Regulation of Cumulus Oophorus Expansion by Gonadotropins in vivo and in vitro

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1 BIOLOGY OF REPRODUCTION 23, (198) Regulation of Cumulus Oophorus Expansion by Gonadotropins in vivo and in vitro JOIINJ. EPPIG The Jackson Laboratory, Bar Harbor, Maine 469 ABSTRACT In previous studies it was shown that highly purified follicle stimulating hormone (FSH), but not highly purified luteinizing hormone (LII) or human chorionic gonadotropin (hcg), stimulated hyaluronic acid synthesis and cumulus expansion in vitro by cumuli oophori isolated from mice primed with pregnant mare serum gonadotropin (PMSG). In contrast, it is shown here that highly purified hcg stimulated hyaluronic acid synthesis, cumulus expansion, and ovulation when injected into PMSG-primed mice. Cumulus expansion was also stimulated by hcg in ovarian organ cultures. Therefore, the action of injected hcg was probably directly on ovarian constituents. Culture of cumuli oophori in medium containing hcg was not effective in completing the cumulus expansion-stimulating processes begun by hcg in vivo. Therefore, the cumuli do not acquire the ability to respond directly to hcg in vitro as a result of the injection of hcg in vivo. This result also suggests that cumuli oophori do not respond directly to hcg in vivo. Preparations of crude follicular fluid (CFF) from PMSG-primed mice were very active in stimulating hyaluronic acid synthesis and cumulus expansion by isolated cumuli oophori. This showed that CFF contained both FSH-like activity that stimulated hyaluronic acid synthesis and the serum-like factor(s) necessary for the retention of the hyaluronic acid within the oocyte-cumulus cell complexes. PMSG stimulated the expansion of cultured cumuli oophori isolated from PMSG-primed mice and from mice not primed with PMSG. However, the PMSG priming itself did not stimulate cumulus expansion in vivo. Therefore, ample cumulus expansion-stimulating activity was potentially available to the oocyte.cumulus cell complexes in vivo as a result of PMSG-priming and in the foil icular fluid, but the cumulus cells did not respond to this activity until after the hcg injection. Taken together, the results of these experiments suggest that hcg stimulates cumulus expansion in vivo by an indirect mechanism. It is hypothesized that some component(s) of the intact Graafian follicles prevents the response of the cumuli oophori to the FSH-like activity present in the follicle until that inhibition is terminated as a result of hcg administration or the endogenous LH surge. INTRODUCTION mus expansion by preparations of both follicle Dramatic changes in the disposition of the stimulating hormone (FSH) and luteinizing cumulus oophorus coincide with gonadotropin- hormone (LU) (Hillensjo et al., 1976). Cumulus induced oocyte meiotic maturation in vivo expansion was also stimulated when isolated rat (Dekel et al., 1979; Schuetz and Schwartz, oocyte-cumulus cell complexes were incubated 1979). The cumulus becomes expanded (muci- in medium containing 1 pg/mi biological grade fied) as a result of the deposition of a hyalur- LI-I or FSH (Dekel and Kraicer, 1978; Dekel onic acid matrix between the cumulus cells. In and Phillips, 1979). However, when oocytemany mammals the matured ovum is ovulated cumulus cell complexes isolated from mice while enclosed in this mucified mass of cumulus were incubated in medium containing highly cells (Brambell, 1956, Austin, 1961). purified gonadotropins, it was found that FSH, The hormonal regulation of cumulus expan- but not LH, stimulated cumulus expansion sion has been the subject of several studies in (Eppig, 1979a) and hyaluronic acid synthesis vitro. Treatment of isolated rat follicles with (Eppig, 1979b). Human chorionic gonadotropin biological grade gonadotropins in defined (hcg) is similar to LH in its biological activity medium resulted in the stimulation of cumu- since it competes for the same receptors as LH (Rajaniemi and Vanha-Perttula, 1972; Kammerman and Canfield, 1972). Since biological grade hcg stimulated cumulus expansion by isolated cumuli oophori, but highly purified Accepted July 9, 198. Received February 29, 198. hcg did not, it was concluded that biological 545 on 3 February 218

2 546 EPPIG grade preparations of hcg may be contaminated with chorionic FSU (Eppig, 1979a,b). Dibutyryl cyclic adenosine monophosphate (DBcAMP), phosphodiesterase inhibitors, and cholera toxin also stimulated cumulus expansion in vitro. Therefore, it has been suggested that the effects of gonadotropins on cumulus expansion are mediated by camp (l-lillensjo et al., 1977; Dekel and Kraicer, 1978; Eppig, 1 979a,b). Cumulus expansion in vitro, but not hyaluronic acid synthcsis, was dependent upon the presence of some high molecular weight (>1, daltons) component of serum (Eppig, 1979a, 198). It was concluded that the role of serum in cumulus expansion in vitro was to promote the retention of hyaluronic acid within the oocyte-cumulus cell complex (Eppig, 198). The purpose of the present study was to relate the findings of studies in vitro to the mechanism for the regulation of cumulus expansion in vivo. This report shows that although highly purified hcg did not stimulate the expansion of isolated oocyte-cumulus cell complexes, it did stimulate hyaluronic acid synthesis, cumulus expansion, and ovulation when injected into immature mice primed with pregnant mare serum gonadotropin (PMSG). Cumulus expansion was also stimulated by hcg in ovarian organ cultures, indicating that the action of injected hcg was probably directly on ovarian constituents. The evidence suggests that purified hcg may stimulate cumulus expansion in vivo and in organ culture via an indirect mechanism. The hcg may act to allow the oocyte-cumulus cell complex to respond to FSH-like activity already present in the follicle at the time of the hcg treatment. MATERIALS AND METHODS Animals and isolation of Oocyte-Cu mu/us Cell Co?nplexes (C57BL/6J X LT/Sv)F1 mice, days old, were primed with 5 IU PMSG 48 h prior to injecting hcg or isolating the oocyte-cumulus cell complexes. Oocyte-cumulus cell compleses were isolated by puncturing the large Graafian follicles with 25 gauge needles in Whitten s medium (Whitten, 1971) containing 5% (v/v) fetal bovine serum (FBS). The complexes were washed by serially transferring them with micropipets four times through 35 mm Corning tissue culture dishes containing 2 ml of medium. They were incubated at 37#{176}Cwith an atmosphere of 5% 2 :5% CO2 :9% N2. hormones The hormones used in this study were highly purified hcg (Canfield preparation CR-119), highly purified FSH (NIAMDD-rat FSH-I-3), and biological grade FSH (NIAMDD-rat FSH-B-1). PMSG was purchased from Organon Inc. For experiments on cumulus expansion in vivo, 5 lu hcg was injected i.p. into PMSG-primed mice in.1 ml saline (.9% NaCl). Controls were injected with.1 ml saline. hlyaluronic Acid Synthesis in vivo and in vitro Synthesis in vivo. Hyaluronic acid synthesis was measured over 3 h intervals from to 12 h after the hcg injection into PMSG primed mice and over a 3 h period in the control animals not receiving hcg. The mice were injected i.p. at, 3, 6, or 9 h after hcg injection with 1 pci of [3 HI -glucosamine hydrochloride, sp act 38 Ci/mmole (Amersham). After the 3 h postinjection period, the animals were killed by cervical dislocation, the ovaries were removed, and the oocyte-cumulus cell complexes were isolated as described above. The incorporation of l HI -glucosamine into cetyl pyridinium chloride (CPC) precipitable Counts was measured as described by Solursh (1976) for groups of 5 complexes. Specificity of incorporation into hyaluronic acid was determined by sensitivity of the CPC-precipitable counts to highly specific Strep tomyces hyaluronidase (Ohya and Kaneko, 197). Synthesis in vitro. Oocyte-cumulus cell complexes were isolated from PMSG-primed mice and cultured in Whitten s medium containing 5% FBS and 1,.tg/ml biological grade FSH. Control cultures contained no FS1I. Hyaluronic acid synthesis was measured over 3 h intervals from to 12 h by adding 25 pci/mi of [3 HI -glucosamine to cultures, 3, 6, or 9 h after the addition of FSH. Control cultures were incubated with isotope for 3 h without adding FSH. After the 3 h labeling periods, hyaluronic acid synthesis was measured in groups of 5 complexes by the method of Solursh (1976) as described previously (Eppig, 1979b). It was shown previously that about 9% of the CPCprecipitable counts incorporated in such cultures was incorporated into hyaluronic acid. At least three assays of 5 complexes each were made for every treatment group. Data are expressed as cpm/5 complexes ± SEM and were subjected to one-way analysis of variance followed by the Student- Newman-Kuels multiple range test. Method for Obtaining a Preparation of Crude Follicular Fluid (CFF) from Mouse Follicles A preparation of CFF was obtained from the ovaries of PMSG-primed mice exsanguinated by decapitation which results in a significant blanching of the ovaries due to blood loss. So that contamination of the CFF preparations with blood could be further minimized, the central region of the ovaries was dissected away with fine forceps so that only a cortical region of large Graafian follicles remained. The ovaries prepared in this manner were rinsed in Whitten s medium, and the Graafian follicles were gently punctured with 25 gauge needles. The follicular fluid could be seen escaping into the medium from the punctured on 3 February 218

3 REGULATION OF CUMULUS EXPANSION 547 follicles. For each experiment, the ovarian follicles of six mice were punctured in 5 ml of medium which was then centrifuged at 15 X g for 1 mm to pellet the granulosa cells. The supernatant fluid is referred to as crude follicular fluid (CFF) since it is probably contaminated with very small amounts of mouse serum and other intercellular fluids that could not be excluded from the prepatations. The concentration of CFF in the medium was determined by the method of Bradford (1976) using the Bio-Rad Protein Assay kit and bovine serum albumin as the standard. Ovarian Organ Culture Ovaries were removed from PMSG-primed mice, exsanguinated by decapitation, and were placed in modified MAB87/3 (Eppig, 1977) containing 1% FBS. Insulin was included as in the original formulation (Gorham and Waymouth, 1965). The central region of the ovaries was dissected away with fine forceps so that only the cortical region containing the large Graafian follicles remained. These preparations were cultured in 5 ml of medium in 35 ml trypsinizing flasks (Vitro) with an atmosphere of 1% CO2 in air. The flasks were agitated at 2 rpm in a gyratory water bath maintained at 37#{176}C.The culture media contained either no added hormones (control) or 5 lu/mi highly purified hcg. After 2, 4, or 6 h of organ culture, samples were washed in Whitten s medium containing 5% FBS but no added hormones. The oocyte-cumulus cell complexes were isolated and cultured overnight in Whitten s medium plus 5% FBS at 37#{176}Cwith an atmosphere of 5% 2 CO2: 9% N2 and the percentage of cumuli that expanded was determined by examination with a dissecting microscope. RESU LTS Stimulation of Cumulus Expansion in vivo When ovaries were examined 8 h after hcg injection, it was found that hcg stimulated the expansion of 43 ± 3 cumuli per animal (n = 7), whereas no expanded cumuli were found in the ovaries of the control mice (n = 7). When the animals were examined 15 h after injection, it was found that those injected with hcg ovulated 32 ± 3 (n = 1) expanded complexes per animal, whereas no ovulations were found in the saline-injected mice (n = 7). Therefore, highly purified hcg, which did not stimulate cumulus expansion in vitro (Eppig, 1979a), stimulated cumulus expansion and ovulation in vivo. Hyaluronic Acid Synthesis in vivo and in vitro The timing of hyaluronic acid synthesis after stimulation by highly purified hcg in vivo and the timing of synthesis after stimulation by FSH in vitro was determined (Fig. 1). No increase in hyaluronic acid synthesis over control levels was detected in the -3 h period in vivo after hcg injection. However, a significant (P<.1) elevation of synthesis occurred within this period in vitro. The peak of hyaluronic acid synthesis in vitro occurred during the 3-6 h period of FSH stimulation. Although there was a significant (P<.1) elevation of hyaluronic acid synthesis during the 3-6 h period in vivo, maximal synthesis did not occur until the 6-9 h period after hcg injection. The hyaluronic acid synthesis during the 9-12 h period was not significantly different from that during the 6-9 h period in vivo. About 8% of the CPC-precipitable counts incorporated after hcg-injection was sensitive to Streptomyces hyaluronidase, indicating that about 8% of the 3 H] -glucosamine incorporated in vivo was specifically incorporated into hyaluronic acid. A similar specificity was reported for incorporation in vitro (Eppig, 1979b). eq a,. a,. E U, N, b eq eq a, C a,. E I, a. C Labeling Periods During Hormone Stimulation FIG. 1. Hyaluronic acid synthesis by oocyte-cumulus cell complexes over 3 h periods after stimulation by highly purified hcg (5 IU) in vivo (top panel) and FSH (1 pg/mi) in Vitro (bottom panel). Controls (C) indicate hyaluronie acid synthesis over a 3 h period without hcg or ESH stimulation. Bars indicate the mean of at least three measurements ± SEM. on 3 February 218

4 548 EPPIG Length of Exposure to Hormone Required to Stimulate Cumulus Expansion in vivo and jn vitro Oocyte-cumulus cell complexes were isolated at various times after the animals were injected with 5 IU highly purified hcg. The complexes were then incubated overnight in Whitten s medium containing 5% FBS to allow those complexes that were sufficiently stimulated in vivo to expand. The results are illustrated in Fig. 2. When the complexes were isolated 1, 1.5, or 2 h after hcg injection, very few of the complexes subsequently expanded. When the complexes were isolated 2.5 h after hcg injection about half of them expanded in culture, whereas 92% expanded in culture when removed 3 h after hcg injection. In contrast, 18% of the cumuli oophori incubated in 1 pg/mi FSII in vitro for 1.5 h expanded during subsequent culture in medium without FSI-I, and 2 h exposure to FSII in vitro was sufficient to stimulate the subsequent expansion of 86% of the cumuli. Very few expanded after a 1 h exposure to FSII in vitro. V C. w C (171) 4 3( 2 (113) (157) (177) Effect of hcg Injection in vivo on the Response of Isolated Oocyte-Cumulus Cell Complexes to hcg in vitro The purpose of this experiment was to determine whether cumulus cells might become responsive to hcg in vitro as a result of hcg treatment in vivo. Oocyte-cumulus cell complexes were isolated 2 or 2.5 h after the injection of 5 lu highly purified hcg. These times were near the end of the hcg-stimulatory period required in vivo (Fig. 2), but there was no evidence that the cumulus had begun expanding at this time. The oocyte-cumulus cell complexes were then cultured in medium containing highly purified hcg (5 lu/mi), highly purified FSH (125 pg/mi), or no added hormones (control). As illustrated in Fig. 3, culture of the cumuli oophori from hcginjected mice in medium containing hcg did not stimulate any more cumuli to expand than did culture of cumuli in control medium. In contrast, culture in medium containing FSH stimulated the expansion of more than 8% of the cumuli. Therefore, treatment of cumuli oophori with hcg in vitro did not complete the stimulatory process initiated by hcg in vivo, and cumulus cells do not become responsive to hcg in vitro after hcg administration in vivo. I r (87) (146) I I Hrs Hormone Treatment FIG. 2. Time of exposure to hormone in vivo and in vitro required to stimulate cumulus expansion. Solid bars indicate the percent of cumuli oophori that expand in vitro as a result of the injection of 5 lu highly purified hcg in vivo. Empty bars indicate the percent of cumuli oophori that expand in Vitro as a result of exposure to 1 pg/mi FSH in vitro. The numbers on top of bars indicate the number of cumuli oophori tested. Stimulation of Cumulus Expansion in Ovarian Organ Culture The purpose of this experiment was to determine whether the cumulus expansion stimulated by injected hcg was the direct result of the action of hcg on the ovary. When ovaries were cultured in 5 lu/mi highly purified hcg for 4 or 6 h, 65%-7% of the oocytecumulus cell complexes expanded when subsequently isolated and cultured overnight in medium without added hormones (Fig. 4). on 3 February 218

5 REGULATION OF CUMULUS EXPANSION a C a 4 3C V V C. * Lii 4- C 4).. 2 I (ic 5 114) (:34) (:43) I URS RGJ CJLTURE FIG. 4. Stimulation of cumulus expansion in organ culture. Bars indicate the percent of cumuli that expand in culture after isolation from ovaries cultured in medium containing 5 lu/mi highly purified hcg (striped bars) or no added hormones (solid bars) for 2, 4, and 6 h. Numbers on top of bars indicate the number of cumuli oophori tested. C HCG FSH C HCG FSH 2Hrs After HCG 25 Hrs After HCG FIG. 3. Effect of incubating cumuli oophori in medium containing 5 lu/mi highly purified hcg (striped bars), 125 ng/ml highly purified FSH (stippled bars), or no added hormones (solid bars) after isolating them from mice injected with 5 lu hcg 2 or 2.5 h prior to isolation. Numbers on top of bars indicate the number of cumuli oophori tested. Culture of isolated oocyte-cumulus cell complexes for 6 h in the same culture medium as used for organ culture, containing 5 lu/mi hcg, did not stimulate subsequent expansion (2%; n = 16). About 1% or less of the oocytecumulus cell complexes expanded after isolation from ovaries cultured in control medium for 2, 4, or 6 h. This experiment showed that highly purified hcg stimulated cumulus expansion in ovarian organ cultures, although hcg did not stimulate cumulus expansion by isolated oocyte-cumulus cell complexes. Therefore, the ovaries, but probably not the oocyte-cumulus cell complexes, were the targets for the cumulus expansion stimulating action of hcg injected in vivo. Effect of CFF on Hyaluronic Acid Synthesis and Cumulus Expansion The purpose of these experiments was to determine whether FSH-like, cumulus expansion-stimulating activity was present in the follicular fluid of PMSG-primed mice and, therefore, potentially available to the oocytecumulus cell complexes prior to hcg treatments. In initial experiments, isolated oocytecumulus cell complexes were incubated in Whitten s medium containing CFF but no FBS or added hormones. The concentration of CFF protein was 56 pg/mi. In these experiments, 99% (n 164) of the isolated cumuli expanded. Therefore, the CFF contained both FSH-like cumulus expansion-stimulating activity and the serum-like factor(s) necessary to retain hyaluronic acid within the complexes. In subsequent experiments, the CFF was serially diluted with Whitten s medium containing 5% FBS to obtain a rough estimate of the FSH-like activity on 3 February 218

6 55 EPPIG contained in the CFF preparation without introducing the additional variable of diluting the serum-like factors necessary for hyaluronic acid retention. As illustrated in Fig. 5, a concentration of - 16 pg/mi of CFF protein was sufficient to stimulate the expansion of 93% of the cumuli oophori. A concentration of about 159 pg/mi of CFF protein was required for maximal stimulation of hyaluronic acid synthesis (Fig. 6). Effect of PMSG on Cumulus Expansion in vitro When oocyte-cumulus cell complexes, isolated from PMSG-primed mice, were incubated in medium containing.1 lu/mi (n = 151) or.5 lu/mi (n = 341) of PMSG, about 87% of the cumuli oophori expanded in both concentrations. Fifteen percent (n = 151) expanded in.1 lu/mi and 3% (n = 115) expanded in.1 lu/mi PMSG. PMSG and FSH also stimulated the expansion of oocyte-cumulus cell complexes isolated from mice not primed with PMSG. The oocytecumulus cell complexes obtained from 25-dayold mice that were not primed with PMSG were very delicate in that the cumulus cells easily fell off the oocytes. Nevertheless, 6% (n = 52) expanded in medium containing.1 lu/mi I, CO jig CFF Protein /ml FIG. 6. Effect of various concentrations of CFF on hyaluronic acid synthesis by isolated oocyte-cumulus cell complexes. Each point is the mean of at least three measurements ± SEM. PMSG and 78% (n = 41) expanded in medium I OC 9 (:5:) ( 35> (1341 containing 1 pg/mi biological grade FSH, while only 2.5% (n = 4) expanded in medium containing no added hormones (control). Since the oocyte-cumulus cell complexes of mice not primed with PMSG expanded when cultured in 7 medium containing PMSG or FSH, there is 6 probably an inhibition of this response of the cumuli when the priming dose of PMSG is a,z 5 injected into the mice. 4C DISCUSSION a- 3C 2 I Ii) ( 34> BD too ug CFF Pcote,n /ml FIG. 5. Effect of various concentrations of CFF on the expansion of isolated oocyte-cumulus cell complexes. The numbers on top of bars indicate the number of cumuli oophori tested. Highly purified LH or hcg did not stimulate hyaluronic acid synthesis or cumulus expansion by oocyte-cumulus cell complexes isolated from the Graafian follicles of PMSG-primed mice, but highly purified FSH did (Eppig, 1979a,b). Paradoxically, it was shown here that highly purified hcg injected into PMSG-primed mice did stimulate hyaluronic acid synthesis, cumulus expansion, and ovulation. It seemed possible that the cumuli oophori might acquire the ability to respond directly to on 3 February 218

7 REGULATION OF CUMULUS EXPANSION 551 hcg in vitro as a result of processes occurring after the injection of hcg in vivo. To test this idea, the oocyte-cumulus cell complexes were isolated from ovaries 2 or 2.5 h after the injection of hcg; these times were late in the hcg stimulatory period as shown in Fig. 2. It was found that incubation of these complexes in medium containing hcg was not effective in completing the stimulatory process that was begun in vivo. These results indicate that cumuli oophori probably do not respond directly to hcg in vivo. It is likely that hcg acts directly on some components of the ovary, other than the cumulus cells, to stimulate cumulus expansion since organ culture of ovaries in medium containing hcg stimulated cumulus expansion. This result suggested that hcg stimulates cumulus expansion in vivo either by activating a factor(s) contained in other follicular components that stimulates cumulus expansion and/or by terminating a cumulus expansioninhibiting function of the intact follicle thus allowing the cumuli to respond to the FSH-like activity already present in the follicle. To help resolve this question, isolated oocyte-cumulus cell complexes were incubated in crude preparations of follicular fluid from PMSG-primed mice. It was found that the CFF contained high concentrations of FSH-like activity. Assuming that the protein concentration of follicular fluid in situ is similar to that of serum (reviewed by McNatty, 1978), it was calculated that follicular fluid contained more than 35 times the concentration of FSH-like activity necessary to stimulate hyaluronic acid synthesis maximally. McNatty (1978) has reported that the follicular fluid of nonatretic human follicles contains substantial quantities of FSH, up to 6% of plasma levels. Since FSH activity is important for granulosa cell proliferation, it is obvious that FSH must be present in the growing follicles. PMSG priming stimulated follicular development but not cumulus expansion in vivo. However, PMSG and FSH stimulated the expansion of cumulus oophori isolated from mice not primed with PMSG, thus indicating that these cumuli were capable of responding to PMSG or FSH even though they did not expand in response to the PMSG priming in vivo. In sum, there must be ample FSH-like cumulus expansion-stimulating activity potentially available to the cumuli oophori in situ. Why, then, does cumulus expansion not occur in the follicle until the administration of hcg? One explanation is that some component(s) of the intact Graafian follicles prevents the response of the cumuli to the available FSH-like activity until that inhibition is terminated as a result of hcg administration or the endogenous LH surge. This hypothesis is supported generally by evidence that low molecular weight fractions of porcine follicular fluid that inhibit oocyte meiotic maturation also inhibit progesterone secretion by cumulus cells and cumulus cell outgrowth (Hillensjo et al., 198). It was suggested that these inhibitory substances may act in a generalized way to maintain the oocytecumulus cell complex in an immature state. In addition, a low molecular weight inhibitor of FSH binding has been found in the follicular fluid of bovine Graafian follicles (Darga and Reichert, 1978). In the experiments described here, mouse follicular fluid stimulated cumulus expansion. If inhibitors of FSH binding are also present in mouse follicular fluid, they may be required at higher concentrations than were present in the CFF preparations to be effective. It is generally thought that LU is the sole gonadotropin required to stimulate the follicular events associated with ovulation (Schwartz, 1974; Sheela Rani, 1977). Yet the findings presented here and elsewhere (Eppig, 1979a,b) indicate that LH cannot directly stimulate hyaluronic acid synthesis or cumulus expansion by isolated cumuli oophori. Rather, LH seems to stimulate cumulus expansion via an indirect mechanism that allows the cumulus cells to respond to FSH-like activity already present in the follicle. Therefore, the hormonal regulation of follicular events associated with ovulation should be considered as a complex regulating mechanism triggered by LH but requiring the interplay of both FSH- and LU-stimulated processes in those mammals where cumulus expansion is a part of the normal sequence of ovulation-associated events. ACKNOWLEDGMENTS This research was supported by a grant from NSF (PCM ). I thank Cindy Grindle and Avis Silva for their excellent assistance in this research. I am grateful to Dr. A. F. Parlow and the Rat Pituitary Distribution Program of the NIAMDD and R. E. Canfleld and the Center for Population Research of the NICHHD for generously providing the gonadotropins. The Jackson Laboratory is fully accredited by the American Association for Accreditation of Laboratory Animal Care. on 3 February 218

8 552 EPPIG REFERENCES Austin. C. R. (1961). The Mammalian Egg. Charles C. Thomas, Springfield. Bradford, M. M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye l)inding. Anal. Biochem. 72, Brambeli, F.W.R. (1956). Ovarian changes. In: Marshall s Physiology of Reproduction. Vol. 1, Part 1. (A. S. Parkes, ed). Longmans, Green and Co., London. pp Darga, M. and Reichert, L. E. (1978). Some properties of the interaction of follicle stimulating hormone with bovine granulosa cells and its inhibition by foilicular fluid. Biol. Reprod. 19, Dekel, N. and Kraicer, P. F. (1978). Induction in vitro of mucification of rat cumulus oophorus by gonadotropins and adenosine 3,5 -m#{244}nophosphate. Endocrinology 12, Dekel, N., I-lillensjo, T. and Kraicer, P. F. (1979). Maturational effects of gonadotropins on the cuniulus-oocyte complex of the rat. Biol. Reprod. 2, l Dekel, N. and Phillips, D. M. (1979). Maturation of the rat cumulus oophorus: A scanning electron microscopic study. Biol. Reprod. 21, Eppig, J. J. (1977). Mouse oocyte development in vitro with various culture systems. Develop. Biol. 6, Eppig, J. J. (1979a). Gonadotropin stimulation of the expansion of cumulus oophori isolated from mice: General conditions for expansion in vitro. J. Exp. Zool. 28, Eppig, J. J. (1979b). FSU stimulates hyaluronic acid synthesis by oocyte-cumulus cell complexes from mouse preovulatory follicles. Nature 281, Eppig, J. J. (198). The role of serum in FSH-stimulated cumulus expansion by mouse oocytecumulus cell complexes in vitro. Biol. Reprod. 22, Gorham, L. W. and Waymouth, C. (1965). DifferentiatiOn in vitro of embryonic cartilage and bone in a chemically defined medium. Proc. Soc. Exp. Biol. Med. 119, Hiiiensjo, T. (1977). Dissociation of preovulatory maturational events in rat oocytes and cumuli in the presence of dibutyryl cyclic AMP. Acta Physioi. Scand. 1, Hiilensjo, T., Dekel, N. and Ahren, K. (1976). Effects of gonadotrophins on the cumulus oophorus of isolated rat Graafian follicles. Acts Physiol. Scand. 96, I-Iiliensjo, T., Pomerantz, S. II., Schwartz-Kripner, A., Anderson, L. D. and Channing, C. P. (198). Inhibition of cumulus cell progesterone secretion by low molecular weight fractions of porcine follicular fluid which also inhibit oocyte maturation. Endocrinology 16, Kammernian, S. and Canfield, R. E. (1972). The inhibition of binding of iodinated human chorionic gonadotropin to mouse ovary in vivo. Endocrinology 9, McNatty, K. P. (1978). Follicular fluid. In: The Vertebrate Ovary: Comparative Biology and Evolution. (Ft. E. Jones, ed.). Plenum Press, New York and London. pp Ohya, T. and Kaneko, V. (197). Novel hyaluronidase from Streptomyces. Biochim. Biophys. Acts 198, Rajaniemi, II. and Vanha-Perttula, T. (1972). Specific receptor for LH in the ovary: Evidence by autoradiography and tissue fractionation. Endocrinology 9, 1-9. Schuetz, A. W. and Schwartz, W. J. (1979). Intrafollicular cumulus cell transformations associated with oocyte maturation following gonadotrophic hormone stimulation of adult mice. J. Exp. Zool. 27, Schwartz, N. B. (1974). The role of FSH and LH and of their antibodies on follicle growth and on ovulation. Biol. Reprod. 1, Sheela Rani, C. S. and Moudgal, N. R. (1977). Examination of the role of FSH in periovulatory events in the hamster. J. Reprod. Fert. 5, Solursh, M. (1976). Glycosaminoglycan synthesis in the chick gastrula. Develop. Biol. 5, Whitten, W. K. (1971). Nutrient requirements for culture of preimpiantation embryos in vitro. Adv. Biosciences 6, on 3 February 218