Oocyte-secreted factors enhance oocyte developmental competence

The capacity of fully grown oocytes to regulate their own microenvironment by paracrine factors secreted by the oocyte (oocyte-secreted factors, OSFs) may in turn contribute to oocyte developmental competence. Here, we investigated if OSFs have a direct influence on oocyte developmental competence during in vitro maturation (IVM). Bovine cumulus-oocyte complexes (COCs) were aspirated from abattoir-derived ovaries and matured in serum-free medium. COCs were either co-cultured with denuded oocytes (DOs) or treated with specific OSFs: recombinant bone morphogenetic protein 15 (BMP15) and/or growth differentiation factor 9 (GDF9). Following maturation, embryos were fertilized and cultured in vitro and blastocyst development and cell number were assessed on day 8. Co-culturing intact COCs with DOs did not affect cleavage rate, but increased (P<0.001) the proportion of cleaved embryos that reached the blastocyst stage post-insemination from 39% to 51%. OSFs also altered blastocyst cell allocation as co-culture of COCs with DOs significantly increased total and trophectoderm cell numbers, compared to control COCs. BMP15 alone, GDF9 alone or the two combined all (P<0.05) increased the proportion of oocytes that reached the blastocyst stage post-insemination from 41% (controls) to 58%, 50% and 55%, respectively. These results were further verified in neutralization experiments of the exogenous growth factors and of the native OSFs. Follistatin and the kinase inhibitor SB-431542, which antagonize BMP15 and GDF9, respectively, neutralized the stimulatory effects of the exogenous growth factors and impaired the developmental competence of control COCs. These results demonstrate that OSFs, and particularly BMP15 and GDF9, enhance oocyte developmental competence and provide evidence that OSF regulation of the COC microenvironment is an important determinant of oocyte developmental programming.     

Effect of ovarian cortex cells on nuclear maturation of sheep oocytes during in vitro maturation

The objective of this study was to investigate the influence of ovarian cortex cells (OCCs) monolayers on the nuclear maturation of sheep oocytes with or without cumulus cells during IVM. Sheep ovaries collected from a local abattoir were transported to the laboratory in warm PBS containing antibiotics within 2-3h after collection. Cumulus-oocyte complexes (COCs) were obtained by aspiration and evaluated in a pre-incubated Hepes-modified TCM 199 medium. The selected COCs were randomly divided into six treatment groups: group 1 (control group): oocytes enclosed by cumulus cells were cultured in maturation medium; group 2 (co-culture group): oocytes enclosed by cumulus cells co-cultured with OCCs monolayers; group 3 (conditioned group): oocytes enclosed by cumulus cells were cultured in OCCs-conditioned medium; group 4 (denuded group): denuded oocytes were cultured in the maturation medium; group 5 (denuded co-culture group): denuded oocytes co-cultured with OCCs monolayers in maturation medium; group 6 (denuded conditioned group): denuded oocytes were cultured in OCCs-conditioned medium. After maturation for 24h, the oocytes in each treatment group were fixed, stained and the nuclear status of the oocytes were assessed under an inverted microscope. The highest percentage of metaphase II (M-II) stage oocyte was observed in group 2 (86.3%) and the lower percentage was observed in the denuded groups (group 4-6). The removal of cumulus cells dramatically decreased the percentage of M-II stage oocyte. The comparison of the nuclear maturation status in group 4-6 showed that the co-culture of oocyte with OCCs monolayers resulted in progression to completing the GVBD stage to reach the M-II stage. The results demonstrated that the presence of OCCs could positively influence the meiotic resumption and progression of sheep oocytes during IVM.     

Developmental stage of the oocyte during antral follicle growth and cumulus investment determines in vitro embryo development of sow oocytes

The aim of the study was to determine the contribution of cumulus cells on the developmental competence of porcine oocytes during follicle growth. Oocytes from large (5-8mm) and small (2-3mm) follicles were cultured with or without follicle stimulating hormone (FSH), subsequently examined for nuclear stage and spindle morphology, or fertilized and cultured for embryo development, or analyzed for glutathione content. Additionally, the significance of cumulus investment, corona radiata cells, cumulus cell number and origin of cumulus cells for oocyte maturation were investigated. Small follicle oocytes cultured without FSH exhibited the highest incidence of spindle aberrations. Oocytes cultured without FSH exhibited reduced sperm penetration and blastocyst rates, and a higher proportion monospermic oocytes developed to the blastocyst stage when derived from large follicles. The glutathione content in oocytes increased during follicle growth and oocyte maturation, but no direct correlation between oocyte glutathione content and oocyte developmental capacity was observed. Oocytes with a bigger cumulus investment exhibited better embryo development. Oocytes with a single corona radiata cell layer (CROs) exhibited similar progression through meiosis to oocytes with more cumulus cell layers, but showed reduced embryo development. More blastocysts were observed when CROs were cultured with disconnected cumulus cells during IVM, but no blastocyst increase was observed when CROs were cocultured with a higher number of cumulus cells or with cumulus cells from large follicles. We conclude that increased developmental capacity of oocytes during follicle growth is intrinsic and whether cumulus cells originate from large or small follicles, their contribution to oocyte maturation remains unchanged. Further, cumulus investment can be used as a variable to predict oocyte developmental capacity.     

Cumulus cells suppress meiotic progression in pig oocytes cultured in vitro

The objective of this study was to examine the effect of cumulus cell removal from cumulusoocyte complexes (COCs) on meiotic progression. In Experiments 1, 2 and 3, pig COCs were cultured for 16, 20 and 24 h, respectively. The cumulus cells were then removed, and the denuded oocytes were incubated in fresh medium for another 32 h in Experiment 1, for 28 h in Experiment 2 and for 24 h in Experiment 3. In Experiment 4, the denuded oocytes and COCs were co-cultured in a drop of fresh medium from 24 h of cultivation to the end of the culture period (48 h). Removal of the cumulus cells after 16 h of cultivation had no effect on the proportions of oocytes both undergoing germinal vesicle breakdown (GVBD) and reaching MII. When the denuded oocytes were further cultured for 24 h, following the removal of their cumulus cells after 24 h of cultivation, the proportion of oocytes undergoing GVBD was significantly higher (90%, P < 0.05) than that of oocytes that were continuously cultured for 48 h without removing the cumulus cells (80%). Removal of the cumulus cells after 20 and 24 h of incubation produced a significant increase in the proportion of oocytes reaching the MII stage (84%, P < 0.05 and 76%, P < 0.01, respectively) as compared with COCs cultured continuously for 48 h without removing cumulus cells (71% and 55%, respectively). The maturation rate of denuded oocytes co-cultured with COCs for the second 24 h of cultivation was comparable to that of denuded oocytes cultured without COCs (77 and 74%, respectively). From these results, it was concluded that cumulus cells surrounding oocytes suppressed meiosis of both the GVBD process and progression from GVBD to MII in pig oocytes cultured in vitro, and that the suppressive factor in meiotic progression produced by the cumulus cells might be transferred to the oocytes through gap junctions rather than through the medium.     

In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes

Female cancer patients who seek fertility preservation but cannot undergo ovarian stimulation and embryo preservation may consider 1) retrieval of immature oocytes followed by in vitro maturation (IVM) or 2) ovarian tissue cryopreservation followed by transplantation or in vitro follicle culture. Conventional IVM is carried out during the follicular phase of menstrual cycle. There is limited evidence demonstrating that immature oocyte retrieved during the luteal phase can mature in vitro and be fertilized to produce viable embryos. While in vitro follicle culture is successful in rodents, its application in nonhuman primates has made limited progress. The objective of this study was to investigate the competence of immature luteal-phase oocytes from baboon and to determine the effect of follicle-stimulating hormone (FSH) on baboon preantral follicle culture and oocyte maturation in vitro. Oocytes from small antral follicle cumulus-oocyte complexes (COCs) with multiple cumulus layers (42%) were more likely to resume meiosis and progress to metaphase II (MII) than oocytes with a single layer of cumulus cells or less (23% vs. 3%, respectively). Twenty-four percent of mature oocytes were successfully fertilized by intracytoplasmic sperm injection, and 25% of these developed to morula-stage embryos. Preantral follicles were encapsulated in fibrin-alginate-matrigel matrices and cultured to small antral stage in an FSH-independent manner. FSH negatively impacted follicle health by disrupting the integrity of oocyte and cumulus cells contact. Follicles grown in the absence of FSH produced MII oocytes with normal spindle structure. In conclusion, baboon luteal-phase COCs and oocytes from cultured preantral follicles can be matured in vitro. Oocyte meiotic competence correlated positively with the number of cumulus cell layers. This study clarifies the parameters of the follicle culture system in nonhuman primates and provides foundational data for future clinical development as a fertility preservation option for women with cancer.     

Apoptosis within bovine follicular cells and its effect on oocyte development during in vitro maturation

Developmental competence of oocytes is compromised if they originate from atretic follicles. Apoptosis is the underlying process of atresia. Apoptotic changes in follicular cells are thought to influence the outcome of IVF. The aim of this study was to investigate apoptosis in different compartments of single bovine follicles (follicular wall, granulosa and cumulus cells (CC)) in relation to COC morphology, and to determine whether the addition, in vitro, of exogenous follicular cells from atretic follicles to maturing cumulus oocyte complexes (COCs) influenced the development of oocytes.Antral follicles were dissected from bovine ovaries and opened to obtain COCs and free floating granulosa cells (GC). The COCs were classified according to morphology. Apoptosis was determined in cumulus and granulosa cells and in homogenates of the remaining follicular wall.For every morphological class of COCs, a large variability of apoptotic expression was found in all follicle compartments. Follicular wall apoptosis was not correlated to COC morphology or to the percentage of apoptotic granulosa or cumulus cells. In grade 1 (best morphology) COCs, the degree of apoptosis in granulosa cells was comparable to cumulus cell apoptosis (P<0.01). The overall expression of apoptosis in granulosa cells of follicles containing grade 3 COCs (median+/-median absolute deviation: 37.8+/-13.8%) was significantly higher (P<0.05) than in follicles with grade 1 (22.7+/-10.4%) or grade 2 COCs (20.0+/-17.0%). About 48.3% of grade 3 COCs possessed strongly apoptotic cumulus cells compared to 27.8 and 28.2% of grade 1 or grade 2 COCs, respectively. Nonapoptotic cumulus complexes were observed in grades 1 and 2 COCs only.Adding exogenous follicular cells from atretic follicles to bovine COCs (grades 1 and 2) during in vitro maturation (IVM) had no impact on fertilization, blastocyst formation or hatching after IVF. This is of particular practical relevance to embryo production after ovum pick up (OPU), as during this process, good quality COCs are cultured together with simultaneously collected slightly atretic COCs.     

The new system of shorter porcine oocyte in vitro maturation (18 hours) using ≥8 mm follicles derived from cumulus-oocyte complexes

Despite recent efforts to improve in vitro maturation (IVM) systems for porcine oocytes, developmental competence of in vitro-matured oocytes is still suboptimal compared with those matured in vivo. In this study, we compared oocytes obtained from large (≥8 mm; LF) and medium (3–7 mm; MF) sized follicles in terms of nuclear maturation, intracellular glutathione and reactive oxygen species levels, gene expression, and embryo developmental competence after IVM. In the control group, cumulus-oocyte complexes (COCs) were aspirated from MF and matured for 22 hours with hormones and subsequently matured for 18 to 20 hours without hormones at 39 °C, 5% CO₂in vitro. In the LF group, COCs were obtained from follicles larger than 8 mm and were subjected to IVM for only 18 hours. The ovaries have LF were averagely obtained with 1.7% per day during 2012 and it was significantly higher in the winter season. The results of the nuclear stage assessment of the COCs from the LFs are as follows: before IVM (0 hours); germinal vesicle stage (15.2%), metaphase I (MI) stage (55.4%), anaphase and telophase I stages (15.8%), and metaphase II (MII) stage (13.6%). After 6 hours IVM; germinal vesicle (4.2%), MI (43.6%), anaphase and telophase I (9.4%), and MII (42.8%). After 18-hour IVM; MI (9.7%) and MII (90.3%). Oocytes from LF showed a significant (P < 0.001) increase in intracellular glutathione (1.41 vs. 1.00) and decrease in reactive oxygen species (0.8 vs. 1.0) levels compared with the control. The cumulus cells derived from LFs showed lower (P < 0.1) mRNA expression of COX-2 and TNFAIP6, and higher (P < 0.1) mRNA expression of PCNA and Nrf2 compared with the control group-derived cumulus cells. After parthenogenetic activation, in vitro fertilization and somatic cell nuclear transfer (SCNT) using matured oocytes from LFs, the embryo development was significantly improved (greater blastocyst formation rates and total cell numbers in blastocysts) compared with the control group. In conclusion, oocytes from LFs require only 18 hours to complete oocyte maturation in vitro and their developmental competence is significantly greater than those obtained from MFs. Although their numbers are limited, oocytes from LFs might offer an alternative source for the efficient production of transgenic pigs using SCNT.     

Bone Morphogenetic Protein 15 in the Pro-Mature Complex Form Enhances Bovine Oocyte Developmental Competence

Developmental competence of in vitro matured (IVM) oocytes needs to be improved and this can potentially be achieved by adding recombinant bone morphogenetic protein 15 (BMP15) or growth differentiation factor (GDF9) to IVM. The aim of this study was to determine the effect of a purified pro-mature complex form of recombinant human BMP15 versus the commercially available bioactive forms of BMP15 and GDF9 (both isolated mature regions) during IVM on bovine embryo development and metabolic activity. Bovine cumulus oocyte complexes (COCs) were matured in vitro in control medium or treated with 100 ng/ml pro-mature BMP15, mature BMP15 or mature GDF9 +/− FSH. Metabolic measures of glucose uptake and lactate production from COCs and autofluorescence of NAD(P)H, FAD and GSH were measured in oocytes after IVM. Following in vitro fertilisation and embryo culture, day 8 blastocysts were stained for cell numbers. COCs matured in medium +/− FSH containing pro-mature BMP15 displayed significantly improved blastocyst development (57.7±3.9%, 43.5±4.2%) compared to controls (43.3±2.4%, 28.9±3.7%) and to mature GDF9+FSH (36.1±3.0%). The mature form of BMP15 produced intermediate levels of blastocyst development; not significantly different to control or pro-mature BMP15 levels. Pro-mature BMP15 increased intra-oocyte NAD(P)H, and reduced glutathione (GSH) levels were increased by both forms of BMP15 in the absence of FSH. Exogenous BMP15 in its pro-mature form during IVM provides a functional source of oocyte-secreted factors to improve bovine blastocyst development. This form of BMP15 may prove useful for improving cattle and human artificial reproductive technologies.     

Denuding and centrifugation of maturing bovine oocytes alters oocyte spindle integrity and the ability of cytoplasm to support parthenogenetic and nuclear transfer embryo development

The effects of cumulus cell removal and centrifugation of maturing bovine oocytes on nuclear maturation and subsequent embryo development after parthenogenetic activation and nuclear transfer were examined. Removal of cumulus cells at 4, 8, and 15 hr after in vitro maturation (IVM) or the centrifugation of denuded oocytes had no effect on maturation rates. Oocytes treated at 0 hr of IVM had a lower expulsion rate (50%) of the first polar body (PB1). The removal of cumulus cells and centrifugation affected the pattern of spindle microtubule distribution and division of chromosomes. There were almost no spindle microtubules allocated to PB1 and the spindles were swollen in anaphase I and telophase I oocytes. Approximately 20% of PB1 oocytes contained tripolar or multipolar spindles. After activation, oocytes denuded with or without centrifugation at 8 hr of IVM resulted in the lowest rate of development (3.0%). Denuded oocytes at 4, 15, and 24 hr of IVM with centrifugation or not resulted in similar blastocyst development rates (9.6%-13.2%). However, centrifugation of oocytes denuded at the beginning of IVM resulted in lower blastocyst development rate (8.1%, P < 0.05) than the noncentrifuged oocytes (17.3%). After nuclear transfer, the blastocyst development rates of oocytes denuded and centrifuged at 0, 4, and 8 hr of IVM were not different when compared to the same patch of noncentrifuged oocytes. However, oocytes denuded and centrifuged at 15 hr of IVM resulted in lower (P < 0.05) blastocyst development rates than the noncentrifuged oocytes. The results of this study suggest that removal of cumulus cells and centrifugation of denuded oocytes affect the spindle pattern. Embryo development of denuded and centrifuged oocytes may differ depending on the time of removal of cumulus cells.     

Changes in cumulus-oocyte complexes of pregnant and non-pregnant camels (Camelus dromedarius) during maturation in vitro

The aim of the present study was to examine the cumulus morphology and the oocyte chromatin quality of camel cumulus-oocyte complexes (COCs) at the time of recovery, and to monitor changes in oocyte chromatin configuration and apoptosis in cumulus cells from camel COCs during in vitro maturation (IVM) (0, 12, 24, 32, 36, 42, and 48 p.IVM) depending on pregnancy of donors. A total of 1023 COCs were isolated from sliced ovaries after slaughtering of 47 pregnant and 43 non-pregnant camels in an abattoir. The mean number of COCs per donor was 10.3 in pregnant and 12.5 in non-pregnant donors. The cumulus morphology of COCs was independent of the type of donor and was divided in COCs with compact (26.9 and 28%), dispersed (39.3 and 46%), corona radiata cumulus investment (27.9 and 21.7%) and without cumulus (6 and 4.2%), respectively for pregnant and non-pregnant donors. The highest proportion of COCs exhibited dispersed cumulus (P<0.05). Oocytes with meiotic stages of diplotene >50% were found only in compact (55 and 56.5%) and in dispersed COCs (58.4 and 60%), respectively for pregnant and non-pregnant donors. During IVM (0-48h) the first significant onset of specific meiotic stages were different in oocytes from pregnant donors: metaphase 1 (24-32h), metaphase 2 (36-42h), versus oocytes from non-pregnant donors: metaphase 1 (24h), metaphase 2 (32-48h) (P<0.05). The level of apoptotic cells in cumuli of matured COCs increased during IVM and was higher in matured COCs from non-pregnant donors for each time point during IVM (P<0.01). Camel oocytes meiosis during IVM is accompanied by a drastic increase of apoptosis in the surrounding cumulus cells 0-32 and 0-24h during IVM, respectively for pregnant and non-pregnant donors. The oocytes of pregnant camels require 36h of maturation to reach levels of >50% metaphase 2 stage in comparison to oocytes from non-pregnant donors where 32h are sufficient. The earlier onset of apoptosis in the COCs derived from non-pregnant donors possibly determines the faster progression of the oocytes through the final stages of meiosis.     

Prepubertal goat oocytes from large follicles result in similar blastocyst production and embryo ploidy than those from adult goats

Developmental competence of oocytes from prepubertal females is lower than those from adult females. Oocyte development competence is positively related to follicular diameter. Most of the follicles of prepubertal goat ovaries are smaller than 3 mm. The aim of this study was to compare oocytes of two follicle sizes (< 3 mm and ≥ 3 mm) from prepubertal goats with oocytes from adult goats in relation to their in vitro production and quality of blastocysts. Oocytes from prepubertal goats were obtained from slaughterhouse ovaries and selected according to the follicle diameter whereas oocytes from adult goats were recovered in vivo by LOPU technique without prior selection of follicle size. COCs were IVM for 27 h, IVF at the conventional conditions with fresh semen and presumptive zygotes were cultured in SOF medium for 8 days. Blastocysts obtained were vitrified and after warming their blastocoele re-expansion and the ploidy by FISH technique were assessed. We found significant differences between blastocysts yield of oocytes recovered from follicles smaller than 3 mm of prepubertal goats compared to those from adult goats (5.45% vs 20. 83%, respectively) however, these differences disappear if oocytes were recovered form large follicles (18.07%). A total of 28 blastocysts were analysed and 96.43% showed mixoploidy. Age did not affect the number of embryos with abnormal ploidy or blastocyst re-expansion after warming. Furthermore, the percentage of diploid blastomeres per embryo was similar in the 3 groups studied, adult, prepubertal from follicles ≥ 3 mm and < 3 mm (68.6%, 80.8% and 73.6%, respectively). In conclusion, IVP of blastocysts coming from follicles larger than 3 mm of goats 45 days old were not different to the blastocysts produced from adult goats, both in terms of quantity and quality.     

Apoptotic index within cumulus cells is a questionable marker of meiotic competence of bovine oocytes matured in vitro

Information gained from most human studies indicate a negative correlation between the apoptotic index (AI) in cumulus cells (CC) and the quality of the corresponding oocytes. However, results obtained in other species are not so consistent. The rate of apoptosis-free COCs (cumulus oocytes complexes) subjected to IVM (in vitro maturation) also varies among studies. The aim of the present study was to investigate whether the AI in cumulus cells of post-IVM COCs is related to the morphology of pre-IVM COCs and to meiotic competence of bovine oocytes. COCs of known morphology (four grade scale) obtained from individual follicles were matured in a well-in-drop system. After IVM, the external layers of CC of each COC were analyzed by TUNEL. In order to determine the meiotic stage, oocytes were stained with DAPI. It was found that 25.6% of bovine COCs contained apoptosis-free cumulus cells. Moreover, the majority of COCs with apoptotic cells were characterized by apoptotic index lower than 15%. The level of apoptosis in CC was related neither to COC morphology nor to the oocyte meiotic stage. It is suggested that the extent of apoptosis in cumulus cells is not a reliable quality marker of the corresponding oocyte after IVM.     

Effect of follicular size on in vitro developmental competence of oocytes and viability of embryos after transfer in the dromedary (Camelus dromedarius)

The aim of this work was to determine the effect of follicle size on camel oocyte quality as measured by developmental competence in vitro and in vivo. Ovaries from a local slaughterhouse were dissected to obtain two classes of follicle size: small (3-6 mm) and large (>6 mm) follicles. Quality of the oocytes was assessed after in vitro maturation (IVM), in vitro fertilization (IVF) and in vitro culture (IVC) of cumulus oocyte complexes (COCs). All cultures were done in four replicates at 38.5 degrees C, under 5% CO(2) and high humidity (>95%). Only COCs with cumulus and homogenous (dark) cytoplasm were used. The COCs were matured for 28 h in TCM-199 medium supplemented with 10% heat-treated fetal calf serum (FCS), 10 ng/mL EGF, and 250 microM cysteamine. Nuclear maturation rate for each class of follicle size was determined by contrast phase microscopy in a sample of COCs (n=30) denuded, fixed and stained with aceto-orcein. In vitro fertilization was performed using fresh semen (0.5 x 10(6)spermatozoa/mL in modified TALP-solution). Fertilized oocytes were cultured in mKSOMaa, under 5% O(2) and 90% N(2). The percentage of COCs reaching metaphase II (MII) after 28 h of maturation was 87% (26/30) and 73% (22/30) for oocytes originating from large and small follicles, respectively (P>0.1). The rate of total cleavage (two cells to blastocyst stage) was greater (P<0.05) for oocytes originating from large follicles (72%; 116/162) than for those derived from small follicles (59%; 140/237). The percentage of fertilized oocytes reaching the blastocyst stage was 35% (57/162) and 20% (48/237) for oocytes collected from large and small follicles, respectively (P<0.05). The viability of in vitro-produced hatched blastocyst from the two groups (15 from 3 to 6mm follicle size and 22 from follicles >6 mm) was assessed by transfer to synchronized recipients. None of the hatched blastocysts from small follicles resulted in a pregnancy whereas 68% (15/22) of the transferred hatched embryos from large follicles developed into a 25-day pregnancy. Of the resulting 15 pregnancies, 53% (n=8) aborted (five between 2 and 4 months and three between 5 and 7 months of pregnancy). The remaining seven pregnant females gave birth to normal healthy offsprings (four females and three males). The present study shows that dromedary oocytes developmental competence is acquired late during the final phase of follicular development and this developmental ability translates into greater pregnancy rates after transfer of in vitro produced hatched blastocysts.     

Oocyte-secreted factor activation of SMAD 2/3 signaling enables initiation of mouse cumulus cell expansion

Expansion of the mouse cumulus-oocyte complex (COC) is dependent on oocyte-secreted paracrine factors. Transforming growth factor beta (TGFB) superfamily molecules are prime candidates for the cumulus expansion-enabling factors (CEEFs), and we have recently determined that growth differentiation factor 9 (GDF9) alone is not the CEEF. The aim of this study was to examine oocyte paracrine factors and their signaling pathways that regulate mouse cumulus expansion. Using RT-PCR, oocytes were found to express the two activin subunits, Inhba and Inhbb, and activin A and activin B both enabled FSH-induced cumulus expansion of oocytectomized (OOX) complexes. Follistatin, an activin-binding protein, neutralized activin-induced expansion but had no effect on oocyte-induced expansion. The type I receptors for GDF9 and activin are activin receptor-like kinase 5 (ALK5) and ALK4, respectively, both of which activate the same SMAD 2/3 signaling pathway. We examined the requirement for this signaling system using an ALK 4/5/7 inhibitor, SB-431542. SB-431542 completely ablated FSH-stimulated GDF9-, activin A-, activin B-, and oocyte-induced cumulus expansion. Moreover, SB-431542 also antagonized epidermal growth factor-stimulated, oocyte-induced cumulus expansion. Using real-time RT-PCR, SB-431542 also attenuated GDF9-, activin A-, and oocyte-induced OOX expression of hyaluronan synthase 2, tumor necrosis factor alpha-induced protein 6, prostaglandin synthase 2, and pentraxin 3. This study provides evidence that the CEEF is composed of TGFB superfamily molecules that signal through SMAD 2/3 to enable the initiation of mouse cumulus expansion.     

Presence of granulosa cells during oocyte maturation improved in vitro development of IVM-IVF bovine oocytes that were collected by ultrasound-guided transvaginal aspiration

The effects of granulosa cells in maturation media on male pronuclear formation and in vitro development of in vitro-matured and fertilized (IVM-IVF) bovine oocytes were examined. In Experiment 1, cumulus-oocyte complexes (COCs) were aspirated from follicles of slaughterhouse ovaries and classified into 4 morphological categories according to the surrounding cumulus cells: Grade 1 (> 4 layers), Grade 2 (3 to 4 layers), Grade 3 (1 to 2 layers) and Grade 4 (denuded). Oocytes were co-cultured with or without granulosa cells (1 x 10(6) cells/ml) for 21 to 22 h. At 18 and 192 h after insemination, the abilities of oocytes to form a male pronucleus and develop up to the blastocyst stage in vitro were determined, respectively. The presence of granulosa cells during maturation did not affect (P < 0.05) the ability of oocytes in Grades 1 and 2 to form a male pronucleus and to develop to the blastocyst stage in Grades 1 and 4. However, the incidence of male pronuclear formation in Grades 3 and 4 and in vitro development to the blastocyst stage in Grades 2 and 3 was higher (P < 0.05) when COCs were cultured in the presence of granulosa cells than when cultured in the absence of granulosa cells. In Experiment 2, COCs collected by ultrasound-guided aspiration were co-cultured with or without granulosa cells, fertilized and cultured as described above. The incidence of blastocysts at 192 h after insemination was higher (P < 0.05) when COCs were cultured for maturation in the presence of granulosa cells (24%) than in the absence of granulosa cells (12%). These results demonstrate that supplementation of maturation medium with granulosa cells improves the quality of oocytes with relatively few cumulus cell layers, as determined by male pronuclear formation and in vitro development. We also conclude that this supplementation effectively improves the developmental ability of bovine IVM-IVF oocytes that were collected by ultrasound-guided transvaginal aspiration.     

The influence of cumulus cells during in vitro fertilization of buffalo (Bubalus bubalis) denuded oocytes that have undergone vitrification

The aim of this work was to evaluate whether providing a support of cumulus cells during IVF of buffalo denuded oocytes submitted to vitrification-warming enhances their fertilizing ability. In vitro matured denuded oocytes were vitrified by Cryotop in 20% EG + 20% of DMSO and 0.5 M sucrose and warmed into decreasing concentrations of sucrose (1.25 M-0.3M). Oocytes that survived vitrification were fertilized: 1) in the absence of a somatic support (DOs); 2) in the presence of bovine cumulus cells in suspension (DOs+susp); 3) on a bovine cumulus monolayer (DOs+monol); and 4) with intact bovine COCs in a 1:1 ratio (DOs+COCs). In vitro matured oocytes were fertilized and cultured to the blastocyst stage as a control.An increased cleavage rate was obtained from DOs+COCs (60.9%) compared to DOs, DOs+susp (43.6 and 38.4, respectively; P < 0.01) and DOs+monol (47.5%; P < 0.05). Interestingly, cleavage rate of DOs+COCs was similar to that of fresh control oocytes (67.8%). However, development to blastocysts significantly decreased in all vitrification groups compared to the control (P < 0.01).In conclusion the co-culture with intact COCs during IVF completely restores fertilizing capability of buffalo denuded vitrified oocytes, without improving blastocyst development.     

Androgens augment the mitogenic effects of oocyte-secreted factors and growth differentiation factor 9 on porcine granulosa cells

In this study, we test the hypothesis that the growth-promoting action of androgens on granulosa cells requires paracrine signaling from the oocyte. Mural granulosa cells (MGCs) from small antral (1-3 mm) prepubertal pig follicles were cultured in the presence or absence of denuded oocytes (DO) from the same follicles to determine whether mitogenic and/or steroidogenic responses, to combinations of FSH, insulin-like growth factor 1 (IGF1), and dihydrotestosterone (DHT) were influenced by oocyte-secreted factors (OSFs). To further explore the identity of such factors we performed the same experiments, substituting growth differentiation factor 9 (GDF9), a known OSF, for the DO. OSFs and GDF9 both potently enhanced IGF1-stimulated proliferation, and inhibited FSH-stimulated progesterone secretion. Alone, DHT had little effect on DNA synthesis, but significantly enhanced the mitogenic effects of OSFs or GDF9 in the presence of IGF1. Denuded oocytes, GDF9, and DHT independently inhibited FSH-stimulated progesterone secretion, and androgen, together with DO or GDF9, caused the most potent steroidogenic inhibition. Focusing on mitogenic effects, we demonstrate that both natural androgen receptor (AR) agonists, testosterone and DHT, dose-dependently augmented the mitogenic activity of DO or GDF9. Antiandrogen (hydroxyflutamide) treatment, which is used to block androgen receptor activity, opposed the interaction between androgen and GDF9. In conclusion, androgens stimulate porcine MGC proliferation in vitro by potentiating the growth-promoting effects of oocytes or GDF9, via a mechanism that involves the AR. These signaling pathways are likely to be important regulators of folliculogenesis in vivo, and may contribute to the excess follicle growth that is observed in androgen-treated female animals.