Effect of in vitro maturation of mouse oocytes on the health and lifespan of adult offspring.

BACKGROUND: In vitro maturation of oocytes can, in some circumstances, provide an alternative approach to gonadotrophin-induced maturation in clinical settings. However, the consequences of these protocols on the long-term health of offspring are unknown. Here, the long-term health status and lifespans of offspring produced by in vitro maturation of mouse oocytes was compared with that of oocytes induced to mature in vivo using gonadotrophin treatment. METHODS: Mouse oocytes were matured in vitro using both an established optimized system and in the absence of amino acids to produce a suboptimal condition for maturation. Oocytes induced to mature in vivo with gonadotrophins constituted the control group. All metaphase II oocytes were fertilized in vitro and transferred at the 2-cell stage to the oviducts of pseudo-pregnant foster mothers for development to term. Offspring were subjected to a wide variety of physiological and behavioral tests for the first year of life and natural lifespan determined. RESULTS: There was no difference among the groups in lifespan or in most of the physiological and behavioral analyses. However, the pulse rate and cardiac output were slightly, but significantly, reduced in the optimized in vitro matured group compared with the in vivo matured group (P = 0.0119 and P = 0.0197, respectively). Surprisingly, these decreases were largely abrogated in the in vitro group matured without amino acids. CONCLUSIONS: Evidence presented here using a mouse model suggests that the in vitro maturation of oocytes has minimal effects on the long-term health of offspring. However, a finding of slight reductions in pulse rate and cardiac output may focus future clinical attention.

An epithelial cell line with chronic polyoma infection established from a spontaneous mouse pancreatic adenocarcinoma.

The establishment of an epithelial cell line from a mouse pancreatic adenocarcinoma is described. The cell line, designated LTPA, was aneuploid and exhibited many transformed growth properties (rapid growth rate, failure to show density-dependent inhibition of growth, ability to grow in defined medium). A type C oncornavirus was isolated from the culture medium, and electron microscopy also revealed the presence of intracisternal type A particles. LTPA cells carried a persistent polyoma infection which produced only low levels of cytopathic effects. A mycoplasmal contamination was also carried. When injected s.c. into Swiss nu/nu mice, LTPA cells formed ductular structures which were destroyed by inflammatory reactions within 3 weeks.

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.

Ovarian glycosaminoglycans: evidence for a role in regulating the response of the oocyte-cumulus cell complex to FSH.

The effects of sulfated glycosaminoglycans (GAGs) on FSH-stimulated cumulus expansion and hyaluronic acid (HA) synthesis were tested using cumuli oophori isolated from mice. It was found that heparin (Hep), heparin sulfate (HeS) and chondroitin sulfates (CSs) A,B, and C inhibited FSH-stimulated cumulus expansion and HA synthesis. The relative potencies of these GAGs in inhibiting HA synthesis was as follows: Hep greater than HeS congruent to CS-B greater than CS-C greater than CS-A. The GAGs did not block spontaneous oocyte meiotic maturation. It is suggested that the GAGs may function in vivo to block the response of the cumulus cells to FSH indigenous to the Graafian follicle prior to the preovulatory gonadotropin surge.

Cyclic adenosine monophosphate and ovarian follicular fluid act synergistically to inhibit mouse oocyte maturation.

The relationship between cAMP and the capacity of two partially-purified fractions of porcine follicular fluid (PFF) to suppress mouse oocyte maturation was investigated. The fractions used were: 1) a low molecular weight filtrate (less than 10,000) derived from Amicon PM10 filtration of PFF (PM10), and 2) a fraction from Bio-Gel P2 chromatography of the PM10 filtrate (Bio-Gel) that has meiotic inhibitory activity in porcine oocytes. PM10 alone produced a transient inhibition of the maturation of both cumulus cell-enclosed and denuded mouse oocytes, as manifested by germinal vesicle breakdown. The addition of FSH or (Bu)2cAMP to medium containing either of the PFF fractions resulted in dramatic synergism of the inhibitory effect of PFF alone in cumulus cell-enclosed oocytes. Forskolin or (Bu)2cAMP promoted a similar synergistic response with either PFF fraction in denuded oocytes. The degree of inhibition was consistently greater in cumulus cell-enclosed than in denuded oocytes. The putative inhibitor was dialyzable through tubing having a nominal molecular weight cutoff of 1,000. Proteolysis, acid hydrolysis, or ether extraction of PM10 did not reduce its inhibitory synergism with (Bu)2cAMP. However, inhibition was completely abolished by charcoal extraction. Steroid hormones did not mimic the PM10-induced synergism when added to (Bu)2cAMP-containing medium in a concentration at least 14-fold greater than that present in PM10-supplemented medium. In conclusion, our results demonstrate the presence of a factor(s) in PFF that acts synergistically with a cAMP-dependent process to inhibit oocyte maturation in vitro. Furthermore, although PFF fractions suppressed the maturation of both denuded and cumulus cell-enclosed oocytes, cumulus cells appear to mediate the inhibitory activity of PFF. The data also suggest that the PFF inhibitor is a small (mol wt less than 1,000) hydrophobic molecule but not a peptide or nonpolar lipid.

Regulation of prostaglandin-endoperoxide synthase 2 messenger ribonucleic acid expression in mouse granulosa cells during ovulation.

Normal ovulation in mice requires PG-endoperoxide synthase 2 (cyclooxygenase-2; COX-2) expression. This study examined the role of the oocyte and other factors in regulating steady state levels of COX-2 messenger RNA (mRNA) in granulosa cells. Multiphasic changes in the expression pattern of COX-2 mRNA were found, with peaks of expression 4 and 12 h after hCG treatment. Changes in relative expression levels in cumulus cells and mural granulosa cells occurred over time, with similar mRNA levels at 4 h, but higher levels in cumulus cells compared with mural granulosa cells at 8 and 12 h post-hCG. In cultured mural granulosa cells, LH, FSH, and oocytes promoted COX-2 mRNA expression concurrent with the first expression peak in vivo. At the same time, FSH, but not LH, treatment of cultured cumulus-oocyte complexes (COC) promoted COX-2 mRNA expression in cumulus cells. This response of cumulus cells to FSH treatment was largely dependent on the presence of either fully grown germinal vesicle stage or maturing oocytes, but not growing oocytes. At 8 h, COX-2 mRNA expression in FSH-stimulated COC was lower than at 4 h; however, oocyte coculture promoted COX-2 mRNA expression in cumulus cells. No second peak in expression occurred in cultured COC. However, coculture of COC with follicle walls promoted COX-2 mRNA expression in cumulus cells 12 h post-hCG; an effect augmented by oocytes. Therefore, the oocyte resident within ovulatory follicles produces a factor(s) that promotes expression of COX-2 mRNA by cumulus cells and possibly by mural granulosa cells. Thus, the oocyte probably plays an important role in promoting ovulation. However, the multiphasic changes in the pattern of COX-2 expression appear orchestrated by non-oocyte-derived factors.

New mouse genetic models for human contraceptive development.

Genetic strategies for the post-genomic sequence age will be designed to provide information about gene function in a myriad of physiological processes. Here an ENU mutagenesis program (http://reprogenomics.jax.org) is described that is generating a large resource of mutant mouse models of infertility; male and female mutants with defects in a wide range of reproductive processes are being recovered. Identification of the genes responsible for these defects, and the pathways in which these genes function, will advance the fields of reproduction research and medicine. Importantly, this program has potential to reveal novel human contraceptive targets.

Conditions that affect acquisition of developmental competence by mouse oocytes in vitro: FSH, insulin, glucose and ascorbic acid.

The simplest unit required for the support of oocyte growth and development is the oocyte-granulosa cell complex. Therefore, a culture system was established that utilizes these complexes to assess mechanisms promoting nuclear, cytoplasmic and genomic maturation in mammalian oocytes. Deletion of serum from the culture, results in increased apoptosis in oocyte-associated granulosa cells (OAGCs), however, addition of ascorbic acid (0.5 mM) significantly reduced the level of apoptosis in the OAGCs, although no improvement of oocyte developmental competence was detected. The effects of reducing glucose during oocyte growth were studied since, under some culture conditions, glucose has deleterious effects on early preimplantation development. Reducing the glucose concentration to 1 mM resulted in the production of oocytes with greatly reduced developmental competence. Deleterious effects of FSH plus insulin during oocyte growth in vitro on preimplantation development are reviewed and discussed in terms of the communication of oocytes with inappropriately developing granulosa cells. Evidence that oocytes promote the appropriate differentiation of OAGCs in intact follicles in vivo is also discussed. It is hypothesized that oocytes control the differentiation of these cells, in order to promote intercellular signaling essential for the acquisition of competence to undergo normal embryogenesis.

Coordination of nuclear and cytoplasmic oocyte maturation in eutherian mammals.

As oocytes near the end of their growth phase, they become competent to undergo two aspects of maturation, cytoplasmic and nuclear. Both are essential for the formation of an egg having the capacity for fertilization and development to live offspring. Nuclear maturation encompasses the processes reversing meiotic arrest at prophase I and driving the progression of meiosis to metaphase II. Cytoplasmic maturation refers to the processes that prepare the egg for activation and preimplantation development. This review focuses on the developmental programmes whereby oocytes at the germinal vesicle (GV) stage acquire competence to undergo nuclear and cytoplasmic maturation, the coordination of programmes regulating the acquisition of these competencies in GV-stage oocytes, and the coordination of the maturational processes themselves. Although the developmental programme of the GV-stage oocyte for acquiring competence to complete preimplantation development does not appear to be tightly linked to the acquisition of competence to complete nuclear maturation, GV breakdown (GVB) is probably essential for activating some critical aspects of cytoplasmic maturation, particularly those related to fertilization and activation. Nuclear and cytoplasmic maturation are normally coordinated by this mechanism requiring the mixing of the GV contents with the cytoplasm at the time of GVB, but some processes of cytoplasmic maturation related to successful preimplantation development probably still occur without coordination with nuclear maturation. Thus, continued differentiation of GV-stage oocytes is necessary after the acquisition of competence to undergo nuclear maturation, to allow for the deposition of the maternal factors required for the development of preimplantation embryos beyond the 2-cell stage.

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.

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.

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.

The role of purines in the maintenance of meiotic arrest in mouse oocytes.

Meiotic arrest of mammalian oocytes within ovarian follicles is maintained by a specific factor(s) within the follicle. There is strong evidence that cAMP plays an important role in the control of meiosis. Purines have also been implicated in the maintenance of meiotic arrest in vivo. Hypoxanthine and/or adenosine have been identified in pig and mouse follicular fluid and exert a meiosis-arresting action on mouse oocytes in culture. While adenosine apparently need not be metabolized to exert its action on oocyte maturation, the action of hypoxanthine is apparently due to the production of guanyl and/or xanthyl compounds by the oocyte-cumulus cell complex. The inosine monophosphate dehydrogenase inhibitors, mycophenolic acid and bredinin, induced maturation in cumulus cell-enclosed oocytes maintained in meiotic arrest by hypoxanthine. Hypoxanthine and adenosine are not toxic to oocytes, because oocytes undergo normal fertilization and pre- and post-implantation development following exposure to these molecules in vitro. It is not known how gonadotropins stimulate the resumption of meiosis within the follicle, but there are several possibilities: (1) the intrafollicular level of an oocyte maturation inhibitor is decreased; (2) the oocyte is uncoupled from surrounding follicle cells; (3) an inhibitory molecule is secreted or metabolized by the oocyte; and/or (4) a positive stimulus is produced by the follicle that overrides the presence of inhibitory molecules. Preliminary evidence suggests that cumulus cells may produce a positive stimulus that induces the maturation of cultured cumulus cell-enclosed oocytes. Whether germinal vesicle breakdown in vivo results from a positive induction, a loss of inhibitory input, or a combination of these two mechanisms remains to be determined.

Oocytes are required for the preantral granulosa cell to cumulus cell transition in mice.

Preantral granulosa cells (PAGCs) differentiate into cumulus cells following antrum formation. Cumulus cells, but not PAGCs, are competent to undergo expansion. Experiments reported here tested the respective roles of both oocytes and FSH in the transition of preantral granulosa cells to cumulus cells competent to undergo expansion. PAGC-oocyte complexes were cultured with or without a low dose of FSH (0.005 IU/ml) and isolated PAGCs were cultured with or without oocytes. At the end of culture, complexes or isolated PAGCs were tested for their ability to undergo cumulus expansion and upregulate expansion transcripts in response to EGF or FSH (0.5 IU/ml). The ability to undergo expansion in response to EGF required the presence of oocytes but not FSH during the culture period. Likewise, complexes isolated from the ovaries of hypogonadal mice, which lack circulating gonadotropins, underwent expansion in response to EGF, but not FSH. In contrast, the ability to activate MAPK3/1 and MAPK14 and undergo expansion in response to FSH required prior exposure to low doses of FSH. However, these low levels (0.005 or 0.025 IU FSH/ml) suppressed expression of Slc38a3 and Amh, two transcripts highly expressed in cumulus cells, suggesting opposing effects of FSH on cumulus cell differentiation. In conclusion, the ability to undergo expansion in response to FSH requires prior exposure to FSH during development, while oocyte-derived factors alone are sufficient to promote the ability to undergo expansion in response to EGF. These results highlight the crucial role of oocytes in driving the differentiation of PAGCs into cumulus cells during the preantral to antral follicle transition.