Granulosa Cell Populations Research Articles

Ultrastructural and metabolic changes associated with reproductive tract atrophy and adiposity in diabetic female mice.

The effect of progressive, diabetes-associated adiposity on reproductive tract structure and function was examined in 4- to 16-week-old C57BL/KsJ, control (+/?) and diabetic (db/db) mice. Uterine and ovarian tissues were analyzed by transmission electron microscopy for ultrastructural changes associated with increased intracellular lipid accumulation. In addition, the same tissues were analyzed for changes in activity of tissue lipoprotein lipase, an enzyme that hydrolyzes lipoprotein-associated triacylglycerols and supports the cellular uptake and storage of free fatty acids. Between 8 and 16 weeks of age, intracellular lipid deposits increased dramatically in the ovarian granulosa, thecal and stromal cell populations, as well as in the uterine epithelium, of diabetic mice compared to controls. By 16 weeks of age, the lipid deposits essentially occupied the entire cytoplasmic area of both the ovarian and uterine cell types in diabetics. The basal lamina underlying the uterine epithelium was expanded in the diabetics relative to controls, and the hyperglycemic condition induced an observable increase in endometrial intercellular space that was occupied by a hyaline type of ground substance of unknown composition and origin. In association with these structural changes, both ovarian and uterine lipase activities were greatly increased in the db/db mice compared with controls. These data suggest that the structural adiposity and functional decline in reproductive tract condition of the db/db mutants are related to the enhanced cellular lipid deposition observed in this species. These changes in structural and metabolic parameters are related to the reproductive incompetence characteristic of this murine model.

Identification of subpopulations of rat granulosa cells: sedimentation properties and hormonal responsiveness.

Ovarian granulosa cells from small follicles have generally been considered to comprise a homogeneous cell population; however, stratified arrangements of hormone receptors have been found in antral and mural granulosa cells of Graafian follicles. Using cultured granulosa cells derived from immature, hypophysectomized, estrogen-treated rats, we have previously shown that vasoactive intestinal peptide (VIP) as well as FSH stimulates steroid production by these cells. Dose-response analysis indicated that the actions of these hormones were additive, suggesting the presence of subpopulations of granulosa cells. Using a continuous (0-30%) Metrizamide density gradient, we have identified three populations of granulosa cells with different sedimentation properties. After centrifugation for 15 min at 1500 X g, cells sedimented at Metrizamide concentrations of 13%, 18%, and 20% (peaks A, B, and C, respectively). The subpopulation with lowest density (peak A) comprised 5% of the total cells, whereas the remaining cells were distributed approximately equally in the other two peaks. The profiles of estrogen and progesterone production by these cells in response to FSH and VIP indicated that FSH preferentially stimulated steroid production in cells with the highest density (peak C), whereas VIP mainly induced steroidogenic responses in cells of intermediate density (peak B). In contrast, cells with the lowest density (peak A) were unresponsive to either hormone. Treatment with forskolin, a universal adenylate cyclase activator, induced steroid production in both subpopulations B and C. Further studies demonstrated that LH/human CG receptors were induced by FSH and forskolin in cells from peak C, whereas VIP treatment did not induce LH/human CG receptors in cells from peak B. In unfractionated cultured cells, GnRH potently antagonized FSH- but not VIP-induced steroidogenesis. Upon density-gradient fractionation, the profile of GnRH receptor content correlated well with GnRH effects since FSH-responsive cells (peak C) contained the majority of GnRH receptors. The present results demonstrate that granulosa cells from immature follicles are heterogeneous and consist of two major subpopulations of cells with differential responsiveness to FSH and VIP. These findings provide the basis for further morphological and biochemical analysis of subpopulations of granulosa cells during follicular development.

Influence of follicular atresia on LH-induced cAMP and steroid synthesis by bovine thecae interna

The aim of the present study was to examine the interrelationships between the luteinizing hormone (LH) receptor, the LH-induced changes in adenosine cyclic 3', 5'-monophosphate (cAMP) and steroid synthesis in theca interna tissue of large antral follicles (> 8 mm diameter) from oestrous cycling cows. Three distinct types of theca interna were identified (types I, II and III), all of which contained an LH receptor: type I was capable of secreting increased amounts of cAMP dehydroepiandrosterone, androstenedione and testosterone when exposed to LH; type II was capable of secreting increased amounts of cAMP and progesterone but not the androgens when exposed to LH; type III was incapable of cAMP or steroid synthesis when exposed to LH. Follicles with type I thecae contained: (a) a full complement of granulosa cells; (b) high intrafollicular concentrations of oestradiol; and (c) granulosa cells with a high capacity to metabolise testosterone to oestradiol. These follicles were considered to be non-atretic structures. Follicles with types III thecae contained: (a) fewer granulosa cells; (b) low intrafollicular concentrations of oestradiol; and (c) granulosa cells with a low capacity to metabolise testosterone to oestradiol. Moreover, follicles with type III thecae contained the highest concentrations of progesterone and the lowest concentrations of androstenedione and testosterone. These follicles were considered to be severely atretic structures. Follicles with type II thecae contained granulosa cell populations and progesterone, and androgen concentrations which were intermediate between those with thecae of types I and III. These follicles were considered to be at an intermediate stage of atresia. Collectively the data suggest that during follicular atresia the LH receptor characteristics in theca interna remain unchanged while the tissue first loses its capacity to respond to LH to synthesize androgens and then loses its ability to respond to LH to synthesize cAMP.

Characterization of porcine granulosa cells by isopycnic gradient centrifugation.

Granulosa cells in developing follicles undergo changes in morphology and steroidogenesis during luteinization. Accordingly, the following questions arise: as granulosa cells secrete increased amounts of steroids during maturation, do their densities change, and might these alterations result in a heterogenous population of granulosa cells? To answer these questions, granulosa cells were separated by isopycnic gradient centrifugation on nonlinear and continuous 30% and 50% polyvinylpyrrolidone- coated silica gradients. 125I-human follicle-stimulating hormone (hFSH) labeled porcine granulosa cells and standardized density marker beads were used to generate a nomogram for conversion of the gradient elution volumes into densities. Separate pools of antral and mural granuiosa cells from large porcine follicles were dispersed in 0.02% EDTA-O.05% trypsin, then layered onto gradients and centrifuged at 800 × g (20 min, 20°C).Gradient aliquots were then cultured for 24 h in medium 199 supplemented with 15% fetal calf serum and antibiotics. Spent media were collected for progesterone determination, and the number of cells in each fraction quantitated by an automated cell counter. Mural cells displayed a uniform density of 1.02–1.03 g/ml, whereas antral cells displayed at least two distinct populations at 1.02–1.03 and 1.05–1.08 g/ml. Antral granulosa cells with higher density produced 7 times more progesterone (3.4 pg/cell per 24 h) than both mural and antral granulosa cells with lower density (0.5 pg/cell per 24 h). When aliquots of granulosa cells from medium sized follicles (3–5 mm) were cultured in the presence of pregnant mare’s serum gonadotropin (PMSG) for 0, 24, 48, and 72 h, a gradual shift in the density distribution was observed from the higher density towards the lower density cells, accompanied by a concomitant increase in both progesterone and estradiol secretion, In conclusion, by equilibrating granulosa cells to their specific gravity using isopycnic gradient centrifugation, we have found that the density of granulosa cells is a function, at least in part, of their steroidogenic capabilities. Further, their specific gravity changed as the cells luteinized in culture, accompanied by consequent changes in steroid secretion. That the developing follicle contains a heterogenous population of granulosa cells was evidenced by the marked difference in steroid secretory capacities of both mural and antra! granuiosa cells, and by their differential elution profiles following isopycnic centrifugation.

Flow cytometric deoxyribonucleic acid analysis of granulosa cells aspirated from human ovarian follicles. A new method to distinguish healthy and atretic ovarian follicles.

Granulosa cell aspirates from human ovarian follicles were analyzed by flow cytometry to determine the fraction of cells in the DNA S-phase of the mitotic cell cycle. The aim of the study was to evaluate if the percentage of granulosa cells in S-phase (the S-fraction) could be used to indicate whether a follicle was healthy or atretic. A highly significant relationship was found between the S-fraction and the concentration of estradiol in the follicular fluid (r = 0.6, P less than 0.001). More than 85% of the follicles having an S-fraction of 16% or greater contained intrafollicular levels of estradiol equal to or greater than 200 ng/ml and had a low androstenedione:estradiol ratio. Conversely, 95% or more of the follicles that had an S-fraction of less than 16% contained low estradiol (less than 200 ng/ml) and had a high androstenedione to estradiol ratio. We conclude that flow cytometric DNA measurements on follicular aspirates provide a reliable and rapid method by which to distinguish healthy and atretic ovarian follicles. Since only a small fraction (less than 5%) of an entire granulosa cell population is required for S-phase analysis, the technique allows the majority of cells to be immediately available or other biochemical studies. Moreover, since excision of ovarian tissue is avoided, the technique may be acceptable for studies on women with normal ovarian function but who are undergoing laparotomy or laparoscopy for some reason.

Divergent effects of prolactin upon steroidogenesis by porcine granulosa cells in vitro: influence of cytodifferentiation.

Ovine PRL (20–1,000 ng/ml) produced dose-dependent inhibition of progesterone production by cultured granulosa cells isolated from small (1–2 mm) immature follicles of porcine ovaries. The inhibition was most prominent during the initial 4 days of culture, when PRL decreased progesterone accumulation by 30–67%. The suppressive effects of PRL persisted when steroid accumulation was corrected per cell [control (0.51 ± 0.03) vs. PRL-treated (0.24 ± 0.03 ng progesterone/105 cells-48 h)]. In contradistinction, with granulosa cells isolated from large (>6 mm) mature follicles, PRL (4–1,000 ng/ml) exerted an opposite effect upon steroidogenesis, stimulating progesterone secretion by 30–100%. Previous studies had suggested that the two granulosa cell populations employed differed with regard to PRL receptor content. To clarify the relationship of PRL binding to its divergent action, specific ovine [125I]PRL ([125I]oPRL) binding was compared serially in immature and mature granulosa cells before and during 6–8 day...

The microenvironment of the human antral follicle: interrelationships among the steroid levels in antral fluid, the population of granulosa cells, and the status of the oocyte in vivo and in vitro.

A number of parameters in human antral follicles were investigated. They were: the number of granulosa cells in each follicle; the concentrations of progesterone, androstenedi-one, testosterone, dihydrotestosterone, estrone, and 17β-estra-diol in antral fluid; and the status of the oocyte immediately after removal from the follicle and again after 48 h in culture. In addition, the phase of the menstrual cycle at the time of ovariectomy and the diameter of each dissected follicle were recorded. The number of granulosa cells present in each follicle and the viability of the oocyte before culture as well as its subsequent in vitro behavior were correlated with the concentration of estra-diol and⁄or the ratio of androgen to estrogen in antral fluid. It is tentatively suggested that follicles with a potential for further development are those with 1) more than 50‰ of their full complement of granulosa cells for their respective diameters, 2) high concentrations of 17β-estradiol (≥1000 ng⁄ml) in their antral fl...

A qualitative and quantitative study of rete ovarii development in the fetal rat: Correlation with the onset of meiosis and follicle cell appearance

AbstractDevelopment of the rete ovarii and its contribution to the cells of the ovary were examined in fetal rats. Histochemical and autoradiographic techniques were used for the observations between days 15 and 21 of gestation. The data presented indicate that the rete system contributes somatic cells to the ovary before birth.The basement membrane which surrounds the cuboidal epithelium of the mesonephric tubules immediately adjacent to the ovary becomes discontinuous on day 15 of gestation. The mesonephric epithelial cells in this region form a knot or clump of pleiomorphic cells, with no apparent tubular organization, and this clump later becomes surrounded by a basement membrane. On day 17 of gestation the newly established fetal rete ovarii is comprised of three regions; (1) the extraovarian mesonephric tubules (ER), (2) intraovarian cords of flattened epithelioid cells which surround the oogonia (IR), and (3) a knot or clump of cells connecting the ER and IR regions (CR). The entire rete system is enclosed by a continuous basement membrane as defined by Periodic Acid‐Schiff Reagent techniques.Autoradiographic and quantitative analyses demonstrate that the ER tubule cells proliferate and are incorporated into the other regions of the rete system. These processes begin on day 17 and continue until at least day 21 of gestation. The role these mesonephric tubule cells may play in the regulation of meiosis and their early contribution to the presumptive granulosa cell population is discussed.

Effects of Fibroblast and Epidermal Growth Factors on Ovarian Cell Proliferationin Vitro. I. Characterization of the Response of Granulosa Cells to FGF and EGF

Despite numerous studies on the effects of gonadotropins on ovarian cells in tissue culture, the factors controlling the proliferation of granulosa cells in vitro remain unknown. We have examined the effect of fibroblast growth factor (FGF) and epidermal growth factor (EGF) on granulosa cell proliferation in vitro in an attempt to clarify their possible roles in the control of ovarian development. FGF and EGF both stimulate DNA synthesis in resting populations of granulosa cells. The half-maximal response forthis effect with FGF was observed at 4 X 10(-11)M and with EGF at 1.5 X 10(-13)M. Autoradiography demonstrated that the whole cell population initiated DNA synthesis in the presence of either EGF or FGF, thus precluding an additive effect of the two mitogens. When cells were maintained at low density (100 cells/cm2) in the presence of low serum (1%) they divided with a doubling time of 48-72 h, but addition of either EGF or FGF accelerated their proliferation. The doubling time observed in the presence of FGF was 16 h versus 20 h with EGF and the final cell density reached in the presence of EGF or FGF was 20 times that of cells maintained in the presence of 1% calf serum alone. In the presence of 10% serum, granulosa cells had a doubling time of 24 h and the final density reached was similar to that observed in 1% serum with EGF and FGF. Addition of EGF or FGF to 10% serum resulted in a final density 3 to 4-fold higher than that observed with 10% serum alone. The ultrastructure of the granulosa cells grown in the presence of EGF or FGF was similar to that of cells maintained in the absence of added mitogens. The only marked difference was that cells grown in the presence of FGF or EGF had a high lipid granule content while cells grown in their absence had a low lipid granule content. The effect of various concentrations of FGF and EGF on the proliferation of granulosa cells has been analyzed. The minimal effective dose of EGF was 3 X 10(-14)M and saturation was observed at 3 X 10(-11)M, with a half-maximal response at 6 X 10(-13)M. With FGF the minimal dose stimulating proliferation was 1.5 X 10(-12)M and saturation was achieved at 1.5 X 10(-10)M, with a half-maximal response at 3 X 10(-11)M. Our results show that EGF and FGF are the most potent mitogens ever observed and are mitogenic for granulosa cells at 300 to 3000-fold lower concentrations than for other cell types which have been studied, such as fibroblasts or lens epithelial cells.

Prostaglandin F production [formula omitted] by granulosa cells from rabbit pre-ovulatory follicles

The concentration of PGF in rabbit Graafian follicles increases at ovulation but the cell type responsible for PGF secretion has not been identified. We have found that a pure population of granulosa cells isolated from pre-ovulatory follicles of estrous rabbits secrete prostaglandin F in tissue culture (total secretion, 446 ng/10 days; 0.09 pg/cell/day). LH/FSH did not influence the rate of PGF secretion, but there was a 50% inhibition after dibutyryl cAMP treatment, and complete inhibition by indomethacin. These results indicate that granulosa cells could secrete the prostaglandin which accumulates in the follicle at ovulation, and that PGF secretion may be modified by the addition of cAMP to the medium.

Analysis of the kinetics of granulosa cell populations in the mouse ovary.

ABSTRACTThe kinetics of granulosa cell populations in two types of follicles in ovaries of 28‐day‐old Bagg mice are investigated. The analysis includes estimations of mean values and standard deviations of the transit times (TG1, TS, TG2 and TC), the doubling time TD, and the proliferative fraction p.First the percentage of labelled mitosis curve (PLM‐curve) and the continuous labelling curve (CL‐curve) are estimated. Then a hypothesis concerning the cell kinetics of the granulosa cells in the two follicle types is set up. The normal distribution is chosen to simulate the probability density functions of the transit times. On the basis of the hypothesis mathematical expressions for the PLM‐ and CL‐curves are worked out. By fitting the calculated PLM‐curve to the experimental one it is possible to estimate mean values and standard deviations of TG1, TS>, and TG2. As a test of the hypothesis the CL‐curve is calculated by means of the estimated parameter values and compared to the experimental one. The calculated PLM‐ and CL‐curves are found to be in good agreement with the experimental data as far as both follicle types are concerned.It is concluded that the method is a useful procedure. The choice of a normal distribution does not imply a significant limitation of the method in these investigations. Moreover it is concluded that the hypothesis is plausible. This means, e.g., that the proliferative fraction is unity in the two follicle types and that there is no cell loss from the cell systems.

Diurnal variations in the number of granulosa cells synthesizing DNA in relation to follicle size in the mouse ovary

Autoradiographs of mouse ovaries prepared after pulse-labelling with 3H-thymidine at 4-hourly intervals over a 24-hour period show, that in the early stages of follicle development there is a diurnal variation in the number of granulosa cells synthesizing DNA. This is not found in later stages. The diurnal variations are correlated with low proliferation rates of the cell populations and with growth fractions below one. It is concluded, that diurnal variations in the number of granulosa cells synthesizing DNA are due to variations in the growth fractions of the granulosa cell populations.