Introduction
Bufo arenarum females exhibit a seasonal sexual cycle characterized by a breeding or reproductive period and a postovulatory one. The former comprises a preovulatory period, before oviposition (end of July–August), in which the gonad presents a great population of follicles with a maximum degree of development (Valdez Toledo & Pisano, Reference Valdez Toledo and Pisanó1980). At this time fully grown oocytes, arrested at the first meiotic division (Masui, Reference Masui, and Monroy1985), are ready to mature (de Romero et al., Reference de Romero, de Atenor and Legname1998) and almost prepared for ovulation, one of the most relevant reproductive events.
The ovulatory or spawning period, which completes the breeding period, takes place during the spring (September–November), at which time environmental conditions such as temperature, humidity and food supply are appropriate to insure normal embryonic development after the eggs have been fertilized.
The postovulatory period, characterized by the recovery of the reproductive system, comprises the early postovulatory phase, during the summer months (December–March), marked by follicular growth and development (folliculogenesis), and the late postovulatory period or hibernation. This period coincides with the fall and winter months (April–June) and is characterized by the completion of oogenesis and the acquisition of the maturation capacity of the oocyte. Toward the end of the winter, the reinitiation of the breeding period completes the cycle.
Under physiological conditions, ovulation is restricted to those oocytes that have completed both cytoplasmic and nuclear maturation. In this way, it is evident that maturation is a process temporally associated with ovulation. In our laboratory, cytoplasmic maturation is expressed through biochemical changes in the intermediary metabolism of carbohydrates that the oocytes exhibit, while nuclear maturation is represented by nuclear membrane dissolution and meiosis reinitiation (Fernández & Ramos, Reference Fernández, Ramos and Jamieson2003).
It is known that, in amphibians, the coordination of the different events in the reproductive activity is under the control of the hypothalamic–pituitary axis (Whittier & Crews, Reference Whittier, Crews, Norris and Jones1987). During the hibernation period, this axis presents a low degree of activity, probably due to a dopaminergic inhibition, as reported for Rana temporaria (Sotowska-Brochocka et al., Reference Sotowska-Brochocka, Martynska and Licht1994) or to the inhibitory action of the pineal gland (de Atenor et al., Reference de Atenor, de Romero, Brauckmann, Pisanó and Legname1994). At the end of hibernation the central nervous system inhibition decreases, causing the release of GnRH. Concurrently, pituitary contents and circulating gonadotropin levels begin to rise gradually (Itoh & Ishii, Reference Itoh and Ishii1990; Sotowska-Brochocka et al., Reference Sotowska-Brochocka, Martynska and Licht1992).
The gonadotropin surge is accompanied by an increase in gonadal steroid production (Polzonetti–Magni et al., 1998). The fully grown follicles mainly secrete progesterone and androgens (dihydrotestosterone and testosterone) (Fortune, Reference Fortune1983), whose high circulating levels characterize the preovulatory period (Medina et al., Reference Medina, Ramos, Crespo, González and Fernández2004).
Although nuclear maturation is a well known process, the complex mechanism involved in ovulation control has not been completely elucidated. Up to now, it is not clear how gonadotropins and sexual hormones regulate this ovarian function. Prostaglandins (PGs) have been demonstrated to play an important role in regulating gonadotropin induced ovulation in several vertebrates (Goetz et al., 1989; Jones et al., Reference Jones, Orlicky, Austin, Rand and Lopez1990). In addition, Espey (Reference Espey1980) reported that the rupture of the ovarian wall required for ovulation could be compared to the inflammatory response in which PGs are involved. The ovary of anuran amphibians such as Rana esculenta releases PGs (PGF2α and PGE2), which exhibit seasonal changes at the basal levels (Gobbetti & Zerani, Reference Gobbetti and Zerani1993) and exert various effects on reproductive functions at the ovarian and oviductal level.
Taking the above into account, the aim of this work was to investigate in vitro the Bufo arenarum ovulation in response to endocrine stimulation and to study the role of PGs in this process.
Materials and Methods
Animals
Sexually mature Bufo arenarum females were collected in the neighbourhood of San Miguel de Tucumán, Argentina. Animals were used immediately after capture or housed for brief periods in boxes with appropriate humidity at room temperature until use.
Ovarian samples
Ovaries, free of extraneous tissue, were rapidly removed from females under ether anaesthesia and placed in amphibian Ringer's solution, pH 7.4. The gonad was cut off in pieces of about 1 ± 0.1 g and washed in the same solution in order to eliminate blood and tissue remains. Each ovary piece was maintained in Ringer's solution containing streptomycin sulphate 0.50 g/l, penicillin G sodium 0.30 g/l and l-ascorbic acid 0.01 mM (hereafter referred to as incubation medium) until use.
Pituitary homogenate
Bufo arenarum pituitary glands were removed from females captured during the breeding period and a pool of them was homogenized in Ringer's solution, using a glass homogenizer, at a ratio of 1 gland/ml. Then, the homogenate was aliquoted and kept at –24 °C. Hereafter the homologous pituitary homogenate is referred to as HPH.
Ovulation induction
Ovulation was induced by incubating ovary pieces in the incubation medium with or without different doses of HPH and/or the hormones or drugs under study. The final incubation volume was 15 ml. After 12 or 24 h of 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 25 ± 1 °C in a shaking incubator agitated at 80 oscillations per min. As controls, ovarian pieces were incubated in medium alone under the same experimental conditions.
Hormones and chemicals
Progesterone, obtained from Sigma Chemical Co. was dissolved in ethanol at a ratio of 1 mg/ml and taken to the appropriate volume with Ringer's solution. PGF2α, PGE1, indomethacin and diclofenac sodium, supplied by Sigma Chemical Co., were dissolved in Ringer's solution before addition to the incubation medium.
Data analysis
Results, expressed as percentage of ovulated oocytes, are presented as mean ± SEM of the number of experiments performed with individual toads. The number of animals used and the duration of each treatment are indicated in the legend to each figure or table.
Statistical analysis of the values was carried out by Student's t-test and results were considered significantly different at p < 0.05.
Results
Preliminary results obtained in our laboratory indicated that the in vitro ovulatory response to HPH was dependent on seasonal variations (Ramos et al., Reference Ramos, Cisint, Medina, Crespo and Fernández2005). These data led us to evaluate the optimal HPH dose in the ovulatory response during the breeding period and the influence of progesterone and PGs as intermediates of the pituitary action in this process.
Figure 1 shows the data obtained when ovary pieces were incubated with HPH at doses of 0.01–0.1 gland/ml of incubation medium. Maximum percentage of ovulated oocytes was obtained with 0.03 gland/ml (p < 0.001), which is highly significant with respect to the lowest dose assayed. Higher HPH doses did not lead to an increase in the percentage of ovulated oocytes. The results obtained also show that during the time analysed there was a strong response within the first 12 h at all doses assayed. No ovulation was obtained without HPH in the incubation medium (control conditions).
Effect of homologous pituitary homogenate (HPH) on in vitro ovulation in Bufo arenarum. Ovary pieces obtained from animals captured during the breeding period were incubated with different doses of HPH. Data obtained from experiments using different animals (n = 7) represent the percentage mean ± SEM of ovulated oocytes after 12 and 24 h of treatment.
Taking into account that the processes of maturation and ovulation are related temporally, in a second set of experiments we analysed the effect of progesterone, the steroid physiologically responsible for inducing meiosis reinitiation or nuclear maturation, on Bufo arenarum ovulation. Figure 2 shows the data obtained when ovary pieces were treated with progesterone at doses of 1–8 μg/ml. Progesterone was able to induce ovulation in a dose-dependent manner at the times analysed. However, the highest percentage of ovulated oocytes obtained with 6 μg/ml of progesterone is low compared with the data obtained after treatment with HPH at 0.03 gland/ml dose (p < 0.001). It is important to note that incubation with 1 μg/ml of progesterone, which proved highly effective in inducing nuclear maturation, had little effect on the ovulatory response.
Ovulatory response in Bufo arenarum under progesterone treatment. Ovary pieces obtained during the breeding period were incubated with progesterone at different doses for 12 and 24 h. Data obtained by experiments performed with different animals (n = 6) represent the percentage mean ± SEM of ovulated oocytes.
In order to determine the relationship between maturation and ovulation, we analysed the presence of the germinal vesicle in ovulated oocytes after 12 h of treatment with HPH and progesterone. Data (not shown) demonstrated that all gametes released were mature oocytes.
For determining the effect of prostaglandins on the ovulatory process, PGF2α and PGE1 were tested.
The results obtained indicated that PGF2α per se was able to induce ovulation in a dose-dependent manner during the breeding period. The highest percentage of ovulated oocytes was observed with the 1–5 μg/ml dose (Fig. 3).
Effect of PGF2α on in vitro ovulation in Bufo arenarum. Ovary pieces were incubated with different doses of PGF2α. Results obtained from experiments using different animals (n = 6) are presented as the percentage mean ± SEM of ovulated oocytes after 12 and 24 h of treatment.
The effect of PGF2α on ovulation was also tested in ovaries from animals captured during all periods of the sexual cycle. The highest effect of PGF2α was visible in gonads obtained from animals captured during the breeding period (Fig. 4), while a marked decrease was observed during both the early and late post ovulatory periods.
Effect of PGF2 α on in vitro ovulation in Bufo arenarum throughout the sexual cycle. Ovary pieces obtained from animals captured monthly during 2 consecutive years were induced to ovulate with PGF2α 1.5 μg/ml. Each bar represents the percentage mean ± SEM of ovulated oocytes after 12 h of treatment (n = 4–7).
In contrast, the results found using PGE1 demonstrated that, in our experimental conditions, it did not affect the ovulation process at the doses assayed (0.25–5.0 μg/ml).
Experiments were carried out to determine whether PGF2α affected the response of HPH- or progesterone-induced ovulation. Ovary pieces were incubated in a medium containing different doses of PGF2α associated with HPH or progesterone, both at suboptimal doses. Under these conditions, a progressive increase in the percentage of ovulated oocytes was observed, a maximum being reached at the dose of 1.0 μg/ml of PGF2α (Fig. 5).
Effect of PGF2α on in vitro ovulation induced by HPH or progesterone in Bufo arenarum. Ovary pieces were incubated in a medium containing different doses of PGF2α associated with subliminal doses of HPH (0.01 gland/ml) or progesterone (2 μg/ml). Each point in the figure represents the percentage mean ± SEM of ovulated oocytes after 12 h of treatment. (n = 6).
The association of PGF2α with HPH had a stronger effect on ovulation (90% ± 12.4) than with progesterone (30% ± 11.2).
When we assayed the association of PGE1 with HPH an inhibition of 70% in the ovulatory response was observed (Fig. 6).
Inhibitory effect of PGE1 on in vitro HPH-induced ovulation in Bufo arenarum. Ovary pieces from individual animals (n = 4) were incubated in the presence of HPH (0.03 gland/ml), PGE1 (1.5 μg/ml) or HPH + PGE1. Data represent the percentage mean ± SEM of ovulated oocytes after 12 h of treatment.
In order to test if the effect of HPH was mediated by PG synthesis, ovary pieces were preincubated for 1 h in the presence of inhibitors of cyclooxygenase such as indomethacin or diclofenac sodium at different doses. After that time HPH (0.03 gland/ml) was added to the incubation medium. Table 1 shows that both inhibitors induced a significant decrease in the percentage of ovulated oocytes in a dose-dependent manner. The highest doses of inhibitors assayed blocked ovulation completely.
Effect of cyclooxygenase inhibitors on HPH-induced ovulation
Ovary pieces were preincubated (1 h) with indomethacin or diclofenac sodium and then HPH (0.03 gland/ml) was added to the incubation medium. Results obtained from experiments performed with different animals (n = 5) are expressed as mean ± SEM of ovulated oocytes after 12 h of treatment.
Discussion
Our results demonstrate that not only HPH, but also progesterone and PGF2α, acting at the ovarian level, were able to induce in vitro ovulation in Bufo arenarum.
The ovulatory response obtained by treatment with HPH from the pituitaries of females captured during the breeding period could be caused by the effect of gonadotropins, especially LH, which is considered the physiological inducer of ovulation in amphibians (Itoh & Ishii, Reference Itoh and Ishii1990; Kim et al., Reference Kim, Im, Choi, Ishii and Kwon1998). In agreement with the above, a preovulatory increase in gonadotropin circulating levels, as well as in pituitary content, has been reported for Rana temporaria (Sotowska-Brochocka et al., Reference Sotowska-Brochocka, Martynska and Licht1992).
At the beginning of the reproductive period, under the influence of gonadotropins, progesterone is secreted by follicle cells that surrounded the fully grown oocytes (Fortune, Reference Fortune1983; Medina et al., Reference Medina, Ramos, Crespo, González and Fernández2004; Morril et al., Reference Morril, Schatz, Kostellow and Bloch2006). It is known that progesterone induces oocyte nuclear maturation (Maller & Krebs, Reference Maller and Krebs1980; de Romero et al., Reference de Romero, de Atenor and Legname1998). Furthermore, our results show that progesterone also induces ovulation per se. Similar results were reported for Rana pipiens (Schuetz, Reference Schuetz1986) and Rana temporaria (Skoblina et al., Reference Skoblina, Kondrat'eva, Nikiforova and Huhtaniemi1997).
In amphibians the exact role of progesterone in this process has not been established yet. It is important to point out that progesterone-induced ovulation could be associated with the interruption of the microvilli and gap junction connections between the follicle cells and the oocytes that takes place previously during nuclear maturation (Schuetz, Reference Schuetz1986; Lessman & Kessel, Reference Lessman and Kessel1992; Ramos et al., Reference Ramos, Winik, Cisint, Crespo, Medina and Fernández1999).
Additional studies performed in mammals have also shown that progesterone is involved in the ovulatory process. Thus, as reported for rat ovary, the use of inhibitors of progesterone synthesis decreases the ovulation rate (Hellberg et al., Reference Hellberg, Larson, Olofsson, Brännström and Hedin1996). Likewise, progesterone receptor (PR)-knockout mice exhibited no ovulation in response to hCG due to an abnormal expression of the proteases involved in follicular rupture (Robker et al., Reference Robker, Russell, Espey, Lydon, O'Malley and Richards2000).
In relation to the quality of the ovulated oocytes obtained after 12 h treatment with HPH or progesterone it is important to note that 100% of them are mature oocytes. These results are in agreement with those obtained for Rana (Chang et al., Reference Chang, Kim, Im, Kang and Kwon1997). These oocytes, fertilizable in a high percentage, exhibited normal embryonic development.
The percentage of ovulatory response observed under progesterone treatment was lower than the ones obtained after incubation with HPH, suggesting that, for the ovulatory process, pituitary action requires not only the action of LH and progesterone secretion but also of other compounds. It is known that the high levels of gonadotropins before ovulation cause an increase in PGs production through the induction of cyclooxygenase, as reported for mammals (Sirois, Reference Sirois1994). This enzyme transforms arachidonic acid into cycle endoperoxide intermediators that are precursors of prostanoids (Murdoch et al., Reference Murdoch, Hansen and McPherson1993).
Experiments performed with PGF2α and PGE1 showed that only the former was able to induce ovulation in a dose dependent manner.
The analysis of the completely sexual cycle demonstrated that the action of PGF2α was significantly higher during the reproductive period than during the postovulatory period (p < 0.001). This positive effect of PGF2α agrees with data for Rana esculenta that report a marked increase in ovary content and plasma circulating level during the ovulatory phase (Gobetti & Zerani, 1993).
As with progesterone, the percentage of ovulation obtained with PGF2α alone was lower than the ones observed after treatment with HPH. This finding suggests that PGs alone are not enough for follicular rupture, which agrees with the results reported for mammals (Espey et al., Reference Espey, Tanaka, Stacy and Okamura1992). However, when ovary pieces were treated with PGF2α associated with HPH or progesterone, both at suboptimal doses, a significant increase (p < 0.001) in the percentage of ovulated oocytes was obtained with respect to progesterone, PGF2α or HPH alone. Our data suggest a synergistic potentiation effect of PGF2α. These results are in agreement with those reported for Rana temporaria, in which PGF2α stimulated in vitro ovulation and potentiated the effect of progesterone (Skoblina et al., Reference Skoblina, Kondrat'eva, Nikiforova and Huhtaniemi1997).
The functional roles of PGs in ovulation have not been fully defined yet. In rats, PGs increased the vascular permeability and caused the activation of collagenase enzymes required for digestion of the follicular wall (Reich et al., Reference Reich, Daphna-Iken, Chun, Popliker, Slager, Adelmann-Grill and Tsafriri1991). An additional mechanism was proposed by Gobetti & Zerani (1993), who suggested that, in Rana esculenta, PGF2α could favour ovulation through the increase in the release of ovarian corticosteroids, whose highest levels characterize the ovulatory phase.
The negative ovulatory response found when PGE1 was assayed could be due to an increase in the cAMP levels produced by this prostaglandin. It is known that the second messenger inhibits not only maturation but also strongly suppresses in vitro ovulation (Kwon et al., Reference Kwon, Chang, Yoo, Lee and Schuetz1992).
In the present work the participation of PGs as a component of the ovulatory process triggered by the pituitary gland was confirmed by using cyclooxygenase inhibitors in the incubation medium. In fact, indomethacin and sodium diclofenac significantly reduced the percentage of HPH-induced ovulation in a dose-dependent manner. These results are in agreement with those obtained with indomethacin in Rana (Skoblina et al., Reference Skoblina, Kondrat'eva, Nikiforova and Huhtaniemi1997) and mouse (Rose et al., Reference Rose, Hanssen and Kloosterboer1999).
In Bufo arenarum the inhibition of ovulation induced by HPH by the action of melatonin (de Atenor et al., Reference de Atenor, de Romero, Brauckmann, Pisanó and Legname1994) supports the above results. In this connection, it is known that the pineal hormone is a potent inhibitor of cyclooxygenase (Takamura & Kogo, Reference Takamura and Kogo1989; Hardeland et al., Reference Hardeland, Pandi-Perumai and Cardinali2006).
The present results suggest that several ovarian factors such as progesterone and PGF2α could be involved in the control of ovulation in Bufo arenarum. It should be noted that, although our results were obtained under in vitro conditions, the experimental model used could have a physiological relevance for the study and understanding of the ovulatory process and the factors involved in it.
Acknowledgements
The present work was supported by Consejo de Investigaciones de la Universidad Nacional de Tucumán (CIUNT).
