Human ovarian granulosa cell culture: determination of blood cell contamination and evaluation of possible culture purification steps*

Transcription

1 FERTLTY AND STERLTY Copyright 1991 The Amerian Fertility Soiety Vol. 56, No. 5, November 1991 Printed on aid-free paper in U.S.A. Human ovarian granulosa ell ulture: determination of blood ell ontamination and evaluation of possible ulture purifiation steps* Matthias W. Bekmann, M.D.t:l: Denise Polaek, Ph.D. Lisa Seung, B.A.t James R. Shreiber, M.D.t The University of Chiago, Chiago, llinois Objetive: To determine the degree of blood ell ontamination in GC preparations; to assess tehniques of GC purifiation; and to determine possible effets of ontaminating ells and purifiation tehniques on ultured GC in terms of steroid hormone prodution. Design: Contamination of GC by white blood ells was assessed by Wright's stain and immunohistohemistry. Purifiation was attempted by: (1) Fioll density entrifugation (to remove polymorphonulear leukoytes [PML]); (2) inubation in tissue ulture plasti dishes (to remove adherent monoyte/marophages); and (3) inubation in the presene of high salt (to remove lymphoytes). Results: Fioll density entrifugation redued PML to 2% of total ells, inubation in plasti dishes redued monoyte/marophage ontamination from 6% to 7% down to <1 %, and high-salt inubation redued lymphoyte ontamination from 1% to 12% down to 4%. Granulosa ells plated after preparation with addition of high salt showed inreased progesterone prodution, whih ould not be entirely explained by the removal of ontaminating lymphoytes. Conlusion: These results indiate that the human GC ulture system is more omplex than is often assumed, and methods that remove a majority of these white blood ells are presented. Fertil Steril 56:881, 1991 Granulosa ells (GCs) are among the most well studied of all endorine ells, 1 and human GCs have been examined beause of their easy availability through tehniques of modern infertility treatment, suh as in vitro fertilization (VF) and gamete in- Reeived April 8, 1991; revised and aepted July 11, * Supported by grant HL 1562 from the National nstitutes of Health, Bethesda, Maryland. t Supported by a fellowship (Be 1215/1-1) from the Deutshe Forshungsgemeinshaft, Bonn, Germany. :j: Department of Obstetris and Gyneology. Present address: Universitiits-Frauenklinik, Frankfurt, Germany. Department of Pathology. 1f Reprint requests and present address: James R. Shreiber, M.D., Department of Obstetris and Gyneology, Washington University Shool of Mediine, 4911 Barnes Hospital Plaza, St. Louis, Missouri trafallopian transfer. 2 5 Different ulture tehniques for human GC have been desribed, and these tehniques vary primarily in the type and number of preparation steps before the final plating ofthe GC. Granulosa ells have been plated: (1) diretly after dilution in ulture medium3.4; (2) after repetitive washes5; or (3) after density-gradient entrifugation with Fioll. 6 7 The different preparation tehniques lead to GC ultures with various amounts of blood ell ontamination. n vitro studies of GC have foused on their role in steroidogenesis and the modulation of their responses by gonadotropins (follilestimulating hormone [FSH] and human horioni gonadotropin [hcg]), androgens, and lipoproteins Furthermore, the regulation of their growth, luteinization, metabolism, and seretory ativities of animal and human ovary ells by regulatory proteins suh as inhibin, ativin, growth Bekmann et al. Purifiation of GCs 881

2 fators inluding epidermal growth fator, insulinlike growth fator, fibroblast growth fator, and transforming growth fator f3 have been extensively studied The possible influene of blood ell ontamination on human GC in ulture has not been ritially assessed. Beause monoytes/marophages are known to produe apolipoprotein E {Apo E), espeially when the medium is enrihed with lipoproteins/ 2 we examined the blood ell ontamination of the GC ulture in our studies of Apo E prodution by ultured human GCY The presene of monoytes/marophages in our ulture ould onfound the experimental results. Monoytes/marophages, as well as lymphoytes, are pluripotent ells with many seretory produts, 14 and therefore have the potential of altering GC properties in ulture. 15 These onsiderations lead us to the aim of the present study, whih was to determine the degree of blood ell ontamination after a basi GC preparation, to assess various tehniques of GC purifiation, and to determine possible effets of blood ells and preparative tehniques on ultured GC as related to steroid hormone prodution. Materials MATERALS AND METHODS Dulbeo's modified Eagle's medium (DMEM), peniillin-streptomyin, L-glutamine, and Dulbeo's phosphate-buffered saline (PBS) were obtained from GBCO Laboratories (Grand sland, NY). Fioll type 4 (Histopaque), hcg, and Wright's stain were obtained from Sigma Chemial (St. Louis, MO). Anti-Leu-M5 ( ll) antibody was purhased from Beton Dikinson (Mountain View, CA). Radioisotope for radioimmunoassay (RA, 3 H progesterone, 48 Ci/mmol) was purhased from Amersham (Arlington Heights, L). Low-density lipoprotein (LDL, density = 1.25 to 1.45 g/ml) was freshly isolated from antioagulated blood of normal human donors by sequential ultraentrifugation flotation as desribed by Polaek et al./ 6 dialyzed against.15 M NaCl,.1% ethylenediaminetetraaeti aid filter sterilized (.22 m), and stored in the dark at +4 o for <3 weeks. Lipoproteins were analyzed for protein onentration by a modifiation of the method of Lowry et al. 17 using bovine serum albumin as standard. Human male donor serum was isolated from the blood of one normal donor, filter sterilized (.22 m), and stored at +4 o for up to 4 weeks. Lymphoytes were prepared by density gradient entrifugation as desribed by B6yum. 18 Methods Granulosa ells were aspirated from preovulatory folliles of women undergoing ovum retrieval for VF at Rush Presbyterian Medial College, Chiago or Mihael Reese Medial Center, Chiago. Women were treated with either urinary menotropins (Pergonal; Serono Laboratories, Randolph, MA) or urinary FSH (Metrodin; Serono Laboratories) at a daily M dose of 15 to 3 U starting on day 2 of their menstrual yle. Exogenous gonadotropin was ontinued until serum estradiol was >5 pg/ml and at least two folliles had a diameter of > 16 mm by abdominal or vaginal ultrasonography. Ten thousand U hcg was given M 34 hours before ovum retrieval. All folliles > 1 mm in diameter were aspirated. After removal of the ovum, ells were isolated from the ombined folliular fluid by entrifugation (25 C, 7 minutes, 5 X g). The pooled ells from a patient were resuspended in 1 ml inubation medium (DMEM ontaining 4 mm L-glutamine, 1 U /ml peniillin, and 1 g/ml streptomyin) and then divided in two 5-mL aliquots for two separate preparations: the basi granulosa ell preparation (Fig. 1, left side) and the salt modifiation (Fig. 1, right side). Basi GC Preparation For the basi GC preparation, one 5-mL aliquot was prepared aording to Go los et al. 7 n brief, ells were resuspended in up to 2 ml of medium, layered onto 15 ml Fioll (6.4% Fioll with 1% sodium diatrizoate), and entrifuged (2 minutes, 1, X g, 25 C) (Fig. 1, left side; step B). This proedure sediments red blood ells and polymorphonulear leukoytes (PML) away from an interfae layer of mononulear ells that inludes GC, lymphoytes, and monoytes/marophages. The ells of this interfae layer were olleted by aspiration, resuspended in 5 ml of inubation medium, and pelleted by entrifugation (15 minutes, 1, X g, 25 C). The ell pellet was resuspended in 1 ml of medium. The total number of ells and their viability were assessed by Trypan blue dye staining. The average viability was 89%, and the ell number varied from 8.2 to 19.5 X 1 6 ells per patient. To determine the type and perentage of blood ell ontamination of the GC, ytospin preparations were analyzed ytohemially by Wright's stain 19 and immunohemially by 882 Bekmann et al. Purifiation of GCs Fertility and Sterility

3 Follile Aspiration and Cell Colletion Figure 1 Flow-hart of different preparation tehniques. Step A was performed in an attempt to remove ontaminating lymphoytes. Step B was performed in an attempt to remove ontaminating erythroytes and PML. Step C was performed to test the loss of lymphoytes on GC steroidogenesis. Step D was performed in an attempt to remove ontaminating marophages. "BASC GRANULOSA CELL PREPARATON" "SALT MODFCATON" A) Sequential Addition of Salt B) Floll Density Gradient Centrifugation B) Floll Density Gridlent Centrifugation Wash and Resuspend Cells Cell Count and Viability (Trypan Blue) 11 Cytospln and Staining of Cells (Wright and Leu-MS) (see Fig. 2A) Wash and Resuspend Cells C) Add Lymphoytes D) Culture 6 min n Plasti Culture Dish. D) Culture 6 min n Plasti Culture Dish. D) Culture 6 min n Plasti Culture Dish. Remove Non-adherent Cells. Remove Non-adherent Cells. Remove Non-adherent Cells. l Cell Count and Cell Count and Viability Viability (Trypan Blue) (Trypan Blue) 12 Cytospln and t3 Cytospln and Staining of Cells Staining of Cells (Wright and (Wright and Leu-M5) Leu MS) (see Fig. 2B) (see Fig. 2C) Culture 72 hours Measure Progesterone Culture 72 hours Measure Progesterone Culture 72 hours Measure Progesterone staining with a monolonal mouse antibody, Anti Leu-M5 ( ll), whih is speifi for a human monoyte/marophage membrane antigen. The antibody was visualized by indiret immunoperoxidase staining2 (Fig. 1; #1 ytospin and staining). Salt Modifiation: Lymphoyte Removal by High-Salt nubation n an effort to remove ontaminating lymphoytes from the GC preparation, a modifiation of the method for monoyte isolation reported by Realde 21 was performed 22 (Fig. 1, right side; step A). This separation tehnique is based on the property of lymphoytes to beome more dense in the presene of high salt. n brief, 25 JLL of 1.54 M NaCl, ph 7.4, was added to the tube ontaining the seond original 5-mL ell suspension, inubated (37 C, 95% humidified air, 5% C 2) for 1 minutes, after whih another 5 JLL of 1.54 M NaCl, ph 7.4, was added. The 1-minute inubation was repeated, followed by a final addition of 5 JLL of 1.54 M NaCl, ph 7.4. At the end of the third 1-minute inubation period (total time at 37 C was 3 minutes), the ells were diluted by the addition of 15 ml of hypertoni PBS (1.54 M NaCl added to PBS at a ratio of 1:36 vol/ vol). Cells were mixed by gentle up and down pipeting, the suspension then layered onto 15-mL Fioll, and entrifuged as desribed previously (Fig. 1, right side; step B). An aliquot of this ell preparation was then analyzed for yield and viability by Trypan blue staining. The average viability was 84%, and the ell number varied from 7.3 X 1 6 ells to 17.5 X 1 6 ells per patient. Monoyte/Marophage Removal by 1-Hour nubation To attempt removal of ontaminating monoytes/ marophages, the 1 ml ell suspensions from both preparations were brought up to 3 ml total volume with inubation medium and plated into a 32-mm diameter plasti tissue ulture dish (Nunlon, Roskilde, Denmark) (Fig. 1; step D). After 1-hour inubation (37 C, 95% humidified air, 5% C 2), nonadherent ells were olleted and pooled by washing the dish three times with inubation medium. Contaminating monoytes/marophages adhere avidly to plasti under these onditions. 23 Trypan blue staining revealed that 75% to 93% of the ells were reovered in the nonadherent fration. The ytospins and stainings #2 and #3 (Fig. 1) were performed as previously desribed. n an additional experiment to assess the diret influene of lymphoytes on the GC ulture, a lymphoyte-depleted GC preparation from the salt modifiation proedure was divided into two aliquots (Fig. 1, right side). To one aliquot, blood lymphoytes prepared from the same patient were re-added at this time point to make up to the loss beause of the salt addition (Fig. 1, right side; step C). The number of lymphoytes added was equal to the number removed by the salt modifiation proedure. This mixture of ells was inubated for 1 hour, step D, as previously desribed. Bekmann et al. Purifiation of GCs 883

4 'il ] ' Culture of Human Ovarian GCs Granulosa ells from the three preparations were plated on 32-mm diameter plasti tissue ulture wells in 2 ml of inubation medium with 2% voljvol human male serum, at a density of.75 to 1 X 16 ells/well. Cell viability at plating was always >92%. The addition of serum was required for GC attahment to the dishes. After 24 hours in ulture, the medium was removed, and replaed with serum-free medium plus LDL added at a onentration of 1 J.lg protein/ml. At this onentration, LDL, whih delivers exogenous holesterol to the ells, provides 1-fold more holesterol than needed for maximum progesterone (P) prodution. 24 After 48 hours in ulture, the medium was hanged again and hcg (1 U /ml) was added to the indiated ultures. Human horioni gonadotropin, whih stimulates steroidogenesis via ativation of yli adenosine monophosphate-dependent A-kinase, added at this onentration and this time in ulture, has been shown to onsistently stimulate P synthesis At 4, 8, 12, and 24 hours after the seond medium hange, aliquots of medium (2 J.LL) were removed from eah well and frozen for subsequent analysis of P by RA. All experiments were performed in tripliate. The amount of P in the tissue ulture medium was determined by RA as previously desribed. 24 Statistis Data are presented as means ± SEM. Analysis of variane was used to test for signifiant differenes between the groups. A P value <.5 was onsidered a signifiant differene between the groups. RESULTS Purity of GC Preparations After Different Preparation Protools After the basi GC preparation (Fig. 1, left side), the #1 ytospin and staining with Wright and Anti Leu-M5 revealed a blood ell ontamination of 15% to 17% (Fig. 2A). The perentages of monoytes/ marophages and PML were the same using either staining method. After the 1-hour inubation in the plasti tissue ulture dish (Fig. 1; step D), the #2 ytospin and staining (Fig. 1, left side) revealed that the perentages of monoytes/marophages and PML were redued to 1.2% and 1.4%, respetively (Fig. 2B). The #1 ytospin and staining showed also that the perentage of lymphoytes was nearly 1% after the basi GC preparation (Fig. 2A). As expeted, the no. 2 ytospin and staining (Fig. 2B) 884 Bekmann et al. Purifiation of GCs ll == 6 <fl :! 6 s 4 <fl !! 8 :! 6 s <fl. 4 2 Marophages Marophages Maro phages Figure 2 Cell ounts after different preparation protools. (A) shows the #1 ell ount of Wright and Anti-Leu-M5 ( ll) stainings with the basi GC preparation after Fioll density gradient entrifugation. (B) shows the #2 ell ount after basi granulosa ell preparation plus the 1-hour inubation. (C) shows the #3 ell ount using the same staining methods after the high-salt lymphoyte separation and the 1-hour inubation. The solid blak olumns are the perentages determined by Wright's stain, and the striped olumns are the perentages determined by the Anti Leu-M5 ( ll) stain. The results represent the average of 12 separate experiments with three repliates in eah experiment (n = 12). Results are means ± SEM. indiated that the perentage of lymphoytes was not signifiantly hanged by the 1-hour inubation period (Fig. 1; step D). Beause the basi GC preparation and 1-hour inubation in plasti did not redue the perentage of the ontaminating lymphoytes, we then tested the effet of the addition of sequential hypertoni salt. After the salt modifiation step, performed in the attempt to remove ontaminating lymphoytes (Fig. 1, right side; step A), followed by preparation steps B Fertility and Sterility

5 B and D, the #3 ytospin and staining (Fig. 1, right side) showed that the perentage of lymphoytes was redued to 3.3% (Fig. 2C). The perentages of monoyte/marophages and PML were idential to those observed in the no. 2 ytospin and staining (Fig. 2B). Progesterone Prodution by Different GC Preparations To test the funtional harateristis of the GC after the different preparation protools, we ompared the basal P and the hcg-stimulated P prodution of GC plated after the basi granulosa ell preparation (Fig. 1, left side and Fig. 3A) to the amounts of P produed by GC plated after the salt modifiation (Fig. 1, right side and Fig. 3B). Amounts of P were measured at 4, 8, 12, and 24 hours after the last medium hange. At all examined time points, the basal P levels as well as the amounts of P produed after hcg stimulation were signifiantly higher from the salt-exposed GC preparations than from the GC prepared aording to the basi protool (ompare results Fig. 3A versus Fig. 3B). The differene between the basal and the hcg-stimulated P prodution of GC prepared aording to the basi protool was signifiant only at the 24-hour time point (Fig. 3A), whereas the salt-exposed GC preparation showed signifiant differenes between basal and hcg-stimulated P prodution at all examined time points (Fig. 3B). However, the ratio of basal P to hcg-stimulated P, assessed at 24 hours, from GC prepared aording to the basi GC preparation was not signifiantly different from the ratio in GC prepared aording to the salt modifiation, with ratios of 1:1.96 and 1:2.14, respetively (Fig. 3). To evaluate whether the inreased basal and hcgstimulated P prodution of salt-exposed GC was beause of the removal of lymphoytes or the addition of salt to the GC ultures, we performed an additional experiment in whih we added bak previously purified lymphoytes prepared from the same patient (Fig. 1, right side; step C). Preparation step D and the GC ulture were performed as desribed for the basi GC preparation and for the salt modifiation (Fig. 1). BasalP and hcg stimulation results from GC prepared simultaneously aording to all three preparation protools were ompared (Fig. 4). The basal P prodution values (Fig. 4A) from GC prepared aording to the salt modifiation, with and without the re-addition of lymphoytes, were not signifiantly different at any examined time point. However, the basal P values from GC prepared aording to the basi GC preparation (i.e., no salt addition step) were signifiantly lower than from ;:: ! 1 "' 1 5 ;:: o; Figure nubation Time nubation Time Basal and hcg-stimulated P prodution after basi GC preparation inluding the 1-hour inubation in plasti tissue ulture dish (Fig. 1, left side) or after lymphoyte separation by salt modifiation proedure inluding 1-hour inubation (Fig. 1, right side). (A) represents the basal P < ) and hcg-stimulated (l!!il) prodution of GC after basi GC preparation inluding the 1-hour inubation. (B) represents the basal P < ) and hcg-stimulated (l!!il) prodution of GC after lymphoyte separation by salt modifiation proedure inluding the 1-hour inubation. Progesterone represents the amount of umulative prodution from 48 to 72 hours of ulture (Control = 1). The results represent the average of five separate experiments with three repliates in eah experiment (n = 5). Results are means± SEM. *, signifies statistial signifiane (AN OVA, P <.5) between ontrol and hcgstimulated ultures. the salt-exposed ells at all examined time points (Fig. 4A). The P prodution values from GC given hcg stimulation (Fig. 4B) paralleled the differenes seen among the various preparation protools for the basal P prodution values (ompare with Fig. 4A). However, P prodution values from hcg-stimulated GC prepared aording to the salt modifiation without re-addition of lymphoytes were signifiantly higher at 12 and 24 hours ompared with P prodution values of GC prepared identially but with there-addition of lymphoytes (Fig. 4B). DSCUSSON Granulosa ell ontamination with blood ells ours during follile puntures at the time of ooyte aspiration. After performing a basi GC preparation aording to Golos et al., 7 Wright's stain revealed a * Bekmann et al. Purifiation of GCs 885

6 3 25 'E : 1 G) u; 1:11 E D. 5 3 B A * * * * nubation Time 25 * * 'E : 1 G) u; 5 1:11 D: nubation Time * * * * Figure 4 Basal and hcg-stimulated P prodution of GC after basi GC preparation and of GC after lymphoyte separation, with or without re-addition of lymphoytes. (A) represents the basal P prodution, whereas (B) represents the P prodution after hcg stimulation. The stippled olumn represents the amount of P produed by GC after basi GC preparation inluding the 1- hour inubation in plasti tissue ulture dishes (see Fig. 1, left side), the striped olumn represents the P prodution of GC after lymphoyte separation by salt modifiation without re-addition of lymphoytes (see Fig. 1, middle), and the blak olumn represents the P prodution of GC after lymphoyte separation by salt modifiation with re-addition of lymphoytes (see Fig. 1, right side). Progesterone represents the amount of umulative prodution from 48 to 72 hours of ulture (Control= 1). The results represent the average of three separate experiments with three repliates in eah experiment (n = 3). Results are means± SEM. *, signifies statistial signifiane (ANOVA, P <.5) between the basi GC preparation and the other preparations. e, signifies statistial signifiane (ANOVA, P <.5) between the lymphoyte separation without and with re-addition of lymphoytes. blood ell ontamination of 15% to 17% at the time of plating. The analyses of the different ell types showed a low ontamination with polymorphonulear ells ( <2%) but a ontamination of nearly 6% with monoytes/marophages (onfirmed by Anti Leu-M5 stain) and more than 1% with lymphoytes. This monoyte/marophage ontamination has to be onsidered in evaluating GC ulture results. These ells are pluripotent ells that serete hydrolyti enzymes, arahidoni aid oxygenation produts, lymphoyte-ativating fator, interferon, growth inhibiting fators, and many other produts.14 The influene of marophages on P prodution by GC in primary ulture has been demon- strated previously by Halme et al.15 and Kirsh et al. 25 Both demonstrated an inreased P prodution by GC in ulture after the addition of peritoneal marophages, suggesting a luteotropi effet of marophages, possibly exerted through a sereted luteotropi ybernin Therefore, the influene of ontaminating marophages on P prodution by GC and an alteration of measured GC seretory produts should be taken into aount when dealing with the human GC ulture system. We, therefore, have previously introdued a 1-hour inubation period in plasti tissue ulture dishes to the basi GC preparation13 to make use of the property that monoytes/marophages stik avidly to plasti after 3 minutes.23 This inubation dereased the amount of ontaminating monoytes/marophages to <1 %, thereby dereasing their possible influene on experimental results. The remaining lymphoytes after the basi GC preparation (inluding the 1-hour inubation) led us to test a further modifiation of the basi GC preparation protool. This modifiation is based on the property of lymphoytes to beome more dense in the presene of high salt.22 By Wright's stain analysis, the high-salt step was shown to result in the signifiant redution of ontaminating lymphoytes to nearly 3%. The testing of basal and hcgstimulated P prodution of both GC preparations revealed that the salt-exposed GC in ulture had signifiantly higher basal and hcg-stimulated P seretion, suggesting an effet of this preparation protool on GC either through the salt itself or, indiretly, through the removal of lymphoytes. To differentiate between these two possibilities, we readded purified lymphoytes to salt-modified GC ultures. There was no signifiant differene in the basal P prodution of the salt-exposed GC ulture, with or without the re-addition of the lymphoytes. However, salt-modified GC preparations exhibited signifiantly higher P prodution than did the basi GC preparation, whih did not inlude the high-salt step. A similar, salt-related inrease of P prodution was observed in the results from the hcg stimulation experiments, exept here the inrease of P prodution in the group with lymphoyte re-addition was not as high as that of the group reeiving no addition of lymphoytes. These results indiate a omplex system of interations between gonadotropins, granulosa ells, and lymphoytes. However, the data are onsistent with both salt exposure and removal of lymphoytes having stimulatory effets on ultured granulosa ell P prodution. To summarize our results, we have shown that the human GC ulture system using ells aspirated 886 Bekmann et al. Purifiation of GCs Fertility and Sterility

7 at the time of ooyte reovery is a more omplex system than is often assumed. There is ontamination of GC ultures with blood ells, whih have the potential of altering experimental results by either (1) their effet on GC produt seretion or (2) their seretion of the produt being examined. We reommend that as a preliminary step in the study of human GC in ulture, an assessment of the degree of blood ell ontamination and its possible effet on the experimental parameters being measured should be undertaken to avoid onfounding results. Aknowledgment. We thank James Vardiman, M.D., Department of Pathology, The University of Chiago, Chiago, llinois, and the personnel of the hematology /onology laboratory for their tehnial support with the ytospin preparations and the stainings. REFERENCES 1. Hsueh AJW, Adashi EY, Jones PBC, Welsh TH: Hormonal regulation of the differentiation of ultured granulosa ells. Endor Rev 5:76, Parinaud J, Perret B, Ribbes H, Chap H, Pontonnier G, Douste-Blazy L: High density lipoprotein and low density lipoprotein utilization by human granulosa ells for progesterone synthesis in serum-free ulture: respetive ontributions of free and esterified holesterol. J Clin Endorinol Metab 64:49, Hillesjo T, Sjogren A, Strander B, Nilsson L, Wikland M, Hamberger L, Roos P: Effet of gonadotropins on progesterone seretion by ultured granulosa ells obtained from human preovulatory folliles. Ata Endorinol (Copenh) 11: 41, Ben-Ze'ev A, Amsterdam A: Regulation of ytoskeletal protein organization and expression in human granulosa ells in response to gonadotropin treatment. Endorinology 124:133, Turek R, Wilburn AB, Gwynne JT, Paavola LG, Strauss JF ll: The role of lipoproteins in steroidogenesis by human luteinized granulosa ells in ulture. J Steroid Biohem 19: 133, Pertoft H, Rubin K, Kjellen L, Laurent TC, Klingeborn B: The viability of ells grown or entrifuged in a new density gradient medium, Peroll(TM). Exp Cell Res 11:449, Golos TG, Soto EA, Turek RW, Strauss JF : Human horioni gonadotropin and 8-bromo-adenosine 3',5'-monophosphate stimulate [ 125 l]low density lipoprotein uptake and metabolism by luteinized human granulosa ells in ulture. J Clin Endorinol Metab 61:633, Bernhisel MA, Holman JF, Haney AF, Shomberg DW: Estrogen and progesterone prodution by granulosa ell monolayers derived from in vitro fertilization proedures: lak of evidene for modulation by androgen. J Clin Endorinol Metab 64:1251, Hillier SG, Yong EL, llingworth P, Baird DT, Shwall RH, Mason AJ: Effet of reombinant inhibin on androgen synthesis in ultured human thea ells. Mol Cell Endorinol 75:R1, Tonetta S, DiZerega G: ntragonadal regulation of folliular maturation. Endor Rev 1:25, Hsueh AJW, Dahl KD, Vaughan J, Tuker E, Rivier J, Bardin CW, Vale W: Heterodimers and homodimers of inhibin subunits have different pararine ation in the modulation of luteinizing hormone-stimulated androgen biosynthesis. Pro Nat! Aad Si USA 84:582, Basu SK, Ho YK, Brown MS, Bilheimer DW, Anderson RGW, Goldstein JL: Biohemial and geneti studies of the apoprotein E sereted by mouse marophages and human monoytes. J Bioi Chern 257:9788, Bekmann MW, Olson LM, Shreiber JR: Apolipoprotein E (Apo E) synthesis by ultured human ovarian granulosa ells (GC): regulation by hcg and holesterol. Fertil Steril. n press 14. Davies P, Bonney RJ: Seretory produts of mononulear phagoytes: a brief review. J Retiuloendothel So 26:37, Halme J, Hammond MG, Syrop GH, Talbert LM: Peritoneal marophages modulate human granulosa-luteal ell progesterone prodution. J Clin Endorinol Metab 61:912, Polaek D, Byrne RE, Sanu AM: Modifiation of low density lipoproteins by polymorphonulear ell elastase leads to enhaned uptake by human monoyte-derived marophages via the low density lipoprotein reeptor pathway. J Lipid Res 29:797, Lowry HR, Rosebrough NJ, Farr L, Randall RJ: Protein measurement with the folin phenol reagent. J Bioi Chern 193:265, Boyum A: solation of mononulear ells and granuloytes from human blood. Sand J Clin Lab nvest 21(Suppl. 97): 77, Wright JH: Methods of staining. n Pathologial Tehnique, 4th edition, Edited by FB Mallory, JH Wright. Philadelphia, W. B. Saunders, 198, p Lanier LL, Arnaout MA, Shwarting R, Warner NL, Ross GD: Third member of the LFA-1/CR 3 polypeptide family identified by Anti-Leu-M5 monolonal antibody. Eur Jmmunol 15:713, Realde HR: A simple method of obtaining monoytes in suspension. J mmunol Methods 69:71, Fogelman AM, Elahi F, Sykes K, Van Lenten BJ, Territo MC, Berliner JA: Modifiation of the Realde method for the isolation of human monoytes. J Lipid Res 29:1243, Fisher DG, Koren HS: solation of human monoytes. n Methods for Studying Mononulear Phagoytes, vol. 5, Edited by DO Adams, PJ Edelson, HS Koren. New York, Aademi Press, 1981, p Lee H-LC, Shangold GA, Larsen AL, Shreiber JR: The role of exogenous alium for gonadotropin-stimulated progesterone prodution by human granulosa-luteal ells. Fertil Steril 52:958, Kirsh TM, Vogel RL, Flikinger GL: Marophages: a soure of luteotropi ybernins. Endorinology 113:191, 1983 Bekmann et al. Purifiation of GCs 887