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In preovulatory follicles, after the endogenous gonadotropin surge, the oocyte-cumulus complexes (OCCs) produce hyaluronan (HA) in a process called "cumulus expansion". During this process, the heavy chains (HCs) of the serum-derived inter-alpha-trypsin inhibitor (IαI) family bind covalently to synthesized HA and form a unique structure of the expanded cumulus HA-rich extracellular matrix. Understanding the biochemical mechanism of the covalent linkage between HA and the HCs of the IαI family is one of the most significant discoveries in reproductive biology, since it explains basis of the cumulus expansion process running in parallel with the oocyte maturation, both essential for ovulation. Two recent studies have supported the above-mentioned findings: in the first, seven components of the extracellular matrix were detected by proteomic, evolutionary, and experimental analyses, and in the second, the essential role of serum in the process of cumulus expansion in vitro was confirmed. We have previously demonstrated the formation of unique structure of the covalent linkage of HA to HCs of IαI in the expanded gonadotropin-stimulated OCC, as well as interactions with several proteins produced by the cumulus cells: tumor necrosis factor-alpha-induced protein 6, pentraxin 3, and versican. Importantly, deletion of these genes in the mice produces female infertility due to defects in the oocyte-cumulus structure., (© 2024 Eva Nagyova et al., published by Sciendo.)

The presence of multiple pathogenic variants in desmosomal genes (DSC2, DSG2, DSP, JUP, and PKP2) in patients with arrhythmogenic right ventricular cardiomyopathy (ARVC) has been linked to a severe phenotype. However, the pathogenicity of variants is reclassified frequently, which may result in a changed clinical risk prediction. Here, we present the collection, reclassification, and clinical outcome correlation for the largest series of ARVC patients carrying multiple desmosomal pathogenic variants to date (n = 331). After reclassification, only 29% of patients remained carriers of two (likely) pathogenic variants. They reached the composite endpoint (ventricular arrhythmias, heart failure, and death) significantly earlier than patients with one or no remaining reclassified variant (hazard ratios of 1.9 and 1.8, respectively). Periodic reclassification of variants contributes to more accurate risk stratification and subsequent clinical management strategy. Graphical Abstract., (© 2023. The Author(s).)

Histopathological studies have revealed key processes of atherosclerotic plaque thrombosis. However, the diversity and complexity of lesion types highlight the need for improved sub-phenotyping. Here we analyze the gene expression profiles of 654 advanced human carotid plaques. The unsupervised, transcriptome-driven clustering revealed five dominant plaque types. These plaque phenotypes were associated with clinical presentation and showed differences in cellular compositions. Validation in coronary segments showed that the molecular signature of these plaques was linked to coronary ischemia. One of the plaque types with the most severe clinical symptoms pointed to both inflammatory and fibrotic cell lineages. Further, we did a preliminary analysis of potential circulating biomarkers that mark the different plaques phenotypes. In conclusion, the definition of the plaque at risk for a thrombotic event can be fine-tuned by in-depth transcriptomic-based phenotyping. These differential plaque phenotypes prove clinically relevant for both carotid and coronary artery plaques and point to distinct underlying biology of symptomatic lesions., Competing Interests: CLM has received funding support from AstraZeneca for work unrelated to this study. GP received funding support from Roche to partly cover the generation of biomarker data. The remaining authors declare no competing interests.

In the mammalian ovary, the hyaluronan (HA)-rich cumulus extracellular matrix (ECM) organized during the gonadotropin-induced process of oocyte maturation is essential for ovulation of the oocyte-cumulus complex (OCC) and fertilization. Versican is an HA-binding proteoglycan that regulates cell function and ECM assembly. Versican cleavage and function remain to be determined in ovarian follicle. We investigated versican expression in porcine ovarian follicles by real-time (RT)-PCR and western blotting. The aims of the present work were to determine whether 1) versican was produced and cleaved by porcine OCCs during gonadotropin stimulation; 2) these processes were autonomous or required the participation of mural granulosa cells (MGCs); and 3) versican cleavage was involved in the formation or degradation of expanded cumulus ECM. We demonstrate two cleavage products of G1 domain of versican (V1) accumulated in the HA-rich cumulus ECM. One of them, a G1-DPEAAE N-terminal fragment (VG1) of ~70 kDa, was generated from V1 during organization of HA in in vivo and in vitro expanded porcine OCCs. Second, the V1-cleaved DPEAAE-positive form of ~65 kDa was the only species detected in MGCs. No versican cleavage products were detected in OCCs cultured without follicular fluid. In summary, porcine OCCs are autonomous in producing and cleaving V1; the cleaved fragment of ~70 kDa VG1 is specific for formation of the expanded cumulus HA-rich ECM., Competing Interests: The authors declare no conflicts of interest.

Although massively parallel sequencing (MPS) is becoming common practice in both research and routine clinical care, confirmation requirements of identified DNA variants using alternative methods are still topics of debate. When evaluating variants directly from MPS data, different read depth statistics, together with specialized genotype quality scores are, therefore, of high relevance. Here we report results of our validation study performed in two different ways: 1) confirmation of MPS identified variants using Sanger sequencing; and 2) simultaneous Sanger and MPS analysis of exons of selected genes. Detailed examination of false-positive and false-negative findings revealed typical error sources connected to low read depth/coverage, incomplete reference genome, indel realignment problems, as well as microsatellite associated amplification errors leading to base miss-calling. However, all these error types were identifiable with thorough manual revision of aligned reads according to specific patterns of distributions of variants and their corresponding reads. Moreover, our results point to dependence of both basic quantitative metrics (such as total read counts, alternative allele read counts and allelic balance) together with specific genotype quality scores on the used bioinformatics pipeline, stressing thus the need for establishing of specific thresholds for these metrics in each laboratory and for each involved pipeline independently., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Objectives: For the first time we used targeted next-generation sequencing to detect candidate pathogenic variants in Slovak cardiomyopathy patients., Background: Targeted next-generation sequencing is considered to be the best practice in genetic diagnostics of cardiomyopathies. However, in Slovakia, with high cardiomyopathies prevalence of 1/440, the current diagnostic tests are still based on Sanger sequencing of a few genes. Consequently, little is known about the exact contribution of pathogenic variants in known cardiomyopathy genes in Slovak patients., Methods: We used a panel of 46 known cardiomyopathy-associated genes to detect genetic variants in 16 Slovak cardiomyopathy patients (6 dilated, 8 hypertrophic, 2 non-compaction subtypes)., Results: We identified candidate pathogenic variants in 11 of 16 patients (69 %). Genes with higher count of candidate pathogenic variants were MYBPC3, MYH and TTN, each with 3 different variants. Seven variants ACTC1 (c.329C>T), ANKRD1 (c.683G>T), MYH7 (c.1025C>T), PKP2 (c.2003delA), TTN (c.51655C>T, c.84841G>T, c.101874_101881delAGAATTTG) have been detected for the first time and might represent Slovak-specific genetic cause., Conclusions: We have performed genetic testing of previously untested Slovak cardiomyopathy patients using next-generation sequencing cardiomyopathy gene panel. Given the high percentage of candidate pathogenic variants it should be recommended to implement this method into routine genetic diagnostic practice in Slovakia (Tab. 4, Ref. 39).

Fertilization of the mammalian oocyte requires interactions between spermatozoa and expanded cumulus extracellular matrix (ECM) that surrounds the oocyte. This review focuses on key molecules that play an important role in the formation of the cumulus ECM, generated by the oocyte-cumulus complex. In particular, the specific inhibitors (AG1478, lapatinib, indomethacin and MG132) and progesterone receptor antagonist (RU486) exerting their effects through the remodeling of the ECM of the cumulus cells surrounding the oocyte have been described. After gonadotropin stimulus, cumulus cells expand and form hyaluronan (HA)-rich cumulus ECM. In pigs, the proper structure of the cumulus ECM depends on the interaction between HA and serum-derived proteins of the inter-alpha-trypsin inhibitor (IαI) protein family. We have demonstrated the synthesis of HA by cumulus cells, and the presence of the IαI, tumor necrosis factor-alpha-induced protein 6 and pentraxin 3 in expanding oocyte-cumulus complexes (OCC). We have evaluated the covalent linkage of heavy chains of IαI proteins to HA, as the principal component of the expanded HA-rich cumulus ECM, in porcine OCC cultured in medium with specific inhibitors: AG1478 and lapatinib (both inhibitors of epidermal growth factor receptor tyrosine kinase activity); MG132 (a specific proteasomal inhibitor), indomethacin (cyclooxygenase inhibitor); and progesterone receptor antagonist (RU486). We have found that both RU486 and indomethacin does not disrupt the formation of the covalent linkage between the heavy chains of IαI to HA in the expanded OCC. In contrast, the inhibitors AG1478 and lapatinib prevent gonadotropin-induced cumulus expansion. Finally, the formation of oocyte-cumulus ECM relying on the covalent transfer of heavy chains of IαI molecules to HA has been inhibited in the presence of MG132., Competing Interests: The author declares no conflict of interest.

Bone morphogenetic protein-15 (BMP-15), an oocyte-derived growth factor, has been shown to play integral roles in regulation of ovarian follicular function in mammals. Despite the recognition of the physiological importance of the BMP system in regulation of gonadotropin action in the ovary, molecular mechanisms of BMP-15 effect on oocyte and somatic follicular cell functions remain poorly understood. The objective of this study was to determine the effect of BMP-15 on the FSH/LH-stimulated synthesis of hyaluronan (HA) by oocyte cumulus complexes (OCC) and progesterone by OCC and granulosa cells (GC) in the presence or absence of serum using primary porcine cultures. In addition, the effect of BMP-15 on oocyte maturation- and steroidogenesis-related transcripts after 4, 8, 16, and 24 hours of cultivation was evaluated using real-time RT-PCR. We demonstrated that the FSH/LH-induced cumulus expansion was accompanied by a significant increase in CD44, PTGS2, CYP11A1 (at 4 h) and AREG, HAS2, TNFAIP6, STAR (at 8 h) mRNAs. While FSH/LH-stimulated total HA synthesis by OCC was not affected by BMP-15 in serum-supplemented medium, its retention within the complex was significantly increased after the action of BMP-15 in comparison to FSH/LH alone (P < 0.001; 65% versus 35%, respectively). Moreover, we detected a significant increase in the expression of AREG and TNFAIP6 (both at 16 h), and CYP11A1 (at 24 h) in FSH/LH-stimulated OCC due to the action of BMP-15 compared to complexes cultured only with FSH/LH. In the presence of serum, BMP-15 markedly increased FSH/LH-stimulated progesterone secretion by OCC (about 69%) and induced a significant decrease in FSH/LH-induced progesterone release by GC (about 35%) compared to FSH/LH alone. The present results indicate that the addition of BMP-15 to the gonadotropin-stimulated OCC cultured in serum-supplemented medium might improve oocyte-cumulus maturation.

Objectives: The aims of this study were to test the utility of benchtop NGS platforms for NIPT for trisomy 21 using previously published z score calculation methods and to optimize the sample preparation and data analysis with use of in silico and physical size selection methods., Methods: Samples from 130 pregnant women were analyzed by whole genome sequencing on benchtop NGS systems Ion Torrent PGM and MiSeq. The targeted yield of 3 million raw reads on each platform was used for z score calculation. The impact of in silico and physical size selection on analytical performance of the test was studied., Results: Using a z score value of 3 as the cut-off, 98.11%-100% (104-106/106) specificity and 100% (24/24) sensitivity and 99.06%-100% (105-106/106) specificity and 100% (24/24) sensitivity were observed for Ion Torrent PGM and MiSeq, respectively. After in silico based size selection both platforms reached 100% specificity and sensitivity. Following the physical size selection z scores of tested trisomic samples increased significantly--p = 0.0141 and p = 0.025 for Ion Torrent PGM and MiSeq, respectively., Conclusions: Noninvasive prenatal testing for chromosome 21 trisomy with the utilization of benchtop NGS systems led to results equivalent to previously published studies performed on high-to-ultrahigh throughput NGS systems. The in silico size selection led to higher specificity of the test. Physical size selection performed on isolated DNA led to significant increase in z scores. The observed results could represent a basis for increasing of cost effectiveness of the test and thus help with its penetration worldwide.

Objectives: Different applications of high-resolution melting (HRM) analysis have been adopted for a wide range of research and clinical applications. This study compares the performance of selected DNA binding fluorescent dyes for their possible application in HRM., Design and Methods: We compared twelve dyes with basic properties considered relevant for PCR amplification and melting curve analysis. These included PCR inhibition, fluorescence intensity, the ability to generate melting curves and their effect on melting temperature (Tm). Seven of these dyes with promising properties were then evaluated for possible use in basic HRM applications; such as small amplicon genotyping, genotyping of a 1 kb insertion/deletion polymorphism, probe-based genotyping and mutation screening., Results: Five dyes failed to exhibit promising properties during the first part of the study, and these were excluded from further testing. Of the remaining dyes, SYTO11, SYTO13 and SYTO16 showed better PCR inhibitory and Tm affecting properties compared to commercial HRM dyes LCGreen Plus, EvaGreen and ResoLight. Although the SYTO dyes generally exhibited good discrimination powers in HRM applications, SYTO11 and SYTO14 gave low signal intensity and lower quality results., Conclusions: Our results suggest that the best performing dyes for HRM are those commercially offered for HRM analyses. However, the performance of SYTO16 and SYTO13 was comparable to the HRM dyes in the majority of our assays, thus demonstrating that they are also quite suitable for both real-time PCR and HRM applications., (Copyright © 2015 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)

Objective: To determine whether inhibition of epidermal growth factor (EGF) receptor tyrosine kinase with lapatinib affects oocyte maturation, expression of the cumulus expansion-associated genes such as tumor necrosis factor alpha-induced protein 6 (TNFAIP6) and prostaglandin-endoperoxide synthase 2 (PTGS2), and synthesis of hyaluronan (HA) and progesterone (P) by porcine oocyte cumulus complexes (OCC)., Design: Our work focuses on lapatinib, an orally active small molecule that selectively inhibits the tyrosine kinase domain of both EGF receptor and human EGF receptor 2, and downstream signaling., Setting: A reproductive biology laboratory., Patient(s): Not applicable., Intervention(s): Porcine OCC were cultured in vitro in a medium with FSH/LH in the presence/absence of lapatinib., Main Outcome Measure(s): Methods performed: real-time reverse transcriptase-polymerase chain reaction (PCR), immunofluorescence, RIA., Result(s): In FSH/LH-stimulated and expanded cumulus oophorus extracellular matrix, HA was detected with biotinylated HA-binding proteins. However, weaker HA- and weaker cytoplasmic TNFAIP6 were detected were detected in lapatinib-pretreated OCC. The expression of the two cumulus expansion-associated gene transcripts was significantly decreased and synthesis of HA by cumulus cells was reduced. Lapatinib (10 μM) inhibited FSH/LH-induced oocyte meiotic maturation. Progesterone production increased after OCC stimulation with FSH/LH and was significantly decreased by lapatinib (10 μM)., Conclusion(s): Lapatinib inhibits oocyte maturation and reduces expression of cumulus expansion-associated transcripts, and synthesis of HA and P in OCC cultured in vitro in FSH/LH-supplemented medium., (Copyright © 2013 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

Several lines of evidence suggest that in mice the activation of SMAD2/3 signaling by oocyte secreted factors, together with epidermal growth factor receptor (EGFR) activation, is essential to induce cumulus expansion. Here we show that inhibition of EGFR kinase in follicle stimulating hormone (FSH)-stimulated porcine oocyte-cumulus cell complex (OCCs) strongly decreases hyaluronan (HA) synthesis and its retention in the matrix, as well as progesterone synthesis. Although porcine cumulus cells undergo expansion independently of oocytes, we use biochemical and gene expression analyses to show that they do require activation of SMAD2/3 for optimal stimulation of HA synthesis and proteins involved in the organization of this polymer in the expanded matrix. Furthermore, FSH-induced progesterone synthesis by porcine cumulus cells was increased by blocking SMAD2/3 activation. In conclusion, these results support the hypothesis that an FSH-EGF autocrine loop is active in porcine OCCs, and provide the first evidence that the SMAD2/3 signaling pathway is induced by paracrine/autocrine factors in porcine cumulus cells and is involved in the control of both cumulus expansion and steroidogenesis., (Copyright © 2011 Wiley-Liss, Inc.)

It has been shown previously that tumor necrosis factor alpha-induced protein 6 (TNFAIP6) is essential for formation of the cumulus extracellular matrix and female fertility. Therefore, we studied the expression of TNFAIP6 mRNA in porcine preovulatory follicles. In addition, we asked whether the expression of TNFAIP6 mRNA changes in mural granulosa cells (MGCs) during the periovulatory period or after culture of oocyte-cumulus complexes (OCCs) or MGCs in vitro. Mural granulosa cells obtained from follicles on day 12 (D 12) and day 15 (D 15) of the estrous cycle, eCG-stimulated follicles, follicles at 4-32 h after hCG stimulation and MGCs and OCCs obtained from immature gilts and cultured for 0-44 h in vitro with gonadotropins were used for extraction of total RNA and assessment of the relative abundance (RA) of TNFAIP6 mRNA by reverse transcription-polymerase chain reaction (RT-PCR). The levels of TNFAIP6 mRNA were low in the follicles on D 12 and D 15 of the estrous cycle and at 66 h after eCG stimulation but were significantly increased at 4 h after hCG. The high level of TNFAIP6 expression was maintained until 16 h after hCG stimulation and gradually decreased at 24 and 32 h after hCG. During in vitro culture, FSH/LH-induced TNFAIP6 mRNA was expressed in both OCCs and MGCs in a similar temporal pattern as seen in vivo. We conclude that TNFAIP6 expression in the pig, like other species, increases in preovulatory follicles following the LH (hCG) surge. The OCC and MGC display similar patterns of TNFAIP6 expression under both in vivo and in vitro conditions.

We have previously shown that the heavy chains (HCs) of inter-alpha-trypsin inhibitor (IalphaI) become covalently linked to hyaluronan (HA) during in vivo and in vitro expansion of porcine oocyte-cumulus cell complexes (OCCs). We have now studied by immunoblotting the synthesis of tumor necrosis factor alpha-induced protein 6 (TNFAIP6), which is essential for catalyzing this reaction in expanding mouse OCCs. Expanding OCCs were collected from preovulatory follicles of naturally cycling pigs and also after in vitro culture (24 or 42 h) in medium supplemented with FSH and pig serum. After isolation, OCCs were treated with Streptomyces hyaluronidase or Chondroitinase ABC. Matrix, cell pellet, and total extracts were analyzed by Western blotting. A band of about 35 kDa and a doublet of about 120 kDa, corresponding to the molecular weight of the native and HC-linked forms of TNFAIP6, respectively, were detected by a rabbit anti-human TNFAIP6 polyclonal antibody in matrix extracts of expanded cumuli. Moreover, we found by using a cell-free assay that porcine follicular fluid collected from follicles at 24 h after hCG stimulation contains HC-HA coupling activity. This activity was abolished by the rat anti-human monoclonal antibody A38, which has an epitope within the Link module domain of TNFAIP6. These experiments suggest that free TNFAIP6 protein was present in follicular fluid aspirated from porcine follicles 24 h after hCG stimulation. In contrast to mouse, we show that the A38 monoclonal antibody does not affect in vitro cumulus expansion of porcine OCCs.

The resumption of oocyte meiosis in mammals encompasses the landmark event of oocyte germinal vesicle (GV) breakdown (GVBD), accompanied by the modification of cell-to-cell communication and adhesion between the oocyte and surrounding cumulus cells. The concomitant cumulus expansion relies on microfilament-cytoskeletal remodeling and extracellular matrix (ECM) deposition. We hypothesized that this multifaceted remodeling event requires substrate-specific proteolysis by the ubiquitin-proteasome pathway (UPP). We evaluated meiotic progression, cytoskeletal dynamics, and the production of cumulus ECM in porcine cumulus-oocyte complexes (COCs) cultured with or without 10-200 microM MG132, a specific proteasomal inhibitor, for the first 22 h of in vitro maturation, followed by 22 h of culture with or without MG132. Treatment with 10 microM MG132 arrested 28.4% of oocytes in GV stage (vs. 1.3% in control), 43.1% in prometaphase I, and 16.2% in metaphase I, whereas 83.7% of control ova reached metaphase II (0% of MG132 reached metaphase II). The proportion of GV-stage ova increased progressively to >90% with increased concentration of MG132 (20-200 microM). Furthermore, MG132 blocked the extrusion of the first polar body and degradation of F-actin-rich transzonal projections (TZP) interconnecting cumulus cells with the oocyte. The microfilament disruptor cytochalasin E (CE) prevented cumulus expansion but accelerated the breakdown of TZPs. Ova treated with a combination of 10 microM MG132 and 10 microM CE underwent GVBD, despite the inhibition of proteasomal activity. However, 90.0% of cumulus-free ova treated with 10 microM MG132 remained in GV stage, compared with 16.7% GV ova in control. Cumulus expansion, retention of hyaluronic acid, and the deposition of cumulus ECM relying on the covalent transfer of heavy chains of inter-alpha trypsin inhibitor (IalphaI) were also inhibited by MG132. Cumulus expansion in control COCs was accompanied by the degradation of ubiquitin-C-terminal hydrolase L3, an important regulator of UPP. RAC1, a UPP-controlled regulator of actin polymerization was maintained at steady levels throughout cumulus expansion. We conclude that proteasomal proteolysis has multiple functions in the progression of oocyte meiosis beyond GV and metaphase I stage, polar body extrusion, and cumulus expansion.

Previous studies have shown that the heavy chains (HCs) of serum-derived inter-alpha-trypsin inhibitor (IalphaI) molecules become covalently linked to hyaluronan (HA) during in vivo mouse cumulus expansion and significantly contribute to cumulus matrix organization. Experiments with mice suggest that the incorporation of such proteins in cumulus matrix appears to be rather complex, involving LH/hCG-induced changes in blood-follicle barrier and functional cooperation between cumulus cells, granulosa cells, and oocyte within the follicle. We demonstrate here that HC-HA covalent complexes are formed during in vivo porcine cumulus expansion as well. Western blot analysis with IalphaI antibody revealed that follicular fluids from medium-sized follicles and those from large follicles unstimulated with hCG contain high levels of all forms of IalphaI family members present in pig serum. The same amount of HCs were covalently transferred from IalphaI molecules to HA when pig oocyte-cumulus complexes (OCCs) were stimulated in vitro with FSH in the presence of pig serum or follicular fluid from unstimulated or hCG-stimulated follicles. In addition, HC-HA coupling activity was stimulated in cumulus cells by FSH treatment also in the absence of oocyte. Collectively, these results indicate that IalphaI molecules can freely cross the blood follicle barrier and that follicular fluid collected at any stage of folliculogenesis can be successfully used instead of serum for improving OCC maturation. Finally, pig cumulus cells show an autonomous ability to promote the incorporation of IalphaI HCs in the cumulus matrix.

We have shown previously that porcine cumulus and mural granulosa cells produce a factor that is very similar, if not identical, to the oocyte-derived cumulus expansion-enabling factor (CEEF). Because growth differentiation factor 9 (GDF9) is the most likely candidate for the CEEF, in the present study we tested the hypothesis that GDF9 is expressed not only in oocytes in the pig but also in somatic follicular cells. In addition, we asked whether the relative abundance (RA) of GDF9 mRNA changes in oocytes and/or follicular cells during the periovulatory period or culture of oocyte-cumulus complexes (OCCs) in vitro. Denuded oocytes, OCCs, cumulus, and mural granulosa cells were isolated from growing and preovulatory follicles. Total RNA was extracted from the cells, and reverse transcription-polymerase chain reaction (RT-PCR) was carried out using specific oligonucleotide primers. The RT-PCR resulted in amplification of a product of expected size (277 base pairs) in samples prepared from all follicular cell types. The identity of the RT-PCR products with GDF9 was confirmed by analysis of their nucleotide sequence, which was 88% and 91% identical to human and ovine GDF9, respectively. The RA of GDF9 mRNA in the somatic follicular cells was approximately fourfold lower than in oocytes. Assessment of the RA of GDF9 mRNA during the periovulatory period and during culture and expansion of OCCs in vitro revealed that it remained stable in oocytes and mural granulosa cells and decreased significantly in expanding cumulus cells. We conclude that GDF9 mRNA can be produced by somatic follicular cells in the pig and that cumulus expansion is not preceded or accompanied by an increase in the RA of GDF9 mRNA in any of the tested cell types.

We have recently shown that epidermal growth factor (EGF) strongly stimulates expansion of porcine oocyte-cumulus complexes (OCCs) isolated from large follicles (>6 mm) and does not promote expansion of OCCs from small (3-4-mm) follicles. In order to elucidate the role of EGF in OCCs expansion, in the present study, we first examined the presence of EGF receptors (EGFRs) in cumulus cells isolated from follicles of different sizes. Surprisingly, immunoblotting showed that cumulus cells obtained from all follicular size categories contained similar amounts of EGFR protein. On the other hand, we found a dramatic difference in the pattern of protein tyrosine phosphorylation in a comparison of cumulus cells isolated from small and large follicles treated by EGF. Furthermore, tyrosine-phosphorylated EGFR was specifically immunoprecipitated with antiphosphotyrosine antibodies from EGF-treated cumulus cells isolated from the large follicles. This result strongly indicates that only OCCs from the large follicles contain mature EGFRs that are capable of becoming activated by EGF. Remarkably, preincubation of cumulus cells from small follicles (3-4 mm) with FSH strongly increased EGF-stimulated tyrosine phosphorylation to levels comparable with OCCs from large follicles. The FSH-dependent activation of EGFRs was beneficial for expansion of OCCs isolated from the small follicles since OCCs treated sequentially by FSH (3 h) and EGF (1 h) underwent expansion significantly better then OCCs cultured in FSH or EGF alone. We conclude that a FSH-dependent pathway has an important role in the maturation of the EGFR in cumulus cells and that activation of EGFR-dependent signaling is sufficient to induce expansion.

Oocytes secrete factors that control cumulus and granulosa functions, including cumulus expansion and steroid hormone production. Some members of the transforming growth factor beta (TGFbeta) superfamily influence these activities, yet it is still not determined conclusively whether any of these superfamily members are the previously reported oocyte-secreted factors. The aim of this study was to examine the effects of TGFbeta1 and growth differentiation factor 9 (GDF-9) on cumulus expansion and progesterone production by mouse oocytectomized (OOX) complexes in culture. TGFbeta1 mimics the effects of oocytes by both enabling cumulus expansion and inhibiting progesterone production; however, neutralizing antibodies to TGFbeta1 in cultures of cumulus-oocyte complexes (COCs) or in co-cultures of OOX complexes failed to inhibit the ability of oocytes to enable cumulus expansion or inhibit progesterone production. Activin A had no effect on progesterone production by OOX complexes. In experiments using oocytes obtained from mice with deficient expression of GDF-9, OOX complexes cultured in the presence of heterozygous oocytes were capable of full expansion, whereas OOX complexes cultured with oocytes from GDF-9 null mice did not expand. Similarly, GDF-9 null oocytes failed to suppress FSH-induced progesterone production by OOX complexes. These results support the hypothesis that GDF-9 is the cumulus expansion enabling factor produced by mouse oocytes and that GDF-9 also inhibits cumulus progesterone production; however, the possibility remains that loss of GDF-9 may indirectly affect the ability of oocytes to produce the factors that regulate cumulus cell activity.

The meiotic competence and meiosis resumption of Blue fox (Alopex lagopus) oocytes from anoestrous animals were followed. Oocyte-cumulus complexes (OCC) were cultured in modified TC 199 medium with or without FSH, recombinant bovine somatotrophin (bST) and okadaic acid (OA). The results showed that oocytes less than 100 microns in diameter did not achieve germinal vesicle breakdown (GFBD) by 72 h of culture, which indicates their meiotic incompetence. Oocytes larger than 100 microns in diameter underwent GVBD after 48 h of culture (27%) and reached metaphase II (MII) after 72 and 96 h (20% and 27%) in control medium. Both bST and OA accelerated resumption of meiosis (bST: 55% GVBD and 42% MII after 48 h; OA: 66% GVBD after 18 h). In contrast, FSH significantly reduced meiosis resumption (only 3% GVBD and MII after 72 h) and induced changes in the shape of cumulus granulosa (CG) cells and F-actin assembly typical for cumulus expansion. However, the innermost layers of CG cells (corona radiata) remained connected with the oocyte via gap junctions until the end of culture. Cumuli of oocytes cultured in control, bST-supplemented or OA-supplemented medium did not expand (changes in cell shape and F-actin redistribution did not occur). Moreover, especially in media with bST and OA an increased detachment and rapid disconnection of their gap junctions with the oocyte were observed. These results suggest that under in vitro conditions FSH stimulates expansion of the CG cells and the attached membrana granulosa cells but in contrast it secures heterologous gap junctions between cytoplasmic processes of the corona radiata cells and oolemma during 3 days of culture. Thus, in agreement with the in vivo situation in which Canidae oocytes are ovulated in the GV stage, the cumulus, mainly corona radiata cells, controls resumption of meiosis in Blue fox oocytes under in vitro conditions also.

The meiosis resumption process has been related to spontaneous cytoplasmic InsP3-dependent calcium oscillations in fully grown mouse oocytes. Our purpose was to determine whether the acquisition of meiotic competence during the growth phase of oogenesis was associated with that of Ca2+ oscillations and whether these oscillations were dependent on the phosphoinositide cycle. We used confocal laser scanning microscopy to image free calcium ions in fluo-3/AM-loaded oocytes recovered from 12- to 26-day-old mice for 15 min following follicular release. As expected, oocytes isolated from 12-day-old mice were totally incompetent to undergo GVB in vitro, whereas the GVB rate increased progressively with mouse age and oocyte diameter. The percentage of oocytes exhibiting spontaneous calcium oscillations and that of oocytes resuming meiosis were similarly correlated with the female age, with incompetent oocytes failing to exhibit spontaneous Ca2+ oscillations. It is noteworthy that regardless of the stage of growth, thapsigargin induced an ooplasmic calcium release from the InsP3-sensitive stores when it was added to the culture medium. However, intracytoplasmic microinjection of InsP3 induced a shorter sequence of Ca2+ oscillations in 12-day-old mouse oocytes than in 15-day-old mouse oocytes and, whereas InsP3 increased the GVB rate at 15 days, it was unable to induce GVB at 12 days. These data lead us to conclude that the acquisition of meiotic competence is related to the functionality of the InsP3 pathway and, correspondingly, to the oocyte's ability to generate spontaneous cytoplasmic InsP3-dependent calcium oscillations.