Lorenzo Colombo

Endocrinology of teleost reproduction: A testicular steroid pheromone in the black goby, Gobius jozo L.

Abstract The male black goby, Gobius jozo, is known to possess a large mesorchial gland which synthesizes large amounts of 5β-reduced androgens conjugates, especially etiocholanolone glucuronide. When gravid females releasing eggs on stripping were exposed singly to this steroid in a tank monitored through a closed television circuit, most of them manifested appetitive behaviour with negative kinesis towards the steroid source and were stimulated to lay their eggs. The steroid stimulus was effective in low concentration (below 2μM), in still water, and in the absence of sensorial reinforcements by a male partner. Females were little or not responsive outside in the interval between ovulation and oviposition. The pheromonal system of the black goby resembles that of the pig where the smell of the testicular 16-androstenes in the boars breath induces the mating stance in the estrous sow. Both species are characterized by overdevelopment of the Leydighian compartment, chemical divergence between hormonal an...

Sertoli and Leydig cell numbers and gonadotropin receptors in rat testis from birth to puberty

SummaryIn testes of rats from 2 to 60 days of age, we examined the number of Sertoli cells (SC) and Leydig cells (LC) as well as the binding of radioiodinated gonadotropins to frozen sections and homogenates. The number of SC per testis increased only during the first 2 postnatal weeks, whereas that of LC was stable up to days 7–10 and increased thereafter. The uptake of 125I-labelled human follicle-stimulating hormone (125I-FSH) to frozen sections was confined to sex cords or seminiferous tubules, while that of 125I-labelled human choriogonadotropin (125I-hCG) matched the distribution of LC in the interstitium. High affinity receptors for FSH and hCG were found in homogenates at all stages studied. The number of FSH receptors per testis increased steadily, whereas that of hCG receptors was low until days 7–10 and rose afterwards. Thus, SC in rat testis appear to proliferate in the presence of fetal LC during the first 2 postnatal weeks and to differentiate concomitantly with the emergence of the adult LC generation after day 10. The complement of FSH receptors in SC remains constant as they proliferate and increases after day 21 as they differentiate. The hCG receptor number is relatively fixed in each LC generation, being higher in adult compared to fetal LC.

Developmentally Regulated Expression and Activity of 17α-Hydroxylase/C-17,20-Lyase Cytochrome P450 in Rat Liver1

We have investigated the developmental pattern of expression and activity of 17α-hydroxylase/C-17,20-lyase cytochrome P450 (cytochrome P450c17) in the liver, stomach, duodenum, and testis of rats from day 18 of pregnancy to adulthood. In the male liver, the enzyme became detectable at birth (135 pmol/mg protein·min) at a level comparable to that in the testis (188 pmol/mg protein·min). The activity then increased dramatically, reaching a peak at 8 days (691 pmol/mg protein·min), which was more than 4-fold the testicular levels in rats of the same age or in adults. Thereafter it declined steadily, becoming undetectable from puberty onward. The hepatic peak followed a depression in testicular activity (58 pmol/mg protein·min) on day 6. Northern and immunoblot analyses showed a good temporal correlation between enzyme activity and the occurrence of P450c17 messenger RNA (mRNA) and protein. The same patterns of mRNA and protein occurrence were observed in female rat liver, indicating that the hepatic CYP17 ex...

Sequence comparison and phylogenetic analysis of fish nodaviruses based on the coat protein gene

Summary. We have amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced a 605-bp fragment covering the variable region of the coat protein gene of fish nodaviruses infecting European sea bass, Dicentrarchus labrax (n = 24), and shi drum, Umbrina cirrosa (n = 2), in the Mediterranean basin.Nine new isolates were identified and their sequences were combined with sequences in the literature to produce three different data sets. The first set, based on amino acid sequences, was used to verify the monophyly of fish nodaviruses. The second and third data sets, based on nucleic acids, were used to resolve the phylogenetic relationships between closely related fish nodaviruses. Phylogenetic analyses were performed according to the maximum parsimony and neighbor-joining methods. Our results support the monophyly of fish nodaviruses. Moreover, they confirm the subdivision of fish nodaviruses into four main clusters, in agreement with the previously suggested phylogeny of the genus Piscinodavirus, that was based on a smaller number of sequences and an alternative phylogenetic approach [14]. All the Mediterranean isolates were clustered in the group of the red-spotted grouper nervous necrosis virus and appear to have a restricted geographic distribution, except for one sequence-type (10 samples) that is widespread throughout the basin.

The knockdown of maternal glucocorticoid receptor mRNA alters embryo development in zebrafish

In zebrafish, ovulated oocytes contain both maternal cortisol and the mRNA for the glucocorticoid receptor (gr), which is spread as granular structures throughout the ooplasm. At 0.2 hpf, this transcript is relocated in the blastodisc area and partitioned among blastomeres. At 6–8 hpf, it is replaced by zygotic transcript. We used morpholinos to block translation of both maternal and zygotic gr transcripts, and a missplicing morpholino to block post‐transcriptionally the zygotic transcript alone. Only knockdown of translation produced an increase of apoptosis and subsequent craniofacial and caudal deformities with severe malformations of neural, vascular, and visceral organs in embryos and 5‐dpf larvae. Such defects were rescued with trout gr2 mRNA. Microarray analysis revealed that 114 and 37 highly expressed transcripts were up‐ and down‐regulated, respectively, by maternal Gr protein deficiency in 5‐hpf embryos. These results indicate that the maternal gr transcript and protein participate in the maternal programming of zebrafish development. Developmental Dynamics 240:874–889, 2011.

Expression analysis of steroid hormone receptor mRNAs during zebrafish embryogenesis

We have analyzed by qRT-PCR and/or RT-PCR the abundance and degradation rate of maternal mRNAs for nine steroid hormone receptors and their possible replacement by corresponding embryonic transcripts in both ovulated oocytes and embryos of zebrafish collected at 0, 1, 2, 4, 8, 12, 24 and 48 h post-fertilization (hpf). The mRNAs encoded the nuclear receptors for progesterone (pr), androgen (ar), estrogen (er alpha, er beta 1 and er beta 2), glucocorticoids (gr), mineralocorticoids (mr) and the membrane progestin receptor-alpha and beta (mpr alpha and beta). gr mRNA was the most abundant maternal transcript in oocytes and early embryos followed by er beta 2 and ar mRNAs. They declined during the first 8 hpf, being replaced, thereafter, by the embryonic messengers. er beta 1 and mr transcript levels were low until 8 hpf, but increased steadily during embryonic transcription from 24 to 48 hpf. pr transcripts were detectable only in ovulated oocytes and at 24 and 48 hpf. At these stages, there was a slight increase of er alpha mRNA that initially was very low. mPr alpha and beta mRNAs were expressed in ovulated oocytes and faintly persisted during the first 4 hpf. There was no subsequent embryonic expression of these transcripts. The possible involvement of maternal mRNAs for glucocorticoid and sex hormone receptors in the programming of early zebrafish development is intriguing, since they mainly occur at stages in which gene replication predominates over transcription.

Induction of spawning in gilthead seabream, Sparus aurata L., by a long-acting GnRH agonist and its effects on egg quality and daily timing of spawning

Ovulation and spawning were induced in gilthead seabream, Sparus aurata L., which had failed to spawn spontaneously after thermal manipulations, using a long-acting commercial preparation of leuprolide acetate, an agonist of gonadotropin releasing hormone (GnRHa), in order to study the effects of this treatment on spawning periodicity, the timing of spawning, daily egg production and embryonic viability. Grouped and single females bearing yolky follicles were implanted once with 20, 40 or 80 μg kg−1 (body weight) of GnRHa encapsulated in a biodegradable copolymer, copoly(dl-lactic/glycolic acid ratio of 3:1). All treated females started to spawn after 48 h and about 40% of the eggs were laid during the initial 10-day period with daily peaks of 105 eggs kg−1. The mean daily production of viable eggs ranged between 71–288.102 eggs kg−1. Embryonic viability was equivalent to that of control fish which were induced to spawn with a rise in water temperature. There was, however, a significantly (P < 0.01) higher egg production in the GnRHa-treated fish. While egg laying occurred regularly at dawn in controls, the long-acting GnRHa tended to shorten the normal 24-h ovulatory cycle of seabream, gradually shifting spawning from dawn to sunset. In tanks with single females, sudden spawning readjustments were observed from sunset to dawn through short 12-h ovulatory cycles. Interestingly, spawning of single females with two males resulted in a greater percentage of non-fertilized eggs in comparison to groups of spawning females maintained with a similar ratio of males, presumably as a result of territorial competition when few males try to occupy the same spawning tank.

Fine structure of the testicular gland of the black goby, Gobius jozo L.

SummaryThe homologue of mammalian Leydig cells in the testis of fish of the genus Gobius is believed to be a glandular mass lying along the mesorchium and quite distinct from the seminiferous region. This gland was studied in Gobius jozo by light and electron microscopy. Histologically, the gland is composed of cords of polyhedral cells located between the main vessels of the testis and the deferent duct. Dark and light cell varieties were observed both after staining with toluidine blue and with respect to their cytoplasmic electron density. The fine structure of the glandular cells is characterized by a well developed agranular endoplasmic reticulum in the form of random anastomosing tubules and vesicles and by very numerous mitochondria with tubulo-vesicular cristae. In some mitochondria the internal organization is paracrystalline. One or two bodies of unknown nature are Found in the Golgi region. They are partially limited by an unusually thick membrane and contain a material subdivided mostly into small vesicles. Liposomes and lipofuscin pigment granules are also present.The ultrastructural features of the glandular cells of the testis of Gobius jozo confirm a specialization in steroid hormone production.

Metabolism of pregnenolone-4-14C by the testis of Gobius paganellus (Teleostei)☆☆☆

Abstract The steroid-synthetic capacity of the mature testis of the teleost Gobius paganellus was studied in vitro by incubating the tissue with pregnenolone-4-14C of four different specific activities for four different time intervals. Metabolic products were identified by their mobilities on thin-layer chromatograms, derivative formation, radio-gas chromatography, and recrystallization to constant specific activity. Both free and conjugated metabolites appeared in the incubates. Among the free metabolites, formation of C19 steroids was observed and two different biosynthetic pathways were suggested by the kinetics of the product yield-time curves: (1) pregnenolone → progesterone → 17α-OH-progesterone → androstenedione → testosterone and (2) pregnenolone → 17α-OH-pregnenolone → dehydroepiandrosterone, the former pathway being predominant. Many polar metabolites and other unknown compounds were also detected but not identified. Conjugation of unidentified products as β-glucuronosides and “sulfate” esters is also reported. These metabolic features are discussed in comparison with those reported for higher vertebrates.

Occurrence of cytochrome P450c17 mRNA and dehydroepiandrosterone biosynthesis in the rat gastrointestinal tract

We have investigated 17 alpha-hydroxylase and C17,20-lyase activities and the presence of cytochrome P450c17 mRNA in the esophagus, stomach, duodenum, and colon of adult rats of both sexes. All tissues converted [4-14C]pregnenolone mainly to dehydroepiandrosterone (DHEA) through the 5-ene-3 beta-hydroxysteroid route as opposed to the 4-ene-3-ketosteroid pathway in a control testicular incubate. Synthesis of dehydroepiandrosterone was particularly high in the duodenum and was found to be lower in the stomach, colon and esophagus, in decreasing order. 20 alpha-Hydroxypregnenolone and progesterone were also formed primarily by the esophagus and colon, respectively. P450c17 mRNA was demonstrated by ribonuclease protection assay in the stomach and duodenum, but not in esophagus and colon. However, a 335 bp-long cDNA fragment, whose sequence corresponded to that of rat P450c17 cDNA, was amplified by reverse transcription (RT) and polymerase chain reaction (PCR) from the poly(A)+ RNAs of all four tissues. This result was further confirmed by Southern blotting using a 794-bp testicular probe. The complete sequence of P450c17 cDNA in the stomach and duodenum was identical to that reported for rat testis P450c17 cDNA. No amplification and no positive signal in Southern blotting were observed with the total RNAs from adult male adrenal and spleen, which were taken as negative controls since they had been previously found unable to form androgens from pregnenolone. Although the levels of transcription in gonads, duodenum and stomach were found to be equivalent, as indicated by the RNase protection assay and semiquantitative RT-PCR assay, P450c17 enzyme activity was much higher in the testis, pointing at a possible dissimilarity in the respective rates of mRNA translation. Thus, P450c17 is differentially expressed in the rat gastrointestinal tract, where it leads to the synthesis of the sex steroid precursor DHEA, especially in the duodenum and stomach.