Beneficial effects of brain-derived neurotropic factor on in vitro maturation of porcine oocytes

In an effort to improve the quality of in vitro produced porcine embryos, we investigated the effect of brain-derived neurotropic factor (BDNF), a neurotropin family member, on in vitro maturation (IVM) of porcine oocytes. The expression of BDNF and truncated isoforms of its receptor, tyrosine kinase B (TrkB), and p75 common neurotropin receptor was detected in both follicular cells and metaphase-I stage oocytes by RT-PCR. However, mRNA of full-length TrkB was not found in oocytes although it was detected in follicular cells. The expression pattern of BDNF and TrkB was confirmed by immunohistochemistry. Supplementation with BDNF (30 ng/ml) during IVM significantly (P < 0.05) increased the first polar body extrusion and glutathione levels in oocytes, whereas the effect of BDNF on nuclear maturation was diminished when gonadotropin and epidermal growth factor (EGF) were added to the culture media. However, treatment with BDNF (30 ng/ml) along with EGF (10 ng/ml) in the presence of gonadotropin significantly (P < 0.05) increased the developmental competence of oocytes to the blastocyst stage after both in vitro fertilization (IVF; 29.1% when compared with control, 15.6%) and somatic cell nuclear transfer (SCNT; 13.6% when compared with control, 3%). This appeared to reflect a stimulatory interaction between BDNF and EGF to enhance the cytoplasmic maturation of oocytes to support successful preimplantation development. In conclusion, BDNFenhanced nuclearand cytoplasmic maturation of oocytes by autocrine and/or paracrine signals. Also, when used together with EGF, BDNF increased the developmental potency of embryos after IVF and SCNT, demonstrating an improved in vitro production protocol for porcine oocytes.     

Proteomic analysis of porcine oocytes during in vitro maturation reveals essential role for the ubiquitin C-terminal hydrolase-L1

In this study, we performed proteomic analysis of porcine oocytes during in vitro maturation. Comparison of oocytes at the initial and final stages of meiotic division characterized candidate proteins that were differentially synthesized during in vitro maturation. While the biosynthesis of many of these proteins was significantly decreased, we found four proteins with increased biosynthetic rate, which are supposed to play an essential role in meiosis. Among them, the ubiquitin C-terminal hydrolase-L1 (UCH-L1) was identified by mass spectrometry. To study the regulatory role of UCH-L1 in the process of meiosis in pig model, we used a specific inhibitor of this enzyme, marked C30, belonging to the class of isatin O-acyl oximes. When germinal vesicle (GV) stage cumulus-enclosed oocytes were treated with C30, GV breakdown was inhibited after 28 h of culture, and most of the oocytes were arrested at the first meiosis after 44 h. The block of metaphase I-anaphase transition was not completely reversible. In addition, the inhibition of UCH-L1 resulted in elevated histone H1 kinase activity, corresponding to cyclin-dependent kinase(CDK1)-cyclin B1 complex, and a low level of monoubiquitin. These results supported the hypothesis that UCH-L1 might play a role in metaphase I-anaphase transition by regulating ubiquitin-dependent proteasome mechanisms. In summary, a proteomic approach coupled with protein verification study revealed an essential role of UCH-L1 in the completion of the first meiosis and its transition to anaphase.     

Apoptosis in cumulus cells during in vitro maturation of bovine cumulus-enclosed oocytes

The aim of this study was to investigate whether apoptosis occurs in cumulus cells during in vitro maturation (IVM) of bovine cumulus-enclosed oocytes (CEOs). The bovine CEOs obtained from ovaries from an abattoir were cultured for 24 h in IVM medium in the presence or absence of 10% (v/v) fetal bovine serum. The developmental competence of enclosed oocytes, as assessed by the development of the blastocyst after IVF, was significantly higher in the serum-treated group than in the control group. The morphological features of apoptosis that were analysed by orcein staining were hardly detectable in the cumulus cells at the start (0 h) of IVM, but were evident at the end (24 h) of IVM both in the control and serum-treated groups. Genomic DNA was extracted from CEOs at 0, 6, 12, 18 and 24 h of IVM and subjected to ligation-mediated PCR (LM-PCR) to detect apoptotic internucleosomal DNA fragmentation. DNA fragmentation was hardly detectable at the start of IVM, but increased in a time-dependent manner as the IVM culture proceeded. DNA fragmentation was not observed in the oocytes, indicating that fragmentation occurs in cumulus cells. The degree of fragmentation was lower in the serum-treated group compared with the control group. The LM-PCR analysis of DNA extracted from CEOs at 24 h of IVM, in which the DNA had been pretreated with Klenow enzyme or T4 DNA polymerase, revealed that the characteristic forms of the DNA ends generated during cumulus cell apoptosis were mainly 3'-overhangs and blunt ends. In conclusion, the results of the present study demonstrate that cumulus cells in bovine CEOs spontaneously undergo apoptosis during IVM. The degree of apoptosis may be correlated with the developmental competence of the enclosed oocytes.     

Inactivation of glucocorticoids by 11beta-hydroxysteroid dehydrogenase enzymes increases during the meiotic maturation of porcine oocytes

Recent reports have shown that glucocorticoids can modulate oocyte maturation in both teleost fish and mammals. Within potential target cells, the actions of physiological glucocorticoids are modulated by 11beta-hydroxysteroid dehydrogenase (HSD11B) isoenzymes that catalyse the interconversion of cortisol and cortisone. Hence, the objective of this study was to establish whether HSD11B enzymes mediate cortisol-cortisone metabolism in porcine oocytes and, if so, whether the rate of glucocorticoid metabolism changes during oocyte maturation. Enzyme activities were measured in cumulus-oocyte complexes (COCs) and denuded oocytes (DOs) using radiometric conversion assays. While COCs and DOs oxidised cortisol to inert cortisone, there was no detectable regeneration of cortisol from cortisone. The rate of cortisol oxidation was higher in expanded COCs than in compact COCs containing germinal vesicle (GV) stage oocytes (111+/-6 vs 2041+/-115 fmol cortisone/oocyte.24 h; P<0.001). Likewise, HSD11B activities were 17+/-1 fold higher in DOs from expanded COCs than in those from compact COCs (P<0.001). When GV stage oocytes were subject to a 48 h in vitro maturation protocol, the enzyme activities were significantly increased from 146+/-18 to 1857+/-276 fmol cortisone/oocyte.24 h in GV versus MII stage oocytes respectively (P<0.001). Cortisol metabolism was inhibited by established pharmacological inhibitors of HSD11B (glycyrrhetinic acid and carbenoxolone), and by porcine follicular and ovarian cyst fluid. We conclude that an HSD11B enzyme (or enzymes) functions within porcine oocytes to oxidise cortisol, and that this enzymatic inactivation of cortisol increases during oocyte maturation.     

Chemical manipulation of glucose metabolism in porcine oocytes: effects on nuclear and cytoplasmic maturation in vitro

The objectives of this study were to manipulate metabolism of glucose through glycolysis and the pentose phosphate pathway (PPP) in porcine oocytes during in vitro maturation, and determine the effects of this manipulation on meiotic progression, intracellular glutathione (GSX) concentrations and embryonic development. Cumulus-oocyte complexes isolated from abattoir ovaries were matured (40-44 h) in Purdue Porcine Medium for maturation alone (control) or supplemented with pyrroline-5 carboxylate (PC, 0.1 microM; PPP stimulator), diphenyleneiodonium (DPI, 0.1 microM; PPP inhibitor), dinitrophenol (DNP, 10 microM; glycolytic stimulator), hexametaphosphate (HMP, 100 microM; glycolytic inhibitor), PC + HMP or DNP + DPI. At the conclusion of in vitro maturation, cumulus cells were removed and oocytes were randomly allocated for analysis of GSX, metabolism and nuclear maturation, or in vitro fertilization and embryo culture. Both DPI and DNP + DPI decreased (P < or = 0.05) the activity of glycolysis and the PPP, increased (P < or = 0.05) the percentage of immature oocytes, and decreased (P < or = 0.05) the proportion of mature oocytes compared with control oocytes and oocytes from the other treatments. Embryonic development (cleavage and blastocyst stage) and the intracellular content of GSX were also decreased (P < or = 0.05) following exposure to DPI or DNP + DPI compared with control oocytes and oocytes from the other treatments. Oocyte metabolism, nuclear maturation, GSX content and embryonic development were unaffected (P > 0.05) following exposure to PC, DNP, HMP or PC + HMP. Our results suggest that metabolism of glucose through the PPP and/or glycolysis plays a key role in the control of nuclear and cytoplasmic maturation of porcine oocytes in vitro.     

Multiple aneuploidies in the oocytes of balanced translocation carriers: a preimplantation genetic diagnosis study using first polar body

Preimplantation genetic diagnosis (PGD) of first polar bodies (1PBs) has been used in carriers of balanced chromosomal reorganizations and also for aneuploidy screening. Although an acceptable number of normal or balanced embryos is usually obtained using PGD in translocation carriers, the pregnancy rate is disappointingly low. To determine whether aneuploidy of chromosomes not involved in the chromosome rearrangements could be the cause of the low pregnancy rates achieved, the present authors analysed the segregation products of three translocation carriers, t(8;13)(q24.1;q22) and two Robertsonian (Rob)(13;14), using 1PBs, and afterwards another eight chromosomes in the same 1PBs, for a total of 10 chromosomes in each 1PB, that is chromosomes 1, 8, 13, 14, 15, 16, 17, 18, 21, 22 and X. In the reciprocal translocation, chromosomes with different chromatids due to meiotic recombination were found. Only one out of nine 1PBs was normal for the reorganization products but no aneuploidies were found after PGD in this case. In the two balanced Rob(13;14), six out of 12 and four out of 11 1PBs were normal or balanced for the reorganization but only one oocyte was euploid for all the chromosomes analysed in each case; a single embryo transfer was made in both but no pregnancy was achieved. The incidence of aneuploidy for the chromosomes not involved in the Robertsonian translocations was extremely high (91.7% and 81.8%). Extra chromosomes were present in most of the aneuploid oocytes (81.8% and 90%). The reason for this increase could be the tendency to non-disjunction related to advanced maternal age combined with an interchromosomal effect resulting in the presence of synaptic errors in other chromosome pairs.     

Improved development by Taxol pretreatment after vitrification of in vitro matured porcine oocytes

This study was designed to examine the effect of Taxol pretreatment on vitrification of porcine oocytes matured in vitro by an open pulled straw (OPS) method. In the first experiment, the effect of Taxol pretreatment and fluorescein diacetate (FDA) staining on parthenogenetic development of oocytes was evaluated. In the second experiment, viability, microtubule organization and embryo development of oocytes were assessed after oocytes were exposed to vitrification/warming solutions or after vitrification with or without Taxol pretreatment. The results showed that Taxol pretreatment and/or FDA staining did not negatively influence the oocyte's developmental competence after parthenogenetic activation. After being exposed to vitrification/warming solutions, the survival rate (83.3%) of the oocytes was significantly (P < 0.05) reduced as compared with that in the control (100%). Vitrification/warming procedures further reduced the survival rates of oocytes regardless of oocytes being treated with (62.1%) or without (53.8%) Taxol. The proportions of oocytes with normal spindle configuration were significantly reduced after the oocytes were exposed to vitrification/warming solutions (38.5%) or after vitrification with (10.3%) or without (4.1%) Taxol pretreatment as compared with that in control (76.8%). The rates of two-cell-stage (5.6-53.2%) embryos at 48 h and blastocysts (0-3.8%) at 144 h after activation were significantly reduced after exposure to vitrification/warming solutions or after vitrification as compared with control (90.9% and 26.6% respectively). However, the proportion of vitrified oocytes developed to two-cell stage was significantly higher when oocytes were pretreated with (24.3%) than without (5.6%) Taxol. These results indicate that pretreatment of oocytes with Taxol before vitrification helps to reduce the damage induced by vitrification and is a potential way to improve the development of vitrified porcine oocytes.     

Development of a reliable in vitro maturation system for zebrafish oocytes

In zebrafish oocytes, it has been reported that a 60 or 75% Leibovitz L-15 medium or simple balanced saline solution containing 17alpha, 20beta-dihydroxy-4-pregnen-3-one (DHP) is effective for nuclear maturation. However, most of the oocytes that matured under these conditions were not fertilized and did not hatch. Thus, these in vitro maturation methods could not support the cytoplasmic maturation of zebrafish oocytes. Therefore, we tried to develop a reliable in vitro maturation method for zebrafish oocytes, which supports their ability to be fertilized and to develop till hatching. When zebrafish oocytes at stage III were cultured in 50-100% Leibovitz L-15 medium supplemented with DHP, the highest rates of cleavage (24%) and hatching (12%) were obtained from oocytes matured in 90% Leibovitz L-15 medium. When we examined the suitable pH (7.5-9.5) of the 90% medium, higher rates of cleavage (45%) and hatching (33%) were obtained in oocytes matured at pH 9.0 than at pH 7.5, 8.5, or 9.5 (cleavage rate, 16-29%; hatching rate, 8-21%). In oocytes matured in 90% Leibovitz L-15 medium at pH 9.0, high rates of cleavage (70%) and hatching (63%) were obtained when oocytes were cultured for 270 min with 0.5 mg/ml BSA. Thus, 90% Leibovitz L-15 medium at pH 9.0 containing 0.5 mg/ml BSA was effective for normal maturation of zebrafish oocytes. This method will become a powerful tool for understanding the mechanism of in vitro maturation in zebrafish oocytes and for the practical use of immature oocytes.     

Effect of beta-mercaptoethanol during in vitro fertilization procedures on sperm penetration into porcine oocytes and the early development in vitro

This study was carried out to determine the effects of beta-mercaptoethanol (bME) during a transient co-culture of gametes for 10 min, and/or the following culture until 6-9 h after insemination, on sperm penetration of porcine in vitro maturation (IVM) oocytes and the early development in vitro. When fresh spermatozoa were cultured in various concentrations of bME for 2 h, bME neutralized the stimulatory effect of caffeine-benzoate on sperm capacitation and the spontaneous acrosome reaction at 50-250 micromol/l. When 50 micromol/l bME were added during a transient co-culture of gametes for 10 min, the sperm penetration rate was reduced 9 h after insemination (70.5-82.0% vs 90.5-94.0% in the absence of bME), but the incidence of monospermic penetration was not affected. When 50 micromol/l bME were supplemented during culture after a transient co-culture, the sperm penetration rate was not affected, but the incidence of monospermy oocytes was increased (43.9-45.8% vs 31.7-34.3% in the absence of bME). The presence of bME following a transient co-culture minimized a decrease of oocyte glutathione content at 6 h after insemination (7.9 pmol/oocyte before in vitro fertilization (IVF), 6.7 pmol/oocyte in the presence of bME vs 5.5 pmol/oocyte in the absence of bME). When the distribution of cortical granules was evaluated 1 h after activation with calcium ionophore, mean pixel intensity of fluorescein isothiocyanate-labeled peanut agglutinin (FITC-PNA) at the cortex region was lower in the oocytes activated and cultured in the presence of 50 micromol/l bME. Although the presence of 50 micromol/l bME during a transient co-culture for 10 min and the following culture did not increased blastocyst formation (29.6-37.7%), 50 micromol/l bME during the following culture significantly increased the mean cell numbers per blastocyst (73.3-76.4 vs 51.2 in the presence and absence of bME respectively). These results demonstrate that supplementation with bME during IVF procedures, except during a transient co-culture period of gametes in the presence of caffeine, has a beneficial effect in maintaining the function of gametes, the incidence of normal fertilization and, consequently, the quality of IVF embryos.     

Retinol improves development of bovine oocytes compromised by heat stress during maturation

The objectives of this study were to evaluate: 1) effects of a physiologically relevant elevated temperature on in vitro development of maturing oocytes, 2) effects of retinol on in vitro development of maturing oocytes, and 3) effects of retinol to improve development of oocytes compromised by an elevated temperature. Bovine oocytes were matured for 24 h at 38.5 or 41.0 degrees C (first 12 h) in 0 or 5 microM retinol. After insemination, cleavage and blastocyst development were assessed on d 3 and 8, respectively. Temperature, retinol, and their interaction were included in the statistical model. Culture of oocytes at 41.0 degrees C decreased the proportion of 8- to 16-cell embryos and increased that of 2-cell embryos. In addition, culture at 41.0 degrees C decreased the ability of oocytes to develop to the blastocyst stage. Blastocysts derived from oocytes cultured at 41.0 degrees C had fewer total nuclei. In 3 of the 7 experimental replicates, effects of 41.0 degrees C to reduce blastocyst development were minimal (difference in the development of the control vs. heat stress group was <20%). To provide a more precise test of our hypothesis (retinol administration may improve development of oocytes compromised by heat stress), data were analyzed, including only those replicates (n = 4) in which heat stress reduced development to blastocyst >20%. When this was done, a significant temperature x retinol interaction was noted. The addition of retinol to the maturation medium prevented heat-induced reductions in development of oocytes to blastocyst stage. Results indicate that retinol may protect oocytes from some of the deleterious effects of heat stress.     

Possible mechanism for acceleration of meiotic progression of bovine follicular oocytes by growth factors in vitro

The mechanism for the accelerating effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the meiotic cell cycle of bovine oocytes cultured in vitro was investigated. Cumulus-oocyte complexes (COCs) were obtained from small (< or = 3 mm in diameter), medium (4-6 mm in diameter) or large (7-10 mm in diameter) ovarian follicles and cultured with or without a combination of EGF and IGF-I (growth factors). Growth factors significantly increased the frequency of first polar body extrusion of oocytes derived from small follicles at 16 h of culture (PB16 oocytes; with growth factors: 75%; without growth factors: 55%), but did not increase the frequency in oocytes from medium or large follicles. COCs from small follicles were cultured with individual growth factors and sampled for kinase activity. The frequencies of polar body extrusion in EGF only (67%) and EGF + IGF-I (75%) treatment groups were significantly higher than those in the control (no growth factor) group (49%), but not significantly higher than in the IGF-I only group (63%). The H1 kinase activity at 6-8 h of culture in each group increased significantly from the baseline value at 0 h of culture, and the H1 kinase activities in the EGF only, IGF-I only and EGF + IGF-I treatment groups were significantly higher than those in the control group at 8 h of culture. MAP kinase activity was significantly higher than the baseline value and significantly higher than that in the control group at 6 h of culture in the EGF treatment group only. In conclusion, EGF and IGF-I act on COCs from small follicles to accelerate the meiotic cell cycle of the oocytes. This accelerating effect may be related to increased H1 and MAP kinase activities during the early stages of maturation.     

Overcoming poor in vitro nuclear maturation and developmental competence of domestic cat oocytes during the non-breeding season

The domestic cat experiences circannual variations in ovarian activity and intrafollicular oocyte quality. One result is poor nuclear and cytoplasmic maturation during in vitro maturation (IVM) conducted during the annual non-breeding season (July through November). In an attempt to overcome this seasonal phenomenon immature oocytes were collected from July through November and cultured in a conventional IVM medium (IVM1) or in IVM1 supplemented with different FSH concentrations and antioxidant (ascorbic acid or cysteine). Nuclear status of oocytes was assessed after IVM or IVF. Embryo stage and blastocyst quality were evaluated after 7 days of in vitro culture. Although the addition of antioxidant alone had no effect, the presence of 10 microg FSH ml(-1) improved nuclear maturation (75.4+/-4.1% versus 48.7+/-8.8% in IVM1; P<0.05) and fertilization success (47.9+/-3.2% versus 35.0+/-5.1% in IVM1; P<0.05). Furthermore, developmental competence of fertilized oocytes was enhanced (P<0.05) only in the presence of ascorbic acid (30.6+/-6.7%) or cysteine (33.6+/-5.1%) compared with IVM1 (8.1+/-8.8%). Consequently, blastocyst yield (17% of total oocytes cultured) was highest when oocytes were matured in medium containing higher FSH concentration and antioxidants. The results of this study demonstrate that meiotic and developmental competences are inherent to the immature cat oocyte collected during the non-breeding season. However, appropriate mechanisms (perhaps seasonal variation in FSH receptors or lack of antioxidant capacity of the cumulus-oocyte complex) are inadequate during this period of gonadal quiescence. Regardless, this compromised oocyte function during the non-breeding season can be overridden by altering in vitro culture conditions to include supplemental FSH and antioxidant.     

Effects of stage of oestrous cycle and progesterone supplementation during culture on maturation of canine oocytes in vitro

The aim of this study was to evaluate the effect of progesterone supplementation and stage of oestrous cycle on in vitro maturation (IVM) of canine oocytes. Oocytes were cultured in medium supplemented with 0, 2000, 4000 or 8000 ng progesterone ml(-1) (Expt 1; n=274 oocytes) or 0, 20, 200 or 2000 ng progesterone ml(-1) (Expt 2; n=789 oocytes). In Expt 3, oocytes (n=1202) were cultured in a bi-phasic system of meiotic arrest followed by IVM, both in the presence of 0, 20, 200 or 2000 ng progesterone ml(-1). Rates of meiotic resumption for Expt 1 ranged from 40.0% to 58.5%; there were no significant differences among groups. In Expt 2, rate of meiotic resumption was significantly lower in the 2000 ng progesterone ml(-1) treatment (35.5%) compared with the 200 ng progesterone ml(-1) treatment (54.0%; P<0.05). There were no significant differences in rates of maturation to metaphase II among treatments in Expt 1 (1.8-8.6%) or Expt 2 (8.4-14.7%); however, oocytes collected from ovaries of bitches in oestrus and dioestrus had higher rates of maturation to metaphase II than did oocytes from bitches at pro-oestrus or anoestrus (P<0.01). In Expt 3, no differences were observed in rates of maturation among treatment groups. Rates of maturation to metaphase II of oocytes from bitches in dioestrus were significantly higher than those from bitches in pro-oestrus (P<0.01). These results indicate that supplementation of culture medium with progesterone either during maturation or during meiotic arrest before maturation does not increase the rate of IVM of canine oocytes. However, stage of oestrous cycle is a key factor in the selection criteria for meiotically competent canine oocytes for use in in vitro experiments.     

Inhibition of in vitro maturation of equine oocytes by interleukin 1 beta via specific IL-1 receptors

Interleukin 1 beta (IL-1 beta) inhibits the LH-induced resumption of meiosis of equine oocytes in vitro. The present study was performed to clarify this inhibitory effect of IL-1 beta by testing increasing concentrations of IL-1 beta, and by measuring the effect of addition of IL-1 receptor antagonist (IL-1RA) to the culture medium. The effect of IL-1 beta on epidermal growth factor (EGF)-induced resumption of meiosis was also studied. Cumulus-oocyte complexes (COCs) were collected from subordinate follicles on ovaries obtained from an abattoir. In five distinct experiments, COCs were cultured for 30 h and nuclear maturation of oocytes was evaluated by DNA staining. In Expt 1, seven different media were tested: medium 1 (TCM199+BSA); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); medium 3 (medium 1+eLH); and media 4, 5, 6 and 7 (medium 3 containing 0.1, 1.0, 10.0 and 50.0 ng IL-1 beta ml(-1), respectively). In Expt 2, four different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and media 3 and 4 (medium 2+IL-1RA at 50 and 100 ng ml(-1), respectively). In Expt 3, three different media were tested: medium 1 (TCM199+BSA+eLH); medium 2 (medium 1+50 ng IL-1RA ml(-1)); and medium 3 (medium 2+50 ng IL-1 beta ml(-1)). In Expt 4, four different media were tested: medium 1 (TCM199+BSA+eLH); and media 2, 3 and 4 (medium 1+IL-1RA at 50, 100 and 150 ng ml(-1), respectively). In Expt 5, three different media were tested: medium 1 (TCM199+BSA+EGF); medium 2 (medium 1+50 ng IL-1 beta ml(-1)); and medium 3 (medium 2+50 ng IL-1RA ml(-1)). In Expt 1, LH alone induced an increase in the rate of in vitro maturation (IVM) of equine oocytes (P<0.05), whereas IL-1 beta alone did not have any effect compared with medium 1. IL-1 beta (50 ng ml(-1)) significantly inhibited the eLH-induced IVM of oocytes (P<0.05) compared with medium 3. A decrease in rate of maturation was observed from a concentration of 10 ng IL-1 beta ml(-1) onwards. In Expt 2, the presence of IL-1RA in the culture medium inhibited the effect of IL-1 beta and restored the rate of oocyte maturation (P<0.05) observed in the presence of LH alone. In Expts 3 and 4 it was demonstrated that IL-1RA alone had no positive effect on the eLH-induced rate of maturation. In Expt 5, IL-1 beta inhibited the EGF-induced resumption of meiosis (P<0.05). The addition of IL-1RA inhibited this effect and restored the rate of oocyte maturation (P<0.05) observed with EGF alone. In conclusion, the present data confirm the inhibitory effect of IL-1 beta on IVM of equine oocytes induced by eLH and demonstrate its inhibitory effect on EGF-induced oocyte maturation. The rate of maturation decreased in a dose-dependent way and the lowest rate of maturation was observed at 50 ng IL-1 beta ml(-1) (P<0.05). The use of IL-1RA inhibited these effects, demonstrating that the action of IL-1 beta is receptor-mediated. Moreover, the results clearly show that, in equine species, IL-1 beta is involved in the physiology of COCs by regulating resumption of meiosis.     

Induction of final maturation by sperm penetration in canine oocytes

In contrast to oocytes of most mammals, the canine oocyte is at the germinal vesicle stage at ovulation. Moreover, the bitch is receptive to mating while immature oocytes are present in the oviducts. The aims of this study were to examine the influence of fertilization in immature oocytes on the resumption of meiosis, and the modification of both male and female chromatin in fertilized oocytes. Canine cumulus-oocyte complexes collected from routine ovariectomies were cultured in medium 199 with 20% fetal calf serum for 24 h, incubated in the same medium with fresh semen for 24 h, washed, cultured for a further 24 h and fixed. Control oocytes were cultured in the same medium but without spermatozoa for 24, 48 or 72 h. After fixation, chromatin was stained with propidium iodide and examined using laser scanning confocal microscopy. The data indicate that sperm penetration can occur in immature canine oocytes and that it induces resumption of meiosis. After 72 h of culture, the percentage of oocytes at the germinal vesicle stage was significantly lower in fertilized oocytes (40% versus 60.3% for control oocytes; P < 0.05) and the percentage of oocytes beyond metaphase I was significantly greater in fertilized oocytes (28.3% metaphase I and II, and two pronuclei versus 10.2% metaphase I and II for control oocytes; P < 0.01). Observation and measurement of the area of chromatin in fertilized oocytes showed an overall parallel condensation-decondensation of both female and male chromatin from the germinal vesicle stage to the pronuclear stage.     

In vitro maturation of bovine cumulus enclosed primary oocytes and their subsequent in vitro fertilization and cleavage

Cumulus enclosed primary oocytes from 2 to 4-mm bovine follicles were matured in vitro in Minimum Essential Medium containing follicle-stimulating hormone (0, .1, 1, 10, 50, or 100 micrograms/ml) or human chorionic gonadotropin (0, .1, 1, or 10 IU/ml) for 48 h at 37 degrees C under paraffin oil. Cumulus mass expansion comparable to that seen in vivo occurred in 18% of the control oocytes, 39% of those cultured in human chorionic gonadotropin, and 56% of those cultured in follicle-stimulating hormone. The optimum follicle-stimulating hormone concentration for cumulus expansion was 1 microgram/ml, and this was then used to mature oocytes individually or in groups of 5 for in vitro fertilization. Ejaculated bovine semen, extended 1:10 with yolk-TES-Tris extender and stored 24 to 48 h at 4 degrees C, was warmed, washed once with Minimum Essential Medium, and 500,000 motile sperm/ml were used to inseminate the matured oocyte-cumulus cell complexes. Criteria for fertilization was cleavage to the two-cell stage 48 h after insemination. Oocytes, inseminated individually, cleaved with a frequency of 5%, whereas 15% of those inseminated in groups of 5 cleaved, perhaps as the result of cumulus factors enhancing capacitation. The cleavage rate for the parthenogenetic control with killed spermatozoa was 0%. Therefore, primary oocytes matured in vitro to secondary oocytes were successfully fertilized in vitro and cleaved to at least the two-cell stage in the Minimum Essential Medium. Individual differences between bulls in ability to fertilize in vitro were noted.     

Disruption of nuclear maturation and rearrangement of cytoskeletal elements in bovine oocytes exposed to heat shock during maturation

Meiotic maturation in mammalian oocytes is a complex process which involves extensive rearrangement of microtubules, actin filaments and chromosomes. Since cytoskeletal elements are sensitive to disruption by heat shock, a series of experiments were performed to determine whether physiologically relevant heat shock disrupts the progression of the oocyte through meiosis, fertilization and zygote formation. Cumulus-oocyte complexes were cultured at 38.5, 40.0 or 41.0 degrees C for the first 12 h of maturation. Incubation during the last 10 h of maturation and 18 h after fertilization was at 38.5 degrees C and in 5% (v/v) CO2 for both treatments. Examination of the cytoskeleton and the chromosome organization in matured oocytes revealed that oocytes matured at 38.5 degrees C were mostly at metaphase II (MII) stage, while the majority of heat-shocked oocytes were blocked at the first metaphase (MI), first anaphase or first telophase stages. A subset of heat-shocked oocytes possessed misshapen MI spindles with disorganized microtubules and unaligned chromosomes. A higher percentage of TUNEL-positive oocytes was noted for oocytes matured at 41.0 degrees C. Addition of 50 nmol/l sphingosine 1-phosphate to maturation medium blocked the effect of heat shock on progression through meiosis and apoptosis and increased the proportion of oocytes matured at 41.0 degrees C that were at MII. Following insemination, a high percentage of heat-shocked oocytes were unfertilized, while the majority of the control zygotes were fertilized and had two visible pronuclei. In conclusion, heat shock disrupts nuclear maturation and induces apoptosis. These alterations are likely to be involved in the mechanism underlying heat-shock-induced disruption of oocyte capacity for fertilization and subsequent development.     

Delay of nuclear maturation and reduction in developmental competence of pig oocytes after mineral oil overlay of in vitro maturation media

Steroid hormones, such as progesterone, oestrogen, androgen and meiosis activating sterols, are secreted from cumulus cells that are stimulated by gonadotrophins during maturation of oocytes in vitro. These steroid hormones may be absorbed by mineral oil or paraffin oil; however, in vitro maturation of pig oocytes is commonly performed using medium covered by oil. In this study, high concentrations of progesterone, oestradiol and testosterone were detected in the culture medium after pig cumulus-oocyte complexes (COCs) were cultured with FSH and LH for 44 h in medium without an oil overlay. However, high concentrations of these steroid hormones were not detected in medium when COCs were cultured with the mineral oil overlay. When high concentrations of these steroid hormones were secreted by COCs, germinal vesicle breakdown (GVBD) and the activation of p34(cdc2) kinase and mitogen-activated protein (MAP) kinase in oocytes occurred earlier in comparison with oocytes cultured in medium covered with mineral oil. Moreover, a decrease in p34(cdc2) kinase activity during meiotic progression beyond metaphase I was observed in oocytes cultured in conditions under which high concentrations of steroid hormones were secreted by COCs. In addition, the rate of development to the blastocyst stage after IVF was higher in oocytes matured in medium without an oil overlay. These adverse effects of oil may be explained by absorption by the oil of cumulus-secreted steroids or by the release of toxic compounds into the medium.     

Effects of follicular cells and FSH on the resumption of meiosis in equine oocytes matured in vitro

It has been suggested that preculturing immature oocytes in a manner that maintains them in meiotic arrest may improve cytoplasmic maturation and, thereby, the eventual developmental competence of oocytes matured in vitro. This study examined the ability of follicular cells to maintain meiotic arrest in equine oocytes. Cumulus-oocyte complexes (COCs) recovered from dead mares were cultured for 38 h in M199 either attached to, or together with, different follicle wall components, as follows: (1) attached to the follicle wall, (2) cocultured with separated follicle wall, (3) attached to membrana granulosa (COCG), (4) COCGs cocultured with sheets of theca cells, (5) COCGs cultured in theca-cell conditioned medium, and (6) control COCs without any follicle wall components. When oocytes were cultured attached to their follicle wall, 79% remained in the GV stage throughout the 38 h incubation. However, when oocytes were cocultured with separate pieces of follicle wall, meiosis resumed and a similar proportion of oocytes progressed to metaphase II (79%) as under control conditions (84%). Only 16% of oocytes cultured while still attached to the membrana granulosa (COCGs) maintained the GV stage, whereas when COCGs were cocultured with theca cells or in theca-cell conditioned medium, significantly more oocytes remained in the GV stage (64 and 52%, respectively), indicating that theca cells secrete a meiosis-inhibiting factor. The effect of FSH on the meiosis-inhibiting activity of follicular cells was investigated by culturing COCs attached to the follicle wall and COCGs in the presence or absence of theca cells in medium containing FSH. Addition of 0.05 iu recombinant human FSH ml(-1) to the culture medium did not affect nuclear maturation and failed to overcome the suppressive effect exerted by the follicle wall or by theca cells, despite the fact that mRNA for the FSH receptor was found using RT-PCR in both cumulus and granulosa cells. These results demonstrate that the maintenance of meiotic arrest in equine oocytes during culture can be promoted by theca cells, which appear to act via a secreted inhibitory factor that cannot be suppressed or counteracted by FSH.     

Quality of porcine blastocysts produced in vitro in the presence or absence of GH

GH receptor (GHR) mRNA is expressed in bovine in vitro produced embryos up to the blastocyst stage and GH improves the quality of bovine embryos by increasing blastocyst cell numbers and reducing the incidence of apoptosis as evaluated by DNA strand-break labelling. Porcine in vitro produced blastocysts have lower cell numbers than in vivo blastocysts and exhibit higher incidences of apoptosis. Therefore we investigated the effects of 100 ng GH/ml NCSU23 medium during in vitro culture of presumptive in vitro fertilized sow zygotes on embryo development and blastocyst quality (defined by diameter, cell number, apoptosis and survival after non-surgical transfer). In vivo produced blastocysts were analysed concurrently as a reference value. GHR was expressed in embryos from the 2-cell to blastocyst stages. GH had no effect on blastocyst development or cell numbers, but increased the mean blastocyst diameter. The incidence of apoptosis, detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), was decreased by GH, but when non-TUNEL-labelled apoptotic fragmented nuclei were included, no difference was seen. GH appeared to slow down the progression of apoptosis though. In vivo produced blastocysts presented no apoptotic nuclei, and contained higher cell numbers and larger diameters. Pregnancy rates on day 11 were similar for all groups, but survival was poorer for in vitro than in vivo produced blastocysts. In this study GH appeared to be beneficial only from the blastocyst stage, but the presence of GHR from early cleavage stages nevertheless indicates a role for GH throughout porcine embryo development and deserves further investigation.