Cumulus cell function during bovine oocyte maturation,fertilization, and embryo development in vitro

Several contemporary micromanipulation techniques, such as sperm microinjection, nuclear transfer, and gene transfer by pronuclear injection, require removal of cumulus cells from oocytes or zygotes at various stages. In humans, the cumulus cells are often removed after 15–18 hr of sperm-oocyte coincubation to assist the identification of the fertilization status. This study was designed to evaluate the function of cumulus cells during oocyte maturation, fertilization, and in vitro development in cattle. Cumulus cells were removed before and after maturation and after fertilization for 0,7,20, and 48 hr. The cumulus-free oocytes or embryos were cultured either alone or on cumulus cell monolayers prepared on the day of maturation culture. Percentages of oocyte maturation, fertilization, and development to cleavage, morula, and blastocyst stages and to expanding or hatched blastocysts were recorded for statistical analysis by categorical data modeling (CATMOD) procedures. Cumulus cells removed before maturation significantly reduced the rate of oocyte maturation (4–26% vs. 93–96%), fertilization (0–9% vs. 91–92%), and in vitro development at all stages evaluated. Cumulus cells removed immediately prior to in vitro fertilization (IVF) or 7 hr after IVF reduced the rates of fertilization (58–60% and 71%, respectively, vs. 91–92% for controls), cleavage development (40–47% and 53–54% vs. 74–78% for controls), and morula plus blastocyst development (15% and 24% vs. 45%, P < 0.05). Cumulus cell co-culture started at various stages had no effect on fertilization and cleavage development but significantly improved rates of embryo development to morula or blastocyst stages (P < 0.05). Cumulus cell removal at 20 hr after IVF resulted in similar development to controls (P > 0.05) at all stages tested in this study. The intact state of surrounding cumulus cells of oocytes or embryos appears to be beneficial before or shortly after insemination (at or before 7 hr of IVF) but not essential at 20 hr after IVF. © 1995 Wiley-Liss, Inc.     

In vitro equine oocyte maturation in pure follicular fluid plus interleukin-1 and fertilization following ICSI

The interleukin-1 (IL-1) system is thought to be involved in periovulatory events in the mare. Previous in vivo studies have demonstrated that IL-1β induces oocyte maturation, but depresses the pregnancy rate 14 days after ovulation. To better understand the role of IL-1 in oocyte maturation and fertilization, the effects of IL-1 on the in vitro maturation rate of equine oocytes in pure follicular fluid were evaluated and fertilization rate assessed following intracytoplasmic sperm injection (ICSI). Oocytes collected from slaughterhouse ovaries were cultured in four different media for 30 h prior to fertilization. Two experiments were performed, each using three maturation media as the experimental treatments. Medium 1 was pure follicular fluid from subordinate follicles. Medium 2 was medium 1 plus 50 ng/ml recombinant human IL-1β. Medium 3 was pure follicular fluid collected from mares administered crude equine gonadotropin (CEG). Medium 4 was medium 2 plus 50 ng/ml of recombinant human IL-1 receptor antagonist. Media 1, 2 and 3 were compared in experiment 1. In experiment 2, media 1, 2 and 4 were compared. After maturation, metaphase II oocytes were submitted to microinjection and assessed for signs of fertilization. In experiment 1, 101 oocytes were evaluated. The rate of polar body extrusion was 66, 51 and 68% and the proportions of normally fertilized oocytes after ICSI were 40, 18 and 38% for media 1, 2 and 3, respectively. In experiment 2, 122 oocytes were evaluated. The rate of polar body extrusion was 55, 48 and 42% and the proportions showing normal fertilization after ICSI were 14, 25 and 29% for media 1, 2 and 4, respectively. There was no positive effect of IL-1β on maturation in both experiments, but the fertilization rate and percentage of embryos reaching four-cell were low in the presence of IL-1β, indicating that this cytokine may interfere with fertilization and early embryo development.     

Factors affecting nuclear maturation, cleavage and embryo development of vitrified bovine cumulus-oocyte complexes

The objective was to investigate the effects of cryodevice, vitrification solutions, and equilibration time on in vitro maturation, cleavage, and embryo development of vitrified bovine oocytes. In Experiment 1, the nuclear maturation (MII) rate of immature bovine COCs vitrified was compared between two equilibration times (0 vs 10 min) in vitrification solution 1 (VS1) and two cryodevices (cryotop vs 0.25 mL straw). The MII rate was higher in the non-vitrified control group than in vitrified groups (61 vs 16%, P < 0.0001). Equilibration time did not affect MII rate (P = 0.964); however, the MII rate was higher for COCs vitrified on cryotops than in straws (23 vs 9%, P = 0.007). In Experiment 2, bovine COCs were vitrified on cryotops using two equilibration times (0 vs 5 min) in VS1 and two kinds of vitrification solutions (freshly prepared vs frozen). Cleavage and blastocyst rates were higher (P < 0.0001) in the non-vitrified control group than vitrified groups (cleavage rate 93 vs 42% and blastocysts rate 31 vs 0.4%). Cleavage rate of COCs vitrified using frozen solutions with 5 min equilibration was higher (P = 0.05) than other treatment groups. However, blastocyst rate did not differ (P = 0.993) among treatment groups. In conclusion, cryotop was a better cryodevice than 0.25 mL straw for vitrification of bovine COCs. Furthermore, 5 min equilibration in VS1 improved cleavage. Compared with control, the vitrification procedure per se damaged bovine COCs, resulting in poor nuclear maturation and embryo development. However, vitrification did not immediately kill oocytes, as the cleavage rate was acceptable.     

Influence of serum and hormones on bovine oocyte maturation and fertilization in vitro

Three approaches were investigated for improvement of in vitro maturation (IVM), in vitro fertilization (IVF), and early embryonic development in cattle. These were: 1) Selection of oocytes, 2) medium supplementation with fetal calf serum (FCS) and cow sera (DO, Dl, D10, and D20 to correspond with estrus, metestrus, diestrus, and proestrus, respectively), and 3) addition of follicle-stimulating hormone (FSH), luteinizing hormone (LH), and estradiol-17β (E₂)during maturation. Greater proportions (percentage) of oocytes initially selected for their compact cumulus cells completed IVM and IVF when compared to unselected oocytes (P < .05). Proportions (percentage) of selected oocytes that matured and cleaved after in vitro insemination according to serum type used for IVM were: FCS: 110/175 (62.9%) and 37/110 (33.6%) and DO: 130/145 (89.7%) and 52/130 (40.0%); D1 127/130 (97.7%) and 41/127 (32.3%); D10 95/98 (96.9%) and 35/95 (36.8%); D20:113/116 (97.4%) and 49/113 (43.4%). A higher proportion (P < .05) of embryos resulting from the D20 group reached four- and eight-cell stages. In FCS-supplemented maturation media with no hormones added during maturation (control), results of IVM and IVF were 157/265 (59.2%) and 39/157 (24.8%), respectively. With E₂ (1 μg/ml) and FSH (5 μg/ml), comparable results were 189/215 (87.9%) and 71/189 (37.6%); with E₂ (1 μg/ml) plus LH (10 μml), 280/327 (85.6%) and 111/280 (39.6%). Added hormones improved IVM results (P < .05) and, when FSH or LH was added with E₂, in vitro development to four- and eight-cell stages was markedly enhanced (P < .05). Selection of oocytes, D20 serum, and added E₂ and FSH or LH for IVM improved in vitro development of bovine embryos after IVF.     

In vitro techniques of bovine oocyte maturation, fertilization and embryo culture resulting in the birth of a calf

Oocyte cumulus complexes were aspirated from 3 to 5 mm follicles of cows prestimulated with 2.000 IU PMSG 24 h before slaughter. Oocytes matured in culture were fertilized in vitro by heparinized freshly ejaculated or epididymal spermatozoa. The cultivation procedure for fertilized eggs was the same as that used for cultivation of oocytes. From 163 matured oocytes, 109 cleaved to the 2-cell stage 24 h after fertilization and after 6 days of cultivation, 18 developed to the late morula and 18 to the blastocyst stages. Eleven blastocyts and 1 late morula were transferred surgically to the uteri of 7 recipient heifers. Two heifers became pregnant: one delivered a bull-calf at term, while the other pregnancy resulted in abortion at the 3rd month. The examination of some embryos by transmission electron microscopy showed an almost normal morphology for most cells. The degenerated cells contained mostly electron-dense residual bodies of unknown origin.     

The effect of the composition of the culture media on bovine oocyte maturation and embryo development in vitro

We studied the capacity of the cattle oocyte for the resumption of meiosis and achievement of metaphase II in various protein-free culture media (DMEM, TCM-199, Ham's F-10, and Ham's F-12) and the pattern of influence of the estrous serum on in vitro development of fertilized cattle oocytes, with special reference to the time of its addition to the synthetic oviduct fluid containing BSA. In the first experimental series, it was shown that the highest number of oocytes (76.1%) resumed meiosis in DMEM medium. Meiosis was not resumed in Ham's F-12. Intermediate results were obtained for TCM-199 (55.1%), which is commonly used for maturation of cattle oocytes in vitro, and for Ham's F-10 (51.7%). The oocytes reached metaphase II in DMEM at a higher rate (45.3%) than in TCM-199 or Ham's F-10 (29.4 and 8.6%, respectively). In the second experimental series, the estrous serum was added to the culture medium within 20 h (control) or 42 h (experiment) after the beginning of fertilization. The estrous serum did not inhibit the first cleavage division (the percentage of cleaving embryos did not differ reliably: 32.7 and 37.9%, respectively). However, a later serum addition to the culture medium (within 42 h after the beginning of fertilization) reliably increased the percentage of embryos that reached the blastocyst stage (6.5% in the control and 17.8% in the experiment) and the hatched blastocyst stage (2 and 9.2%, respectively).     

Ammonia concentration in bovine follicular fluid and its effect during in vitro maturation on subsequent embryo development

10 mm) to determine ammonia concentration. Ammonia concentration was greater (P<0.001) in the bFF from smaller follicles compared with bFF from larger follicles, and ammonia concentration decreased (P10 mm follicles, respectively. In Experiment 2, abattoir oocytes (n=2885) were matured in vitro in media containing ammonia at the concentrations of 0 microM (Control), 29 microM (A1), 88 microM (A2), 132 microM (A3), 176 microM (A4) and 356 microM (A5), followed by in vitro fertilization and culture. There was no difference (P0.05) in cleavage rates and morula or blastocyst development between treatments, indicating that in vitro exposure of bovine oocytes to ammonia does not adversely influence subsequent embryonic development.     

The effect of oviductal epithelial cell co-culture during in vitro maturation on sow oocyte morphology,fertilization and embryo development

In vitro embryo production in the sow is challenged by poor cytoplasmic maturation, low sperm penetration and low normal fertilization, leading to the development of poor quality blastocysts containing a small number of nuclei. In prepubertal gilt oocytes, the presence of porcine oviductal epithelial cells (pOECs) during maturation increases cytoplasmic maturation and blastocyst development. These aspects, as well as blastocyst quality, may be improved when adult sow oocytes are matured with pOEC. Therefore, the effect of the presence of pOEC on sow oocyte morphology, fertilization and the progression of embryo development was evaluated. The pOEC were cultured in M199 for 18 h, then cultured in NCSU23 for 4 h before the oocytes were added. Oocytes from 2 to 6 mm follicles were matured in 500 microl NCSU23, with eCG and hCG, for 24 h, and then cultured with or without pOEC, in NCSU23 without hormones, for 18 h. In vitro fertilization took place in modified Tris-buffered medium, for 6 h, and the presumptive zygotes were then cultured for 162 h in NCSU23. Morphology of the IVM oocytes was compared to that of immature oocytes and in vivo matured MII oocytes from slaughtered sows in estrus. The in vitro matured oocytes had a greater diameter and a wider perivitelline space than the immature and in vivo matured MII oocytes (P < 0.01). Penetration, polyspermy and pronucleus formation did not differ between the pOEC and Control groups, although the total penetration rate was higher for the Control oocytes (26% versus 39%; P < 0.01). Fewer blastocysts developed in the pOEC group than in the Control group (19% versus 27%; P < 0.01), but blastocyst growth was accelerated, leading to a higher percentage of hatched blastocysts (3% versus 10%; P < 0.01). Finally, the average blastocyst cell number was higher in the pOEC group (47 versus 40; P < 0.05) and a greater percentage of blastocysts contained a superior number of nuclei. In conclusion, the addition of pOEC during the second half of in vitro maturation resulted in fewer blastocysts formed, but of those blastocysts that did form the quality was improved.     

Effect of adiponectin on bovine granulosa cell steroidogenesis,oocyte maturation and embryo development

Background  

Adiponectin is an adipokine, mainly produced by adipose tissue. It regulates several reproductive processes. The protein expression of the adiponectin system (adiponectin, its receptors, AdipoR1 and AdipoR2 and the APPL1 adaptor) in bovine ovary and its role on ovarian cells and embryo, remain however to be determined.     

Protein kinase C (PKC) role in bovine oocyte maturation and early embryo development

The aims of the present study were to determine the role of protein kinase C (PKC) on meiotic resumption and its effects on pronuclear formation and cleavage in the bovine. Oocytes were matured in the presence of 0, 1, 10 and 100 nM of phorbol 12-myristate 13-acetate (PMA), to evaluate the percentage of germinal vesicle breakdown. To study pronuclear formation and cleavage, oocytes were randomly distributed in four groups and matured in modified TCM-199 with LH and FSH (negative control); 10% of estrous cow serum (positive control); 100 nM of PMA (treatment); 100 nM of 4alpha-PDD (phorbol ester control). Oocytes were also matured in positive control medium, fertilized and transferred to KSOM with increasing concentrations of a PKC inhibitor. The protein profile and the presence of PKC at the end of maturation period were determined by SDS-PAGE followed by Silver Stain and Western blot, respectively. PMA stimulated meiotic resumption in a concentration-dependent manner. PKC stimulation during oocyte maturation caused an increase in pronuclear formation and did not cause parthenogenetic activation. Inhibitor of PKC (MyrPKC) inhibited cleavage in a dose-dependent and irreversible manner. A protein band around 74 kDa was not detected in PMA-treated oocytes and PKC was not detected by Western blot at the end of the maturation period. In conclusion, meiotic resumption was accelerated and the rate of oocytes with two pronuclei was increased when PKC was activated during oocyte maturation. Moreover, cleavage was inhibited in the presence of PMA.     

Current concepts of cell-cycle regulation and its relationship to oocyte maturation, fertilization and embryo development

Genetic and biochemical approaches have contributed to an explosion of literature on cell-cycle control. Regulation of the cell-cycle is controlled by a series of kinases and phosphatases. Key control points are during the G(1)-S and G(2)-M transitions. During both transitions, cyclins interact with a specific kinase to allow a cell to pass through that phase. The meiotic maturation of oocytes, fertilization and embryo development are all events influenced by cell-cycle regulation. Understanding cell-cycle control should provide new ways for gamete and embryo biologists to approach culture and development problems.     

Bovine oocyte maturation, fertilization and further development in vitro and after transfer into recipients

Bovine oocytes were aspirated from ovaries within 1.6 to 2 hours after slaughter. They were then matured in TCM-199 medium drops under oil in CO(2)/air incubator at 39 degrees C. Spermatozoa were capacitated in SP-TALP medium with heparin. The percentage of embryos that developed in vitro to the 4- and 6- cell stages 48 hours post insemination and then reached the morula or blastocyst stage was 64.3% and 59.2%, respectively, while only 3.6% of the embryos that reached the 2-cell stage became morula or blastocysts. An average of 6.3+/-3.2 total in vitro fertilized embryos per cow were obtained (range 2 to 11). Maturation of bovine oocytes in vitro for 18 or 24 hours did not influence the percentage of cleaved embryos (71.0 and 75.9%, respectively) or that developed to the blastocyst stage (25.6 and 24.2%, respectively). The use of reindeer blood serum for in vitro culture of immature bovine oocytes and of dividing of embryos gave the following results: 57.4% of the oocytes cleaved after fertilization and 16.2% developed further to the blastocyst stage. In contrast in the control group, where cow serum was used, the values were 73.4% and 24.8%, respectively. Rabbit oviduct epithelium cell monolayers were able to support the development of 16.3% of the cleaved bovine embryos to the blastocyst stage as compared with 24.0% of the embryos on cow oviduct epithelium cell monolayers. After nonsurgical transplantation, 12 calves were produced from 91 in vitro fertilized embryos.     

Surface alterations of the bovine oocyte and its investments during and after maturation and fertilization in vitro

Surface characteristics of the bovine oocyte and its investments before, during, and after maturation, and fertilization in vitro were evaluated by scanning electron microscopy (SEM). Oocyte diameters were also measured during SEM analysis of the oocyte. The cumulus cells manifested a compact structure with minimal intercellular spaces among them in the immature oocytes. These became fully expanded with increased intercellular spaces after maturation in vitro, but contracted again after fertilization. The zona pellucida (ZP) showed a fibrous, open mesh-like structure in the maturing and matured oocytes. The size and number of meshes on the ZP decreased dramatically after fertilization. The vitelline surface of immature oocytes was characterized by distribution of tongue-shaped protrusions (TSPs) varying in density. After 10 and 22 hr of maturation incubation, oocyte surface microvilli (MV) increased to become the predominant surface structure, and TSPs decreased substantially. The vitelline surface of fertilized oocytes (at 6 and 20 hr) was similar to that of the matured oocytes, but unfertilized oocytes had less dense MV than did fertilized oocytes (at 20 hr). The diameter of the oocytes decreased from 99 to 80 μm during maturation and increased to 106 μm after insemination (P < 0.05). Membrane maturation was characterized by surface changes from a TSP-predominant pattern to a MV-predominant pattern. Thus, the bovine oocyte maturation process was found to involve the expansion of cumulus cells and the maturation of the ZP, which changes dramatically upon fertilization. Also, volumetric changes occurred in ooplasm processed for SEM following oocyte maturation and insemination. © 1994 Wiley-Liss, Inc.     

Effect of follicle-stimulating hormone and luteinizing hormone during bovine in vitro maturation on development following in vitro fertilization and nuclear transfer

The effects of luteinizing hormone (LH) (0, 100, 10,000 lU/ml) and follicle-stimulating hormone (FSH) (20 μg/ml) supplementation during in vitro maturation of slaughterhouse-derived oocytes on polar body formation and embryo development subsequent to in vitro fertilization and nuclear transfer were evaluated. Go-nadotropin supplementation of maturation medium in the presence of serum neither enhanced the proportion of oocytes forming a polar body nor significantly affected development following in vitro fertilization or nuclear transfer, except at the highest LH concentration. A very high concentration of LH (10,000 lU/ml) significantly decreased polar body formation, initial cleavage, and blastocyst development (P < 0.05). © 1993 Wiley-Liss, Inc.     

Mouse zona pellucida dynamically changes its elasticity during oocyte maturation, fertilization and early embryo development

A change in the elasticity of mouse zona pellucida was quantitatively evaluated during oocyte maturation, fertilization and early embryo development. Young's modulus of zona pellucida of germinal vesicle (GV), metaphase-II (MII), pronuclear (PN), 2cell, 4cell, 8cell, morulae (M) and early blastocyst (EB) stages was measured using a micro tactile sensor (MTS) and a chamber exclusively designed for the measurement. The MTS has very high sensitivity and a deformation of only 5 microm was sufficient to calculate the Young's modulus and the oocyte/embryo maintained its original spherical shape during the measurement. The Young's modulus of GV, MII, PN, 2cell, 4cell, 8cell, M and EB was 22.8+/-10.4 kPa (n=30), 8.26+/-5.22 kPa (n=74), 22.3+/-10.5 kPa (n=66), 13.8+/-3.54 kPa (n=41), 12.6+/-3.34 kPa (n=19), 5.97+/-4.97 kPa (n=6), 1.88+/-1.34 kPa (n=8) and 3.39+/-1.86 kPa (n=4), respectively. Experimental results clearly demonstrated that the mouse zona pellucida hardened following fertilization. Interestingly, once the zona pellucida hardened at the PN stage, it gradually softened as the embryo developed (i.e. it was found that the zona hardening is a transient phenomenon). Furthermore, the zona pellucida of the GV oocyte was as hard as that of the PN embryo and became soft as it matured to the MII stage. In addition, the safety of the MTS measurement for oocytes and embryos was discussed both theoretically and experimentally.     

Structural aspects of bovine oocyte maturation in vitro.

Bovine cumulus-oocyte complexes (COCs) were collected from 4-8 mm follicles and graded into four categories on their morphological characteristics. These four categories were matured in vitro and processed for transmission electron microscopy at 24 h after the onset of culture. The morphology of the four groups of oocytes was analysed and compared with that of oocytes that had matured in vivo and were collected 20-23 h after the preovulatory luteinizing hormone peak. After in vivo maturation, oocytes formed a homogeneous group with respect to their morphological characteristics. After in vitro maturation, the oocytes formed a heterogeneous group with respect to their morphology between as well as within the four categories of oocytes. Oocytes from the first three categories showed the same morphology after in vitro maturation. The fourth category showed some specific characteristics: 1) vacuolization, 2) flattening of cumulus cells, and 3) almost complete lack of cortical granules in some category 4 oocytes. These characteristics are interpreted as signs of degeneration. Besides these aspects of degeneration, other deviations from normal development were seen: 1) retraction of cumulus cell process endings from the oocyte without the breaking down of these processes, 2) retardation of some aspects of the cytoplasmic maturation, and 3) incomplete cumulus expansion. It is concluded that oocytes capable of development in vitro show a large morphological variability before the onset of culture. In vitro maturation systems can support normal development, but many oocytes show signs of degeneration and deviant development after in vitro maturation.     

In vitro maturation of bovine cumulus-oocyte complexes in undiluted follicular fluid: effect on nuclear maturation,pronucleus formation and embryo development

Since resumption of meiosis and cytoplasmic maturation of bovine oocytes takes place in close association with follicular fluid, it would be logical to assume that this might be a perfect maturation medium. To test the hypothesis, abattoir-derived cumulus-oocyte complexes (COCs) were in vitro matured in undiluted (i) mixed follicular fluid (FF) from 3 to 15 mm follicles from abattoir ovaries, (ii) preovulatory follicular fluid (POF) from the dominant follicle from a cyclic unstimulated heifer, (iii) preovulatory follicular fluid (OPU) from synchronised and superovulated heifers 60 h after prostaglandin and 20 h after GnRH treatment, and in (iv) TCM-199 with 5% serum. Subsequent to IVM, the COC were subjected to IVF and IVC, and embryo development was followed until the blastocyst stage at Day 8 after insemination. The MII rates in the TCM-199 (69%), POF (69%) and OPU (72%) groups were not different from each other but different from the FF (41%) group (P<0.05). In spite of the high MII rates, none of the follicular fluids supported embryo development: the FF, POF and OPU blastocyst rates were alike (3%, 3%, 2%) and different (P<0.05) from the rates in the TCM-199 (19%). During IVM in follicular fluids but not in TCM-199, the expanded cumulus masses became trapped in a coagulum. Although it could be prevented by the presence of heparin during IVM, it did not improve the blastocyst rates. In conclusion, undiluted preovulatory follicular fluids supported nuclear maturation but not further embryonic development as judged by the high MII and low blastocyst rates.     

High oxygen tension during in vitro oocyte maturation improves in vitro development of porcine oocytes after fertilization

This study was conducted to evaluate the effect of oxygen tension during IVM and/or IVC on developmental competence of porcine follicular oocytes. Prospective, randomized experiments were designed, and oocytes were matured, inseminated and cultured in vitro in the designated condition. In experiment 1, either high (20%) or low (7%) oxygen tension was used for IVM. The high oxygen significantly improved blastocyst formation (23% versus 13%; P<0.01) after IVF than the low oxygen. Such treatment, however, did not significantly (P>0.05) improve the rates of nuclear maturation (89% in each treatment), sperm penetration (62-72%), monospermic fertilization (56-67%), pronuclear formation (90-96%), cleavage (49-53%) and blastocyst cell number (31-32 cells). In experiment 2, the combined effect of oxygen tension during IVM and IVC of embryos was evaluated by a 2 x 2 factorial arrangement. Again, the high oxygen tension during IVM supported blastocyst formation more efficiently (P<0.01) than the low oxygen, and this was independent of oxygen tension during IVC (26-28% versus 15-16%). In oocytes matured under the high oxygen, a tendency to increase blastomere number (P=0.0630) was found, when the low oxygen was used for IVC after insemination (39-45 cells/blastocyst). In conclusion, the use of high oxygen tension (20% maintained by exposure to 5% CO2 in air) for IVM of porcine oocytes promoted blastocyst formation in vitro.