Use of individual human follicles to compare oocyte in vitro fertilization to granulosa cell in vitro luteinization*†

FERTILITY AND STERILITY Copyright c 1987 The American Fertility Society

Vol. 48, No. 2, August 1987 Printed in U.S.A.

Use of individual human follicles to compare oocyte in vitro fertilization to granulosa cell in vitro luteinization*t

George A. Hill, M.D.t Carl M. Herbert, III, M.D. Anne Colston Wentz, M.D. Kevin G. Osteen, Ph.D. Center for Fertility and Reproductive Research, Department of Obstetrics and Gynecology, Vanderbilt University Medical Center, Nashville, Tennessee

Granulosa-lutein (G-L) cells from individual follicles aspirated during cycles of in vitro fertilization-embryo transfer were examined after 3 and 6 days in culture. G-L cells from follicles that contained an oocyte that fertilized in vitro were compared with G-L cells from follicles that contained an oocyte that did not fertilize in vitro. Spent culture media was assayed for progesterone at days 3 and 6 of culture and luteinizing hormone/human chorionic gonadotropin (LH/hCG) receptor content of G-L cells was determined at day 6. G-L cell cultures from follicles that contained an oocyte that fertilized in vitro produced significantly more progesterone over 3 and 6 days of culture than those obtained from follicles in which the oocyte did not fertilize. Furthermore, LH/hCG receptor content after 6 days was significantly higher in G-L cells obtained from follicles with fertilized oocytes compared with follicles with unfertilized oocytes. Increased progesterone output and LH/ hCG receptor acquisition demonstrate more maturation or "luteinization" by G-L cells aspirated from individual follicles that contain oocytes that fertilized in vitro. Fertil Steril 48:258, 1987

Ovarian granulosa cells secrete steroids that influence the microenvironment of the developing follicle and may reflect the functional maturity of both the oocyte and the granulosa cells. 1- 3 The maturity of the oocyte determines its ability to resume ~eiosis and to be subsequently fertilized in vitro. 3 The maturity of granulosa cells may also determine their ability to eventually luteinize. Higher concentrations of estrogen in follicular fluid (FF) are

Received December 1, 1986; revised and accepted April 14, 1987. * Supported in part by Biomedical Research Support grant RR-05424 to K.G.O. t Presented in part at the thirty-third annual meeting of the Society for Gynecologic Investigation, March 19 to 22, 1986, Toronto, Ontario, Canada. t Reprint requests: George A. Hill, M.D., Center for Fertility and Reproductive Research, Department of Obstetrics and Gynecology, Vanderbilt University, Nashville, Tennessee 37232. 258

Hill et al. Granulosa cell luteinization in vitro

associated with mature follicles, while atretic follicles are associated with higher levels of androgens.3·4 The level of maturity of the follicle also may be reflected in histologic grading of the granulosa cells or oocyte, granulosa cell number, or granulosa cell steroid output.3-7 Very high concentrations of progesterone (P), androstenedione (Ll4A), and 17(j-estradiol (E 2 ) are found in the FF surrounding an oocyte. 3 It is likely that these steroids are synthesized by the granulosa and/or theca cells,3·4•8 although they may also enter the FF as a transudate of blood. 2 A marked increase is found in the level of P in antral fluid during the late proliferative phase (days 10 to 14) compared with the early proliferative phase (days 4 to 9) of the menstrual cycle.2 E 2 is also elevated in the late proliferative phase and reaches its peak in FF on cycle day 12.2 E 2 and Ll4A levels in FF of ovulatory human follicles fall drastically, while the P level rises sharply, after the administration of an Fertility and Sterility

ovulatory dose of human chorionic gonadotropin (hCG) during the late follicular phase of the cycle.9 Animal data have also shown that a marked shift occurs in preovulatory follicular steroidogenesis after the luteinizing hormone (LH) surge. 1 This ability to increase P secretion after the administration of hCG may reflect the state of functional maturity of the entire follicle, which includes the granulosa cell compartment and the oocyte. In addition, LH activity appears to be important in reinitiating meiosis in the oocyte and LH/hCG appears to be the major luteotrophic factor for the human corpus luteum during both the luteal phase of the menstrual cycle and early pregnancy. 10•11 Thus, the maturational state of both the oocyte and granulosa cells at the time of exposure to an ovulatory stimulus may be linked and relate to the continued biologic functions of these cells. Once granulosa cells are exposed to the ovulatory stimulus of hCG, they become granulosa-lutein (G-L) cells. In this study, fertilization or nonfertilization of oocytes aspirated from individual preovulatory follicles was used as an indicator of their functional maturity. The G-L cells obtained from these individual follicles were cultured. P secretion and LH/ hCG receptor acquisition in culture were compared between G-L cells from follicles with oocytes that fertilized in vitro and G-L cells obtained from follicles containing an oocyte that did not fertilize in vitro. MATERIALS AND METHODS

Materials for this study were obtained from patients undergoing in vitro fertilization-embryo transfer (IVF-ET) at Vanderbilt University Medical Center in Nashville, Tennessee. Twenty-nine follicles containing an oocyte that subsequently fertilized in vitro were compared with 13 follicles from these same patients that contained an oocyte that did not fertilize in vitro. Only patients who had at least one oocyte fertilized were included in the study. Patients with male factor infertility (sperm count < 20 X 106 /ml) were excluded from the study. Patients underwent ovarian hyperstimulation by one of three protocols: human menopausal gonadotropin (hMG; Pergonal, Serono Laboratories, Inc., Randolph, MA); combination clomiphene citrate (CC; Serophene, Serono Laboratories, Inc., Randolph, MA), and hMG (CC/hMG); or pure folliclestimulating hormone (FSH; Metrodin, Serono Laboratories, Inc., Randolph, MA). The hMG and CCI Vol. 48, No.2, August 1987

hMG protocol are as previously describedP The pure FSH protocol consisted of administering 2 ampules/day pure FSH (75 miU FSH, <1 miU LH/ampule), starting on cycle day 2. hCG was administered when the serum E 2 level approximated 600 pg/ml and at least one follicle measured 15 mm in mean diameter. Laparoscopy was performed 35 hours after hCG administration. Isolation of Granulosa Cells

At the time of laparoscopy for oocyte retrieval, FF containing the oocyte and G-L cells was aspirated into a sterile polyethylene trap at 37°C and transported under sterile conditions to the embryology laboratory for oocyte identification and its subsequent IVF. Additional G-L cells were obtained from a heparinized buffer wash (Dulbecco, phosphate-buffered saline [PBS], pH 7.4, Gibco, Grand Island, NY) of the follicle to enhance recovery of oocytes. Once the oocytes were identified, they were graded by the criteria of Veeck et al. 5 Briefly, oocytes were considered mature if the cumulus mass and coronal cells were expanded and the accompanying G-L cells were in loosely aggregated clumps and had an increased cytoplasmic/nuclear ratio. Oocytes with a small compact cumulus mass surrounded only by coronal cells were considered immature. Atretic oocytes were identified by their conspicuous lack of cumulus or coronal cells and usually displayed degenerative changes in their cytoplasm. Oocytes were inseminated in vitro in Ham's F-10 media (Gibco, Grand Island, NY) containing 15% fetal cord serum. Mature oocytes were inseminated 6 hours after aspiration, while immature oocytes were allowed to mature in culture for 24 hours and then inseminated. The remaining G-L cell and FF components were processed immediately after oocyte retrieval. G-L cells were separated from FF by low-speed centrifugation (200 X g) for 5 minutes. FF was frozen at -20°C for future analysis of steroid content by radioimmunoassay (RIA). Following low-speed centrifugation, the G-L cells were transferred to a 15-ml sterile centrifugation tube and resuspended gently in 3 ml of Dulbecco's Modified Eagle's medium/Ham's F-12 (DME/F-12; 50:50; containing 12.5 mM/HEPES [pH 7.4]). The G-L cells were further separated from red blood cells by additional centrifugation (1500 X g) for 20 minutes on a 70% percoll "cushion." The G-L cell layer was removed Hill et al. Granulosa ceU luteinization in vitro

259

and washed in fresh DME/F-12. After the wash, the G-L cells were resuspended in 500 #Ll DME/F12 and an aliquot counted on a hemocytometer in 0.04% Trypan Blue (Gibco, Grand Island, NY) for determination of cell number and viability. G-L cells were adjusted to a concentration of 50,000 viable cells/150 #Ll in DME/F-12 with 10% charcoal-stripped fetal calf serum. Since at least 5 wells were cultured on each follicle, a minimum of 250,000 viable cells was required. All follicles in these patients which contained at least 250,000 viable cells were included in the study. The G-L cells were plated in 96-well tissue culture plates (Costar, Cambridge, MA) at a concentration of 50,000 viable cells/well and maintained at 37°C in 95% air:5% C02 for 3 or 6 days. Media was collected on day 3 and day 6 and stored at -20°C until assayed in triplicate by RIA. After 3 or 6 days in culture, the cells were collected and stored in liquid nitrogen for receptor assay. Steroid Assays

E 2 and ll4A were evaluated by RIA after extraction of FF with diethyl ether, and P was measured by RIA after extraction of FF with petroleum ether. Culture media was analyzed for P content by RIA without prior extraction. Antisera to E 2 (antiestradiol-1713-6-BSA) and P (antiprogesterone-11-BSA) were supplied by Dr. Gordon Niswender (Colorado State University, Fort Collins, Colorado). Antiserum for ll4A (antiandrostenedione-7-BSA) was supplied by Drs. Fortune Kohen and Hans Linder (The Weitzmann Institute of Science, Rehovot, Israel). The characteristics of these antisera have been described previously in detail. 13-15 RIA was performed as reported previously .16 The lower limits of sensitivity for the E 2, ll4A, and P assays were 10, 10, and 25 pg, respectively, with interassay coefficients of variation of 9.6, 12.4, and 9.9%, respectively. Steroid values were corrected for recovery and blanks were below the sensitivity of each RIA. Luteinizing Hormone/Human Chorionic Gonadotropin Receptor Analysis

Our preliminary observations indicate that, after stimulation of follicle development for IVF-ET, optimal levels of LH/hCG receptors in G-L cells are observed in vitro on day 6 (unpublished data). Also, since exposure of granulosa cells to LH or hCG has been shown to decrease levels of LH receptors17 the current experiments were performed 260

Hill et al. Granulosa cell luteinization in vitro

on cells grown under basal conditions. After 6 days of culture, cells were collected by gently scraping the cells in PBS buffer with 0.01% BSA. Cells were aliquoted at 50,000 cells/tube in polypropylene microcentrifuge tubes and frozen in liquid nitrogen (vapor-phase) for subsequent measurement of LH/hCG receptor content. Highly purified hCG (CR-121), obtained from Dr. Robert Canfield, was iodinated with 1 mCi 1251 (New England Nuclear, Boston, MA) according to the lactoperoxidase method. 18 The hCG was iodinated to a specific activity of 50 to 60 #LCii#Lg. Binding was measured according to the method described by Osteen et al. 19 and was modified for a smaller number of cells available from individual human follicles. Briefly, five tubes per follicle, frozen at day 6 of culture, were rapidly thawed at 37°C. Triplicate tubes for total binding contained cells and 3 to 4 ng 1251-hCG, while duplicate nonspecific binding (NSB) tubes contained additionally a 100-fold molar excess of unlabeled hCG (Ayerst Laboratories, New York, NY). Tubes were incubated for 2 hours in a shaking water bath and the binding reaction terminated by dilution with ice-cold PBS containing 0.01% BSA. Bound hormone was separated from unbound hormone by high-speed centrifugation. Specifically bound 1251-hCG was determined by subtracting the radioactivity in NSB tubes from that in the total tubes. Statistical Analysis

Statistical analysis was performed using analysis of variance and Student's t-test, where appropriate. Significance was defined as P < 0.05.

RESULTS

Twenty-nine individual follicles (18 patients) contained an oocyte that subsequently fertilized in vitro. Twenty-seven of these oocytes were graded as mature and two were immature. These were compared with 13 individual follicles (from 8 of these 18 patients) that contained oocytes that did not fertilize in vitro. Nine of these oocytes were graded as mature and four were immature. Table 1 shows the FF P, E 2, and ll4A content of follicles containing fertilized oocytes compared with follicles containing nonfertilized oocytes. Table 2 shows the FF steroid values obtained from follicles that contained a mature oocyte. Fertility and Sterility

Table 1 Characteristics of FF from Follicles with an Oocyte that Fertilized In Vitro Compared with FF from Follicles with an Oocyte that Did Not Fertilize In Vitro (i ± Standard Error of the Mean [SEM]) Fertilized P (ng/ml) E2 (ng/ml) !:. 4 A (ng/ml) P:E2 ratio E2:!:. 4 A ratio

17182 ± 2274 375 ± 30 408 ±57 51± 10 1.3 ± 0.1

N onfertilized 9660 ± 196 ± 782 ± 60 ± 0.9 ±

3131 39 411 29 0.2

~Fertilized Oocrtes

0

12000

p

NS" <0.01 NS NS NS

Unr.rtltized Oocytes

10000

8000

• NS, not significant.

8000

4000

P output of G-L cells after 3 and 6 days in culture is shown in Figure 1. At days 3 and 6, P output was significantly higher in media from G-L cells obtained from follicles with oocytes that subsequently fertilized compared with those that contained oocytes that did not fertilize. Figure 2 shows the P output in culture of G-L cells according to the maturity of the oocyte contained in the follicle. When the follicles containing a mature oocyte were compared, G-L cells from follicles in which the oocyte fertilized in vitro produced significantly more P after 3 days in culture than G-L cells from follicles that contained an oocyte that did not fertilize in vitro. While the G-L cells from follicles with a mature, fertilized oocyte continued to produce more P after 6 days in culture, the difference was not statistically significant. This pattern also was true for the G-L cells obtained from follicles containing an immature oocyte. After 3 days in culture, P output was significantly higher from G-L cells from follicles in which the immature oocyte fertilized in vitro compared with G-L cells from follicles in which the immature oocyte did not fertilize in vitro. The P output of G-L cells from follicles with a fertilized immature oocyte .was also higher after 6 days; however, this was not significant.

2000

DAY I

DAY.

Figure 1 P output of G-L cells obtained from follicles with either a fertilized or unfertilized oocyte after 3 and 6 days of culture (*P < 0.001; **P < 0.05).

The LH/hCG receptor content of G-L cells from follicles that contained an oocyte that fertilized in vitro was significantly greater (P < 0.05) than the LH/hCG receptor content of G-L cells contained in follicles that produced an oocyte that did not fertilize in vitro (Fig. 3).

12000

~Ftrfii1Zid0ocrtes

D

Nanftrt•lired Oocrt••

Table 2 Characteristics of Follicular Fluid from Follicles Containing a Mature Oocyte that Fertilized In Vitro Compared with Follicles Containing a Mature Oocyte that Did Not Fertilize In Vitro (i ±Standard Error of the Mean [SEM])

P (ng/ml) E 2 (ng/ml) !:. 4A (ng/ml) P:E2 ratio E2:!:. 4 A ratio

Fertilized

N onfertilized

p

16603 ± 2380 381 ± 31 410 ±59 51± 11 2.3 ± 1

9878 ± 3713 186 ± 47 376 ±54 83 ± 39 0.3 ± 0.1

NS" <0.01 NS NS NS

• NS, not significant. Vol. 48, No. 2, August 1987

MATURE

IMMATURE

DAY 3

MATURE

IMMATURE

DAY 6

Figure 2 Progesterone output after 3 or 6 days in culture of G-L cells obtained from follicles that contained either a mature or immature oocyte, and separated by whether the oocyte fertilized or did not fertilize in vitro (*P < 0.02, **P < 0.01).

Hill et al. Granulosa cell luteinization in vitro

261

~ Fertilized Oocrtet C.•51

CJ U..fertlllzed Oocyte• tn•5)

DAYI .

Figure 3 LH/hCG receptor content of G-L cells obtained from a follicle with an oocyte that fertilized in vitro compared with G-L cells obtained from a follicle with an oocyte that did not fertilize (**P < 0.05).

DISCUSSION

FF steroid concentrations represent a composite of granulosa cell secretion over several days' time. Because of this, the FF steroid concentration may not be entirely representative of the secretory capability of the granulosa cells at the time of aspiration of the oocyte for IVF. Several authors3.4 have reported higher levels of estrogens in the FF of follicles with mature oocytes, and higher levels of androgens in the FF of follicles with atretric oocytes. We demonstrated significantly higher E 2 levels in FF from all follicles that contained an oocyte that fertilized and also from follicles containing a mature oocyte that fertilized in vitro. P was approximately 2-fold higher and !!..4A was approximately 2-fold lower in FF from follicles that contained a fertilized oocyte; however, these did not reach statistical significance because of the marked variability in individual follicle steroid levels, even within the same patient. E 2 was also . significantly higher in the FF from follicles containing mature oocytes that fertilized compared with those containing mature oocytes that did not fertilize. P was again higher in the FF from follicles containing mature oocytes that fertilized; however, this did not reach statistical significance because of the wide variation among individual follicles. When the FF steroid content from follicles containing an immature oocyte that fertilized in vitro was com262

Hill et al. Granulosa ceU luteinization in vitro

pared with those that contained an immature oocyte that did not fertilize in vitro, there were no statistically significant differences in P, E 2 , or !!..4A (data not shown). FF steroid values did not appear to be totally predictive ofthe ability ofthe oocyte to be fertilized in vitro, therefore, other parameters which might be predictive of the ability of the oocyte to fertilize in vitro were examined. The granulosa cells and oocyte from an individual follicle should theoretically be at the same level of maturity. Therefore, the outcome of oocyte fertilization was used as the parameter to judge the G-L cell response in culture. It has been shown that follicular steroidogenesis after the LH surge mainly involves production of P. Based on this observation, we sought to determine the capability of G-L cells to secrete P in culture and also to determine the content of LH/hCG receptor on these cells. These parameters were examined to determine whether G-L cells from follicles with an oocyte that fertilized in vitro were distinguishable from G-L cells obtained from a follicle that contained an oocyte that did not fertilize in vitro. Even though three different ovarian stimulation protocols were used, it has been shown that there is no statistically significant difference in mean P production by G-L cells in vitro among these three protocols. 20 Controversy exists in the literature between the relationship of P secretion by G-L cells and the ability of the oocyte to subsequently fertilize in vitro. Numerous authors 6 •7 have examined P output by G-L cells and have tried to correlate this with oocyte maturity. Dlugi and collegues6 examined the P output of G-L cells cultured for 2 hours. They compared the P output of pooled G-L cells obtained from follicles associated with an oocyte that fertilized in vitro to the P output of pooled G-L cells associated with follicles containing an oocyte that did not fertilize in vitro. These authors found that P output was significantly less in G-L cells from follicles that contained oocytes that fertilized in vitro compared with those that did not fertilize. Furthermore, they found that the G-L cells derived from the follicles of patients who conceived produced even less P than those G-L cells associated with fertilization, but nonconception oocytes. Laufer et al. 7 found increased P output in culture when fertilized versus nonfertilized oocyte-coronacumulus complexes (OCCC) were compared. Laufer and colleagues 7 used 24-hour cultures as opposed to the 2-hour culture reported by Dlugi et al., 6 and used the OCCC rather than G-L cells. The Fertility and Sterility

results reported at 3 and 6 days of culture in the current study tend to support the conclusions of Laufer and colleagues7 in that mean P output was higher at 3 and 6 days of culture when the G-L cells from follicles with oocytes that fertilized were compared with the G-L cells from follicles with oocytes that did not fertilize. However, differences in the study exist because Laufer and colleagues 7 used the OCCC, whereas G-L cells aspirated from individual follicles were used in the present study. The differences observed among these three studies may be due to differences in culture technique (pooled G-L cells versus individual follicle G-L cells), to different responses of G-L cells in shortterm cultures compared with long-term cultures, or to differences in the response of G-L cells as compared with OCCC. The results of the present study confirm work reported by Veldhuis et al. 8 These investigators demonstrated increased P production in 4% serum on day 4 and a decline in P production at day 6. Despite the decline of P on day 6 in the current study by G-L cells associated with both fertilized and nonfertilized oocytes, the mean P output by G-L cells from follicles with fertilized oocytes was still higher than the mean P output of G-L cells from follicles that contained nonfertilized oocytes. All of these studies use G-L cells from follicles during hyperstimulated cycles in patients undergoing IVF. Whether this reflects the physiology in natural human cycles is not known at this time. The content of LH/hCG receptor on the granulosa cell may be an important parameter determining the ability of the granulosa cell to respond to the LH/hCG surge with an increase in the secretion of P. Rajaniemi et al. 21 showed that, during the menstrual cycle, LH/hCG receptor content on granulosa cells increased 1.6-fold during the follicular phase, and increased 4-fold after ovulation. The maximum receptor content was reached about cycle day 17 and remained elevated until the end of the cycle. Yamoto et al. 22 showed that specific binding of 1251-labeled hCG peaked on cycle day 21 and decreased slightly throughout the remainder of the cycle. Based on these studies, day 6 of culture should be representative of maximal LH/hCG receptor content on G-L cells. In the present study, the mean level of LH/hCG receptor on the G-L cells after 6 days in culture was significantly higher in the G-L cells from follicles that contained an oocyte that subsequently fertilized in vitro compared with the mean LH/hCG receptor content on G-L cells from follicles that contained an oocyte Vol. 48, No. 2, August 1987

that did not fertilize in vitro. The increased mean LH/hCG receptor content on G-L cells from follicles that contained oocytes that fertilized in vitro correlates well with the increased mean P secretion seen in those same cells after 3 or 6 days in culture. Ultrastructural characteristics of human granulosa cells in stimulated cycles have been examined and correlated with oocyte fertilizability. 23 Granulosa cells from follicles that contained an oocyte that fertilized contained long microvillus projections on cell surfaces that were markedly different from granulosa cells obtained from follicles that contained an oocyte that did not fertilize. The emergence of microvilli on cell surfaces of granulosa cells has been reported to coincide with LH/ hCG receptor appearance 24 and in vivo or in vitro luteinization. 25 Rotmensch et al. 23 suggested that, because these granulosa cells from follicles that contained an oocyte that subsequently fertilized exhibited numerous microvilli, they must also contain more LH/hCG receptors than granulosa cells from follicles that contained an oocyte that did not fertilize in vitro. These cells were therefore more responsive to the administration of hCG. The data reported in the current study showing increased LH/hCG receptor content on G-L cells obtained from follicles that contained an oocyte that subsequently fertilized in vitro confirms these suggestions. Rotmensch et al. 23 further concluded that decreased LH receptor content might explain a relative unresponsiveness of the G-L cells and the decreased P secretion in culture, and that this might eventually affect oocyte fertilizability. This conclusion also would be supported by the work reported in this study. In conclusion, these data suggest that FF parameters may not satisfactorily differentiate those oocytes destined to fertilize in vitro from those that do not fertilize in vitro. However, G-L cells from follicles that contain an oocyte that subsequently fertilizes in vitro are functionally different from G-L cells obtained from follicles that contain an oocyte that does not fertilize in vitro. This is reflected by the in vitro steroid output, where G-L cells from follicles that contain an oocyte that fertilizes in vitro produce more P in culture at both 3 and 6 days than G-L cells from follicles that contain oocytes that do not fertilize in vitro. Furthermore, G-L cells obtained from follicles that contain a fertilizable oocyte have higher LH/hCG receptor content after 6 days in culture compared with G-L cells obtained from follicles that contain a nonfertilizable oocyte. The G-L cell P output and LH/ Hill et al.

Granulosa cell luteinization in vitro

263

hCG receptor content in vitro seems to reflect the functional maturity of the oocyte when judged by the oocyte's ability to fertilize in vitro.

REFERENCES 1. Leung PCK, Armstrong DT: Interactions of steroids and gonadotropins in the control of steroidogenesis in the ovarian follicle. Ann Rev Physiol 42:71, 1980 2. Sanyal MK, Borger MJ, Thompson IE, Taymor ML, Horne HW Jr: Development of graafian follicles in adult human ovary. I. Correlation of estrogen and progesterone concentration in antral fluid with growth of follicles. J Clin Endocrinol Metab 38:828, 1974 3. McNatty KP, Smith DM, Makris A, Osathanondh R, Ryan KJ: 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. J Glin Endocrinol Metab 49:851, 1979 4. Bomsel-Helmreich 0, Gougeon A, Thebault A, Saltarelli D, Milgrom E, Frydman R, Papiernick E: Healthy and atretric human follicles in the preovulatory phase: differences in evolution of follicular morphology and steroid content of follicular fluid. J Clin Endocrinol Metab 48:686, 1979 5. Veeck LL, Wortham JWE Jr, Whitmyer J, Sandow BA, Acosta AA, Garcia JE, Jones GS, Jones HW Jr: Maturation and fertilization of morphologically immature human oocytes in a program of in vitro fertilization. Fertil Steril 39:594, 1983 6. Dlugi MA, Laufer N, Polen ML, DeCherney AH, Tarlatzis BC, MacLusky NJ, Behrman HR: 17i3-estradiol and progesterone production by human granulosa-luteal cells isolated from human menopausal gonadotropin-stimulated cycles for in vitro fertilization. J Clin Endocrinol Metab 59:986, 1984 7. Laufer N, Tarlatzis BC, DeCherney AH, Behrman HR: Steroid secretion by human oocyte-corona-cumulus complexes associated with conceptions following in vitro fertilization. J In Vitro Fert Embryo Transfer 2:156, 1985 8. Veldhuis JD, Klase PA, Sandow BA, Kolp LA: Progesterone secretion by highly differentiated human granulosa cells isolated from preovulatory graafian follicles induced by exogenous gonadotropins and human chorionic gonadotropin. J Clin Endocrinol Metab 57:87, 1983 9. Fowler RE, Edwards RG, Walters DE, Chan STH, Steptoe PC: Steroidogenesis in preovulatory follicles of patients given human menopausal and chorionic gonadotropins as judged by the radioimmunoassay of steroids in follicular fluid. J Endocrinol 77:161, 1978 10. Rice BF, Hammerstein J, Savard K: Steroid hormone formation in the human ovary. II. Action of gonadotropins in vitro in the corpus luteum. J Clin Endocrinol Metab 24:606, 1964 11. Hanson FW, Powel JE, Stevens VC: Effects of hCG and human pituitary LH on steroid secretion and functional life of the human corpus luteum. J Clin Endocrinol Metab 32:211, 1971

264

Hill

e~

al. Granulosa cell luteinization in vitro

12. Diamond MP, Hill GA, Webster BW, Herbert CM, Rogers BJ, Osteen KG, Maxson WS, Vaughn WK, Wentz AC: Comparison of human menopausal gonadotropin, clomiphene citrate, and combined human menopausal gonadotropin-clomiphene citrate stimulation protocols for in vitro fertilization. Fertil Steril 46:1108, 1986 13. Korenman SG, Stevens RH, Carpenter LA, Robb M, Niswender GD, Sherman BM: Estradiol radioimmunossay without chromatography: procedure, validation and normal values. J Clin Endocrinol Metab 38:718, 1974 14. Gibori B, Antczak E, Rothchild I: The role of estrogen in the regulation of luteal progesterone secretion in the rat after day 12 of pregnancy. Endocrinology 100:1483, 1977 15. Channing CP, Gagliano P, Hoover J, Tanabe K, Batta SK, Sulewaski J, Lebeck P: Relationship between human follicular fluid inhibin F activity and steroid content. J Clin Endocrinol Metab 52:1193, 1981 16. Osteen KG, Mils TM: In vivo and in vitro steroidogenic activity of postovulation ovarian follicles of the rabbit. J Reprod Fertil 70:683, 1984 17. Schwall RH, Erickson GF: Inhibition of synthesis of luteinizing hormone (LH) receptors by a down-regulating dose of LH. Endocrinology 114:1114, 1984 18. Papaonannou S, Gospodarowicz D: Comparison of the binding of human chorionic gonadotropin to isolated bovine luteal cells and bovine plasma membranes. Endocrinology 97:114, 1975 19. Osteen KG, Anderson LD, Reichert LE Jr, Channing CP: Follicular fluid modulation of functional LH receptor induction in pig granulosa cells. J Reprod Fertil 74:407, 1985 20. Osteen KG, Hill GA, Wentz AC: In vitro steroid output of granulosa-lutein cells from individual follicles among different stimulation protocols for in vitro fertilization-embryo transfer (IVF/ET) (Abstr 344). Presented at the sixtyeighth annual meeting of The Endocrine Society, Anaheim, California, June 25-27, 1986. Published by The Endocrine Society, Bethesda, Maryland, p 117 21. Rajaniemi HJ, Ronnbert L, Kauppila A, Ylostalo P, Jalkanen M, Saastamoinen J, Selander K, Pystynen P, Vihko R: Luteinizing hormone receptors in human ovarian follicles and corpora lutea during menstrual cycle and pregnancy. J Clin Endocrinol Metab 108:307, 1981 22. Yamoto M, Nalcano R, Iwasaki M, Ikoma H, Furukawa K: Luteinizing hormone receptors in human ovarian follicles and corpora lutea during the menstrual cycle. Obstet Gynecol 68:200, 1986 23. Rotmensch S, Dor J, Furman A, Rudak E, Mashiach S, Amsterdam A: Ultrastructural characterization of human granulosa cells in stimulated cycles: correlation with oocyte fertilizability. Fertil Steril 45:671, 1986 24. Amsterdam A, Naor Z, Knecht M, Dufau ML, Catt KJ: Hormone action and receptor redistribution in endocrine target cells: gonadotropins and gonadotropin releasing hormone. In Receptor-Mediated Binding and Internalization of Toxins and Hormones, Edited by JL Middlebrook, LD Cone. New York, Academic Press, 1981, p 283 25. Gulyas BJ: Fine structure ofthe luteal tissue. In Ultrastructure of Endocrine Cells and Tissues, Edited by PM Motta. Boston-The Hague, Martinus Nijhoff Publishers, 1984, p 238

Fertility and Sterility