[Elective single embryo transfer - the Scandinavian experiment]. / Transfert électif d'un embryon - le modèle scandinave.

Limiting the number of embryos transferred from three to two does not reduce the high risk of twin pregnancy (between 21 and 40%). Scandinavian centers have proposed in the 2000s the elective single embryo transfer (eSET) as the only means to reduce maternal, neonatal and psychological consequences related to multiple births. Pooled results from prospective randomized controlled trials and prospective cohort studies comparing eSET and transfer of two embryos (DET) in a selected population have confirmed the almost complete disappearance of twins when eSET was effective but the compromising effect of eSET upon live birth rates was discussed. Optimizing the eSET overall pregnancy rate need to associate a freezing policy and to define risk factors for increased chance of multiple birth (patient age, diagnosis, number of top-embryos or unsuccessful attempts). The extension of eSET practice to an unselected population irrespective of embryo quality is still controversial. The choice between offering one cycle of SET or DET in an unselected patient population depends on the society's willingness to optimize the in vitro fertilization results according to a defined health care policy: the first one is the twins disappearance with reduced overall pregnancy rate and the second one is a reduced twin birth rate with maintain of the total pregnancy percentage. The real question is to define what percentage of twin pregnancy could be considered as acceptable.

[Apoptosis of the follicular cells: its implication in ovarian induction protocols]. / L'apoptose des cellules folliculaires: son implication dans les protocoles de stimulation ovarienne.

Atresia, a degenerative process through which many follicles are removed from the grown pool of follicles involves apoptotic changes in the follicular cells. This review analyses the endocrine regulation of apoptotic cell death in ovarian follicle. FSH is the major survival factor for preovulatory follicle but follicle integrity, in vitro, was necessary to its action on granulosa cell. The role of LH is more ambivalent. FSH and LH exert their activity via activation of the cAMP signal. High levels of intracellular cAMP could enhance steroidogenesis and in the same time induce apoptosis in granulosa cells. Moreover, no correlation between steroidogenesis and apoptosis can be established. During ovarian stimulation in IVF protocol, the use of LH, of coasting and of GnRH agonists and antagonists could be deleterious in follicle survival.

Involvement of theca cells and steroids in the regulation of granulosa cell apoptosis in rabbit preovulatory follicles.

Follicular atresia is characterized by a rapid loss of granulosa cells and, to a lesser extent, theca cells, via apoptosis. The aim of this study was to investigate the possible involvement of theca cell secretions in the regulation of apoptosis of rabbit granulosa cells. The annexin-V binding method based on externalization of phosphatidylserine to the outer layer of plasma membrane during apoptosis was used to detect apoptotic granulosa cells in flow cytometry. Regulation of apoptosis of granulosa cells was studied in three different culture systems: (i) isolated cultured granulosa cells, (ii) granulosa cells obtained from cultured preovulatory follicles and (iii) granulosa cells co-cultured with theca cells. The results of this study indicate that: (i) the rate of apoptosis of granulosa cells was significantly reduced when granulosa cells were co-cultured with theca cells or obtained from cultured preovulatory follicles in comparison with isolated cultured granulosa cells; (ii) FSH exerts its anti-apoptotic effect only on granulosa cells issued from cultured preovulatory follicles; (iii) ovarian steroids do not affect the percentage of isolated apoptotic granulosa cells; and (iv) the occurrence of an apoptotic process in rabbit theca cells could be upregulated in vitro by hCG and an analogue of the gonadotrophin second messenger cAMP. The results of this study indicate that in rabbits (i) steroids were ineffective in vitro in protecting isolated granulosa cells against apoptosis in comparison with observations in vivo in rats, and (ii) the presence of theca cells was efficient to reduce granulosa cell apoptosis but not sufficient to allow the anti-apoptotic effect of gonadotrophins observed in cultured follicles.

Quantification of cytochrome P450 aromatase transcripts before and during luteal phase in rabbit.

The aim of the present study was to quantify the promoter II- and I.r-derived transcripts of p450 aromatase gene during follicular stages and during corpus luteum formation in the rabbit. An ovulatory dose of hCG induced, first the disappearance of 90% of aromatase transcripts since 6 h before ovulation, and second a gradual decrease during pseudopregnancy. Individual quantification of both the promoter-derived transcripts showed that promoter II-derived transcript was the main transcript expressed both during follicular phase and pseudopregnancy, but kinetics of disappearance were not similar between both the promoter-derived transcripts. Moreover, hCG up-regulates aromatase expression in vitro in luteal tissue but estradiol, which was without effect on aromatase expression in preovulatory granulosa cells, down-regulates this expression in luteal tissue. In conclusion, the regulation of P450 aromatase in rabbit is mainly under control of promoter II regardless of which cyclic stage is studied. Moreover, we reported an opposite effect of estradiol on aromatase expression in vitro between follicular and luteal cells.

Hormonal regulation of apoptosis in rabbit granulosa cells in vitro: evaluation by flow cytometric detection of plasma membrane phosphatidylserine externalization.

Annexin V and propidium iodide bivariate analysis and the TUNEL method were used to quantify hormonal regulation of apoptosis in rabbit granulosa cells from preovulatory follicles in vitro. The aim of this study was to analyse comparatively the effects of gonadotrophins and their second messenger in the regulation of granulosa cell apoptosis in (i) cultured isolated granulosa cells and (ii) granulosa cells scraped from cultured follicles. The results showed that increasing doses of FSH had no effect on apoptosis of cultured isolated cells but caused a decrease in the number of apoptotic granulosa cells from preovulatory follicles cultured in serum-free conditions. Unlike FSH, addition of hCG did not modify apoptosis of granulosa cells significantly. In contrast, dibutyryl cAMP had an apoptotic effect in the two cellular models in the presence of serum. Moreover, a biphasic effect of dibutyryl cAMP in isolated granulosa cells was observed with an increase in the incorporation of [(3)H]thymidine into DNA at the lowest dose and an increase in apoptotic cell death at the highest dose. It was concluded that, in rabbits: (i) FSH requires follicle integrity to exert its anti-apoptotic effect in granulosa cells; (ii) dibutyryl cAMP induces a dose-dependent apoptotic effect in granulosa cells cultured alone or obtained from cultured preovulatory follicles; and (iii) cAMP signals induce opposite effects on growth and apoptosis in granulosa cells.

Expression of the rabbit cytochrome P450 aromatase encoding gene uses alternative tissue-specific promoters.

The aim of the present study was to analyse the tissue-specific expression of various promoter-derived transcripts from the gene encoding rabbit aromatase cytochrome P450. A new promoter, named I.r, was identified, and promoters II and I.r were sequenced. Promoter I.r-derived transcripts were found in preovulatory granulosa cells, corpus luteum, placenta and adipose tissue. An alternative splice variant of this transcript was found with tissue-specific preference. Tissue-specific expression of promoter-derived variants was studied in the ovary before and after ovulation. While the level of promoter II-derived transcript decreased dramatically after ovulation, that of promoter I.r-derived transcript remained unchanged, indicating that promoter II and promoter I.r were not controlled by a single regulation system. The existence of this dual system of regulation suggests that the rabbit ovary could be a useful model to study the promoter-specific regulation of aromatase.

Rabbit ovarian production of interleukin-6 and its potential effects on gonadotropin-induced progesterone secretion in granulosa and theca cells.

Recent studies suggest that non-steroid factors, such as cytokines, may play a role in ovarian processes. The purpose of this study was to explore cellular sites of interleukin (IL)-6 biosynthesis in rabbit follicles and to investigate IL-6 modulation in granulosa and theca cell functions. In this report development of rabbit preovulatory follicles was induced by 200 mIU equine chorionic gonadotropin (eCG) daily for 2 days. Seventy-two hours after the last injection ovaries were excised and granulosa and theca cells isolated. The two types of cells were preincubated for 24 h in Minimum Essential Medium (MEM) with 5% fetal calf serum (FCS), and then incubated for 24 h in MEM-2.5% FCS with appropriate stimulants. Results showed that rabbit granulosa and theca cell culture supernatants contained IL-6 bioactivity and that its production was inhibited by FSH and human CG and stimulated by IL-1. IL-6 inhibited gonadotropin-induced progesterone production, but not basal secretion, in both cell types, without a cytotoxic effect. IL-6 affected cAMP generation and steps distal to cAMP formation, but the mechanism of IL-6 action on progesterone differed in granulosa and theca cells. Taken together our results suggest that gonadotropins, by inhibiting IL-6 production, could control, in our model, IL-6 modulation of gonadotropin action on steroidogenesis.

Inhibition by gonadotropins of interleukin-1 production by rabbit granulosa and theca cells: effects on gonadotropin-induced progesterone production.

Increasing evidence suggests that cytokines may play a role in ovarian processes. The purpose of this study was to determine if interleukin-1 (IL-1) could modulate rabbit pre-ovulatory follicular function and to explore cellular sites of IL-1 biosynthesis in rabbit follicles. Development of rabbit pre-ovulatory follicles was induced by 200 mIU equine chorionic gonadotropin daily for 2 days. Seventy-two hours after the last injection, ovaries were excised and granulosa and theca cells isolated. The two types of cell were pre-incubated for 24 h in Minimum Essential Medium (MEM) with 5% fetal calf serum (FCS), and then incubated for 24 h in MEM with 2.5% FCS with appropriate stimulants. Results showed that rabbit granulosa and theca cell culture supernatants contain IL-1 bioactivity and that this bioactivity was diminished by gonadotropins, FSH and human chorionic gonadotropin, in a dose-dependent manner. Low doses of IL-1 (1 ng/ml) inhibited gonadotropin-induced progesterone production in both cell types and at the same time increased cell numbers. A study of the mechanism of IL-1 action demonstrated that it affects cAMP generation, and also steps distal to cAMP formation. We conclude that in our model gonadotropins, by inhibiting IL-1 production, could control IL-1 modulation of gonadotropin action on steroidogenesis.

High frequency of IgG antagonizing follicle-stimulating hormone-stimulated steroidogenesis in infertile women with a good response to exogenous gonadotropins.

OBJECTIVE: To investigate the presence of FSH-blocking IgG in infertile women. DESIGN: Retrospective study. Sera from patients and controls were processed for IgG purification, and purified IgG were tested at various concentrations for their ability to inhibit the recombinant human FSH-induced P production in vitro by human granulosa cells. SETTING: Departments of Endocrinology, and Obstetrics and Gynecology, University of Caen. PATIENT(S): Fifty-seven infertile women including 14 women with premature ovarian failure (POF), 29 women with a poor response to IVF-ET, and 14 women with a good response to IVF-ET. Controls consisted of 22 healthy age-matched women. INTERVENTION(S): IVF-ET allowed human granulosa cell pooling and culture for FSH bioassay. MAIN OUTCOME MEASURE(S): Inhibition by purified IgG of the in vitro recombinant human FSH-induced P production by human granulosa cells. RESULT(S): Blocking IgG were identified in only 3 of 14 POF and in 2 of 29 women with a poor response to IVF-ET. In contrast, IgG from women with a good response to IVF-ET inhibited significantly P production, and blocking IgG were detected in 85% women with a good response to IVF-ET. CONCLUSION(S): This study identified FSH-blocking IgG in a high proportion of women with a good response to IVF-ET. The significance of this remains questionable.

Luteotrophic factors in hyperstimulated pseudopregnant rabbit: I--Evidence for aromatase activity in luteal tissue and luteal cells.

It is well established that the rabbit corpus luteum (CL) function depends upon endogenous oestradiol, the major source of which in the rabbit ovary is considered to be the ovarian follicles. The absence of oestradiol formation by the rabbit CL has been previously reported. In a hyperstimulated pseudopregnant rabbit model used in our laboratory which developed a large number of corpora lutea in response to chorionic gonadotrophin (eCG)/hCG, we observed the survival of corpora lutea in vivo, and normal levels of plasma progesterone throughout pseudopregnancy (PP), despite the scarcity or the absence of follicles as a source of the luteotrophic hormone. Measurement of oestradiol in the plasma indicated that it was at high levels and correlated with the number of corpora lutea. This led us to investigate the luteal origin of oestradiol in this model. PP was induced in rabbits by i.m. injection of 200 IU eCG daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue obtained at days 5, 9 and 12 of PP and cultured for 24 h synthesized oestradiol and testosterone in addition to progesterone. However, under the same conditions, follicles had limited capacity to secrete oestradiol. The presence of an aromatase activity in luteal tissue was confirmed when exogenous testosterone was added to the culture medium. P450aromatase (P450arom) mRNA was found in luteal tissue at days 5, 9 and 12 of PP. Small or large luteal cells, obtained by enzymatic digestion of the tissue followed by centrifugation in a Percoll density gradient, were cultured during several days with or without gonadotrophin or dibutyryl cAMP (dbcAMP). Both types of cells secreted oestradiol. In small cells and luteal tissue, aromatase activity was stimulated (1.5-2-fold) by hCG and dbcAMP. Large cells exhibited a greater capacity to aromatize testosterone than small cells, but aromatase activity was not modified by hCG or by dbcAMP. FSH had no effect on aromatase activity of either luteal cell type. This intrinsic luteal tissue aromatase capacity and the absence of premature regression of corpora lutea despite the limited support of follicular oestrogen, suggest an autocrine and luteotrophic role for this luteal oestrogen.

Luteotrophic factors in hyperstimulated pseudopregnant rabbit: II--High sensitivity to hCG of luteal tissue and small luteal cells.

Previous studies on rabbit corpus luteum (CL) led to the conclusion that the luteotrophic complex, in rabbit, may include LH as well as oestradiol for normal luteal function. However, the requirement for LH is controversial. We have recently demonstrated the existence of a human chorionic gonadotrophin (hCG)-stimulated aromatase activity in cultured corpora lutea from a hyperstimulated pseudopregnant rabbit model, which develops a large number of corpora lutea, with only a few or no follicles in the ovaries. The present study was undertaken to investigate the in vitro responsiveness to hCG, dibutyryl cAMP (dbcAMP) and oestradiol of those corpora lutea. Pseudopregnancy (PP) was induced in rabbits by i.m. injection of 200 IU equine chorionic gonadotrophin daily for 2 days followed on day 4 by i.m. injection of 200 IU hCG (day 0 of PP). Luteal tissue and small and large luteal cells obtained at days 5 and 9 of PP were cultured for 24 h or during several days respectively with or without hCG, dbcAMP or oestradiol. Basal progesterone secretion was 3.6- and 22-fold higher in large cells compared with small ones at day 5 and 9 of PP respectively. When stimulated by small doses of hCG, luteal tissue responded by a 5-fold increase in progesterone secretion. Small cells produced four times higher amounts of progesterone than controls in the presence of 1 mIU/ml hCG and more than ten times in the presence of 0.1 IU/ml hCG, whereas large cells were insensitive to hCG stimulation. dbcAMP mimicked the effect of hCG on progesterone secretion by luteal tissue and luteal cells and oestradiol stimulated basal progesterone secretion in both small and large luteal cells. Given the large contribution of non stimulated large cells to luteal progesterone production and the remarkably high sensitivity of luteal tissue to gonadotrophin in vitro it seems that interactions between the two types of cells might occur during LH stimulation. Our results suggest that LH could participate in the luteotrophic complex at least in part through the stimulation of endogenous oestradiol production by luteal cells.

Angiotensin II receptor type 1 on granulosa and thecal cells of rabbit preovulatory follicles.

Specific, high-affinity angiotensin II (A II) receptors were observed on granulosa and thecal cells of preovulatory ovarian follicles from immature PMSG-treated rabbits. Scatchard analysis of 125I-[Sar1,Ile8]A II binding to freshly prepared cells was indicative of only one class of binding sites. Kd values were 0.26 +/- 0.11 nM and 0.18 +/- 0.02 nM, densities of A II receptors were 0.06 +/- 0.02 fmol/10(5) cells and 0.08 +/- 0.01 fmol/10(5) cells for granulosa and thecal cells, respectively. When cells were incubated for 48 h with hCG, Kd values were of the same order of magnitude, but the amount of A II receptors was increased 2-fold in granulosa and 4-fold in theca. Using subtype specific ligands (Losartan for AT1 and PD 123319 for AT2) in competitive binding experiments, A II receptors were found to be of the AT1 type on both granulosa and thecal cells freshly prepared or incubated 48 h in vitro. These results establishing the existence of high affinity AT1 receptors on the two cell types of the rabbit preovulatory follicles contrast with previous observations showing the presence of AT2 receptors on granulosa or theca from several species.

Rapid sequencing of rabbit aromatase cDNA using RACE PCR.

The sequencing of aromatase cDNA from rabbit granulosa cells was obtained by RACE PCR. This cDNA is 2.9 kb long. The first 119 nucleotides correspond to the first untranslated exon. Nucleotides 120 to 1,629 correspond to the coding region (1,509 nucleotides) and the rest of the sequence is non coding and contains a polyadenylation signal. Translation of the cDNA sequence indicates that the protein is composed of 503 amino acids, like in human aromatase. Its molecular weight is 57.4 kDa. The alignment between the rabbit aromatase amino acid sequence and other aromatases already described in the human, mouse, rat, cow, pig, chicken, rainbow trout and teleost fish shows that the rabbit protein exhibits the highest homology with the human one (85%).

Progesterone secretion and proliferation in cultured rabbit granulosa cells under conditions of beta-D-xyloside-induced inhibition of proteoglycan synthesis.

Proteoglycans present in follicular fluid are synthesized by granulosa cells under gonadotropin control. An inhibitor of proteoglycan synthesis, p-nitrophenyl-beta-D-xyloside (beta-D-xyloside) was used as a probe to study rabbit granulosa cell steroidogenesis and proliferation under abrogated proteoglycan synthesis. Granulosa cells isolated from rabbit preovulatory follicles were cultured 24 h in Minimum Essential Medium plus 2.5% fetal calf serum in the presence or absence of beta-D-xyloside and were then treated with FSH or dibutyryl cAMP (db-cAMP) alone or in combination with beta-D-xyloside for a further 24 h. The exposure for 48 h of granulosa cells to 1 mM beta-D-xyloside in the absence or presence of FSH inhibited proteoglycan synthesis and increased the amount of glycosaminoglycans (GAG). FSH-stimulated progesterone production was significantly correlated only with proteoglycan synthesis and not with GAG production. The addition of various concentrations of beta-D-xyloside (0.1-4 mM) for 48 h to granulosa cells induced a dose-dependent inhibition of FSH-stimulated progesterone secretion and [3H]thymidine incorporation into DNA. beta-D-Xyloside concentrations lower than 1 mM induced an inhibition of FSH-stimulated progesterone secretion but had no significant effect on FSH-induced proliferation. One millimolar beta-D-xyloside did not modify basal progesterone production, but in the presence of various doses (0.1-2.5 ng/ml) of FSH or hCG (0.1-1 IU/ml) it exerted a significant inhibitory effect on steroid secretion. Fifty percent inhibition was obtained for doses of FSH above 0.5 ng/ml.(ABSTRACT TRUNCATED AT 250 WORDS)

Uterine luteinizing hormone/human chorionic gonadotropin-binding sites in the early pregnant rat uterus: evidence for total occupancy in the periimplantation period.

We have investigated the presence of high affinity LH/hCG-binding sites (RLH) in crude membranes from early pregnant rats uteri. The uterine concentration of available RLH increased from day 1 to day 3 (1.3 +/- 0.2 vs. 2.8 +/- 0.4 fmol/mg protein) without a change in the affinity constant (approximately 5 x 10(10) M-1). However, unoccupied uterine RLH disappeared in the periimplantation period (days 4-6). To determine if the drop in available RLH was consecutive to their occupancy, uterine membranes were treated with acidified medium (25 mM Tris-HCl, and 5 mM MgCl2, pH 2.5) to remove endogenous ligand. The number and affinity of total (occupied plus available) RLH in acid-eluted membranes were estimated by Scatchard analysis of [125I]hCG binding and compared with those of available RLH in untreated membranes from the same uterine preparation. The uterine concentration of total RLH increased first between days 1 and 2 (2.2 +/- 0.5 vs. 4.2 +/- 0.8 fmol/mg protein), then between days 3 and 4 (4.2 +/- 0.6 vs. 6.5 +/- 0.8 fmol/mg protein), before plateauing until day 6. Thus, the reduction in the available uterine RLH in the periimplantation period is largely due to occupancy, rather than down-regulation, of RLH. The occupancy of uterine RLH 1) increased during early pregnancy (day 1, approximately 20%; days 2-3, approximately 40%; days 4-6, approximately 100%), 2) paralleled the increase in total RLH number, and 3) was probably due to pituitary LH only. However, the blastocyst itself seemed to influence uterine RLH occupancy, since available uterine RLH were detected on day 5 of pseudopregnancy. The increase in total uterine RLH as well as the perfect synchrony between their occupancy and the previously described pattern of uterine cAMP concentration during rat early pregnancy suggest that the response of uterine (and more precisely luminal epithelium) adenylate cyclase to LH (and/or related substance originating from embryo) may determine uterine receptivity for ovoimplantation and subsequent decidualization.

Changes of progesterone content of rat uterine flushings in relation to serum concentrations of progesterone during the oestrous cycle.

Uterine fluid was collected from four-day cyclic rats at each stage of the oestrous cycle and assayed for progesterone and protein content. Progesterone was determined by radioimmunoassay either after ethanol (or 2.5% NaOH) denaturation of proteins from uterine flushings ('total' progesterone) or without protein denaturation ('ether-extractable' progesterone). The amount of 'ether-extractable' progesterone in the lumen was constant from metoestrus to pro-oestrus (340 pg per uterus) but lower in oestrus (200 pg per uterus). However, 'total' progesterone content of uterine fluid was subject to cyclic variations and was highest in dioestrus (890 pg per uterus) and lowest in oestrus (350 pg per uterus), in contrast to serum progesterone which is lowest in dioestrus and highest in oestrus. Protein content of uterine flushings peaked to 780 micrograms per uterus in pro-oestrus then fell to about 140 micrograms per uterus until the end of the oestrous cycle. Changes in protein content of the lumen were followed by qualitative variations since the mean amount of 'bound' progesterone ('total' progesterone minus 'ether-extractable' progesterone) released per milligram of denatured lumen protein rose from 1.8 pmol in pro-oestrus to 18.2 pmol in dioestrus. The changes of luminal 'bound' progesterone during the oestrous cycle suggest that progesterone binding to luminal proteins could be an important modulator of progesterone action in rat uterus. Moreover, the variations in progesterone content of the lumen, irrespective of serum progesterone concentrations, are consistent with the hypothesis that progesterone synthesis occurs in the uterus.

Inhibition of progesterone synthesis and cAMP accumulation in small bovine luteal cells by a low molecular weight component of bovine follicular fluid.

Follicular fluid from large follicles of cows was extracted with charcoal and filtered through an Amicon XM-50 membrane. The XM-50 filtrate was further fractionated on a column of Fractogel TSK HW-40 (s) using Krebs-Ringer-phosphate buffer (1/100th dilution), pH 7.2, as an eluant. Two fractions (1 and 2) were obtained. Inhibition of progesterone secretion by small luteal cells was associated with the XM-50 filtrate and Fraction 2. Whole follicular fluid, the XM-50 retentate and Fraction 1 had no significant inhibitory activity. Fraction 2, which contained about 1/100,000th of the original follicular fluid proteins, inhibited the LH- or (Bu)2cAMP-induced progesterone production during a 2-h incubation. This inhibition was dose-dependent. Fraction 2 also inhibited LH-induced cAMP accumulation, but did not affect the conversion of pregnenolone to progesterone or the basal progesterone production. The molecular weight of the inhibitory factor was estimated to be less than 10,000 and its ability to inhibit steroidogenesis was lost after digestion with protease but retained after heating for 60 min at 75 degrees C. These results demonstrate that bovine follicular fluid contains a heat-stable factor likely to be a polypeptide and which suppresses the steroidogenic response of small luteal cells to LH. The action of this inhibitory factor could involve both an inhibition of the LH-induced synthesis of cAMP and an inhibition of the action of cAMP.

Acute stimulation by lutropin of mitochondrial protein synthesis in small luteal cells.

A two-dimensional electrophoretic technique was used to study the effect of acute stimulation of bovine luteal cells with lutropin on protein synthesis. Cells were incubated for 30 min with [35S]methionine in the presence of stimulating levels of luteinizing hormone (lutropin), after which the proteins were analyzed by autoradiography. Lutropin or N6,2'-O-dibutyryl-adenosine 3',5'-phosphate (Bt2cAMP) induced the labelling of three proteins, referred to as proteins A, B and C. Protein A, had a molecular mass of 28 kDa and an isoelectric point (pI) of 6.7. Proteins B and C had a molecular mass of 27 kDa and pI of 6.2 and 6.4 respectively. After subcellular fractionation, the three proteins were found to be markedly concentrated in the only fraction enriched in an established mitochondrial marker. Moreover, protein A was one of the major mitochondrial newly synthesized proteins. Its appearance was observed after a 5-min incubation and was prevented by 100 microM cycloheximide. The acute accumulation of proteins A, B and C in mitochondria, the site of the rate-limiting step of steroidogenesis, suggest that they could be involved in the mechanism of stimulation by lutropin of progesterone synthesis.

Estrous cycle-related changes of high affinity luteinizing hormone/human chorionic gonadotropin binding sites in the rat uterus.

LH/human CG (LH/hCG) high affinity binding sites were detected in crude membrane preparations of rat uteri. There was little competition for receptor occupancy between hCG and ovine FSH (oFSH) (0.05%) and no competition between hCG and ovine PRL (less than 0.01%). No similar binding sites were detected in crude membrane preparation of heart, kidney, skeletal muscle, liver, and lung tissues. Concentrations of uterine unoccupied binding sites (RLH) were determined for each stage of the 4-day estrous cycle. The RLH were found in all preparations of metestrus uteri (n = 10) but only in some preparations from the other stages of the estrous cycle (1 of 7 on proestrus, 3 of 4 on estrus, 5 of 7 on diestrus). The concentration of uterine RLH varied throughout the estrous cycle with highest values during the metestrus (1.50 +/- 0.15 fmol/mg protein) and lowest values during the proestrus (less than 0.2 fmol/mg protein). The affinity constant for hCG of uterine RLH remained constant during the estrous cycle (about 0.8 x 10(11) M-1) and was nearly identical to that of rat ovarian receptors. On metestrus, RLH concentration appeared to be approximately 35-fold lower in the uterus than in the ovaries when expressed per mg protein (1.50 +/- 0.15 vs. 52.83 +/- 3.61 fmol/mg protein) but only 20 times lower when expressed per organ (2.2 vs. 48.3 fmol/organ). The estrous cycle-related changes of uterine RLH concentration, together with our data establishing an in vitro influence of hCG on progesterone metabolism in rat uterus, suggest that some uterine functions could be directly regulated either by LH from the pituitary or during early pregnancy by an LH-like substance originating from the embryo.

Involvement of the phospholipase C second messenger system in the regulation of steroidogenesis in small bovine luteal cells.

We have previously suggested that the interaction between luteinizing hormone (LH) and its receptor, in addition to stimulating adenylate cyclase, is able to trigger a negative regulatory signal at a step beyond cAMP synthesis (Benhaim et al. (1987) FEBS Lett. 223, 321-326). The present study was conducted to determine whether the phospholipase C system is involved in this phenomenon. Small bovine luteal cells from pregnant cows were incubated with phospholipase C, A23187, an ionophore of calcium and/or phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C (PKC), in the presence or absence of bovine luteinizing hormone or dibutyryl cyclic AMP (dbcAMP). A23187 associated with PMA was able to mimic the stimulatory effect of phospholipase C on basal progesterone production, whereas neither A23187 nor PMA alone had any effect. In the presence of high doses of LH, phospholipase C inhibited progesterone and cAMP production in a dose-dependent manner. A23187 and PMA were able to mimic the inhibition of progesterone synthesis but stimulated LH-induced cAMP accumulation. When cells were stimulated by high doses of dbcAMP, phospholipase C and A23187 but not PMA inhibited progesterone synthesis. These observations suggest that (1) phospholipase C can mimic the post-cAMP negative regulatory signal induced in vitro by high doses of LH, in the presence of an activation of PKC; (2) phospholipase C is also able to mimic in vitro the luteolytic properties of prostaglandin F2 alpha that we previously described (Benhaim et al. (1987) Prostaglandins 33, 227-239); and (3) under basal conditions or in the presence of low doses of LH, the phospholipase C system slightly stimulates steroidogenesis.