Shalom Bar-Ami

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.

Gonadotropin- and estrogen-induced increase of peripheral-type benzodiazepine binding sites in the hypophyseal-genital axis of rats

Peripheral-type benzodiazepine binding sites (PBS) were demonstrated in the cell membranes of various organs (ovary, uterus, oviduct, pituitary and kidney) of mature and immature female rats by using the PBS-specific ligand [3H]PK 11195. The equilibrium dissociation constants of [3H]PK 11195 for PBS in mature rats ranged from 3 to 4 nM. The specific binding of [3H]PK 11195 (2 nM) in the hypophyseal-genital axis of immature (19-27 days old) female rats was found to be significantly increased in the ovary and uterus, concurrently with the increase in age. Administration of pregnant mare serum gonadotropin or diethylstilbestrol to immature rats increased the density of PBS in the ovary and uterus 2- to 3-fold but no change was found in the kidney. The affinity of [3H]PK 11195 to these tissues did not change following hormonal treatment. These results suggest that gonadotropin and estrogen are involved in the induction of PBS in the organs of the hypophyseal-genital axis in female rats.

Increasing progesterone secretion and 3β-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.

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)

Prospective evaluation of early follicular ovarian stromal blood flow in infertile women undergoing IVF-ET treatment

Background: To evaluate the role of early follicular stromal flow studies in predicting ovarian response during IVF-ET treatment and to assess their correlation with ovarian reserve parameters and clinical pregnancy achievement. Materials & Methods: One hundred and sixty-eight consecutive and unselected infertile women undergoing their first IVF-ET treatment were included in the study. Basal ovarian reserve and stromal Doppler flow studies were performed in a natural cycle before starting treatment. Four Doppler indices were measured; peak systolic velocity (PSV), pulsatility index (PI), resistance index (RI) and systole/diastole ratio (S/D). Following completion of IVF-ET treatment Pearson’s correlation analysis was performed to examine the correlation between Doppler indices, ovarian response, basal ovarian reserve parameters and clinical pregnancy achievement. Results: A positive correlation was found between the number of ≥14 mm follicles on hCG day and PSV. The number of ≥14 mm follicles and retrieved oocytes had a significant negative correlation with RI and S/D ratio. As well, the number of fertilized oocytes had a significant negative correlation with S/D ratio. Absence of a Doppler signal in one or both ovaries was significantly higher in the women with poor response (31%) as compared to women with normal response (16%). In addition, RI correlated positively with basal FSH as well as FSH/LH ratio and negatively with AFC. The S/D ratio had a negative correlation with AFC (p = 0.027). A significant positive correlation between PSV, total ovarian volume (p = 0.011) and mean ovarian volume (p = 0.019) was detected. However, no correlation between all four Doppler indices and age was detected. Moreover, Doppler indices did not differ significantly between conception and non-conception cycles following IVF-ET treatment. Conclusions: Early follicular stromal Doppler signals is correlated with ovarian response as well as basal ovarian reserve parameters, but have no correlation with age neither with clinical pregnancy achievement in infertile women undergoing IVF-ET treatment.

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.

Improved development of very-poor-quality human preembryos by coculture with human fallopian ampullary cells.

AbstractPurpose: To determine whether a confluent culture of fallopian ampullary epithelial cells, taken from women at the end of their reproductive life, is capable of rescuing very-poor-quality preembryos from cleavage arrest and/or degeneration. Methods: Human preembryos, rejected for transfer or freezing because of very poor quality, and arrested within 24 h of cleavage, were cultured for 5 days in medium alone or over a confluent culture of fallopian ampullary epithelial cells. Morphological criteria were utilized to assess preembryo degeneration and stage of development. Results: The described coculture rescued preembryos from degeneration, enhancing development to the blastocyst stage 2.2-fold, compared with cultures in medium alone. Furthermore, fully expanded and hatching blastocysts were observed only under coculture conditions. Conclusions: Very-poor-quality human preembryos may be rescued from degeneration, and their growth and development dramatically improved, when cocultured with a confluent culture of fallopian ampullary epithelial cells.

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)

Increased Progesterone Secretion and 3β-Hydroxysteroid Dehydrogenase Activity in Human Cumulus Cells by Pregnenolone Is Limited to the High Steroidogenic Active Cumuli

AbstractPurpose: Several reports imply that lower progesteronesecretion by cumulus-oocyte complexes (COCs) isassociated with lower fertilization in the corresponding oocyte.The possible role of progesterone in oocyte fertilization inhumans was studied using two approaches: (a) increasingthe total progesterone secretion by culturing more than oneCOC per dish; and (b) increasing the cumulus cell progesterone secretion by providing pregnenolone as a substrate. Methods: Mature COCs were cultured individually orcocultured in groups. Oocyte fertilization and progesteronesecretion were tested after 20 hr and 3 days in culture, respectively.The cumuli from individually plated COCs were cultured inthe absence of oocyte for an additional 3 days in order totest the effects of pregnenolone on progesterone secretionand the 3β-hydroxysteroid dehydrogenase (3β-HSD)activity. A comparable study with pregnenolone was performedon the corresponding granulosa-lutein cells. Results: Increasing the number of COC to two instead ofone led to a significant increase in both fertilization rateand progesterone secretion. The addition of pregnenoloneduring days 3–6 increased significantly both progesteronesecretion and 3β-HSD activity. Comparable results wereobserved in granulosa-lutein cells subjected to pregnenolonetreatment. Following the first 3 days culture, cumulus masseswere categorized as secreting high or low progesteronelevels. Adding pregnenolone had a greater effect on bothprogesterone secretion and 3β-HSD activity in thehigh-progesterone-secreting cumuli. Conclusions: Addition of pregnenolone increased progesterone secretion and 3β-HSD more efficiently in thehigher-progesterone-secreting cumuli. Coculture of two COCsinstead of one led to a higher fertilization rate and greaterprogesterone secretion.

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-17β secretion was significantly higher in type A than type C/D cumulus.Purpose: Our purpose was to assess whether the decreased estradiol-17β 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-17β, 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-17β 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-17β/testosterone and progesterone/estradiol-17β 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-17β secretion in the presence of various androgens suggests that, under basal conditions, androgen is less available for estradiol-17β 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.