Synergetic effects of epidermal growth factor and estradiol on cytoplasmic maturation of porcine oocytes

This study was conducted to examine the effect of epidermal growth factor (EGF) and 17beta-estradiol (E2) on nuclear and cytoplasmic (male pronuclear formation and early embryo development) maturation of porcine oocytes. Oocytes were aspirated from antral follicles and cultured in modified TCM-199 medium supplemented with 0.57 mM cysteine, 10 IU/ml eCG, 10 IU/ml hCG, with or without EGF and/or E2. In vitro fertilisation of matured oocytes was performed in a modified Tris-buffered medium (mTBM) with frozen-thawed ejaculated spermatozoa. Oocytes were transferred to NCSU-23 supplemented with 0.4% bovine serum albumin at 6 h after in vitro fertilisation. Significantly higher (p < 0.05) rates of nuclear maturation, pronuclear formation and cleavage (91.7%, 65.2% and 37.3%, respectively) were observed when oocytes were cultured in the medium containing both EGF (10 ng/ml) and E2 (1 microg/ml) than in the medium supplemented with either EGF or E2 or without both. Intracellular glutathione concentration in the oocytes cultured in the medium containing both E2 and EGF was also significantly higher (12.1 pmol per oocyte) than that of oocytes cultured in the medium with E2 or EGF alone or without both. These findings suggested that EGF and E2 have a synergestic effect on both nuclear and cytoplasmic maturation of porcine oocytes.     

The effects of 17beta-estradiol and protein supplement on the response to purified and recombinant follicle stimulating hormone in bovine oocytes

During in vitro maturation of bovine oocytes, effects of gonadotropins (FSH, LH) and growth factors such as epidermal growth factor (EGF) vary among studies. Now that we can use defined or semi-defined medium, it becomes possible to evaluate recombinant products to assess their roles. Therefore, this study was designed to evaluate the effect of purified porcine (pFSH) or recombinant human (r-hFSH; 5, 50, or 500 ng/ml) follicle stimulating hormone, luteinising hormone (LH; 50,500 or 5000 ng/ml) and epidermal growth factor (EGF; 5, 10, 30 or 50 ng/ml) on subsequent embryonic development of in vitro matured bovine oocytes. In addition, the presence of bovine serum albumin (BSA; 8 mg/ml) as a protein supplement during in vitro maturation (IVM) was studied. For all treatments, cumulus-oocyte complexes were matured in defined maturation medium consisting of synthetic oviduct fluid. Addition of LH to the maturation medium at all concentrations studied did not increase the proportion of oocytes developing to the morula and blastocyst stages. However, morula and blastocyst yield were improved (p < 0.05) after addition of EGF (30 ng/ml) as compared with maturation medium alone (29.3% vs 18.0%, respectively). Addition of r-hFSH to the maturation medium in the presence of 17beta-estradiol (E2) significantly (p < 0.0001) increased the morula and blastocyst rate compared with maturation medium alone (40.3% vs 19.3%, respectively). The presence of BSA alone during IVM significantly reduced the developmental competence of oocytes as reflected by the morula and blastocyst yield. These results demonstrate the essential role of FSH, EGF and E2 on the kinetics of nuclear and cytoplasmic maturation that are essential for the formation of an egg capable of fertilisation and development. Also, supplementation of r-hFSH and E2 during IVM under our conditions increases morula and blastocyst yield following in vitro fertilisation and in vitro culture in defined medium. Finally, the presence of BSA as the only protein supplement during IVM may be detrimental to oocyte maturation.     

Developmental competence of porcine oocytes selected by brilliant cresyl blue and matured individually in a chemically defined culture medium

The objective of this study was to investigate the effects of oocyte selection using brilliant cresyl blue (BCB) and culture density during individual in vitro maturation (IVM) on porcine oocyte maturity and subsequent embryo development using a chemically defined medium. Cumulus-oocyte complexes (COCs) were classified as BCB-positive or BCB-negative after exposure to a BCB solution for 90 min. The classified COCs were matured in a group (15 COCs per 100-μL droplet) or individually (1 COC per 1-, 2.5-, 5-, or 10-μL droplet). Meiotic competence, intraoocyte glutathione concentration, and developmental competence after intracytoplasmic sperm injection were monitored. The BCB selected oocytes competent for nuclear and cytoplasmic maturation. Furthermore, meiotic competence for oocytes matured individually in a 5-μL droplet was superior (P < 0.05) to that of oocytes matured in a 1-μL droplet. Also, the culture density in a 5-μL droplet during IVM resulted in a higher (P < 0.05) rate of cleaved embryos than that in a 1-μL droplet and produced a similar rate of blastocysts compared with that of a group culture system. Conversely, BCB selection did not improve cleavage and blastocyst formation. In conclusion, it was possible to predict porcine oocytes competent for maturation using oocyte selection with BCB. Moreover, a 5-μL droplet during the individual IVM culture was most suitable for oocyte maturation and subsequent embryo development, although every culture density used in this study supported development up to the blastocyst stage.     

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.     

Serial nuclear transfer improves the developmental potential of mouse embryos cloned from oocytes matured in a protein-free medium

Germinal vesicle (GV) oocytes matured in vitro are an alternative source for cytoplasmic recipients of nuclear transfer (NT). However, the developmental potential of oocytes matured in vitro is limited. In this study, we developed a protein-free maturation medium for mouse GV oocytes. Following parthenogenetic activation, the oocytes matured in the protein-free medium develop to blastocyst stage with a high efficiency, even up to the rate obtained from in vivo MII-oocytes (90.6% vs. 92.8%). Using the oocytes matured in the protein-free medium as the recipient, NT embryos develop to the blastocyst stage (17.6%). To further improve the developmental potential of NT embryos, we performed serial NT and compared the effect of three different activated cytoplasm samples derived from in vitro matured oocytes as the second recipient, that is, the effect of in vitro fertilized (IVF) zygote, the preactivated cytoplast and the IVF cytoplast, on the development of NT embryos. We found that when the pronucleus of NT zygote was transferred into the cytoplasm of the IVF zygote, the blastocyst formation increased to 39.4%. This is the first report to demonstrate the IVF zygote from oocytes matured in protein-free medium can be used successfully as the recipient for serial NT to enhance the developmental potential of mouse NT embryos from oocytes matured in the protein-free medium.     

Effects of hexoses on in vitro oocyte maturation and embryo development in pigs

The objective was to determine the effects of supplementing hexoses in oocyte maturation and embryo culture medium on in vitro maturation (IVM) and in vitro fertilization (IVF) of porcine oocytes and in vitro development of in vitro produced (IVP) porcine embryos. In the first experiment, oocytes were matured in vitro in modified North Carolina State University (NCSU)-37 medium, supplemented with hexoses (glucose, fructose or galactose) at various concentrations: 0 (control), 2.5, 5.5 and 10 mM. Supplementing the maturation medium with either glucose or fructose (5.5 mM) increased the percentages of oocytes that matured to metaphase II (79.4 and 70.2%, respectively), as compared with the control group (P < 0.05). However, supplementing galactose had no effects on meiotic maturation and fertilization. In the second experiment, cleaved embryos were collected 3 days after IVF of oocytes matured in the maturation medium supplemented with 5.5 mM of glucose; they were cultured for an additional 4 days in modified NCSU-37 medium, supplemented with 5.5mM of glucose, fructose or galactose. The incidence of blastocyst formation was higher (P < 0.05) in the glucose and fructose groups (18.6 and 18.2%, respectively) than in the galactose group and non-supplemented control group (12.9 and 9.2%). Moreover, fructose supplementation increased the total cell number/blastocyst (48.0 versus 37.6) and reduced the index of DNA-fragmented nucleus in the blastocysts (7.6% versus 11.8%), as compared with glucose supplementation (P < 0.05). In conclusion, fructose was a practical alternative to glucose for supporting IVM of porcine oocytes and fructose was superior to glucose for producing high-quality porcine embryos in vitro.     

New technique to quantify the lipid composition of lipid droplets in porcine oocytes and pre-implantation embryos using Nile Red fluorescent probe

The principal objective of this study was to develop a novel method based on confocal microscopy and a solvatochromic fluorescent dye, Nile red (NR) to quantify the main types of lipids in a single mammalian oocyte and embryo. We hypothesize that NR staining followed by the decomposition of NR-spectra identifies and quantifies the triglycerides, phospholipids, and cholesterol in a single oocyte and embryo. We analyzed the lipid droplets in porcine oocytes and pre-implantation embryos up to the hatched blastocyst stage developed in vivo and in cultured blastocysts. The emission spectrum of NR-stained mixture of different lipid types is a convolution of several component spectra. The principal component analysis (PCA) and a multivariate curve resolution-alternating least squares method (MCR-ALS) allowed to decompose the emission spectrum and quantify the relative amount of each lipid type present in mixture. We reported here that the level of the triglycerides, phospholipids and cholesterol in lipid droplets significantly decreases by 17.7%, 26.4% and 23.9%, respectively, from immature to mature porcine oocytes. The content of triglycerides and phospholipids remains unchanged in droplets of embryos from the zygote up to the morula stage. Then the triglyceride level decreases in the blastocyst by 15.1% and in the hatched blastocyst by 37.3%, whereas the amount of phospholipids decreases by 10.5% and 12.5% at the blastocyst and hatched blastocyst stages, respectively. In contrast, the content of cholesterol in droplets does not change during embryo cleavage. The lipid droplets in the blastocyst produced in vivo contain lower amounts of triglycerides (by 26.1%), phospholipids (by 14.2%) and cholesterol (by 34.8%) than those in the blastocyst cultured in NCSU-23 medium. In conclusion, our new technique is suitable to quantify the content of triglycerides, phospholipids and cholesterol in individual mammalian oocytes and embryos. Our findings indicate an important role for lipids during porcine oocyte maturation and early embryonic development, and suggest an altered lipid metabolism in cultured embryos.     

Duration of in vitro maturation of recipient oocytes affects blastocyst development of cloned porcine embryos

This study investigated the effects of different incubation periods for oocyte maturation and contact inhibition of donor cells as well as different osmolarities for storage of recipient oocytes on fusion rates, cleavage rates, and blastocyst yields of porcine somatic nuclear transfer (SCNT) derived embryos. In addition, the in vivo developmental potential of cloned embryos derived from the most promising SCNT protocol was tested by transfer to recipient gilts. Storage of in vitro-matured oocytes for 7.5 h in calcium-free TL-HEPES medium at 295 or 320 mOsmol prior to activation yielded significantly (p < 0.05) higher parthenogenetic blastocyst rates compared to storage in TL-HEPES with an osmolarity of 270 mOsmol (24.4 +/- 3.0% and 26.2 +/- 4.3% vs. 18.3 +/- 6.4%, respectively, mean +/- SD) and improved the visibility of the polar body. Electrical fusion of fibroblasts to enucleated oocytes matured for 38, 40, or 42 h resulted in similar fusion and cleavage rates (74.8-84.4%). However, nuclear transfer with oocytes matured for 40 h in vitro yielded significantly higher (p < 0.05) development to the blastocyst stage after 7 days of culture (14.7 +/- 1.7%) than with oocytes matured for 38 h (9.5 +/- 2.1%) or 42 h (5.1 +/- 2.1%). Contact inhibition for 24, 48, or 72 h significantly (p < 0.05) increased the proportion of cells at G0/G1 compared with cycling fibroblasts. However, duration of contact inhibition of the donor cells for either 24, 48, or 72 h had no effect on blastocyst rates of SCNT embryos. Four gilts received an average of 150 SCNT embryos (range 138-161) reconstructed with oocytes matured for 40 h; two of these became pregnant; one of them went to term and farrowed four piglets on day 115 of pregnancy. Microsatellite analysis confirmed that the clones were genetically identical with the donor cells. These results show that changes of the in vitro maturation protocol may affect in vitro development of reconstructed porcine embryos, while duration of the contact inhibition period plays a minor role for the success of porcine SCNT. The effects on in vivo development are yet to be determined.     

Gas6在猪卵母细胞及早期胚胎中表达模式与功能的初步研究

该研究通过免疫组化和QRT-PCR等方法系统分析了Gas6（growth arrest-special gene 6）基因在猪卵泡发生及早期胚胎发育过程中的表达规律,并提出了一种改良猪卵母细胞体外成熟系统的方法。研究结果显示,Gas6基因表达于猪卵巢中的卵母细胞细胞核及其周围的卵丘细胞,在卵母细胞体外成熟及早期胚胎发育过程中始终有表达,且在囊胚中表达最高。Gas6 mRNA在卵母细胞成熟过程中始终存在,但在孤雌激活后迅速消失,直到发育到囊胚时再次出现。在猪卵母细胞体外培养系统中添加不同浓度的Gas6重组蛋白培养卵母细胞,发现添加Gas6重组蛋白对卵母细胞的极体率无显著影响;但是当添加浓度为100 ng/mL时,培养的卵母细胞孤雌激活后分裂率、囊胚率及囊胚细胞数都显著增高。Gas6可能是通过改善卵母细胞细胞质的成熟质量提高卵母细胞的发育潜能,从而获得了更多、更好的胚胎。     

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.     

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.     

Inactivation of p34cdc2 kinase by the accumulation of its phosphorylated forms in porcine oocytes matured and aged in vitro.

Culturing of matured porcine oocytes in vitro results in the enhancement of their cytoplasmic ability for oocyte activation (so-called ageing), although they are arrested at metaphase II. The enhanced ability for oocyte activation is related to decreased activity of the maturation promoting factor (MPF). In the present study we clarified the molecular mechanism of MPF inactivation during ageing, especially the changes in the phosphorylation status of p34cdc2, a catalytic subunit of MPF, compared with that in fertilised oocytes. The MPF activity decreased gradually when maturation culture was prolonged from 36 to 72 h, confirming the decreasing MPF activity in aged oocytes. The activity of 48 h matured oocytes also decreased after in vitro fertilisation. Immunoblotting of p34cdc2 with anti-PSTAIRE antibody revealed that the culturing of matured oocytes induces a gradual increase in pre-MPF, which is a p34cdc2 and cyclin B complex inactivated by phosphorylation at the inhibitory phosphorylation site of p34cdc2. In contrast, pre-MPF decreased after fertilisation, indicating the degradation of cyclin B. These results suggest that the molecular mechanisms of inactivation of MPF are different between oocyte activation and ageing, and that the mechanism during ageing might be based on the inhibitory phosphorylation of p34cdc2, whereas that of oocyte activation is based on the degradation of cyclin B.     

Glutathione content and embryo development after in vitro fertilisation of pig oocytes matured in the presence of a thiol compound and various concentrations of cysteine.

The present study examined the effect of different concentrations of cysteine in the presence of a thiol compound, beta-mercaptoethanol (BME), during in vitro maturation (IVM) of pig oocytes on cumulus expansion, nuclear maturation, intracellular glutathione (GSH) level and subsequent embryonic development after in vitro fertilisation (IVF). In experiment 1, oocytes were matured in NCSU 23 medium containing 10% porcine follicular fluid, 25 microM BME, 0.5 microgram/ml LH, 0.5 microgram/ml FSH and 0, 0.1, 0.2 or 0.4 mg/ml cysteine for 20-22 h and then without hormonal supplements for an additional 20-22 h. After culture, cumulus cells were removed and a proportion of oocytes fixed to examine the rate of nuclear maturation. The remaining oocytes were co-incubated with spermatozoa for 5-6 h and putative zygotes were transferred to NCSU 23 medium containing 0.4% bovine serum albumin for 144 h. A proportion of putative zygotes were fixed 12 h after insemination to examine fertilisation parameters. In experiment 2, oocytes were matured as in experiment 1 and the GSH content was measured by a DTNB-GSSG reductase recycling assay. No mean differences among treatments were observed in nuclear maturation (78-89%). The mean differences in penetration rate (69-77%), polyspermy rate (31-40%), male pronuclear formation rate (93-96%) or mean number of sperm per oocyte (1.5-1.8) were not affected by the presence or absence of cysteine during oocyte maturation. Also no difference was observed in cleavage rates 48 h after insemination. However, compared with no addition (19%), the presence of 0.1-0.4 mg/ml cysteine during IVM increased (p < 0.001) the proportion of blastocysts (32-39%) at 144 h. In comparison with controls (5.6 pmol/oocyte), the GSH content of oocytes matured in the presence of cysteine was significantly (p < 0.001) higher (13-15 pmol/oocyte) with no mean differences among different cysteine concentrations. The results indicate that in the presence of a thiol compound, supplementation of IVM medium with cysteine can increase the GSH level and improve the developmental competence of pig oocytes following fertilisation. Further, no effect on either GSH level or embryo development was observed by increasing the levels of cysteine supplementation from 0.1 to 0.4 mg/ml.     

In vitro maturation of porcine oocytes using a defined medium and developmental capacity after intracytoplasmic sperm injection

To establish a defined in vitro maturation culture system for porcine oocytes, we examined the effects of adding cysteine (Cys) and epidermal growth factor (EGF) to the maturation medium. Furthermore, to evaluate cytoplasmic maturation, we investigated GSH concentrations and embryo development after intracytoplasmic sperm injection (ICSI). The basic media for IVM were modified TCM199 containing 10% newborn calf serum (NBCS) or 0.1% polyvinyl alcohol (PVA), supplemented with amino acids. Adding EGF (10 ng/ml) or EGF + Cys (0.57 mM) to the defined medium (0.1% PVA + amino acids) increased (P < 0.05) the rate of nuclear maturation relative to the defined medium (without these additives). After ICSI, oocytes matured in a medium supplemented with NBCS, Cys and EGF had a higher (P < 0.05) rate of pronuclear formation rate than oocytes matured in the defined IVM medium. Although there was no significant difference in cleavage rates between NBCS- and PVA-containing media supplemented with both Cys and EGF, the rate of blastocyst development was lower (P < 0.05) in the defined medium than in the NBCS-containing medium. Intracellular GSH concentrations of oocytes matured in the NBCS- and PVA-containing media supplemented with both Cys and EGF were higher (P < 0.05) than in oocytes matured in PVA alone or in oocytes before maturation. Adding Cys and EGF to a defined medium for porcine IVM improved rates of nuclear maturation and cleaved oocytes following ICSI, probably due to increased GSH concentrations. Also, embryos derived from oocytes matured in the defined medium (with the addition of Cys and EGF) developed into blastocysts after ICSI.     

Effects of protein synthesis on maturation, sperm penetration, and pronuclear development in porcine oocytes.

In vitro matured porcine oocytes were used to test the importance of protein synthesis for sperm penetration, the second meiotic division, and pronuclear development. Experiments were carried out to measure rates of protein synthesis in the presence of protein synthesis inhibitors (35 microM or 350 microM cycloheximide or a combination of inhibitors) (study 1); to test for sperm penetration and pronuclear development when protein synthesis was inhibited during fertilization (study 2); to test for oocyte meiosis, sperm penetration, and female and male pronuclear development when protein synthesis was inhibited during maturation (oocyte maturation in vitro with addition of inhibitor at 0, 24, or 36 hr of culture) (study 3); and to analyze the changes in the pattern of protein synthesis during these phases. Sperm penetration, oocyte meiosis, and female pronuclear development were not affected by the total inhibition of protein synthesis during fertilization. By contrast, inhibiting protein synthesis during maturation severely impaired the completion of meiosis and pronuclear development. Although inhibition of protein synthesis after 36 hr of maturation culture did not totally block male pronuclear development (MPN), the rate of MPN formation was lower than for controls (52% vs. 72%, P less than 0.05). However, protein synthesis was absolutely essential between 24 and 36 hr for the formation of MPN after decondensation. This period of maturation coincided with the dominant phase of protein reprogramming in the oocyte.     

Role of follicle stimulating hormone and epidermal growth factor in the development of porcine preantral follicle in vitro

The aim of the present study was to assess the role of follicle stimulating hormone (FSH), epidermal growth factor (EGF) or a combination of EGF and FSH on the in vitro growth of porcine preantral follicles, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. Porcine preantral follicles were cultured for 3 days in the absence or in the presence of FSH or EGF. Oocytes from these follicles were then matured, fertilized in vitro and embryos were cultured. Estradiol secretion and histological analysis of cultured follicles were also carried out. The results showed that when FSH, or a combination of EGF and FSH, was added to the culture medium, most of preantral follicles grew to antral follicles with high estradiol secretion and the oocytes from these antral follicles could mature, fertilize and develop to the blastocyst stage. Without FSH, or a combination of EGF and FSH, preantral follicles were unable to develop to the antral stage. Histology demonstrated that the resulting follicles were nonantral, estradiol production was reduced and none of their oocytes matured after in vitro maturation. The results indicate the essential role of FSH in promoting in vitro growth of porcine preantral follicle, estradiol secretion, antrum formation, oocyte maturation and subsequent embryonic development. EGF with FSH treatment of porcine preantral follicles improves the quality of oocytes, shown by a higher frequency of embryonic development.     

Preovulatory follicular fluid during in vitro maturation decreases polyspermic fertilization of cumulus-intact porcine oocytes in vitro maturation of porcine oocytes

Porcine follicular fluid (pFF), as a supplement of maturation media, has been shown several times to improve the in vitro production (IVP) of porcine embryos. As a transudate of serum, pFF contains locally produced factors in addition to the ones derived from serum. The objective of this study was to determine the additional positive effects of these pFF specific factors on the nuclear and cytoplasmic maturation of porcine oocytes. Follicular fluid and autologous serum were collected from sows in the preovulatory phase of the estrous cycle. Subsequently, oocytes from prepubertal gilts were matured in NCSU23 supplemented with either 10% pFF or 10% autologous serum derived from the same sow. Oocytes were then fertilized and the putative zygotes were cultured for 7 days. Nuclear maturation and cumulus expansion were assessed after the maturation culture. For evaluation of cytoplasmic maturation, oocyte glutathione (GSH) content, fertilization parameters and embryonic development were evaluated. After in vitro maturation (IVM) of the oocytes, both cumulus expansion rate and oocyte GSH content were increased for oocytes matured in pFF (P<0.05). More monospermic penetration was found when cumulus-intact oocytes had been matured in 10% pFF but this effect was lost after fertilization of cumulus denuded oocytes indicating that the pFF was acting through the cumulus. We speculate that the increased cumulus expansion and increased glutathione content, which were prevalent after IVM in pFF, are responsible for the positive effects on fertilization and the pre-implantation development of the embryos.     

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.     

Advances on in vitro production and cryopreservation of porcine embryos

There have been intensive attempts to establish reliable in vitro production (IVP) and cryopreservation methods of embryos in pigs. Although a great deal of progress has been made, current IVP systems and cryopreservation still suffer from insufficient cytoplasmic abilities of in vitro matured oocytes, polyspermic fertilization, poor quality of in vitro produced embryos and low efficiency of embryo cryopreservation. Compared to other mammalian species, pig oocytes and embryos are characterized by large amounts of lipid content stored mainly in the form of lipid droplets in the cytoplasm. This fact has a negative influence on biotechnological applications on porcine oocytes and embryos. In this review, we will discuss recent studies about methods and techniques for modifying porcine embryo IVP system and embryo cryopreservation that produces high quality of pig blastocysts using in vitro maturation, in vitro fertilization, in vitro culture, microsurgical manipulation, addition of protein, the use of cytoskeleton stabilizing agents and various physical methods. The presented methods and techniques make it possible to modify the characteristics of oocytes and embryos and thus may become major tools in mammalian gamete and embryo agricultural or biotechnological applications in the future.     

Developmental ability of porcine oocytes matured and fertilized in vitro

The developmental abilities of porcine oocytes matured and fertilized in vitro were examined in vivo and in vitro. Cumulus-oocyte complexes were cultured in mM199 supplemented with 10% porcine follicular fluid (PFF) and hormonal supplements (PMSG, hCG and estradiol-17beta) for 20 h and then without hormonal supplements for an additional 20 h. In Experiment 1, oocytes were then co-cultured for 6 h with spermatozoa which had been preincubated with 1% PFF (PFF-treated) or without (control). Oocytes were transferred to oviducts of gilts or cultured in modified Whitten's medium for 5 d. The percentages of oocytes with monospermic penetration (59%, 42 71 ) and with monospermic penetration and male and female pronuclei (32%, 23 71 ) were higher (P < 0.01) in the PFF-treated group than in controls (25%, 18 71 and 8%, 6 71 , respectively). After 5 d, the percentages of oocytes that developed to the morula or blastocyst stages in vitro and in vivo in the PFF-treated group (10%, 28 288 and 13%, 41 318 , respectively) were also higher (P < 0.05) than in controls (2%, 6 284 and 6%, 16 248 , respectively). Whereas some oocytes that were matured and fertilized in vitro developed to the blastocyst stage after 5 d in vivo culture (3%, 9 288 in PFF-treated group and 2%, 6 284 in control), no blastocysts were observed after 5 d when oocytes were cultured in vitro. When the progression of in vitro development of porcine oocytes that were matured and fertilized in vitro was examined in Experiment 2, morulae appeared after 72 h of culture, and 3% (3 100 ) of the oocytes developed to the blastocyst stage after 144 h (6 d) of culture. These results demonstrate that decreasing polyspermic penetration and increasing monospermic male pronuclear formation, as a result of PFF treatment of maturing spermatozoa, improved the developmental ability of porcine oocytes matured and fertilized in vitro. However, development in vitro was delayed by approximately 24 h compared with in vivo development, most of the embryos were blocked at the morula stage.