A potential role for triglyceride as an energy source during bovine oocyte maturation and early embryo development

The potential role of endogenous triglyceride in bovine oocyte maturation and preimplantation development has been investigated. Bovine immature oocytes were recovered from abattoir-derived ovaries, matured and fertilised in vitro and the zygotes grown to the blastocyst stage in SOFaaBSA. Methyl palmoxirate (MP) blocks the oxidation of fatty acids by inhibiting mitochondrial carnitine palmitoyltransferase A. The development of zygotes exposed to MP during oocyte maturation, and of zygotes exposed to MP during embryo culture has been assessed in terms of oxygen consumption by oocytes and embryos during a 4-6 hr incubation period in the presence of MP and as blastocyst formation and cell number. Immature oocytes exposed to MP during maturation had reduced capacity to form blastocysts after fertilisation; the same effect was apparent, but to a lesser extent, in zygotes exposed to MP during embryo development. Oxygen consumption values of oocytes and blastocysts in the absence of exogenous substrates were similar to those in control medium containing nutrients. MP-inhibited oxygen consumption of immature oocytes, mature oocytes, cleavage stages embryos and blastocysts by 64, 45, 12 and 13%, respectively. The data are consistent with a role for triglyceride as a key energy source during bovine oocyte maturation and potentially, during preimplantation embryo development.     

The importance of mitochondrial metabolic activity and mitochondrial DNA replication during oocyte maturation in vitro on oocyte quality and subsequent embryo developmental competence

Mitochondrial metabolic capacity and DNA replication have both been shown to affect oocyte quality, but it is unclear which one is more critical. In this study, immature oocytes were treated with FCCP or ddC to independently inhibit the respective mitochondrial metabolic capacity or DNA replication of oocytes during in vitro maturation. To differentiate their roles, we evaluated various parameters related to oocyte maturation (germinal vesicle break down and nuclear maturation), quality (spindle formation, chromosome alignment, and mitochondrial distribution pattern), fertilization capability, and subsequent embryo developmental competence (blastocyst formation and cell number of blastocyst). Inhibition of mitochondrial metabolic capacity with FCCP resulted in a reduced percent of oocytes with nuclear maturation; normal spindle formation and chromosome alignment; evenly distributed mitochondria; and an ability to form blastocysts. Inhibition of mtDNA replication with ddC has no detectable effect on oocyte maturation and mitochondrial distribution, although high-dose ddC increased the percent of oocytes showing abnormal spindle formation and chromosome alignment. ddC did, however, reduce blastocyst formation significantly. Neither FCCP nor ddC exposure had an effect on the rate of fertilization. These findings suggest that the effects associated with lower mitochondrial DNA copy number do not coincide with the effects seen with reduced mitochondrial metabolic activity in oocytes. Inhibiting mitochondrial metabolic activity during oocyte maturation has a negative impact on oocyte maturation and subsequent embryo developmental competence. A reduction in mitochondrial DNA copy number, on the other hand, mainly affects embryonic development potential, but has little effect on oocyte maturation and in vitro fertilization.     

Control of oocyte maturation in cows--biological factors

Since bovine in vitro fertilization became possible in the early 80s, a lot of effort has been done to clarify the mechanisms of what seems more and more one of the crucial steps in this procedure, being oocyte maturation. Undoubtedly, many biological factors act together to prepare the immature oocyte for a successful development to a competent embryo after fertilization. Defects in oocyte maturation can possibly be caused by an inadequate nuclear or cytoplasmic maturation or even by a failure of both. There is a general agreement upon the fact that the origin of the oocyte can play an important role. Oocytes derived from very small follicles show a lower rate of maturation and lower blastocyst development with currently used maturation protocols. Parthenogenetic activation of small size follicle derived oocytes suggests that their poor development was not caused by fertilization problems but more likely by intrinsic oocyte factors. Similar developmental rates achieved through nuclear transfer and parthenogenetic activation suggests that the nucleus of the incompetent oocyte may not be the sole reason for a poor development. Another important factor appears to be the donor animal age. The younger the donor animal, the more impaired is its oocyte's developmental competence in most of the embryo IVP systems. Treatment with exogeneous gonadotropins can be beneficial in young donors on the oocyte cleavage rates but does not always increase the final blastocyst outcome. This review briefly documents some of the biological factors and their possible effects on the developmental capacities of the bovine oocyte in vitro.     

Effects of oocyte quality, oxygen tension, embryo density, cumulus cells and energy substrates on cleavage and morula/blastocyst formation of bovine embryos

Various factors, such as quality of the oocyte, oxygen tension, embryo density, and kind of energy substrate during in vitro production of embryos may affect the rate of preimplantation embryo development. In the present study we used 12553 bovine oocytes aspirated from slaughterhouse ovaries to evaluate various culture conditions that would increase in vitro production of advanced stages of preimplantation embryos. The morphological quality of the oocyte based on the compactness and number of layers of cumulus cells had significant positive effects on the rates of in vitro maturation, fertilization and development to the morula and blastocyst stages. None of the corona-enclosed or nude oocytes progressed beyond the 8- to 16-cell stage. The level of oxygen (5 or 20%) did not affect the proportion of one-cell embryos undergoing cleavage or progressing to morula and blastocyst stages. The rate of development of one-cell embryos originating from inferior quality oocytes was significantly improved when cultured in groups of 40 instead of 20 embryos per 0.5 mL medium. In the presence of cumulus cells, glucose had beneficial effects on in vitro maturation and subsequent development of IVM-IVF zygotes. The presence of serum improved the rate of in vitro development of one-cell embryos. Minimum Essential Medium supplemented with energy substrates according to the findings of metabolic studies was less effective in supporting in vitro maturation and subsequent development than TCM-199. In conclusion, morphological grading of immature oocytes is an appropriate selection criterion for their developmental ability. Embryo yields from low quality oocytes can be increased by culturing them in large groups. Serum is not essential for in vitro generation of embryos but its addition improves rates of success.     

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.     

Cytoplasmic changes and developmental competence of bovine oocytes cryopreserved without cumulus cells

The cryopreservation of female gametes is still an open problem because of their structural sensitivity to the cooling-and-freezing process and to the exposure to cryoprotectants. The present work was aimed to study the effect of vitrification on immature bovine oocytes freed of cumulus cell investment before freezing. To verify the feasibility and efficiency of denuded oocyte (DO) cryopreservation, the cytoplasmic alterations eventually induced either by cell removal or by the vitrification process were analyzed. In particular, the migration of cortical granules and Ca++ localization were studied. In addition, the localization and distribution of microtubules and microfilaments in immature fresh and vitrified DOs were evaluated. Finally, to establish whether the removal of cumulus cells influenced developmental competence, DOs were thawed after vitrification, matured in vitro and fertilized; then presumptive zygotes were cultured to reach the blastocyst stage. The results indicate that mechanical removal of cumulus cells from immature bovine oocytes does not affect their maturation competence but reduces the blastocyst rate when compared with intact cumulus oocyte complexes (COCs). The findings indicate further that the vitrification process induces changes of cytoplasmic components. However, the composition of the manipulation medium used to remove cumulus cells plays a crucial role in reducing the injuries caused by cryopreservation in both cytoplasmic and nuclear compartments. In fact, the presence of serum exerts a sort of protection, significantly improving both oocyte maturation and blastocyst rates. In conclusion, we demonstrate that denuded immature oocytes can be vitrified after cumulus cells removal and successfully develop up, after thawing, to the blastocyst stage, following in vitro maturation and fertilization.     

Nuclear and cytoplasmic maturation of sow oocytes are not synchronized by specific meiotic inhibition with roscovitine during in vitro maturation

The effect of roscovitine exposure prior to IVM was studied on cumulus expansion, on changes of cumulus-oocyte contacts and on nuclear and cytoplasmic maturation of sow oocytes. It was hypothesized that delayed nuclear maturation and prolonged contact with cumulus cells allows prolonged cytoplasmic differentiation and therefore improves oocyte developmental potential. Cumulus-oocyte complexes (COCs) were exposed for 22 h or 44 h to 0, 25 or 50 microM of roscovitine and subsequently cultured for 22, 29 or 44 h without roscovitine. COCs were examined for cumulus expansion and oocytes for nuclear status and dynamics of transzonal microfilaments. Oocyte developmental potential was assessed by blastocyst formation after IVF. Fifty muM of roscovitine inhibited cumulus expansion for the first 22 h of culture, and maintained oocytes in meiotic arrest for 44 h. Roscovitine treatment during 22 h prior to culture for 44 h without roscovitine did not increase embryo development, but oocytes cultured for 66 h without roscovitine had reduced blastocyst formation. Oocytes cultured for 29 h after roscovitine exposure showed reduced blastocyst rates compared with their counterparts cultured for 44 h. Roscovitine treatment during 44 h prior to culture for 22 h or 44 h without roscovitine reduced embryo development. Transzonal microfilaments were reduced after culture with roscovitine, and disappeared during culture without roscovitine. It is concluded that prolonged contact with cumulus cells does not improve oocyte developmental potential. Furthermore, it is suggested that nuclear and cytoplasmic maturation in vitro cannot be seen as two independent processes.     

Meiosis-activating sterol promotes the metaphase I to metaphase II transition and preimplantation developmental competence of mouse oocytes maturing in vitro

The objective of this study was to determine the effects of a sterol found in ovarian follicular fluid, known as meiosis-activating sterol (FF-MAS), on the maturation of mouse oocytes in vitro. Possible effects of FF-MAS in promoting the metaphase I (MI) to metaphase II (MII) transition (nuclear maturation) and the competence of oocytes to complete preimplantation embryo development to the blastocyst stage (cytoplasmic maturation) were assessed. Cumulus cell-enclosed oocytes that were compromised in their ability to undergo nuclear maturation and subsequent development because of the age or genotype of the female were isolated at the germinal vesicle stage and matured in vitro using media supplemented with 0 to 20 microM FF-MAS. Oocytes that progressed to MII were inseminated in vitro, and the percentages developing to the 2-cell and blastocyst stages were determined. The sterol was omitted from the media used for oocyte insemination or preimplantation development. FF-MAS promoted a significantly higher percentage of oocytes in all groups to progress to MII in vitro. Moreover, FF-MAS treatment of oocytes maturing in vitro dramatically increased the competence of all but one of the groups of oocytes to complete preimplantation development. Therefore, FF-MAS improved mouse oocyte quality by promoting both nuclear and cytoplasmic maturation in vitro.     

Effect of prematuration, meiosis activating sterol and enriched maturation medium on the nuclear maturation and competence to development of calf oocytes

New strategies were proposed to improve the developmental competence of calf oocytes through in vitro technologies. Cumulus-oocyte complexes were first prematured for 24 h in the presence of meiosis inhibitors. Both Roscovitine alone (50 microM) or in combination with Butyrolactone-I (12.5 microM Rosco+6.25 microM BL-I) prevented the progression of meiosis. Their effect on nuclear maturation was reversible after a further 17 or 24 h maturation step. However, a dramatic decrease in embryo development was observed after fertilization (abattoir oocytes: 4-9% blastocyst rate versus 14-17% for control embryos). Similar results were obtained with oocytes collected by Ovum Pick Up from living donors. No pregnancy was obtained after single transfer of two blastocysts obtained from prematured oocytes (0/2 versus 4/12 for control embryos). Adding low concentrations (1, 3 or 10 microM) of follicular fluid-meiosis activating sterol (FF-MAS) during the maturation step had a beneficial effect on nuclear maturation (73-86% metaphase II versus 58% for control oocytes). However, subsequent embryo development was not improved. Enriching the maturation medium, namely with hormones, growth factors and precursors of glutathione, induced a sixfold increase in glutathione in the oocyte and had a beneficial effect on embryo development (38% increase in blastocyst rate). In conclusion, in opposition to the results reported with adult oocytes, prematuring calf oocytes had a negative impact on their developmental potential. Although FF-MAS improved nuclear maturation, its addition in the maturation medium did not increase embryo development. However, enriching the maturation medium had a positive effect on embryo development, indicating that cytoplasmic maturation was improved.     

Capacity of adult and prepubertal mouse oocytes to undergo embryo development in the presence of cysteamine

The present study was carried out to study de novo glutathione (GSH) synthesis and to evaluate the effect of stimulating GSH synthesis during in vitro maturation (IVM) of adult and prepubertal mouse oocytes on the embryo developmental rate. Adult (8 weeks old) and prepubertal mice (24-26 days old) were primed with 5 IU of PMSG and oocytes were retrieved from the ovary 48 hr later for IVM. After IVM (18 hr) Cumulus oocyte complexes (COC) were in vitro fertilized (IVF) and in vitro culture (IVC) in order to observe embryo development. The IVM medium was supplemented with: 0, 25, 50, 100, or 200 microM of cysteamine. To study the novo GSH synthesis, 5 mM BSO was added during IVM of adult or prepubertal oocyte. Developmental rates up to blastocyst were recorded for each group. Experiments also included a group of ovulated oocytes (in vivo matured) after priming with PMSG and HCG. After IVM of adult mice oocytes, an improvement was observed on embryo development in all the supplemented groups when compared with the untreated group (P < 0.05). No differences were observed in blastocyst rate among IVM oocytes with cysteamine and ovulated oocytes. Prepubertal IVM mouse oocytes had a lower cleavage rate compared with ovulated oocytes (P < 0.05). Cysteamine failed to improve prepubertal oocytes developmental rates (P > 0,05). 2-cell embryos, coming from IVM prepubertal oocytes and ovulated oocytes had the same preimplantation developmental rate up to the blastocyst stage. In prepubertal, and adult oocytes an inhibition of embryo development was observed when buthionine sulfoximide (BSO), a specific inhibitor of the gamma-glutamylcysteine synthetase, was added during oocyte maturation (P < 0.01). In conclusion, an improvement in mouse embryo development was observed when cysteamine was added to the IVM medium of adult mice oocytes. In prepubertal oocytes cysteamine addition during oocyte maturation failed to improve embryo developmental rates. The presence of BSO lowered or completely blocked blastocyst development. This proves that, de novo GSH synthesis during oocyte maturation of adult and prepubertal oocytes undoubtedly plays an important role in embryo development. The improvement on oocyte competence observed in adult mice oocytes is probably related to intracellular GSH synthesis stimulated by cysteamine. Nevertheless the reason why cysteamine failed to improve prepubertal oocytes competence remains as an open question.     

The mycotoxin beauvericin induces oocyte mitochondrial dysfunction and affects embryo development in the juvenile sheep

Beauvericin (BEA) is a mycotoxin produced by Beauveria bassiana and Fusarium species recently reported as toxic on porcine oocyte maturation and embryo development. The aim of this study was to assess, in the juvenile sheep, whether its effects are due to alterations of oocyte and/or embryo bioenergetic/oxidative status. Cumulus‐oocyte‐complexes (COCs) were exposed to BEA during in vitro maturation (IVM), evaluated for cumulus cell (CC) apoptosis, oocyte maturation and bioenergetic/oxidative status or subjected to in vitro fertilization (IVF) and embryo culture (IVEC). Oocyte nuclear maturation and embryo development were assessed after Hoechst staining and CC apoptosis was analysed by terminal deoxynucleotidyl transferase‐mediated dUTP nick‐End labeling assay and chromatin morphology after Hoechst staining by epifluorescence microscopy. Oocyte and blastocyst bioenergetic/oxidative status were assessed by confocal microscopy after mitochondria and reactive oxygen species labelling with specific probes. BEA showed various toxic effects, that is, short‐term effects on somatic and germinal compartment of the COC (CCs and the oocyte) and long‐term carry‐over effects on developing embryos. In detail, at 5 µM, it significantly reduced oocyte maturation and immature oocytes showed increased late‐stage (Type C) CC apoptosis and DNA fragmentation while matured oocytes showed unaffected CC viability but abnormal mitochondrial distribution patterns. At lower tested concentrations (3–0.5 µM), BEA did not affect oocyte maturation, but matured oocytes showed reduced mitochondrial activity. At low concentrations, BEA impaired embryo developmental capacity and blastocyst quality after IVF and IVEC. In conclusion, in the juvenile sheep, COC exposure to BEA induces CC apoptosis and oocyte mitochondrial dysfunction with negative impact on embryo development.     

Leptin accelerates pronuclear formation following intracytoplasmic sperm injection of porcine oocytes: Possible role for MAP kinase inactivation

Leptin, a multifunctional hormone, is present in mammalian oocytes and follicular fluids and cumulus cells. While leptin modulates oocyte maturation in vitro which seems to result in enhancement of embryo development, it is unclear whether leptin treatment of oocytes affects cytoplasmic maturation and fertilization processes. In order to gain a better understanding of the role of leptin during oocyte maturation, we examined microtubule and microfilament assembly following oocyte maturation and blastocyst formation, mitogen-activated protein kinase (MAPK) activity, and pronuclear formation following parthenogenetic stimuli or intracytoplasmic sperm injection (ICSI) in leptin-treated oocytes. Addition of 10 or 100 ng/ml leptin during oocyte maturation did not increase the proportion of metaphase II oocytes, but enhanced development to blastocyst stage by day 7 (P < 0.01) after parthenogenetic activation (PA), accompanied by increased cell number. However there was no effect on the number of apoptotic cells in blastocysts. Following maturation in the presence of leptin, there were more oocytes with normal spindle formation. MAPK activity decreased more rapidly, and pronuclear formation was accelerated after parthenogenetic activation or ICSI of leptin-treated oocytes. These results suggested that exogeneous leptin enhanced spindle assembly and accelerated pronuclear formation following fertilization, possibly via the MAPK pathway.     

Morphological features of lipid droplet transition during porcine oocyte fertilisation and early embryonic development to blastocyst in vivo and in vitro

Lipid content in mammalian oocytes or embryos differs among species, with bovine and porcine oocytes and embryos showing large cytoplasmic droplets. These droplets are considered to play important roles in energy metabolism during oocyte maturation, fertilisation and early embryonic development, and also in the freezing ability of oocytes or embryos; however, their detailed distribution or function is not well understood. In the present study, changes in the distribution and morphology of porcine lipid droplets during in vivo and in vitro fertilisation, in contrast to parthenogenetic oocyte activation, as well as during their development to blastocyst stage, were evaluated by transmission electron microscopy (TEM). The analysis of semi-thin and ultra-thin sections by TEM showed conspicuous, large, electron-dense lipid droplets, sometimes associated with mitochondrial aggregates in the oocytes, irrespective of whether the oocytes had been matured in vivo or in vitro. Immediately after sperm penetration, the electron density of the lipid droplets was lost in both the in vivo and in vitro oocytes, the reduction being most evident in the oocytes developed in vitro. Density was restored in the pronculear oocytes, fully in the in vivo specimens but only partially in the in vitro ones. The number and size of the droplets seemed, however, to have decreased. At 2- to 4-cell and blastocyst stages, the features of the lipid droplets were almost the same as those of pronuclear oocytes, showing a homogeneous or saturated density in the in vivo embryos but a marbled or partially saturated appearance in the in vitro embryos. In vitro matured oocytes undergoing parthenogenesis had lipid droplets that resembled those of fertilised oocytes until the pronuclear stage. Overall, results indicate variations in both the morphology and amount of cytoplasmic lipid droplets during porcine oocyte maturation, fertilisation and early embryo development as well as differences between in vivo and in vitro development, suggesting both different energy status during preimplantation development in pigs and substantial differences between in vitro and in vivo development.     

The equine oocyte: Factors affecting meiotic and developmental competence

There is currently much interest in assisted reproduction techniques in the horse, however, many aspects of oocyte maturation, fertilization, and embryo development in the horse differ from those in other species. Because of the close attachment of the equine oocyte to the follicle wall, scraping of the follicle is the most effective method for oocyte recovery. A notable feature of equine oocytes is that those with expanded cumuli (Ex oocytes), which originate from atretic follicles, have higher meiotic competence (ability to mature to metaphase II in vitro) than do oocytes with compact cumuli (Cp oocytes). Cp oocytes originate in viable follicles but are largely juvenile. Recovery and culture of equine oocytes immediately after slaughter yields a higher maturation rate than that obtained from oocytes after ovary storage; this is related to damage to chromatin in Cp oocytes during storage. In contrast, developmental competence (rate of blastocyst development in vitro) is higher in oocytes recovered from the ovary after a delay. The optimum duration of maturation varies based on cumulus morphology and time of recovery from the ovary, but there is no difference in developmental competence between Ex and Cp oocytes. Because standard in vitro fertilization is not repeatable in the horse, oocyte transfer (surgical transfer of oocytes to the oviducts of inseminated mares) has been developed to allow fertilization of isolated oocytes. Fertilization in vitro may be achieved using intracytoplasmic sperm injection; culture of injected oocytes in a medium with high glucose can yield over 30% blastocyst development. Mol. Reprod. Dev. 77: 651–661, 2010. © 2010 Wiley‐Liss, Inc.     

Effect of specific phosphodiesterase isoenzyme inhibitors during in vitro maturation of bovine oocytes on meiotic and developmental capacity

Compared with oocytes matured in vivo, in vitro-matured oocytes are compromised in their capacity to support early embryo development. Delaying spontaneous in vitro meiotic maturation using specific phosphodiesterase (PDE) isoenzyme inhibitors may permit more complete oocyte cytoplasmic maturation, possibly by prolonging cumulus cell (CC)-oocyte gap junctional communication during meiotic resumption. This study aimed to investigate the effect of the isoenzyme 3- (oocyte) and isoenzyme 4- (granulosa cell) specific PDE inhibitors on the kinetics of in vitro maturation and on subsequent oocyte developmental competence. Cumulus-oocyte complexes from antral bovine follicles were isolated and cultured in the presence of the specific PDE inhibitors milrinone (type 3) or rolipram (type 4) (100 microM). In the presence of FSH, both PDE inhibitors only slightly extended CC-oocyte gap junctional communication over the first 9 h, but they completely blocked meiotic resumption during this period (P < 0.001). The indefinite inhibitory effect of milrinone on meiotic resumption (30% at germinal vesicle stage after 48 h) was overridden by 24 h when treated with FSH, but not with hCG, suggesting a form of induced meiotic resumption. Oocytes treated with FSH with or without either PDE inhibitor were inseminated at either 24, 26, or 28 h. Treated with either the type 3 or type 4 PDE inhibitor significantly (P < 0.05) increased embryo development to the blastocyst stage by 33%-39% (to an average of 52% blastocysts) compared with control oocytes (38%) after insemination at 28 h, and significantly (P < 0.05) increased blastocyst cell numbers when inseminated at 24 h. These results suggest that delayed spontaneous meiotic maturation, coupled with extended gap junctional communication between the CCs and the oocyte has a positive effect on oocyte cytoplasmic maturation, thereby improving oocyte developmental potential.     

Ion currents involved in oocyte maturation, fertilization and early developmental stages of the ascidian Ciona intestinalis

Electrophysiological techniques were used to study the role of ion currents in the ascidian Ciona intestinalis oocyte plasma membrane during different stages of growth, meiosis, fertilization and early development. Three stages of immature oocytes were discriminated in the ovary, with the germinal vesicle showing specific different features of growth and maturation. Stage-A (pre-vitellogenic) oocytes exhibited the highest L-type calcium current activity and were incompetent for meiosis resumption. Stage-B (vitellogenic) oocytes showed a progressive disappearance of calcium currents and the first appearance of sodium currents that remained high during the maturation process, up to the post-vitellogenic stage-C oocytes. The latter had acquired meiotic competence, undergoing spontaneous in vitro maturation and interacting with the spermatozoon. However, fertilized oocytes did not produce normal larvae, suggesting that cytoplasmic maturation may affect embryo development. In mature oocytes at the metaphase I stage, sodium currents were present and remained high up to the zygote stage. Oocytes fertilized in the absence of sodium showed significant reduction of the fertilization current amplitude and high development of anomalous "rosette" embryos. Current amplitudes became negligible in embryos at the 2- and 4-cell stage, whereas resumption of all the current activities occurred at the 8-cell embryo. Taken together, these results suggest: (i) an involvement of L-type calcium currents in initial oocyte meiotic progression and growth; (ii) a role of sodium currents at fertilization; (iii) a role of the fertilization current in ensuring normal embryo development.     

Role of hyaluronic acid in maturation and further early embryo development of bovine oocytes

Hyaluronic acid (HA), an important component of the extracellular matrix, plays a crucial role for cumulus cell expansion. Genes and proteins involved in HA synthesis and its receptor CD44 are expressed in cumulus oocyte complexes (COCs) in different animal species and increase during maturation. Hyaluronidase enzymes (Hyal) degrade HA into smaller biologically active HA fragments. To investigate the effects of the molecular size and concentration of HA on oocyte maturation and further embryo development, bovine oocytes were matured in vitro in the presence or absence of HA, Hyal-2 or 4-methylumbelliferone (4-MU); an HA synthesis inhibitor. The rates of oocyte nuclear maturation to metaphase II stage and development of embryos to blastocyst stage and blastocyst quality were recorded. Hyal-2 inhibited cumulus cell expansion without affecting oocyte maturation and further embryo development. Whereas, 4-MU at 1 mm reduced cumulus cell expansion, oocyte maturation rate and further embryo development; an effect which was partially abrogated by exogenous HA supplementation. These data suggest that HA production by cumulus cells during maturation is essential not only for cumulus cell expansion, but also for oocyte maturation and further embryo development. This effect is not affected by HA-degradation by Hyal-2.