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.

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.

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.

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.

Radiological "metamorphosis" in a patient with severe congenital osteogenesis imperfecta.

Congenital osteogenesis imperfecta (OI) was diagnosed by ultrasound in a 31-week-old fetus, and the diagnosis confirmed after delivery by caesarean section at week 36. The baby survived the neonatal period, but failed to thrive, had recurrent respiratory infections and ultimately died at 8 months. Cultured fibroblasts synthesized both normal type I collagen and unstable type I collagen harbouring a structural defect in the alpha 1 (I) cyanogen bromide-derived peptide number 8 (CB8) region of the molecule, indicating a heterozygous dominant mutation. At birth, the radiological picture was that of the "thin bone"-type of congenital OI (OI type IIB/III in the Sillence classification); at the age of 12 weeks ribs and long bones had undergone a marked expansion giving a very different picture, that of the "thick bone"-type congenital OI (OI type IIA). The mechanism responsible for this change in bone structure is not known, but fractures and callus formation are unlikely to be the only factors. Caution is needed in the interpretation of radiographs of newborns with OI for prognostic or genetic purposes.

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.

Comparison of recombinant growth differentiation factor-9 and oocyte regulation of KIT ligand messenger ribonucleic acid expression in mouse ovarian follicles.

Oocytes secrete factors that regulate the development of the surrounding granulosa cells in ovarian follicles. KIT ligand (KL) mRNA expression in granulosa cells is thought to be regulated by oocytes; however, the factor(s) that mediate this effect are not known. One candidate is the oocyte-specific gene product growth differentiation factor-9 (GDF-9). This study examined the effect of recombinant GDF-9 (rGDF-9) on steady-state KL mRNA expression levels in preantral and mural granulosa cells in vitro. Furthermore, the study compared the effect of rGDF-9 with that of coculture with oocytes at different developmental stages. As determined by RNase protection assay, both KL-1 and KL-2 mRNA levels in preantral and mural granulosa cells were suppressed by 25-250 ng/ml rGDF-9. Fully grown oocytes also suppressed both KL-1 and KL-2 mRNA expression levels. Partly grown oocytes isolated from 7-, 10-, or 12-day-old mice either had no effect on KL mRNA levels or promoted KL-1 mRNA steady-state expression. It is concluded that GDF-9 is likely to mediate the action of fully grown, but not partly grown, oocytes on granulosa cell KL mRNA expression.

Oocyte regulation of kit ligand expression in mouse ovarian follicles.

Kit ligand (KL), a product of granulosa cells in ovarian follicles, is a putative regulator of oocyte development. However, the factors that regulate KL mRNA levels in granulosa cells remain unclear. This study tested the hypothesis that oocytes regulate granulosa cell steady-state KL mRNA expression levels and that the characteristics of this regulation are dependent on the stage of growth and development of both oocytes and follicles. Levels of mRNA for the KL splice variants (KL-1 and KL-2) were shown to be high in granulosa cells from preantral follicles and then decline after follicular antrum formation. Preovulatory follicular development was associated with a dramatic increase in steady-state levels of KL-1 mRNA in mural granulosa but not cumulus cells. Regulation of these changes was examined in vitro using partly grown oocytes isolated from preantral follicles and fully grown oocytes isolated from preovulatory follicles. FSH increased the steady-state KL mRNA levels in preantral granulosa cells in vitro. Partly grown oocytes either increased or decreased KL mRNA levels in preantral granulosa cells depending on the absence or presence of FSH stimulation, respectively. Fully grown oocytes reduced the KL mRNA level in preantral granulosa cells and increased the ratio of KL-1 to KL-2 mRNA. In mural granulosa cell culture, FSH augmented testosterone-dependent elevation of the steady-state KL mRNA level, but had no effect alone. Fully grown oocytes reduced KL-2 but not KL-1 mRNA levels in mural granulosa cells treated with testosterone plus FSH, whereas fully grown oocytes reduced levels of both KL transcripts in cumulus cell culture. These effects of oocytes on steady-state KL mRNA expression levels in vitro explain the changes in granulosa cell KL mRNA levels observed during follicle development in vivo. The results therefore support the hypothesis that oocytes regulate granulosa cell kit ligand mRNA levels in a way that is characteristic of the stage of growth and development of the oocyte. Moreover, the results suggest that oocytes play a major role in promoting dynamic changes in gene expression by granulosa cells appropriate to the stage of follicular development.

Oosp1 encodes a novel mouse oocyte-secreted protein.

Oocyte-somatic cell communication is necessary for normal ovarian function. However, the identities of the majority of oocyte-secreted proteins remain unknown. A novel cDNA encoding mouse oocyte-secreted protein 1 (OOSP1) was identified using a modified subtractive hybridization screen. The Oosp1 cDNA encodes a 202-amino acid protein that contains a 21-amino acid signal peptide sequence, 5 putative N-linked glycosylation consensus sequences, and 6 cysteines that are predicted to form 3 disulfide bonds. OOSP1 shares amino acid identity with placental-specific protein 1 (PLAC1), a secreted protein expressed in the placenta and the ectoplacental cone. The Oosp1 mRNA is approximately 1.0 kb and is present at high levels in the oocytes of adult ovaries and at lower levels in the spleen. The mouse Oosp1 gene is 5 exons, spans greater than 16.4 kb, and localizes to chromosome 19 at a position that shares synteny with human chromosome 11q12-11q13. The identification of OOSP1 as a new oocyte-secreted protein permits future in vitro and in vivo functional analyses to define its role in ovarian folliculogenesis.

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.

[Osteopetrosis: description of 2 cases, non-familial, of the fatal infantile form and of a case of the mild adult form. Impossibility of performing early prenatal diagnosis]. / Osteopetrosi: descrizione di due casi, non famigliari, della forma infantile letale e di un caso della forma media dell'adulto. Impossibilità di effettuare diagnosi prenatale precoce.

Two non familial cases of the juvenile lethal form of osteopetrosis and one case of the mild adult type are reported. In one case at risk of lethal osteopetrosis an early prenatal diagnosis was attempted, after informing the parents about the extreme difficulty to obtain conclusive results by such investigation. Although several ultrasonographic and one radiological examinations showed an apparently normal foetus, the newborn was affected by the lethal form of osteopetrosis.

Phenotypic variability and abnormal type I collagen unstable at body temperature in a family with mild dominant osteogenesis imperfecta.

Autosomal dominant inheritance of a mild form of osteogenesis imperfecta (osteogenesis imperfecta type I) with different phenotypic expression was found in a family. Phenotypic expression was different for the affected mother and son, in the presence of the same biochemical results. Dermal fibroblast cultures synthesized normal and mutant type I collagen alpha chains. Collagen heterotrimers containing abnormal chains were overmodified along the entire triple helical domain and showed an unusually low denaturation temperature, so far found only in lethal cases. The mild phenotype in the family is probably due to the fact that abnormal type I collagen molecules are more likely to be degraded than utilized in the extracellular matrix.