The role of MAP kinases in rapid gene induction after lesioning of the rat sciatic nerve.

Lesion of the sciatic nerve caused a rapid activation of p38MAP kinase in the injured nerve adjacent to the site of transection. This activation was detectable 3 min after lesioning, increased during the next 15 min and remained high for several hours. Erk1/2 activation was also observed as early as 15 min after lesioning. Activation of these MAP kinases was seen in both the external sheaths and the endoneurium. The separation of the external sheaths from the endoneurium accelerated the p38MAP kinase activation. To evaluate whether the injury-activated MAP kinase cascades are implicated in the rapid gene induction observed after nerve lesion, experiments were performed with an ex vivo model. Segments of sciatic nerves were incubated in oxygenated Krebs-Ringer buffer. MAP kinases were activated at 15 min and remained active after 6 h. Induction of mRNA was also observed for nerve growth factor (NGF), interleukin 6 (IL-6), leukaemia inhibitory factor (LIF) and deiodinases of type 2 (D2) and type 3 (D3). Thus, the ex vivo model mimics events occurring in the animal after nerve section. Finally, nerve segments were incubated in the presence of specific inhibitors of Erk1/2 activation (U0126) and of p38MAP kinase activity (SB203580). U0126 inhibited D3, LIF and to a lesser extent NGF mRNA induction, but did not affect significantly the induction of D2 and IL-6 mRNAs. SB203580 inhibited the expression of the genes for D3 and LIF. We conclude that MAP kinase cascades, activated by nerve transection, are involved in the rapid gene induction in the nerve.

Induction of type 3 iodothyronine deiodinase by nerve injury in the rat peripheral nervous system.

Thyroid hormones are essential for the development and repair of the peripheral nervous system. The type 2 deiodinase, which is responsible for the activation of T(4) into T(3), is induced in injured sciatic nerve. To obtain information on the type 3 deiodinase (D3) responsible for the degradation of thyroid hormones, we looked for its expression (mRNA and activity) in the sciatic nerve after injury. D3 was undetectable in the intact sciatic nerve of adult rats, but was rapidly and highly increased in the distal and proximal segments after nerve lesion. After cryolesion, D3 up-regulation disappeared after 3 d in the proximal segment, whereas it was sustained for 10 d in the distal segment, then declined to reach basal levels after 28 d, when functional recovery was completed. After a transsection preventing the nerve regeneration, up-regulation of D3 persisted up to 28 d at high levels in the distal segment. D3 was expressed in peripheral connective sheaths and in the internal endoneural compartment. D3 mRNA was inducible by 12-O-tetradecanoylphorbol-13-acetate in cultured fibroblasts or Schwann cells. In conclusion, induction of D3 in the peripheral nervous system after injury may play an important role during the regeneration process by adjusting intracellular T(3) levels.

Type 2 deiodinase in the peripheral nervous system: induction in the sciatic nerve after injury.

Thyroid hormones are essential for the development and function of the brain and also for the maturation and repair of the peripheral nervous system. In the brain, most of the 3,5,3'-triiodothyronine is locally produced by 5'-deiodination of thyroxine catalyzed by the type 2 deiodinase. The absence of any information about thyroid hormone metabolism in the peripheral nervous system prompted us to study the expression of type 2 deiodinase (mRNA and activity) in the peripheral nervous system. Expression of type 2 deiodinase mRNA was very low in the sciatic nerve of rats until day 5 after birth, then increased from day 10 to 35-45 and gradually decreased afterwards, down to the low basal levels observed in the adult. A lesion of the sciatic nerve in the adult induced an increase in type 2 deiodinase mRNA and activity. After a cryolesion, the stimulation was observed as early as 4 h and mRNA levels increased until 24-48 h, then gradually declined down to basal levels around 28 days, when regeneration and functional recovery were completed. After a permanent transection, up-regulation of type 2 deiodinase persisted in both proximal and distal segments until the end of the experiment (28 days). Transection and cryolesion were also followed by increased type 2 deiodinase mRNA expression in the ipsilateral L4/L6 dorsal root ganglia within 24 h. Both mRNA and activity were found in the peripheral nerve sheaths but not in the internal compartment of the intact or injured nerve. Cultured fibroblasts from the sciatic nerve expressed type 2 deiodinase 4 h after stimulation by 10 microM forskolin, whereas purified Schwann cells did not. The present study provides evidence that the peripheral nervous system has its own system responsible for the local production of 3,5,3'-triiodothyronine, which may play a key role during the regeneration process.

Reversible induction of rat hepatoma cell polarity with bile acids.

A dynamic model for inducing and isolating polarized cell colonies from differentiated rat hepatoma was established with chenodeoxycholic acid (CDCA). Cells were treated with 75 microM CDCA in a 1% solvent mix (DMSO/ethanol: 0.5%/0.5%) for 11 days and positive Fao-BA1 and C2rev7-BA1 clones were isolated, respectively, from Fao and C2rev7. Cell polarization in these two clones was demonstrated by (i) the detection of (gamma)-glutamyl transpeptidase activity (gamma)-GT) and the presence of specific proteins, namely aminopeptidase N (APN), bile acid export pump (Bsep), multidrug resistance-associated protein 2 (Mrp2) at the canalicular pole, (ii) the expression of tight junction (ZO-1) and basolateral (1-18) marker proteins, (iii) the presence of regular microvilli in the cavities sealed by tight junctions, and (iv) functional bile canaliculi-like structures with the capacity to metabolise and secrete carboxyfluorescein diacetate dye. The polarized phenotype was maintained for more than 200 cell generations in the presence of CDCA and could be modulated by cell density or omitting the inducing agent. Hence this cellular model is well suited for studies on hepatic differentiation, polarization and bile salt trafficking with therapeutic implications.

Neurosteroid progesterone is up-regulated in the brain of jimpy and shiverer mice.

Concentrations of neurosteroids have been measured in the brains of postnatal myelin mutants jimpy (jp) and shiverer (shi) mice and of their normal controls. Progesterone (PROG) concentrations were increased more than threefold in the brains of mutant mice. Marked astroglial reaction occurs in the brains of jp mice and to a much smaller extent in shi ones. Whereas the mitochondrial benzodiazepine/diazepam binding inhibitor (DBI) receptor (MBR) was below the immunohistochemical detection limit in normal mice (except in the choroid plexus and ependyma cells), it was significantly expressed in many reactive astrocytes of jp and shi mice brains. DBI-like peptides, investigated either by immunohistochemistry or by radioimmunoassay, were expressed to similar extents in mutant and control mice. Reversed-phase HPLC indicated that DBI-like peptides were almost exclusively of the triakontatetraneuropeptide size. It was concluded that the increased expression of MBR (involved in the intramitochondrial delivery of cholesterol to P450scc) likely accounts for the large PROG content in mutant mice brain. The role of PROG in myelin repair is discussed.

Stage-specific expression of the immunophilin FKBP59 messenger ribonucleic acid and protein during differentiation of male germ cells in rabbits and rats.

FKBP59 is an immunophilin that binds the immunosuppressant drugs FK506 and rapamycin. It is a 90-kDa heat shock protein (hsp90)-binding protein that was originally discovered as a member of steroid receptor complexes. FKBP59 is ubiquitous and well conserved, and it appears to be a multifunctional protein. It has peptidylprolyl cis-trans-isomerase activity and therefore may be involved in protein folding as a molecular chaperon. FKBP59 also includes in its structure a tetratricopeptide repeat (TPR) motif and could have a function in the cell division process. In situ hybridization experiments revealed an overexpression of FKBP59 mRNA in rabbit and rat testes in comparison with other organs. This high level of expression was restricted to germ cells of the seminiferous epithelium. Increasing levels of FKBP59 mRNA became obvious from the midpachytene stage, and the strongest signal was observed in the late pachytene, diplotene, and diakinesis primary spermatocytes. The expression then declined progressively in postmeiotic early spermatids. High expression of FKBP59 mRNA did not occur in earlier and later germ cell stages. During prepubertal development, Northern blot and in situ hybridization of rat testes examined at various postnatal ages revealed that FKBP59 mRNA was not expressed at over a basal level until the pachytene stage. High expression of the FKBP59 protein was demonstrated in the rabbit testis by Western blot and was localized by immunohistochemistry from late pachytene spermatocytes to round spermatids. The cell type-specific and developmental stage-specific expression of FKBP59 at a restricted period of male germ cell differentiation suggests that FKBP59 is involved in a specific function during the cell division process.

Lead affects steroidogenesis in rat Leydig cells in vivo and in vitro.

Lead is known to impede the male reproductive function, however, the mechanisms through which the adverse effects are mediated are not clearly elucidated. In order to get insight into those mechanisms, we have examined the effects of lead on the biosynthesis of steroid hormones by Leydig cells in the rat. To determine whether lead has a direct action on Leydig cells, we have compared the concentrations of testosterone secreted by Leydig cells in ex vivo experiments after animals had been injected with high doses of lead and in vitro experiments with Leydig cells from normal rats maintained in culture in presence or absence of lead. In ex vivo experiments male Spargue-Dawley rats were injected i.p. with lead acetate (8 mg lead/kg/day, 5 days a week for 5 weeks) or with sodium acetate. Testosterone production by Leydig cells isolated and maintained in culture for 48 h was then assessed under basal conditions or after stimulation by human chorionic gonadotrophin (hCG). Both basal and hCG-stimulated testosterone production dropped by 59% and 37%, respectively, with Leydig cells from lead-exposed rats. For in vitro experiments, cultures of Leydig cells from control rats were exposed to various concentrations of lead acetate for different periods. Dose and time-dependent reductions of testosterone level were observed in the culture medium. The effective doses of hCG for maximal and half-maximal testosterone production did not change, indicating that the sensitivity of Leydig cells to hCG was not impaired by exposure to lead in vitro. Progesterone production was also decreased after this exposure. The negative effect of lead on testosterone and progesterone production was correlated with the lower expression of the enzymes cytochromes P450scc (CYP11A1) and P450c17 (CYP17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) involved in steroid hormone biosynthesis, as shown by immunohistochemistry. Ultrastructural alterations of the smooth endoplasmic reticulum observed after lead administration might be correlated with the lower expression of the microsomal enzymes P450c17 and 3 beta-HSD. Our results indicate that lead can adversely affect the Leydig cell function by impairing directly steroidogenesis.

Androgen biosynthesis in the stomach: expression of cytochrome P450 17 alpha-hydroxylase/17,20-lyase messenger ribonucleic acid and protein, and metabolism of pregnenolone and progesterone by parietal cells of the rat gastric mucosa.

Dehydroepiandrosterone (DHEA) and its conjugates persist in the rat brain, for up to 1 month after ablation of both adrenals and gonads. Since DHEA synthesis in brain from pregnenolone (PREG) was excluded, we have considered other tissular sources including the digestive tract. In situ hybridization with specific oligonucleotide probes showed that the parietal cells of the gastric mucosa, contrary to other cell types, strongly expressed P450(17) alpha messenger RNA. Expression of the enzyme in the parietal cells was confirmed by immunocytochemistry with specific antibodies. An intense reaction was observed in the stomach of adult males and of cyclic or pregnant females. Access to food did not influence the intensity of immunostaining. It appeared at postnatal days 16-21, then the number of positive cells increased rapidly and leveled off at adult age. Parietal cells were released by pronase digestion of everted stomachs from adult male and female rats and were purified by density gradient centrifugation on Nycodenz. 5 x 10(4) to 1.6 x 10(6) cells were incubated with either 1 microM 14C-PREG or 14C-progesterone (14C-PROG) at 37 C under 95% O2-5% CO2, for 10-180 min. PREG was converted to 17-OH PREG and to androstenediol, whereas PROG was converted to 17-OH PROG and to testosterone. Only minute amounts of either DHEA or androstenedione, respectively, were detected at any incubation time, indicating their fast conversion to the corresponding 17 beta-hydroxysteroids. 3H-25-OH cholesterol was not metabolized to 3H-PREG, and 14C-PREG was not converted to 14C-PROG, in accordance with negative immunocytochemical results with antibodies to cytochrome P450scc and 3 beta-hydroxysteroid dehydrogenase delta 5-->4-isomerase (3 beta-HSD). In conclusion, the parietal cells, which are known as the source of gastric acid secretion, can synthesize testosterone from PROG and androstenediol from PREG. The physiological relevance of such conversions remains to be established.

Corticotropin-releasing hormone (CRH) is expressed at the implantation sites of early pregnant rat uterus.

We have shown previously that the epithelial cells of human endometrium produce CRH. The biological role of endometrial CRH is not yet known. Among other things, CRH appears to be involved in the inflammatory process, acting as an autocrine/paracrine proinflammatory regulator. Since the reaction of endometrium to the invading blastocyst has characteristics of an aseptic inflammatory reaction, we have hypothesized that endometrial CRH may participate in the inflammatory phenomena taking place at the implantation site of blastocyst. In the present study we demonstrate a higher content of immunoreactive (IR)-CRH and CRH mRNA in the implantation sites of early pregnant rat uterus compared to the inter-implantation regions. Specifically we have found that: a) rat uterus contained a 1.3 kb CRH transcript, similar or identical in size to that present in human placenta, b) the size of the IR-CRH present in uterine extracts was similar to authentic hypothalamic CRH, c) Northern blot analysis showed that the content of CRH mRNA in uterus at the implantation sites was 3.5 fold higher compared to that in the inter-implantation regions and finally, d) immunohistochemical localization of IR-CRH in early pregnant rat uterus revealed positive staining of the luminal epithelial cells in both implantation and inter-implantation uterine regions, while decidualized stromal cells were positive only at the implantation sites. Our data suggest that endometrial CRH may play a role in the implantation of blastocyst.

Suppressed expression of the cytochrome P45017 alpha protein in the testicular feminized (Tfm) mouse testes.

The testes of testicular feminized (Tfm) mice synthesize and secrete abnormally low amounts of testosterone, as a consequence of selectively decreased cytochrome P450(17 alpha) activity. To investigate the mechanism of this deficiency, three steroidogenic enzymes were immunolabeled in the testes of normal and Tfm adult (2.5-6 month old) mice. Cholesterol side-chain cleavage cytochrome P450 (P450scc) and delta 5-3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) were detected in the Leydig cells of both normal and Tfm mice whereas, in contrast to normal mice, only a small proportion of Leydig cells were immunostained for cytochrome P450-17 alpha-hydroxylase, C17-->20 lyase (P450(17 alpha)) in the testes of Tfm mice. The numbers of cells differed from male to male and interestingly were markedly higher in the right testis. Explants of testes from Tfm mice were kept in organ culture at 32 degrees C for 45 h, with or without dibutyryl cyclic AMP (100 or 500 mumol/l). All Leydig cells remained positive for P450scc and 3 beta-HSD, and P450(17 alpha) became detectable in the majority of Leydig cells in both left and right testes, showing that the lack of expression of P450(17 alpha) protein in Tfm mouse testes in vivo is not structural but is a regulatory phenomenon.

Astrocytes and neurosteroids: metabolism of pregnenolone and dehydroepiandrosterone. Regulation by cell density.

The rat central nervous system (CNS) has previously been shown to synthesize pregnenolone (PREG) and convert it to progesterone (PROG) and 7 alpha-hydroxy-PREG (7 alpha-OH PREG). Astrocytes, which participate to the regulation of the CNS function, might be involved in the metabolism of neurosteroids. Purified type 1 astrocytes were obtained from fetal rat forebrain with the use of selective culture conditions and were identified by immunostaining with specific antibodies (GFAP+, A2B5-). They were plated at low, intermediate, or high densities (2.5-5 x 10(5), 1-2 x 10(6), or 4-8 x 10(6) cells/dish, respectively) and maintained for 21 d. They were then incubated with 14C-PREG and 14C-DHEA for 24 h and the steroids extracted from cells and media were analyzed. Most radioactive derivatives were released into incubation media. Two metabolic pathways were mainly observed. PREG and DHEA were oxidized to PROG and androstenedione (ADIONE), respectively, [3 beta-hydroxysteroid-dehydrogenase, delta 5-->4 3-ketosteroid-isomerase (3 beta-HSD) activity], and converted to 7 alpha-OH PREG and 7 alpha-OH DHEA, respectively (7 alpha-hydroxylase activity). After low density plating, the formation of PROG and ADIONE was approximately 10% of incubated radioactivity, tenfold larger than that of 7 alpha-hydroxylated metabolites. In contrast, after high density plating, low levels of PROG and ADIONE were formed, whereas the conversion to either 7 alpha-OH PREG or 7 alpha-OH DHEA was > or = 50%. The results expressed per cell indicated that the 3 beta-HSD activity was almost completely inhibited at high cell density, in contrast to the 7 alpha-hydroxylation which was maintained or increased. The pattern of steroid metabolism was related to cell density at the time of measurement and not to an early commitment of cells: when primary cultures were plated at high density (8 x 10(6) cells/dish), then subcultured after several dilutions (3-, 9-, or 27-fold), the 3 beta-HSD activity was recovered only at low density. Furthermore, when 5 x 10(5) cells were centrifuged and the resulting clusters were plated, 3 beta-HSD activity was decreased, whereas steroid 7 alpha-hydroxylation was enhanced. This implies that cell density per se, but neither cell number nor a diffusible factor(s) is involved in the regulation of steroid metabolism. We conclude that astrocytes in culture metabolize PREG and DHEA, and that the metabolic conversions and, therefore, the related enzymatic activities depend on cell-to-cell contacts.(ABSTRACT TRUNCATED AT 400 WORDS)

Immunoreactive cytochrome P-450(17 alpha) in rat and guinea-pig gonads, adrenal glands and brain.

The cytochrome P-450(17 alpha)-hydroxylase, 17----20 lyase (P-450(17 alpha)) is the key enzyme responsible for the biosynthesis of androgens in steroidogenic organs. Its cellular localization has been examined with an immunohistochemical technique. In immature rat ovary, P-450(17 alpha) was first detected in sparse interstitial cells on postnatal Day 8. The number of immunoreactive interstitial cells increased thereafter and the intensity of P-450(17 alpha) staining in these cells was highest at 3 weeks of age. The intensity of staining then started to decline and was very faint at Day 35. From 6 weeks on, the distribution of immunoreactive P-450(17 alpha) was of the adult type: it was detected exclusively in the thecal cells of the large antral, preovulatory, follicles. P-450(17 alpha) was not detectable during pregnancy except on the day of parturition, when thecal cells were transiently immunoreactive. The staining had vanished 24 h after delivery. Human chorionic gonadotrophin (hCG), injected into immature females on Days 24 to 26, induced P-450(17 alpha) prematurely in thecal cells. When injected on Days 12 to 14 of pregnancy, hCG also induced P-450(17 alpha) in the thecal cells surrounding the largest follicles, whereas the interstitial and luteal cells were not immunostained. The antiprogestin RU486, injected on Day 16 of pregnancy, reinstated P-450(17 alpha) (and P-450scc) immunoreactivity in the thecal cells. Oestradiol selectively suppressed P-450(17 alpha) expression in the thecal cells of RU486-treated females. In immature guinea-pig ovary, P-450(17 alpha) was immunostained in thecal cells, not in interstitial cells, although the interstitial cells expressed the delta 5-3 beta-hydroxysteroid dehydrogenase. P-450(17 alpha) was also immunolocalized in the Leydig cells of rat and guinea-pig testes, and in the guinea-pig adrenal cortex (zonae fasciculata and reticularis), but not in the rat adrenal cortex. P-450(17 alpha) was not detectable in the brain of either rat or guinea-pig.

Neurosteroids: biosynthesis, metabolism and function of pregnenolone and dehydroepiandrosterone in the brain.

Pregnenolone (P) and dehydroepiandrosterone (D) accumulate in the brain as unconjugated steroids and their sulfate (S) and fatty acid (L) esters. The microsomal acyl-transferase activity is highest in immature (1-3 weeks old) male rats. The immunocytochemical and biochemical evidence for P biosynthesis by differentiated oligodendrocytes is reviewed. The importance of P synthesis for its brain accumulation is assessed by the intracysternal injection of the inhibitor aminoglutethimide. Primary glial cell cultures convert P to 20-OH-P, PL, progesterone, 5 alpha-pregnane-3,20-dione and 3 alpha-hydroxy-5 alpha-pregnane-20-one (Polone). Astroglial cell cultures also produce these metabolites, whereas neurons from 17-day mouse embryos only form 20-OH-P. P and D are converted to the corresponding 7 alpha-hydroxylated metabolites by a very active P-450 enzyme from rat brain microsomes. Several functions of neurosteroids are documented. P decreases in olfactory bulb of intact male rats exposed to the scent of estrous females. D inhibits the aggressive behavior of castrated male mice towards lactating female intruders. The D analog 3 beta-methyl-androst-5-en-17-one, which cannot be metabolized into sex steroids and is not demonstrably androgenic or estrogenic is at least as efficient as D. Both compounds elicit a marked decrease of PS in rat brain. The Cl- conductance of gamma-aminobutyric (GABAA) receptor is stimulated by GABA agonists, an effect which is enhanced by Polone and antagonized by PS. Thus, P metabolites in brain as well as steroids of extraencephalic sources may be involved physiologically in GABAA receptor function. The neurosteroids accumulated in brain may be precursors of sex steroid hormones and progesterone receptors have been localized in glial cells. P and D do not bind to any known intracellular receptor. A heat stable P binding protein has been found in brain cytosol with distinct ligand specificity. A binding component specific for steroids sulfates, including Polone S, DS and PS, in the order of decreasing affinity is localized in adult rat brain synaptosomal membranes. Its relationship to the GABAA receptor is under current investigation.

Cell-specific variations and hormonal regulation of immunoreactive cytochrome P-450scc in the rat ovary.

Specific rabbit antibodies to the bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc) were used to cross-react with the enzyme in the rat ovary. The luteal cells of cyclic, pregnant, and pseudopregnant rats were immunostained. P-450scc was also expressed in the interstitial cells of prepubertal and cyclic adult rats, and in the thecal cells lining the preovulatory follicles. In cyclic females, RU 486 and oestradiol increased the intensity of P-450scc immunostaining. The granulosa cells of ovarian follicles whatever their stage of development, including preovulatory follicles, were not labelled, except after ovulation. The intensity of immunostaining of thecal and interstitial cells decreased during early pregnancy or pseudopregnancy, and disappeared after Day 9, whereas these cells were intensely labelled 24 h after parturition. The immunostaining of thecal and interstitial cells was again detected in 18-day pregnant rats, treated with the antiprogesterone RU 486. It is therefore concluded that both oestradiol and progesterone are involved in P-450scc regulation.

Neurosteroids: cytochrome P-450scc in rat brain.

The steroid hormones corticosterone and testosterone are supplied to the central nervous system by endocrine glands, the adrenals and gonads. In contrast, the 3 beta-hydroxy-delta 5-derivatives of cholesterol, pregnenolone and dehydroepiandrosterone, accumulate in the rat brain through mechanisms independent of peripheral sources. Immunohistochemical studies have been performed with specific antibodies to bovine adrenal cytochrome P-450scc, which is involved in cholesterol side-chain cleavage and pregnenolone formation. The enzyme was localized in the white matter throughout the brain. Scarce clusters of cell bodies were also stained in the entorhinal and cingulate cortex and in the olfactory bulb. These observations strongly support the existence of "neurosteroids," which have been posited on the basis of biochemical, physiological, and behavioral studies.

Alkaline phosphatase activity in the membrane of Xenopus laevis oocytes: effects of steroids, insulin, and inhibitors during meiosis reinitiation.

The mechanism of steroid hormone-induced reinitiation of meiosis in Xenopus laevis oocytes in vitro involves interaction of the hormone with an ooplasma membrane receptor and early changes of enzymatic activities (adenylate cyclase, p48 protein kinase). In full-grown (stage 6) oocytes, we have observed cytochemically, at the ultrastructural level, alkaline phosphatase activity in the ooplasma membrane of microvilli, its decrease by 2 hr of progesterone action, and its complete disappearance at the time of germinal vesicle breakdown (GVBD). Insulin (30 micrograms/ml) also provoked a decrease of phosphatase activity, although it did not promote GVBD under these circumstances. When oocytes were exposed simultaneously to progesterone (1 microM) and insulin (30 micrograms/ml), the enzymatic activity disappeared earlier than with any one of them, correlating with the faster occurrence of GVBD. Inhibitors of alkaline phosphatase activity and competitive substrates potentiated progesterone action on GVBD. Insulin and beta-glycerophosphate potentiating activities were additive. These results suggest that the ooplasma membrane alkaline phosphatase may be implicated in the course of reinitiation of meiosis in X. laevis oocytes.

Testosterone-induced meiotic maturation of Xenopus laevis oocytes: evidence for an early effect in the synergistic action of insulin.

Meiosis reinitiation in oocytes (stage 5-6 of Dumont) isolated free of follicle cells by collagenase treatment from ovarian pieces of Xenopus laevis, was studied in observing the germinal vesicle breakdown (GVBD) provoked by progesterone and testosterone (0.1 nM-1 microM), alone or in association with insulin (30 micrograms/ml). Testosterone, was more active than progesterone to elicit GVBD in vitro, raising the question of the relative roles of both steroids in the physiological maturation process in vivo. The potentiating effect of insulin, already observed on progesterone action, was also demonstrated upon testosterone effect; the results suggested that it occurs during the early phase of hormone-induced meiosis reinitiation.

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: synergistic action of progesterone and insulin.

Progesterone is the hormone that reinitiates the meiotic division of amphibian oocytes and insulin and insulin-like growth factors are also active on defolliculated oocytes in vitro. We have studied Xenopus laevis oocytes (stage 5-6) of different hormonal sensitivities, obtained from unstimulated and from human chorionic gonadotrophin-stimulated females. Some oocytes from unstimulated females were also precultured with a subthreshold level of progesterone. A dose-dependent potentiating effect of the action of progesterone was observed with insulin, and this was particularly remarkable in low-sensitivity oocytes. Since in the presence of insulin, the optimally effective concentration of progesterone was much reduced (as an example from 1 mumol/1 to 50 nmol/l), it is suggested that an insulin-like growth factor may play a physiological role in the reinitiation of meiosis in ovaries.

[Potentiation of the effect of steroid hormones (progesterone and testosterone) inducing the reinitiation of meiosis in Xenopus oocytes in vitro, by inhibitors of phosphatase activity]. / Potentialisation de l'effet des hormones stéroïdes (progestérone et testostérone) inductrices de la reprise de la méiose de l'ovocyte de Xénope in vitro, par des inhibiteurs de l'activité phosphatasique.

The induction of meiosis reinitiation by steroid hormones (progesterone and testosterone) in Xenopus laevis oocytes was studied in vitro in presence of inhibitors of phosphatase activity such as beta-glycerophosphate, considered as a competitive inhibitor, and the two ions, Zn++ and MoO--4. Kinetics of the germinal vesicle breakdown indicating the reinitiation of meiosis, have shown that while these phosphatase inhibitors were not active by themselves under the present experimental conditions, they enhanced the process elicited by progesterone or testosterone.

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin.

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.