S. Fasano

Stimulatory effect of a GnRH agonist (buserelin) in in vitro and in vivo testosterone production by the frog (Rana esculenta) testis

The summary testicular effects of an agonistic analogue of gonadotropin-releasing hormone (buserelin, GnRHa) have been studied in vitro and in vivo in the frog, Rana esculenta. During 3 h incubation GnRHa (8 X 10(-7) M) potentiated pituitary factors in stimulating testosterone production by minced testes in vitro. After 6 h of incubation 8 X 10(-7) M GnRHa stimulated maximal testosterone output. Testes of 10-day hypophysectomized animals did not show any GnRHa effect in vitro. In vivo, a direct effect of GnRHa on testicular testosterone production was demonstrated in hypophysectomized animals, although this effect was temperature-dependent, requiring the frog to be maintained at a high temperature (24 degrees C). No effect of GnRHa was detectable in frogs kept at a low temperature (4 degrees C).

Evolutionary Aspects of Cellular Communication in the Vertebrate Hypothalamo–Hypophysio–Gonadal Axis

This review emphasizes the comparative approach for developing insight into knowledge related to cellular communications occurring in the hypothalamus-pituitary-gonadal axis. Indeed, research on adaptive phenomena leads to evolutionary tracks. Thus, going through recent results, we suggest that pheromonal communication precedes local communication which, in turn, precedes communication via the blood stream. Furthermore, the use of different routes of communication by a certain mediator leads to a conceptual change related to what hormones are. Nevertheless, endocrine communication should leave out of consideration the source (glandular or not) of mediator. Finally, we point out that the use of lower vertebrate animal models is fundamental to understanding general physiological mechanisms. In fact, different anatomical organization permits access to tissues not readily approachable in mammals.

Plasma and testicular estradiol and plasma androgen profile in the male frog Rana esculenta during the annual cycle.

Seasonal plasma and testicular estradiol levels were measured in the male frogs, Rana esculenta, by radioimmunoassay. In plasma samples a simultaneous measurement of androgens was carried out in order to investigate a possible relationship between androgens and estradiol-17 beta. Concomitantly with the estradiol-17 beta peak in plasma and testes during the April-May period, plasma androgens sharply decreased.

The amphibian testis as model to study germ cell progression during spermatogenesis.

Testicular morphology of vertebrate testis indicates requirement of local control. In urodeles, the testis is organized in lobes of increasing maturity throughout the cephalocaudal axis. The anuran testis is organized in tubules. Spermatogenesis occurs in cysts composed by Sertoli cells enveloping germ cells at synchronous stages. Moreover, in numerous species germ cell progression lasts a year which defines the sexual cycle. Due to the above quoted features, research on factors regulating germ cell progression in amphibians may reach greater insight as compared with mammalian animal models. In particular, studies on endocrine and paracrine/autocrine factors involved in the regulation of germ cell functions reveal that fos activation and a J protein, previously specifically found in mouse testis, exert an important role in spermatogonial proliferation and maturation of post-meiotic stages, respectively.

Regulation of androgen production by frog (Rana esculenta) testis: An in vitro study on the effects exerted by estradiol, 5α-dihydrotestosterone, testosterone, melatonin, and serotonin

The possible role of estradiol-17 beta (E2), testosterone (T), 5 alpha-dihydrotestosterone (DHT), melatonin, and serotonin on the regulation of androgen (A) production by the frog, Rana esculenta, testes was studied in vitro. E2 (10(-6) M) inhibited A production whether alone or in combination with oLH (20 micrograms) after 6 hr incubation. After 24 hr incubation. A production was reduced by E2 concentration of around 10(-6) and 10(-9) M. Melatonin and serotonin did not induce any change whichever experimental condition was used. Preincubation for 6 hr with 10(-6) M T or DHT enhanced the oLH-stimulated A production after 18 hr incubation. These data suggest that steroids may regulate their intratesticular levels without passing into the blood stream.

Characterization of gonadotropin-releasing hormone (GnRH) binding sites in the pituitary and testis of the frog, Ranaesculenta

Frog, Rana esculenta, pituitary and testis gonadotropin-releasing hormone (GnRH) receptors were characterized by using 125I-chicken IIGnRH (cIIGnRH) as radiolabeled ligand. At 4 C equilibrium binding of 125I-cIIGnRH to pituitary homogenates was achieved after 90 min of incubation; binding of 125I-cIIGnRH to testis membrane fractions reached its maximum at 60 min of incubation. Binding of the radioligand was a function of tissue concentration, with a positive correlation over the range 0.5-2 tissue equivalents per tube. One pituitary and one testis per tube were used as standard experimental condition. Incubation of the pituitary homogenate with increasing concentrations of 125I-cIIGnRH indicated saturable binding at radioligand concentrations of 1 nM and above while for the testis membrane preparation saturation was achieved using 5 nM 125I-cIIGnRH. The binding of 125I-cIIGnRH was found to be reversible after addition of the cold analog and the displacement curves could be resolved into one linear component for both tissues. Scatchard analysis suggested the presence of one class of binding sites for both pituitary and testis (Pituitary: Kd = 1.25 +/- 0.14 nM and Bmax = 8.55 +/- 2.72 fmol/mg protein; testis: Kd = 2.23 +/- 0.89 nM and Bmax = 26.48 +/- 7.39 fmol/mg protein). Buserelin displaced the labeled 125I-cIIGnRH with a lower IC50 as compared with cIIGnRH cold standard, while Arg-vasopressin (AVP) was completely ineffective, confirming the specificity of binding.

Hypothalamus-hypophysis and testicular GnRH control of gonadal activity in the frog, Rana esculenta: Seasonal GnRH profiles and annual variations of in vitro androgen output by pituitary-stimulated testes ☆

The binding of a gonadotrophin-releasing hormone (GnRH) long acting analog (GnRHA), D-Ser (But)6,Pro9-NEt GnRH (HOE 766), to pituitary and testicular extracts and the presence of GnRH-like material in testes and hypothalamuses were measured in the frog, Rana esculenta. Also, the cellular localization of immunoreactive GnRH was investigated in testes by immunohistochemical staining. Furthermore, lyophilized preparations of pituitary crude homogenates from animals caught monthly were tested in vitro for their ability to stimulate androgen production by December testes. Satisfactory results on specific 125I-GnRH binding were difficult to obtain in view of its low binding capacity. Moreover, binding in testicular homogenates was of the same order of magnitude (about 2%) as that found in pituitaries. In a cospecific radioimmunoassay for GnRH nonapeptide, both hypothalamic and testicular extracts gave displacement parallel to the standard curve. Immunoreactive GnRH did not significantly fluctuate in hypothalamuses, while it peaked in testes during December and July. Immunoreactive GnRH was evidenced in June and September testes employing immunohistochemical staining. In particular, the interstitial cells and the Sertoli cells were faintly stained. Testes of December animals stimulated by February pituitaries produced larger quantities of androgens as compared with testes stimulated with hypophyseal preparations from the remaining periods of the year. In conclusion, the present results are consistent with the idea that seasonal changes of the hypothalamus-hypophyseal activity play an important role in regulating the hormonal response in vertebrate testes. Moreover, we report that, in addition to rats, GnRH-like material is present in frog testes and for the first time it has been shown that such putative intratesticular material undergoes seasonal fluctuations in a vertebrate.

Fos localization in cytosolic and nuclear compartments in neurones of the frog, Rana esculenta, brain: an analysis carried out in parallel with GnRH molecular forms.

C‐fos activity was determined in the brain of the frog, Rana esculenta, during the annual sexual cycle. The localization of GnRH molecular forms (mammalian‐ and chicken‐GnRHII) was also carried out to determine whether or not the proto‐oncogene and the peptides showed a functional relationship. Northern blot analysis of total RNA revealed the presence of a single strong signal of c‐fos like mRNA of 1.9u2003Kb during February and April. This was followed by expression of c‐Fos protein (Fos) in several brain areas during March and July shown by immunocytochemistry. In particular, the olfactory region, the lateral and medial pallium, the nucleus lateralis septi, the ventral striatum, the caudal region of the anterior preoptic area, the suprachiasmatic nucleus, the ventral thalamus, tori semicircularis and ependymal layers of the tectum were immunostained. There was no overlap between Fos immunoreactive perikarya and GnRH immunoreactive perikarya (e.g. gonadotrophin‐releasing hormone (GnRH) in the rostral part and Fos in the caudal region of the anterior preoptic area). Interestingly, a cytoplasmic localization of Fos was also observed by immunocytochemistry and gel retardation experiments supported this observation. Cytoplasmic extracts from September–October animals bound the AP1 oligonucleotide. The complex was not available in the nuclear extracts from the same preparation, suggesting that, besides Fos, Jun products were also present. Conversely, nuclear but not cytosolic binding was detected in the brain of animals collected in July. In conclusion, we show that Fos and GnRH activity does not correlate in the frog brain and, for the first time in a vertebrate species, we give evidence of a cytoplasmic AP1 complex in neuronal cells.

Plasma and follicular tissue steroid levels in the elasmobranch fish, Torpedo marmorata☆

Steroid concentrations in plasma and follicular tissues (theca plus granulosa layers) were determined by radioimmunoassay in the aplacental viviparous ray, Torpedo marmorata, during various stages of the reproductive cycle. Steroids in the uterine fluid of pregnant animals and in preovulatory atretic follicles were also measured. In the follicular tissue of cyclic animals, levels of progesterone were always lower than those of estradiol-17 beta and androgens (testosterone plus 5 alpha-dihydrotestosterone). Estradiol-17 beta and androgen levels increased as the animals approached the ultimate maturational stage before ovulation. Androgens were not detectable in plasma, while estradiol-17 beta increased dramatically before ovulation. In pregnant animals, only small ovarian follicles (less than 5 mm in diameter) were observed, and these had hormone concentrations that were similar to those of the small follicles of cyclic animals. Progesterone was the only steroid detected in the uterine fluid of pregnant animals. In completely sclerotic atretic follicles of pregnant animals, steroids were not detected. Progesterone was the main hormone in atretic follicles undergoing yolk resorption. This suggests that the latter may contribute to the elevated plasma progesterone concentrations of pregnant animals.

Effect of temperature and darkness on testosterone concentration in the testes of intact frogs (Rana esculenta) treated with gonadotrophin-releasing hormone analog (HOE 766).

Testicular testosterone was determined by radioimmunoassay in the frog (Rana esculenta) kept in total darkness, at a high or a low temperature (24 or 4 degrees C), and treated with a gonadotrophin-releasing hormone analog (GnRHa, HOE 766). Prolonged exposure to dark conditions seemed to inhibit hypotalamic functions. Moreover, it is shown that high temperature interacts positively with GnRHa treatment on testicular testosterone concentration.