Primary structure of three forms of gonadotropin-releasing hormone (GnRH) from the pacu brain.

Perchlike fish are a vast group of advanced teleosts. The species examined to date have three forms of gonadotropin-releasing hormone (GnRH) within a single species, but the origin of the third GnRH peptide is unknown. In this study, the primary structure of three GnRH peptides is determined from the brain of the pacu, Piaractus mesopotamicus, an example of a teleost that is less advanced than the perchlike fish. The GnRH was purified from pacu brain extracts using high performance liquid chromatography (HPLC) and radioimmunoassay (RIA). The three forms identified by chemical sequencing and mass spectrometry are sea bream GnRH (pGlu-His-Trip-Ser-Tyr-Gly-Leu-Ser -Pro-Gly-NH2, 1113.4 Da); chicken GnRH-II (pGlu-His-Trp-Ser-His-Gly-Trp-Tyr-Pro-Gly-NH2, 1236.6 Da); and salmon GnRH (pGlu-His-Trp-Ser-Tyr-Gly-Trp-Leu-Pro-Gly-NH2, 1212.3 Da). In addition the number of forms of GnRH in the brains of male and female fish was determined separately. The same three forms of GnRH were present in the brains of both sexes as determined by antisera cross-reactivity and elution position from the HPLC column. The results indicate that the pacu brain has the identical forms of GnRH identified in perchlike fish and hence, the origin of three forms occurred earlier in evolution than previously thought.

Plasma steroid and corticosteroid levels in female pacu Piaractus mesopotamicus, Teleostei-Characidae.

We report the plasma levels of estradiol-17 beta (E2), testosterone (T), 17 alpha-20 beta-dihydroxy-4-pregnen-3-one (17-20P), and cortisol (F) in female pacu during the reproductive cycle (N = 44) and in females induced to ovulate with an analogue of luteinizing hormone releasing hormone (LHRHa; 10 micrograms/kg) (N = 24). The plasma hormone levels were determined by validated radioimmunoassays. Females sampled during the reproductive cycle were grouped into 4 gonadal stages: resting, early maturation, advanced maturation and regression. The calculated gonadosomatic index varied from 0.5 +/- 0.1% in resting stage to 8.1 +/- 0.6% in advanced maturation stage. The E2 and T values were highest during the early maturation stage (E2 = 2172 +/- 7.1 pg/ml; T = 412 +/- 58 pg/ml) and the F values were highest during the advanced maturation stage (132 +/- 5 ng/ml). Females induced to ovulate by LHRHa injection were sampled at 0.6, and 12 h after injection of LHRHa. Two additional groups were sampled at ovulation and 24 h after ovulation. The E2 values were highest at 6 h (2917 +/- 65 pg/ml). The T and F values were highest at ovulation (T = 3498 +/- 77 pg/ml; F = 387 +/- 16 ng/ml) and 17-20P was detected only at ovulation (2163 +/- 80 pg/ml).

Plasma steroid and corticosteroid levels in female pacu Piaractus mesopotamicus, Teleostei-Characidae

We report the plasma levels of estradiol-l7Beta (E2), testosterone (T), 17(alpha-2Obeta-dihydroxy-4-pregnen-3-one (l7-2OP), and cortisol (F) in female pacu during the reproductive cycle (N = 44) and in females induced to ovulate with an analogue of luteinizing hormone releasing hormone (LHRHa; 10 mug/kg) (N = 24). The plasma hormone levels were determined by validated radioimmunoassays. Females sampled during the reproductive cycle were grouped into 4 gonadal stages: resting, early maturation, advanced maturation and regression. The calculated gonadosomatic index varied from 0.5 ñ 0.1 per cent in resting stage to 8.1 ñ 0.6 per cent in advanced maturation stage. The E2 and T values were highest during the early maturation stage (E2 = 2172 ñ 7.1 pg/ml; T = 412 ñ 58 pg/ml) and the F values were highest during the advanced maturation stage (l32 ñ 5 ng/ml). Females induced to ovulate by LHRHa injection were sampled at 0, 6, and 12 h after injection of LHRHa. Two additional groups were sampled at ovulation and 24 h after ovulation. The E2 values were highest at 6 h (2917 + 65 pg/ml). The T and F values were highest at ovulation (T = 3498 + 77 pg/ml; F = 387 ñ 16 ng/ml) and 17-20P was detected only at vulation (2163 ñ 80 pg/ml).

Comparative histochemistry of the mucoproteic cells of the hypophysis from Rhamdia hilarii, Hypostomus punctatus, Prochilodus scrofa and Cyprinus carpio (Teleostei). Immunohistochemical identification of the gonadotropic cells.

Adenohypophyseal cells showing positive histochemical reactions for mucosubstances were classified as type I-IV in Hypostomus (Plecostomus) punctatus (Loricariidae), Rhamdia hilarii (Pimelolidae), Prochilodus scrofa (Prochilodontidae) and Cyprinus carpio (Cyprinidae) according to cell shape, size, cytological characteristics and adenohypophyseal distribution. Cell types I and II are common to the four species, with each cell type showing very similar cytological and histochemical characteristics, in spite of different adenohypophyseal distribution of cell type II, according to the teleost species. Type I cells are globular basophils located in the proximal pars ditalis and are positive to PAS and Alcian blue pH 2.5 (AB) reactions, showing cytoplasmic vacuoles and changes in granule concentration in the mature phase of the gonadal cycle. The smaller type II cells are fusiform or oval basophils exhibiting a strong AB reaction but also reacting to PAS. Type III cells are located in the pars intermedia showing PAS-positive reaction. Considering different teleost species, these cells exhibit some variations specially in relation to cell size and shape which are not detected in mature male C. carpio. Otherwise cell type IV is only present in the rostral pars distalis of P. scrofa. They are weakly basophilic and negative to PAS, reacting strongly to AB. Only cell type I showed unequivocally positive immunohistochemical results with anti-salmon gonadotropin.

Thin section and freeze fracture studies of the hypophyseal proximal pars distalis in a teleost (Rhamdia hilarii Val.) during different stages of the reproductive cycle.

Chromophilic cells in the proximal pars distalis of the adenohypophysis of Rhamdia hilarii were studied in thin section and freeze fracture preparations. The gonadotropic cells (GTH-cells) exhibit a diversity of form, the frequency of which can be related to stages (maturation, mature and spent) in the sexual cycle. GTH-cells showing a cytoplasm filled with electron dense polymorphic secretory granules and small rough endoplasmic reticulum (RER) vesicles, have been termed non-vacuolated. During the mature gonadal stage, such cells become increasingly vacuolated. The small RER vesicles become dilated and/or fuse, forming a single enormous cisternum (4--11 micrometer diameter), the contents of which show direct contact with the inner nuclear membrane. These morphological aspects support the idea that Rhamdia hilarii possesses only one GTH-cell type. Evidence from freeze fracture replicas suggests that membrane-associated events precursory to exocytosis take place in regions where the cell and secretory granule membranes are in close apposition. Thin section analysis of secretory granule formation revealed their derivation from the dilated extremities of the inner Golgi saccule which appears to resemble the rigid lamella described in other cells. After detachment of the inner saccule, the immature secretory granules appear to enlarge by microvesicular transport. Freeze fracture and ultrastructural data on the morphology of the cells that presumably synthetise growth hormone are also presented.

Cytology and cytochemistry of the hypophysis of the freshwater teleost Rhamdia hilarii (Val., 1840).

The characterization of cell types in the rostral pars distalis, proximal pars distalis and pars intermedia of the Bagre fish hypophysis during the reproductive cycles, as well as a cytochemical study of mucosubstances were undertaken. The cytophysiological relationship among the different acidophilic, basophilic and chromophobic cells with the several hypophyseal hormones was also considered.

Morphology of the hypophysis of the freshwater teleost rhamdia hilarii (val., 1840).

The anatomical relationship between hypophysis and encephalon of the Bagre fish (Rhamdia hilarii, Val.) was studied. The subdivisions, morphology and the relative volume (%) of the different hypophyseal regions during the reproductive stage of the fish were also considered.