On the role of luteinizing hormone-releasing hormone in the in vitro synthesis of bioactive human chorionic gonadotropin in human pregnancies

The dynamics of luteinizing hormone-releasing hormone (LHRH) induced human chorionic gonadotropin (hCG) production were studied in isolated placental cells from normal and anencephalic midterm and term gestations. A spontaneous release of immunoreactive hCG was first detected after 24-36 h of preparation in term control cells. The addition of LHRH at a concentration ranging from 10(-9) to 10(-6) M induced a threefold increase in this output of hCG. Placental cell responsiveness to LHRH varied according to the number of days of cell cultures, with maximal response on days 1 and 6. Placental cells from normal pregnancies incubated with 1 X 10(-6) M LHRH showed a release of both immuno- and bio-assayable hCG, which was four- to six-fold higher at midgestation than at term (p less than 0.001). In contrast, placental cells from pregnancies with anencephalic fetuses showed, at both stages of gestation, an hCG production that was comparable to that observed with normal term placental cells. We conclude that LHRH at a concentration appropriate for its placental receptor binding affinity induces a production of bioactive hCG in humans. Furthermore, our data suggest that anencephaly changes the placental response of hCG to LHRH stimulation.     

Placental lactogen as a regulator of luteal cells function during pregnancy in sheep

The luteotropic activity of ovine placental lactogen (oPL) on different days of gestation in ewes was assessed using in vitro methods. Corpora lutea (CL) harvested on Days 45, 70, 95, 120 and 135 of gestation and during parturition were enzymatically dispersed and plated on multiwell plates. After 48 h of incubation, all cultures were terminated and media were frozen for further steroid analysis. Cells were cultured in control medium, with addition of oPL alone, or in combination with PGE2 or PGF2alpha. Supplementation of culture media with oPL increased basal progesterone secretion by cells isolated on Days 45 and 70 of gestation. There was no effect on progesterone secretion by cells isolated on other days of gestation; PGE2 added to the culture media increased progesterone production only by cells isolated on Day 70 of pregnancy. Simultaneous oPL treatment with PGE2 had a statistically significant and stimulatory effect on progesterone production by luteal cells collected on Days 70 and 95 of pregnancy. In contrast, PGF2alpha alone in culture media decreased progesterone secretion by cells isolated on Days 45, 70 and 95 of gestation, while oPL plus PGF2alpha on Days 70 and 95 of gestation protected against luteolytic action of PGF2alpha. The results showed 1) a direct effect of the oPL on luteal cells isolated on Days 45 and 70 of gestation; 2) synergism between PL and PGE2 in progesterone production; by cells isolated on Day 70; 3) and a luteoprotective effect of oPL against the luteolytic action of prostaglandin F (PGF2alpha) observed on Days 70 and 95 of gestation.     

Preservation of human chorionic gonadotropin and placental lactogen secretion and normal morphology following long-term culture of normal human placental cells

In order to study the regulation of hCG and hPL secretion during gestation, a system for the preservation of the functional integrity of normal placental cells in long-term culture was established. Normal term placental cells were dispersed with 0.25% trypsin-500 units DNAse I and cultured in a monolayer in Dulbecco's modified Eagle medium with 10% fetal bovine serum. Normal cell morphology, basal hCG and hPL production and hCG responses to dibutyryl cAMP were preserved till 54 days of culture. This model may be useful for the study of long-term regulation of normal placental hCG and hPL synthesis and secretion.     

Progesterone production by monkey luteal cell subpopulations at different stages of the menstrual cycle: changes in agonist responsiveness.

Small (less than or equal to 15 microns diameter) and large (greater than 20 microns diam.) luteal cells of the rhesus monkey have been separated by flow cytometry based on light scatter properties. To determine whether the steroidogenic ability and agonist responsiveness of luteal cell subpopulations vary during the life span of the corpus luteum, small and large cells were obtained at early (Days 3-5), mid (Days 7-8), mid-late (Days 11-12), and late (Days 14-15) luteal phase of the cycle. Cells (n = 4 exp./group) were incubated in Ham's F-10 medium + 0.1% BSA for 3 h at 37 degrees C with or without hCG (100 ng/ml), prostaglandin E2 (PGE2; 14 microM), dibutyryl-cAMP (db-cAMP; 5 mM), or pregnenolone (1 microM). Basal progesterone (P) production by large cells was up to 30-fold that by small cells depending on the stage of the cycle. HCG stimulated (p less than 0.05) P secretion by both small (1.8 +/- 0.2-fold) and large (3.7 +/- 0.7-fold) cells in the early luteal phase. HCG responsiveness declined during the luteal lifespan; P production by small cells was not significantly enhanced by hCG by mid luteal phase, whereas that by large cells was stimulated 1.7 +/- 0.2-fold (p less than 0.05) even at late luteal phase. Cell responses to db-cAMP were similar to those for hCG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of maternal serum on viability and function of early human placental explants

Summary Fetal bovine serum (FBS) is frequently used to supplement chemically defined media such as Ham’s F10 when studying placental explant cultures. However in vitro production of hormones is usually declining by the 2nd or 3rd day and is short-lived (7 to 10 days). In this study we explored the use of human maternal serum (HMS) from early gestation as the medium supplement to Ham’s F10. Early placental hormone production was compared using two concentrations of FBS and HMS. On Day 3 of incubation, progesterone production in 10% HMS was 12-fold increased over that in 10% FBS, estradiol production was increased 10-fold, and βhCG production more than 3-fold. When the serum concentrations were increased to 40%, the results in all cases were similar to those at 10%. Preliminary characterization studies revealed that the stimulatory activity of HMS is heat-labile, neither extractable into organic solvent (diethyl ether) nor dialyzable, suggesting that it is protein in nature. In a long-term incubation, compared with FBS (7 days), HMS permitted survival of culture up to 30 days, judged both histologically and biochemically. We conclude that HMS provides substance(s), probably protein in nature, not present in FBS or non-pregnant human serum, which are important for human placental viability and function in vivo.     

Gonadotropin induction of a regulatory mechanism of steroidogenesis in fetal Leydig cell cultures

Studies were conducted to define further the development of the gonadotropin induced, E2 mediated steroidogenic lesion (17-alpha-hydroxylase/17,20-desmolase) in fetal Leydig cell cultures. Analysis of dispersed fetal testes purified by centrifugal elutriation demonstrated a group of cells with sedimentation velocity 12 less than to less than 16.8 mm/h.g containing a small population of adult like "transitional" Leydig cells and homogeneous "fetal" Leydig cell population collected at greater than 19.3 mm/h.g. After cells were cultured for 3 days with addition of 1 microgram oLH at 3 day intervals, the transitional cells showed testosterone accumulation comparable to the fetal cells. In contrast, transitional cells had 10-fold higher basal and hCG-stimulated aromatase activity than fetal cells, and a lack of testosterone response to acute (3 h) hCG stimulation. At day 6, transitional cells steroidogenic ability declined markedly. The fetal population maintained in culture with LH additions every 3 days, showed typical immature Leydig cell response, with enhancement of acute testosterone response to hCG at 3 day (1-fold) and at 6 day of culture (5-fold). Higher doses of LH (5 micrograms/day) or daily treatment of 1 microgram to fetal cultures, induced a lesion of 17 alpha-hydroxylase/17,20-desmolase with reduction of enzymatic activities (P less than 0.01) and impaired testosterone production (P less than 0.01) in response to acute hCG stimulation. Also aromatase was stimulated by hCG + 140% and 50% and E2 receptors were increased by 100 and 180% at 3 days and 6 days of cultures with daily or high dose LH addition, findings consistent with the observation of the E2-mediated lesion during LH action. In conclusion, the cultured fetal Leydig cell provides a useful model to elucidate molecular mechanisms involved in the development of gonadotropin-induced estradiol-mediated desensitization. Treatment of fetal Leydig cell cultures with multiple or frequent doses of LH elevate aromatase activity to necessary levels for the induction of desensitization. We have isolated small population of transitional Leydig cells with morphological characteristics of cells found in 15 day post-natal testis but functional capabilities of adult cells. We have also demonstrated the emergence of a functional adult-like population from the fetal Leydig cell.     

Gonadotropin-releasing hormone effects on placental hormones during gestation: I. Alpha-human chorionic gonadotropin, human chorionic gonadotropin and human chorionic somatomammotropin

The release of alpha-human chorionic gonadotropin (alpha hCG), gonadotropin human chorionic gonadotropin (hCG) and human chorionic somatomammotropin (hCS) in vitro from placentas of different gestational ages was studied. In addition, the effect of gonadotropin-releasing hormone (GnRH) on these hormonal releases, as related to the gestational age of the placenta cultured and the dose of GnRH, was determined. The basal release of alpha hCG and hCG was greatest at 9-13 wk of gestation (1000-1500 ng/mg and 250-350 ng/mg, respectively). Lowest release rates were at term (28 ng/mg and 20 ng/mg, respectively). Hormonal release declined with extended culture, except from the cultures of 13- and 15-wk placentas, in which the initially high release continued throughout the 8 days of culture. The initial release of hCS was low at 6 wk, increased to maximum rates by 15 wk, and was similar to the initial rate of release at term. Gonadotropin-releasing hormone stimulated the release of alpha hCG and hCG most dramatically in cultures of 16-wk and 17-wk placentas, where as much as a 400- and 250-fold increase, respectively, on Day 6 of culture was observed (p less than 0.0001). In term placenta cultures after 6 days in vitro, a 20-fold stimulation of alpha hCG and a 10-fold increase of hCG was effected by GnRH (p less than 0.001). The largest responses of alpha hCG and hCG to GnRH were observed when estrogen levels were low. Dose-related responses were observed in some placentas, yet in some instances, maximal effects were attained with all doses utilized in these studies (0.2 to 50 micrograms/ml). These data demonstrate that human placentas of different gestational ages have varying hormonogenic capabilities in vitro. The data also establish that synthetic GnRH is capable of stimulating alpha hCG and hCG production, but the degree and pattern of response to GnRH stimulation are related to the gestational age of the placental tissue and its time in culture. The most responsive period to exogenous GnRH stimulation of alpha hCG and hCG release was on Days 5 and 6 of culture, when basal estrogen release was very low. These data support the hypothesis that hCG release might be controlled by a chorionic GnRH stimulation and suggest that local steroid levels may modulate the hCG response to GnRH stimulation.     

Effects of follicle regulatory protein on thecal aromatase and 3 beta-hydroxysteroid dehydrogenase activity in medium- and large-sized pig follicles

Theca was excised from large (greater than 8 mm) and medium-sized (3-6 mm) pig follicles and prepared as monolayer cultures in serum-free media. After 24 h cells were treated with (1) M199 (control), (2) 5 i.u. hCG, (3) 100 micrograms or 100 ng FRP or (4) hCG (5 i.u.) + FRP (100 micrograms or 100 ng). At 3, 6, 12, 24 and 48 h after treatment, progesterone, oestradiol, androstenedione and testosterone were measured in media. Formation of progesterone by microsomal fractions incubated (37 degrees C) with 1 microM-pregnenolone + 5-microM-NAD+ for 1 h was used as a measure of 3 beta-HSD activity. Aromatase activity was determined by incubating cells with [3H]testosterone for 3 h (37 degrees C) and measuring 3H2O release. In theca from large follicles, hCG enhanced 3 beta-HSD activity after 48 h (P less than 0.05) and secretion of progesterone after 36 h. FRP alone inhibited 3 beta-HSD activity at 36 and 72 h, but had little effect on progesterone secretion. FRP inhibited (P less than 0.05) the hCG-induced increase in 3 beta-HSD activity at 36, 48 and 72 h. HCG enhanced aromatase activity after 48 h while FRP prevented (P less than 0.05) the hCG-induced increase in aromatase activity at 48 and 72 h. Secretion of oestradiol was enhanced (P less than 0.05) at 48 h but inhibited at 72 h by hCG. FRP alone had little effect on secretion of oestradiol but hCG + FRP was inhibitory at 72 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Co-culture of ovine eggs with oviductal cells and trophoblastic vesicles

Three experiments were conducted to determine whether coculture of early sheep eggs with oviductal cells would improve the ability of eggs to survive in culture. Eggs recovered from superovulated ewes were cultured in Ham's F-10 medium supplemented with 10% fetal calf serum (F10FCS) at 37.5 degrees C in 95% air:5% CO(2). In Experiment 1, eggs with one to eight cells were either transferred into recipient ewes immediately after collection or were cultured for 24 h in 5 ml Ham's F10 medium supplemented with 10% fetal calf serum (F10FCS), 5 ml F10FCS on a confluent monolayer of oviductal cells; in 25 ml of fresh F10FCS; or in 25 ml of F10FCS removed cultures of oviductal cells, 25 mul of fresh F10FCS or 25 mul of F10FCS removed from cultures of oviductal cells. After 24 h, the cultured eggs were transferred to recipient ewes synchronous with donors and subsequently recovered at necropsy on Day 8 post estrus. Coculture of sheep eggs with oviductal cells improved (P < 0.05) the development of transferred eggs compared to culture in F10FCS alone. In Experiment 2, eggs recovered from superovulated ewes on Days 3 to 6 after estrus had undergone 1.8 cleavages by Day 3 and 4.1 cleavages by Day 6. In Experiment 3, single-cell eggs were cultured for 3 d in 5 ml F10FCS, cocultured with ovine trophoblastic vesicles or cultured on a confluent monolayer of oviductal cells. Coculture of eggs in F10FCS on a monolayer of oviductal cells supported in vitro egg cleavage to a greater degree than did F10FCS alone or F10FCS with trophoblastic vesicles (P < 0.05). In Experiment 4, single-cell eggs were cultured for 3 d then transferred to recipients. Eggs were cultured in 5 ml F10FCS on confluent monolayers of oviductal cells from luteal or estrous ewes or on cells that had been frozen after recovery from a culture of oviductal cells. After culture, the eggs were transferred to oviducts of recipients and recovered 3 d later at necropsy. Coculture of eggs for 72 h with oviductal cell monolayers did not increase the in vitro, or subsequent in vivo, cleavage rate regardless of the type of oviductal cells.     

Effects of lymphokines and immune complexes on murine placental cell growth in vitro

Isolated murine placental cells obtained at Day 16 of allogeneic gestation (C3H x DBA/2J) were cultured for 3 days alone or in coculture with irradiated mouse splenocytes at the end of which 3H-thymidine was added for an additional 18-h culture to assess cell proliferation. Placental cell proliferation was significantly enhanced at spleen cell:placental cell ratios of 10:1 and 25:1 above that observed in the absence of added spleen cells. The stimulatory effect of irradiated allogeneic (C3H plus Balb/cJ) spleen cell cultures was significantly greater (approximately 2-fold) than that of isogeneic spleen cells (C3H alone). Conditioned medium from murine spleen cells cultured with concanavalin A (ConA) to induce lymphokine production had dose-dependent inhibitory effects on proliferation when added to placental cell cultures over a range of concentrations from 10 to 40% (vol:vol). Addition of pseudo "immune complexes" in the form of heat-aggregated human gamma globulin (AHGG) to culture medium failed to alter placental cell proliferation over a range of concentrations from 2 to 200 micrograms/ml either in the absence or presence of ConA-conditioned medium. In contrast to late-gestational stage placental cells, cell suspensions obtained from Days 8-9 murine ectoplacental cone (EPC) outgrowths, or from earlier stage placentas (Days 12-14) responded to low concentrations of conditioned medium from ConA-stimulated splenocytes with increased proliferation. The effect was less impressive on placental cells at gestational ages later than 12 days than on earlier stage preparations. On all placental cell suspensions tested, as well as EPC cells, a clear-cut inhibition of growth was observed at high doses of conditioned medium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of steroid and steroid sulfate production and aromatase activity in cultured human granulosa-luteal cells

The regulation of the production of steroids and steroid sulfates and the activity of aromatase in human luteinized granulosa cells were investigated. The cells were cultured for 48 h in the presence or absence of hCG and FSH. Basal production of pregnenolone (Pre, 0.3 +/- 0.03 ng/micrograms protein) and progesterone (P, 19.3 +/- 1.7 ng/micrograms protein) were high compared with that of other steroids beyond P in the steroidogenic pathway. The concentration of 17 alpha-hydroxyprogesterone (17-OHP) was lower 0.17 +/- 0.06 ng/micrograms and that of other steroids in the 4-ene and 5-ene pathways and steroid sulfates less than 0.05 ng/micrograms. Both hCG and FSH (100 ng/ml) stimulated the production of Pre and P 3- to 5-fold, but only minimal stimulation of other steroids and steroid sulfates was observed. Aromatase activity of granulosa-luteal cells was measured from the rate of formation of 3H2O from 1 beta-[3H]androstenedione (1 beta[3H]A) after exposing the cells to hCG, FSH or estradiol (E2) for 48 h. Basal aromatase activity was relatively low, but hCG and FSH stimulated aromatase 8- and 4-fold, respectively. The incubation of granulosa-luteal cells with E2 did not affect basal aromatase activity, but E2 augmented FSH-stimulated aromatase 1.4-fold (P less than 0.025). The results suggest that there is low 17 alpha-hydroxylase and steroid sulfokinase activity in human granulosa-luteal cells. Aromatase activity in these cells is regulated by both hCG and FSH, and intra-ovarian estrogens may regulate granulosa cell aromatase activity.     

Modulation of porcine thecal cell aromatase activity by human chorionic gonadotropin, progesterone, estradiol-17 beta, and dihydrotestosterone

Porcine thecal cells synthesize estradiol, which may function as an intraovarian regulator of follicular growth. Production of estradiol by granulosa-cell aromatase is modulated by gonadotropins and local steroidal and nonsteroidal factors. Therefore, the effect of human chorionic gonadotropin (hCG) and physiological concentrations of steroids on aromatase activity of the thecal cells was determined. Theca was excised from large porcine follicles (greater than 10 mm diameter) and plated as monolayer cultures in 1 ml of serum-free medium. Twenty-four hours after culture, cells were treated as follows: 1) control; 2) hCG (5 IU); 3) progesterone (P, 3 micrograms), estradiol-17 beta (E, 4 micrograms), or dihydrotestosterone (DHT, 1 microgram); 4) hCG + P, E, or DHT. After 27, 30, 36, 48, and 72 h of culture, media were assessed for levels of P and E. Aromatase activity was determined by a radiometric assay. Levels of P in control media increased from 27 to 72 h. hCH significantly (p less than 0.01) increased P levels from 27 to 72 h of culture. Estrogen decreased (p less than 0.05) P levels at 36, 48, and 72 h compared to controls and also prevented the hCG-induced increase in P levels at these times. DHT significantly increased (p less than 0.05) P levels at 48 and 72 h. DHT + hCG reduced the hCG-associated increase in P concentration at 36 h and 72 h, but enhanced the hCG-induced increase in P levels at 48 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Dose response of luteinized porcine granulosa cells in vitro to prolactin: dependency on pre-exposure to human chorionic gonadotrophin

This study examined the hypothesis that human chorionic gonadotrophin (hCG) increases prolactin (PRL) stimulation of the utilization of lipoprotein-borne cholesterol by pig luteinized granulosa cells in culture. These cells, which luteinize in culture, were harvested from 6-mm or greater diameter follicles and cultured in the presence of 1% fetal calf serum and 1 microgram/mL insulin for 48 h. On the third day, the media were replaced with fresh serum-free media, with the same dose of insulin, and on the following day (day 4) the media were replaced with serum- and insulin-free media. At this time (day 4) hCG was added to some cultures. On day 5, cells from the group with hCG and cells from the group without hCG were treated with graded doses of ovine PRL (0.1-3.0 micrograms/mL). To a second set of cells, likewise treated, 100 micrograms of porcine low density lipoprotein (LDL) was added. Two days later (day 7) media were sampled and replaced with media alone or media containing hormones and (or) LDL. On day 9 cultures were terminated. In the cells pre-exposed to hCG, PRL (1 microgram/mL) in the presence of LDL increased progesterone production 1.7-fold (p less than 0.01) on day 7 and 2.2-fold (p less than 0.01) on day 9. In the granulosa cells in culture pre-exposed to hCG, the effect of PRL on LDL utilization was dose dependent and saturable at 1 microgram/mL on days 7 and 9. We conclude that brief pretreatment of luteinized pig granulosa cells with hCG results in a dose-dependent PRL-induced utilization of LDL for progesterone synthesis.     

Exogenous activin increases the proportion of insulin cells in the developing chick pancreas in culture

As activin is believed to be a key signalling factor during early pancreatic development, its influence on the proliferation and/or determination of insulin cells in the developing chick dorsal pancreatic bud was investigated. Dorsal pancreatic buds of 5-day-old chick embryos were explanted on to Matrigel and cultured in serum-free medium (Ham's F12.ITS), to which 1 or 10ng/ml activin was added. After 7 days in culture, the explants were processed for immunocytochemistry and the insulin-positive cells were scored and expressed as a proportion of the sum of insulin and glucagon cells. When compared to the control cultures (Hams F12.ITS alone), activin treatment resulted in respective increases in the proportion of insulin cells of 1.6 and 1.9 fold. It is suggested that activin treatment favours differentiation of the insulin cell pathway relative to glucagon cells.     

Growth of H-35 rat hepatoma cells in unsupplemented serum-free media: Effect of transferrin,insulin and cell density

Summary Serum-free tissue culture medium consisting of a 1∶1 mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F12 medium is herein shown to support growth of Reuber H-35 cells over several days in culture. Cells were initially plated in serum containing DMEM medium for 3 h. After cell attachment, serum is removed and replaced with a serum-free 1∶1 mixture of these two commercially available tissue culture media. The doubling time of cell growth in this unsupplemented serum-free medium was 46 h in lightly plated cultures over the first 5 d. The presence of transferrin (5 μg/ml) and insulin (3.3 nM) results in a cell doubling time of 17 h, which equaled the growth rate in medium containing 10% fetal bovine serum. In the absence of transferrin, growth rates in serum-free medium were correlated with the cell density of cultures. Conditioned medium from dense, serum-free cultures has growth-stimulating activity in recipient lightly plated cultures. This simple, serum-free culture medium will facilitate studies on the growth regulation of H-35 rat hepatoma cells. This work was funded by a feasibility grant from the American Diabetes Association, as well as by the National Institutes of Health grants CA 24604-09 and CA 16463-14.     

Co-culture of rabbit one-cell embryos with rabbit oviduct epithelial cells

Summary Rabbit 1-cell embryos were co-cultured with rabbit oviduct epithelial cells (ROEC) to determine if ROEC can enhance embryo development in vitro. Primary ROEC were cultured in serum-free media at 39°C in a 5% CO₂:95% air environment. In experiment 1, 1-cell embryos were co-cultured in Ham's F10 with freshly collected or 4-d-old cultures of ROEC seeded in plastic culture wells or on collagen membranes. One-cell embryos cultured without ROEC served as controls. After 65 h in culture, embryos were stained with Hoechst 33342 to determine the number of cells per embryo. Cell numbers were higher (P<0.035) in all co-culture treatments when compared to controls. Optimal development was obtained by co-culture with 4-d-old ROEC grown on plastic (P<0.003). In experiment 2, Ham's F10, Medium 199, and CZB with glucose medium were compared for their ability to support embryo development in the presence or absence of 4-d-old ROEC growth on plastic. Cell number and the percentage of embryos becoming blastocysts were significantly (P<0.002) higher for embryos cultured in Medium 199 compared to the other media tested. In Medium 199, co-culture with ROEC resulted in only a slight, nonsignificant increase in cell number over culture in Medium 199 alone (110 vs. 96 cells). However, the percentage of embryos reaching the blastocyst stage when co-cultured in Medium 199 with ROEC (49%) was nearly twice (P=0.01) that of embryos in Medium 199 without ROEC (26%). In experiment 3, transfer of embryos cultured in Medium 199 with or without ROEC of 24 or 48 h resulted in no significant differences in posttransfer development. These data indicate a beneficial effect of ROEC on blastogenesis and a salvage effect of ROEC on cell proliferation in embryos grown in a less supportive medium such as Ham's F10. This work was supported by a Multicenter Cooperative Program on Non-HumanIn Vitro Fertilization and Preimplantation Development and was funded by the National Institute of Child Health and Human Development, NIH, Bethesda, MD, through Cooperative Agreement HD 21939.     

Isolation of human Leydig cell mesenchymal precursors from patients with the androgen insensitivity syndrome: testosterone production and response to human chorionic gonadotropin stimulation in culture.

Mature Leydig cells, the main source of testicular testosterone in mammals, arise from immature mesenchymal precursors through an LH-dependent differentiation process. In order to study the steroidogenic potential of these precursors, undifferentiated mesenchymal cells were obtained from the testicular interstitium of two patients with androgen insensitivity syndrome. After double digestion with collagenase and separation of the suspensions in a Percoll density gradient, the cells were cultured in Ham's F12 medium: Dulbecco's Modified Eagle Medium (1:1) supplemented with antibiotics, transferrin, insulin, hydrocortisone, and vitamin E with or without 1 IU of hCG/ml. At 11 days in culture, samples were removed for morphological characterization and determination of 3 beta-hydroxysteroid dehydrogenase activity (3 beta-HSD). Testosterone concentration was determined by RIA in the culture medium at different intervals. Cultured cells were mesenchymal in appearance, elongated in shape, with numerous processes running in different directions. No mature Leydig cells were present. In basal conditions, the percentages of 3 beta-HSD-positive cells at 11 days on patients 1 and 2 were 33% and 28%, respectively, and the testosterone concentrations in the culture media were 4.8 and 8.4 ng.10(6) cells.24 h, respectively. In cultures stimulated with hCG, there was an increase of histochemical reactivity (47% and 42% in patients 1 and 2, respectively) and in the amount of testosterone secreted (10.2 and 12.0 ng.10(6) cells, respectively). Electron microscopic studies of cultures grown in the absence of hCG demonstrated a homogenous population of poorly differentiated, fibroblastic-type mesenchymal cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Human granulosa cells in vitro: characteristics of growth, morphology and influence of some cytokines on steroidogenesis.

Granulosa cells (GCs) were characterised morphologically by light and electron microscopy. The steroidogenic capability of GCs in vitro was estimated by radioimmunoassay (RIA): oestradiol (E2), progesterone (P) and androstenedione (A) secreted into the culture medium were measured. The influence of several culture media and anchorage of the cell either to plastic vessels (monolayer) or to collagen fibrils (in gel) were studied. As the various culture media were assayed with regard to their suitability for IVF, it was found that Ham's F10 is quite satisfactory (in agreement with other observations on embryo cultures). A chemically defined medium BM 86 was found to be inadequate. In addition to the two cell types which are known, a third cell type which can perform efficient aromatisation (E2 production) in vitro is characterised here. The influence of cytokines/growth factors (GF) like insulin-like GF (IGF-1), epidermal GF (EGF), platelet-derived GF (PDGF) and fibroblast GF (FGF) on steroidogenesis was tested either alone or with human chorionic gonadotrophin (hCG). Except for oestradiol (E2) from early GCs, hCG generally stimulated progesterone (P) and E2 secretion. EGF by itself enhanced the secretion of P but not of E2. EGF did not affect hCG stimulation of P, but reduced that of E2. In contrast, in pre-ovulatory GCs IGF-I reduced the stimulatory effect of hCG on both E2 and P. In early GCs IGF-I potentiated hCG stimulation of P. In early GCs, neither hCG nor IGF-I nor a combination of IGF-I with hCG had any effect on E2 production.