prl) on nuclear maturation Incubation time (h) 4 6 0 50 0 0 45 52 8 0 64 0 0 70 68 10 0 75 0 0 77 79 12 0 83 0 0 80 85 24 0 96 0 0 93 95

Tucum an. Animals were used immediately after capture or kept for brief periods in boxes with appropriate humidity at room temperature until use. Nuclear maturation induction Follicles, constituted by fully grown oocytes surrounded by one layer of follicle cells, were dissected from the ovary with watchmakers forceps. Samples of 100 follicles were distributed at random into wells containing 15 ml of amphibian Ringers solution. Nuclear maturation was induced by incubating follicles in the presence of progesterone (Sigma Chemical) dissolved in ethanol at a ratio of 1 mg/ml. Oocyte maturation was veried by dissecting follicles after xation for 48 h in Ancel and Vintembergers solution and the result was expressed as the percentage of nuclear or germinal vesicle breakdown (% GVBD). The GVBD was checked at different times throughout a 24 h period. Ovulation Ovulation was induced by incubating pieces of ovary (1 g) in 15 ml of Ringers solution in the presence of the pituitary homologous homogenate (PHH) at 0.033 and 0.066 pituitary equivalents/ml. These doses were obtained by homogenizing a pool of homologous pituitaries in Ringers solution at a ratio of 1 gland/ml. After a 12 or 24 h treatment the percentage of ovulation was scored by determining the number of ovulated oocytes/total number of oocytes present in the tissue sample 100. All incubations were performed in duplicate at 26 C in the presence of antibiotics (penicillin G sodium 0.3 g/l and streptomycin sulfate 0.5 g/l). Control samples were incubated in Ringers solution alone under the same experimental conditions. Statistical analysis was carried out using Students test, p < 0.05 being considered statistically signicant.

Control Progesterone (0.1 g/ml) Prl (0.1 g/ml) Prl (1 g/ml) Prl (0.1 g/ml) + Prog. 0.1 g/ml) Prl (1 g/ml) + Prog. 0.1 g/ml)

0 22 0 0 0 0

The results are expressed as the mean of the percentage of germinal vesicle breakdown (% GVBD) obtained from experiments carried out with different animals (n = 4).

Results and discussion

In order to analyse the inuence of Prl on nuclear maturation, follicles obtained from animals captured during the breeding period (end of July to October) were either incubated in the presence of only ovine Prl (Sigma Chemical) dissolved in Ringers solution or preincubated for 1 h with the hormone followed by the addition of 0.1 g/ml progesterone to the medium as a nuclear maturation inducer. Follicles incubated in Ringers solution were considered as controls. The data obtained (Table 1) indicate that Prl per se, at the doses assayed, did not affect nuclear maturation, nor did preincubation with the hormone followed by progesterone treatment. These results are probably due

to the prolonged time of meiosis and the lower activity of histone H1 and MAP-kinases induced by Prl, as reported for bovine oocytes (Torner et al., 2001). The in vitro ovulatory response under homologous pituitary stimulation was determined during the whole reproductive cycle. As shown in Fig. 1, the effect of PHH on the ovulation process is dose- and monthdependent. The maximum ovulation percentages at the lower dose used were observed during the breeding period, from the end of July until October. The strong sensitivity of oocytes to this ovulation inducer would be explained by the endogenous increase in the pituitary activity of Bufo arenarum females and the resulting high levels of circulating gonadotropins (de Romero et al., 1998). In addition, gonads display a population of fully grown oocytes which characteristically show both a high sensitivity to progesterone effect on maturation (de Romero et al., 1998) and the capacity to ovulate. From November, the beginning of the postovulatory period, ovulation percentages decreased. The lowest values were found during the summer months (DecemberMarch), concurrently with the period of gonadal recovery. A slow increase in the ovulatory response was observed from April to June, a time span which is almost coincident with the quiescent or hibernation period of the species. This low ovulatory response obtained with the higher dose used would be related to the presence in the gonad of medium-sized oocytes (late vitellogenic and auxocytosic) which are not physiologically competent to mature and ovulate. In order to determine the inuence of Prl on ovulation, pieces of ovary obtained from breeding animals were incubated in the presence of the hormone alone and in a medium containing Prl plus PHH at the more effective dose found during the reproductive period. The results in Fig. 2 show that Prl by itself, at the doses assayed, did not affect ovulation, although a signicant enhancement (p < 0.01) in ovulation

Prl in oocyte maturation and ovulation

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50
PHH 0.033 Equiv./ml PHH 0.066 Equiv./ml

% Ovulated Oocytes

40

30

20

10

0
Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Post-ovulatory Quiescent Breeding Post-ovulatory

Months Periods

Figure 1 Monthly variations in the in vitro ovulation in Bufo arenarum. Pieces of ovary were incubated in the presence of the pituitary homologous homogenate (PHH). Data represent the percentage mean SEM of ovulated oocytes after 24 h of treatment (n = 68).

80
PHH 12 h treatment PHH 24 h treatment Prl Prl + PHH 12 h treatment Prl + PHH 24 h treatment

60

% Ovulated Oocytes

40

20

0
PHH (0.033 Equiv/ml)

Prl (g/ml)

Figure 2 Prolactin effect on Bufo arenarum oocytes ovulation. Results are expressed as the percentage mean SEM of ovulated oocytes (n = 5) after 12 and 24 h of treatment. Prl, prolactin; PHH, pituitary homologous homogenate.

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Ishii, S., Yoneyama, H., Inoue, M., Yamamoto & Kikuyama, S. (1989). Changes in plasma and pituitary levels of prolactin in the toad, Bufo japonicus, throughout the year with special reference to the breeding migration. Gen. Comp. Endocrinol. 74, 36572. R. (1993). High-afnity binding of progesLiu, Z. & Patino, terone to the plasma membrane of Xenopus oocytes: characteristics of binding and hormonal and developmental control. Biol. Reprod. 49, 9808. Kikuyama, S., Yazawa, T., Abe, S., Yamamoto, K., Iwata, T., Hoshi, K., Hasunuma, I., Mosconi, G. & PolzonettiMagni, A.M. (2000). Newt prolactin and its involvement in reproduction. Can. J. Physiol. Pharmacol. 78, 98493. Masui, Y. (1985). Meiotic arrest in animal oocytes. In Biology of Fertilization, vol 1 (ed. C.B. Metz & A. Monroy), pp.189219. Orlando, FL: Academic Press. Ramos, I., Cisint, S., Alcaide, M. & Campos Casal, F. (1998). Morphological and cytochemical changes during nuclear maturation in Bufo arenarum oocytes. Biocell 22, 16775. Sadler, S.E. & Maller, J.L. (1981). Progesterone inhibits adenylate cyclase in Xenopus oocytes: action on the guanine nucleotide regulatory protein. J. Biol. Chem. 256, 636873. Torner, H., Kubelka, M., Helei, B., Tomek, W., Aim, H., Kuzmina, T. & Guiard, V. (2001). Dynamics of meiosis and protein kinase activities in bovine oocytes correlated to prolactin treatment and follicle size. Theriogenology 55, 88599. A. (1980). Fases de la ovog Valdez Toledo, C. & Pisano, enesis en Bufo arenarum. Reproducci on 4, 31530. Whittier, J.M. & Crews, D. (1987). Seasonal reproduction: patterns and control. In Hormone and Reproduction in Fishes, Amphibians and Reptiles (ed. D.O. Norris & R.E. Jones), pp. 385409. New York: Plenum Press.

percentages was obtained when Prl was added simultaneously with PHH to the incubation medium compared with the effect of PHH alone. The increase in ovulated oocytes suggests a direct sensitizing effect of Prl on the ovary and would be related to the endogenous circulating levels of the hormone. In fact, the maximum Prl concentration was detected during the breeding period as reported for Bufo japonicus (Ishii et al., 1989). In addition, the role of Prl in ovulation is supported by its effect on the secretion of the oviductal jelly that surrounds the ovulated oocytes during their transit through the oviduct, as reported for amphibian urodeles (Kikuyama et al., 2000). In conclusion, our results suggest that Prl does not participate in oocyte maturation; however, the hormone may play a role in inducing the development of ovarian sensitivity to pituitary ovulatory hormones.

Acknowledgements
The present work was supported by Consejo de Investigaciones de la Universidad Nacional de Tucum an (CIUNT). References
de Romero, I.R., de Atenor, M.B. & Legname, A.H. (1998). Nuclear maturation inhibitors in Bufo arenarum oocytes. Biocell 22, 2734.

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