Increased progesterone secretion and 3 beta-hydroxysteroid dehydrogenase activity in human cumulus cells by pregnenolone is limited to the high steroidogenic active cumuli.

PURPOSE: Several reports imply that lower progesterone secretion by cumulus-oocyte complexes (COCs) is associated with lower fertilization in the corresponding oocyte. The possible role of progesterone in oocyte fertilization in humans was studied using two approaches: (a) increasing the total progesterone secretion by culturing more than one COC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. METHODS: Mature COCs were cultured individually or cocultured in groups. Oocyte fertilization and progesterone secretion were tested after 20 hr and 3 days in culture, respectively. The cumuli from individually plated COCs were cultured in the absence of oocyte for an additional 3 days in order to test the effects of pregnenolone on progesterone secretion and the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity. A comparable study with pregnenolone was performed on the corresponding granulosa-lutein cells. RESULTS: Increasing the number of COC to two instead of one led to a significant increase in both fertilization rate and progesterone secretion. The addition of pregnenolone during days 3-6 increased significantly both progesterone secretion and 3 beta-HSD activity. Comparable results were observed in granulosa-lutein cells subjected to pregnenolone treatment. Following the first 3 days culture, cumulus masses were categorized as secreting high or low progesterone levels. Adding pregnenolone had a greater effect on both progesterone secretion and 3 beta-HSD activity in the high-progesterone-secreting cumuli. CONCLUSIONS: Addition of pregnenolone increased progesterone secretion and 3 beta-HSD more efficiently in the higher-progesterone-secreting cumuli. Coculture of two COCs instead of one led to a higher fertilization rate and greater progesterone secretion.

Expression of Wilms' tumor suppressor gene (WT1) in term human trophoblast: regulation by cyclic adenosine 3',5'-monophosphate.

The Wilms' tumor suppressor gene (WT1), which is deleted in some Wilms' tumors, encodes a zinc finger transcription factor. We studied WT1 messenger ribonucleic acid (mRNA) in human term placenta and cytotrophoblasts differentiating into syncytiotrophoblasts in vitro by RT-PCR. The results suggest that WT1 mRNA is expressed in the trophoblasts in a cell-specific fashion. WT1 mRNA expression has been observed to decline remarkably in trophoblast cells after 72 h, when these cells are morphologically differentiated into multinucleated syncytiotrophoblasts. As it is well known that cAMP as a second messenger plays a significant role in cellular proliferation and differentiation of placental cells, we examined the effect of 8-bromo-cAMP on WT1 mRNA expression in undifferentiated cytotrophoblasts and differentiated syncytiotrophoblasts. We observed that cAMP enhanced WT1 mRNA expression in cytotrophoblasts, but remained ineffective in altering WT1 mRNA in syncytiotrophoblasts. In summary, the results of this investigation demonstrate that the WT1 gene is developmentally regulated during trophoblast differentiation. An involvement of the cAMP-mediated system in regulating the WT1 gene in the trophoblast is suggested.

Effect of androgen substrates on the steroidogenic pattern of cumulus cells: correlation with cumulus culture morphology.

BACKGROUND: In previous studies, higher progesterone secretion was observed in mature versus immature cumulusoocyte complexes. In mature cumulus mass that become homogeneously spread in culture (type C/D) progesterone secretion was higher than in partially (type B) or totally (type A) aggregated morphology. In sharp contrast, estradiol-17 beta secretion was significantly higher in type A than type C/D cumulus. PURPOSE: Our purpose was to assess whether the decreased estradiol-17 beta level in type C/D cumulus culture is caused by deficiency of substrates. METHODS: The different cumulus types were incubated with or without 10(-7) M dehydroepiandrosterone, 4-androstane-3, 17-dione, or testosterone. The levels of estradiol-17 beta, testosterone, and progesterone, were measured after 24 hr of culture. RESULTS: The addition of dehydroepiandrosterone or 4-androstane-3, 17-dione significantly increased the estradiol-17 beta levels in all types of cumulus cells, whereas the addition of testosterone was less effective. In all types of cumulus cells the testosterone levels increased significantly on adding these androgen substrates. In the type C/D cumulus, the testosterone increased to lower levels compared to type A cumulus cells. In the presence of these androgens progesterone secretion is significantly reduced in type A cumulus cells. In type C/D cumulus cells, however, progesterone levels were significantly higher than in type A. The estradiol-17 beta/ testosterone and progesterone/estradiol-17 beta ratios, which partially resemble the degree of aromatase activity and the degree of selectivity for progesterone secretion, respectively, were higher in type C/D than in type A cumulus cells. CONCLUSIONS: In type C/D cumulus the significant increase in estradiol-17 beta secretion in the presence of various androgens suggests that, under basal conditions, androgen is less available for estradiol-17 beta biosynthesis compared to type A cumulus. Furthermore, the higher progesterone secretion in type C/D cumulus may suggest that the follicles yielding type C/D cumulus cells are more mature than the follicles yielding type A cumulus.

Increasing progesterone secretion and 3 beta-hydroxysteroid dehydrogenase activity of human cumulus cells and granulosa-lutein cells concurrent with successful fertilization of the corresponding oocyte.

In many studies it has been documented that the induction of multiple follicular growth in humans results in an asynchrony between the degree of cumulus mucification, oocyte meiotic maturation, fertilizability, and follicular cell progesterone (P4) secretion. The present study was carried out on oocytes enclosed in fully mucified cumulus. Thus, oocyte fertilizability was correlated to human cumulus cell (hCC) and human granulosa-lutein (G-L) cell competence for P4 secretion in culture. In the G-L cells, P4 secretion and percentage of cells manifesting 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) activity increased concurrently with the period of culture. In the hCC, however, P4 secretion decreased concurrently with elongation of the culture period, whereas the percentage of 3 beta-HSD-positive cells increased. In hCC corresponding to the fertilized oocytes, P4 accumulation in culture medium was 1.9-fold (P < 0.001) and 1.6-fold (P < 0.02) higher on days 0-3 and 3-5 of culture, respectively, as compared to P4 accumulation in hCC of unfertilized oocytes. Also, in hCC corresponding to the fertilized oocytes, the degree of 3 beta-HSD activity was found to be significantly higher shortly after aspiration and after either 3 or 5 days, compared to hCC of unfertilized oocytes. In the G-L cells pooled from all follicles yielding mature cumulus-oocyte complexes, P4 accumulation and percentage of 3 beta-HSD-positive cells increased concurrently with the increase in percentage of fertilized eggs of each individual woman. These results indicate that in stimulated cycles, follicles yielding mature cumulus-oocyte complex, oocyte fertilizability, and G-L cell or hCC competence for P4 secretion are correlated and synchronous.

Induction of maturation of cumulus-oocyte complex by gonadotropin-releasing hormone analog is associated with lower progesterone secretion.

Induction of ovulation by CG in women subjected to in vitro fertilization and embryo transfer results in maturation of the cumulus-oocyte complex (COC) in terms of oocyte meiotic maturation, cumulus mucification, and an increase in progesterone (P4) secretion. Recently an alternative approach, in which the ovulatory processes are induced by the administration of GnRH analog (GnRHa), has resulted in COCs yielding viable embryos. In the present study the effect of GnRHa administration on oocyte meiotic maturation and cumulus cell steroidogenesis was evaluated in 27 women undergoing ovarian stimulation with gonadotropin for the purpose of in vitro fertilization and embryo transfer. In GnRHa- or CG-treated women, 79.5 +/- 4.2% and 72.0 +/- 8.4% of the oocytes, respectively, manifested the first polar body. The percentages of atretic oocytes and of oocytes failing to resume meiosis were 5.8 +/- 2.1% and 6.0 +/- 2.4%, respectively, in GnRHa-treated women, and were 9.3 +/- 4.7% and 7.9 +/- 4.1%, respectively, in CG-treated women. P4 was secreted in high quantity in human cumulus cells (CCs) during a 7-day culture period. However, in CCs collected from GnRHa-treated women, P4 secretion was more than 50% less than in CCs of CG-treated women (P < 0.005). Addition of 20 alpha-hydroxycholesterol significantly increased P4 secretion in CCs collected from GnRHa-treated women, similar to P4 secretion in CCs collected from CG-treated women. This study evaluated for the first time the effectiveness of GnRHa administration on COC maturation. It seems that although oocyte meiotic maturation in GnRHa-treated women proceeds as in CG-stimulated women, CC steroidogenic activity is marked by cholesterol deficiency or availability.

Effect of cell-plating density on the steroidogenic activity of human cumulus cells.

Expansion of the cumulus mass results in dilution of cumulus cells in accumulated hyaluronic acid. This process is associated with an increase in progesterone secretion. The present study was carried out to evaluate the relationship between these two processes in vitro. Dilution of human cumulus cells was attempted by changing the cell-plating density of cumulus cells in culture. Concurrent with an increase in cell-plating density from 0.25 x 10(4) to 8 x 10(4), progesterone secretion increased by 10.5 times (P < 0.001), 9 times (P < 0.001), and 5.9 times (P < 0.001), and oestradiol secretion increased by 1.4 times (P < 0.001), 1.1 times (P > 0.05), and 2.6 times (P < 0.005) during days 0-3, 3-5, and 5-7 of culture, respectively. However, when steroid secretion was expressed in terms of ng per number of cells, the increase in cell-plating density from 0.25 x 10(4) to 8 x 10(4) coincided with a decrease in progesterone secretion of 3 times (P < 0.001), 3.5 times (P < 0.001), 30 times (P < 0.001), and 12.5 times (P < 0.001) during days 0-3, 3-5, and 5-7 of culture, respectively. The progesterone:oestradiol ratio increased gradually with the increase in cell-plating density. However, at the higher range of cell-plating density the progesterone:oestradiol ratio decreased with extension of the culture period. Addition of human FSH (hFSH) or hCG increased progesterone secretion when the cumulus cells were plated at 2 x 10(4), but in most cases not at 0.25 x 10(4) or 8 x 10(4) cells per dish.(ABSTRACT TRUNCATED AT 250 WORDS)

Failure of meiotic competence in human oocytes.

The administration of hCG to women undergoing in vitro fertilization and embryo transfer (IVF/ET) results in the meiotic maturation of cumulus-oocyte complexes (COC). Sometimes oocytes being aspirated for IVF/ET fail to resume meiosis in vivo and even after a subsequent 20-h incubation in vitro and are thus defined as meiotic competence failure (MCF) oocytes. The relationship between the proportion of MCF oocytes and other IVF/ET outcomes was studied over 3 years in 703 tested cycles of 487 women. Women yielding one or more MCF oocytes in at least one menstrual cycle represented 8.6% of this population and were defined as MCF women. Cumulus state in the MCF oocyte population was characterized as mature in 57.4 +/- 6.7%, intermediate in 13.9 +/- 4.0%, immature in 24.1 +/- 8.7%, and atretic in 4.6 +/- 2.7%. These values differed significantly, by 0.6-, 2.9-, 7.1-, and 4.6-fold, respectively, as compared to the corresponding COC aspirated from women yielding only meiotically competent (MC) oocytes. In a menstrual cycle yielding both MC and MCF oocytes, the IVF/ET variables were evaluated in the MC oocytes. Thus, in such cases the incidence of fertilization or cleavage and the number of blastomeres per embryo were significantly reduced concomitant with the increase in percentage of MCF oocytes. When the percentage of MCF oocytes was 25% or more, no pregnancy was achieved. Various follicular parameters and serum 17 beta-estradiol (E2) and progesterone (P4) levels were compared in MC and MCF women over the four days preceding day of aspiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Modulation of peripheral benzodiazepine receptors in female rat genital organs by various gonadal steroids.

Peripheral benzodiazepine receptors (PBR) in the ovary, oviduct, uterus, and kidney of immature rats were studied under short- and long-term treatment with testosterone (T), progesterone (P4), and diethylstilbestrol (DES). A significant increase in PBR specific binding was observed after 4 days' treatment with T in the ovary (1.6-fold), oviduct (2.0-fold), and uterus (1.4-fold) compared with intact rats. Four days' treatment with P4 increased PBR specific binding in the ovary (1.5-fold), but no changes were detected in the oviduct or uterus. In contrast, PBR specific binding was significantly reduced by 10 days' treatment with T or P4: 40 and 12%, respectively, in the ovary and 35 and 40%, respectively, in the oviduct. Ten days' treatment with T reduced PBR specific binding in the uterus by 25%, but the same interval of treatment with P4 did not alter specific binding in the uterus. Four or 10 days' treatment with DES significantly increased PBR specific binding in the ovary (1.5-fold), oviduct (2.4-fold), and uterus (1.9-fold). Scatchard analysis revealed that the changes in the PBR specific binding were due to a change in PBR density values rather than PBR affinity values. No change in PBR specific binding was found in the kidney following any of these treatments. Taken together, it is suggested that PBR density in the ovary is altered by exogenously administered steroids that usually are biosynthesized in the ovary. Additionally, the altered PBR density in the oviduct and uterus via the various steroids employed may imply that changes occurring in ovarian steroidogenesis should affect PBR density in these organs.

Long-term testosterone or diethylstilbestrol treatment affects gamma-aminobutyric acid and central-type benzodiazepine receptors but not peripheral-type benzodiazepine receptors in the female rat brain.

Steroids have often been associated with modulation of the GABAergic system in the central nervous system, mainly in ovariectomized rats. In the present study, the effect of the synthetic estrogen diethylstilbestrol (DES) and testosterone (T) on the density of peripheral and central benzodiazepine (BZ) and gamma-aminobutyric acid (GABAA) receptors was evaluated in the frontoparietal cortex and whole cerebellum of female rats during the peripubertal period. The density of peripheral-type BZ receptors was not altered in either of these organs, whether or not treated with DES or T. The density of central BZ and GABAA receptors in either frontoparietal cortex or whole cerebellum was significantly reduced following treatment with DES or T; however, the effect of DES was much more pronounced. The similarity of the effect of T to that of DES may suggest that the effect of T is mediated at least partially by intraovarian biosynthesis of estradiol-17 beta from the exogenously administered T. Collectively, these results may suggest that in female rats during the peripubertal period, sex steroids produce a down-regulatory effect on expression of the brain GABAA/BZ complex, in contrast to their well-established up-regulatory effect in adult ovariectomized rats.

Increasing progesterone secretion in human granulosa-luteal cells induced by human follicular fluid.

Increasing evidence suggests that local ovarian agents play a central role in the regulation of follicular maturation and corpus luteum formation. In previous studies, we have shown that porcine follicular fluid induces granulosa cell luteinization in sow, human and rat. In the present study, the effect was investigated of either human follicular fluid (FF) alone, human follicle-stimulating hormone (FSH) alone, or both upon progesterone secretion of human granulosa-luteal cells. Granulosa-luteal cells were cultured in the presence of either FSH (5, 50 and 250 ng/ml), lyophilized FF (50 and 250 micrograms/ml) or both. Secretion of progesterone increased from a minimum of 2.5-fold to a maximum of 23-fold in the presence of FSH alone and, significantly less (approximately 2-fold) in the presence of FF alone, compared to cells cultured in medium alone. The co-administration of FSH and FF was significantly more effective than either alone, while addition of both FSH (250 ng/ml) and FF (250 micrograms/ml) gave maximal progesterone secretion. In granulosa-luteal cells pre-cultured with both FSH and FF, subsequent exposure to human chorionic gonadotrophin (HCG) alone increased progesterone secretion 1.6-fold to 11-fold, compared to cells pre-cultured with FSH alone. The effect of FF from individual follicles was also studied. FF from follicles yielding mature cumulus-oocyte complexes was 4.2-fold more effective, than FF obtained from follicles yielding immature cumulus-oocyte complexes in enhancing the FSH stimulation of progesterone secretion.(ABSTRACT TRUNCATED AT 250 WORDS)

Altered steroidogenic activity of human granulosa-lutein cells at different cell densities in culture.

In the present study, the relationship between human granulosa-lutein cell (hGLC)-plating density and steroidogenic activity was evaluated. Increasing hGLC-plating density 32-fold, from 0.25 x 10(4) to 8 x 10(4) cells/well, was associated with a concomitant increase in the total amount of progesterone (P4), testosterone (T), and estradiol-17 beta (E2) secretion. The daily amount of each steroid (P4, T, and E2) secreted by hGLC at different cell-plating densities was further normalized per 10(3) cells. Thus, an increase in hGLC-plating density from 0.25 x 10(4) to 1 x 10(4) cells/well was associated with approximate increases of 1.3-fold in P4 and 3-fold in T and a 50% decrease in E2 secretion, per 10(3) cells. A further increase in hGLC-plating density, from 1 x 10(4) to 8 x 10(4) cells/well, was associated with a significant decrease of approximately 3.7-fold in P4 and 6-fold in T per 10(3) cells. A similar increase in hGLC-plating density was associated with no change or a 2-fold decrease, per 10(3) cells, in E2 secretion during days 0-3 or days 3-5 of culture, respectively. The P4/E2 ratio was increased and the E2/T ratio decreased with extension of the culture period. These two ratios had a tendency to be altered inversely, concurrent with the increase in cell-plating densities. At 1-2 x 10(4) cells/well, P4/E2 was maximal, whereas E2/T was minimal.(ABSTRACT TRUNCATED AT 250 WORDS)

Biochemical, physiological, and pathological aspects of the peripheral benzodiazepine receptor.

The PBR is a mitochondrial protein composed of at least two subunits, an approximately 30-kDa subunit that contains the site for BZs and an approximately 18-kDa subunit that binds isoquinoline carboxamide derivatives. Porphyrins and diazepam binding inhibitor are putative endogenous ligands for these receptors, which are under neural and hormonal control. Alterations in the density of PBR seem to be a sensitive indicator of stress: up-regulation after acute stress and down-regulation induced by repeated stress. PBR-specific ligands are involved in the control of cell proliferation and differentiation, and their binding is increased in some cancer tumors. Numerous studies in various endocrine organs have revealed that PBR are located in specific regions or tissues in the organs. Furthermore, PBR densities in various organs subject to hormonal control are regulated by organotropic hormones. At least in some cases, BZ ligands do not exert a specific effect in an organ, but rather modulate the well-documented effects of that particular hormone. To the best of our knowledge, BZ ligand action in peripheral tissues is dependent on recognition of PBR, which may suggest a receptor-mediated action.

Altered peripheral benzodiazepine receptor density in human granulosa-lutein cells in relation to follicular maturity.

Since the introduction of treatment by in vitro fertilization/embryo transfer (IVF-ET) in humans, a harmless approach to identifying the ovum with a greater chance of producing an implantable embryo has been sought worldwide. Our previous studies indicated a high correlation between degree of follicular maturation and benzodiazepine (BZ) receptor density in rats. We hypothesized that if such a correlation existed in humans, the latter might be used to evaluate degree of follicular maturation and, consequently, its corresponding oocyte competence. We used [3H]PK 11195, a ligand selective to peripheral BZ receptors, to determine their density in human granulosa-lutein (G-L) cells. [3H]PK 11195 bound to G-L cells with high affinity in a saturable manner. Scatchard analysis revealed the presence of a single population of receptors. The average equilibrium dissociation constant and maximal number of receptors (Bmax) measured in G-L cells were 3.4 +/- 0.2 nM and 608 +/- 61 fmol/mg protein, respectively. In G-L cells obtained from individual follicles, a significant increase in the specific binding to peripheral BZ receptors was noted in G-L cells of follicles yielding oocytes at an advanced stage of cumulus maturation or oocytes competent for fertilization and subsequent cleavage. When G-L cells were pooled from all the follicles of each individual woman, the average Bmax value of [3H]PK 11195 in G-L cells was 900 +/- 127 fmol/mg protein in women who conceived, which was 80% higher (P less than 0.01) than in women who did not conceive.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory and stimulatory action of porcine follicular fluid on FSH-induced 125I-hCG specific binding in rat granulosa cells.

In previous studies, follicular fluid (FF) of large antral follicles (LFF) manifested a stimulatory effect on granulosa cell (GC) luteinization in domestic livestock, but was found to have an inhibitory effect on rodent GC. In the present study, the type of LFF effect on the luteinizing of rat GC was reevaluated in two different models. GC obtained from immature hypophysectomized rats treated with diethylstilbestrol were cultured with increasing concentrations of FSH alone or FSH + estradiol-17 beta (E2), either for 3 or 4 successive days of culture (model 1) or for 4 days of culture with medium change after 2 days of culture (model 2). The addition of FSH increased 125I-hCG specific binding in a dose-dependent manner to a maximum of approximately 110-fold (model 1) or 45-fold (model 2) compared with GC culture in medium alone. At the maximal effective dose of FSH, addition of E2 increased the 125I-hCG binding 2-fold (model 1) and 3.5-fold (model 2). 125I-hCG specific binding induced by FSH or FSH + E2 in model 1 was decreased by concurrent treatment (added on the day of cell inoculation) with porcine LFF (approximately 3-fold) or porcine serum (approximately 4.5-fold). In model 2, however, porcine LFF increased FSH-induced 125I-hCG specific binding 3-fold, provided LFF was added only after GC were primed with FSH alone for 2 days. When porcine serum was added instead of porcine LFF, only a permissive action was observed. These data may suggest that an initial GC differentiation is indispensible for obtaining the FF stimulatory effect on FSH-induced 125I-hCG specific binding in rat GC.

Altered steroidogenic pattern of human granulosa-lutein cells in relation to cumulus cell culture morphology.

It has been reported that human-oocyte complexes (COC) retrieved at a stimulated cycle manifest an asynchrony between oocyte meiotic maturation and cumulus mucification. However, when mature COC were subdivided into subtypes marked by the culture morphology of their cumulus cells following 3 days' culture, successful fertilization and cleavage were approximately 1.5-fold lower in mature COC yielding cumulus cells aggregated into clumps (type A and B COC) than in mature COC yielding homogeneously spread cells (type C-D COC). To determine whether the existing relationship between cumulus culture morphology and oocyte functionality in the various COC types (A-D) could be extended to another follicular compartment--the granulosa-lutein (G-L) cells--basal steroid secretion by the corresponding G-L cells was evaluated within 5 days of culture. Over the first 3 days of culture, secretion of progesterone was 3-fold lower and secretion of testosterone (T) was 2.5-fold higher in cultures of G-L cells from follicles yielding type A COC than in type C-D COC. During days 4 and 5 of culture, G-L cells were incubated with or without 10(-7) M 3 beta-hydroxy-5-pregnen-20-one (pregnenolone), dehydroepiandrosterone (DHA), 4-androstene-3,17-dione (androstenedione), or T. The pattern of progesterone level noted over the first 3 days of culture was not altered in the presence of pregnanolone. DHA, androstenedione, or T. Addition of pregnenolone, DHA, androstenedione, or T increased T level 2.5-, 5.6-, 7.3-, and 17.7-fold, respectively, in cultures of G-L cells from follicles yielding type A COC, but did not significantly alter T level in cultures of G-L cells from follicles yielding type C-D COC. In cultures of G-L cells from follicles yielding type A COC, addition of androgens unsaturated at position 4 preferentially increased oestradiol-17 beta (E2) level, whereas in cultures of G-L cells of type C-D COC, DHA and androstenedione preferentially increased E2 level. Taken together, the asynchrony between oocyte and cumulus activity could be diminished when the activity of various follicular cell compartments is evaluated according to cumulus culture morphology rather than cumulus expansion and mucification. The present study suggests that follicles yielding mature COC represent a non-homogenous population in which G-L cells from follicles yielding type A-B COC manifest a less luteinized state than those from follicles yielding type C-D COC.

Increasing 125I-human chorionic gonadotrophin specific binding in human granulosa cells by follicle-stimulating hormone and follicular fluid.

It has been reported that success at in-vitro fertilization/embryo transfer (IVF/ET) treatment is increased by follicular fluid (FF) re-injected into the abdomen. In the present study a possible direct effect of FF on human granulosa cell (GC) progesterone (P4) secretion and LH/human chorionic gonadotrophin (HCG) receptor content was studied in the presence and absence of FSH. Human GC cultured for 8 days in medium alone showed a 40-fold decrease in P4 secretion. Addition of human FSH increased P4 secretion and [125I]HCG specific binding by 12- and 8-fold, respectively, compared to human GC cultured in medium alone. The effect of FF was evaluated in a heterologous system by the addition of FF from large antral porcine follicles (LFF) to human GC in culture. The decline in human GC-P4 secretion after 8 days of culture was not altered by either porcine serum alone or porcine LFF alone. However, the concomitant addition of FSH and LFF significantly increased [125I]HCG specific binding, but did not alter the FSH-induced P4 secretion when both parameters were compared to GC cultured in FSH + porcine serum. Furthermore, the addition of HCG alone significantly increased P4 secretion 33- and 70-fold in GC pre-cultured with either FSH alone or FSH + LFF respectively compared with the stimulatory effect of HCG on GC pre-cultured in medium alone. These results may suggest that FSH and LFF increase the functional content of LH/HCG receptor in luteinized human GC.

Different morphological and steroidogenic patterns in oocyte/cumulus-corona cell complexes aspirated at in vitro fertilization.

We attempted to correlate distinct morphological data on cumulus cells to oocyte and cumulus cell activity. Oocyte/cumulus-corona cell complexes, which were mature 4 h after aspiration, were divided into four subgroups designated according to the type of cumulus culture morphology after 3 days of culture: type A, compact clumps; type B, partially spread clumps; type C, nonhomogeneously spread cells; and type D, homogeneously spread cells. Fertilization and cleavage rates of mature oocytes appeared to differ according to their prospective cumulus culture morphology. Fertilization and cleavage rates were 81.5 and 62.6%, respectively, in oocyte/cumulus-corona cell complexes yielding type D cumulus cells, versus 54 and 34%, respectively, in those yielding type A cumulus cells. Basal secretion of progesterone in type A cumulus cells was 105.2 +/- 10.3 ng/ml compared to 231.8 +/- 22.5 ng/ml in type D cumulus cells (p less than 0.001). Testosterone and estradiol secretion exhibited a significant difference as well: testosterone was 293 +/- 10 pg/ml in type A cumulus cells versus 224 +/- 11 pg/ml in type D cumulus cells (p less than 0.001), and estradiol was 4.6 +/- 0.4 ng/ml in type A cumulus cells versus 3.5 +/- 0.3 ng/ml in type D cumulus cells (p less than 0.05). The present study demonstrated by indirect means that oocyte/cumulus-corona cell complexes, characterized as mature a few hours after aspiration, are composed of a heterogeneous population and differ in their potential for fertilization and consequent cleavage.

Hormonal regulation of peripheral benzodiazepine binding sites in female rat adrenal gland and kidney.

The effect of hypophysectomy and hormonal replacement on the density of peripheral benzodiazepine binding sites (PBS) in rat adrenal gland and kidney was studied. In the adrenal gland, hypophysectomy caused a significant decrease of 3-fold in PBS density. In the kidney, in contrast, hypophysectomy did not affect PBS density. In the adrenal gland, adrenocorticotropic hormone (ACTH) administered to hypophysectomized rats caused a significant increase of more than 5-fold in PBS density compared to untreated hypophysectomized rats, and of more than 1.6-fold compared to intact rats. In contrast, the hormones pregnant mare serum gonadotropin (PMSG), diethylstilbestrol (DES), and hydrocortisone (HC), administered to hypophysectomized rats, failed to restore PBS density in the adrenal gland. In the kidney, HC administered to hypophysectomized rats caused an increase of 1.4-fold in PBS density compared to untreated hypophysectomized and intact rats. In contrast, the hormones ACTH, PMSG, and DES, administered to hypophysectomized rats, did not affect PBS density in the kidney. None of the hormones tested altered the equilibrium dissociation constant of PBS in either the adrenal gland or the kidney. These findings indicate that PBS density in rat adrenal gland and kidney is hormonally modulated.