Etienne-Emile Baulieu

Progesterone-induced meiosis in xenopus laevis oocytes: a role for cAMP at the “maturation-promoting factor” level

Cholera toxin inhibition of progesterone-induced meiosis of Xenopus laevis oocytes in vitro has been correlated with increased cAMP levels. Inhibition of germinal vesicle breakdown (Gvbd) and cAMP increase occurred after a lag period of 2 hr, when cholera toxin was injected, or 4--5 hr, when applied externally. The ability of the maturation-promoting factor (Mpf) to provoke Gvbd when injected into recipient oocytes was found to be dependent upon whether the oocytes had been exposed to cholera toxin alone or to toxin and progesterone. With the former, cAMP levels were elevated and Mpf activity was abolished, whereas with the latter, the increase in cAMP was less pronounced and Mpf activity was observed. Injection of cAMP or its 8-thio derivatives shortly before the appearance of progesterone-induced Mpf abolished Gvbd. If injected earlier or later, no inhibition was observed. In contrast, cholera toxin inhibited maturation even when added several hours before progesterone, suggesting a sustained accumulation of cAMP. No Gvbd occurred when 8-thio-methyl-cAMP was injected together with Mpf. These data suggest that cAMP is involved in the control of the formation/amplification and/or activity of Mpf-a result which may be of general significance in cell division mechanisms.

Growth Factors Acting Via Tyrosine Kinase Receptors Induce HSP90a Gene Expression

Hsp90 is a heat-shock protein constitutively expressed in most cells. Besides regulation by thermal stress, the expression of hsp90 is also positively regulated by developmental and mitogenic stimuli. The effect of serum and insulin on protein and hsp90 alpha-mRNA levels has been studied in the chicken hepatoma cell line DU249. The culture of cells in serum-free medium resulted in a decrease of hsp90 alpha-mRNA level. A transient increase was observed at 6-9 h after serum restimulation. The expression of hsp90 gene was also increased by insulin alone in a dose-dependent manner and was maximum between 6 and 9 h treatment. The insulin induced increase of hsp90 alpha-mRNA was suppressed by cycloheximide (10 micrograms/ml) but not by an inhibitor of DNA synthesis, demonstrating that this induction requires protein neosynthesis. In serum starved cells, other growth factors (IGF1, EGF and bFGF) showed a positive effect on hsp90 alpha-mRNA level which took place before DNA synthesis with the same time-course as that of insulin. With PDGF, the induction of hsp90 alpha-mRNA occurred earlier. The time interval between the maximum of hsp90 alpha-mRNA induction and that of DNA synthesis was the same for all growth factors studied. From these results, we conclude that growth factors acting via tyrosine kinase receptors up-regulate hsp90 alpha-mRNA level in a DNA synthesis independent manner, possibly in late G1.

DISCUSSION PAPER: INDUCTION BY PROGESTERONE AND A “MATURATION-PROMOTING FACTOR” OF SOLUBLE PROTEINS IN XENOPUS LAEVIS OOCYTES IN VITRO*

In vitro incubation of isolated X. laevis oocytes with progesterone induces the specific labeling of proteins during maturation. They are detected as several discrete peaks with a double-labeling technique that involves the injection of [3H] leucine into hormone-treated and [14C] leucine into control cells. The peaks are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Identical proteins are obtained with two other steroids that provoke maturation and with p-chloromercuribenzoate. The same progesterone-induced proteins are observed even if oocytes have been treated with actinomycin D or ethidium bromide or have been enucleated. Cycloheximide suppresses the labeling of proteins that are induced by progesterone, if administered either at the time of radioactive amino acid exposure or after prelabeling the oocytes to determine whether the hormone-induced peaks are due to compartmental changes of the proteins. A cytoplasmic fraction, obtained from maturing oocytes before GVBD, promotes maturation when injected into resting oocytes and therefore contains an MPF. After this injection, GVBD occurs earlier than during incubation with progesterone and is accompanied by the formation of the same proteins and of MPF, both suppressed by cycloheximide. The possibility that MPF is an induced protein is then discussed. It has been also found that there is a decrease in membrane permeability, as evidenced by decreased leucine uptake into proteins of oocytes incubated in radioactive amino acids and exposed to progesterone. The same decrease occurs after injection of MPF-containing cytoplasm into oocytes.

Chicken oviduct nuclear oestrogen receptors: Aspects of steroid hormone action

I. The Scatchard plots as well as competition experiments carried out with several compounds binding to the chick oviduct oestrogen receptor suggested existence of a single class of binding sites while the dissociation kinetics displayed biphasic pattern. This apparent paradox can be resolved by a proposed model where two interconvertible forms of the receptor-ligand complex are assumed. II. The cumulative (24 h) oestrogenic action on withdrawn chicken magnum in vivo was dependent on the length of retention of the receptor-agonist complex in the nuclear fraction. It was found to be in a better correlation with the rate of dissociation (“slow” component) of the receptor-ligand complexes than with their apparent equilibrium affinity constant. The antagonists (tamoxifen and monohydroxytamoxifen), in spite of their ability to maintain elevated nuclear receptor concentration for long periods of time (> 24 h), had no oestrogenic activity. Remarkably, the apparent equilibrium affinity constant of monohydroxytamoxifen was found to be 1.8 times greater than that of oestradiol. III. Following administration of various doses of oestradiol benzoate, the induction of ovalbumin and conalbumin synthesis followed sigmoidal-shaped curves rather than showing a definite “lag”; the initial part of the curves was dose-independent within the range yielding maximum receptor translocation at 1 h post-injection (0.1–3.0 mg/kg). When tamoxifen was given 2–4 h after the oestradiol benzoate, the relative rate of ovalbumin synthesis continued to increase up to 4 h while that of conalbumin synthesis was arrested virtually immediately. IV. Models of oestrogen action on transcription level were analysed in respect to the experimental data concerning the induction of ovalbumin and conalbumin synthesis. Three different models were shown to lead to the expression for specific induced mRNA concentration y=ymax(1+μk−μ)exp(−kt)−kk−μexp(−μt) where µ is the degradation rate constant of the mRNA, k having different meaning for different models: A. Model involving an intermediary product catalyzing transcription of the DNA sequence concerned (k-degradation rate constant of the intermediary product); B. The hormone-receptor molecule forms a complex with a regulatory site and activates the gene transcription (k-rate constant of dissociation of this complex); C. The receptor-hormone molecule binds to a “repressor” molecule causing it to dissociate (with the rate constant k) releasing the gene for transcription. The above expression can be made fit the available experimental data for oestrogen induction of ovalbumin and conalbumin synthesis.

Aspects of steroid hormone-target cell interactions.

Being asked to present this Gregory Pincus memorial lecture naturally brings back many memories of this great man. The first time I saw him was in Paris when he visited Max Jayle’s laboratory twenty years ago. Later, during the period I spent in the Lieberman laboratory, I went to the Worcester Foundation where I spoke about dehydroepiandrosterone sulfate, which was my favorite topic at that time. This work was subsequently presented at the 1964 Laurentian Hormone Conference at Lake George, New York. As a relatively young worker, I was at that time almost only interested in my own research. Pincus was a real leader, helping science by motivating individuals to expand their fields of interest. He enlarged my vision, drawing my attention to the overall significance of oral contraception. I consider that Dr. Pincus made an exceptional contribution by introducing his contemporaries to one of the most important tools that science has given to man. He sent me to the Puerto Rico clinics, asked me to participate in the organization of Steroid Congresses, and to sit on WHO Committees on Contraceptive Development. Thanks to him, I learned a lot, and even believe that my own work in the laboratory benefited from this sort of social activity. Needless to say, I am really happy that the Gregory Pincus Award Committee members have this year honoured the domain of steroid receptors which embraces both fundamental and applied research as Pincus would have liked. I am proud to have been selected and happy to share the Award with my very good friend Elwood Jensen whose pioneering contributions are known to all (1).

STEROIDAL AND PEPTIDIC CONTROL MECHANISMS IN MEMBRANE OF XENOPUS LAEVIS OOCYTES RESUMING MEIOTIC DIVISION

Stage 5-6 Xenopus laevis oocytes are arrested in the prophase of the first meiotic division, and can be studied in vitro after removal from their follicle cell environment. While they do not mature spontaneously, they demonstrate germinal vesicle (nucleus) breakdown (GVBD) if exposed to approximately 1 microM progesterone, the hormone released in vivo at the time of ovulation and maturation. They become an egg ready to be fertilized. The progesterone-oocyte interaction, contrary to what is observed in endocrine steroid target organs so far studied, takes place at the surface membrane level and is not narrowly progesterone specific, since other hormones such as cortisol or testosterone also can resume meiosis in vitro. This is the first description of such a paracrine steroid system, which depends however on a receptor mechanism, as revealed by physico-chemical experiments, studies with antagonistic (competitive) steroids, and cell-free specific inhibitory effects on membrane bound adenylate cyclase. It was found that insulin and related growth factors (MSA, IGF) are also meiosis reinitiators. Insulin potentiates low progesterone concentration (approximately 1 nM), also in completely denuded oocytes (free of the vitelline membrane). From these observations, it is suggested that there may be a physiological cooperative involvement of a steroid (progesterone) and an insulin-like peptidic factor within the ovaries to promote oocyte maturation in vivo. The molecular mechanisms implicated in the hormone-dependent changes of c-AMP and Ca2+ remain to be elucidated in detail, as well as the respective role of these two sets of metabolic events.

Current Approaches to Steroid Hormone-Cell Interactions

In this paper, we give a summary of the findings which have led to the present understanding of steroid hormone receptors (Parts I, II and III), and we discuss a new physiological approach to receptor pharmacology, based on differential hormone binding properties and on the cellular plurality of receptors (Part IV), and a new frontier in steroid hormone action, the plasma membrane (Part V).

A novel approach to human fertility control: contragestion by the anti-progesterone RU 486.

The introductory presentation for a session on antiprogestins summarizes the action of RU 486 relating to its use as a contragestive agent. RU 486, or mifepristone (Roussel-Uclaf), is a progestin analogue resembling norethindrone with a longer 17-alpha side chain and a 11-beta phenyl group that confers the progesterone antagonist activity. It binds the progesterone receptor with a half-life of about 20 hours. Since progesterone receptors are only located in a few tissues, notably the decidualized endometrium and the corpus luteum, its action can be targeted. RU 486 interrupts the cycle regardless of whether fertilization occurred. A single 600 mg dose arrests over 80% of pregnancies up to 41 days, then a decreasing proportion, down to 60% at 10 weeks, due to a greater chance of incomplete evacuation. There have been no recorded side effects in 4000 trails. The effect of prostaglandins is magnified when progesterone action is suppressed. 400 trials have confirmed that over 98% successful termination of pregnancy can be obtained if 600 mg RU 486 is followed by 1 mg synthetic prostaglandin 36 hours later. RU 486 has also tested successfully for delivery of dead fetuses, cervical dilation, a postcoital contraceptive, and as an antiglucocorticoid agent. It has been submitted to the French Ministry of Health, and will probably be approved for marketing in 1988. Clinical trials are underway in 15 countries. This drug is a useful contragestive agent, active in the beginning of the continuum of pregnancy, and may defuse the abortion issue.

Trans-membrane Signals During Progesterone-mediated Meiosis in Xenopus laevis Oocytes: Their Modulation by Other Inducers or by Inhibitors of Steroid Action

In target cells, hormone-receptor complex interacts with an effector structure according to two major patterns. The first one, for polypeptide hormones and neurotransmitters, involves an interaction at the plasma membrane level with concomitant modulation of membrane enzyme activity (for ex-ample that of adenylate cyclase) or of ion permeability. In the other case, steroid and thyroid receptor complexes interact in the cell nucleus with elements of the gene expression machinery, as yet poorly defined, and appear to modify transcription (see Jensen and DeSombre 1972, for review). Nevertheless, it has been suggested a long time ago that steroids may exert their pharmacological actions and their physiological functions, at least in part, by interacting with cell membranes (Bangham et al. 1965).

STEROID-INDUCED MEIOSIS REINITIATION IN XENOPUS LAEVIS OOCYTES: ROLE OF MEMBRANE CONSTITUENTS AND CYCLIC AMP

Publisher Summary Xenopus laewis oocytes are arrested in prophase of the first meiotic division. They are induced to resume meiosis when exposed to a steroid hormone, progesterone. Actinomycin or enucleation do not block progesterone action but cycloheximide inhibits maturation. The first step is followed by the appearance of a cytoplasmic factor, known as maturation promoting factor (MPF), which is formed after 3-5 hours of external progesterone treatment. MPF is neither progesterone nor a metabolite of progesterone. A variety of experiments have strengthened the hypothesis that Ca 2+ translocation from membrane sites may be the first requirement for meiosis reinitiation. These experiments involved Ca 2+ iontophoresis, treatment with the ionophore A 23187 or calcium chelators, and recently aequorin injections to monitor free Ca 2+ changes during the meiotic process. However, a large body of data indicate that cyclic AMP plays also a crucial role which may be anterior, complementary or subsequent to that of calcium.