Gonadal remodeling and hormonal regulation during sex change of juvenile dusky grouper Epinephelus marginatus (Teleostei, Serranidae), an endangered protogynous hermaphrodite fish.

Sex change was induced in Epinephelus marginatus juveniles using a nonsteroidal aromatase inhibitor (AI), a synthetic androgen (17α-methyltestosterone; MT), and a combination of both (MT + AI) in a 90-day experiment. A detailed remodeling of the gonads, the plasma level of gonadal steroids, and immunostaining of pituitary follicle-stimulating hormone (FSH), luteinizing hormone (LH), and somatolactin (SL) cells were analyzed. Sex inversion reached the final spermatogenesis stages using MT, while AI triggered spermatogenesis, but reaching only the spermatid stage. Estradiol (E2) levels did not change in fish treated with AI but decreased throughout the experimental period in animals treated with MT and MT + AI. Testosterone (T) levels increased in animals treated with MT during the first 60 days (and combined with AI in the first 30 days), decreasing in all experimental groups at 90 days, while AI-treated animals had increased plasma 11-ketotestosterone (11-KT) levels after 90 days. In control fish, FSH- and SL-producing cells (ir-FSH and ir-SL) were restricted to pars intermedia (PI) of the adenohypophysis. Pituitary ir-FSH cells were decreased at the end of the experimental period in all treatments compared with the CT animals. LH-producing cells (ir-LH) were present in proximal pars distalis (PPD) and pars intermedia (PI) of adenohypophysis and did not change after the experimental period. The decreased number of ir-FSH cells at the end of the experiment in all treatments could be related to the negative feedback loop triggered by the increase in natural and/or synthetic androgens.

Testes of Astyanax altiparanae: the Sertoli cell functions in a semicystic spermatogenesis.

The Astyanax altiparanae (lambari) is a South American freshwater fish belonging to the family Characidae. Although some authors have described reproductive aspects of this species, this is the first study about the morphology of the testes throughout the annual reproductive cycle of A. altiparanae. Fish spermatogenesis differs from that in mammals as it occurs in cysts whose borders are defined by cytoplasmic processes of Sertoli cells, thus creating a favorable environment for spermatogenesis. The functions commonly attributed to fish Sertoli cells were investigated using stereological, light and electron microscopy in A. altiparanae. Results showed that when the Sertoli cells of A. altiparanae are in contact with germ cells, they plan a support function that culminates in the production of spermatozoa. After releasing spermatozoa, modified Sertoli cells form the duct epithelium, transform into secretory cells and release a secretion into the duct lumen where spermatids and sperm are located. Thus, the present study revealed important aspects of the testes of A. altiparanae, and propose a sequence of functions played by the Sertoli cells in this species.

Distribution of GnRH in the brain of the freshwater teleost Astyanax altiparanae.

GnRH is well known as a key decapeptide neurohormone involved in reproduction, stimulating the pituitary gland to release gonadotropins (LH and FSH), which, in turn, regulate steroidogenesis and gametogenesis. However, in addition to its reproductive functions, GnRH displays neuromodulatory roles with implications for sexual behavior. The pattern of distribution in the brain of GnRH may help reveal GnRH specific functions. Therefore, the main emphasis of this study is to detect the presence and distribution of GnRH in the brain of the freshwater teleost Astyanax altiparanae ("lambari"). The immunohistochemical method of peroxidase with an antibody raised against GnRH3 was used to detect the location of GnRHs in the brain and pituitary gland. Immunoreactivity to GnRH was found in the following encephalic areas: olfactory bulb, terminal nerve ganglion, preoptic area, nucleus of midbrain tegmentum, but also in torus longitudinalis, glomerular nucleus, and central and dorsal posterior nuclei of dorsal thalamus. In addition, cell bodies from neurons in the parvocellular and magnocellular periventricular nuclei and ventral tuberal nucleus along with many fibers including ones innervating the neurohypophysis were immunoreactive to a GnRH antiserum that detects all known eight GnRH peptides in teleosts. This is the first study describing the distribution of the complete GnRH system in the brain of A. altiparanae, which has great importance for aquaculture and ecology, and represents one of the major orders of South American teleosts--the Characiformes.

Patterns of oocyte development in natural habitat and captive Salminus hilarii Valenciennes, 1850 (Teleostei: Characidae).

Fecundity and oocyte development in Salminus hilarii female brood stock were analyzed with the aim of investigating the impact of migration impediment on oogenesis. Histological analyses of the ovaries were performed in adult females caught in two different environments--the Tietê River (natural) and captivity--and the gonadossomatic index, oocyte diameter and fecundity determined. Five germ cell development stages (oogonium, perinucleolar, cortical alveoli, vitellogenic, ripe) and two other structures (postovulatory follicles and atretic oocytes) were observed in females caught in the river. Captive animals lacked the ripe oocytes and postovulatory follicles and had a relatively higher number of atretic oocytes. Females in captivity are known to produce larger oocytes, and they release fewer eggs in each spawn (absolute fecundity) when compared with animals that are able to migrate. Our results suggest that the Tietê River is undergoing alterations which are being reflected in the reproductive performance of S. hilarii, mainly due to the presence of atretic oocytes in females caught in the river. The lack of postovulatory follicles and ripe oocytes in captive animals reveals that migratory impediment negatively impacts final oocyte maturation. However, the stage of maturation reached is adequate for ovulation induction with hormone manipulation.

Immunocytochemical identification of adenohypophyseal cells in the pirarucu (Arapaima gigas), an Amazonian basal teleost.

The adenohypophysis (AH) of juvenile pirarucu (Arapaima gigas), a representative species of the Osteoglossomorpha (bonytongue fishes, one of the oldest living groups of the teleosts), was studied using histochemical and immunocytochemical methods. The AH is comprised of the pars distalis (PD), without a clear distinction between rostral pars distalis (RPD) and proximal pars distalis (PPD), and the pars intermedia (PI). The neurohypophysis (NH) is positioned on top of the PD and penetrates and branches into the PI. In the most rostral dorsal portion of the PD, adrenocorticotropic cells and fusiform gonadotropic cells were found. In the central PD, scarce prolactin-producing cells and growth-hormone-producing cells were located mainly in the dorsal part, whereas round gonadotropic cells were abundant in the ventral portion of this region. Human thyrotropin immunoreactive cells were not found in the entire AH. In the PI, melanotropic, some adrenocorticotropic, and somatolactin-producing cells were located intermingled surrounding the neurohypophyseal branches. Our results showed that the A. gigas pituitary has some basal characteristics between the ancient Actinopterygii and the more derived teleosts.

Occurrence and significance of ovarian and follicular regression in cachara Pseudoplatystoma fasciatum (Linnaeus, 1766): a histology approach

The processes of ovarian regression and follicular atresia which reproduction was not induced by hormone in confined cachara, Pseudoplatystoma fasciatum, were investigated. The macro and microscopic characteristics (oocytes diameter and histology) of the ovaries were described every 20 days, in four stages: initial regression (Rg I = first 20 days), intermediate regression (Rg II = from 21st to 40th day), final regression (Rg III = from 41st to 80th day) and the recovering stage, called resting II (R II = from 81st to 150th day). The experiment was conducted from late January (summer - longer days) to May (autumn - shorter days). In the beginning, A0 samples showed oocyte diameters ranging from 437.5 to 1,187.5mm, suggesting that oocytes were in perinucleolar, at final maturation and atretic phases. After 150 days, the diameters reached the lowest values and a ruptured zona radiata, as well as the nearly complete reabsorption of the yolk could be visualized. At the same time, a sharp decrease in the mean values of the gonadosomatic index (GSI), water temperature, photophase and rainfall was observed. The gradual involution of this long process was dynamic and complex, affecting the spawning success (fertilization, eclosion and larvae survival rates) and, consequently, the whole productive system.

Estudaram-se os processos de regressão ovariana e atresia folicular em cachara, Pseudoplatystoma fasciatum, mantida em cativeiro, na reprodução não induzida por hormônios. As características macro e microscópicas (diâmetro dos ovócitos e histologia) dos ovários foram descritas a cada 20 dias, em quatro estádios: na regressão inicial (Rg I - os primeiros 20 dias), na regressão intermediária (Rg II - do 21° ao 40° dia), na regressão final (Rg III - do 41° ao 80° dia) e na fase de recuperação ou de repouso II (R II - do 81° ao 150° dia). O experimento foi realizado do final de janeiro (verão-dias longos) a maio (outono-dias curtos). No início do experimento, as amostras apresentaram ovócitos com diâmetros que variaram de 437,5 a 1.187,5mm, sugerindo encontrarem-se nas fases perinucleolar, de maturação final e atrésicos. Aos 150 dias, os diâmetros atingiram os menores valores e pôde-se visualizar a zona radiata rompida e o vitelo reabsorvido. Concomitantemente, houve diminuição abrupta dos valores médios do índice gonadossomático, da temperatura da água, das horas de luz e de chuva. A involução gradual do longo processo foi dinâmica e complexa, afetando o êxito da desova (taxas de fertilização, de eclosão e de sobrevivência de larvas) e, conseqüentemente, o sistema produtivo.

The reproductive cycle of male catfish Pseudoplatystoma fasciatum (Teleostei, Pimelodidae) revealed by changes of the germinal epithelium. An approach addressed to aquaculture.

The aims of the present study were to analyze the gonadal structure of Pseudoplatystoma fasciatum males during their annual cycle to enhance understanding of their reproductive biology and to improve the hormonally induced reproduction and culture of this species in hatcheries. We adopted the recently proposed method that establishes reproductive classes that are based on variations of the germinal epithelium within the year. Five reproductive classes were established: maturation (early, middle, and late), regression and recrudescence. Our observations revealed that in the spawning season P. fasciatum testes display two main functions: sperm production and sperm storage. We also concluded that the analysis of the variation of germinal epithelium was satisfactory when applied to this freshwater catfish and should be adopted for other fish species.

Light and electron microscopy oogenesis in matrinxã, Brycon cephalus.

Oocyte development has been divided into 5 distinct stages in the reared matrinxã, Brycon cephalus, based on morphological criteria by light and transmission electron microscopy: I) chromatin-nucleolus; II) perinucleolar; III) cortical alveoli; IV) vitellogenesis; V) final maturation. In stages I and II (primary growth), oocytes reside in nests close to other oocytes (chromatin-nucleolus phase) and then within a definitive follicle (perinucleolar phase) where they greatly increase in size (the Balbiani vitelline body is the main cytoplasmic component in these latter oocytes), respectively. In stage III (cortical alveolus phase) oocytes are distinguished by the appearance of variably sized cortical alveoli and the number of these structures increases steadily towards hydration. The vitelline envelope becomes prominent. In the process of vitellogenesis (stage IV) one major accumulation of yolk proteins occurs in oocytes. In stage V (final maturation), oocyte increase slightly in size. Follicle cells go through a primordial stage and later change to a squamous and to a cubical shape. The chorion grows to a tripartite structure: an outer thin porous layer, an intermediate homogenous layer and an inner thick helicoidal layer. The ovulation of females matrinxã, required hormonal stimulation and this occurred 6 and 8 h after the second application.

Ultrastructure of the germ cells in the testis of matrinxã, Brycon cephalus (Teleostei, Characidae).

This study describes at ultrastructural level the germ cells in the testis of matrinxã (Brycon cephalus) raised in captivity. The specimens 'matrinxã' were maintained in four breeding tanks of 200 m(2), at the Aquaculture Research Center at Vale do Ribeira-CEPAR, from Fishery Institute, in Pariquera-Açu City, São Paulo, Brazil. The samples were collected from March 1994 to February 1996. The testis has been classified as tubular unrestricted spermatogonial type, in which four stages of germ cells can be distinguished as follows: spermatogonia, spermatocytes (primary and secondary); spermatids and spermatozoa.

Plasma testosterone and 11-ketotestosterone levels of male pacu Piaractus mesopotamicus (Cypriniformes, Characidae)

The levels of testosterone (T) and 11-ketotestosterone (11-KT) of the South American pacu Piaractus mesopotamicus were determined by radioimmunoassay during two stages of the reproductive cycle, i.e., resting and maturation, and the gonadosomatic index (GSI) was calculated. The highest levels of T and 11-KT were reached during the maturation stage (T= 2400 + 56 pg/ml; 11-KT= 2300 + 60 pg/ml) and lower levels were maintained during the resting period. The rise in androgen levels occurred with the appearance of spermatozoa in the maturation stage, when GSI was highest.

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 testosterone and 11-ketotestosterone levels of male pacu Piaractus mesopotamicus (Cypriniformes, Characidae).

The levels of testosterone (T) and 11-ketotestosterone (11-KT) of the South American pacu Piaractus mesopotamicus were determined by radioimmunoassay during two stages of the reproductive cycle, i.e., resting and maturation, and the gonadosomatic index (GSI) was calculated. The highest levels of T and 11-KT were reached during the maturation stage (T = 2400 +/- 56 pg/ml; 11-KT = 2300 +/- 60 pg/ml) and lower levels were maintained during the resting period. The rise in androgen levels occurred with the appearance of spermatozoa in the maturation stage, when GSI was highest.

Morphological and cytochemical characterization of cell types of the adenohypophysis of Manjuba, Anchoviella lepidentostole (Fowler, 1911) (Osteichthyes, Engraulidae)

A hipófise de Anchoviella lepidentostole apresenta-se dividida em neuro-hipófise e adeno-hipófise, sendo que a caracterizaçäo morfológica e citoquímica dos tipos celulares desta regiäo foi a proposta deste trabalho. A adeno-hipófise divide-se em pars intermedia (PI) e pars distalis (PD), sendo que esta última se divide em pars distalis rostralis (PDR) e pars distalis proximalis (PDP). As células da PDR organizam-se em folículos. No epitélio folicular foram caracterizados quatro tipos celulares: I-PDR (basófilo), II-PDR (positivo à hematoxilina-chumbo/HPb+), III-PDR (PAS+, AB pH2,5+ e AF+), e IV-PDR (acidófilas). A PDP possui dois tipos celulares: I-PDP (PAS+, AB pH2,5+ e AF+) e II-PDP (acidófilas). Na PI também foram caracterizados dois tipos celulares: I-PI (HPb+) e II-PI (cromófobo aos métodos empregados)

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).

Bursa of Fabricius--mitotic index in the follicles of immunized and non-immunized chicks (Gallus domesticus).

The mitotic index in the cortical compartment of the follicles of the bursa of Fabricius from chicks immunized with sheep red blood cells (SRBC) is always higher when compaired with non-immunized ones. This mitotic index reachs its maximum 6 days after the SRBC injection, coincident with the highest serum antibody titer. The mitotic activity in the cortex of the follicles of the bursa of Fabricius is always higher than that of the medulla during the postembryonic development of chickens (PROCHAZKA, RODAK, KREJCI 1967). Otherwise it is almost established that the cortex is a zone of continuous lymphocyte proliferation, not occuring the same with the medulla. In addition these bursal histological structures are considered as 2 distinct compartments (GROSSI et al. 1974). The purpose of this paper is to study the response in the mitotic index of the cortical and medullary compartments of the follicles of the bursa of immunized and non-immunized chicks. To correlate possible changes in the mitotic index with circulating antibody levels, the serum antibody titer from the same birds was also recorded.