87, 163-170 (1992)

A Possible Involvement of Prostaglandin’ F,, (PGF,,) in Rana esculenta Ovulation: Effects of Mammalian Gonadotropin-Releasin Hormone on in Vitro PGF,, and I’-/@Estradiol Production from Ovar and Oviduct ANNA Department

of Molecular,




and Animal Biology, University 62032 Camerino MC, Italy

ZERANI of Camerino,

via F. Camerini


Accepted November 5, 1991 The present work studied the PGF,, and 17P-estradiol plasma levels during ovulation, and the in vitro effects of mammalian gonadotropin-releasing hormone (mGnRH) on ovarian and oviductal production of prostaglandin F,, (PGF,,) and 17P-estradiol during four different stages of the annual sexual cycle in water frog, Rana esculenta. Plasma levels of PGF,, increased in ovulating frogs, with respect to preovulatory and postovulatory levels, while estradiol did not change. In addition, mGnRH increased PGF,, and 17@-estradiol in the incubation media of ovaries taken during the recovery stage. Moreover, mGnRH increased PGF,, in incubation media of oviducts collected during the reproductive stage. These findings suggest that PGF,, could be involved in the control of egg deposition in the female R. @SCUkFZk2.

0 1992 Academic

Press, Inc.

Gonadotropin-releasing hormone (GnRH) and/or related molecules are localized in extrahypothalamic areas of the central and peripheral nervous system and in several nonneural tissues (Sherwood, 1986; King and Millar, 1987). In mammals, ovarian GnRH binding sites occur in granulosa, theta, and luteal cells at all stages of differentiation (Clayton et al., 1979; Jones et al., 1980; Pieper et al., 1981; Pelletier et al., 1982). Locally synthesized mammalian GnRH (mGnRH) and/or mGnRH-like molecules, moreover, are believed to act as paracrine and/or autocrine hormones in ovarian regulation, including steroidogenesis and prostaglandin synthesis (Clark, 1982; Gore-Langton and Armstrong, 1988). Prostaglandins, however, do not seem to mediate GnRH direct effects on steroidogenesis (Zilberstein et al., 1984; Naor, 1990). Mammalian GnRH is present in hypothalamus and other brain tissues of several species of amphibians (King and Millar,

1987); more recently, GnRH-like snbstances were found in the testis and ovary of the lizard Podarcis s. sicula (Ciarcia and Botte, 1990) and an immunoreactive, mGnRH-like substance was detected in hypothalamus and testis of the frog Rana esculenta (Cariello et al., 1989). In viva, this peptide induces an increase in prostaglandin F,, (PGF,,) plasma level in the water frog, R. esculenta. In this species, therefore, mGnRH-dependent PGF*,-secreting tissues exist (Gobbetti et al., 199fa). Recent studies support this assumption, since cultured frog interrenals release PGF, in response to mGnRH (Gobbetti and Zerani, 1991). In female frogs, reproductive organs like the ovary and oviduct could have sensitivity to mGnRH as was found in interrenals, since plasma PGF,, levels change in relation to the sexual cycle (Gobbetti et al., 1990). To ‘test this hypothesis, we studied the effects of mGnRH on PGF,, and I?‘& estradiol release in vitro from the ovary and 163 0016-6480/92 $4.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.



oviduct of R. esculenta obtained at several stages of the annual sexual cycle; in addition, the involvement of PGF,, and 17pestradiol during ovulation was observed. MATERIALS


Animals. The annual sexual cycle of the R. esculenta population living in the mountain pond of Colfiorito (Umbria, Italy, 870 m above sea level) consists of four major stages. Frogs breed in May (reproductive stage), when the temperature increases. In the summer (postreproductive stage), breeding is interrupted. The recrudescence of sex organs (recovery stage) starts at the end of the summer, when the temperature decreases, and stops at the end of autumn. The animals disappear in ground shelters to avoid the very cold winter months until the next spring. In fact, the animals are particularly scarce and inactive from December to February. At the beginning of spring, the frogs return to the pond (prereproductive stage). Adult female water frogs, R. esculenta, were collected in the mountain pond of Colfiorito during four stages of their annual sexual cycle: prereproduction (April), reproduction (May), postreproduction (June), and recovery (October) stages in 1989 and 1990. The animals were kept in laboratory aquaria under natural photothermal conditions and fed ad libitum on meal worms and beef liver for 1 week. Animals were sacrificed between 9:30 and lo:30 AM to obtain ovaries and oviducts. Samples of ovary and oviduct were placed in Bouin’s fluid for histological examination. In May 1989 and 1990, 10 preovulatory, 10 ovulating, and 10 postovulatory frogs were captured in the field and at once anesthetized (7 mitt) with S-aminobenzoic acid ethyl ester (tricaine; Sigma). The animals were bled through a heparinized microtube inserted in the heart and blood samples were collected into chilled tubes containing acetylsalicylic acid (Aspirin, Sigma) and EDTA (5 pg and 7 p&ml of blood, respectively) (Gobbetti et al., 1990). After centrifugation, plasma samples were kept at - 70” until use. In vitro incubation. From frogs sacrificed by decapitation, ovaries and oviducts were rapidly excised and placed in cold Dulbecco’s modified Eagle’s medium (DME; Sigma) containing 10 miW Hepes, 0.1 mg penicillin G/ml, and 0.1 mg streptomycin/ml (Gobbetti and Zerani, 1991). Ovaries or oviducts obtained from one animal were divided into equally sized fragments, pooled, and equally distributed in eight incubation wells each containing 900 pl of incubation medium. A multiwell tissue culture plate (Becton-Dickinson) was utilized. In each incubation set, four wells received 100 ul of DME plus mGnRH (Sigma; 2.5 x lo-’ M); the other four wells received 100 (~1of DME alane (control). Culture

AND ZERANI plates were wrapped in aluminum foil and put in a shaking water bath (W), set at 30 revolutions/min. Two wells, one treated and one control, were removed after 30, 60, 120, and 180 min of incubation. The incubation medium samples were stored immediately at - 70” to be later used for PGF,, and 17B-estradiol determination. Tissues were homogenized rapidly in amphibian saline, and protein content was determined by the Protein Assay method (Bio-Rad). In addition, parallel incubation sets, without ovary or oviduct, were made with medium alone or with medium plus mGnRH. For each of the four stages examined, tests on 10 parallel incubation sets were carried out for ovary or oviduct. One female was used for each incubation set. Preliminary evidence led us to choose the incubation conditions, and in the dose-response rela-

k3 10-d mGnRH



1. In vitro effects of different doses of mGnRH on PGF,, and 17B-estradiol release from Rana esculenta ovary (A, B) and oviduct (C), incubated for 120 mitt, during prereproductive (W), reproductive (El), ), and recovery (IS) stages. Statistical analysis: each mean refers to 7 determinations 2 SD. One-way ANOVA: (A) recovery stage, F(5,36) = 26.12, P < 0.01; (B) recovery stage, Fo361 = 31.81, P < 0.01; (C) reproductive stage, Fo,36) = 32.33, P < 0.01. Duncan’s multiple range test: *P < 0.01 vs mGnRH 0. FIG.





each hormone determination were submitted to an analysis of variance (ANOVA) followed by Duncan’s multiple range test (Duncan, 1955; Sokal and Rohlf, 1981).

tionship study, we found that 2.5 x lo-’ M mGnRH was the minimally effective dose (Fig. 1). PCF,, and 17/3-estrudiul determination. A radioimmunological method (RIA) was used for the measurement of the PGF,, content of incubation media (Gobbetti and Zerani, 1991) and plasma samples (Gobbetti et al., 1990, 1991a). The 17&estradiol content of ovarian incubation media and plasma samples was determined by RIA according to previously reported methods (d’Istria et al., 1974; Gobbetti and Zerani, 1991). The following sensitivities were recorded: PGF,, 18 pg (intraassay variability, 5%; interassay variability, 8%); 17p-estradiol, 7 pg (intraassay variability, 4%; interassay variability, 9%). The two antisera were provided by Dr. G. F. Bolelli (CNR-Physiopathology of Reproduction Service, University of Bologna, Italy) . Tritiated PGF,, and 17E-estradiol were purchased from Amersham International (England), nonradioactive PGF,, and 17B-estradiol from Sigma. Statisfical analysis. For each year, data relative to

RESULTS Histological sections of ovary and oviduct showed that the animals used underwent a normal cycle, as described in Rastogi et al. (1983). In vitro incubation. Ovarian PGF, release was lower during the postreproduction (P < 0.01) with respect to the other stages (Fig. 2). mGnRH significantly increased PGF,, release from ovary of frogs obtained during the recovery stage at all incubation times (P < 0.01); it was ineffective








ll 180

a 30




INCUBATION TIMES (minutes) FIG. 2. PGF,, production by Rana esculenta ovary in vitro incubated with medium alone (m) or with medium plus mGnRH (2.5 x lo-’ M) (Ci), in four different stages (A, prereproductive; B, reproductive; C, postreproductive; D, recovery) of the annual sexual cycle, during 1989 and 1990, Statistical analysis: each mean refers to 10 determinations -C SD. Two-way ANOVA (treatments x stages interaction F values): Year 1989, Fo72j = 15.83 (30 min), 18.01(60min), 20.07 (120 min), 23.12 (180 min), P < 0.01; Year 1990, Fo72j = 15.64 (30 min), 18.89 (60 min), 19.10 (120 min), 24.19 (180 mm), P < 0.01. Duncan’s multiple range test: *P < 0.01 vs same time medium alone; **P < 0.01 vs prereproduction, reproduction, and recovery medium alone.



when tested on gonads obtained in other stages (Fig. 2). In oviductal incubates, PGF,, release was higher during the reproduction stage (P < 0.01) with respect to the other stages (Fig. 3). mGnRH significantly increased PGF,, release only from oviduct obtained during the reproductive stage at 120 and 180 min of incubation (P < 0.01). mGnRH, on the contrary, slightly inhibited PGF,, release from oviducts obtained during the prereproductive and recovery stages at 60, 120, and 180 min of incubation (P < 0.05) (Fig. 3). Release of 17lSestradiol from incubated ovary was lower in the postreproduction stage (P < 0.01) with respect to the other yYEAR:1989-,

0 !x


stages (Fig. 4). mGnRH induced a significant hormone release only from ovary obtained during the recovery stage at all incubation times (P < 0.01) (Fig. 4). Field experiment. Plasma levels of PGF,, were greater in ovulating frogs, with respect to preovulatory and postovulatory frogs (P < O.Ol), while plasma estradiol levels did not change during the three different phases of the reproductive stage (Fig. 5). DISCUSSION

Our in vitro tests provide evidence of a direct influence of mGnRH on R. escufenta ovary, since the hormone induces both PGF,, and 17@estradiol production, alyYEAR:1990-,

3oo] D





INCUBATION TIMES (minutes) FIG. 3. PGF, production by Rana esculenta oviduct in vitro incubated with medium alone (m) or with medium plus mGnRH (2.5 X lo-’ M) (U), in four different stages (A, prereproductive; B, reproductive; C, postreproductive; D; recovery) of the annual sexual cycle, during 1989 and 1990. Statistical analysis: each mean refers to 10 determinations + SD. Two-way ANOVA (treatments x stages interaction F values): Year 1989, Fo7*) = 15.02 (30 min), 17.63 (60 min), 20.97 (120 min), 24.10 (180 min), P < 0.01; Year 1990, Fo72j = 15.11 (30 min), 18.78 (60 min), 21.12 (120 min), 25.07 (180 mm), P < 0.01. Duncan’s multipIe range test: *P < 0.01 vs same time medium alone; **P < 0.01 vs prereproduction, postreproduction, and recovery medium alone; ***P < 0.05 vs same time medium alone.









400: c








1000. 1500. 11


z -














FIG. 4. 17B-estradiol production by Rum esculenta ovary in vitro incubated with medium alone (m) or with medium plus mGnRH (2.5 X lo-’ M) (II!), in four different stages (A, prereproductive; B, reproductive; C, postreproductive; D, recovery) of the annual sexual annual cycle, during 1989 and 1990. Statistical analysis: each mean refers to 10 determinations -+ SD. Two-way ANOVA (treatments X stages interaction F values): Year 1989, Fc3,72j = 15.48 (30 min), 17.62 (60 mm), 20.64 (120 min), 25.81 (180 mm), P < 0.01; Year 1990, Fc3,,*) = 15.69 (30 min), 18.91 (60 min), 20.18 (120 mm), 24.34 (180 min), P < 0.01. Duncan’s multiple range test: *P < 0.01 vs same time medium alone; **P < 0.01 vs prereproduction, reproduction, and recovery medium alone.

though only during the recovery stage, when vitellogenesis occurs. These data are consistent with the plasma PGF, increase recorded for adult R. esculenta females during the recovery stage (Gobbetti et al., 1990). A PGF,, mediation of mGnRHdependent 17@-estradiol synthesis should be excluded, since PGF,, administration to vitellogenetic frogs does not modify the 17P-estradiol plasma levels (Gobbetti et al., 1990). The direct mGnRH effect on gonadal steroidogenesis in R. esculenta is in line with the similar in vivo and in vitro results obtained in the same species (Pierantoni et al., 1984a,b; Zerani and Gobbetti, 1990; Gobbetti and Zerani, 1990; Zerani et al., 1991), and support a role for GnRH as a

paracrine or autocrine hormone in vertebrate gonads (Licht and Porter, 1987; King and Millar, 1987; Gore-Langton and Armstrong, 1988). However, mGnRH in our in vitro studies stimulated estradiol release in the recovery stage only, in accordance with previous studies (Zerani et al., 1991). This phenomenon could be due to a different sensitivity to mGnRH stimulation, depending on ovarian developmental phase, as has been proposed by Hubbard and Licht (1985). Finally, the lower in vitro production of PGF,, and estradiol in the postreproduction stage with respect to the other stages could depend on the quiescent status of the gonads; in fact, several amphibians and reptiles show a transient refractoriness to environmental cues during the post-









6000 1 g 4




z 5 8 2 8 L





3 I CA P

4000 2000





5. PGF, (H) and 17@estradiol(O) plasma levels in female Rana esculenta in three different phases of the reproductive stage, during 1989 and 1990. Statistical analysis: each mean refers to 10 determinations +- SD. One-way ANOVA: Year 1989, PGF,,, Fo2,) = 22.63, P < 0.01; Year 1990, PGF,, Fo2,) = 23.23, P < 0.01. Duncan’s multiple range test: *P < 0.01 vs preovulation and postovulation. FIG.

reproduction stage, at which time this refractory status induces the interruption of the reproductive process (Salthe and Mecham, 1974; Botte and Angelini, 1980; Polzonetti-Magni et al., 1984). In reptiles, as in mammals, prostaglandins are involved in corpora lutea lysis and in the regulation of egg deposition (Guillette, 1990). Recently, Gobbetti et al. (1991b) found that PGF,, was responsible for a decrease in progesterone plasma levels at the end of the reproductive stage in the female newt, Triturus carnifex, and this result seemed to suggest a PGF,, luteolytic action. Our in vitro tests show that R. esculenta oviduct releases the highest PGF, levels during the reproductive stage when ovulation and egg deposition occur (Rastogi et al., 1983; Lofts, 1984). In addition, mGnRH stimulates oviductal PGF,, production only in this stage. This finding agrees with the increase in PGF,, plasma


levels seen during ovulation and supports a PGF,, involvement in both corpora lutea regression and egg laying. Estradiol did not seem to be involved in these processes, since the plasma estradiol did not modify during ovulation. Therefore, at present, it is difficult to assign any physiological role to this steroid in this phase of the reproductive stage. In female R. esculenta, as in other oviparous lower vertebrate species (Xavier, 1987), short-lived corpora lutea develop and show steroidogenetic activity. They are probably involved in timing of egg transit through the oviduct (Botte and Cottino, 1964). Oviductal prostaglandin, moreover, could be responsible for, as in reptiles (Guillette et al., 1984; Guillette, 1990), the timing of egg deposition through regulation of oviductal contractility. Experimental evidence of such functions, however, is lacking in amphibians. In several reptilian species, the prostaglandin effects on oviduct are under the control of arginine vasotocin (AVT) (Guillette, 1990); moreover, this peptide is known to stimulate the oviductal contractions in the salamander, Ambystoma tigrinum (Guillette et al., 1985). Our findings in R. esculenta seem to assign to mGnRH the AVT role, although tests with AVT have not been carried out. It is not known, moreover, whether amphibian oviducts synthesize either GnRH or related molecules and whether they possess GnRH-specific receptors. In this context, we recall that various GnRH forms have been localized in frog nonneural tissues: salmon-GnRH in inter-renal tissue (Eiden et al., 1982)) and mGnRH and chicken IIGnRH in the testis (Cariello et al., 1989). Finally, it is surprising that mGnRH slightly but significantly inhibits the oviductal PGF,, synthesis in recovery and prereproductive animals. Even if this result seems very interesting, the meaning of this phenomenon is, at present, unknown. ACKNOWLEDGMENTS This work was supported by a grant to Prof. Alberta

mGnRH EFFECTS Polzonetti CNR.

of the Italian Ministry


of Education


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A possible involvement of prostaglandin F2 alpha (PGF2 alpha) in Rana esculenta ovulation: effects of mammalian gonadotropin-releasing hormone on in vitro PGF2 alpha and 17 beta-estradiol production from ovary and oviduct.

The present work studied the PGF2 alpha and 17 beta-estradiol plasma levels during ovulation, and the in vitro effects of mammalian gonadotropin-relea...
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