Relationship between follicle-stimulating hormone levels at the beginning of the human menstrual cycle, length of the follicular phase and excreted estrogens: the FREEDOM study.

Although reproductive aging has been separately related to elevated FSH and shorter follicular phase (FP), the direct association between both parameters has not been investigated. Also, the exact effects of increased FSH on estrogen production are yet to be established.A large database of daily urinary concentrations of FSH, LH, and estrone 3-glucuronide (E1G) from 37 regularly menstruating women (median 11 cycles per patient) was used. Initial FSH levels (iFSH) were estimated as the mean value of d 1-5. The day of E1G take-off (ETO) was determined by an algorithm, and accordingly, the FP was divided into early (d 1 to ETO) and late (ETO+1 to LH peak). FP maximum and integrated E1G were calculated. Subjects were distributed according to their mean iFSH into three categories (5 to 10, and >10 IU/liter). There was a gradual decrease in FP length with increasing category (15.2 +/- 3.8, 14.1 +/- 3.6, and 13 +/- 2.6 d, respectively; P < 0.0001). A similar effect occurred in early FP (7.5 +/- 4, 6.4 +/- 3.7, and 5.4 +/- 2.7; P < 0.0001); in contrast, late FP was unaffected (7.7 +/- 2.1, 7.7 +/- 2.1, and 7.6 +/- 2.4; P = 0.86). No consistent increase in E1G was found with advancing iFSH category; however, women with mean initial LH higher than 6 IU/liter had significantly elevated maximum (P < 0.0001) and integrated (P = 0.002) E1G.FP length decreases in parallel with increasing iFSH, with a selective effect on the early FP. Increased FSH does not affect E1G; however, elevated initial LH level was related to higher E1G.

The carboxy-terminal domain of Grp94 binds to protein kinase CK2 alpha but not to CK2 holoenzyme.

Surface plasmon resonance analysis shows that the carboxy-terminal domain of Grp94 (Grp94-CT, residues 518-803) physically interacts with the catalytic subunit of protein kinase CK2 (CK2 alpha) under non-stressed conditions. A K(D) of 4 x 10(-7) was determined for this binding. Heparin competed with Grp94-CT for binding to CK2 alpha. CK2 beta also inhibited the binding of Grp94-CT to CK2 alpha, and CK2 holoenzyme reconstituted in vitro was unable to bind Grp94-CT. The use of CK2 alpha mutants made it possible to map the Grp94-CT binding site to the four lysine stretch (residues 74-77) present in helix C of CK2 alpha. Grp94-CT stimulated the activity of CK2 alpha wild-type but was ineffective on the CK2 alpha K74-77A mutant.

Multiple idiopathic small bowel strictures: report of three cases.

Multiple strictures of the small bowel are relatively rare. In many cases, a distinct cause can be defined, but some strictures are unexplainable by any specific mechanism and have been termed 'idiopathic' small bowel strictures. We present 3 cases of multiple small bowel strictures in which the affected segments were studied with perioperative photoplethysmography, in vivo specimen angiography and pathology. Neither photoplethysmographic alterations nor structural vascular lesions were found.

Osmotic stress regulates the stability of cyclin D1 in a p38SAPK2-dependent manner.

We report here that different cell stresses regulate the stability of cyclin D1 protein. Exposition of Granta 519 cells to osmotic shock, oxidative stress, and arsenite induced the post-transcriptional down-regulation of cyclin D1. In the case of osmotic shock, this effect was completely reversed by the addition of p38(SAPK2)-specific inhibitors (SB203580 or SB220025), indicating that this effect is dependent on p38(SAPK2) activity. Moreover, the use of proteasome inhibitors prevented this down-regulation. Thus, osmotic shock induces proteasomal degradation of cyclin D1 protein by a p38(SAPK2)-dependent pathway. The effect of p38(SAPK2) on cyclin D1 stability might be mediated by direct phosphorylation at specific sites. We found that p38(SAPK2) phosphorylates cyclin D1 in vitro at Thr(286) and that this phosphorylation triggers the ubiquitination of cyclin D1. These results link for the first time a stress-induced MAP kinase pathway to cyclin D1 protein stability, and they will help to understand the molecular mechanisms by which stress transduction pathways regulate the cell cycle machinery and take control over cell proliferation.

Tumour suppressor protein p53 released by nuclease digestion increases at the onset of rat liver regeneration.

BACKGROUND/AIMS: Protein kinase CK2 (CK2) increases when cells are committed to proliferate, as in liver regeneration. This enzyme phosphorylates the tumour suppressor protein p53, whose expression controls the levels of many other cell cycle proteins. The aim of this study was to determine if CK2 was affected by p53. METHODS: Male Sprague-Dawley rats (200-250 g) were subjected to either partial hepatectomy or laparotomy and the levels and subcellular distribution of p53 were studied, following the approach used earlier for CK2. The levels of both proteins were also studied in the human cell lines HL-60 (devoid of p53) and HepG2 (with normal p53 levels) and in fibroblasts from transgenic p53-deficient mice (p53-/-) or homozygous for wild-type p53 (p53+/+). Computer-assisted search was used to detect p53 consensus sequences in genes for CK2 subunits Binding of p53 protein to some of these sequences was assayed by electrophoretic mobility shift assay. RESULTS: Rat liver p53 protein was present mainly in the fraction extracted from intact nuclei by nucleases (S1) and showed a transient increase at 6 h post partial hepatectomy, as observed previously with nuclear CK2. The human CK2a gene presents the consensus sequence for trans-activation by p53 and specific binding of p53 protein to some of these sequences was detected in vitro. Total CK2a was higher in HepG2 than in HL-60 cells but total CK2 and its cytosolic/ nuclear distribution was similar in mice (p53+/+) fibroblasts and (p53-/-) fibroblasts. CONCLUSIONS: p53 is present in the nuclease-extracted S1 fraction from liver cells, as described for CK2, and undergoes similar changes at the beginning of rat liver regeneration. However, the data on cultured cells suggest that the expression of CK2 and its subcellular localization are p53-independent events.

Association of protein kinase CK2 with eukaryotic translation initiation factor eIF-2 and with grp94/endoplasmin.

Protein kinase CK2 forms complexes with some protein substrates what may be relevant for the physiological control of this protein kinase. In previous studies in rat liver cytosol we had detected that the trimeric form of eukaryotic translation initiation factor 2 (eIF-2) co-eluted with protein kinase CK2. We have now observed that the ratio between eIF-2 and cytosolic CK2 contents in testis, liver and brain is quite similar, being eIF-2 levels about 5-fold higher than those of CK2. Furthermore eIF-2 was present in liver samples immunoprecipitated with anti-CK2alpha/alpha' antibodies, confirming the existence of complexes containing both proteins. Nonetheless, these complexes would represent only a fraction of total cytosolic CK2 and eIF-2. We had also observed that rat liver membrane glycoproteins obtained through chromatography on wheat-germ lectin-Sepharose contain CK2 activity which copurifies with grp94/endoplasmin. We have now confirmed that this activity was due to the presence of protein kinase CK2 as evidenced by immunodetection with antibodies against CK2alpha/alpha'. The fractions enriched in grp94/endoplasmin and CK2 also contained another 55-kDa polypeptide (p55) phosphorylated by CK2 which has been identified as calreticulin by N-terminal sequencing. Calreticulin and grp94/endoplasmin could be partially resolved from CK2 by chromatography on heparin-agarose and almost completely on ConA-Sepharose. However, phosphorylation of immunoprecipitated grp94/endoplasmin was enhanced by its preincubation with purified CK2 prior to immunoprecipitation, what confirms the easy reassociation between these proteins. The association of protein kinase CK2 with eIF-2 and with grp94/endoplasmin may serve to locate the enzyme in the cellular machinery involved in protein synthesis and folding, and reinforces the possible involvement of CK2 in these processes.

Substrates for protein kinase CK2 in insulin receptor preparations from rat liver membranes: identification of a 210-kDa protein substrate as the dimeric form of endoplasmin.

Chromatography of extracts from rat liver membranes on wheat-germ lectin-Sepharose resulted in a partial resolution of the insulin receptor from other phosphorylatable proteins. Among the latter, a protein (p210, with an apparent M(r) of 210 kDa on SDS/PAGE under nonreducing conditions) was found to be phosphorylated by protein kinase CK2 on Thr and Ser residues. Under reducing conditions p210 was resolved into two phosphopolypeptides with apparent M(r) of 95 and 105 kDa. Neither the 95-kDa nor the 105-kDa polypeptides were recognized by antibodies against the beta-subunit of the insulin receptor. Both polypeptides gave identical phosphopeptide maps after protease V8 digestion and contained the same N-terminal amino acid sequence. This sequence coincided with that of endoplasmin, and both polypeptides as well as p210 were recognized by antibodies against this protein. This shows that p210 corresponds to the dimeric form of rat liver endoplasmin. DEAE-Sepharose chromatography of p210 preparations removed most other contaminating proteins and revealed the presence of a protein kinase activity that coeluted with p210. This protein kinase possessed the properties (substrate specificity and inhibition by heparin) that are characteristic of the protein kinase CK2 enzymes. Furthermore, phosphoamino acid analysis and phosphopeptide maps of the 95/105-kDa polypeptides phosphorylated either by the endogenous protein kinase or by exogenous protein kinase CK2 gave similar results. The phosphorylation of p210/endoplasmin by protein kinase CK2 and its coelution gives support to the involvement of this protein kinase in membrane-associated processes.

GnRH-induced calcium mobilisation and inositol phosphate production in immature and mature rat ovarian granulosa cells.

In rat ovarian granulosa cells the effects of GnRH are determined by the state of granulosa cell development with mainly inhibitory actions in immature cells and stimulatory actions in differentiated mature cells. These developmentally related effects of GnRH may arise from changes in either one or more of the signal transduction pathways activated by GnRH. The present study therefore measured downstream signalling events associated with the activation of the phospholipase C (PLC) signal transduction pathway in both mature and immature rat ovarian granulosa cells. Results showed that GnRH produced similar total inositol phosphate and intracellular calcium ([Ca2+]i) responses in both immature and mature granulosa cells. In contrast to the biphasic GnRH-induced [Ca2+]i response in pituitary gonadotropes, stimulation of the endogenously expressed GnRH receptor in both immature and mature granulosa cells produced a prompt monophasic rise in [Ca2+]i. This calcium transient was abolished by pretreating either cell type with a potent GnRH receptor antagonist or the PLC inhibitor U73122, demonstrating a GnRH receptor-specific activation of PLC. Similarly, pretreatment of cells with the [Ca2+]i antagonists thapsigargin or cyclopiazonic acid abolished the GnRH-induced calcium transient, whereas EGTA and nifedipine, a voltage-operated calcium channel (VOCC) antagonist, had no effect. These results suggest that in either immature or mature granulosa cells GnRH mobilises calcium from thapsigargin/cyclopiazonic acid-sensitive [Ca2+]i stores but does not involve the influx of extracellular calcium through VOCCs. We conclude that GnRH-induced stimulation of the PLC signal transduction pathway is independent of the stage of granulosa cell maturity and that alternative mechanisms account for the opposite effects of GnRH on gonadotrophin-induced steroidogenesis in mature and immature rat granulosa cells in vitro.

Modulation of granulosa cell deoxyribonucleic acid synthesis and differentiation by activin.

FSH stimulates follicular growth through inducing granulosa cell proliferation. Our working hypothesis is that mitogenesis is facilitated by a locally produced growth/differentiation factor(s) that modulates FSH action in developing granulosa cells. The present study was undertaken to examine the effect of the related gonadal peptides activin and inhibin on granulosa cell proliferation. Monolayer cultures of granulosa cells isolated from preantral/early antral follicles in immature rat ovaries were established by incubation overnight in serum-free medium 199. Treatment was initiated with serum-free medium containing recombinant human (rh) activin and/or rh-inhibin in the presence or absence of FSH. After incubation for 18 h, medium was collected for progesterone determination (as a marker of cell differentiation), and the cell monolayers were incubated for 2 h in the presence of [3H]thymidine to measure DNA synthesis. Activin dose dependently (1-100 ng/ml) stimulated DNA synthesis (minimal effective dose, 1 ng/ml), whereas inhibin or FSH alone was without effect. When activin (1 ng/ml), but not inhibin, was present with FSH, the gonadotropin caused dose-dependent increases in [3H]DNA synthesis over a wide range of FSH concentrations (1-100 ng/ml). This property of activin was unaltered by the additional presence of inhibin (1-100 ng/ml). To analyze the role of cAMP in mediating the mitogenic action of FSH in the presence of activin, experiments were repeated substituting a membrane-permeable cAMP agonist, 8-bromo-cAMP (8br-cAMP; 0.1-3 mM). Similar to FSH, 8br-cAMP had no effect on granulosa cell DNA synthesis in the absence of activin. However, in the presence of activin (1 ng/ml) 8br-cAMP was stimulatory. The dose response to 8br-cAMP revealed a biphasic effect on DNA synthesis and differentiation: DNA synthesis rose to a maximum in the presence of 0.5 mM 8br-cAMP and declined thereafter. Progesterone synthesis only started to increase in the presence of 0.1 mM 8br-cAMP, rising to a maximum at 3 mM 8br-cAMP, at which concentration DNA synthesis was fully suppressed. We conclude that activin induces DNA replication in rat granulosa cells. In the presence of activin, FSH and 8br-cAMP are mitogens. These actions of FSH and 8br-cAMP occur at doses too low to stimulate steroidogenesis, emphasizing the role of intracellular cAMP tone in granulosa cell proliferation and differentiation.

Effects of growth hormone releasing hormone on rat ovarian steroidogenesis.

During the last decade, it has been shown that each part of the somatotrophic axis can influence granulosa cell function. Growth hormone releasing hormone (GHRH) may be effective through the release of hypophyseal growth hormone (GH) and the subsequent increase of insulin-like growth factors (IGF). There is also some evidence that GHRH could act directly on ovarian function. The aim of this study was to determine the mechanism through which GHRH affects granulosa cell steroidogenesis in the ovary. Granulosa cells were obtained from immature, oestrogen- treated rats supplemented with or without follicle stimulating hormone (FSH) in vivo and were cultured for 48 h to evaluate steroid production. GHRH was administered either in vivo at the same time as FSH, or in vitro in the presence or absence of testosterone and FSH. Our results show that co-treatment with GHRH and FSH in vivo induced significant increases in plasma IGF-I concentrations and steroid production by cultured granulosa cells. The addition of GHRH to culture medium did not significantly alter steroid production by either non-differentiated (no FSH in vivo) or differentiated (FSH in vivo granulosa cells. In contrast, treatment in vitro with IGF-I significantly increased steroidogenesis in both cases. Our results suggest that any physiologically significant effect of GHRH on ovarian function is probably to be exerted via activation of the somatotrophic axis and the subsequent amplification of ovarian FSH responsiveness by IGF-I.

The role of luteinizing hormone in human follicle development and oocyte fertility: evidence from in-vitro fertilization in a woman with long-standing hypogonadotrophic hypogonadism and using recombinant human follicle stimulating hormone.

To evaluate the relative importance of follicle stimulating hormone (FSH) and luteinizing hormone (LH) in follicular development and oocyte fertility in the human species, the use of recombinant human FSH, human menopausal gonadotrophin (HMG), and very highly purified urinary human FSH (FSH-HP) plus oestradiol valerate for ovarian stimulation and in-vitro fertilization (IVF) were compared in three cycles in a woman with isolated congenital gonadotrophin deficiency who had never been treated with ovarian stimulating agents. The total number of ampoules of gonadotrophins used was lower in the HMG treatment cycle. Ovarian response and IVF outcome in the three treatment cycles were as follows: (i) HMG cycle: normal follicular growth, normal pattern of oestradiol and inhibin through the menstrual cycle, high fertilization rate (93%); (ii) recombinant FSH cycle: normal follicular growth, low oestradiol and abnormal inhibin, finally poor rate of fertilization (28%); (iii) FSH-HP plus oestradiol valerate cycle: normal follicular growth, normal pattern of inhibin and poor fertilization rate (27%). Luteal plasma progesterone concentrations were much higher in the HMG treatment cycle. This case shows that FSH is the only factor required in order to induce follicular growth in the human, although LH or a product derived from its action may assist in order to achieve full follicular maturity and oocytes capable of fertilization. Though oestradiol might have a mediatory role in the process of follicular maturation, our results favour a direct primary role of LH in complete maturation of the follicle.

Effect of luteinizing hormone on follicle stimulating hormone-activated paracrine signalling in rat ovary.

'Pure' follicle stimulating hormone (FSH) and luteinizing hormone (LH) are expected shortly to become available for pharmaceutical use in the clinical setting. To test the contribution of LH to optimal ovarian responsiveness to FSH, 21-day-old hypophysectomized, immature, female rats received four s.c. injections of recombinant human LH (rhLH; total dose 1-10 IU) and/or rhFSH (total dose 30-72 IU) given at 12-hourly intervals. At 48 h after the first injection, ovaries were removed, weighed and used to isolate granulosa and thecal/interstitial cells for assessment of basal and gonadotrophin-responsive steroidogenesis in vitro, or homogenized to extract total RNA for Northern analysis of 17-hydroxylase/C17-20-lyase (cytochrome P-450c17 alpha) mRNA. Serum oestradiol and uterine weight were measured as indices of ovarian oestrogen production; androstenedione was measured to reflect ovarian androgen production. Consistent with the two-cell, two-gonadotrophin model of oestrogen synthesis, increased ovarian oestrogen secretion only occurred if both rhFSH and rhLH were given simultaneously. Treatment with rhFSH alone stimulated ovarian weight gain and granulosa cell aromatase activity without oestrogen secretion, whereas rhLH alone stimulated thecal androgen synthesis and androgen secretion. When the total rhLH dose was fixed at 1 IU, giving rise to an unmeasurably low serum concentration of rhLH, additional treatment with rhFSH (30-72 IU) dose-dependently stimulated serum androgen concentrations as well as oestrogen concentrations. The approximately 2.0 kb-sized P-450c17 alpha mRNA transcript was undetectable in the ovaries of untreated control animals but was abundant in the ovaries of positive controls treated with 15 IU of pregnant mare serum gonadotrophin.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibin, activin, and follistatin. Potential roles in ovarian physiology.

The physiological significance of these results will not become clear until patterns of activin and inhibin protein production and the expression of their receptors have been more thoroughly characterized in relation to follicular development. Meanwhile, in situ hybridization studies on rat and monkey ovaries suggest that inhibin/activin beta-subunit mRNA (favoring synthesis of activin) is relatively abundant in granulosa cells of immature antral follicles, whereas alpha-subunit mRNA (favoring synthesis of inhibin) predominates in Graafian follicles. The increased production of follistatin associated with advanced preovulatory development would serve to further reduce the activin "tone" relative to inhibin (Fig. 1). At the level of protein action in vitro, the pattern emerging is that inhibin minimally affects granulosa cell steroidogenesis at any stage of follicular development, whereas activin has pronounced modulatory effects that alter with follicular maturity. As suggested previously,60 the ability of activin to enhance gonadotropin-responsive aromatase activity and simultaneously suppress progesterone production by mature granulosa cells has physiological implications in that it hints at a mechanism for promoting estrogen synthesis and simultaneously suppressing progesterone synthesis, which is precisely what occurs in the preovulatory follicle. The effects of inhibin and activin on human thecal androgen synthesis observed in vitro suggest how these proteins might act locally to modulate preovulatory follicular growth and estrogen synthesis in vivo (Fig. 2).57 In essence, we propose that activin acting at early stages of antral follicular development plays a role in follicular recruitment through sensitizing immature granulosa cells to the cytodifferentiative action of FSH. On the other hand, inhibin is more likely to play a role in preovulatory follicular selection and maintenance of follicular dominance. Studies of follicular fluid levels of androgen and estrogen in relation to granulosa cell aromatase activity indicate that the capacity of the theca interna to generate aromatase substrate (androstenedione) increases hand in hand with aromatase activity in the human preovulatory follicle. It has therefore been suggested that a positive feedback loop (granulosa on theca) exists that promotes thecal androgen synthesis and hence estrogen synthesis in this follicle.64 The discovery that inhibin production in vitro is greatest by granulosa cells isolated from preovulatory follicles strongly implicates inhibin as a component of this feedback loop.

Skeletal muscle histochemistry in male and female Andalusian and Arabian horses of different ages.

Muscle biopsies were taken from the middle gluteal muscle of 143 untrained horses (83 Andalusians [AN] and 60 Arabians [AR]) ranging from 10 days to 24 years old. The horses were separated according to breed and sex and allotted to five age groups: A, 0 to three months; B, yearlings; C, two to three years; D, five to 10 years; and E, 11 to 24 years. There was an increase in the percentage of type I fibres (about 100 per cent) as well as a decrease in the percentage of type IIB fibres (AN, 50 per cent; AR, 40 per cent) over the five age groups. The percentage of type IIA fibres rose significantly over the first two or three age groups. The overall decrease in the subgroup IIB fibres with age was proportionally greater for IIB oxidative fibres (AN, 72 per cent; AR, 68 per cent) than for IIB non-oxidative fibres (AN, 5 per cent; AR, 34 per cent). The mean cross-sectional area of all three fibre types increased significantly with age. In any given age group, the mean relative cross-sectional area occupied by IIA fibres in the biopsy specimens was significantly greater in stallions than in mares, at the expense of IIB fibres.

Relative effects of activin and inhibin on steroid hormone synthesis in primate granulosa cells.

Ovarian granulosa cells produce inhibin and activin, structurally related proteins with potentials to directly modulate follicular steroidogenesis. The aim of the present study was to compare development-related effects of inhibin-A and activin-A on steroidogenesis in marmoset monkey (Callithrix jacchus) granulosa cells. Granulosa cells from "immature" (< 1.0 mm diameter) and "mature" (> 2 mm diameter) follicles were incubated in serum-free culture medium for 96 h with and without peptide (1-100 ng/mL), in the presence and absence of gonadotropins [human (h) FSH or hLH] (10 ng/mL). Spent medium was collected and stored frozen for progesterone assay. Aromatase activity was determined by incubating cells for a further 6 h in the presence of 1 mumol testosterone and assaying accumulation of oestradiol. Granulosa cells from immature follicles showed characteristically low basal rates of steroid synthesis that were unaffected by treatment alone with either inhibin or activin. Treatment with hFSH stimulated both progesterone production and aromatase activity. Cotreatment with activin and hFSH further enhanced aromatase activity by up to 4-fold. The progesterone response to activin plus hFSH was related to the effect of hFSH in the absence of activin: high-level responsiveness to hFSH was suppressed by activin while low-level responsiveness was enhanced. Inhibin had no significant effect on FSH-responsive progesterone production, but at high concentrations (> 10 ng/mL) it caused slight (up to 30%) reduction in FSH-induced aromatase activity. Granulosa cells from mature follicles showed relatively high basal rates of steroidogenesis, and treatment with inhibin did not influence either basal or gonadotropin responsive steroidogenesis. Treatment with activin had divergent effects on aromatase activity and progesterone synthesis in that it increased both basal and hLH-responsive aromatase activity (up to 11-fold), had no effect on basal progesterone production, and markedly suppressed (by more than 50%) the progesterone response to hLH. These data reveal development-dependent effects of inhibin and activin on granulosa cell steroidogenesis that are likely to have physiological relevance to ovarian function in vivo.

Steroidogenesis in vitro of human granulosa-luteal cells pretreated in vivo with two gonadotropin releasing hormone analogs employing different protocols.

We have previously observed impaired progesterone accumulation in vitro in response to human chorionic gonadotropin (hCG) by cells pretreated in vivo with a gonadotropin releasing hormone analog (GnRH-a). The present study was conducted in order to evaluate different protocols for GnRH-a in in vitro fertilization (IVF), employing two different available analogs. Granulosa-luteal cells were collected at ovum pick-up and stimulated with hCG. Buserelin (Bus) was employed as long (Bus-L) and short (Bus-S) protocol, and Leuprolide (Leu) was also used as long (Leu-L) and short (Leu-S) protocol. Progesterone accumulation in vitro was compared with cells treated with clomiphene citrate (CC) and gonadotropins. Maximal progesterone production was observed on culture day 6 using Bus-L in comparison to day 4 when clomiphene citrate was employed. While Leu-S showed a similar pattern of progesterone accumulation to clomiphene citrate, Leu-L and Bus-S had an intermediate pattern. The response to hCG was maximal on day 4 for the clomiphene citrate- and Leu-S-treated cells, while the rest of the protocols had a peak on day 6. In addition, hCG consistently stimulated progesterone production in all protocols except in Bus-L. These results confirm an altered progesterone accumulation in vitro when GnRH-a are used. The effect seems to be more evident in long protocols, especially when buserelin is used, suggesting a higher accumulation of the analog in follicular fluid.

The use of gonadotrophin releasing-hormone analogues (GnRHa), in in-vitro fertilization: some clinical and experimental investigations of a direct effect on the human ovary.

Several lines of investigation suggest a direct modulatory role for gonadotrophin-releasing hormone (GnRH) on granulosa cell functions. Also, GnRH and its analogues (GnRHa) have been implicated in the resumption of meiosis, both in vivo and in vitro. Despite the presence of specific receptors for GnRH on human granulosa and luteal cells, very little is known about the possible effects of this hormone on the ovary. The use of GnRHa for long periods of time in patients undergoing in vitro fertilization (IVF) may influence granulosa cell function and/or oocyte maturation. We describe our clinical and experimental data in which we have searched for evidence of a direct action of GnRHa on the ovary. We have found that the retrieval of higher numbers of oocytes in women treated with GnRHa is correlated with oocytes of lower quality, manifested by a decreased fertilization and implantation rate. This impairment seems to be the consequence of oocyte immaturity, as ascertained by cytogenetic analysis of unfertilized oocytes in which an increase in diploidy, as well as prematurely condensed sperm chromosomes of the G1 phase, was observed in women with an excessive response to the stimulating drugs. Follicular atresia was not increased in women treated with GnRHa. Thus, there was no evidence for a direct effect of GnRHa on the human oocyte. Rather, the observations reflect the harmful effect of pushing follicles in early stages of development using this stimulation protocol. We have also searched for possible effects of GnRHa on granulosa-luteal cells obtained at ovum collection. In vitro culture of these cells has shown that the steroidogenic pathway is affected.(ABSTRACT TRUNCATED AT 250 WORDS)

Development-related effects of recombinant activin on steroid synthesis in rat granulosa cells.

Activin is structurally related to polypeptide growth factors such as transforming-growth factor-beta, which may have paracrine and/or autocrine functions in the ovaries. We have investigated the action of activin on granulosa cell steroidogenesis in vitro in relation to preovulatory follicular development in vivo. Estrogen-primed immature female rats received no other treatment (nondifferentiated granulosa cells), treatment with ovine (o) FSH (differentiated granulosa cells), or treatment with oFSH followed by human (h) CG (preovulatory granulosa cells) to stimulate preovulatory follicular development. Granulosa cells were isolated and cultured in the presence and absence of recombinant human activin-A using serum-free medium supplemented with 1.0 microM testosterone as an aromatase substrate and hFSH, hLH, forskolin, or 8-bromo-cAMP to stimulate steroid synthesis in vitro. After 48 h, medium was collected for measurement of estradiol (aromatase activity), progesterone, and cAMP. Basal steroid synthesis in nondifferentiated granulosa cells was unaffected by activin, but both aromatase activity and progesterone production induced by treatment with FSH in vitro were dosedependently enhanced up to 10-fold by the presence of activin. FSH-stimulated cAMP production was not measurably altered by activin; however, steroidogenesis induced by forskolin or 8-bromo-cAMP was significantly enhanced by the factor. Thus the effect of activin on steroidogenesis includes action at a subcellular level(s) distal to the production of cAMP. After gonadotropin treatment in vivo, granulosa cell aromatase activity and progesterone production showed divergent responses to activin in vitro. Basal-, FSH-, and LH-stimulated aromatase activity were all enhanced by activin in cultures of differentiated and preovulatory granulosa cells. However, whereas basal progesterone production was stimulated by activin in cultures of differentiated granulosa cells, in preovulatory granulosa cells it was inhibited. Moreover, in vitro stimulation of progesterone production by treatment of both differentiated and preovulatory granulosa cells with FSH or LH was suppressed by the presence of activin. Thus rat granulosa cells display development-related steroidogenic responses to activin, aromatase production becoming enhanced and progesterone production suppressed as follicular maturation progresses. These results further implicate activin as a local modulator of granulosa cell steroid synthesis in the ovaries, although its functional significance has yet to be established.

In vitro fertilization as a diagnostic and therapeutic tool in a patient with partial 17,20-desmolase deficiency.

OBJECTIVE: To present a case with 17,20-desmolase activity deficiency in which in vitro fertilization (IVF) served not only as a therapeutic approach but also as a diagnostic tool for the specificity of the enzymatic deficiency. DESIGN: IVF in the patient under study compared with a control group. All women treated with pure follicle-stimulating hormone (FSH). SETTING: IVF program at the Instituto Valenciano de Infertilidad. PATIENTS, PARTICIPANTS: A patient with primary amenorrhea, who was the subject under study, and seven normally cycling control patients undergoing IVF in the same series. INTERVENTIONS: IVF, steroidogenesis in vitro of granulosa-luteal cell obtained at ovum pick-up. MAIN OUTCOME MEASURE(S): Oocyte fertilization and embryo cleavage. Serum and follicular fluid (FF) levels of estradiol (E2), progesterone (P), testosterone (T), androstendione (A), 17 alpha-hydroxyprogesterone (17-OHP). In vitro accumulation of E2 and P. RESULTS: Ovulation induction with FSH was successful in achieving follicular development despite low circulating E2. Fertilization and cleavage rates were similar to the control subjects. The patient developed ovarian hyperstimulation. The lack of 17,20-desmolase activity was detected by normal P levels in serum and FF, high 17-OHP, and low T, A, and E2 levels in serum and FF. Granulosaluteal cell cultures in the presence of T restored normal E2 and P production in response to gonadotropins. CONCLUSIONS: In patients with 17,20-desmolase deficiency, follicular development, oocyte maturation, and fertilization can take place in a low estrogenic environment.

Development of ovarian cysts during gonadotrophin-releasing hormone agonists (GnRHa) administration.

The incidence of ovarian cyst formation during stimulation with additional pituitary suppression was retrospectively studied in 359 patients included in our in-vitro fertilization (IVF) programme. Women were classified according to the type of pituitary desensitization with subcutaneous buserelin used in group A (long protocol; n = 285) and group B (short protocol; n = 74). The rate of appearance of single follicular ovarian cysts for group A was 9.82% and for group B 22.97% (P less than 0.005). Ovarian cystic formations were usually asymptomatic and nonfunctional. The presence of these cysts did not seem to interfere with the ovarian response to stimulation treatment. Oocyte retrieval and pregnancy rate were similar between patients who developed ovarian cysts during gonadotrophin-releasing hormone analogue (GnRHa) therapy and those without cyst formation. These results suggest that ovarian cysts developing during GnRHa treatment are probably the consequence of the initial gonadotrophin rise and that the presence of ovarian cysts in these conditions should not be considered a necessary cause of cancellation for IVF patients.