Steroid metabolism in vitro during final oocyte maturation in white croaker Micropogonias furnieri (Pisces: Sciaenidae).

Final oocyte maturation (FOM) is a process involving a complex set of genetical, biochemical, and morphological mechanisms. FOM involves the shift of a post-vitellogenic follicle to a pre-ovulated oocyte, which is necessary for fertilization by spermatozoan to occur. This process is regulated by a maturation-inducing steroid (MIS) at the follicular level. In other species of scienids fish the MIS, a hydroxilated derivatives of progestagen 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S), was identified. Although Micropogonias furnieri is the second fishery resource of Uruguay, basic knowledge about its endocrine process is very scarce. The aim of this work was to investigate what steroids are synthesized in vitro by the oocyte follicle of M. furnieri during the maturation process. Fragments of ovary (1 g) in three stages: post-vitellogenic (PV), maturing (Mtg), and mature (M) were incubated with 1 microg x g(-1) of tritiated progesterone (P) at 30, 60, and 180 min. After extraction with ethanol and dichloromethane, steroid metabolites were purified by TLC and rpHPLC. Two progesterone derivatives with identical chromatographic properties of 20beta-S and 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P) were purified. In other Teleost fish these steroids are biologically active as MIS. The 17,20beta-P was clearly detected in Mtg and M stages and confirmed by enzymatic oxidation with enzyme 20beta-HSD. The 20beta-S was strongly detected in all Mtg oocytes. The results do not corroborate 20beta-S as a major hormone synthesized in the ovary in FOM as occurs in other scienid fish. A differential steroid synthesis in the advanced oocyte stages suggests that the 20beta-S is acting as a MIS in M. furnieri.

Induction of oocyte maturation in the white croaker Micropogonias furnieri (Pisces: Sciaenidae) by human chorionic gonadotropin.

The present work aimed to identify the best doses of human chorionic gonadotropin (hCG) needed to induce oocyte maturation of Micropogonias furnieri and to characterize ovarian dynamics during the periovulatory period. Adult M. furnieri females with fully developed ovaries were injected intraperitoneally with four different doses of hCG. The gonadotropin response was succeeded by analyzing morphologically gonadal biopsies and following the postinjection changes in follicle diameter. Oocyte maturation was induced by three doses used: 100, 300, and 500 IU of hCG kg bw-1, and was reached 48 h after treatment with 300 and 500 IU of hCG kg bw-1, and 72 h after treatment with 100 IU of hCG kg bw-1. Concerning ovarian dynamics, only 100 and 300 IU of hCG kg bw-1 mimicked the natural ones which have a synchronic group maturation. In conclusion, the dose mimicking natural ovarian dynamics and inducing oocyte maturation more quickly is 300 IU of hCG kg bw-1.

Steroid metabolism in vitro during final oocyte maturation in white croaker Micropogonias furnieri (Pisces: Scianidae)

A maturação ovocitária final (FOM) é um processo complexo que envolve mecanismos genéticos, bioquímicos e morfológicos que conduzem à transformação de um ovócito pós-vitelogênico em um ovócito apto a ser fertilizado. Esse processo está regulado pelo hormônio esteróide indutor da maturação ovocitária final (MIS), o qual é sintetizado no folículo. Em outras espécies de Sciaenidae, o MIS foi identificado como um derivado hidroxilado da progesterona 17, 20beta, 21-trihydroxy-4-pregnen-3-one (20beta-S). Micropogonias furnieri é um recurso superexplorado na costa uruguaia, contudo, seus processos endócrinos são pouco conhecidos. O objetivo deste trabalho foi pesquisar quais esteróides são sintetizados in vitro pelos folículos em maturação de M. furnieri. Fragmentos de ovários (1 g) foram incubados em três estágios diferentes: pós-vitelogênese (PV), em maturação (Mtg) e maduros (M) com 1 mg/g de progesterona tritriada (P) durante 30, 60 e 180 min. Depois da extração dos esteróides com etanol e diclorometano, esses foram purificados e identificados utilizando-se TLC, rpHPLC e oxidação enzimática. Foram identificados dois derivados de progesterona com idênticas propriedades cromatográficas ao 20beta-S e 17,20beta-dihydroxy-4-pregnen-3-one (17,20beta-P), os quais, em outras espécies de peixes, apresentam atividade biológica como o MIS. A 17,20beta-P foi observada claramente nos estágios Mtg e M e confirmada pela oxidação com a enzima 20beta-HSD. A 20beta-S foi claramente observada nos ovários em maturação (Mtg). Os resultados não permitiram confirmar que 20beta-S é o hormônio mais sintetizado nos estágios estudados, como ocorre em outras espécies de ciaenídeos, mas a presença de uma síntese diferencial no estágio de maturação sugere que 20beta-S esteja atuando como o MIS em M. furnieri.

Induction of oocyte maturation in the white croaker Micropogonias furnieri (Pisces: Sciaenidae) by human chorionic gonadotropin

O presente estudo teve por objetivo identificar as melhores doses de gonadotrofina cariônica humana (hCG) necessárias para maturação de oócitos de Micropogonias furnieri e caracterizar a dinâmica do ovário ao longo do período pré-ovulatório. Fêmeas adultas de M. furnieri, com ovários completamente desenvolvidos, receberam intraperitonealmente quatro doses diferentes de hCG. A resposta à gonadotrofina foi acompanhada de análise morfológica de biópsias gonadais e mudanças no diâmetro dos folículos após estimulação. A maturação dos oócitos foi induzida por três doses de hCG kg bwû1, 100, 300 e 500 UI. A maturação dos oócitos foi atingida após 48 h de tratamento com 300 e 500 UI de hCG kg bwû1 e após 72 h de tratamento com 100 UI de hCG kg bwû1. Em relação à dinâmica do ovário, apenas os tratamentos com 100 e 300 UI de hCG kg bwû1 reproduziram sua dinâmica natural, apresentando maturação em grupo sincronizada. Esses resultados permitem concluir que a dose capaz de reproduzir a dinâmica do ovário e induzir maturação dos oócitos em curtos períodos é de 300 UI de hCG kg bwû1.

Effect of 17 beta-estradiol, testosterone, and 11-ketotestosterone on 17,20 beta-dihydroxy-4-pregnen-3-one production in the rainbow trout testis.

The principal hormone related to spermiation in Oncorhynchus mykiss is 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP). In the present study we analyzed the possible paracrine/autocrine effects of three other testicular steroids (17 beta-estradiol, 11-ketotestosterone, and testosterone) on the synthesis and secretion of this progestin in male rainbow trout. Pieces of testis at various stages of spermatogenesis were incubated for 24 or 48 hr with one of these steroids (5 to 800 ng ml-1) either alone or with the gonadotropin GtH II. Following incubation, 17,20 beta OHP was measured by RIA in the culture media. In vitro, only 17 beta-estradiol (E2) decreased 17,20 beta OHP secretion repeatedly and significantly when doses higher than or equal to 50 ng ml-1 were used. This effect was observed mainly at the spermiating stage and under gonadotropic stimulation. In turn, E2 did not seem to modify the testicular capacity to convert 17-hydroxyprogesterone into 17,20 beta OHP. In vivo, the circulating levels of E2 decreased at the beginning of spermiation, concomitantly with an increase of 17,20 beta OHP in plasma. These in vitro and in vivo data suggest a possible role for E2 in the regulation of 17,20 beta OHP secretion by testes, in particular during the spermiating period.

20 beta-hydroxysteroid dehydrogenase activity in nonflagellated germ cells of rainbow trout testis.

Nonflagellated germ cells were isolated from rainbow trout testis to determine their ability to synthesize 17,20 beta-dihydroxy-4-pregnen-3-one (17,20 beta OHP), a progestin involved in the control of the release of sperm. Germ cells were obtained by enzymatic dissociation (collagenase; 3 mg.ml-1, 4.5 h, 12 degrees C) from testes that were immature and at the beginning of spermatogenesis. Somatic cells were eliminated by adhesion to the culture plates. Dose-related amounts of 17,20 beta OHP were measured by RIA in culture media of germ cells incubated with increasing dosages of 17-hydroxyprogesterone (17OHP; 0.05-10 micrograms.ml-1) for 20 h at 12 degrees C. Furthermore, 3H-17,20 beta OHP was identified by chromatography and co-crystallization with a reference in incubating cells provided by 3H-17OHP (2.5 and 4 h, 12 degrees C). Other metabolites were detected but not identified. 11-Ketotestosterone (11KT) was either nondetectable by RIA in control cultures or, when detected, was found at very low levels. In no case was 11KT stimulated by addition of 17OHP or gonadotropin II (GtH II; 400 ng.ml-1); this indicated the absence of contamination by Leydig cells. Thus, to our knowledge, this report demonstrates steroidogenic activities in nonflagellated germ cells of fish testis for the first time. 20 beta-Hydroxysteroid dehydrogenase (20 beta HSD) activity was identified, showing that germ cells are able to synthesize 17,20 beta OHP at an early stage in rainbow trout testis.

Synthesis and regulation of 17α-hydroxy-20ß-dihydroprogesterone in immature males of Oncorhynchus mykiss.

Three experimental approaches were chosen to study the question if the progestin 17α-hydroxy-20ß-dihydroprogesterone (17α20ßOHP) is synthesised in testes of young Oncorhynchus mykiss, in which the absence of spermatozoa was verified histologically: first, in order to detect 20ß-hydroxysteroid dehydrogenase activity (20ßHSD), testes homogenates were incubated with (3)H-labeled 17αOHP.Metabolites were analysed by TLC, HPLC, and repeated crystallization to constant isotope ratios. One of the metabolites was identified as 17α20ßOHP-(3)H, indicating that already immature testes contain 20ßHSD activity and are able to produce 20ß-reduced steroids. Second, 17α20ßOHP was quantified by radioimmunoassay in incubates of testes fragments. The sensitivity of the gonads to gonadotropin II (GtH II) became evident when comparing incubations in the absence and presence of GtH II. Third, plasma levels of 17α20ßOHP were significantly higher in animals injected with partially purified salmon gonadotropin, compared to controls. Thus, for the first time, it could be shown that 20ßHSD is present in testicular cells other than spermatozoa. Furthermore, 17α20ßOHP is indeed secreted at a very early stage of testicular development; 17α20ßOHP secretion is also responsive to GtH II. Future studies will have to show if the functions of this progestin include the stimulation of spermatogenesis.

[Histology of the ovary of Macrodon ancylodon (Bloch and Schneider, 1801) Teleostei: Sciaenidae). Oogenesis. Post-ovulatory follicles. Atresia]. / Histología del ovario de Macrodon ancylodon (Bloch y Schneider, 1801) Teleostei: Sciaenidae) ovogénesis. Folículos post-ovulatorios. Atresia.

The reproductive cycle of Teleostean fishes may be studied from different points of view. One of them is to examine the histological changes that take place in the gonad. A histological description of the gonad is to be done first. In this work we have studied the oogenesis, atresia and post-ovulatory follicles of Macrodon ancylodon. Specimens were collected from April 1982 to May 1983 on the coast of Río de la Plata (Montevideo, Uruguay). Material used was preserved in neutralized 10% formalin inbedded in paraffin and paraffin-celloidin, sectioned at 5 - 10 microns and stained with haematoxilin-eosin. Pas-haematoxilin, and specific techniques for lipid detection were used. Six oogenetic stages were recognized: oogonias, and basophilic, lipid yolk vesicles, protein yolk vesicles, matures and ripe hydrated oocytes. Theca and granulosa are negative to lipid and cholesterol histochemic reaction techniques. Post-ovulatory follicles show structural degenerative changes. Two types of atresia are described: hypertrophic and nonhypertrophic, which apparently would not have an endocrine function. It is important to recognize post-ovulatory follicles to establish the spawning pattern and also to recognize atretic follicles due to their incidence in the fecundity of a species.