Control of mouse cumulus cell-oocyte complex integrity before and after ovulation: plasminogen activator synthesis and matrix degradation.

During the preovulatory period, cumulus cells (CCs) form a hyaluronan-protein extracellular matrix (cumulus expansion) that positively influences oocyte fertilization. Degradation of this matrix and CC-oocyte complex (COC) dissociation occurs within a few hours of ovulation and parallels the aging of oocytes. Modulation of CC proteolytic activity by gonadotropins and oocyte soluble factors has been hypothesized to determine such cumulus matrix changes. In the present study, we investigated plasminogen activator (PA) synthesis by COCs during the expansion and disassembly processes. Our results show that the secretion of tissue type PA and urokinase type PA (uPA) by oocytes and CCs, respectively, does not change significantly during expansion but dramatically increases thereafter. Compact COCs were isolated from immature mice, primed 48 h earlier with 5 IU PMSGs, and were induced to expand in vitro with 100 ng/ml FSH in the presence of 1% FCS. Full expansion was achieved at 16 h, when hyaluronan synthesis ceased. Release of hyaluronan and CCs from the COC matrix began between 18 and 20 h of culture, which indicates that matrix degradation started at this time. PA activities in culture media were determined by SDS-PAGE, followed by a zymography at various time intervals between 4 and 32 h of culture. Secreted tissue type PA and uPA activity abruptly increased between 16 and 20 h after FSH stimulation. Slot blot hybridization of CC messenger RNA showed that uPA messenger RNA levels correlated with the increase in uPA activity. Similar temporal patterns of PA synthesis and matrix degradation were found in COCs induced to expand in vivo by injection of 5 IU human CG into PMSG-primed mice. Cultures of CCs, both in the presence and absence of oocytes, revealed that uPA synthesis is repressed in FSH-stimulated CCs by an oocyte-soluble factor for the first 16 h of culture, whereas CC responsiveness to this factor is lost thereafter. In conclusion, the data show that a sophisticated interplay between oocyte and CCs causes the two cell types to simultaneously secrete PA activity after ovulation. The fact that matrix degradation parallels PA production strongly supports the hypothesis that these enzymes may destabilize the expanded COC matrix.

Urokinase redistribution from the secreted to the cell-bound fraction in granulosa cells of rat preovulatory follicles.

Plasminogen activators (PAs) have been shown to be synthesized in ovarian follicles of several mammalian species, where they contribute to the ovulation process. The type of PA secreted by granulosa cells is species-specific. In fact, whereas in the rat, gonadotropins stimulate tissue-type PA (tPA) production, the same hormonal stimulation induces urokinase PA (uPA) secretion in mouse cells. To investigate in more detail the hormonal regulation of this system, we used the rat ovary as a model in which we analyzed the production of PAs by theca-interstitial (TI) and granulosa cells obtained from preovulatory follicles after gonadotropin stimulation. In untreated rats, uPA was the predominant enzyme in both TI and granulosa cells. After hormonal stimulation, an increase in uPA and tPA activity was observed in both cell types. Surprisingly, only tPA mRNA increased in a time-dependent manner in both cell types, while uPA mRNA increased only in TI cells and actually decreased in granulosa cells. These divergent results between uPA enzyme activity and mRNA levels in granulosa cells were explained by studying the localization of the enzyme. Analysis of granulosa cell lysates showed that after hormonal stimulation, 60-70% of the uPA behaved as a cell-associated protein, suggesting that uPA, already present in the follicle, accumulates on the granulosa cell surface through binding to specific uPA receptors. The redistribution of uPA in granulosa cells and the differing regulation of the two PAs by gonadotropins in the rat ovary suggest that the two enzymes might have different functions during the ovulation process. Moreover, the ability of antibodies anti-tPA and anti-uPA to significantly inhibit ovulation only when coinjected with hCG confirmed that the PA contribution to ovulation occurs at the initial steps.

Targeted integration of adeno-associated virus-derived plasmids in transfected human cells.

Adeno-associated virus (AAV) integrates its genomic DNA into a defined region of human chromosome 19 (AAVS1). The specificity of integration is dependent on the presence of the inverted terminal repeats (ITR) and on expression of the rep gene. To develop vectors capable of targeting the insertion of a selected DNA sequence into a specific location of human chromosome, we determined whether the rep gene can mediate site-specific integration when cloned outside of an ITR-flanked transgene cassette. HeLa and Huh-7 cells were transfected with a plasmid containing the rep gene, as well as the green fluorescent protein (GFP) and neomycin (neo) resistance gene inserted between the ITRs of AAV. Southern blot analysis of individual clones detected Rep-mediated site-specific integration of the ITR-flanked DNA in 25% and 12% of the HeLa and Huh-7 clones, respectively. The localization of the GFP-Neo sequence on chromosome 19 also was confirmed by fluorescent in situ hybridization analysis of the transfected HeLa clones. Sequence analysis of the ITR-AAVS1 junction of one of the transfected Huh-7 clones indicated that the insertion of the ITR DNA fragment had occurred at nucleotide 1003. These results have implications for the development of AAV-derived vectors capable of directing the site-specific integration of a gene of interest.

The open duration of fetal ACh receptor-channel changes during mouse muscle development.

1. We performed an RNase protection assay on cultured C2C12 mouse myotubes, demonstrating that the gamma subunit of the fetal muscle acetylcholine receptor (AChR) exists as two splice variants, which differ in the presence of the amino terminal exon 5. 2. We studied unitary ACh-evoked events in fibres acutely dissociated from the hindlimb flexor digitorum brevis muscle of BALB/C mice aged between embryonic day 16 (E16) and postnatal day 6 (P6). 3. At all ages, the channel conductance was about 30 pS, typical of the fetal form of the AChR. The mean open time increased significantly from 6 ms at E16 to 9 ms at E19, then decreased to about 5 ms during the first postnatal week. The lengthening of the open time was considerably delayed in hypothyroid mice. Data were recorded at 24-26 degrees C. 4. On the basis of previously reported experiments in heterologous expression systems, we suggest that the modulation of channel open time is related to the expression of the AChR incorporating the gammas subunit. These events might be coupled to the crucial modifications in muscle innervation that take place during the same developmental period.

Production of plasminogen activator and plasminogen activator inhibitors by alveolar macrophages in control subjects and AIDS patients.

OBJECTIVE: To reveal a possible impairment of the plasminogen activator system in the pulmonary infections of AIDS patients. DESIGN: To test the plasminogen activator system functionality in alveolar macrophages and bronchoalveolar lavage fluid (BALF) in control subjects and AIDS patients. Procedures were designed to detect the presence of imbalance in plasminogen activator activity and to ascertain if this imbalance is due to a direct effect of the HIV virus on macrophages or to superimposed opportunistic infection. METHODS: Alveolar macrophages obtained by bronchoalveolar lavage (BAL) were either lysed with Triton X-100 or cultured for 24 h. Plasminogen activators and plasminogen activator inhibitors (PAI) were measured by chromogenic substrate assay and binding to 125I-urokinase followed by 10% sodium dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE), respectively. RESULTS: Plasminogen activator activity in BALF and in alveolar macrophages from AIDS patients was decreased. This reduction was independent of the presence of an infectious pulmonary process. In contrast, free PAI was increased in AIDS patients with Pneumocystis carinii infection. This increase is possibly caused by a different glycosylated form of PAI-2. CONCLUSIONS: Our data support the view that the pulmonary fibrogenic response is in part secondary to an imbalance within the plasminogen activator system and provide the basis for clarifying the role of these alterations in the pathophysiology of AIDS-related pulmonary infections.

Mouse Zp1 encodes a zona pellucida protein homologous to egg envelope proteins in mammals and fish.

Zp1 encodes one of the three major glycoproteins of the zona pellucida, an extracellular matrix that surrounds growing oocytes, ovulated eggs, and preimplantation embryos. The mouse gene is composed of 12 exons ranging in size from 82 to 364 base pairs and spans 6.5 kilobase pairs on chromosome 19 (2.13 +/- 1.5 centimorgans distal to D19Bir1). The Zp1 exon map is similar to ZPB, a human orthologue, and an E-box (CANNTG), implicated in oocyte-specific gene expression of mouse Zp2 and Zp3, is similarly located upstream of the transcription start site. The single copy Zp1 gene encodes a 623-amino acid protein, the carboxyl-terminal half of which is significantly similar to a corresponding region of mouse ZP2. The conservation of this same region in a fish egg envelope protein suggests that not only has this protein domain been duplicated in mammals but that it has been conserved and used as an egg envelope protein in species that diverged 650 million years ago.

Coordinate expression of the three zona pellucida genes during mouse oogenesis.

The mammalian zona pellucida is an extracellular matrix that surrounds growing oocytes, ovulated eggs and early embryos. The mouse zona is composed of three sulfated glycoproteins: ZP1, ZP2 and ZP3. Each is critically involved in fertilization, the postfertilization block to polyspermy and protection of the preimplantation embryo. We have previously isolated cDNAs encoding mouse ZP2 and ZP3 and now report the isolation of a full-length cDNA encoding ZP1. Mouse ZP1 is composed of a 623 amino acid polypeptide chain with a signal peptide and a carboxyl terminal transmembrane domain, typical of all zona proteins. Sequence comparison demonstrate that mouse ZP1 is an orthologue of a rabbit zona protein, R55. The expression of R55 has been reported previously in both oocytes and granulosa cells. However, by northern analysis and in situ hybridization with 33P-labelled antisense probes to each of the three mouse zona mRNAs, we have determined that the expression of each mouse zona gene is restricted to the oocyte. ZP2 transcripts, but not ZP1 or ZP3, are detected in resting (15 microns diameter) oocytes, and all three zona transcripts coordinately accumulate as oocytes begin to grow. Together they represent approximately 1.5% of the total poly(A)+ RNA in 50-60 microns oocytes. In the latter stages of oogenesis, their abundance declines and each zona transcript is present in ovulated eggs at less than 5% of its maximal level. No zona transcripts were detected above background signal in granulosa cells. We conclude that, in mice, the three zona pellucida genes are expressed in a coordinate, oocyte-specific manner during the growth phase of oogenesis. Our data support the hypothesis that the transcription of the zona genes is controlled, in part, by shared regulatory element(s).

Mouse oocytes inhibit plasminogen activator production by ovarian cumulus and granulosa cells.

Following the preovulatory surge of gonadotropins, the compact layer of cumulus cells in the antral follicle secretes a hyaluronic acid-enriched extracellular matrix and undergoes a morphological change referred to as cumulus expansion. It has been previously shown that a soluble factor(s) produced by the oocyte is required, in combination with FSH, to promote this process. Since such matrix is sensitive to proteases we have now studied the effect of the oocyte on another gonadotropin-controlled follicle cell function, i.e., the synthesis of plasminogen activator (PA). Our data indicate that isolated cumulus cells secrete uPA in the medium and that FSH or dbcAMP increases this production. The presence of the oocyte or the oocyte-conditioned medium greatly reduces uPA synthesis induced by FSH and dbcAMP in cumulus cells by modulating the abundance of its mRNA. The ability of the mouse oocyte to produce such a factor(s) is dependent upon its stage of development, with fully grown oocytes but not growing oocytes or two-cell embryos being able to inhibit uPA synthesis. A preliminary characterization of this factor suggests that it is a heat-unstable protein with an apparent molecular weight above 100 kDa. Thus, the mouse oocytes appear to promote preovulatory matrix accumulation that occurs just prior ovulation by modulating the gonadotropin action on both the synthesis and the degradation of specific matrix component.

Biology and structure of the zona pellucida: a target for immunocontraception.

Although reversible interference of sperm-egg interactions with pharmacological agents has not yet been achieved, animal models have provided increasing evidence that immunological reagents directed against mammalian gametes can effectively inhibit fertilization. One potential target of immunocontraception is the zona pellucida, an extracellular matrix that surrounds the growing oocyte and ovulated egg. Recent advances in our knowledge of the biosynthesis and molecular biology of the zona pellucida have provided much information useful in the rational design of immunocontraceptive vaccines. There remain, however, major obstacles to using immunological reagents to prevent fertilization, including potential toxic side effects, the lack of adequate delivery systems and the possibility of incomplete reversibility. This review summarizes current understanding of the production of the zona pellucida during folliculogenesis, the structure of the conserved proteins and genes in the zona pellucida, and the progress made in the development of immunocontraceptive strategies that focus on this oocyte-specific structure.

In vitro production of plasminogen activator by human granulosa cells.

Plasminogen activator (PA) production by granulosa cells has been demonstrated in several species. In the human ovary, tissue-type PA and urokinase-type PA antigens have been found in the follicular fluids, but neither PA activity nor mRNA for both enzymes was found in granulosa cells of preovulatory follicles. All of these studies were performed on granulosa cells collected from follicles immediately before ovulation, when the cells were already in the luteal phase. In the attempt to better characterize the PA/plasminogen system in the human ovary, we examined PA and PA inhibitor (PAI) production in cultures of granulosa cells obtained from normally cycling untreated women at different stages of the cycle. In addition, we analyzed granulosa-luteal cells obtained from hormonally stimulated women undergoing gamete intrafallopian tube transfer, as a model of late phase follicular development. Zymographic analysis as well as immunoprecipitation with specific antisera revealed that granulosa cells from follicles at early phases of antral stages secreted high levels of PA of the urokinase type in the medium. No free tissue-type PA activity was found in any of the examined samples. On the contrary, free PAI was undetectable in medium obtained from granulosa cell cultures, and it was abundant in granulosa-luteal cell cultures, where it was found in two forms. These data show that in the human ovary as in that of the rat, PAs and PAIs are tightly time regulated. The timing of PA production in human granulosa cells suggests a role for PA activity at early stages of follicular maturation.

In vitro production of estradiol by ovarian granulosa cells in a case of McCune-Albright syndrome.

A 14-year-old girl presented with classic McCune-Albright syndrome. She underwent the ablation of several cysts during a laparotomy performed on the basis of persistent and intense pelvic pain and recurrent episodes of menometrorrhagia not responsive to pharmacologic therapy. Granulosa cells obtained from an isolated follicle and a cyst were cultured and estradiol (E2) secreted in the culture medium measured. Granulosa cells, obtained from the follicle, produced much higher levels of estradiol compared to those of cells coming both from follicles of equivalent size and preovulatory follicles of normal patients. Secretion of E2 by granulosa cells from the cyst was comparable to that of normal preovulatory follicles. We conclude that in this patient, ovaries are hyperfunctioning in terms of E2 production. This high production of estradiol and the fact that several cysts were found in the ovaries can justify the high levels of estradiol found in the serum of this patient at the moment of the operation.