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J J Eppig

Publications and source records attributed to J J Eppig.

At least 37 records · Page 2Linked to original sources

Comparison of preimplantation developmental competence after mouse oocyte growth and development in vitro and in vivo.

Culture systems for oocytes are essential for the experimental analysis of the basic mechanisms of oocyte development and, moreover, they will eventually find wide application in agriculture, the clinic, and wildlife preservation. Here, progress in mouse oocyte growth and development in vitro using oocyte-granulosa cell complexes from preantral follicles is reviewed. Oocyte-granulosa cell complexes were isolated from preantral (secondary) follicles of 12 day old mice, grown in vitro for 10 days, then matured and fertilized in vitro. The developmental competence of these oocytes was compared with oocytes grown in vivo and isolated from 22 day old mice, then matured and fertilized in vitro. In vitro-grown oocytes did not achieve the same size as their in vivo-grown counterparts. However, when oocytes were grown in medium containing fetal bovine serum, their preimplantation developmental competence was equivalent to that of in vivo-grown oocytes. Surprisingly, more blastocysts per animal were produced when oocytes were grown in vitro than in vivo. There was no correlation between oocyte size and either preimplantation developmental competence or number of cells per blastocyst. Oocytes grown in serum-free medium did not achieve the same developmental competence as oocytes grown in medium supplemented with serum. Lastly, the health status as an adult of the only animal born after complete oocyte development in vitro is described and discussed.

Animals↗

Mouse oocytes suppress cAMP-induced expression of LH receptor mRNA by granulosa cells in vitro.

Mouse oocytes suppress follicle-stimulating hormone (FSH)-induced luteinizing hormone receptor (LHR) messenger ribonucleic acid (mRNA) expression in cultured granulosa cells. The objective of this study was to assess the mechanism by which oocytes suppress FSH-induced LHR expression. The effect of cumulus cell-denuded, germinal-vesicle-stage oocytes, isolated from antral follicles, on FSH-induced cyclic adenosine monophosphate (cAMP) production by cultured granulosa cells was determined by radioimmunoassays. In addition, the effect of oocytes on 8Br-cAMP-induced LHR mRNA steady-state expression by granulosa cells was assessed by RNase protection assays. Oocytes had no detectable effect on FSH-induced cAMP production. However, oocytes dramatically suppressed 8Br-cAMP-induced LHR mRNA steady-state expression by granulosa cells. It was concluded that the mechanism by which oocytes suppress FSH-induced steady-state expression of LHR mRNA is not by inactivating FSH, preventing functional interactions of FSH with its granulosa cell receptors, or by interfering with the signal-transduction mechanisms required for FSH-dependent cAMP production. In addition, since oocytes suppressed the 8Br-cAMP-induced increase in steady-state expression of mRNA for LHR, oocyte-derived factors probably suppress expression by acting downstream of FSH-induced elevation of granulosa cell cAMP.

Animals↗

SPIN, a substrate in the MAP kinase pathway in mouse oocytes.

The newly cloned gene Spin encodes a 30-kDa protein, a well-defined abundant molecule found in mouse oocytes and early embryos. This protein SPIN undergoes metaphase-specific phosphorylation and binds to the spindle. To understand the role of SPIN in oocyte meiosis, oocytes were treated with drugs that affect the cell cycle by activating or inactivating specific kinases. The posttranslational modification of SPIN in the treated oocytes was then investigated by one- and two-dimensional gel electrophoresis. Modification of SPIN is inhibited by treatment with 6-dimethylaminopurine (DMAP), suggesting that SPIN is phosphorylated by a serine-threonine kinase. Furthermore, SPIN from cycloheximide-treated oocytes that lack detectable MAP kinase activity is only partially phosphorylated, indicating that SPIN may be phosphorylated by the MOS/MAP kinase pathway. To confirm this observation, SPIN was analyzed in Mos-null mutant mice lacking MAP kinase activity. Normal posttranslational modification of SPIN did not occur in Mos-null mutant oocytes. In addition, there is reduced association of SPIN with the metaphase I spindle in Mos-null mutant oocytes, as determined by immunohistochemical analysis. These findings suggest that SPIN is a substrate in the MOS/ MAP kinase pathway and further that this phosphorylation of SPIN may be essential for its interaction with the spindle.

Adenine↗

Mouse primary spermatocytes can complete two meiotic divisions within the oocyte cytoplasm.

This study was undertaken to determine whether primary spermatocyte nuclei can complete meiosis after transfer into maturing or mature oocytes and can participate in normal embryogenesis. When injected into maturing mouse oocytes at prometaphase of the first meiotic division, spermatocyte chromosomes became arranged on a first meiotic metaphase (Met-I) spindle. Thus, oocytes contained two sets of Met-I chromosomes. When these oocytes were matured in vitro and artificially activated, pronuclei and polar bodies of both maternal and paternal origin were formed, and zygotes began development. When single spermatocyte nuclei were injected into fully mature oocytes at metaphase of the second meiosis (Met-II), the spermatocyte nuclei transformed into a Met-I configuration, resulting in the formation of oocytes with both maternal (Met-II) and paternal (Met-I) chromosome complements. After activation of these oocytes, half of each chromosome set was separated into polar bodies. Transfer of nuclei from polar bodies of "paternal" origin into other Met-II oocytes resulted in the formation of oocytes with two sets of Met-II chromosomes, one maternal and one paternal in origin. When activated, two pronuclei and two polar bodies were formed and zygotes began development. It is concluded that nuclei of primary spermatocytes are able to undergo two successive meiotic divisions within oocyte cytoplasm. Thus, factors that drive chromosome condensation, organization of metaphase, and chromosome separation at anaphase in oocytes can drive these same maturation processes in primary spermatocyte nuclei. When a total of 258 two-cell embryos were transferred to foster mothers, only two live pups were born to two mothers. One died shortly after birth and the other 3 wk after birth. The reasons for poor embryonic and neonatal development remain to be determined.

Animals↗

Mammalian male and female germ cells express a germ cell-specific Y-Box protein, MSY2.

Here we report the isolation and characterization of mouse testicular cDNAs encoding the mammalian homologue of the Xenopus germ cell-specific nucleic acid-binding protein FRGY2 (mRNP3+4), hereafter designated MSY2. MSY2 is a member of the Y box multigene family of proteins; it contains the cold shock domain that is highly conserved among all Y box proteins and four basic/aromatic islands that are closely related to the other known germline Y box proteins from Xenopus, FRGY2, and goldfish, GFYP2. Msy2 undergoes alternative splicing to yield alternate N-terminal regions upstream of the cold shock domain. Although MSY2 is a member of a large family of nucleic acid-binding proteins, Southern blotting detects only a limited number of genomic DNA fragments, suggesting that Msy2 is a single copy gene. By Northern blotting and immunoblotting, MSY2 appears to be a germ cell-specific protein in the testis. Analysis of Msy2 mRNA expression in prepubertal and adult mouse testes, and in isolated populations of germ cells, reveals maximal expression in postmeiotic round spermatids, a cell type with abundant amounts of stored messenger ribonucleoproteins. In the ovary, MSY2 is present exclusively in diplotene-stage and mature oocytes. MSY2 is maternally inherited in the one-cell-stage embryo but is not detected in the late two-cell-stage embryo. This loss of MSY2 is coincident with the bulk degradation of maternal mRNAs in the two-cell embryo.

Alternative Splicing↗

Factors affecting the developmental competence of mouse oocytes grown in vitro: follicle-stimulating hormone and insulin.

This study was undertaken to test the hypothesis that FSH treatment of cultured oocyte-granulosa cell complexes promotes acquisition of competence to complete preimplantation embryo development. Oocyte-granulosa cell complexes were isolated from the preantral follicles of 12-day-old mice and cultured for 10 days in serum-free medium, supplemented with insulin (5 microgram/ml), transferrin (5 microgram/ml), and selenium (5 ng/ml) and containing a highly potent preparation of FSH (0-5 ng/ml). Oocytes were matured and fertilized in vitro and embryos cultured to determine the frequency of development to the blastocyst stage. There was no effect of FSH on oocyte size, general morphology, or competence to resume meiosis. However, addition of FSH to medium containing insulin had a deleterious effect on the percentage of mature oocytes competent to develop to the blastocyst stage. Deletion of insulin from the medium for culture of oocyte-granulosa cell complexes prevented the deleterious effect of FSH, but FSH still did not promote acquisition of competence to complete preimplantation development. Culture of oocyte-granulosa cell complexes with FSH resulted in elevated expression of LH receptor (LHR) mRNA by granulosa cells and stimulated the production of functional LHRs, whether or not insulin was present. However, FSH-induced expression of LHR mRNA reached a maximum steady-state level by 4 days of culture in the presence of insulin, but this level was not reached until 10 days of culture without insulin. Granulosa cells encompassing growing mouse oocytes in vivo do not express LHR mRNA. Thus, expression of LHR mRNA by granulosa cells closely associated with growing oocytes in vitro indicates inappropriate or ambiguous development. In conclusion, conditions occurring during oocyte growth can have profound detrimental effects on oocyte developmental competence to complete preimplantation development, even when oocyte growth, general morphology, and competence to resume meiosis appear unaffected.

Animals↗

Sterols affecting meiosis: novel chemical syntheses and the biological activity and spectral properties of the synthetic sterols.

4,4-Dimethyl-5alpha-cholesta-8,14,24-trien-3beta-ol (I) from human follicular fluid and 4,4-dimethyl-5alpha-cholesta-8,24-dien-3beta-ol (II) from bull testes have been reported to activate meiosis in mouse oocytes (Byskov et al., 1995. Nature. 374: 559-562). Described herein are new chemical syntheses of I, II, and the delta8(14),24 analog XXII. A critical step in these syntheses was a remarkably high yield side chain oxidation of 3beta-acetoxy-4,4-dimethyl-5alpha-cholest-8(14)-en-15-one to the corresponding C24 24-hydroxy compound VI. Oxidation of VI to the aldehyde, followed by Wittig olefination gave 3beta -acetoxy4,4-dimethyl-5alpha-cholesta-8(14),24-dien-15- one. Reduction with sodium borohydride to the 15beta-hydroxysteryl ester, dehydration with sulfuric acid in CHCl3, and saponification furnished I in high purity. Reduction of VI with sodium borohydride to the 15-hydroxysteroid followed by dehydration gave 3beta-acetoxy-4,4-dimethyl-5alpha-chola-8,14-dien-24-o l. Hydrogenation over Raney nickel gave the monounsaturated delta8(14) and delta8 compounds. Oxidation to the corresponding aldehydes followed by Wittig olefination and saponification gave II and XXII. Chromatographic, mass spectral, and 1H and 13C nuclear magnetic resonance spectral data have been presented for the synthetic sterols and their derivatives. I, II, XXII, and their delta8,14 and delta7,14 analogs, at 3 microg per ml, caused a resumption of meiosis in mouse oocytes in the presence of hypoxanthine (3.5 mM). Under the same conditions, delta5 and delta5,7 sterols were inactive.

Animals↗

Parthenogenetic development of Mos-deficient mouse oocytes.

Ovarian teratomas develop in Mos-/- mutant mice produced by homologous recombination. These teratomas are probably derived from oocytes that undergo spontaneous parthenogenetic activation within the ovaries. However, it is not clear how the activated eggs develop into teratomas since embryonic development beyond the four-cell stage was not observed either in vitro or in vivo. In this study, Mos-/- parthenotes derived from in vitro-matured oocytes were cultured using a recently developed medium, KSOM/AA, which promotes a high frequency of preimplantation development by normal embryos. In total, 5% of the Mos-/- oocytes developed to the blastocyst stage. Preimplantation-like and early postimplantation-like embryos were observed in the ovaries of 60-63-day-old Mos-/- mice. These observations support the hypothesis that Mos-/- teratomas are derived from parthenogenetically activated oocytes that undergo early embryonic development up to early postimplantation-like stages within the ovaries. Aberrant meiotic divisions commonly observed in Mos-/- oocytes in vitro may adversely affect preimplantation development and reduce the frequency of blastocyst formation even under the best culture conditions.

Animals↗

Initiation of growth of baboon primordial follicles in vitro.

Factors that cause some primordial follicles to enter the growth phase while the others remain quiescent are unknown. The hypothesis was tested that primate primordial follicles can survive and initiate growth in vitro in serum-free medium. Superficial pieces of ovarian cortex, containing mostly primordial follicles, were obtained from baboon fetuses during late gestation and cultured for 0, 2, 4, 7, 10 or 20 days in Waymouth MB 752/1 medium supplemented with insulin, transferrin, selenium, linoleic acid, and bovine serum albumin (ITS +). Histological examination of cortical pieces revealed that after 2 and 4 days in culture, the total number of primordial follicles had decreased by 55 and 76% (P < 0.01) respectively, relative to day 0 of culture. This was associated with a sustained, 5- to 8-fold increase in total primary follicles (P < 0.01) beginning on day 2 of culture. There was also a gradual increase in the total number of early secondary and secondary follicles. The average diameter of follicles and oocytes increased gradually throughout culture for all follicular categories (P < 0.01), except secondary follicles and oocytes. Immunohistochemical localization of proliferating cell nuclear antigen (PCNA), a marker for cell proliferation and growth, showed that PCNA was generally absent in primordial follicles on day 0, but was observed after 2 or 4 days in culture in both granulosa cells and oocytes of most growing follicles. Comparison of cortical pieces cultured for 10 or 20 days with ITS + versus 10% fetal bovine serum (FBS) showed a more pronounced decrease in the numbers of primordial follicles and more primary, early secondary and secondary follicles in ITS + compared to FBS-treated cortical pieces (P < 0.01 at 20 days). These results show that primordial follicles from non-human primates can survive and develop to the secondary stage in vitro in serum-free conditions.

Animals↗

Murine oocytes suppress expression of luteinizing hormone receptor messenger ribonucleic acid by granulosa cells.

This study tested the hypothesis that murine oocytes participate in the establishment of granulosa cell phenotypic heterogeneity in preovulatory follicles. In these follicles, mural granulosa cells express LH receptors (LHR) and LHR mRNA, but expression of these molecules is low or undetectable in cumulus cells. Thus, the expression of LHR mRNA is a marker of the mural granulosa cell phenotype in preovulatory follicles. Cumulus cells expressed elevated steady-state levels of LHR mRNA when oocytes were microsurgically removed from oocyte-cumulus cell complexes, and this was prevented by paracrine factor(s) secreted by isolated oocytes. These factors also suppressed FSH-induced elevation of the level of LHR mRNA expression by mural granulosa cells isolated from small antral follicles, even when expression was augmented by culturing granulosa cells on components of basal lamina. Moreover, factor(s) secreted by oocytes suppressed the expression of LHR mRNA in mural granulosa cells isolated from preovulatory follicles already expressing elevated levels of these transcripts. The ability of oocytes to suppress the LHR mRNA expression by granulosa cells was developmentally regulated. Oocytes from preantral follicles and mature (metaphase II arrested) oocytes were less effective in suppressing expression than fully grown, germinal vesicle (GV)-stage oocytes. Furthermore, two-cell-stage embryos did not suppress LHR mRNA levels. Coculture of isolated oocytes with granulosa cells affected the synthesis of very few granulosa cell proteins detected by fluorography of two-dimensional gels after 35S-methionine labeling. Thus, oocyte suppression of FSH-induced LHR mRNA expression is specific in both the suppressing cell type and the effects on granulosa cells. It is concluded that the default pathway of granulosa cell differentiation produces the mural granulosa cell phenotype, as represented by the expression of LHR mRNA. This pathway is abrogated by oocytes. Thus, oocytes play a dominant role in establishing the fundamental heterogeneity of the granulosa cell population of preovulatory follicles.

1-Methyl-3-isobutylxanthine↗

Analysis of the mechanism(s) of metaphase I arrest in strain LT mouse oocytes: participation of MOS.

Oocytes of almost all vertebrates become arrested at metaphase II to await fertilization. Arrest is achieved with the participation of a protein complex known as cytostatic factor (CSF) that stabilizes histone H1 kinase activity. MOS and mitogen-activated protein kinase (MAPK) are important components of CSF. Strain LT/Sv mice, and strains related to LT/Sv, produce a high percentage of atypical oocytes that are arrested at metaphase I when normal oocytes have progressed to metaphase II. The potential role of MOS in metaphase I arrest was investigated using strain LT/Sv and LT-related recombinant inbred strains, LTXBO and CX8-4. MOS and MAPK are produced and functional in maturing LT oocytes. Two experimental paradigms were used to reduce or delete MOS in LT oocytes and assess effects on metaphase I arrest. First, sense and antisense Mos oligonucleotides were microinjected into metaphase I-arrested oocytes. Antisense, but not sense, Mos oligonucleotides promoted the activation of metaphase I-arrested oocytes. Second, mice carrying a Mos null mutation were crossed with LT mice, the null mutation was backcrossed three times to LT mice, and Mos(+/-) N3 mice were intercrossed to produce Mos(-/-), Mos(+/-) and Mos(+/+) N3F1 mice. Oocytes of all three Mos genotypes of N3F1 mice sustained meiotic arrest for 17 hours indicating that metaphase I arrest is not initiated by a MOS-dependent mechanism. However, unlike Mos(+/+) and Mos(+/-) CX8-4 N3F1 oocytes, metaphase I arrest of Mos(-/-) CX8-4 N3F1 oocytes was not sustained after 17 hours and became reversed gradually. These results, like the antisense Mos oligonucleotide microinjection experiments, suggest that MOS participates in sustaining metaphase I arrest in LT oocytes.

Animals↗

Oocyte control of granulosa cell development: how and why.

Interaction between oocytes and granulosa cells is complex and involves both gap junctions and paracrine signalling factors. Oocyte development in antral follicles is highly dependent on communication with cumulus cells, a subset of granulosa cells that is intimately associated with oocytes. Cumulus cells express characteristics distinct from the mural granulosa cells of preovulatory follicles. The thesis of this paper is that, without the influence of oocytes, the pathway of granulosa cell differentiation in antral follicles leads to the establishment of the mural granulosa cell phenotype. Oocytes in antral follicles abrogate that pathway of granulosa cell differentiation and promote the development of the cumulus cell phenotype. Oocytes may do this in order to control their own microenvironment by regulating differentiation of the supporting cells that are in direct communication with them. Possibly, some aspects of the mural granulosa cell phenotype are antagonistic to, or insufficient for, supporting the final stages of oocyte development. We present evidence that oocytes control their environment by suppressing differentiation of the mural granulosa cell phenotype and promoting differentiation of the cumulus cell phenotype. They achieve this suppression via the secretion of labile paracrine signalling factors. Errors in this regulatory mechanism, whether instigated by defects in the production of oocyte-derived ligands or granulosa cell responses to them, may result in the production of oocytes unable to undergo embryo development or that undergo abnormal follicular development.

Cell Differentiation↗

Cyclin D2 is an FSH-responsive gene involved in gonadal cell proliferation and oncogenesis.

THE D-type cyclins (D1, D2 and D3) are critical governors of the cell-cycle clock apparatus during the G1 phase of the mammalian cell cycle. These three D-type cyclins are expressed in overlapping, apparently redundant fashion in the proliferating tissues. To investigate why mammalian cells need three distinct D-type cyclins, we have generated mice bearing a disrupted cyclin D2 gene by using gene targeting in embryonic stem cells. Cyclin D2-deficient females are sterile owing to the inability of ovarian granulosa cells to proliferate normally in response to follicle-stimulating hormone (FSH), whereas mutant males display hypoplastic testes. In ovarian granulosa cells, cyclin D2 is specifically induced by FSH via a cyclic-AMP-dependent pathway, indicating that expression of the various D-type cyclins is under control of distinct intracellular signalling pathways. The hypoplasia seen in cyclin D2(-/-) ovaries and testes prompted us to examine human cancers deriving from corresponding tissues. We find that some human ovarian and testicular tumours contain high levels of cyclin D2 messenger RNA.

Animals↗

Genetic regulation of traits essential for spontaneous ovarian teratocarcinogenesis in strain LT/Sv mice: aberrant meiotic cell cycle, oocyte activation, and parthenogenetic development.

Strain LT/Sv female mice show a high frequency of spontaneous ovarian teratomas arising from parthenogenetically activated follicular oocytes. LT/Sv oocytes also arrest at metaphase of meiosis I, rather than progressing through to metaphase II, as do almost all fully grown oocytes from most other strains. We investigated a new set of recombinant inbred strains derived from BALB/c and C58 (the progenitor strains of LT/Sv) and crosses of these two progenitor strains and found that metaphase I arrest is necessary, but not sufficient, to cause parthenogenetic activation. Occurrence of progeny with phenotypes more extreme than either parent (transgressive variation) suggests that these traits are polygenic and that LT/Sv mice inherited a novel combination of permissive alleles from their progenitor strains. Absence of teratomas from some LT-related strains demonstrate that metaphase I arrest and parthenogenetic activation are not sufficient for teratoma formation and that additional permissive alleles are required for teratocarcinogenesis. Finally, segregation analysis of teratoma formation in these strains suggests that a single autosomal gene derived from C57BL/6J mice is responsible for the high tumor incidence in one of these strains, LTXBO. Together these results show that metaphase I arrest, parthenogenetic activation of oocytes, and teratoma formation are multigenic traits involving a modest number of permissive alleles.

Animals↗

Mammalian oocyte growth and development in vitro.

This paper is a review of the current status of technology for mammalian oocyte growth and development in vitro. It compares and contrasts the characteristics of the various culture systems that have been devised for the culture of either isolated preantral follicles or the oocyte-granulosa cell complexes form preantral follicles. The advantages and disadvantages of these various systems are discussed. Endpoints for the evaluation of oocyte development in vitro, including oocyte maturation and embryogenesis, are described. Considerations for the improvement of the culture systems are also presented. These include discussions of the possible effects of apoptosis and inappropriate differentiation of oocyte-associated granulosa cells on oocyte development. Finally, the potential applications of the technology for oocyte growth and development in vitro are discussed. For example, studies of oocyte development in vitro could help to identify specific molecules produced during oocyte development that are essential for normal early embryogenesis and perhaps recognize defects leading to infertility or abnormalities in embryonic development. Moreover, the culture systems may provide the methods necessary to enlarge the populations of valuable agricultural, pharmaceutical product-producing, and endangered animals, and to rescue the oocytes of women about to undergo clinical procedures that place oocytes at risk.

Animals↗

FSH and growth factors affect the growth and endocrine function in vitro of granulosa cells of bovine preantral follicles.

The hypothesis was tested that bovine preantral follicles can be stimulated to grow in vitro by FSH and by the mitogens, epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF), but not by transforming growth factor-beta (TGFbeta), which generally inhibits EGF and bFGF action. Preantral follicles, 60 to 179 mum in diameter, were isolated from fetal ovaries by treatment with collagenase and DNase and cultured for 6 d in serum-free medium, with or without FSH and growth factors. Basic FGF (50 ng/ml), and to a lesser extent FSH (100 ng/ml) and EGF (50 ng/ml), stimulated thymidine incorporation by granulosa cells in bovine preantral follicles compared to control cultures (8-, 4- and 2.5-fold the labeling index of the controls; P < 0.05). Alone TGFbeta (10 ng/ml) had no effect on (3)H-thymidine incorporation, but it completely inhibited the bFGF- but not the FSH-stimulated increase in the labeling index and mean follicular diameter of preantral follicles (P < 0.05). By the end of the culture period oocytes in most treatments had degenerated, and the few surviving oocytes were in preantral follicles cultured with FSH or bFGF. Progesterone accumulation was greater (P < 0.05) in the presence of FSH (100 ng/ml) or EGF (50 ng/ml) than with bFGF, TGFbeta or control medium. Basic FGF strongly inhibited the effect of FSH on progesterone secretion (P < 0.05). Only FSH stimulated the conversion of exogenous testosterone to estradiol and both bFGF and TGFbeta markedly inhibited FSH-stimulated estradiol accumulation. These results indicate that proliferation of granulosa cells of bovine preantral follicles can be stimulated by bFGF, FSH and EGF, whereas TGFbeta inhibits growth, and that they are steroidogenically active in culture. Basic FGF and TGFbeta antagonize FSH-stimulated steroid production by granulosa cells of cultured bovine preantral follicles.

Journal Article↗