Role of secreted proteins and gonadotrophins in promoting full maturation of porcine oocytes in vitro

Experiments were designed to identify the extent to which follicle cells and hormones contribute to the developmental competence of porcine oocytes matured in vitro. Oocyte-cumulus complexes were collected from ovaries by dissection and cultured in 2 ml of TCM199-based medium in 5% CO₂ in humidified air at 38.5°C. This basic maturation system was supplemented, for either the first 24 hr only or for the 48-hr culture period, with 1) everted follicle shell alone, 2) gonadotrophic hormones alone, or 3) both follicle shells and hormones. The effect of these treatments was evaluated on 1) meiotic maturation rates, 2) the capacity of matured eggs to undergo activation and early cleavage, and 3) changes to the profile of proteins secreted into the culture medium. The results showed that 1) supplementation with either follicle shell or hormones alone increased the rates of meiotic maturation over the nonsupplemented control group, and 2) combined follicle shell and hormonal supplementation yielded the highest rates for maturation, activation, and cleavage but only when hormonal supplementation was removed after the first 24 hr of culture. Proteins of 30, 37, 45, and 46 kD, but of unknown function, were secreted during the first 24 hr into the culture medium in groups supplemented with follicle shells. The addition of hormones did not affect this pattern of secreted proteins. It is possible that some secreted proteins may act to facilitate full maturation of pig oocytes. Mol. Reprod. Dev. 47:191–199, 1997. © 1997 Wiley-Liss, Inc.     

Vitellogenin Incorporation into Oocytes of Rainbow Trout, Oncorhynchus mykiss, in Vitro: Effect of Hormones on Denuded Oocytes

An in vitro culture procedure to measure vitellogenin (VTG) incorporation into oocytes without follicle cell layers was developed using oocytes of the rainbow trout, Oncorhynchus mykiss . Oocytes incorporated VTG specifically and linearly for up to 24 hr. The maximum incorporation observed was 314 μg/24 hr/oocyte, using vitellogenic (3.6 mm diameter) oocytes.
The effect of hormones was examined by adding insulin, growth hormone, prolactin, gonadotropins (GTH-I, GTH-II), thyroid hormones, testosterone, estradiol-17β, or 17α, 20β-dihydroxy-4-pregnen-3-one to the medium. The results indicated that insulin and thyroxine stimulated uptake of VTG by 13% and 12%, respectively. Insulin specifically stimulated VTG incorporation and did not cause any change in background uptake of insulin. The lack of an effect of gonadotropins hormones on denuded oocytes suggests that the previously observed stimulation of VTG incorporation into follicle cell-enclosed oocytes in vivo and in vitro by GTH-I is most likely mediated by the somatic cells of the ovarian follicle.     

Follicle cell regulation of protein synthesis and developmental competence in sheep oocytes

The developmental capacity of sheep oocytes cultured outside the follicle was greatly increased by the presence of high concentrations of gonadotrophins (10 micrograms/ml) in the medium. However, even under these conditions, the developmental capacity of the oocytes was only half that of oocytes cultured within the intact follicle. The presence of the cumulus was essential for development; nearly all denuded oocytes failed to undergo cleavage. Maturational changes in the oocyte involving increased amino acid uptake increased incorporation and specific changes in protein synthesis were inhibited by the follicle cells; this suppression was alleviated by gonadotrophic hormones. The cumulus cells suppressed amino acid incorporation and, to some extent, the changes in protein synthesis. However, the suppression of amino acid uptake required the presence of the whole follicle. Patterns of protein synthesis by oocytes cultured outside the follicle differed from those in oocytes cultured within the follicle, irrespective of the presence of the cumulus or gonadotrophins. Analysis of single oocytes cultured outside the follicle showed that the protein profiles varied markedly even under identical culture conditions.     

In vitro and in vivo maturation of oocytes from gonadotrophin-treated brushtail possums

The time course of nuclear maturation of oocytes was examined in brushtail possums, Trichosurus vulpecula. Oocytes were recovered from ovarian follicles > 2 mm in diameter after pregnant mares' serum gonadotrophin/porcine luteinizing hormone (PMSG/LH) treatment (in vivo matured) or 72 hr after PMSG treatment (in vitro matured). Oocytes recovered from small (< 2 mm) and large (> 2 mm) follicles were also assessed for their ability to mature in vitro. Staining with the DNA-specific dye Hoechst 33342 was used to assess the stage of nuclear development by fluorescence microscopy. The process of nuclear maturation progressed rapidly in vivo, as oocytes collected at 20-27 hr post-LH all had a GV, but by 28-29.5 hr post-LH approximately a third of eggs were MII. By 30-hr post-LH, more than 70% of oocytes had reached MII stage and all ovulated eggs were MII. In vitro, all oocytes were at germinal vesicle stage at the start of culture. After 24 hr of culture, 67% of oocytes had progressed to metaphase I/anaphase I of meiosis. After 36 hr, 25% of oocytes had completed maturation to metaphase II, increasing to 52% after 48 hr. Maturation of oocytes after 48 hr in culture was unaffected by the presence or absence of granulosa cells, PMSG or LH/porcine follicle stimulating hormone (FSH). More oocytes from large follicles (55%) completed maturation by 48 hr than from small follicles (15%). The potential of oocytes to mature after 48 hr in culture was dependent on the follicle harvested having reaching a critical diameter of 1.5 mm.     

Involvement of the metabolic hormones leptin, ghrelin, obestatin, IGF-I and of MAP kinase in control of porcine oocyte maturation

The general aim of our in vitro experiments was to study the role of the metabolic hormones leptin, ghrelin, obestatin and IGF-I and mitogen-activated protein kinase (MAPK)-dependent intracellular mechanisms in the control of nuclear maturation of porcine oocytes. For this purpose, porcine oocytes were isolated from the ovary and cultured in the presence of leptin, ghrelin, obestatin, IGF-I, MAPK blocker PD98059 and the combinations of hormones with PD98059. Proportions of matured oocytes (at metaphase II of meiosis, determined by DAPI staining) and of oocytes containing MAPK/ERK1-2 (determined by immunocytochemistry) were measured before and after culture. It was observed that the majority of oocytes isolated from the ovary before culture were immature and did not contain visible MAPK, but some oocytes were mature, and the majority of these oocytes contained MAPK. Incubation of oocytes resulted in a significant increase in the proportion of matured oocytes and in the percentage of oocytes containing MAPK in both the matured and not matured groups. Addition of IGF-I to the culture medium increased the proportion of matured oocytes, addition of leptin decreased it, and ghrelin and obestatin did not oocyte maturation. Addition of hormones did not affect the expression of MAPK in either immature or mature oocytes. PD98059, when given alone, suppressed the maturation and accumulation of MAPK in both mature and immature oocytes. When given together with hormones, PD98059 was able to reduce the stimulatory effect of IGF-I, to invert the inhibitory action of leptin to stimulatory and to induce the stimulatory action of ghrelin and obestatin on meiosis. IGF-I, ghrelin and obestatin, but not leptin, when given together with PD98059, increased the accumulation of MAPK in both immature and mature oocytes. Association of nuclear maturation and expression of MAPK in oocytes before, but not after culture, as well as the prevention of oocyte maturation by MAPK blocker suggests the involvement of MAPK-dependent intracellular mechanisms in the promotion of reinitiation, but not completion of meiosis. The effect of hormonal additions on meiosis of oocytes suggests that IGF-I is a stimulator, leptin can be an inhibitor, while ghrelin and obestatin probably do not control oocyte maturation. The ability of PD98059 to modify the effect of hormones on oocyte maturation and on MAPK expression suggests possible interference of hormones and MAPK-dependent intracellular mechanisms in oocytes. However, no influence of hormones on MAPK and lack of association between action of hormones and PD98059 on MAPK and meiosis suggest that MAPK is probably not a mediator of effect of IGF-I, leptin, ghrelin and obestatin on porcine oocyte nuclear maturation.     

Influence of mating on ovarian follicle development in Triatoma infestans (Klug, 1834).

This work examines the influence of mating on ovarian follicle development in Triatoma infestans. The observations were carried out on both virgin and mated females, which were killed at various times after their emergence. There was no difference in the ovarian development of both experimental groups during the first gonadotrophic cycle. By the 7th day mated females as well as virgin females showed vitellogenic oocytes. The coriogenesis and ovulation process began on the 13th day after imaginal moulting. However we could observe that egg-laying was dependent on mating. Mated females laid eggs whereas virgin females did not lay eggs. However ovarian production was significantly greater in the mated females. It is suggested that in T. infestans mating stimulates egg-laying but it does no influence the oogenesis and ovulation process.     

Estrogen is not directly required for oocyte developmental competence

Oocyte maturation and ovulation require a coordinated interaction between gonadotrophs, steroid hormones, and growth factors. The extent to which estrogen is required in this process, however, remains unclear. To better understand the role of estrogen in maintaining developmental competence of mammalian oocytes, we studied the Aromatase knockout (ArKO) mouse, which has been genetically engineered to be incapable of synthesizing endogenous estrogen. Previous studies have established that ArKO female mice are anovulatory with ovaries that progressively degenerate, developing hemorrhagic cystic follicles. In young ArKO females, however, apparently healthy follicles and oocytes have been observed. We investigated if these oocytes could be induced to ovulate, then mature, fertilize, and develop in vitro. Following a standard superovulation protocol, ArKO oocytes did not ovulate. When recovered manually from the ovary, however, ArKO oocytes successfully progressed through in vitro maturation, fertilization, and development to the blastocyst stage at the same rate as wild-type and heterozygote littermates. Therefore, it appears that estrogen is not required for the production and growth of oocytes capable of maturation and complete preimplantation development but is required for continued follicle growth and feedback regulation of ovulation.     

Effects of ovarian stimulation, with and without human chorionic gonadotrophin, on oocyte meiotic and developmental competence in the marmoset monkey (Callithrix jacchus)

A reliable ovarian stimulation protocol for marmosets is needed to enhance their use as a model for studying human and non-human primate oocyte biology. In this species, a standard dose of hCG did not effectively induce oocyte maturation in vivo. The objectives of this study were to characterize ovarian response to an FSH priming regimen in marmosets, given without or with a high dose of hCG, and to determine the meiotic and developmental competence of the oocytes isolated. Ovaries were removed from synchronized marmosets treated with FSH alone (50 IU/d for 6 d) or the same FSH treatment combined with a single injection of hCG (500 IU). Cumulus-oocyte complexes (COCs) were isolated from large (>1.5mm) and small (0.7-1.5mm) antral follicles. In vivo-matured oocytes were subsequently activated parthenogenetically or fertilized in vitro. Immature oocytes were subjected to in vitro maturation and then activated parthenogenetically. Treatment with FSH and hCG combined increased the number of expanded COCs from large antral follicles compared with FSH alone (23.5 +/- 9.3 versus 6.4 +/- 2.7, mean +/- S.E.M.). Approximately 90% of oocytes surrounded by expanded cumulus cells at the time of isolation were meiotically mature. A blastocyst formation rate of 47% was achieved following fertilization of in vivo-matured oocytes, whereas parthenogenetic activation failed to induce development to the blastocyst stage. The capacity of oocytes to complete meiosis in vitro and cleave was positively correlated with follicle diameter. A dramatic effect of follicle size on spindle formation was observed in oocytes that failed to complete meiosis in vitro. Using the combined FSH and hCG regimen described in this study, large numbers of in vivo matured marmoset oocytes could be reliably collected in a single cycle, making the marmoset a valuable model for studying oocyte maturation in human and non-human primates.     

Stimulation of parthenogenesis in mouse ovarian follicles by inhibitors of inosine monophosphate dehydrogenase

The effects of hormonal priming and inosine monophosphate (IMP) dehydrogenase inhibitors on the meiotic maturation and parthenogenetic activation of mouse oocytes were examined in this study. In the first series of experiments, unprimed mice or mice primed 24 h with equine chorionic gonadotropin (eCG) received injections of the IMP dehydrogenase inhibitors, bredinin (Br) or mycophenolic acid (MA), followed by histological examination at 24 h, 48 h, and 72 h after drug administration. In both treatment groups, oocytes from nonatretic antral follicles were stimulated to undergo germinal vesicle breakdown by 24 h and became parthenogenetically activated as manifested by pronuclear formation and early cleavage divisions. The parthenotes underwent degeneration by 72 h. In the second part of this study, the effects of priming and drug treatment on parthenogenetic activation and subsequent developmental potential in vitro were examined. Mice were primed with eCG, and 24 or 48 h later received injections of Br or MA. Cumulus cell-enclosed oocytes were isolated 21-22 h later and assessed for maturation; those having undergone germinal vesicle breakdown were cultured and subsequently examined for embryonic development. In mice primed for 24 h, but not 48 h, Br and MA stimulated a significant number of oocytes to resume maturation in vivo; these subsequently underwent activation and developed to blastocysts in vitro. In another series of experiments, germinal vesicle-stage oocytes were isolated from primed or unprimed mice and cultured in vitro to permit spontaneous meiotic maturation. Nine percent of mature ova from 24-h-primed mice developed to 2-cell parthenotes; activation in ova from unprimed and 48-h-primed mice was considerably lower. A time-course experiment demonstrated that the extent of parthenogenetic activation in vivo following Br treatment was related to the period of time between drug injection and isolation of ova, the optimal period being 12 h. Neither Br nor MA had a direct activating effect on the oocytes as evidenced by an inability to induce parthenogenesis in vitro. Simultaneous injection of hCG with either Br or MA stimulated ovulation and prevented the parthenogenetic response. These data are consistent with the idea that conditions within the follicle promote parthenogenetic activation when the oocyte matures in the absence of gonadotropin stimulation.     

Hormone-controlled culture of secondary follicles of domestic cats

Ovaries were obtained from domestic cats during ovariohysterectomy. Large, preantral follicles were freed by dissection and mechanical crushing, and were cultured in TCM 199 + 10% FCS medium in the presence or absence of hormones (FSH, hydrocortisone and Insulin-Transferrin-Selenite) as well as in hypoxanthine. A decline in growth potential along with increasing follicle size were observed after one week, with no FSH added. Hormone-supplemented medium was found to induce growth to 2 or 3 times the original volume in more than 80% of follicles of all sizes. Oocyte diameters were continuously increasing, depending on follicle size, and reached 90 mum (80 %) at the point of antrum formation. Nuclear configuration of oocytes from follicles which had been cultured without addition of hormones up to the antral stage indicated a high rate of degeneration which, however, could be reduced by gonadotrophic stimulation. Meiosis at the germinal vesicle stage was found to be inhibited by hypoxanthine. For some oocytes, evidence was provided to meiotic maturation up to extrusion of the first polar body.     

Regulation of mouse oocyte growth: probable nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth

Intercellular communication, as determined by two different assay procedures, was established in vitro between mouse oocytes free of adhering follicle cells and monolayers of either follicle or 3T3 cells. Both of these cell types are known to be able to form homologous gap junctions, and follicle cells naturally form heterologous gap junctions with oocytes in vivo. Monolayers of L cells that are communication deficient did not establish intercellular communication with oocytes as determined by the two different assays for intercellular communication. The diameter of oocytes cultured for 4 days in medium or on monolayers of L cells decreased markedly, 9.7 and 13.1 micron, respectively. In contrast, oocytes cultured for 4 days on follicle cell monolayers increased on the average about 4.7 micron in diameter. Oocytes cultured for 4 days on monolayers of 3T3 cells decreased slightly in diameter, i.e., 2.1 micron. Results from these experiments support a nutritional role for intercellular communication between follicle cells and oocytes in oocyte growth.     

Progression of oocyte maturation from metaphase I to metaphase II is disturbed by previous immunological interference with cumulus cell function.

The effects of an antibody preparation reacting with preovulatory mouse cumuli oophori (anticumulus Ig) on oocyte maturation in vivo and in vitro were studied. Continuous presence of anticumulus Ig in culture medium did not impair oocyte maturation in vitro. Similarly, no effect on oocyte maturation in vivo was observed when anticumulus Ig was given to females superovulated with pregnant mare serum gonadotropin (PMSG) and human chorionic gonadotropin (hCG) at the time of hCG treatment. However, when administered earlier, anticumulus Ig brought about serious disturbances of oocyte meiotic competence, since only immature oocytes were ovulated after anticumulus Ig injection at the time of PMSG treatment and as much as 70% of the ovulated oocytes were immature when the antibody was applied 24 hr later. Previous absorption of anticumulus Ig with isolated cumulus cells removed the inhibitory effect of this preparation on oocyte meiotic competence to the same extent as absorption with whole cumuli oophori, despite the persistence of a strong reactivity of the cumulus cell-absorbed antibody preparations with the cumulus intercellular matrix. The ability of oocytes obtained from antibody-injected animals to mature in vitro was also considerably impaired when the injection was made at the time of PMSG treatment. In all cases the maturation defect concerned the progression of meiosis from metaphase I to metaphase II, while the ability of oocytes to undergo germinal vesicle breakdown (GVBD) was unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

In-vivo and in-vitro maturation rate of oocytes from two strains of mice

Female mice of the KE and CBA strains were used to examine the rate of oocyte maturation in vivo and in vitro. In CBA females killed just before ovulation most preovulatory oocytes were already in the metaphase II stage, while the oocytes of KE mice were arrested at metaphase I until the time of ovulation, and further stages of maturation occurred in the oviduct, reaching the metaphase II stage 3-5 h later. A similar strain difference in oocyte maturation rate was observed from in-vitro culture of cumulus-free oocytes, isolated from the ovaries of PMSG-primed females and intact females killed at the metoestrous phase of the cycle. This indicates that the strain-specific course of maturation is determined in the oocyte by a few days before ovulation. Therefore, if the rate of oocyte maturation is influenced by somatic components of the follicle, this must occur at some earlier stages of follicle development.     

Maturation of pig and rat oocytes transplanted into surrogate pig follicles in vitro

Pig oocytes obtained from slaughterhouse material and rat oocytes obtained from PMSG-treated immature females were incubated as isolated oocytes or injected into explanted pig follicles (5–8 mm). Free oocytes of both species, with or without their cumulus investment or gonadotropins during culture, matured at high rates after 30 hr or 9–10 hr of culture, respectively. Gonadotropic stimulation was necessary for maturation of both the native and injected cumulus-intact pig oocytes in follicle culture. Cumulus-free pig oocytes injected into follicle failed to mature in response to gonadotropic stimulation, suggesting an inability to perceive or respond to stimulation. Injected rat oocytes, however, matured irrespective of cumulus investment or gonadotropic stimulation. Their maturation was delayed and reduced at 9 hr. These results in the rat suggest that the pig follicular environment is incapable of regulating rat oocyte maturation but rather presents a permissive or supportive environment for their maturation. The explanted surrogate follicles from the pig or other species may provide a useful model for the study of oocyte-follicle interactions in oocyte maturation within or between species.     

Accumulation of secretory granules in pituitary clonal cells derived from the epithelium of Rathke's pouch

Summary 2A8 clonal cells derived from the epithelium of Rathke's pouch of the fetal rat are essentially agranular when grown in vitro, in spite of their active secretion of hormones, i.e., ACTH, prolactin and growth hormone. These cells do not produce detectable amounts of thyrotrophic or gonadotrophic hormones in vitro. When the growth medium (Ham's F10) was supplemented with rat median eminence extract (MEE), l-thyroxin and fresh serum from hypophysectomized rats, some of the 2A8 cells accumulated and stored secretory granules which were characteristic of the cells of the intact pituitary gland. Typical thyrotrophic and gonadotrophic cells also became recognizable and all six anterior pituitary hormones were released into the culture medium. Growth of 2A8 cells in this modified culture medium resulted in an increased production of all hormones with increasing time in culture.These results indicate that the processes that lead to the accumulation of typical mature secretory granules which characterize the individual pituitary cell types are initiated or promoted by some unidentified factor or factors which are present in fresh rat serum. It is also apparent that fresh rat serum can promote the differentiation of gonadotrophs and thyrotrophs in vitro.Supported by USPHS Grant Am 12583 and Institutional Research Grant of The University of Texas Health Science Center at San Antonio, TexasThe authors wish to thank Mrs. Pauline Polette for her skillful technical assistance     

Offspring from one-month-old lambs: studies on the developmental capability of prepubertal oocytes

A wave of follicular growth in lamb ovaries occurs at about 4 weeks of age, generating a life-time peak in follicle numbers. In order to take advantage of the large number of oocytes available, and to substantially decrease the generation interval, embryos were derived from oocytes collected from 1-mo-old lambs. Animals were subjected to one of 3 regimes of hormonal stimulation: groups 1 and 2 were treated to obtain germinal vesicle-stage oocytes, and group 3 to produce mature metaphase II oocytes. Adult sheep stimulated by an appropriate dose of FSH served as control. The developmental ability of collected oocytes was evaluated by either in vivo or in vitro culture to the blastocyst stage after in vitro maturation and/or fertilization. Blastocysts were transferred immediately or after cryopreservation to suitable recipient sheep. In order to investigate the full developmental potential of these embryos, pregnancies were allowed to go to term. The results show significant differences (P < 0.001) between all experimental groups in blastocyst numbers produced. Embryos derived from group 1 animals produced the greatest number of blastocysts, under both in vivo (36. 7%), and in vitro (22.9%) culture systems. Group 2 gave lowest blastocyst production (5.0%), while group 3 yielded 13.2% blastocysts. The number of pregnant recipients carrying to term lamb-derived embryos was severely reduced for both in vivo- (2 of 9; 22.2%) and in vitro-cultured, fresh (3 of 10; 30.0%) and cryopreserved (1 of 6; 16.7%) lamb embryos. This study is the first report of the birth of live lambs derived from oocytes obtained from donors as young as 4 wk. Defects in the competence of lamb-derived embryos may account for the increased fetal loss during pregnancy and the occurrence of mummified fetuses delivered alongside normal healthy lambs.     

Comparison of exogenous energy sources for in vitro maintenance of follicle cell-freeXenopus laevis oocytes

Summary The purpose of these experiments was to determine which exogenous energy sources are suitable for isolated follicle cell-free oocytes from the frog,Xenopus laevis. In order to compare prospective energy sources, follicle cell-free oocytes from 0.4 to 1.3 mm in diameter were incubated in a 1 mM concentration of each of a variety of energy sources and scored daily for the maintenance of morphological characteristics. Vitellogenic oocytes placed in succinate or fumarate deteriorated at the same time as those in saline alone. Oocytes incubated in oxaloacetate (OAA) appeared to remain in the best morphological condition, followed by oocytes maintained in pyruvate or glucose. Fully grown oocytes were tested at various times of incubation for their ability to respond to progesterone by undergoing germinal vesicle breakdown. These experiments showed that oocytes placed in OAA or pyruvate retained the ability to respond to progesterone longer than those in the other energy sources. Increased respiratory rates were stimulated in isolated oocyte mitochondria by succinate as well as pyruvate and OAA. However, oocytes incubated in labelled pyruvate evolved 80 to 140 times as much labelled CO₂ as oocytes incubated in labelled glucose or succinate. In addition, it was found that the rate of uptake of pyruvate is 20 to 25 times greater than the rate of uptake of glucose or succinate. It is concluded from these experiments that OAA and pyruvate are the most effective exogenous energy sources for the in vitro maintenance ofXenopus oocytes. One possible explanation for the ineffectiveness of glucose or succinate as exogenous energy sources is a restriction in their uptake into the oocytes. Research supported by grants 10179 from the Research Foundation of the City University of New York and BMS 74-18790 from the National Science Foundation.     

Induction of germinal vesicle breakdown in Xenopus laevis oocytes: response of denuded oocytes to progesterone and insulin

Denuded oocytes freed of their vitelline envelope have been prepared by two methods, enzymatically with pronase and manually by microdissection. The response of denuded oocytes to progesterone, in terms of germinal vesicle breakdown (GVBD), was similar to that obtained with defolliculated oocytes (separated with collagenase from follicle cells, but still keeping their vitelline membrane). The same conclusion was drawn with respect to morphological features of the oocyte surface observed by transmission and scanning electron microscopy, before and after progesterone-induced GVBD. The synergistic effect of insulin and progesterone in denuded oocytes was comparable to that observed in defolliculated oocytes. Multiplication stimulating activity (MSA) had the same effect as insulin. These observations indicate that hormones act directly upon oocytes, without interference of the surrounding vitelline envelope and follicle cells.