Acta Physiol Scand 1991, 142, 495-501

ADONIS

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/n vitro steroid production by follicles of frog Rana esculenta :mamma Iia n gonad ot ropin- releasing hormone effects M. Z E R A N I , A. G O B B E T T I and A. P O L Z O N E T T I - M A G N I Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy

ZERANI, M., GOBBETTI, A. & POLZONETTI-MAGNI, A. 1991. In vitro steroid production by follicles of frog Rana esculenta : mammalian gonadropin-releasing hormone effects. Acta Physiol Scand 142,495-501. Received 19 November 1990, accepted 19 March 1991. ISSN 0001-6772. Department of Molecular, Cellular and Animal Biology, University of Camerino, Italy. The effects of mammalian gonadotropin-releasing hormone on ovarian release of progesterone, androgens and estradiol-17P were studied in vitro by a superfusion system carried out on follicles of adult female Rana esculenta, collected at different periods of the annual reproductive cycle. The follicles were superfused with medium alone, pituitary, mammalian gonadotropin-releasing hormone, or pituitary plus mammalian gonadotropin-releasing hormone. For follicles obtained in the prereproductive period, pituitary plus mammalian gonadotropin-releasing hormone increased the estradiol values much more than pituitary alone. In the reproductive period, pituitary alone increased the estradiol values much more than pituitary plus mammalian gonadotropin-releasing hormone. For follicles obtained in the recovery period, mammalian gonadotropin-releasing hormone alone stimulated the highest estradiol production, and pituitary plus mammalian gonadotropin-releasing hormone increased the estradiol values much more than pituitary alone. The results reported here suggest that mammalian gonadotropin-releasing hormone and/or pituitary have a direct effect on ovarian estradiol secretion, and that this effect varies with the annual reproductive cycle. Key words : estradiol-17/$ follicle, frog, mammalian gonadotropin-releasing hormone. T h e brain peptide, gonadotropin-releasing hormone (GnRH), is known to act on pituitary gonadotropin release in all vertebrate species so far studied. Moreover, in mammals, G n R H may have various direct effects on the gonad: inhibitory actions on testicular and ovarian steroidogenesis, blockage of gonadotropininduced progesterone, and inhibition of folliclestimulation hormone and prolactin stimulation function in the ovary (Hsueh & Jones 1981). I n contrast, other studies indicate that G n R H also has stimulatory actions on the mammalian ovary including progesterone and prostaglandins proCorrespondence : Massimo Zerani, Dipartimento di Biologia Molecolare, Cellulare e Animale, Via F. Camerini 2, 62032 Camerino MC, Italy.

duction (Clark 1982), meiotic maturation (Hillensjo & LeMaire 1980) and induction of ovulation in the rat (Corbin & Bex 1981). In addition, GnRH-receptors have been characterized in the rat ovary (Pieper et al. 1981) and testis (Clayton & Catt 1981). Few studies, however, have explored potential extrapituitary actions of G n R H in nonmammalian species. Recent works report evidence that GnRH-like substances are produced by anamniote vertebrate gonads. These peptides, in fact, have been found in the testis of the green frog, Rana esculenta (Fasano et al. 1988, Cariello et ul. 1989) and both in the testis and ovary of the lizard, Podurczs s. siculu (Garcia & Botte 1990). Some of these endogenous gonadal GnRH-like substances seem to exert both endocrine and

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paracrine regulation (4Iillar & K i n g 1987). Mammalian GnRH ( m G n R H ) has been found in amphibians ( K i n g & klillar 1 9 8 i ) ; this peptide or its analogues have variable stimulatory effects on testis either alone or in combination with gonadotropins both in zizo (Pierantoni et r r / . 1984a, b, Zerani & Gobbetti 1990), and in zrtro (%gal& iidejuw-on 1979, Pierantoni et al. 1984a, Alonso-Betade ei NI. 1987) in different Rana species. Recently, in addition, Gobbetti & Zerani (1990) suggested a n in zizo direct effect of m G n R H o n Rana esczilentii ovary, since this peptide increased the plasmatic estradiol in hypophysectomized animals. However, m G n R H proved ineffective when tested in zitro o n the ovary of the anuran Rana ratesbeiana and Ram prpiens ( H u b b a r d & I.icht 1985). -%n in e.itro approach was utilized in this study to examine the direct action of GnRH o n ovarian function in the preen frog Rana e s d e n t i z .

M A T E R I . 4 L S A N D METHODS .4nimals. ..idult female Rrrnri esculeniu were collected in a small mountain pond (Colfiorito, Cmbria; 820 m above sea level in the Apennines of Central Itall-), in different periods of the reproductive annual cycle : prereproduction (April), reproduction (Ma>-), and recoverj- (October). The animals were transferred to our laborator)-, kept at natural photothermal conditions in tanks with running water, fed with fly larvae, and used 24 h after capture. The ovary and the pituitary, after decapitation, w-ere immediately removed and placed in the medium. The experiment was performed in 1989 and 1990. . h e method previously In vitro stiperfirsion s ~ v i e m T described by Porter & Licht (1985) was appropriately modified (Fig. 1). A bottle, containing the medium was connected with the inflow of a peristaltic pump; the pump outflow was connected with the cap of a column (A) (glass barrel column, 5 mm diameter x 4 cm length, with a porous polyethylene disc at the bottom; Bio-Rad, USA). Column A was then directed toward a fraction collector; in some experimental groups another column (B) was connected between the pump and column A ; thus column B was superfused before column A. The effective volume of the columns was 500pI. The flow rate for the superfusion was 200 p1 min-' and fractions were collected at 30 min intervals; at this rate, a bolus of material passed completely through the first column in ca 5 min and through the second in ca 10 min. The bottle u-ith medium was placed in a water bath at 10 "C, and the columns were kept at room temperature (ca 19 "C). The collected medium fractions were frozen and kept at -20 O C until assa>-. Ten full-

growth follicles (2 mm diameter) were manually removed from the ovarj-, under dissecting microscope, and immediately transferred into column A. 'The pituitary gland was placed in column B. Dulbecco's modified Eagle medium (Flow, Italy), containing glutamine, 10 mM hepes and 45 mM NaHCO,, diluted 2 to 1 with buffer RIA (74.5 mM Na,HPO,, 12.5 mbi EDTA-Na,, 0.1 o/o gelatine, 0. l",,, NaN,), was used for all superfusions. Previous studies of ours showed that a medium diluted with RIA buffer improved the steroid extraction, compared with a medium diluted with water as described by Porter & Licht (1985). Synthetic mammalian gonadotropinreleasing hormone (mGnRH, Sigma, USA; 100 ng ml-' medium) was used. In the dose-response relationship study, we found that 100 ng of mGnKI-I was the minimal effective dose. T h e experimental groups \yere : (1) follicles superfused with medium alone; ( 2 ) follicles superfused with medium plus mGnRH ; ( 3 ) pituitary and follicles superfused with medium alone; (4) pituitary and follicles superfused with medium plus mGnRH; (5) pituitary superfused with medium alone; (6) pituitary superfused with medium plus mGnRH; (7) medium alone; and (8) medium plus mGnRH. I n all groups with mGnRI~i, this hormone was added 90 min after the beginning of follicles superfusion with medium alone. In all groups with pituitary column B, containing this gland, was connected 90 min after the beginning of follicles superfusion with medium alone. The superfusions were stopped after 12 h and pituitary and follicles were plunged in Bouin's fluid for histological examination immediately. T h e follicles used in parallel treatments were from the same frog while the pituitary glands were randomly chosen. All the experimental groups were replicated 10 times for each of the three examined periods. Hormone determination. Progesterone, testosterone and estradiol- 178 determinations were carried out by RI.1 method according to d'Istria et al. (1974). T h e following sensitivities were recorded : progesterone, 7 pg (intraassap variability, 4O/;,; interassay variability, 8",); testosterone, 5 pg (intraassay variability, 5.5$/,; interassay variability, 107;); estradiol-17/~', 7 pg (intraassay variability, 4.5O/b; interassay variability, 7.5Oo). Steroid antisera were provided by Dr G. F. Bolelli (CNR-Physiopathology of Reproduction Service, University of Bologna, Italy) ; tritium labelled steroids were purchased from Amersham International (England) and cold steroids were obtained from Sigma. Testosterone antibody crossreacted ( > 800/,) with dihydrotestosterone and since the two steroids were not separated, they are expressed as androgens. Staristiral anabsis. Data were analysed for significance by ANOVA followed by Duncan's test for media differences (Duncan 1955, Sokal & Rohlf 1981). The curves were drawn using two software packages,

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Fig. 1. Schema of the superfusion apparatus. For the description of the superfusion system see

Materials and Methods. MacPaint I1 (Clark Corporation, USA) and

In vitro steroid production by follicles of frog Rana esculenta: mammalian gonadotropin-releasing hormone effects.

The effects of mammalian gonadotropin-releasing hormone on ovarian release of progesterone, androgens and estradiol-17 beta were studied in vitro by a...
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