Impact of media supplements FGF2, LIF and IGF1 on the genome activity of porcine embryos produced in vitro.

In this article, we focused on the impact of precisely chemically modified FLI maturation medium enriched with fibroblast growth factor 2 (FGF2), leukemia inhibitory factor (LIF), insulin-like growth factor 1 (IGF1), and polyvinyl alcohol (PVA) and its potential to improve the efficiency of in vitro production of porcine embryos. We hypothesized that enhancing the composition of the maturation medium could result in an elevated production of embryos in vitro and can affect EGA. FLI medium resulted in a significantly higher rate of oocyte blastocyst maturation and formation compared to the control DMEM medium. In addition, immunocytochemical labelling confirmed the detection of UBF in 4-cell FLI parthenogenic embryos, suggesting similarities with natural embryo development. Through RNAseq analysis, upregulated genes present in 4-cell FLI embryos were found to play key roles in important biological processes such as cell proliferation, cell differentiation, and transcriptional regulation. Based on our findings, we demonstrated the positive influence of FLI medium in the evaluation of in vitro embryo production, EGA detection, transcriptomic and proteomic profile, which was confirmed by the positive activation of the embryonal genome in the 4-cell stage of parthenogenetically activated embryos.

The Role of MAPK3/1 and AKT in the Acquisition of High Meiotic and Developmental Competence of Porcine Oocytes Cultured In Vitro in FLI Medium.

The developmental potential of porcine oocytes cultured in vitro was remarkably enhanced in a medium containing FGF2, LIF and IGF1 (FLI) when compared to a medium supplemented with gonadotropins and EGF (control). We analyzed the molecular background of the enhanced oocyte quality by comparing the time course of MAPK3/1 and AKT activation, and the expression of genes controlled by these kinases in cumulus-oocyte complexes (COCs) cultured in FLI and the control medium. The pattern of MAPK3/1 activation in COCs was very similar in both media, except for a robust increase in MAPK3/1 phosphorylation during the first hour of culture in the FLI medium. The COCs cultured in the FLI medium exhibited significantly higher activity of AKT than in the control medium from the beginning up to 16 h of culture; afterwards a deregulation of AKT activity occurred in the FLI medium, which was not observed in the control medium. The expression of cumulus cell genes controlled by both kinases was also modulated in the FLI medium, and in particular the genes related to cumulus-expansion, signaling, apoptosis, antioxidants, cell-to-cell communication, proliferation, and translation were significantly overexpressed. Collectively, these data indicate that both MAPK3/1 and AKT are implicated in the enhanced quality of oocytes cultured in FLI medium.

Global transcriptome analysis of porcine oocytes in correlation with follicle size.

Although our knowledge regarding oocyte quality and development has improved significantly, the molecular mechanisms that regulate and determine oocyte developmental competence are still unclear. Therefore, the objective of this study was to identify and analyze the transcriptome profiles of porcine oocytes derived from large or small follicles using RNA high-throughput sequencing technology. RNA libraries were constructed from oocytes of large (LO; 3-6 mm) or small (SO; 1.5-1.9 mm) ovarian follicles and then sequenced in an Illumina HiSeq4000. Transcriptome analysis showed a total of 14,557 genes were commonly detected in both oocyte groups. Genes related to the cell cycle, oocyte meiosis, and quality were among the top highly expressed genes in both groups. Differential expression analysis revealed 60 up- and 262 downregulated genes in the LO compared with the SO group. BRCA2, GPLD1, ZP3, ND3, and ND4L were among the highly abundant and highly significant differentially expressed genes (DEGs). The ontological classification of DEGs indicated that protein processing in endoplasmic reticulum was the top enriched pathway. In addition, biological processes related to cell growth and signaling, gene expression regulations, cytoskeleton, and extracellular matrix organization were among the highly enriched processes. In conclusion, this study provides new insights into the global transcriptome changes and the abundance of specific transcripts in porcine oocytes in correlation with follicle size.

Strain Range Dependent Cyclic Hardening of 08Ch18N10T Stainless Steel-Experiments and Simulations.

This paper describes and presents an experimental program of low-cycle fatigue tests of austenitic stainless steel 08Ch18N10T at room temperature. The low-cycle tests include uniaxial and torsional tests for various specimen geometries and for a vast range of strain amplitude. The experimental data was used to validate the proposed cyclic plasticity model for predicting the strain-range dependent behavior of austenitic steels. The proposed model uses a virtual back-stress variable corresponding to a cyclically stable material under strain control. This internal variable is defined by means of a memory surface introduced in the stress space. The linear isotropic hardening rule is also superposed. A modification is presented that enables the cyclic hardening response of 08Ch18N10T to be simulated correctly under torsional loading conditions. A comparison is made between the real experimental results and the numerical simulation results, demonstrating the robustness of the proposed cyclic plasticity model.

Mechanisms of FSH- and Amphiregulin-Induced MAP Kinase 3/1 Activation in Pig Cumulus-Oocyte Complexes During Maturation In Vitro.

The maturation of mammalian oocytes in vitro can be stimulated by gonadotropins (follicle-stimulating hormone, FSH) or their intrafollicular mediator, epidermal growth factor (EGF)-like peptide-amphiregulin (AREG). We have shown previously that in pig cumulus-oocyte complexes (COCs), FSH induces expression and the synthesis of AREG that binds to EGF receptor (EGFR) and activates the mitogen-activated protein kinase 3/1 (MAPK3/1) signaling pathway. However, in this study we found that FSH also caused a rapid activation of MAPK3/1 in the cumulus cells, which cannot be explained by the de novo synthesis of AREG. The rapid MAPK3/1 activation required EGFR tyrosine kinase (TK) activity, was sensitive to SRC proto-oncogene non-receptor tyrosine kinase (SRC)-family and protein kinase C (PKC) inhibitors, and was resistant to inhibitors of protein kinase A (PKA) and metalloproteinases. AREG also induced the rapid activation of MAPK3/1 in cumulus cells, but this activation was only dependent on the EGFR TK activity. We conclude that in cumulus cells, FSH induces a rapid activation of MAPK3/1 by the ligand-independent transactivation of EGFR, requiring SRC and PKC activities. This rapid activation of MAPK3/1 precedes the second mechanism participating in the generation and maintenance of active MAPK3/1-the ligand-dependent activation of EGFR depending on the synthesis of EGF-like peptides.

Significance of epidermal growth factor receptor signaling for acquisition of meiotic and developmental competence in mammalian oocytes.

The surge in luteinizing hormone (LH) in preovulatory ovarian follicles triggers the resumption of oocyte meiosis accompanied by expansion of surrounding cumulus cells and ovulation of cumulus-oocyte complexes (COCs) into the oviduct. Over the last 15 years, substantial progress has been made in elucidating the key pathways by which the LH signal spreads within the preovulatory follicle and in identifying the molecules responsible for maintaining oocyte arrest and meiosis resumption. It is now clear that the adenylcyclase-mediated rise in intracellular cyclic adenosine monophosphate leads to activation of the epidermal growth factor receptor (EGFR) network in granulosa and cumulus cells. This signaling network can control the transcription of key genes required for cell metabolism, cumulus expansion, and oocyte meiosis resumption. In addition, EGFR signaling is involved in the regulation of gap junctional communication within follicular somatic cells, and in this way it can control the diffusion of meiosis-arresting molecules as well as energy substrates into the oocyte. Thus, the proper functioning of the follicular EGFR network is a vital precondition for the production of matured and developmentally competent oocytes. However, most current in vitro maturation systems are based on a culture of COCs isolated from growing follicles, in which function of the EGFR network may be insufficient for promoting oocyte meiotic and developmental competence. This review focuses on research identifying the importance of the EGFR signaling in somatic follicular cells for oocyte meiotic and developmental competence, and on special approaches to the culture of COCs isolated from growing follicles to promote oocyte quality.

Prostaglandin E2 stimulates the expression of cumulus expansion-related genes in pigs: the role of protein kinase B.

The production of prostaglandin E2 (PGE2) seems to play an important role in the ovulation process. PGE2 was found to induce cumulus expansion and meiosis resumption in mice, but little is known about its role in pigs. The goals of this study were (a) to assess the effect of PGE2 on the expression levels of cumulus expansion-related genes, (b) to define the signaling pathways that drive the PGE2-stimulated expression of cumulus expansion-related genes, (c) to measure the effect of PGE2 on the activation of key signaling molecules (MAPK3/1, PKB) and on hyaluronan production in cumulus cells, and (d) to assess the effect of PGE2 on meiosis resumption. We documented that PGE2 is able to induce the expression of cumulus expansion-related genes (HAS2, TNFAIP6) as well as genes involved in steroidogenesis (CYP11A1) or prostaglandin production (PTGS2). PGE2 is able to activate PKB and MAPK3/1 and induce mild cumulus expansion and meiosis resumption, but less efficiently than FSH.

Endogenously produced hydrogen sulfide is involved in porcine oocyte maturation in vitro.

Hydrogen sulfide, one of three known gasotransmitters, is involved in physiological processes, including reproductive functions. Oocyte maturation and surrounding cumulus cell expansion play an essential role in female reproduction and subsequent embryonic development. Although the positive effects of exogenous hydrogen sulfide on maturing oocytes are well known, the role of endogenous hydrogen sulfide, which is physiologically released by enzymes, has not yet been described in oocytes. In this study, we observed the presence of Cystathionine ß-Synthase (CBS), Cystathionine γ-Lyase (CTH) and 3-Mercaptopyruvate Sulfurtransferase (3-MPST), hydrogen sulfide-releasing enzymes, in porcine oocytes. Endogenous hydrogen sulfide production was detected in immature and matured oocytes as well as its requirement for meiotic maturation. Individual hydrogen sulfide-releasing enzymes seem to be capable of substituting for each other in hydrogen sulfide production. However, meiosis suppression by inhibition of all hydrogen sulfide-releasing enzymes is not irreversible and this effect is a result of M-Phase/Maturation Promoting Factor (MPF) and Mitogen-Activated Protein Kinase (MAPK) activity inhibition. Futhermore, cumulus expansion expressed by hyaluronic acid (HA) production is affected by the inhibition of hydrogen sulfide production. Moreover, quality changes of the expanded cumuli are indicated. These results demonstrate hydrogen sulfide involvement in oocyte maturation as well as cumulus expansion. As such, hydrogen sulfide appears to be an important cell messenger during mammalian oocyte meiosis and adequate cumulus expansion.

Gene expression analysis of pig cumulus-oocyte complexes stimulated in vitro with follicle stimulating hormone or epidermal growth factor-like peptides.

BACKGROUND: The gonadotropin-induced resumption of oocyte meiosis in preovulatory follicles is preceded by expression of epidermal growth factor (EGF)-like peptides, amphiregulin (AREG) and epiregulin (EREG), in mural granulosa and cumulus cells. Both the gonadotropins and the EGF-like peptides possess the capacity to stimulate resumption of oocyte meiosis in vitro via activation of a broad signaling network in cumulus cells. To better understand the rapid genomic actions of gonadotropins (FSH) and EGF-like peptides, we analyzed transcriptomes of cumulus cells at 3 h after their stimulation. METHODS: We hybridized aRNA from cumulus cells to a pig oligonucleotide microarray and compared the transcriptomes of FSH- and AREG/EREG-stimulated cumulus cells with untreated control cells and vice versa. The identified over- and underexpressed genes were subjected to functional genomic analysis according to their molecular and cellular functions. The expression pattern of 50 selected genes with a known or potential function in ovarian development was verified by real-time qRT-PCR. RESULTS: Both FSH and AREG/EREG increased the expression of genes associated with regulation of cell proliferation, cell migration, blood coagulation and extracellular matrix remodeling. FSH alone induced the expression of genes involved in inflammatory response and in the response to reactive oxygen species. Moreover, FSH stimulated the expression of genes closely related to some ovulatory events either exclusively or significantly more than AREG/EREG (AREG, ADAMTS1, HAS2, TNFAIP6, PLAUR, PLAT, and HSD17B7). In contrast to AREG/EREG, FSH also increased the expression of genes coding for key transcription factors (CEBPB, FOS, ID1/3, and NR5A2), which may contribute to the differing expression profiles of FSH- and AREG/EREG-treated cumulus cells. CONCLUSIONS: The impact of FSH on cumulus cell gene transcription was higher than the impact of EGF-like factors in terms of the number of cell functions affected as well as the number of over- and underexpressed genes. Both FSH and EGF-like factors overexpressed genes involved in the post-ovulatory switch in steroidogenesis and tissue remodelling. However, FSH was remarkably more efficient in the up-regulation of several specific genes essential for ovulation of matured oocytes and also genes that been reported to play an important role in maturation of cumulus-enclosed oocytes in vitro.

Activation of cumulus cell SMAD2/3 and epidermal growth factor receptor pathways are involved in porcine oocyte-cumulus cell expansion and steroidogenesis.

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.

Analysis of dosing regimen and reproducibility of intraspinal grafting of human spinal stem cells in immunosuppressed minipigs.

In recent studies using a rat aortic balloon occlusion model, we have demonstrated that spinal grafting of rat or human neuronal precursors or human postmitotic hNT neurons leads to progressive amelioration of spasticity and rigidity and corresponding improvement in ambulatory function. In the present study, we characterized the optimal dosing regimen and safety profile of human spinal stem cells (HSSC) when grafted into the lumbar spinal cord segments of naive immunosuppressed minipigs. Gottingen-Minnesota minipigs (18-23 kg) were anesthetized with halothane, mounted into a spine-immobilization apparatus, and received five bilateral injections of HSSC delivered in 2, 4, 6, 8, or 10 µl of media targeted into L2-L5 central gray matter (lamina VII). The total number of delivered cells ranged between 2,500 and 100,000 per injection. Animals were immunosuppressed with Prograf® for the duration of study. After cell grafting, ambulatory function was monitored daily using a Tarlov's score. Sensory functions were assessed by mechanically evoked skin twitch test. Animals survived for 6-7 weeks. Three days before sacrifice animals received daily injections of bromodeoxyuridine (100 mg/kg; IV) and were then transcardially perfused with 4% paraformaldehyde. Th12-L6 spinal column was then dissected; the spinal cord was removed and scanned with MRI. Lumbar transverse spinal cord sections were then cut and stained with a combination of human-specific (hNUMA, hMOC, hNSE, hSYN) or nonspecific (DCX, MAP2, GABA, CHAT) antibodies. The total number of surviving cells was estimated using stereological quantification. During the first 12-24 h after cell grafting, a modest motor weakness was observed in three of eight animals but was no longer present at 4 days to 7 weeks. No sensory dysfunction was seen at any time point. Postmortem MRI scans revealed the presence of the individual grafts in the targeted spinal cord areas. Histological examination of spinal cord sections revealed the presence of hNUMA-immunoreactive grafted cells distributed between the base of the dorsal horn and the ventral horn. In all grafts intense hMOC, DCX, and hSYN immunoreactivity in grafted cells was seen. In addition, a rich axodendritic network of DCX-positive processes was identified extending 300-700 µm from the grafts. On average, 45% of hNUMA-positive neurons were GABA immunoreactive. Stereological analysis of hNUMA-positive cells showed an average of 2.5- to 3-fold increase in number of surviving cells compared with the number of injected cells. Analysis of spinal structural morphology showed that in animals injected with more than 50,000 cells/injection or volumes of injectate higher than 6 µl/injection there was tissue expansion and disruption of the local axodendritic network. Based on these data the safe total number of injected cells and volume of injectate were determined to be 30,000 cells delivered in ≤6 µl of media. These data demonstrate that highly reproducible delivery of a potential cell therapeutic candidate into spinal parenchyma can be achieved across a wide range of cell doses by direct intraspinal injections. The resulting grafts uniformly showed robust cell survival and progressive neuronal maturation.

Development of functional LH Receptors on pig cumulus-oocyte complexes cultured in vitro by a novel two-step culture system.

We show in the present study that freshly isolated pig cumulus-oocyte complexes (COCs) display a limited response to LH, as assessed by the expression of hyaluronan synthase 2 (Has2) mRNA, activation of protein kinase A (PKA), production of hyaluronic acid (HA) and progesterone, cumulus cell expansion and resumption of meiosis. These data indicate that freshly isolated COCs do not possess a sufficient number of functional LH receptors (LHR). However, the expression of Lhr significantly increased during the culture of COCs in vitro in a medium supplemented with FSH. Assuming that the effect of FSH on LHR induction is mediated via cAMP signaling pathways, we developed a new culture system, in which the COCs were pre-cultured for 72 hr in a medium supplemented with dbcAMP. The pre-cultured COCs remained in the germinal vesicle stage, their cumulus investment underwent a dramatic increase in size and gap junctions between the cumulus cells were preserved. The stimulation of such COCs with either FSH or LH led to the resumption and completion of meiosis, activation of PKA, expression of Has2, synthesis of large amounts of HA and progesterone, and extensive expansion of cumulus cells. We conclude that the formation of functional LHR is stimulated in cumulus cells during the culture in vitro in a cAMP-dependent pathway. The dbcAMP-treated COCs thus represent a new model in which the resumption of meiosis and cumulus expansion can be induced exclusively by the action of recombinant LH.

Expression of tumor necrosis factor alpha-induced protein 6 messenger RNA in porcine preovulatory ovarian follicles.

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.

Synthesis of tumor necrosis factor alpha-induced protein 6 in porcine preovulatory follicles: a study with A38 antibody.

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.

Molecular mechanisms of insulin-like growth factor 1 promoted synthesis and retention of hyaluronic acid in porcine oocyte-cumulus complexes.

The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.

Timing of Plk1 and MPF activation during porcine oocyte maturation.

A Polo-like kinase 1 (Plk1) appears involved in an autocatalytic loop between CDC25C phosphatase and M phase promoting factor (MPF) in Xenopus oocytes and leads to activation of MPF that is required for germinal vesicle breakdown (GVBD). Although similar evidence for such a role of Plk1 in MPF activation during maturation of mammalian oocytes is absent, changes in Plk1 enzyme activity correlate with MPF activation, Plk1 co-localizes with MPF, and microinjection of antibodies neutralizing Plk1 delays GVBD. In this study, we exploited the prolonged time required for maturation of porcine oocytes to define precisely the timing of Plk1 and MPF activation during maturation. GVBD typically occurs between 24 and 26 hr of culture in vitro and meiotic maturation is completed after 40-44-hr culture. We find that Plk1 is activated before MPF, which is consistent with its role in activating MPF in mammalian oocytes.

Epidermal growth factor-receptor tyrosine kinase activity regulates expansion of porcine oocyte-cumulus cell complexes in vitro.

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.