Oocyte-specific deletion of eukaryotic translation initiation factor 5 causes apoptosis of mouse oocytes within the early-growing follicles by mitochondrial fission defect-reactive oxygen species-DNA damage.

BACKGROUND: Mutations in several translation initiation factors are closely associated with premature ovarian insufficiency (POI), but the underlying pathogenesis remains largely unknown. METHODS AND RESULTS: We generated eukaryotic translation initiation factor 5 (Eif5) conditional knockout mice aiming to investigate the function of eIF5 during oocyte growth and follicle development. Here, we demonstrated that Eif5 deletion in mouse primordial and growing oocytes both resulted in the apoptosis of oocytes within the early-growing follicles. Further studies revealed that Eif5 deletion in oocytes downregulated the levels of mitochondrial fission-related proteins (p-DRP1, FIS1, MFF and MTFR) and upregulated the levels of the integrated stress response-related proteins (AARS1, SHMT2 and SLC7A1) and genes (Atf4, Ddit3 and Fgf21). Consistent with this, Eif5 deletion in oocytes resulted in mitochondrial dysfunction characterized by elongated form, aggregated distribution beneath the oocyte membrane, decreased adenosine triphosphate content and mtDNA copy numbers, and excessive accumulation of reactive oxygen species (ROS) and mitochondrial superoxide. Meanwhile, Eif5 deletion in oocytes led to a significant increase in the levels of DNA damage response proteins (γH2AX, p-CHK2 and p-p53) and proapoptotic proteins (PUMA and BAX), as well as a significant decrease in the levels of anti-apoptotic protein BCL-xL. CONCLUSION: These findings indicate that Eif5 deletion in mouse oocytes results in the apoptosis of oocytes within the early-growing follicles via mitochondrial fission defects, excessive ROS accumulation and DNA damage. This study provides new insights into pathogenesis, genetic diagnosis and potential therapeutic targets for POI. KEY POINTS: Eif5 deletion in oocytes leads to arrest in oocyte growth and follicle development. Eif5 deletion in oocytes impairs the translation of mitochondrial fission-related proteins, followed by mitochondrial dysfunction. Depletion of Eif5 causes oocyte apoptosis via ROS accumulation and DNA damage response pathway.

Pre-maturational culture promotes the developmental competence of bovine oocytes derived from an 8-day in vitro growth culture system.

In this study, we evaluated the effects of pre-maturational culture (pre-IVM) on the developmental competence of bovine oocytes derived from an 8-day in vitro growth (IVG) culture system. IVG oocytes were subjected to 5 h pre-IVM before in vitro maturation, followed by in vitro fertilization (IVF). The proportion of oocytes that progressed to the germinal vesicle breakdown stage was similar between groups with and without pre-IVM. Although metaphase II oocytes and cleavage rates after IVF were similar regardless of pre-IVM culture, the blastocyst rate was significantly higher in the group with pre-IVM (22.5%) than without pre-IVM (11.0%, P < 0.05). In conclusion, pre-IVM culture improved the developmental competence of bovine oocytes derived from an 8-day IVG system.

New Mutations in the 5' Region of the Notch Gene Affect Drosophila melanogaster Oogenesis.

The Notch pathway is an important and evolutionarily conserved signaling system involved in the development of multicellular organisms. Notch signaling plays an important role in the regulation of proliferation and differentiation of many cell types. In this study, we report new aspects of Notch gene participation in oogenesis using our previously generated mutations. The mutations consist of an insertion of an auxiliary element of a transgene construct into the first intron of the gene and a series of directed deletions within the 5' regulatory region of Notch. We showed that some of these mutations affect Drosophila oogenesis. This insertion, either alone or in combination with the deletion of an insulator sequence, led to lower expression of Notch in the ovaries. As a result, the formation of egg chambers was disturbed in middle oogenesis. These abnormalities have not been described previously and imply one more function of Notch in oogenesis. It can be assumed that Notch is associated with not only follicular epithelium morphogenesis but also cellular mechanisms of oocyte growth.

Regulation of mouse primordial follicle formation by signaling through the PI3K pathway†.

Cell signaling mediated by the KIT receptor is critical for many aspects of oogenesis including the proliferation and migration of primordial germ cells, as well as the survival, growth, and maturation of ovarian follicles. We previously showed that KIT regulates cyst breakdown and primordial follicle formation, and in this study, have investigated the mechanisms downstream of the receptor by modulating the activity of two downstream signaling cascades: the phosphoinositide 3-kinase (PI3K) and the mitogen-activated protein kinase pathways. E17.5 ovaries were cultured for 5 days with a daily dose of media supplemented with either the PI3K inhibitor LY294002, the MEK inhibitor U0126, or a DMSO vehicle control. Our histological observations aligned with the established role of PI3K in oocyte growth and primordial follicle activation but also revealed that LY294002 treatment delayed the processes of cyst breakdown and primordial follicle formation. U0126 treatment also led to a reduction in oocyte growth and follicle development but did not appear to affect cyst breakdown. The delay in cyst breakdown was mitigated when ovaries were dually dosed with LY294002 and KITL, suggesting that while KIT may signal through PI3K to promote cyst breakdown, other signaling networks downstream of the receptor could compensate. These observations unearth a role for PI3K signaling in the establishment of the ovarian reserve and suggest that PI3K might be the primary mediator of KIT-induced cyst breakdown and primordial follicle formation in the mouse ovary.

Effect of docosahexaenoic acid on in vitro growth of bovine oocytes.

PURPOSE: The present study investigated the effects of docosahexaenoic acid (DHA) on the growth of bovine oocytes. METHODS: Oocytes and granulosa cell complexes (OGCs) were collected from early antral follicles (0.4-0.7 mm) on the surface of ovaries harvested from a slaughterhouse. The OGCs were cultured with 0, 1, and 10 µmol/L docosahexanoic acid (DHA) for 16 days. RESULTS: Antrum formation of the OGCs and the number of granulosa cells (GCs) surrounding the oocytes were comparable among groups, whereas supplementation of 0.1 µmol/L of DHA significantly improved oocyte growth. Oocytes grown with DHA had a higher fertilization rate, acetylation levels of H4K12, and ATP contents, as well as a lower lipid content compared with those grown without DHA. In addition, GCs surrounding OGCs grown with DHA had low lipid content compared with vehicle counterparts. Furthermore, when GCs were cultured in vitro, DHA increased ATP production, mitochondrial membrane potential, and reduced lipid content and levels of reactive oxygen species. RNA-seq of GCs revealed that DHA increased CPT1A expression levels and affect genes associated with focal adhesion, oxidative phosphorylation, and PI3K-AKT etc. CONCLUSION: The results suggest that DHA supplementation affects granulosa cell characteristics and supports oocyte growth in vitro.

Optimal conditions for mouse follicle culture.

Mammalian ovaries contain a large number of immature follicles. Follicular culture can contribute to the production of fertile oocytes from latent immature follicles, providing a useful tool for exploring the developmental competencies and related factors that oocytes acquire during growth. However, the potential of oocytes produced by follicular culture is limited. Herein, the optimal follicular culture conditions for the addition of polyvinylpyrrolidone to the medium and oxygen concentration were investigated. Polyvinylpyrrolidone with a high molecular weight (≥ 360,000) and a 7% oxygen concentration were found to increase the blastocyst formation rate by more than 20% compared with conventional culture conditions. Although the developmental ability of oocytes produced by follicular culture remained inferior to that of in vivo-derived oocytes, these findings may pave the way for enhanced production of fertile oocytes in vitro and for studying the process of full developmental potency acquisition by oocytes.

Effects of oocyte-derived growth factors on the growth of porcine oocytes and oocyte-cumulus cell complexes in vitro.

During oocyte growth and follicle development, oocytes closely communicate with cumulus cells. We examined the effects of oocyte-derived growth factors, growth differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), on the growth and acquisition of meiotic competence of porcine oocytes collected from early antral follicles (1.2-1.5 mm). First, we confirmed that GDF9 and BMP15 mRNAs were expressed almost exclusively in the oocytes. Oocyte-cumulus cell complexes (OCCs) collected from early antral follicles were cultured in growth medium supplemented with 0-100 ng/ml of GDF9 or BMP15 for 5 days. GDF9 dose-dependently increased the OCC diameter, while BMP15 did not. GDF9 and BMP15 had no significant effects on oocyte growth (P > 0.05). When OCCs that had been cultured with 50 and 100 ng/ml BMP15 were subjected to a subsequent maturation culture, they expanded fully by gonadotropic stimulation and 49% and 61% of oocytes matured to metaphase II (MII), respectively. In contrast, GDF9 did not promote cumulus expansion, and < 10% of oocytes matured to MII. Based on the difference in cumulus expansion, we compared the expression of luteinizing hormone/choriogonadotropin receptor (LHCGR) and follicle stimulating hormone receptor (FSHR) mRNAs in cumulus cells. The level of LHCGR mRNA was increased in cumulus cells of the BMP15 group, although there were no significant differences in FSHR mRNA levels among the groups. These results suggest that GDF9 promotes the growth of OCCs and that BMP15 promotes LHCGR mRNA expression in cumulus cells during oocyte growth culture, which may contribute to cumulus expansion and oocyte maturation.

Mural granulosa cells support to maintain the viability of growing porcine oocytes and its developmental competence after insemination.

PURPOSE: To enhance the in vitro growth of porcine oocytes, we studied the effect of mural granulosa cells (MGCs) on the viability of oocytes attached to granulosa cells (oocyte-granulosa cell complexes, OGCs) that were obtained from early antral follicles. METHODS AND RESULTS: When OGCs were cultured with MGCs for 12 days, there were significant improvement (P < 0.05) in the robustness of gap junctional communication between the oocyte and the granulosa cells (82% vs. 59%), the survival rate of oocytes (57% vs. 39%), and the diameter of survived oocytes (118 µm vs. 112 µm). The rate of oocyte release of OGCs cultured with MGCs on the 12th day (1.9%) was significantly lower than that of OGCs cultured without MGCs (26%). Complete meiotic arrest was maintained in the group with MGCs (100%), while partial resumption of spontaneous meiosis was noticed in the absence of MGCs (10-19%). Furthermore, the presence of MGCs increased the oocyte maturation rate after maturation culture in both 12- and 14-day culture groups (P < 0.05, 85-88%) compared to OGCs cultured without MGCs (48-60%). MGCs also significantly improved the blastocyst formation rate (day 7) after ICSI (P < 0.05). CONCLUSIONS: The data of this study thus shows that the presence of MGCs during in vitro oocyte growth plays a crucial role in supporting the developmental competence of growing porcine oocytes attached to the granulosa cells via enhancement of their viability.

Reduced Vitellogenesis and Female Fertility in Gper Knockout Zebrafish.

The role G-protein coupled estrogen receptor (GPER) plays in vertebrate reproduction remains controversial. To investigate GPER's reproductive role, we generated a gper zebrafish mutant line (gper-/- ) using TALENs. Gper mutant females exhibited reduced fertility with a 40.85% decrease in embryo production which was associated with a significant decrease in the number of Stage V (730-750 µm) ovulated oocytes. Correspondingly, the number of early vitellogenic follicles (Stage III, 400-450 µm) in gper-/- ovaries was greater than that in wildtypes (wt), suggesting that subsequent follicle development was retarded in the gper-/- fish. Moreover, plasma vitellogenin levels were decreased in gper-/- females, and epidermal growth factor receptor (Egfr) expression was lower in Stage III vitellogenic oocytes than in wt counterparts. However, hepatic nuclear estrogen receptor levels were not altered, and estrogen levels were elevated in ovarian follicles. These results suggest that Gper is involved in the control of ovarian follicle development via regulation of vitellogenesis and Egfr expression in zebrafish.

Recreating the Follicular Environment: A Customized Approach for In Vitro Culture of Bovine Oocytes Based on the Origin and Differentiation State.

The mammalian ovary is a large source of oocytes organized into follicles at various stages of folliculogenesis. However, only a limited number of them can be used for in vitro embryo production (IVEP), while most have yet to complete growth and development to attain full meiotic and embryonic developmental competence. While the in vitro growth of primordial follicles in the ovarian cortex has the potential to produce mature oocytes, it is still at an experimental stage. The population of early antral follicles (EAFs), instead, may represent a reserve of oocytes close to completing the growth phase, which might be more easily exploited in vitro and could increase the number of female gametes dedicated to IVEP.Here we present in vitro culture strategies that have been developed utilizing physiological parameters to support the specific needs of oocytes at distinct stages of differentiation, in order to expand the source of female gametes for IVEP by maximizing the attainment of fertilizable oocytes. Furthermore, these culture systems provide powerful tools to dissect the molecular processes that direct the final differentiation of the mammalian oocyte.

Effect of cryopreservation on the ability of granulosa cells to support in vitro development of oocytes derived from porcine early antral follicles.

Granulosa cells (GCs) contribute to oocyte development. The present study addressed the effect of cryopreservation on the ability of GCs to support oocyte development. GCs were collected from antral follicles. Oocyte granulosa cell complexes (OGCs) derived from early antral follicles were cultured with additional fresh-GCs or frozen-thawed-GCs for 14 days, and the developmental ability and characteristics of the oocytes grown in vitro were examined. Furthermore, fresh- or frozen-thawed-GCs were cultured for two days, and the effects of cryopreservation on the characteristics of GCs were examined. The developmental ability of blastocysts and the acetylation levels of H4K12 in oocytes grown in vitro did not significantly differ among the three culture conditions: OGCs cultured with additional fresh-GCs, frozen-thawed-GCs, or without additional GCs. Although both fresh- and frozen-thawed-GCs exhibited increased ATP content compared with that in oocytes developed without additional GCs, only fresh-GCs showed significantly increased lipid content in oocytes grown in vitro. ATP content, reactive oxygen content, mitochondrial membrane potential, and mitochondrial DNA copy number were greater in cultured frozen-thawed-GCs compared with fresh-GCs. In contrast, lipid content of cultured frozen-thawed-GCs was lower than that of fresh-GCs. Both fresh- and frozen-GCs support oocyte growth, but cryopreservation changes the properties of GCs in a manner that affects the energy status of oocytes grown in vitro.

Reproductive physiological characteristics of tropical Celebes eels Anguilla celebesensis in relation to downstream migration and ovarian development.

Downstream-migrating (n = 64) and non-migrating (n = 21) female Celebes eels Anguilla celebesensis were captured from the Poso Lake-River system on Sulawesi Island, Indonesia, and their reproductive physiological characteristics were examined. A histological observation of the ovaries revealed that most non-migrating eels were at the perinucleolus (43%) or oil-droplet (48%) stage, whereas most migrating eels were at the early vitellogenic (36%) or midvitellogenic (61%) stage. Transcript levels of gonadotropin genes (fshb, lhb) in the pituitary gland and concentrations of sex steroids [11-ketotestosterone (11-KT), testosterone, 17ß-oestradiol (E2 )] in blood plasma of migrating eels were significantly higher than those of non-migrating eels. The fshb messenger (m)RNA levels were lower in perinucleolus and oil-droplet stages and then significantly increased in the early vitellogenic stage. The lhb mRNA levels in vitellogenic-stage eels were significantly higher than those in perinucleolus- and oil-droplet-stage eels. The 11-KT levels of eels at the oil-droplet and vitellogenic stages were significantly higher than those of eels at the perinucleolus stage. The E2 levels at the vitellogenic stage were significantly higher than those at the perinucleolus and oil-droplet stages. These dynamics of the reproductive hormones represented the physiological background of oogenesis in A. celebesensis that has remarkably well-developed oocytes just before downstream migration.

Mechanism of palmitic acid-induced deterioration of in vitro development of porcine oocytes and granulosa cells.

The concentration of fatty acids in follicular fluid reflect the physical condition of donors, and palmitic acid (PA) is a major component of follicular fluid. The present study examined the effect of PA on in vitro oocyte growth and investigated the molecular backgrounds of the PA induced-low quality oocytes. Oocyte-granulosa cell complexes (OGCs) were collected from early antral follicles of gilts. The OGCs were cultured for 14 days in a medium containing 0.5 mM PA or vehicle (BSA). PA was found to reduce granulosa cell (GCs) proliferation (0.73 fold) and viability (93.9% vs. 85.8%) and increase lipid content in oocytes and GCs. Oocytes developed in the presence of PA had low developmental ability to the blastocyst stage. In addition, PA affected developmental and epigenetic markers of histone modifications in oocytes; levels of H4K12 acetylation and H3K9 demethylation. PA affected cellular proliferation, apoptosis and endoplasmic reticulum stress markers along with reducing the phosphor-AKT/AKT levels and increasing the expression levels of caspase-3 and CHOP in GCs. Incubation of OGCs with PA increased ceramide content in the GC, and addition of ceramide to the culture medium inhibited GC proliferation. In conclusion, it is suggested that high PA content in the medium reduces viability and proliferation through ceramide accumulation, and PA impaires the developmental ability of oocytes grown in vitro. In addition, high-fat conditions induce changes in the histone modifications of oocytes grown in vitro.

In vitro culture of secondary follicles and prematuration of cumulus-oocyte complexes from antral follicles increase the levels of maturation-related transcripts in bovine oocytes.

This study evaluates the levels of messenger RNA (mRNA) for eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1 in oocytes from secondary and antral follicles at different stages of development. The effects of in vitro culture, in vitro prematuration, and in vitro maturation on the expression of these genes on oocytes were also analyzed. The results showed that mRNA levels for H1FOO, GDF9, and PARN were higher in oocytes from small, medium, and large antral follicles, respectively, than those seen in secondary follicles. Oocytes from small, medium, and large antral follicles had higher levels of CCNB1 than oocytes from secondary follicles. Oocytes from cultured secondary follicles had higher levels of GDF9, CMOS, PARN, eIF4E, CCNB1, and H1FOO than before culture. Prematured oocytes from small antral follicles had higher levels of mRNA for GDF9, PARN, and eIF4E than before culture. In addition, higher levels of cMOS and H1FOO were identified in prematured oocytes from medium antral follicles. In conclusion, follicular growth is associated with an increase in the expression of H1FOO, GDF9, CCNB1, and PARN. The culture of secondary follicles, prematuration, and maturation of oocytes from antral follicles increase the expression of eIF4E, PARN, H1FOO, cMOS, GDF9, and CCNB1.

Follicular factors determining the developmental competence of porcine oocyte.

PURPOSE: This study aimed to examine the relationship between granulosa cells (GCs), number of follicles, and the ability of follicular fluid to support in vitro growth of oocytes. METHODS: The culture medium was supplemented with follicular fluid (FF) collected from GC-rich ovaries and GC-poor ovaries, and its effect on in vitro growth and quality of oocytes derived from early antral follicles (EAFs) was assessed. RESULTS: GC-rich FF treatment enhanced oocyte growth and augmented changes in the chromatin configuration and lipid content of oocytes when compared to oocytes treated with GC-poor FF. Moreover, oocytes treated with GC-rich FF had a higher ability to progress to the blastocyst stage, than oocytes derived from large antral follicles (3-5 mm in diameter). In addition, supplementation of the culture medium with either GC-rich or GC-poor FF enhanced histone acetylation in oocytes grown in vitro. CONCLUSION: GC-rich FF contains key factors that support in vitro oocyte growth; hence, oocytes grown in GC-rich FF medium had high developmental competence, which was comparative to the oocytes grown in vivo.

Modification of the medium volume and gel substrate under in vitro culture conditions improves growth of porcine oocytes derived from early antral follicles.

This study compared the effects of different volumes of culture medium for the in vitro growth of oocytes derived from porcine early antral follicles (EAFs). Oocyte granulosa cell complexes (OGCs) were collected from EAFs (0.5-0.7 mm in diameter) and individually cultured for 14 days. When OGCs were cultured in 1 ml of medium with or without polyacrylamide gel (PAG), the presence of PAG supported granulosa cell (GC) proliferation and oocyte growth. When OGCs were cultured in 0.2 or 1 ml of medium on PAG, the number of GC in the OGC culture and the developmental ability of the oocytes cultured in vitro were significantly higher for the 1 ml of culture medium group than for the 0.2 ml group. In conclusion, a combination of a large volume of culture medium with PAG improved the growth and developmental ability of the oocytes cultured in vitro, which were comparable to the oocytes collected from large antral follicles.

Developmental ability of oocytes retrieved from Meishan neonatal ovarian tissue grafted into nude mice.

Ovarian xenografting makes it possible to obtain oocytes with fertilization ability from immature pigs of Western breeds. In this study, we applied these methods to the Meishan, an indigenous Chinese pig breed, and investigated the developmental competence of oocytes grown in their neonatal tissue after grafting into nude mice. First, mice harboring neonatal ovarian tissue were infused with follicle stimulating hormone (FSH) (62.5 U/ml) for 13 days starting at 10, 30, and 60 days after vaginal opening (D10-, D30-, and D60-FSH groups, respectively). Development of antral follicles and their oocytes was most enhanced in the D60-FSH group. For the next step, we examined the in vitro maturation ability of the oocytes recovered from host mice after infusion with FSH at a dose of 62.5 U/ml or 125 U/ml (FSH-62.5 or -125 group) for 13 days starting at 60 days after vaginal opening. Many more oocytes with maturation ability were obtained from the FSH-125 group. The FSH-125 mature oocytes were fertilized in vitro, as shown by formation of male and female pronuclei, but did not reach the blastocyst stage. These results indicate that Meishan neonatal ovaries are able to produce oocytes with fertilization ability after being grafted into nude mice.

Various macromolecules in in vitro growth medium influence growth, maturation, and parthenogenetic development of pig oocytes derived from small antral follicles / 대한수의학회지

This study was performed to examine the effects of various macromolecules in in vitro growth (IVG) media on the growth, maturation, and parthenogenesis (PA) of pig oocytes derived from small antral follicles (SAF). Immature oocytes were cultured for two days in IVG medium supplemented with 10% (v/v) fetal bovine serum (FBS), 10% (v/v) pig follicular fluid (PFF), 0.4% (w/v) bovine serum albumin (BSA), or 0.1% (w/v) polyvinyl alcohol (PVA) and then maintained for 44 h for maturation. After IVG, the mean diameters of the SAF treated with FBS, PVA, and no IVG-MAF (113.0–114.8 µm) were significantly larger than that of no IVG-SAF (111.8 µm). The proportion of metaphase II oocytes was higher in PFF (73.6%) than in BSA (43.5%) and PVA (53.7%) but similar to that in the FBS treatment (61.5%). FBS and PFF increased cumulus expansion significantly compared to PVA and BSA while the intraoocyte glutathione content was not influenced by the macromolecules. Blastocyst formation of PA oocytes treated with FBS (51.8%), PFF (50.4%), and PVA (45.2%) was significantly higher than that of the BSA-treated oocytes (20.6%). These results show that the PFF and FBS treatments during IVG improved the growth, maturation, and embryonic development of SAF.

Morphological study of the ovary in Hanseniella caldaria (Myriapoda; Symphyla): The position of oocyte-growth and evolution of ovarian structure in Arthropoda.

In Arthropoda, the ovary is classified into Chelicerata-type and Mandibulata-type, based on the oocyte-growth position within the ovary. By contrast, oocytes of Diplopoda and Chilopoda grow within the hemocoelic space. However, as the position of oocyte-growth in Symphyla and Pauropoda has not been confirmed, whether the hemocoelic nature of oocyte-growth is common among myriapods remains ambiguous. This study described the ovarian structure of Hanseniella caldaria to reveal the oocyte-growth position in Symphyla. The oocyte is surrounded by the follicle epithelium, and the inner surface of the follicle epithelium, i.e., the space between follicle cells and oocytes, is lined with a basement membrane. The follicle epithelial layer continues to the ovarian epithelium via the follicle extension with a continuous layer of basement membrane. Data on the architecture of the follicle suggest that the follicle pouch opens to the hemocoel. Hence, the oocyte of H. caldaria grows within the hemocoelic space. Based on our findings in H. caldaria and previous studies in a millipede and in centipedes, the hemocoelic nature of oocyte-growth is considered as a common feature among myriapods and a synapomorphy of the Myriapoda for which morphological synapomorphies have been ambiguous.

Macromolecular crowded conditions strengthen contacts between mouse oocytes and companion granulosa cells during in vitro growth.

Macromolecular crowded culture medium formed by addition of polyvinylpyrrolidone (PVP; molecular weight = 360 000), positively influences the viability, growth, and development of bovine oocytes. Owing to its apparently various effects, uncovering the specific mechanisms of crowding responsible for these outcomes is important. The present study was conducted to determine the effects of crowding on oocytes with a particular focus on the intimacy of contacts between oocyte and cumulus/granulosa cells. Growing mouse oocyte-granulosa cell complexes were cultured for 10 days in a modified α-minimum essential medium, supplemented with PVP at a concentration of 0%, 1%, 2%, or 3% (w/v). Although the complexes developed in all groups, 2% and 3% PVP medium induced a substantial morphological modification, and a larger proportion of oocytes associated with cumulus cells survived in 3% PVP medium than in the 0% or 1% PVP medium. No significant difference was found in the frequencies of polar body extrusion (78-88%) and blastocyst formation (approximately 40%) after in vitro fertilization among the experimental groups. Confocal laser scanning microscopy indicated a higher number of transzonal processes reaching the oocyte from cumulus cells in 2% PVP medium than in 0% PVP medium. Transmission electron microscopy depicted close adhesion of the oocyte with cumulus cells in 2% PVP medium -bearing a resemblance to their in vivo counterparts- and loose adhesion in 0% PVP medium. In conclusion, we found that a mechanism for the action of crowded conditions involves the strengthening of contacts and communication between oocytes and companion cumulus/granulosa cells.