Paola Belvedere

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.

Isolation of a mRNA encoding a glycine‐proline–rich β‐keratin expressed in the regenerating epidermis of lizard

During scale regeneration in lizard tail, an active differentiation of β‐keratin synthesizing cells occurs. The cDNA and amino acid sequence of a lizard β‐keratin has been obtained from mRNA isolated from regenerating epidermis. Degenerate oligonucleotides, selected from the translated amino acid sequence of a lizard claw protein, were used to amplify a specific lizard keratin cDNA fragment from the mRNA after reverse transcription with poly dT primer and subsequent polymerase chain reaction (3′‐rapid amplification of cDNA ends analysis, 3′‐RACE). The new sequence was used to design specific primers to obtain the complete cDNA sequence by 5′‐RACE. The 835‐nucleotide cDNA sequence encodes a glycine‐proline–rich protein containing 163 amino acids with a molecular mass of 15.5 kDa; 4.3% of its amino acids is represented by cysteine, 4.9% by tyrosine, 8.0% by proline, and 29.4% by glycine. Tyrosine is linked to glycine, and proline is present mainly in the central region of the protein. Repeated glycine–glycine‐X and glycine‐X amino acid sequences are localized near the N‐amino and C‐terminal regions. The protein has the central amino acid region similar to that of claw–feather, whereas the head and tail regions are similar to glycine‐tyrosine–rich proteins of mammalian hairs. In situ hybridization analysis at light and electron microscope reveals that the corresponding mRNA is expressed in cells of the differentiating β‐layers of the regenerating scales. The synthesis of β‐keratin from its mRNA occurs among ribosomes or is associated with the surface of β‐keratin filaments. Developmental Dynamics 234:934–947, 2005.

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.

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.

Changes in the electrophoretic pattern of yolk proteins during vitellogenesis in the gilthead sea bream Sparus aurata L.

Abstract 1. 1. To some extent, oocyte growth within a follicle is due, as well as to the accumulation of normal cytoplasmic components, to that of the cortical alveoli, and of hepatic-derived protein (vitellogenins). 2. 2. Yolk proteins of pre-maturational oocytes at different stages and ovulated eggs were compared by SDS-gel electrophoresis. The largest components stained by Coomassie Blue and those stained by Stains-all, which had formed during vitellogenesis, either disappeared or diminished, whilst smaller components appeared. 3. 3. The distinct changes in yolk-protein banding patterns during oocyte maturation are suggestive of extensive secondary proteolysis of yolk proteins.

Steroid biosynthesis by the ovary of the European eel, Anguilla anguilla L., at the silver stage

Abstract The ovary of the European eel, Anguilla anguilla, at the silver stage, was incubated either as an intact tissue preparation or as a homogenate with and without cofactors in the presence of [4-14C] pregnenolone and [4-14C]progesterone. Intact tissue incubates displayed a more complex metabolite profile than reinforced homogenates, and deprivation of exogenous cofactors reduced the profile even further. Among the metabolites derived from pregnenolone, the following steroids were identified by their isopolarity and isomorphicity with standard compounds: 17α-hydroxypregnenolone; dehydroepiandrosterone; progesterone; 17α-hydroxyprogesterone; and androstenedione. The last three steroids plus testosterone, 17β-hydroxyandrostenedione, and adrenosterone were identified using progesterone as a precursor. Metopirone inhibited the formation of 11-oxygenated androgens. 11-Deoxycorticosteroids were not found, indicating the absence of steroid 21-hydroxylase activity in the eel ovary. Integration of the product yield-time curves demonstrates that in vitro the activities of the enzymes 3β-, 17β-, and 11β-hydroxysteroid dehydrogenases were less apparent than those of steroid 17α,20-C21-desmolase, and 17α-, and to a lesser extent 11β-hydroxylase. Irrespective of the incubation conditions, pregnenolone produced more Δ5-3β-hydroxy-thanΔ4-3-ketosteroids, suggesting a predominance of the former biosynthetic pathway. Among the unidentified metabolites, water-soluble compounds were formed from both precursors in intact tissue incubates.

Aldosterone and the conquest of land

The sequence of the phylogenetic events that preceded the appearance of aldosterone in vertebrates is described, starting from the ancestral conversion of cytochrome P450s from oxygen detoxification to xenobiotic detoxification and synthesis of oxygenated endobiotics with useful functions in intercellular signalling, such as steroid hormones. At the end of the Silurian period [438–408 million yr ago, (Mya)], a complete set of cytochrome P450s for corticoid synthesis was presumably already available, except for mitochondrial cytochrome P450c18 or aldosterone synthase encoded by CYP11B2. This gene arose by duplication of the CYP11B gene in the sarcopterygian or lobe-finned fish/tetrapod line after its divergence from the actinopterygian or ray-finned fish line 420 Mya, but before the beginning of the colonization of land by tetrapodsin the late Devonian period, around 370 Mya. The fact that aldosterone is already present in Dipnoi, which occupy an evolutionary transition between water- and air-breathing but are fully aquatic, suggests that the role of this steroid was to potentiate the corticoid response to hypoxia, rather than to prevent dehydration out of the water. In terrestrial amphibians, there is no differentiation between the secretion rates and gluco- and mineralocorticoid effects of aldosterone and corticosterone. In sauropsids, plasma aldosterone concentrations are much lower than in amphibians, but regulation of salt/water balance is dependent upon both aldosterone and corticosterone, though sometimes with opposed actions. In terrestrial mammals, aldosterone acquires a specific mineralocorticoid function, because its interaction with the mineralocorticoid receptor is protected by the coexpression of the enzyme 11β-hydroxysteroid dehydrogenase type 2, which inactivates both cortisol and corticosterone. There is evidence that aldosterone can be also synthesized extra-adrenally in brain neurons and cardiac myocytes, which lack this protection and where the effects of aldosterone oppose those of glucocorticoids. In conclusion, the phylogenetic history of aldosterone documents the erratic progression of evolutionary changes in the course of the strenuous struggle for environmental resources and survival.

Transcriptional control of human steroid sulfatase

Steroid sulfatase (STS) is a membrane-bound microsomal enzyme that hydrolyzes various alkyl and aryl steroid sulfates, leading to the in situ formation of biologically active hormones. The entire human STS gene spans over approximately 200kbp of which the first 100kbp include the regulatory region, while the STS-coding region is located downstream. Previous studies indicated that STS expression, in different human tissues, could be regulated by at least six different promoters associated with alternative first exons. Here, we describe two new splicing patterns: the first, found in the prostatic cell line PC3, is based upon a partially coding new first exon (0d) that is spliced to a new second exon (1e). The second variant was found in the ovary and it is characterized by the novel splicing of the untranslated exon 0b to exon 0c, which is then spliced to the common exon 1b. We also report the results of a multiplex ligation-dependent probe amplification (RT-MLPA) analysis for the simultaneous detection, in qualitative and/or semi-quantitative terms, of the transcription patterns of STS in different tissues.