Responsiveness of Adenylate Cyclase to Pituitary Gonadotropins and Evidence of a Hormone-Induced Desensitization in the Lizard Ovary

General and Comparative Endocrinology 107, 23–31 (1997) Article No. GC976893

Responsiveness of Adenylate Cyclase to Pituitary Gonadotropins and Evidence of a Hormone-Induced Desensitization in the Lizard Ovary L. Borrelli, R. De Stasio,* V. Bovenzi,1 E. Parisi,* and S. Filosa Department of Evolutive and Comparative Biology, Naples, Italy; and *CNR Institute of Protein Biochemistry and Enzymology, Naples, Italy Accepted January 27, 1997

Gonadotropins (FSH and LH) affect several mammalian gonadal functions. In particular, FSH stimulates oogonial proliferation and oocyte growth, while LH regulates ovulation and progesterone secretion. In lacertilian reptiles, gonadal function is also regulated by pituitary gonadotropins, but which hormone controls ovarian activities and the mechanisms of action are unknown. The present study aimed to clarify mechanisms of action of pituitary gonadotropins on the ovary of Podarcis sicula (Lacertilia). The data demonstrate that mammalian gonadotropins FSH and LH produce a threefold stimulation of adenylate cyclase activity in follicular membranes, while hCG and TSH are less effective, causing a twofold increase in adenylate cyclase activity. Neurotrasmitters such as dopamine, serotonin, and catecholamines have no effect on enzyme activity. The action of mammalian FSH and LH on the ovary mimics the effect of homologous hormones: in lizard ovaries incubated in vitro in the presence of isolated homologous pituitary glands, the intracellular cAMP level increased by 50% with respect to control ovaries. Mammalian gonadotropins appear homologous to lizard gonadotropin(s): Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian bFSH and bLH. Both homologous and heterologous desensitization of adenylate cyclase activity occurs

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Present address: Pediatric Research Center, St-Justine Hospital, 3175 Co˜te St-Catherine, Montreal, Quebec H3T1C5, Canada. 0016-6480/97 $25.00 Copyright r 1997 by Academic Press All rights of reproduction in any form reserved.

in the lizard ovary. In fact, responsiveness of adenylate cyclase to gonadotropin stimulation is abolished in animals 2 hr after in vivo treatment with FSH. Sensitivity to gonadotropin stimulation is restored 2 weeks after the beginning of the in vivo treatment. Desensitization was also observed in ovaries incubated in vitro with mammalian FSH or with isolated pituitary glands. r 1997 Academic Press In eutherian mammals, two hypophysial gonadotropins, follicle-stimulating hormone (FSH) and luteinizing hormone (LH), regulate gonadal function. In females, FSH controls ovarian activity, stimulating oogonial proliferation, oocyte growth, vitellogenesis, and proliferation and differentiation of the follicular epithelium surrounding the oocyte. LH is involved in the control of ovulation and postovulatory progesterone secretion (Pierce and Parson, 1981). FSH and LH, together with the thyroid-stimulating hormone (TSH) and chorionic gonadotropin (CG), contain glycoprotein moieties; each of these hormones is composed of two distinct, non-covalently associated, a and b subunits. Within a single species, all the a subunits have the same primary structure, whereas the amino acid sequences of the b subunits differ (Pierce and Parsons, 1981). Pituitary gonadotropins interact with gonadal membrane receptors to activate adenylate cyclase (Kolena and Channing, 1972; Lamprecht et al., 1973; Hunzicker-Dunn and Birnbaumer, 1976). The binding of the hormone to the receptor is possible only

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if the a and b subunits are linked (Papkoff et al., 1977): the a subunit is involved in receptor recognition, while the b subunit controls receptor binding specificity (Combarnous and Henge`, 1981). The hormone–receptor binding causes a conformation change in the receptor, thus allowing its interaction with the G protein; thus, the hormonal signal is transduced from the receptor to adenylate cyclase (Gilman, 1987; Casey and Gilman, 1988; Tang and Gilman, 1991; Hepler and Gilman, 1992) to catalyze the transformation of ATP into cAMP. Cyclic AMP acts as an intracellular secondary messenger, regulating a number of specific metabolic functions (Berridge, 1985). In the mammalian ovary, after exposure to saturating concentrations of gonadotropins, there is phosphorylation of the receptors, which prevents activation of adenylate cyclase (Hunzicker-Dunn and Birnbaumer, 1976). This process of ‘‘desensitization’’ reflects a temporary refractoriness of the tissue to the hormone and is due to the loss of responsiveness or to the complete insensitivity of the receptor to further stimulation (Ezra and Salomon, 1980, 1981; Jonassen et al., 1982; Ekstrom and Hunzicker-Dunn, 1990). The desensitized receptors, however, can recover and return to a responsive state. Such a process of resensitization has been investigated in detail for the b-adrenergic receptor (Yu et al., 1993) and for the hCG-sensitive adenylate cyclase in the pig ovarian follicle (Ekstrom et al., 1992). Information on the hormonal regulatory systems in other vertebrates is limited. Among reptiles, the number of gonadotropins is uncertain: in turtles, two distinct gonadotropins, homologous to those of mammals with respect to chemical features and biological activity, exist (Papkoff et al., 1976b). In snakes, a single gonadotropin, with an amino acid composition similar to both mammalian FSH and LH, has been suggested (Licht et al., 1979). In Podarcis sicula, the common field lizard, it is uncertain whether one or two distinct gonadotropins control gonadal activity. In this species, as in other reptiles (Licht and Stockell Hartree, 1971; Licht, 1972a, b, c; Licht et al., 1977d; Jones, 1978), the administration of mammalian FSH produces effects comparable to those occurring during the natural reproductive cycle (Filosa et al., 1979; Limatola and Filosa, 1989). The present work aimed to clarify the mechanism of action of heterologous pituitary gonadotropins on the ovarian follicle of P. sicula.

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Borrelli et al.

MATERIALS AND METHODS Animals Sexually mature specimens of P. sicula were collected in the outskirts of Naples, kept in a terrarium, and fed with meal worms ad libitum. Animals were first anesthetized with ether and then killed.

Chemicals [a-32P]ATP (sp act 1.11 TBq/mmol) and [2.8-3H]cAMP (1.2 TBq/mmol) were obtained from Dupont-NEN. Dopamine (DA), serotonin (5HT), epinephrine (A), norepinephrine (NA), porcine FSH, equine LH, porcine TSH, and human chorionic gonadotropin (hCG) were from Sigma (Sigma Chemicals, Poole, England). All the other reagents were of the highest purity and were purchased from standard commercial sources.

Adenylate Cyclase Assay External connective theca was removed from ovarian follicles under a dissection microscope; the latter were homogenized in 5 3 1023 M Tris–HCl buffer, pH 7.4, containing 0.25 M sucrose, using a Dounce homogenizer. The homogenate was centrifuged at 12,000g for 15 min and the resulting pellet containing the membrane fraction was resuspended in the homogenization buffer. In some experiments, follicular cells were separated from the oocyte membranes as follows: ovarian follicles were suspended in Tris–sucrose buffer and subjected to repeated vortex shaking (usually three cycles of 15 sec each). Following this treatment, follicular cells were dissociated and the oocyte membrane was disrupted. The cells were manually separated from oocyte membranes by carefully pipetting off the medium with a Pasteur pipette. The residual material consisted of oocyte membranes without follicular cells. The membrane fraction was resuspended in Tris–sucrose buffer and processed as above. Follicular cells were recovered from the medium by centrifugation at 600g for 5 min and used for membrane preparation as described above. Adenylate cyclase activity was measured from the conversion of [a-32P] ATP into cyclic AMP according to Salomon et al. (1974). The reaction mixture contained:

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Effect of Gonadotropins on Lizard Ovary

0.03 M Tris–HCl, pH 7.4; 7.5 3 1024 M EDTA; 0.01 M caffein; 4 3 1023 M MgCl2; 5 3 1024 M dithiothreitol; 0.02 M creatine phosphate; 1 3 1024 M cAMP; 2 Units of creatine phosphokinase; 1 3 1024 M ATP; 92.5–45.0 kBq [a-32P]ATP (NEN). The reaction was carried out at 30° for 60 min. Radioactivity was measured by liquid scintillation counting. Determinations were carried out in duplicate and expressed as the mean 6 SD. Protein content was analyzed on membrane preparations following Lowry and Rosenbrough (1951).

Desensitizing Treatments in Vivo Sexually mature females were injected ip with a single dose of 125 µg of porcine FSH (Sigma) and killed after 2 hr and 9–14 and 18 days. A second control group was injected with a saline solution. Adenylate cyclase activity was assayed on ovaries from control and treated animals in the presence and in the absence of 1026 M FSH or LH.

Desensitizing Treatments in Vitro Dissected ovaries were incubated at 28° in sterile petri dishes containing 2 ml of phosphate-buffered saline (PBS), pH 7.4 for 20–40 min in the presence or the absence of 20 µg of FSH. After incubation, ovaries were washed three times with PBS, and the follicles were processed for membrane preparation and adenylate cyclase assay, as described.

Determination of Intracellular cAMP Content Dissected ovaries were incubated at 28° for 15, 30, and 60 min in Dulbecco’s phosphate-buffered saline in the presence or the absence of homologous pituitary glands. After washing three times with the culture buffer, the ovaries were homogenized in cold 5% CCl3COOH. The pellet was removed by centrifugation at 8000g for 10 min, and the supernatant was rendered 0.1 M in HCl and extracted six times with watersaturated ether to eliminate CCl3COOH. Samples were then lyophilized and reconstituted in the assay buffer. The intracellular content of cAMP was measured by an enzyme immunoassay (EIA) using a kit from Amersham (Amersham Int. plc, Buckinghamshire, England). The assay itself followed exactly the manufacturer’s instructions.

Southern Blot Hybridization Genomic DNA was isolated from lizard testis following Sambrook et al. (1989). Restriction enzymedigested DNA was electrophoresed on a 0.8% agarose gel and transferred to nitrocellulose (Sambrook et al., 1989). The filter-immobilized DNA was hybridized with cDNA probes labeled using the random priming method (Sambrook et al., 1989). A 400-bp probe, corresponding to a fragment from position 25 to position 404, was obtained by digesting the plasmid pGEM3, containing the complete coding sequence of bovine FSH b subunit, with EcoRI and ApaI (Maurer and Beck, 1986). A 340-bp probe, corresponding to the fragment from position 22 to position 331, was obtained by digesting the plasmid pBR322, containing the complete coding sequence of ovine LH b subunit, with the same enzymes (d’Angelo-Bernard et al., 1990). DNA fragments were separated by electrophoresis on 1% agarose gel. The probes were recovered from the gel by electroelution. Hybridization with the FSH probe was carried out overnight at 55° in 63 SSC (13 SSC: 150 mM NaCl, 15 mM sodium citrate, pH 7.0), 53 Denhardt’s reagent (13 Denhardt’s reagent: 0.02% Ficoll, 0.02% bovine serum albumin, 0.02% polyvinylpyrrolidone), 0.5% sodium dodecyl sulfate (SDS), 100 µg/ml salmon sperm DNA. Hybridization with the LH probe was carried out overnight at 60° in 50% dextran sulfate and 100 µg/ml denatured salmon sperm DNA. The filter hybridized with the FSH probe was washed at 37° in 43 SSC and 0.5% SDS; the filter hybridized with the LH probe was washed at 20–22° in 13 SPC (100 mM NaCl, 30 mM Na2HPO4, 1 mM EDTA, pH 6.2) and 1% SDS. Autoradiographs were obtained by exposing the filters overnight at 280° in the presence of intensifying screens.

Data Analysis Statistical analysis was made by using the program package Systat (SYSTAT, Intelligent Software, Evanston, IL).

RESULTS Adenylate Cyclase Activity in Ovarian Follicles The ovarian follicles of P. sicula contained adenylate cyclase activity which was proportional to incubation

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Borrelli et al.

TABLE 1 Effect of Various Hormones and Neurotrasmitters on Adenylate Cyclase Activity in Podarcis sicula a Hormone

Activity (pmol cAMP/mg protein)

None FSH LH hCG TSH DA 5HT Epinephrine Norephinefrine

96.3 6 1.2 270.5 6 3.4** 315.3 6 2.6** 190.0 6 2.3** 205.7 6 3.6** 110.4 6 5.5* 118.2 6 3.7* 125.6 6 2.2* 115.8 6 6.1*

a Adenylate cyclase activity was measured in membrane fractions from ovarian follicles in the absence and in the presence of various neurotrasmitters and hormones. Neurotrasmitter concentration was 1025 M; peptide hormones were used at 1027 M. The results were analyzed by the test of variance. ** P , 0.001. * P . 0.05.

time and protein concentration in the assay. Table 1 shows the effects of hormones and neurotransmitters on adenylate cyclase activity: enzyme activity was significantly stimulated by the gonadotropins FSH, LH, and hCG and by TSH (P , 0.001), while DA, 5HT, A, and NA had no significant effect on adenylate cyclase activity. Figure 1 shows that stimulation by FSH, LH, hCG, and TSH was dose-dependent: the IC50 for LH was of the order of 1029 M, whereas for the other hormones it was 1028 M. The effects of FSH combined with LH, hCG, or TSH on adenylate cyclase activity were not additive: stimulation of enzyme

FIG. 1. Dependence of adenylate cyclase activity on increasing concentrations of FSH, LH, TSH, and hCG. Assays were carried out at 30° for 60 min, as described under Materials and Methods.

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FIG. 2. Effect of equimolar mixtures of FSH and LH, FSH and TSH, and FSH and hCG on adenylate cyclase activity. Assays were carried out at 30° for 60 min, as described under Materials and Methods.

activity by FSH combined in equimolar amounts with LH, TSH, and hCG (Fig. 2) was similar to that obtained in the presence of each hormone separately (Fig. 1).

Localization of Adenylate Cyclase Activity To establish which cell type is the target of pituitary gonadotropins in ovarian follicles of P. sicula, adenylate cyclase activity was assayed on membranes prepared both from the oocyte and from isolated follicular cells. Figure 3 shows that the basal level of adenylate cyclase activity is approximately the same in follicular cells as in the oocyte; however, only the adenylate cyclase activity in follicular cells was sensitive to gonadotropin stimulation. Such a lack of stimulation

FIG. 3. Gonadotropin stimulation of adenylate cyclase activity in the oocyte and in follicular cells. Follicular cells were dissociated from the oocyte as described under Materials and Methods. Adenylate cyclase activity was measured on isolated membrane fractions in the absence and in the presence of 1026 M FSH or LH.

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Effect of Gonadotropins on Lizard Ovary

FIG. 4. NaF stimulation of adenylate cyclase activity in oocyte membranes. Adenylate cyclase activity was measured on membrane oocytes in the absence and in the presence of 1026 M FSH or LH or of 1022 M NaF.

of gonadotropins in oocyte membranes did not reflect an impaired function of G proteins due to cell manipulation, as the enzyme activity in oocyte membranes was sensitive to NaF stimulation (Fig. 4).

Desensitization of Adenylate Cyclase in Ovarian Follicles: Effect of FSH in Vivo and in Vitro To investigate a possible desensitization response in lizard follicles, basal and FSH-stimulated activities were measured in crude membranes of follicles isolated from animals killed 2 hr after ip injection of saline (control) or porcine FSH. Table 2 shows adenylate

TABLE 2 Desensitization of Adenylate Cyclase by in Vivo FSH Treatment a Adenylate cyclase activity (pmol cAMP/mg protein) Control

FSH-injected

Basal

1FSH

Basal

1FSH

175.8 6 2.2 35.5 6 1.2 131.9 6 3.3 273.1 6 1.1 271.6 6 1.8 238.9 6 2.4 252.8 6 1.5

334.8 6 3.4 76.0 6 2.7 227.2 6 2.1 462.7 6 2.6 480.3 6 1.7 466.1 6 2.3 499.9 6 1.2

60.8 6 1.7 117.9 6 2.3 78.9 6 1.1 217.6 6 3.5 170.0 6 3.4 149.1 6 2.3 —

55.8 6 4.4 149.4 6 1.9 72.1 6 2.0 204.0 6 3.2 174.8 6 1.5 134.9 6 2.4 —

a

One group of animals was treated with a single dose of FSH and another, used as control, received only saline. Adenylate cyclase activity was measured in the absence and in the presence of 1026 M FSH.

FIG. 5. Recovery of sensitivity to FSH and LH stimulation in adenylate cyclase from animals desensitized with FSH. Thirty-five animals were divided into five groups of 7 animals: group I received only saline; groups II to V received a single dose of FSH. Animals were killed 2 hr (group II), 9 days (group III), 14 days (group IV), and 18 days (group V) after the injection. Adenylate cyclase activity was measured in membrane fractions in the absence and in the presence of 1026 M FSH or LH. The basal levels of adenylate cyclase activities were 197.1 6 3.2, 132.4 6 2.4, 97.7 6 2.8, 115.6 6 1.5, and 102.5 6 2.6 pmol cAMP/mg protein for groups I–V, respectively. The results were analyzed by the test of variance. Activities measured with FSH and LH were not significantly different within the five groups tested.

cyclase activity measured in follicles from individual animals: FSH added to the reaction mixtures stimulated adenylate cyclase activity in follicles of control animals, whereas no stimulation was observed in FSH-injected animals. The analysis of covariance demonstrated that the stimulation of adenylate cyclase activity significantly differed (P 5 0.002) in the two groups of animals. To determine whether ovarian follicles exhibit heterologous as well as homologous desensitization, the responsiveness of adenylate cyclase to LH was tested in animals killed 2 hr or several days after the ip injection of FSH (Fig. 5). In untreated animals, adenylate cyclase activity, measured over the basal level, was equally sensitive to both FSH and LH added to the reaction mixture. In animals killed 2 hr after FSH injection, the responsiveness to both LH and FSH was attenuated. Hormone sensitivity gradually reappeared during the first 2 weeks after FSH injection and returned to normal after 18 days. In vitro hormone-dependent desensitization of gonadotropin-sensitive adenylate cyclase was examined in ovaries incubated in the presence of saline or FSH. Figure 6 shows that the sensitivity of adenylate cyclase

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Borrelli et al.

to FSH added to the reaction mixture was relatively high in untreated ovaries (relative stimulation of 1.4 times over the basal level). FSH-stimulated activity decreased only slightly after a 20-min exposure to the hormone, but it was almost completely abolished after 40 min.

Action of Pituitary Gland on the Ovary The stimulation of adenylate cyclase activity so far described employed heterologous hormones. Since reptilian gonadotropins are not at present available, the effects of whole homologous pituitaries were tested on the intracellular cAMP content in ovaries. Ovaries were cocultured with pituitary glands, and the ovarian cAMP content measured. Figure 7 shows that cAMP levels were increased about 30 and 60% after 15 and 30 min of culture. Control ovaries were incubated for the same times in the absence of pituitary glands. In ovaries incubated for 60 min in the presence of pituitary glands the intracellular cAMP content fell to levels similar to those of control ovaries.

Southern Blot Analysis To determine whether the genes of mammalian gonadotropins are homologous to the lizard counterparts, Southern blot analysis was performed using

FIG. 7. Effect of isolated pituitary gland on the ovary of P. sicula. Dissected ovaries were kept in culture with or without homologous pituitary glands for 15 min (group I), 30 min (group II), and 60 min (group III). Intracellular cAMP content was measured as described under Materials and Methods.

lizard testis DNA digested with different restriction enzymes. The two distinct probes used for hybridization were two cDNA fragments of 400 and 340 bp derived from the CDS of the b subunits of bovine FSH and ovine LH, respectively. Figure 8a shows that, on lizard genomic DNA, the probe derived from FSH cDNA recognized an EcoRIdigested 1.5-kb fragment, a BamHI-digested 3.8-kb fragment, and two fragments, of 2.9 and of 1.7 kb, derived by digestion with EcoRI plus BamHI. Figure 8b illustrates the results obtained with the probe derived from LH cDNA. This probe hybridized with a XbaIdigested 8.2-kb fragment and with a HindIII-digested 2-kb fragment of the lizard genomic DNA.

DISCUSSION

FIG. 6. Effect of in vitro FSH treatment on adenylate cyclase from ovarian follicles. The ovaries of 18 animals were divided into three groups. Group I was incubated for 40 min in the absence of FSH; groups II and III were incubated in the presence of 20 µg of FSH for 20 and 40 min, respectively. At the end of the incubation, adenylate cyclase was measured on membrane fractions in the presence and in the absence of 1026 M FSH.

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The present paper clarifies some aspects of the mechanism of action of pituitary gonadotropins in the lizard P. sicula. Morphologically, the ovary responds to exogenous gonadotropins in a way similar to that observed in the animal during the reproductive cycle. In particular, FSH administered during the nonbreeding period, when there is reduced ovarian activity, produces oogonial proliferation and oocyte growth

Effect of Gonadotropins on Lizard Ovary

FIG. 8. Southern blot analysis of genomic DNA of P. sicula probed with bovine bFSH (a) and ovine bLH (b). Ten micrograms of DNA was digested with the indicated restriction enzymes, electrophoresed on a 0.8% agarose gel, and transferred to nitrocellulose paper. Filter a was hybridized with a fragment of porcine bFSH cDNA; filter b was hybridized with a fragment of ovine bLH cDNA. Blots were washed using stringent conditions, as described under Materials and Methods. (a) Lane 1 refers to molecular weight standard; the other lanes are the products obtained with the following restrictions enzymes: EcoRI (lane 2), BamHI (lane 3), EcoRI plus BamHI (lane 4). (b) Digestion products obtained with the enzymes XbaI (lane 1) and HindIII (lane 2). Lane 3 refers to molecular weight standard.

with vitellogenesis and differentiation of the follicular epithelium (Filosa et al., 1979; Limatola and Filosa, 1989). The present data demonstrate that, in lizards, as in mammalian systems (Kolena and Channing, 1972; Lamprecht et al., 1973; Hunzicker-Dunn and Birnbaumer, 1976), the action of pituitary gonadotropins is mediated by a stimulation of adenylate cyclase and the subsequent production of cAMP. In the lizard ovary, adenylate cyclase responds to stimulation by mammalian FSH, LH, and, to a lesser extent, by hCG and TSH. Unlike observations in mammals (Hunzicker-Dunn and Birnbaumer, 1976), neurotransmitters, including catecholamines, had no effect on adenylate cyclase activity. Only adenylate cyclase localized on the follicular membranes is sensitive to gonadotropic stimulation. The lack of additive stimulatory effects on enzyme activity by FSH used in combination with LH, TSH, or hCG requires explaining. Several hormones including gonadotropins, hCG, TSH, interleukin-8, and tumor necrosis factor display flexible binding specificity

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(Moyle et al., 1994). Apart from mammalian LH and hGC, which share a common receptor, the receptor specificity for FSH and LH is apparently altered when their activities are compared in homologous and heterologous assays. For example, porcine FSH receptors recognize turtle LH better than turtle FSH, while equine LH has high affinity for both rat LH and FSH receptors (Moyle et al., 1994). Such a flexibility in binding seems to reflect a lack of specific contacts between much of the hormone and the receptor (Moyle et al., 1995). In lizards, the ovarian follicle is not always responsive to gonadotropins. The present results demonstrate that 2 hr after FSH injection the ovarian follicle undergoes a refractory period, in which adenylate cyclase activity is no longer sensitive to stimulation by either FSH or LH. Such a refractoriness of lizard adenylate cyclase to gonadotropins might be due to a desensitization process of membrane receptors, as in the rat ovary (Jonassen et al., 1982). Desensitization of gonadotropinsensitive adenylate cyclase does not seem to affect the functional activity of the enzyme, nor that of the G protein (Gudermann et al., 1995). The presence of a cross-desensitization phenomenon for FSH and LH may be explained by the inability of lizard receptors to distinguish between them. In lizard ovarian follicles, the desensitization is followed by a process of resensitization: indeed, 18 days after ip FSH treatment, the ovarian follicle again become sensitive to the stimulatory effect of gonadotropins. As in other system (Yu et al., 1993), resensitization may result from a structural reorganization and/or reappearance of hormone receptors. The desensitization process also occurs when isolated ovarian follicles are cultured in the presence of FSH. In this case, the time required for desensitization is markedly reduced: adenylate cyclase activity becomes insensitive to gonadotropin stimulation when follicles are cultured for 40 min in the presence of FSH. A longer time (2 hr) is required for desensitization to occur in vivo; this may depend on the time required for exogenous FSH to reach the gonad. In the present system, the heterologous gonadotropins mimic the in vitro effect exerted by the homologous pituitary gland on the ovaries: in fact, the lizard hypophyses can stimulate cAMP production in cocultured follicles. Moreover, under these experimental

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conditions, the desensitization takes place in 60 min, a time comparable to that measured in ovaries incubated with heterologous FSH. Mammalian gonadotropins are seemingly closely homologous to lizard gonadotropin(s). The Southern blot analyses show that the lizard genome contains nucleotide sequences homologous to those encoding for mammalian bFSH and bLH. However, it remains unclear whether these lizard nucleotidic sequences code for two different gonadotropic b subunits or for a single gonadotropic b subunit having common features with both mammalian b FSH and b LH. In conclusion, mammalian gonadotropins display a structural relationship with the lizard gonadotropic hormone(s) and mimic the natural action of lizard hypophyseal secretion on the gonad, via cAMP. In lizards, the stimulation of oogonial proliferation, oocyte growth, vitellogenesis, and proliferation of the follicular epithelium surrounding the oocyte (Filosa et al., 1979; Limatola and Filosa, 1989) may be primarily due to gonadotropin(s) interaction with membrane receptors and to activation of the adenylate cyclase system.

ACKNOWLEDGMENTS We are grateful to Dr. M. Ciaramella for her advice and support. We thank Professor R. Counis and Professor R. Maurer for bLH and bFSH cDNA probes. This work was supported by a 40% grant from the M.P.I. This paper is dedicated to Professor G. Ghiara.

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