Developmental competence of bovine oocytes after specific inhibition of MPF kinase activity: effect of estradiol supplementation and follicle size.

In the bovine, the concentration of 17beta-estradiol (E2) in the follicular fluid of the dominant follicle is high, indicating a possible role of E2 on the cytoplasmic maturation that occurs before the LH surge. The aim of this study was to investigate the role of E2 on the developmental competence of bovine oocytes originating from different sized follicles and temporarily maintained at the germinal vesicle stage with roscovitine (ROS). First, the efficiency of ROS to inhibit germinal vesicle breakdown (GVBD) in oocytes harvested from small (3-4 mm diameter) and medium (5-8 mm diameter) sized follicles was demonstrated. Next, the effect of E2 during temporary inhibition of GVBD by ROS on the subsequent nuclear maturation was evaluated. Oocytes from small and medium sized follicles were cultured in the presence of ROS, FSH and with or without E2 for 24 h. After this period, oocytes were cultured for another 24 h with FSH but without ROS and E2, after which the nuclear stages and the developmental competence of oocytes were assessed. In conclusion, it is demonstrated that exposure to E2, during temporary inhibition of the GVBD with ROS, affected neither nuclear nor cytoplasmic maturation of oocytes originating from small and medium sized follicles. It might be that in vivo, the increase of E2 during follicle growth is more related to selection of the dominant follicle than to the cytoplamsic maturation of the oocyte as such.

Follicle size-dependent effects of sow follicular fluid on in vitro cumulus expansion, nuclear maturation and blastocyst formation of sow cumulus oocytes complexes.

Follicular fluid from 2 to 4 and 5 to 8 mm diameter non-atretic follicles (SFF and LFF, respectively) of sows was added during IVM of cumulus oocytes complexes (COCs) to study its effects on cumulus expansion, nuclear maturation, and subsequent fertilization and embryo development in presence or absence of recombinant human FSH. COCs aspirated from 2 to 5 mm follicles of sow ovaries, were cultured for the first 22 h in TCM-199 and 100 microM cysteamine, with or without 10% pFF and/or 0.05 IU/ml recombinant hFSH. For the next 22 h, the COCs were cultured in the same medium, but without pFF and FSH. After culture, cumulus cells were removed and the oocytes were either fixed and stained to evaluate nuclear stages or co-incubated with fresh sperm. Twenty-four hours after fertilization, presumptive zygotes were fixed to examine fertilization or cultured for 6 days to allow blastocyst formation. Subsequently, embryos were evaluated and the blastocysts were fixed and stained to determine cell numbers. When LFF was added to maturation medium, cumulus expansion and percentage of nuclear maturation (277 +/- 61 microm and 72%, respectively) of COCs were significantly higher (P < 0.05) than those in SFF (238 +/- 33 microm and 55%, respectively). However, in the presence of FSH both FF stimulated cumulus expansion and nuclear maturation to a similar degree. No differences were observed with regards to sperm penetration, male pronucleus formation, and to polyspermia between fertilized oocytes matured either in SFF or LFF. Fertilized oocytes matured in the presence of LFF without or with FSH showed a higher cleavage (45 +/- 7% and 51 +/- 7%, respectively) and blastocyst (14 +/- 4% and 22 +/- 6%, respectively) formation rate compared to SFF (cleavage, 35 +/- 8% and 41 +/- 4%, blastocyst: 8 +/- 3 and 13 +/-3, respectively; P < 0.05). The mean number of cells per blastocyst did not differ significantly between treatments. These findings indicate that factor(s) within follicles at later stages of development play an important role during oocyte maturation and thereby enhance developmental competence to occur.

Estradiol and its membrane-impermeable conjugate (estradiol-bovine serum albumin) during in vitro maturation of bovine oocytes: effects on nuclear and cytoplasmic maturation, cytoskeleton, and embryo quality.

In various cell types, there is increasing evidence for nongenomic steroid effects, i.e., effects that are not mediated via the classical steroid receptors. However, little is known about the involvement of the nongenomic pathway of estradiol (E2) on mammalian oocyte in vitro maturation (IVM). The aim of this study was to investigate whether the effects of E2 on bovine oocyte IVM are mediated via a plasma membrane receptor (nongenomic). First, we investigated the expression of estradiol (classical) receptor alpha (ERalpha) and beta (ERbeta) mRNA in oocytes and cumulus cells (CC). We also studied the effects of different exposure times to E2 (before and after germinal vesicle breakdown, GVBD) on nuclear maturation. To study the possible involvement of the putative estradiol plasma membrane receptor on the IVM of oocytes, we used E2 conjugated with bovine serum albumin (E2-BSA), which cannot cross the plasma membranes. Our results demonstrate that oocytes expressed ERbeta mRNA, while CC expressed both ERalpha and ERbeta mRNA. Exposure to E2 during the first 8 h of culture (before GVBD) induced a block at the metaphase I stage (MI). However, the presence of E2 after GVBD induced an increase of oocytes with nuclear aberrations. Meiotic spindle organization was severely affected by E2 during IVM and multipolar spindle was the most frequently observed aberration. Exposure of oocytes to E2-BSA did not affect nuclear maturation, blastocyst formation rate, nor embryo quality. Our results suggest that the detrimental effects of E2 on in vitro nuclear maturation of bovine oocyte are not exerted via a plasma membrane receptor.

Effects of in vitro production on horse embryo morphology, cytoskeletal characteristics, and blastocyst capsule formation.

Blastocyst formation rates during horse embryo in vitro production (IVP) are disappointing, and embryos that blastulate in culture fail to produce the characteristic and vital glycoprotein capsule. The aim of this study was to evaluate the impact of IVP on horse embryo development and capsule formation. IVP embryos were produced by intracytoplasmic sperm injection of in vitro matured oocytes and either culture in synthetic oviduct fluid (SOF) or temporary transfer to the oviduct of a ewe. Control embryos were flushed from the uterus of mares 6-9 days after ovulation. Embryo morphology was evaluated with light microscopy, and multiphoton scanning confocal microscopy was used to examine the distribution of microfilaments (AlexaFluor-Phalloidin stained) and the rate of apoptosis (cells with fragmented or terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling-positive nuclei). To examine the influence of culture on capsule formation, conceptuses were stained with a monoclonal antibody specific for capsular glycoproteins (OC-1). The blastocyst rate was higher for zygotes transferred to a sheep's oviduct (16%) than for those cultured in SOF (6.3%). Day 7 IVP embryos were small and compact with relatively few cells, little or no blastocoele, and an indistinct inner cell mass. IVP embryos had high percentages of apoptotic cells (10% versus 0.3% for in vivo embryos) and irregularly distributed microfilaments. Although they secreted capsular glycoproteins, the latter did not form a normal capsule but instead permeated into the zona pellucida or remained in patches on the trophectodermal surface. These results demonstrate that the initial layer of capsule is composed of OC-1-reactive glycoproteins and that embryo development ex vivo is retarded and aberrant, with capsule formation failing as a result of failed glycoprotein aggregation.

Cytoskeleton and chromatin reorganization in horse oocytes following intracytoplasmic sperm injection: patterns associated with normal and defective fertilization.

Intracytoplasmic sperm injection (ICSI) is the method of choice for fertilizing horse oocytes in vitro. Nevertheless, for reasons that are not yet clear, embryo development rates are low. The aims of this study were to examine cytoskeletal and chromatin reorganization in horse oocytes fertilized by ICSI or activated parthenogenetically. Additional oocytes were injected with a sperm labeled with a mitochondrion-specific vital dye to help identify the contribution of the sperm to zygotic structures, in particular the centrosome. Oocytes were fixed at set intervals after sperm injection and examined by confocal laser scanning microscopy. In unfertilized oocytes, microtubules were present only in the metaphase-arrested second meiotic spindle and the first polar body. After sperm injection, an aster of microtubules formed adjacent to the sperm head and subsequently enlarged such that at the time of pronucleus migration and apposition it filled the entire cytoplasm. During syngamy, the microtubule matrix reorganized to form a mitotic spindle on which the chromatin of both parents aligned. Finally, after nuclear and cellular cleavage were complete, the microtubule asters dispersed into the interphase daughter cells. Sham injection induced parthenogenetic activation of 76% of oocytes, marked by the formation of multiple cytoplasmic microtubular foci that later developed into a dense microtubule network surrounding the female pronucleus. The finding that a parthenote alone can produce a microtubule aster, whereas the aster invariably forms at the base of the sperm head during normal fertilization, indicates that both gametes contribute to the formation of the zygotic centrosome in the horse. Finally, 25% of sperm-injected oocytes failed to complete fertilization, mostly due to absence of oocyte activation (65%), which was often accompanied by failure of sperm decondensation. In conclusion, this study demonstrated that union of the parental genomes in horse zygotes is accompanied by a series of integrated cytoskeleton-mediated events, failure of which results in developmental arrest.

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.