Induction of DNA polymerase in mouse L cells

Two molecular species of DNA polymerase are found in mouse L cells. This study is concerned with the variation of these two species of enzyme with the rate of cell growth and DNA synthesis. The 3.4 S DNA polymerase, found in both nuclear and cytoplasmic fractions of mouse L cells, remains relatively constant during different stages of the growth curve. The heterogeneous 6 to 8 S DNA polymerase, found only in the cytoplasmic fractions, varies about 5 to 12-fold in correlation with DNA synthesis, as measured by [³H]thymidine incorporation.     

Follicle-stimulating hormone stimulated differentiation and progesterone production of bovine granulosa cells in culture

Progesterone production of granulosa cells cultured in vitro is stimulated and cell differentiation increased, by follicle-stimulating hormone (FSH). This study examined whether the increased progesterone production observed when bovine granulosa cells are cultured occurs because (1) progesterone production by undifferentiated and/or differentiated cells is increased or (2) the differentiation of granulosa cells is stimulated. Viable bovine granulosa cells (2−3×10⁵) from follicles 5–8 mm in diameter were cultured in the presence of 0, 1, 10 and 100 μu FSH (1 μu ≡ 1 μg NIH-FSH-S1) for 6 days at 37°C in a humidified atmosphere of 5% CO₂ in air in 1 ml of a 1:1 mixture of Dulbecco's modified Eagle medium: Ham's F10 medium supplemented with 365 μg ml⁻¹ l-glutamine, 100 U ml⁻¹ penicillin and 100 μg ml⁻¹ streptomycin. Progesterone production, total DNA and protein, and cell diameter were determined sequentially over the culture period. The increases in progesterone production (ng μg⁻¹ DNA per 24 h), cytoplasmic:nuclear ratio (μg protein μg⁻¹ DNA) and cell diameter (μm) over 6 days culture indicated that granulosa cells underwent differentiation in the presence of FSH. Progesterone production of undifferentiated granulosa cells (diameter 14 μm or less) was stimulated by FSH (P < 0.01) in a dose dependent manner (1.0±0.2, 2.9±0.3, 3.7±0.3 and 4.9±0.4 ng μg⁻¹ DNA per 24 h for 0, 1, 10 and 100 μu ml⁻¹ FSH respectively) but remained constant within dose (P > 0.05) during a 6 day culture period. FSH stimulated (P < 0.05) the rate of granulosa cell differentiation (10±3%, 53±13%, 74±21% and 82±10% differentiating cells per well for 0 μu, 1 μu, 10 μu and 100 μu ml⁻¹ FSH respectively) but did not stimulate (P > 0.05) progesterone production by differentiating granulosa cells (8.7±0.5 ng μg⁻¹ DNA per 24 h). In conclusion, the increase in progesterone production of FSH-stimulated granulosa cells cultured in vitro appears to be mainly due to an increase in the number of differentiating cells with a constant rather than an increasing progesterone production per cell.     

Role of cell division in the cytodifferentiation of rat heart cells in culture

BrdU and irradiation with visible light eliminates dividing cells from rat heart cell cultures. The lethal effect of light on the cultures is dependent upon the prior integration of BrdU into the DNA. Elimination of dividing cells is shown by the decreased uptake of ³H TdR in the remaining cells. Cultures in which the dividing cells were eliminated displayed a loss in myosin ATPase activity and a decrease in the rate of myosin ATPase activity increase, normally seen in control cultures. These results are consistent with the existence of cells at three stages of development; premyoblasts, which divide and contain no myosin; myoblasts which divide and contain myosin; and myocytes, which cannot divide and contain myosin. The results also indicate that the increase in myosin ATPase activity normally seen in heart cell cultures is a result of an increase in myosin in fully developed cells and in the increase in the number of cells capable of synthesizing myosin.     

Populations of granulosa cells in small follicles of the sheep ovary.

The aim of this study in sheep ovaries was to determine the total number of granulosa cells in primordial follicles and at subsequent stages of growth to early antrum formation. The second aim was to examine the interrelationships among the total number of granulosa cells in the follicles, the number of granulosa cells in the section through the oocyte nucleolus, and the diameter of the oocyte. A third aim was to examine whether proliferating cell nuclear antigen labelling occurred in flattened granulosa cells in primordial follicles or was confined to follicles containing cuboidal granulosa cells. The follicles were classified using the section through the oocyte nucleolus by the configuration of granulosa cells around the oocyte as type 1 (primordial), type 1a (transitory), type 2 (primary), type 3 (small preantral), type 4 (large preantral), and type 5 (small antral). In type 1 follicles, the number of granulosa cells and oocyte diameter were highly variable in both fetal and adult ovaries. Each type of follicle was significantly different from the others (all P < 0.05) with respect to oocyte diameter, number of granulosa cells in the section through the oocyte nucleolus and total number of granulosa cells. Follicles classified as type 2, 3, 4 or 5 each corresponded to two doublings of the total granulosa cell population. The relationships between oocyte diameter and the number of granulosa cells (that is, in the section through the oocyte nucleous or total population per follicle) could all be described by the regression equation loge chi = a + b loge gamma with the correlation coefficients R always > 0.93. For each pair of variables the slopes (b) for each type of follicle were not different from the overall slope for all types of follicle pooled. Immunostaining for proliferating cell nuclear antigen was observed in granulosa cells in type 1 follicles, as well as in the other types of follicle. These findings indicate that 'flattened' granulosa cells in type 1 follicles express an essential nuclear protein involved in cell proliferation before assuming the cuboidal shape. Thus, when considering factors that regulate specific phases of early follicular growth, it is important to consider: (i) the follicle classification system used; (ii) the animal model studied; (iii) whether type 1 follicles are all quiescent; and (iv) the likelihood that each follicle type represents more than one doubling of the population of granulosa cells.     

Sites of transcription of the Müllerian inhibiting substance gene in mouse testis

The factors involved in the inhibition of ovarian follicular cellular growth after the luteinizing hormone (LH) surge are poorly established. The aim of this study was to investigate the production of an inhibitory growth factor by human ovarian cells. Luteinized granulosa cells were obtained from an assisted fertilization program and were cultured in the presence or absence of follicle-stimulating hormone (FSH) and estradiol. Data obtained by cell counting showed that the number of human luteinized granulosa cells cultured in the presence of fetal bovine serum (10%) increased 1.8-fold within a 2-day period. In serum-free medium, human luteinized granulosa cells were able to incorporate ³H-thymidine, measured during consecutive 48 h periods. During all the periods tested (up to 7 days), low basal levels of thymidine incorporation were measured and were further reduced in the presence of FSH (200 ng/ml) and estradiol (500 ng/ml). To elucidate the possible production of an inhibitory growth factor, ³H-thymidine incorporation by rat granulosa cell cultures was measured in the presence of conditioned media (CM; from human granulosa cell cultures). In this system, FSH and estradiol elicited a tenfold increase in thymidine incorporation. The addition of CM (10% v/v collected on day 2) to FSH- and estradiol-treated granulosa cell cultures produced an inhibition (61%) of thymidine incorporation. The active factor in CM withstood freeze-thawing, was stable for several weeks at – 20°C, became unstable at 4°C, and was heat labile and sensitive to proteolysis. Ultrafiltration using membranes with different molecular weight cutoffs suggested that the factor had a molecular weight >30,000 dalton. We suggest that an inhibitory growth factor produced by human luteinized granulosa cells could be involved in the differentiation of growing follicles to corpus luteum. © 1993 Wiley-Liss, Inc.     

Separation and analysis of cell types involved in early stages of carrot somatic embryogenesis

The occurrence of the polarized synthesis of DNA in embryogenic cell clusters of carrot on the third and fourth days after transfer to an embryogenesis-inducing medium was observed by labeling with [³H]thymidine and autoradiography. The cells that were actively synthesizing DNA were separated from cells that were not synthesizing DNA by maceration of cell clusters into individual protoplasts and centrifugation in a Percoll density gradient. [³⁵S]Methionine-labeled proteins extracted from the two types of cell were analyzed by SDS-PAGE and fluorography. Three polypeptides (of 69, 98 and 108 kD, respectively) were found only in cells that were actively synthesizing DNA and could be candidates for markers of the polarity of DNA synthesis that is specific to embryogenesis.     

Estrous cycle-controlled cell proliferation in the adrenal cortex of female rats

Summary In a total of 120 rats the number of ³H-Tdr-labelled nuclei of the adrenocortex was counted during various phases of the estrous cycle. In two separate experiments cyclic variations in the number of DNA synthesizing cells were found. The labelling index of the zona glomerulosa showed a significant maximum in proestrus whereas in zona fasciculata a minimum was revealed in estrus. In the glomerulosa the maximum of labelled cells was followed by a mitotic peak in estrus. Due to very low numbers of labelled cells no rhythm was found in the reticularis. The possible endocrine regulations of this endogenous rhythm of adrenocortical cell replication are discussed.The authors wish to express their appreciation for the expert assistance in the statistical evaluation of data rendered by Dr. Trieb and for the reliable technical aid by Mr. Majer     

A comparative kinetic analysis of proliferation in vitro of Con-A-treated splenocytes and syngeneic leukaemia cells

Abstract. The growth kinetics of Con-A-treated mouse splenocytes and syngeneic leukaemia cells cultured in vitro were compared with respect to (i) the total cell number, (ii) the rate of [¹⁴C]thymidine incorporation (measured by pulse-labelling the cells at various times of incubation), and (iii) the labelling index of the cell populations. By correlating the thymidine incorporation, labelling index and cell number data, it has been established that, for both types of cells, the rate of [¹⁴C]thymidine incorporation is directly proportional to the number of cells synthesizing DNA. A new approach to cytokinetic analysis has been developed, showing that important information can be obtained by determining the cumulative kinetics of [¹⁴C]thymidine incorporation. The latter has been calculated by integrating the area underneath the time course of the rate of thymidine incorporation, and was directly proportional to the overall growth of both leukaemia cells and Con-A-stimulated splenocytes. Based on this proportionality, an estimate of the average duration of the S phase for both types of cells was calculated, suggesting that normal and neoplastic blasts maintain this parameter at a constant value (7.6 and 5.9 hr, respectively) throughout different stages of growth. The percentage of Con-A-responsive cells within the initial splenocyte population and their overall proliferation in vitro have been determined by a procedure which measures the cumulative kinetics of thymidine incorporation and the kinetics of cell total number in the presence or in the absence of the lectin, as well as in the presence of Con-A plus colcemid. A minor fraction (11%) of the initial splenocytes is recruited into cycle by Con-A, proliferating with similar kinetics to that of leukaemia cells in the same conditions. The great majority of the initial splenocyte population is unaffected by Con-A, decaying exponentially throughout the incubation with the same half-life (28 hr), both in the presence or in the absence of the lectin.     

Transforming growth factor-alpha in the adult bovine ovary: identification in growing ovarian follicles

The pituitary gonadotropins and gonadal steroids are required for normal follicular growth and development but neither has been shown to act directly as a granulosa cell mitogen in vitro. A number of polypeptide growth factors, however, are known to have pronounced mitogenic effects on the cells of the follicle. We have localized transforming growth factor-alpha (TGF-alpha), a potent mitogen, in bovine thecal cells via immunoperoxidase staining using a monoclonal antibody for TGF-alpha that does not cross-react with epidermal growth factor. TGF-alpha staining is most intense in the theca of follicles at the discrete physiological stages known to show rapid granulosa cell growth (small follicles of 0.7-2.0 mm diameter). Staining intensity for TGF-alpha declines in large preovulatory follicles, coincident with the known decline in granulosa cell mitosis. These studies provide further evidence for paracrine interactions in the ovary and show that TGF-alpha may play an important role in the regulation of follicular development in the adult bovine ovary.     

Inhibition of pig granulosa cell adhesion and growth in vitro by immunoneutralization of epithelial cadherin

Epithelial cadherin (E-cadherin) is a member of the cadherin family of calcium-dependent cell adhesion molecules and is present in the ovary. Although expression of E-cadherin is high in healthy pig granulosa cells and low in granulosa cells of atretic follicles, the importance of E-cadherin-mediated adhesion in granulosa cell function is unclear. The aim of the present study was to determine the impact of immunoneutralization of E-cadherin on granulosa cell adhesion, DNA synthesis and cell proliferation in vitro. Before attachment, pig granulosa cells were exposed to a monoclonal E-cadherin antibody (DECMA-1) which blocks E-cadherin function. Controls included substitution of the antibody with either mouse ascites fluid or another E-cadherin antibody directed against the cytoplasmic domain and which was therefore inaccessible in intact cells. Both granulosa cell proliferation and insulin-like growth factor I-induced DNA synthesis were inhibited significantly in the presence of DECMA-1 compared with controls (P < 0.05). Control granulosa cells in culture formed large clusters with many cells packed tightly together. However, after 48 h exposure to the function-perturbing E-cadherin antibody, there was a significant decrease in the size of the granulosa cell clusters (P < 0.05) and the degree of cell-cell contact was reduced compared with control cultures. No effects on DNA synthesis, cell proliferation or cell adhesion were observed when DECMA-1 was substituted with either mouse ascites fluid or the antibody specific for the cytoplasmic domain of E-cadherin. In conclusion, these data provide evidence to support the hypothesis that E-cadherin is important for maintaining granulosa cell contact, DNA synthesis and cell proliferation in vitro. These results indicate that E-cadherin plays a fundamental role in maintaining both the structure and function of ovarian follicles.     

Flow cytometric analysis of granulosa cells from developing rat follicles.

Immature rats were treated with diethylstilboestrol (DES) or pregnant mares' serum gonadotropin (PMSG) and forward angle light-scatter (FALS) and 90 degrees light-scatter (90 degrees LS) signals were used to measure the size and the granularity (internal organization) of the granulosa cells, respectively. The results confirmed the presence of two major populations of granulosa cells in the ovaries of both groups of rats, with the same percentage of larger cells in both treatments (52.3% in DES, 49.5% in PMSG). Since DES treatment brings about granulosa cell growth while PMSG treatment causes growth and differentiation, it is evident that there is heterogeneity in granulosa cell sizes during different states of growth and differentiation. There was also heterogeneity in sizes of granulosa cells harvested from follicles of small (less than 210 microns), medium (210-420 microns) and large (greater than 420 microns) diameter. Quadrant analysis of granulosa cells in various fractions collected from Percoll gradients suggested an increase in granularity in the small and large granulosa cell populations. Cell cycle analysis of small and large granulosa cell populations collected from large follicles of rats treated with PMSG indicated that each population was distributed in G0/G1, S and G2/M phases. These results demonstrate that populations of small and large granulosa cells exist in rat ovarian follicles during various stages of growth and differentiation.     

THE RELATIONSHIP BETWEEN DIURNAL VARIATION OF THE NUMBER OF CELLS IN MITOSIS AND OF THE NUMBER OF CELLS SYNTHESIZING DNA IN THE EPITHELIUM OF THE HAMSTER CHEEK POUCH

1. The numbers of cells in mitosis and in DNA synthesis in the epithelium of the hamster cheek pouch have been studied at different times of the day and night. 2. By accumulation of mitotic cells using colcemid, both the rate of entry of cells into mitosis and the duration of mitosis have been estimated at two different times of day. 3. A diurnal variation has been demonstrated in both the mitotic index and in the tritiated thymidine labelling index. Although these variations are of different amplitude and timing, the experimental data fit closely to the hypothesis that the diurnal mitotic variation is the result of a partially synchronous population moving through the DNA synthetic period. No direct action on the mitotic process need be postulated. 4. From the results of mitotic accumulation, it is clear that the rate of entry of cells into mitosis depends on the time of day at which this is studied. There is also the possibility that the duration of mitosis is slightly longer when the mitotic index is high. 5. It is concluded that, at least in the epithelium of the hamster cheek pouch, the diurnal rhythm in the number of mitoses present is a reflection of the diurnal variation in the number of cells synthesizing DNA at some time earlier. Small fluctuations in the mitotic pattern imposed by this partially synchronous population moving from S into mitosis, could be caused by slight variations in the duration of mitosis.     

DNA SYNTHESIS AND CELL TURNOVER IN RANA PIPIENS TADPOLE LIVER DURING SPONTANEOUS METAMORPHOSIS1

The relationship of DNA synthesis and cellular turnover to biochemical differentiation during metamorphosis of R. pipiens liver was investigated. Average DNA/cell was constant at 11.6 pg/ nucleus through stage XXV; but increased during juvenile growth; during metamorphosis stages, changes in total DNA content must correspond to changes in cell number. Rates of DNA synthesis were estimated by rates of ³H-thymidine incorporated into the acid-precipitable fractions, corrected for both precursor uptake into the acid-soluble pool, and for endogenous thymine pool size. DNA content increased steadily from premetamorphosis until late prometamorphosis; at preclimax stages XVIII and XX there were two successive decreases in DNA content of approximately 30%. Fluctuations in synthesis rates preceded corresponding fluctuations in content; DNA synthesis was maximal at stages XVI and XVIII, decreased nearly ten-fold at metamorphic climax, and then gradually rose again during late climax stages. The size of the endogenous thymine pool increased transitorily during spontaneous metamorphosis corresponding to a stage of maximal DNA synthesis. These results indicate that both DNA synthesis and cellular turnover play a significant role in determining net DNA synthesis rates and content during metamorphosis. Metamorphosis of the tadpole liver appears to be associated with both proliferation and cellular death, perhaps a replacement of “larval” by “adult” cells. Metamorphosis of the liver cannot be occuring in a “fixed population of cells” as is commonly assumed. An interpretation of the population dynamics of the metamorphic liver is presented.     

Comparative studies between freshly isolated and spontaneously immortalized bovine granulosa cells: Protein secretion,steroid metabolism,and responsiveness to growth factors

A bovine granulosa cell line (BGC-1) has been obtained by spontaneous immortalization of primary cultures. BGC-1 cells have retained some characteristics of primary cultures, such as the hormonal regulation of fibronectin biosynthesis. In the present study we have compaed BGC-1 cells and primary cultures of bovine granulosa cells in terms of protein secretion, steroid metabolism, and mitogenic responses to growth factors. The pattern of protein secretion in BGC-1 cells was qualitatively similar to that of primary cultures. The main differences were a higher proportion of fibronectin and the relative amounts of several other unidentified proteins. Progesterone levels in BGC-1 cultures were undetectable. When BGC-1 cells and primary cultures were incubated with [³H]-pregnenolone, the former showed a lower conversion rate to progesterone. In contrast, the conversion rate of [³H]-progesterone to 5α-reduced metabolites was markedly increased in BGC-1 cells. We also examined the effects of epidermal growth factor (EGF), insulin like growth factor-1 (IGF-I), and transforming growth factor-b? (TGF-b?) on DNA synthesis under serum-free conditions. Both primary cultures and BGC-1 cells exhibited a stimulatory response to EGF and IGF-I on [³H]-thymidine incorporation. Neither BGC-1 cells nor primary cultures showed a significant response to TGF-b? when added alone. However, in the presence of a combination of EGF and IGF-I, TGF-b? displayed an inhibitory effect on primary cultures while it stimulated DNA synthesis in BGC-1 cells even further. The addition of conditioned medium from BGC-1 cells (BGC-1-CM) stimulated DNA synthesis on primary cultures to a greater extent than the addition of conditioned medium from primary cultures. These results suggest that BGC-1 cells may be a useful model to study the regulation of granulosa cell function during the period previous to the preovulatory stage of follicular development. The differential responses of the immortalized cells to growth regulators may offer some clues on the mechanisms that control cell proliferation in normal tissues. © 1995 Wiley-Liss, Inc.     

Biochemical analysis of meiosis in the male mouse

Spermatogenic cells of the mouse have been separated by gravity sedimentation using a modification of a previously published method. Details are given for the collection of purified samples of specific meiotic stages and for the collection of labelled cell fractions following injection of ³H-thymidine. Suppression of semi-conservative meiotic DNA synthesis, essential to the biochemical analysis of pachytene DNA metabolism, has been achieved by in vivo administration of 1 M hydroxyurea.     

Bovine thecal cells secrete factor(s) that promote granulosa cell proliferation

To determine if soluble factors (other than steroids) secreted by bovine thecal cells may be involved in local regulation of follicular development, we examined the effects of thecal cell secretory products on the growth of granulosa cells obtained from the same follicles. DNA synthesis (assessed by the incorporation of 3H-thymidine) by granulosa cells plated on coverslips and cocultured with, but not directly in contact with, thecal cells in organ culture dishes in a serum-free medium was 5-fold greater than controls. The effect of the thecal cell-secreted products on DNA synthesis by granulosa cells was significantly higher than the maximum response produced by epidermal growth factor (EGF). Thecal cell-conditioned medium stimulated 3H-thymidine incorporation into the DNA of granulosa cells and a normal rat kidney cell line in a dose-dependent manner. The increases in 3H-thymidine incorporation into granulosa cell DNA subsequently lead to an increase in cell number. Preliminary characterization studies using ultrafiltration membranes indicated that the mitogenic factor was retained in the greater than 10,000 molecular weight fraction. The activity was stable to heating at 90 degrees C for 5 min and was not extracted in ether. The thecal cell-generated growth factor may act as a paracrine regulator of granulosa cell growth, thus providing the dominant follicle with autonomy over other follicles in the cohort.     

Alterations in the cell cycle characteristics of granulosa cells during the periovulatory period: evidence of ovarian and oviductal influences

Granulosa cells at different stages of differentiation were collected from ovarian follicles and oviducts during the periovulatory period, and their nuclear DNA content was monitored by flow cytometry to establish their cell cycle characteristics (G0 + G1, S, G2 + M). The proportion of cells in the three phases of the cell cycle varied in characteristics patterns depending upon the time they were collected, before or following ovulation. Granulosa (cumulus) cells recovered from ovulated oocytes were mitotically inactive as shown by the large proportion of cells with a 2C amount of DNA and the absence of cells in S phase. The proportion of granulosa cells in G2 + M decreased when recovery from the oviducts was delayed. In contrast, granulosa (cumulus and/or mural) cells recovered from preovulatory follicles prior to luteinizing hormone (LH) exposure contained a considerable population of cells undergoing DNA synthesis, and a decreased proportion of cells with a 2C DNA content. Our findings indicate that granulosa cells undergo dynamic and characteristics changes in all cell cycle phases during the periovulatory period, within follicular and oviductal environments. Intrafollicular events appear to play a major role in controlling DNA synthesis, proliferation, and related cell cycle events in the granulosa cells. Flow cytometric techniques provide objective and detailed information on the cell cycle characteristics of granulosa cell populations at different stages of differentiation. Elucidation of the mechanisms regulating cell cycle parameters of granulosa cells and their physiological significance thus seems feasible.     

Aging of human fibroblasts is a succession of subtle changes in the cell cycle and has a final short stage with abrupt events

The proliferation of diploid human embryonic lung fibroblasts was analysed at different population doubling levels with growth curves, autoradiography after tritiated thymidine ([³H]TdR) labelling and staining of mitoses after incorporation of bromodeoxyuridine (BrdU). The parameters analysed allow for the first time the distinction between different changes in cell growth that occur during in vitro aging. The percent of slowly and rapidly dividing cells is steady during most of the lifespan; the number of cells capable of synthesizing DNA during a 24-h period is always high with a subtle decline during the second half of the lifespan up to the last 4–5 population doublings when the fall becomes pronounced. There is a constant decline of the rate of entrance into DNA synthesis and a stepwise increase in the sensitivity to cell cycle inhibition during cell crowding. Towards the middle of the lifespan, the population becomes more sensitive to low inocula which fail to accelerate the rate of entrance into the cycle. These changes lead to a final, abrupt stage of profound disorganization of proliferation taking place during the last 4–5 doublings.     

Identification of gamma-aminobutyric acid and its binding sites in the rat ovary

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 μg/mg protein) was less than that in the brain (1.2–3.4 μg/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15–21 nM), specific GABA binding sites were identified in the ovaries by specific [³H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.     

Progesterone receptor-mediated inhibition of apoptosis in granulosa cells isolated from rats treated with human chorionic gonadotropin

Almost all ovarian follicles undergo atresia during follicular development. However, the number of corpora lutea roughly equals the number of preovulatory follicles in the ovary. Because apoptosis is the cellular mechanism behind follicle and luteal cell demise, this suggests a change in apoptosis susceptibility during the periovulatory period. Sex steroids are important regulators of follicular cell survival and apoptosis. The aim of the present work was to study the role of progesterone receptor-mediated effects in the regulation of granulosa cell apoptosis. The levels of internucleosomal DNA fragmentation were evaluated in rat granulosa cells before and after induction of the nuclear progesterone receptor, using hCG treatment to eCG-primed rats to mimic the naturally occurring LH surge. Granulosa cells isolated from hCG-treated rats showed a several-fold increase in the expression of progesterone receptor mRNA and a 47% decrease (P < 0.01) in DNA fragmentation after 24 h incubation in serum-free medium compared to granulosa cells isolated from rats treated with eCG only. The effect of hCG treatment in vivo was dose-dependently reversed in vitro by addition of antiprogestins (Org 31710 or RU 486) to the culture medium, demonstrated by increased DNA fragmentation as well as increased caspase-3 activity. Addition of antiprogestins to granulosa cells isolated from immature or eCG-treated rats did not result in increased DNA fragmentation. The results suggest that progesterone receptor-mediated effects are involved in regulating the susceptibility to apoptosis in LH receptor-stimulated preovulatory rat granulosa cells.