Involvement of the Serine Protease Inhibitor, SERPINE2, and the Urokinase Plasminogen Activator in Cumulus Expansion and Oocyte Maturation (original) (raw)
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Molecular and Cellular Endocrinology, 2013
a b s t r a c t 27 Pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptor PAC1-R (PACAP type 1 28 receptor) are transiently expressed in granulosa cells (GCs) of mouse preovulatory follicles and affect 29 several parameters associated with the ovulatory process. We investigated the expression of PACAP 30 and its receptors in cumulus cells (CCs) after the LH surge and their role on cumulus expansion/apoptosis 31 and oocyte maturation. PACAP and PAC1-R expression increased in CCs isolated at different times after 32 treatment with human chorionic gonadotropin (hCG). Moreover, PACAP was able to reverse the inhibition 33 of oocyte meiotic maturation caused by hypoxantine in cumulus cell-oocyte complexes (COCs) and effi-34 ciently promoted male pronuclear formation after fertilisation. PACAP was also able to induce cumulus 35 expansion and prevent CC apoptosis. Our results demonstrated the induction of PACAP and its receptors 36 in CCs by LH and EGF, suggesting that PACAP may play a significant role in the complex interactions of 37 gonadotropin and growth factors during ovulation and fertilisation.
Biology of Reproduction, 1999
We examined whether plasminogen activators (PAs) are produced by bovine cumulus-oocyte complexes (COCs) during maturation in vitro. The effects of epidermal growth factor (EGF) on production of PAs in oocytes and cumulus cells were also examined. When COCs were cultured for 24 h with 30 ng/ml EGF, three plasminogen-dependent lytic zones (58.5 ؎ 3.5 kDa, 79.0 ؎ 3.0 kDa, and 113.5 ؎ 6.5 kDa) were observed. Addition of amiloride, a competitive inhibitor of urokinase-type PA (uPA), to the zymogram eliminated the activity of the 58.5 ؎ 3.5-kDa zone, suggesting that this band is a uPA. However, since the activity of the remaining two bands was not eliminated, it was suggested that the 79.0 ؎ 3.0-kDa band is a tissue-type PA (tPA) and the 113.5 ؎ 6.5-kDa band is possibly a tPA-PA inhibitor (tPA-PAI) complex. In COCs before culture, however, no activity of PAs was detected. At 6 h of culture, the same level of uPA activity was detected in COCs cultured both in the absence and in the presence of EGF. The uPA activity was increased at 12 h of culture but without further increase at 24 h of culture, with higher activity in the presence than in the absence of EGF. The activity of tPA and tPA-PAI was first detected at 24 h of culture in the absence of EGF. In the presence of EGF, however, some activity of tPA-PAI was detected at 12 h of culture. At 24 h of culture, the activity of all PAs was detected in cumulus cells, but only uPA activity was detected in oocytes, with higher activity in the presence than in the absence of EGF. The uPA activity in oocytes was not detected when they were cultured without cumulus cells in either the presence or absence of EGF, although cumulus expansion was stimulated by EGF, exhibiting a time-course similar to that observed in PA production. These results suggest that uPA, tPA, and tPA-PAI are all produced by bovine COCs, but only uPA by oocytes, during maturation in vitro. However, cumulus cells play an essential role or roles in the production of uPA by oocytes, and EGF enhances the roles of cumulus cells.
GENE expression in human cumulus cells: a way to approach oocyte competence
Fertility and Sterility, 2007
BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.
Biology of Reproduction, 2000
To demonstrate secretion of cumulus expansion-enabling factor (CEEF) by porcine oocytes, we used an interspecies testing system. Porcine oocytes were used to condition culture medium, and the presence of CEEF was tested using mouse oocytectomized complexes (OOX), which require CEEF for expansion. Follicle-stimulating hormone-stimulated expansion and synthesis of hyaluronic acid (HA) by mouse OOX were assessed after 18 h of culture in media conditioned by porcine oocytes: 1) at different stages of maturation and 2) in which maturation was inhibited with a specific inhibitor of cdk-kinases, butyrolactone I.
Gene expression in human cumulus cells: one approach to oocyte competence
Human Reproduction, 2007
BACKGROUND: Dialogue between the oocyte and cumulus cells is essential for oocyte maturation. A prospective laboratory research project was designed to evaluate transcription of specific genes in cumulus cells harvested before intracytoplasmic sperm injection from pre-ovulatory follicles, according to individual oocyte nuclear maturity and developmental competence. Genes were chosen because their expression was induced by the LH peak [Steroidogenic Acute Regulatory protein (STAR), Cyclooxygenase 2 (COX2 or PTGS2), Amphiregulin (AREG)] or because they were involved in oocyte lipidic metabolism [Stearoyl-Coenzyme A Desaturase 1 and 5 (SCD1 and SCD5)] or in gap-junctions [Connexin 43 (CX43 or GJA1)]. METHODS: mRNA levels in cumulus cells were assessed by real-time PCR. RESULTS: Expression levels of all genes investigated, except Cx43, were increased after resumption of meiosis. Nuclear maturation was thus associated with increased expression of STAR, COX2, AREG, SCD1 and SCD5 by cumulus cells. When considering only cumulus associated with metaphase II oocytes, gene expression was independent of morphological status at Day 2. In contrast, transcript levels were lower and distributed over a narrower range in cumulus enclosing oocytes achieving blastocyst development at Day 5/6 than in cumulus enclosing oocytes unable to develop beyond the embryo stage. CONCLUSION: Further developmental potential from embryo to blastocyst stage was associated with lower expression in a narrow range for these genes.
Background: GJA1 and PTX3 were proposed as gene markers for oocyte and embryo developmental competence, while SERPINE2 was reported to be associated with pregnancy outcome. PRSS35, which is exclusively expressed in the ovary, may be correlated with oocyte competence. This study was conducted to evaluate the correlation of cumulus GJA1, PRSS35, PTX3, and SERPINE2 gene expression levels with oocyte maturation, fertilization, and early embryo development.
Inhibition of meiotic maturation in growing pig oocytes by factor(s) from cumulus cells
Reproduction Nutrition Development, 1994
― Total inhibition of germinal vesicle breakdown (GVBD) was observed in growing pig oocytes (internal diameter 80, 90 and 100 11m) when they were cultured in a medium conditioned by cumulus oocyte complexes (COCs) of fully grown oocytes. In denuded growing oocytes, only partial inhibition was observed. The inhibitory effect was fully reversible. The addition of heparin (300 IU/ml) could overcome the effect of the conditioned medium. Transient exposure (6 h) of oocytes to dibutyryl cyclic adenosine monophosphate (dbcAMP) (1 mg/mi) could also partly reverse the effect of factor (s) produced by cumulus cells of fully grown oocytes. Follicle-stimulating hormone (5 I1g/ml) was able to increase the percentage of maturing oocytes. The addition of luteinizing hormone (5 I1g/ml) had no effect on GVBD inhibition by cumulus-conditioned medium. growing oocyte / maturation / cumulus cells / pig Résumé ― Inhibitition de la maturation méiotique dans les ovocytes en croissance par des facteurs des cellules du cumulus. La rupture de la vésicule germinative est inhibée dans des ovocytes en croissance de porc (diamètre interne: 80, 90 et 100 ¡lM) cultivés dans un milieu conditionné avec des complexes cumuluslovocytes après croissance. Dans les ovocytes en croissance dénudés, c'est seulement une inhibition partielle qui se produit. L'inhibition est complètement réversible : l'addition d'héparine (300 UIlml) peut renverser l'effet du milieu conditionné. Une exposition transitoire (6 h) des ovocytes à 1 mglml de dibutyryl AMPc (dibutyryl cyclic adénosine monophosphate) est aussi capable de renverser partiellement l'effet des facteurs produits par les cellules du cumulus d'ovocytes après croissance. L'hormone folliculo-stimulante (5 wglml) augmente le taux de maturation des ovocytes. L'addition d'hormone lutéinisante (5¡ l g/ml) n'a pas d'effet sur l'inhibition de la rupture de la vésicule germinative par le milieu conditionné.
At present, oocyte selection is mainly based upon morphological criteria but it is generally acknowledged that its reliability requires further improvement. The aim of this study was to determine whether transcript levels in cumulus cells can provide a useful marker of oocyte developmental competence in vitro. A retrospective study was performed on cumulus cells isolated from 90 oocytes retrieved from 45 patients. Upon fertilization, 35 oocytes originated good-quality embryos and 36 developed into poor-quality embryos, whereas 19 failed to be fertilized. Semi-quantitative measurement of hyaluronic acid synthase 2 (HAS2), gremlin1 (GREM1), and pentraxin 3 (PTX3) mRNAs was performed and data for all genes were obtained from all the samples. Cumulus cells isolated from oocytes that originated high-quality embryos on day 3 of culture had HAS2 and GREM1 transcript levels higher than those detected in cells from oocytes that did not fertilize or developed into poor-quality embryos. No differences were observed in PTX3 levels. Results indicate that the measurement of HAS2 and GREM1 levels in cumulus cells would reliably complement the morphological evaluation providing a useful tool for selecting oocytes with greater chances to be fertilized and develop in vitro.
Domestic Animal Endocrinology, 2012
Porcine oocyte-cumulus complexes (OCCs) form an expanded cumulus extracellular matrix (ECM) in response to gonadotropins during meiotic maturation. Essential components of ECM are hyaluronan (HA), tumor necrosis factor ␣-induced protein 6 (TNFAIP6) and heavy chains (HC) of interalpha-trypsin inhibitor. To form expanded cumulus ECM, intermediate complexes (TNFAIP6-HC) must bind to HA to allow HC transfer onto HA. Protein turnover by the ubiquitin-proteasome pathway is poorly characterized in this process. It is known that the specific proteasomal inhibitor MG132 prevents cumulus expansion and formation of ECM. To determine whether inhibition of proteasomal proteolysis with MG132 affects cumulus cell steroidogenesis and expression of the cumulus expansion-related components (hyaluronan synthase type 2, HAS2, TNFAIP6) we cultured porcine OCCs and granulosa cells (GCs) in a medium supplemented with FSH/LH. Methods performed included real-time reverse transcription PCR, immunofluorescence and RIAs. The expression of TNFAIP6 and HAS2 transcripts increased significantly after the stimulation of OCCs and GCs with FSH/LH. In contrast, treatment with MG132 reduced the expression of TNFAIP6 and HAS2. Hyaluronan was detected with biotinylated HA-binding proteins within FSH/LH-stimulated expanded OCCs but not in those treated with MG132. Progesterone production, although increased almost three times after OCCs stimulation with FSH/LH, was significantly suppressed by MG132. The FSH/LH-stimulated a 40-fold increase in progesterone secretion by GCs was inhibited in the presence of MG132. In conclusion, MG132 affects progesterone secretion and expression of cumulus expansion-related components by cumulus and GCs, suggesting the requirement of ubiquitin-proteasome pathway-regulated protein turnover for formation of ECM during cumulus expansion in the preovulatory period in the pig.
Toxicology in Vitro, 2009
In most mammals, before ovulation, cumulus cells synthesize a large amount of hyaluronan (HA) that is organized into an extracellular matrix (ECM), which provides an essential microenvironment for in vivo oocyte fertilization. This process is called cumulus expansion. The present study assessed effects of selected endocrine disruptors (bisphenol A, BPA; 4-chloro-3-methyl phenol, CMP; di(2-ethylhexyl) phthalate, DEHP; and benzyl butyl phthalate, BBP) in a range of 100 pM-100 lM, on follicle-stimulating hormone (FSH)-induced meiotic maturation and cumulus expansion of porcine oocyte-cumulus complexes (OCC) cultured in vitro. Moreover, FSH-stimulated production of hyaluronic acid (HA) and progesterone by cumulus cells was measured. Both phenols, BPA and CMP (100 lM), significantly affected meiotic maturation of oocytes. The number of oocytes that underwent germinal vesicle breakdown (GVBD) (78.7% and 72.4%, respectively) as well as the rate of oocytes that reached metaphase II stage (MII) (50% and 53.6%, respectively) after 44 h culture were decreased compared to control (89.6% for GVBD and 81.5% for MII). FSH-stimulated expansion of cumulus was altered by the highest concentration of BPA and CMP (70% and 64%, respectively vs. 80.3% in control). Although BPA did not alter FSH-stimulated HA synthesis by cumulus cells, its incorporation within the complex was reduced to a half of control value. Progesterone production by OCC was significantly changed in the presence of BPA or DEHP. Finally, our results provide valuable information that oocyte meiotic progression was adversely affected during in vitro culture with endocrine disruptors.