Biomedical subjects

J J Eppig

Publications and source records attributed to J J Eppig.

At least 19 recordsLinked to original sources

PubMed1992

Modulation of meiotic arrest in mouse oocytes by guanyl nucleotides and modifiers of G-proteins.

Guanyl nucleotide binding-proteins, or G-proteins, are ubiquitous molecules that are involved in cellular signal transduction mechanisms. Because a role has been established for cAMP in meiosis and G-proteins participate in cAMP-generating systems by stimulating or inhibiting adenylate cyclase, the present study was conducted to examine the possible involvement of G-proteins in the resumption of meiotic maturation. Cumulus cell-free mouse oocytes (denuded oocytes) were maintained in meiotic arrest in a transient and dose-dependent manner when microinjected with the nonhydrolyzable GTP analog, GTP gamma S. This effect was specific for GTP gamma S, because GppNHp, GTP, and ATP gamma S were without effect. Three compounds, known to interact with G-proteins, were tested for their ability to modulate meiotic maturation: pertussis toxin, cholera toxin, and aluminum fluoride (AlF4-). Pertussis toxin had little effect on maturation in either cumulus cell-enclosed oocytes or denuded oocytes when meiotic arrest was maintained with dibutyryl cAMP (dbcAMP) or hypoxanthine. Cholera toxin stimulated germinal vesicle breakdown (GVB) in cumulus cell-enclosed oocytes during long-term culture, but its action was inhibitory in denuded oocytes. AlF4- stimulated GVB in both cumulus cell-enclosed oocytes and denuded oocytes when meiotic arrest was maintained with hypoxanthine but was much less effective in dbcAMP-arrested oocytes. In addition, AlF4- abrogated the inhibitory action of cholera toxin in denuded oocytes and also that of follicle-stimulating hormone (FSH) in cumulus cell-enclosed oocytes. Cholera toxin or FSH alone each stimulated the synthesis of cAMP in oocyte-cumulus cell complexes, whereas pertussis toxin or AlF4- alone were without effect. Both cholera toxin and AlF4- augmented the stimulatory action of FSH on cAMP. These data suggest the involvement of guanyl nucleotides and G-proteins in the regulation of GVB, although different G-proteins and mediators may be involved at the oocyte and cumulus cell levels. Cholera toxin most likely acts by ADP ribosylation of the alpha subunit of Gs and increased generation of cAMP, whereas AlF4- appears to act by antagonizing a cAMP-dependent step.

Adenylate Cyclase Toxin↗

PubMed1992

Comparison of embryonic developmental competence of mouse oocytes grown with and without serum.

The first objective of this study was to determine whether oocyte growth in serum-free medium affects the solubility of the zona pellucida to alpha-chymotrypsin digestion, which is an index of zona pellucida "hardening" and reflects the potential penetrability of the zona pellucida by sperm. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in medium containing 5% fetal bovine serum (FBS) or in serum-free medium. The zonae pellucidae of oocytes grown in serum-free medium were four times as hard as freshly isolated germinal vesicle (GV)-stage oocytes grown in vivo or oocytes grown in vitro in FBS-containing medium. The hardening of the zonae pellucidae of oocytes grown in serum-free medium was prevented by addition of fetuin. The second objective was to compare the competence to undergo embryogenesis of oocytes that grew in serum-free vs. FBS-containing medium. Approximately 70% of the oocytes underwent maturation regardless of whether the medium was serum-free or contained FBS. Of the mature ova grown in medium containing FBS, 53% cleaved to the two-cell stage after insemination compared with only 6% of the ova grown in serum-free medium. Addition of fetuin to the serum-free medium used for oocyte growth increased the frequency of cleavage to the two-cell stage. Of the embryos derived from oocytes that grew in FBS-containing medium, 70% completed the two-cell stage to blastocyst transition.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Mating stimulates estradiol production by ovaries of the musk shrew (Suncus murinus).

Asian musk shrews (Suncus murinus) are induced ovulators, but exhibit no cyclic changes in reproductive structures or in sexual behavior. Mating behavior is induced by contact with a male. To determine if mating induces changes in ovarian steroidogenesis, ovaries removed from unmated animals and at 3, 10, 15, and 36 h after mating were cultured for 4 h in the presence or absence of gonadotropins (LH + FSH, 1 microgram/ml). Histological analysis revealed no obvious changes in follicular size or appearance at the end of culture in ovaries cultured at 3 and 10 h post-mating, as compared with ovaries from unmated shrews, and mating did not stimulate any discernable changes in steroid secretion in these two groups. However, at the end of the culture period, ovulation had occurred or was occurring in ovaries from 35% of the animals ovariectomized at 15 h after mating, and corpora lutea (CLs) were present in 39% of ovarian pairs obtained 36 h after mating. At 15 h post-mating, ovaries with ovulations secreted three times more estradiol than did ovaries that showed no evidence of stimulation by mating, but there were no differences in testosterone or progesterone production. In contrast, ovaries isolated 36 h post-mating with CLs secreted dramatically more of all three steroids than ovaries without CLs (23, 13, and 52 times more estradiol, testosterone, and progesterone, respectively). These data are consistent with plasma concentrations of estradiol at the time of ovariectomy, which were twice as high at both 15 and 36 h after mating, in animals whose ovaries showed evidence of ovarian stimulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Mouse oocytes promote proliferation of granulosa cells from preantral and antral follicles in vitro.

Evidence is now emerging that the oocyte plays a role in the development and function of granulosa cells. This study focuses on the role of the oocyte in the proliferation of (1) undifferentiated granulosa cells from preantral follicles and (2) more differentiated mural granulosa cells and cumulus granulosa cells from antral follicles. Preantral follicles were isolated from 12-day-old mice, and mural granulosa cells and oocyte-cumulus complexes were obtained from gonadotropin-primed 22-day-old mice. Cell proliferation was quantified by autoradiographic determination of the 3H-thymidine labeling index. To determine the role of the oocyte in granulosa cell proliferation, oocyte-cumulus cell complexes and preantral follicles were oocytectomized (OOX), oocytectomy being a microsurgical procedure that removes the oocyte while retaining the three-dimensional structure of the complex or follicle. Mural granulosa cells as well as intact and OOX complexes and follicles were cultured with or without FSH in unconditioned medium or oocyte-conditioned medium (1 oocyte/microliter of medium). Preantral follicles were cultured for 4 days, after which 3H-thymidine was added to each group for a further 24 h. Mural granulosa cells were cultured as monolayers for an equilibration period of 24 h and then treated for a 48-h period, with 3H-thymidine added for the last 24 h. Oocyte-cumulus cell complexes were incubated for 4 h and then 3H-thymidine was added to each group for an additional 3-h period. FSH and/or oocyte-conditioned medium caused an increase in the labeling index of mural granulosa cells in monolayer culture; however, no differences were found among treatment groups.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Developmental abnormalities in Steel17H mice result from a splicing defect in the steel factor cytoplasmic tail.

The murine dominant White spotting (W) and Steel (Sl) loci encode the c-kit tyrosine kinase receptor and its cognate ligand steel factor (SLF), respectively. Mutations at either locus produce deficiencies in the same three migratory cell populations--those giving rise to pigment cells, germ cells, and blood cells. The identification of the gene products of these two loci combined with the plethora of W and Sl mutations available for molecular analysis offers a unique opportunity to dissect the role of a tyrosine kinase receptor and its cognate ligand during development in a fashion not possible for most other mammalian genes. Among the most interesting Sl mutations available for study are those that induce sterility in only one sex. In studies described here, we show that one of these alleles, Sl17H, which in the homozygous condition induces sterility in males but not females, is the result of a splicing defect in the SLF cytoplasmic tail. We also characterize the nature of the germ cell defects in male and female Sl17H mice and show that both sexes are affected equally during embryonic but not postnatal development. These studies provide new insights into the role of SLF in germ cell development and indicate that the cytoplasmic domain of SLF is important for its normal biological function.

Amino Acid Sequence↗

PubMed1992

Developmental capacity of mouse oocytes matured in vitro: effects of gonadotrophic stimulation, follicular origin and oocyte size.

Development of mammalian oocytes is usually correlated with ovarian follicular development. This correlation was tested by determining whether gonadotrophic stimulation of follicular development in immature mice resulted in a coordinated increase in the embryonic developmental capacity of the oocytes. Oocyte cumulus cell complexes were isolated at the germinal vesicle stage from small, medium and large antral follicles of 26-day-old mice and matured and fertilized in vitro. The frequency with which embryos from oocytes from small follicles completed the two-cell to blastocyst transition was lower than for embryos from oocytes from large follicles (33% and 79%, respectively). Germinal-vesicle stage oocyte-cumulus cell complexes were isolated from 22-26-day-old mice that were unprimed or primed by injection of equine chorionic gonadotrophin 48 h before isolation. Oocytes were matured in control medium, or in medium containing 1 microgram follicle-stimulating hormone (FSH) ml-1, and then fertilized in vitro. Priming did not increase the number of embryos completing the two-cell stage to blastocyst transition in the 22-day-old group nor did FSH treatment of maturing oocytes when the oocytes were isolated from unprimed 22-day-old mice. In contrast, priming increased the percentage of embryos completing the two-cell stage to blastocyst transition in the 26-day-old group by 20%. FSH treatment of maturing oocytes from the unprimed, 26-day-old group increased the number of embryos completing the transition to the same level as those in the primed 26-day-old group, but FSH did not increase the frequency of transition in the primed 26-day-old group.(ABSTRACT TRUNCATED AT 250 WORDS)

Age Factors↗

PubMed1992

Growth and development of mammalian oocytes in vitro.

In mammalian ovaries, most of the oocytes are in early stages of development and only a small number of these will complete their development, ovulate, and be available for fertilization. Systems for the culture of oocytes at these early stages, when they are present in preantral follicles, are described herein. Results show that oocytes can undergo a major part of their development in vitro. As methods for oocyte development in vitro improve, these culture systems will allow the efficient use of the large available pool of preantral follicles for clinical, agricultural, or zoological purposes.

Animals↗

PubMed1991

Intercommunication between mammalian oocytes and companion somatic cells.

Cellular interactions in the mammalian ovarian follicle between its germ-line and somatic cell components are crucial for its development and function. These interactions are mediated by both membrane gap junctions and paracrine factors. Somatic cell-to-oocyte communication is essential for oocyte growth and the regulation of meiotic maturation. In particular, granulosa cells provide nutrients and molecular signals that regulate oocyte development. Oocytes, on the other hand, promote the organization of the follicle, the proliferation of granulosa cells, and the differentiation and function of cumulus cells, a subset of granulosa cells. Determining the nature of the oocyte-to-granulosa cell signals remains a key challenge for future work.

Animals↗

PubMed1991

Reversal of postmortem degeneration of mouse oocytes during meiotic maturation in vitro.

The developmental capacity of oocytes matured in vitro following isolation at the germinal vesicle stage from freshly killed mice (control) was compared with that of oocytes isolated from the carcasses of mice killed 3, 6, 9, and 12 hr earlier. The yield of intact, cumulus cell-enclosed oocytes decreased as the interval between death of the animal and removal of the ovary increased. After 15-16 hr of culture of medium containing follicle-stimulating hormone, the frequency of germinal vesicle breakdown, extrusion of a polar body, and cumulus expansion was equivalent in oocytes of all groups. The frequency of development of inseminated ova to 2-cell stage embryos in the control, 3, and 6 hr postmortem groups was the same but declined markedly in the 9 and 12 hr groups. There was also no difference in the frequency of blastocyst development from 2-cell stage embryos between the control, 3, 6, and 9 hr postmortem groups, but the 2-cell embryos in the 12 hr postmortem group did not develop to blastocysts. Thirty-six percent of the 2-cell stage embryos from the 6 hr postmortem group developed to live young after transfer to foster mothers. Follicles of 6 hr postmortem ovaries showed degeneration manifested as prominent crystalline inclusions within the oocytes and many pyknotic granulosa cells. The crystals disappeared within 1 hr of culture and the secondary oocytes appeared normal. The cultured oocyte-cumulus cell complexes, therefore, reversed degenerative changes induced by the death of the animal. This study demonstrates the feasibility of recovering developmentally competent oocytes from valuable recently deceased zoological, agricultural, and endangered mammals.

Animals↗

PubMed1991

Maintenance of meiotic arrest and the induction of oocyte maturation in mouse oocyte-granulosa cell complexes developed in vitro from preantral follicles.

During the development of oocyte-granulosa cell complexes from preantral follicles in vitro, oocytes grow and acquire competence to undergo germinal vesicle breakdown (GVB). In the culture system used here, GVB-competent oocytes were maintained in meiotic arrest solely by endogenous physiological mechanisms of the granulosa cells without supplementation with meiosis-arresting substances. Addition of mycophenolic acid, an inhibitor of inosine monophosphate (IMP) dehydrogenase, induced GVB in about 70% of the GVB-competent oocytes grown in vitro. The mechanism for meiotic arrest in this system is, therefore, similar to that for arrest in vivo insofar as it requires the participation of the IMP dehydrogenase pathway. Rp-cyclic adenosine monophosphothioate, a membrane-permeable antagonist to cAMP, induced GVB by about 30% of the competent oocytes. Cyclic AMP-dependent pathways, therefore, participate in the physiological mechanism by which mouse granulosa cells maintain meiotic arrest. Complexes were grown for 10 days in medium containing 0, 1, 5, or 10 ng/ml FSH, were stimulated with either 1 microgram/ml FSH or LH, and were assessed for GVB and cumulus expansion. GVB was stimulated by FSH whether or not the complexes were grown in medium containing FSH, but LH or hCG induced GVB only when the complexes were grown in medium containing FSH. Cumulus expansion occurred in response to either FSH or LH only when complexes were grown in medium containing FSH. FSH, therefore, promotes the differentiation of granulosa cells from preantral follicles in vitro so that LH can stimulate GVB and cumulus expansion.

Animals↗

PubMed1990

mos proto-oncogene function.

Maturation promoting factor (MPF) is a cytoplasmic activity that causes oocytes arrested in prophase to resume meiosis. An inactive form of MPF termed pre-MPF exists in fully grown oocytes. In Xenopus oocytes, progesterone induces maturation and pre-MPF activation. These early maturation events require protein synthesis. We have shown that p39mos synthesis is rapidly induced in progesterone-treated Xenopus oocytes during the protein synthesis sensitive period and prior to activation of pre-MPF or germinal vesicle breakdown (GVBD). p39mos may qualify, therefore, as an 'initiator' of maturation. Mouse oocytes undergoing meiotic maturation also express p39mos. Microinjection of antisense mos oligodeoxynucleotides into fully grown mouse and Xenopus oocytes results in the block of meiotic maturation. In Xenopus, antisense-injected oocytes not only lack p39mos, but also lack MPF and fail to undergo GVBD. In the mouse, the microinjected oocytes progress through GVBD, but fail to produce the first polar body; cytogenetic analysis shows they are arrested at the bivalent chromosome stage of metaphase I. This and additional studies with Xenopus oocytes indicate that p39mos is also required throughout maturation. We have shown that p39mos is indistinguishable from the protein product constitutively expressed in NIH/3T3 cells transformed with activated c-mos. It is likely that its activity as a transforming gene may be due to activation of pre-MPF activities in interphase in the somatic cell cycle.

Animals↗

PubMed1990

Culture media for mouse oocyte maturation affect subsequent embryonic development.

These experiments were done to determine whether the culture medium used for the spontaneous maturation of mouse oocytes can affect the subsequent capacity of the ova to become fertilized and complete preimplantation development in vitro and development to live young. Oocytes obtained from antral follicles of gonadotropin-primed immature mice underwent spontaneous maturation in control medium, i.e. Eagle's Minimum Essential Medium (MEM) supplemented with 5% fetal bovine serum, or in one of eight different media which were also supplemented with serum. All of the ova were fertilized in Whitten's medium and were assessed for cleavage to the 2-cell stage and for further preimplantation development to blastocysts during culture in Whitten's medium. Three of the eight media used for oocyte maturation improved the capacity of the ova to develop to the blastocyst stage when compared with the control: Waymouth MB 752/1, MEM with non-essential amino acids, and MEM Alpha; Waymouth medium promoted the highest frequency of development of ova to the blastocyst stage. Moreover, the blastocysts derived from oocytes that matured in Waymouth medium contained more cells than blastocysts derived from oocytes that matured in control medium. Although BGJb medium promoted the cleavage of eggs to the 2-cell stage when present during oocyte maturation, it had a detrimental effect on their subsequent preimplantation developmental capacity. Following transfer to foster mothers, more 2-cell stage embryos developed to live young after oocyte maturation in Waymouth medium (21%) than in control medium (13%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1990

Developmental pattern of the secretion of cumulus expansion-enabling factor by mouse oocytes and the role of oocytes in promoting granulosa cell differentiation.

The expansion, or mucification, of the mouse cumulus oophorus in vitro requires the presence of an enabling factor secreted by the oocyte as well as stimulation with follicle-stimulating hormone (FSH). This study focuses on (1) the ability of mouse oocytes to secrete the enabling factor at various times during oocyte growth and maturation, (2) the temporal relationships between the development of the capacity of the oocyte to undergo germinal vesicle breakdown, the ability of the oocyte to secrete cumulus expansion-enabling factor, and the capacity of the cumulus oophorus to undergo expansion, and (3) the role of the oocyte in the differentiation of granulosa cells as functional cumulus cells. Growing, meiotically incompetent oocytes did not produce detectable amounts of cumulus expansion-enabling factor, but fully grown meiosis-arrested oocytes, maturing oocytes, and metaphase II oocytes did. Detectable quantities of enabling factor were produced by zygotes, but not by two-cell stage to morula embryos. The ability of oocytes to secrete cumulus expansion enabling factor and the capacity of cumulus cells to respond to FSH and the enabling factor are temporally correlated with the acquisition of oocyte competence to undergo germinal vesicle breakdown. Mural granulosa cells of antral follicles do not expand in response to FSH even in the presence of cumulus expansion-enabling factor, showing that mural granulosa cells and cumulus cells are functionally distinct cell types. The perioocytic granulosa cells of preantral follicles isolated from 12-day-old mice differentiate into functional cumulus cells during a 7-day period in culture. Oocytectomized granulosa cell complexes grown in medium conditioned by either growing or fully grown oocytes were comparable in size to intact complexes and maintained their 3-dimensional integrity to a greater degree than oocytectomized complexes grown in unconditioned medium. After 7 days, the oocytectomized complexes were stimulated with FSH in the presence of enabling factor, but no expansion was observed whether or not the oocytectomized complexes grew in the presence of oocyte-conditioned medium. These results suggest that a factor(s) secreted by the oocyte affects granulosa cell proliferation and the structural organization of the follicle, but continual close association with the oocyte appears necessary for the differentiation of granulosa cells into functional cumulus cells, insofar as they are capable of undergoing expansion.

Animals↗

PubMed1990

FSH-induced expansion of the mouse cumulus oophorus in vitro is dependent upon a specific factor(s) secreted by the oocyte.

Although it has been shown that granulosa cells regulate the growth and meiotic maturation of mammalian oocytes, there is little evidence of a role for the oocyte in the differentiation or function of granulosa cells. To test the hypothesis that the oocyte participates in the regulation of granulosa cell function, oocytes were removed from isolated oocyte-cumulus cell complexes by a microsurgical procedure and oocytectomized complexes were tested for their ability to undergo expansion in response to follicle-stimulating hormone (FSH). FSH increased the levels of intracellular cAMP, the activity of the hyaluronic acid-synthesizing enzyme system, and induced cumulus expansion in intact complexes. In contrast, FSH did not induce increased hyaluronic acid-synthesizing enzyme activity or cumulus expansion in oocytectomized complexes. Therefore, the participation of the oocyte is necessary for the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in vitro in response to stimulation with FSH. FSH induced the elevation of intracellular cAMP to the same extent in both intact and oocytectomized complexes and the cAMP analog 8-bromo cyclic adenosine monophosphate (8Br-cAMP) did not stimulate expansion in oocytectomized complexes. Therefore, the influence of the oocyte on cumulus expansion occurs downstream from the elevation of cAMP levels in the cumulus cells. Epidermal growth factor (EGF), a potent stimulator of cumulus expansion in intact complexes, which probably acts by a mechanism at least initially different from FSH, failed to stimulate cumulus expansion after oocytectomy. Next, oocytectomized complexes were either cocultured with germinal vesicle stage denuded oocytes or cultured in medium conditioned by denuded oocytes. In both cases, FSH or EGF stimulated expansion by oocytectomized complexes. The degree of expansion was directly correlated to the number of oocytes used to condition the medium. Contact between the oocyte and the cumulus cells is not necessary for cumulus expansion. Rather, a factor(s) secreted by the oocyte is necessary for the cumulus cells to undergo expansion in response to either FSH or EGF. FSH did not induce expansion of oocytectomized complexes in media conditioned by various somatic cells such as granulosa cells, fibroblasts, and Sertoli cells; by a mixed population of male germ cells; or by spermatozoa. This suggests that the expansion enabling activity is specific to the oocyte. These results demonstrate that the oocyte participates in the regulation of cumulus cell function.

Cells, Cultured↗

PubMed1990

Interactions between somatic cells and germ cells throughout mammalian oogenesis.

Oocytes and their companion somatic cells maintain a close association throughout oogenesis and this association is essential for normal oocyte and follicular development. This review summarizes current concepts of the role of the somatic cells in the regulation of mammalian oocyte growth, the maintenance of meiotic arrest, the induction of oocyte maturation, and the acquisition of full embryonic developmental competence during oocyte maturation in vitro. Gap junctions appear to mediate these regulatory processes. The regulatory interaction of oocytes and somatic cells, however, is not unidirectional; the oocyte participates in the proliferation, development, and function of the follicular somatic cells. The oocyte secretes factors that enable the cumulus cells to synthesize hyaluronic acid and undergo cumulus expansion in response to hormonal stimulation. In addition, the oocyte produces factors that promote the proliferation of granulosa cells. These interactions in vitro do not appear to require the mediation of gap junctions. The oocyte also promotes the differentiation of granulosa cells into functional cumulus cells, but this function of the oocyte appears to require the continued presence and close association of the oocyte and granulosa cells. Therefore, oocytes and follicular somatic cells are interdependent for development and function.

Animals↗

PubMed1990

Fetuin inhibits zona pellucida hardening and conversion of ZP2 to ZP2f during spontaneous mouse oocyte maturation in vitro in the absence of serum.

The zona pellucida of mouse oocytes becomes resistant to chymotrypsin digestion, or "hardened", when spontaneous maturation occurs in serum-free medium (De Felici and Siracusa, Gam Res 1982; 6:107). The hardened zona pellucida is refractory to sperm penetration, thus preventing fertilization. Conversion of the zona pellucida glycoprotein ZP2 to ZP2f by a protease from precociously released oocyte cortical granules appears to be a major contributory factor of zona pellucida hardening (Ducibella et al., Dev Biol 1990; 137:46). Fetal bovine serum (FBS) prevents zona hardening and the ZP2 to ZP2f conversion during oocyte maturation in vitro (Downs et al., Gam Res 1986; 15:115; Ducibella et al., Dev Biol 1990; 137:46). This study was conducted to determine whether fetuin, a major glycoprotein constituent of FBS and a protease inhibitor, could prevent zona pellucida hardening during murine oocyte maturation in serum-free medium. Commercially available preparations of fetuin purified by three different methods were all active in inhibiting zona pellucida hardening in a concentration-dependent manner. Further chromatographic purification of one of these preparations indicated that the activity preventing zona pellucida hardening was associated specifically with fetuin. Fetuin also inhibited the conversion of ZP2 to ZP2f in a concentration-dependent manner during oocyte maturation in serum-free medium. Moreover, oocytes that matured in serum-free medium containing fetuin could be fertilized and could undergo preimplantation development to the blastocyst stage. These results indicate that fetuin, a component of FBS, inhibits zona pellucida hardening during oocyte maturation, and suggest that fetuin acts by preventing the proteolytic conversion of ZP2 to ZP2f by precociously released cortical granules.

Animals↗

PubMed1990

Developmental capacity of mouse oocytes cryopreserved before and after maturation in vitro.

The survival and developmental capacity of cumulus cell-enclosed oocytes frozen (1) at the germinal vesicle (GV) stage, after maturation in vitro with (2) and without (3) FSH, and (4) after gonadotrophin-stimulated ovulation were assessed. Survival, defined as the number of morphologically normal oocytes, after freeze-thaw at the GV stage (69%), was lower than for oocytes frozen after ovulation (84%), and after maturation in vitro with FSH (88%) and without FSH (81%). Treatment with DMSO without freezing had no effect on survival when compared with untreated controls except in immature GV-stage oocytes for which there was a slight reduction. After insemination in vitro, 9% of frozen-thawed GV-stage oocytes cleaved to two equal blastomeres, but none developed to blastocysts. Of oocytes matured in vitro before freezing, 17% cleaved to the 2-cell stage and 18% of these developed to blastocysts. When oocytes were matured in vitro in the presence of FSH, however, the percentage cleaving to the 2-cell stage after freeze-thaw was improved to 55%, and 77% of 2-cell stage embryos developed to blastocysts. When ovulated cumulus cell-enclosed oocytes were frozen, 88% cleaved and 67% of the cleaved embryos developed to blastocysts. When 158 two-cell embryos resulting from oocytes matured in vitro with FSH were transferred to the oviducts of pseudopregnant foster mothers, 41 genetically marked live young were produced (26%).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1989

Maintenance of meiotic arrest in mouse oocytes by purines: modulation of cAMP levels and cAMP phosphodiesterase activity.

Hypoxanthine and adenosine are present in preparations of mouse ovarian follicular fluid, and these purines maintain mouse oocytes in meiotic arrest in vitro (Eppig et al.: Biology of Reproduction 33:1041-1049. 1985). The first hypothesis tested in this study is that purines which maintain meiotic arrest act by maintaining meiosis-arresting levels of cyclic adenosine monophosphate (cAMP) in the oocyte. Oocyte-cumulus cell complexes were incubated in control medium (no added purines), or medium containing 0.75 mM adenosine, 4 mM hypoxanthine, or both for 3 hr and the percentage of the oocytes that underwent germinal vesicle breakdown (GVB) and the cAMP content of the intact complexes and the oocytes were determined. Adenosine alone had little inhibitory effect on GVB at this time point but sustained higher levels of cAMP in the oocytes. Hypoxanthine maintained 80% of cumulus cell-enclosed oocytes in meiotic arrest and also sustained higher cAMP levels in the oocytes. The addition of adenosine to hypoxanthine-containing medium increased the percentage of oocytes maintained in meiotic arrest, and increased the amount of cAMP in the oocytes above that maintained by either hypoxanthine or adenosine alone. Neither hypoxanthine, adenosine, nor hypoxanthine plus adenosine altered the cAMP content of intact complexes when assayed after 3 hr culture. Microinjection of an inhibitor of the catalytic subunit of cAMP-dependent protein kinase induced GVB in denuded oocytes cultured in medium containing hypoxanthine. This purine, therefore, maintained meiotic arrest by sustaining elevated cAMP levels within the oocytes. The second hypothesis tested in this study is that purines maintain meiosis-arresting levels of cAMP, at least in part, by inhibiting cAMP phosphodiesterase activity. In descending order of potency, 3-isobutyl-1-methylxanthine (IBMX), guanosine, hypoxanthine, adenosine, and xanthosine inhibited cAMP phosphodiesterase in oocyte lysates. Moreover, like the potent phosphodiesterase inhibitor IBMX, hypoxanthine augmented the meiotic arrest and cAMP accumulation mediated by follicle-stimulating hormone (FSH) in intact complexes. Therefore, inhibition of oocyte phosphodiesterase appears to be one mechanism by which the purines could maintain meiosis-arresting levels of cAMP.

1-Methyl-3-isobutylxanthine↗