The activity of three glycosidases (ß-Ν-acetyloglucosaminidase, α-mannosidase, and ß-galactosidase) in the follicular fluid and in the maturation medium affects bovine oocyte maturation.

We studied the role of follicular fluid's (FF) glycosidase (α-mannosidase [α-ΜΑΝ], ß-Ν-acetyloglucosaminidase [NAGASE], ß-galactosidase [ß-GAL]) activity during IVM of bovine oocytes. Oocytes were allocated into two groups according to the follicular size (small follicle [SF]: 2-5 mm, large follicle [LF]: >5-8 mm). In experiment 1, cumulus-oocyte complexes (COCs) quality was evaluated according to morphologic criteria (grades A, B-C, D); oocyte (n = 801) nuclear maturation was assessed after 24 hours of incubation. Bovine embryos were produced in vitro in groups (experiment 2, n = 1503 oocytes) or individually (experiment 3, n = 50 oocytes). More grade-A and -BC COCs were collected from SF and LF groups, respectively (P < 0.05). Maturation rate (experiment 1) and cleavage rate (experiments 2 and 3) were similar in SF and LF groups. Activity of all glycosidases in FF was higher (P < 0.05) in SF group than in LF group, whereas in maturation medium of SF group it was, overall, significantly lower than in that of LF (experiments 2 and 3). In FF of SF group, NAGASE positively associated with grade-A oocytes and negatively with BC oocytes; increased ß-GAL was associated with degenerated oocytes. Cleavage rate in LF group, related negatively to NAGASE and positively to α-MAN in maturation medium. These results indicate that during maturation, COCs release NAGASE and consume ß-GAL, but differences probably exist between individual and group maturation.

Preimplantation genetic diagnosis in Welsh pony embryos after biopsy and cryopreservation.

Preimplantation genetic diagnosis and embryo cryopreservation are important tools to improve genetic management in equine species with marked consequences on the economic value, health, biodiversity, and preservation of the animals. This study aimed to develop a biopsy method at the blastocyst stage that provides viable genotyped cryopreserved Welsh pony embryos. Embryos were collected at d 6.75 to 7 after ovulation. Biopsies were performed with either a microblade or a micropipette. After biopsy, embryos were cryopreserved. The survival rate of biopsied embryos was evaluated on fresh and cryopreserved embryos either 24 h after in vitro culture or after transfer to recipients. Fresh and nonbiopsied embryos were used as controls. Sex, coat color genes, myotony (neuromuscular disorder) diagnosis, and markers of parentage were investigated using PCR on biopsied cells after whole-genome amplification and on remaining embryos. The embryo survival rate after transfer was not affected by the micropipette biopsy (50%, = 8; 43%, = 7; and 50%, = 12, at d 30 for fresh biopsied embryos, vitrified biopsied embryos, and control embryos, respectively) but was significantly reduced by the use of microblade biopsy: 9 ( = 11) vs. 67% ( = 12) for control embryos. Successful sex determination was achieved for 82% ( = 28) of the micropipette biopsies and 100% ( = 50) of the microblade biopsies. Sex determined on biopsied cells was found to correspond completely (100%) with that determined on the remaining embryo ( = 37). More than 90% of the parentage checking markers, coat color, and myotony diagnosis were successfully determined on biopsies obtained with either a micropipette or a microblade. Mendelian incompatibility (7.5 and 5.5%) and embryo genotyping errors (6.6 and 8.6%) were low and not significantly different between the 2 methods. In conclusion, for the first time, pregnancy at Day 30 was obtained after transfer of Welsh pony biopsied and vitrified embryos >300 µm in diameter to recipient pony mares. The biopsied cells collected enabled multigenetic embryo diagnoses to be performed to a high degree of accuracy. The micropipette biopsy is the better method to apply on Welsh pony embryos.

Bovine Oviduct Epithelial Cells Dedifferentiate Partly in Culture, While Maintaining their Ability to Improve Early Embryo Development Rate and Quality.

There are convincing arguments to suggest that the success of early reproductive events is reliant on a satisfactory dialogue between gametes-embryo and the oviduct epithelium. The aim of this study was to develop and characterize an in vitro model to study these interactions. Cattle zygotes produced in vitro were cultured in either SOF or TCM-199 in the presence or absence of bovine oviduct cell monolayers (BOEC), under 20% or 5% O2 . The embryonic development rate and its quality (cell numbers, cryosurvival) were evaluated, as were the BOEC contents in 11 candidate transcripts (real-time PCR) at different time points. A BOEC co-culture did indeed increase the rate of development in both media under 5% O2 (41 vs 27% and 28 vs 10% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). The effect of BOEC on the developmental rate was more pronounced under 20% O2 (35 vs 6% and 27 vs 4% of Day 8 blastocysts in SOF and TCM-199, respectively; p < 0.05). BOEC significantly increased the embryonic cell count in TCM-199 (122.5 ± 11.1 vs 70.3 ± 9.6; p < 0.05) and embryonic cryosurvival in both media. The expression levels of SOD, FGF2 and TGF-ß1 in BOEC remained steady during culture, although mRNA levels of OGP, C3, PGR and ESR2 were clearly reduced, suggesting a dedifferentiation of BOEC during culture. However, SSP1 and GPX4 transcripts were slightly increased during culture, this rise becoming significant by the end of the culture period. In conclusion, our co-culture system with bovine oviduct epithelial cells used for the development of bovine zygotes produced in vitro enhanced blastocyst formation and above all the quality of the resulting embryos, which was associated with specific transcriptomic changes.

Development rate and gene expression of IVP bovine embryos cocultured with bovine oviduct epithelial cells at early or late stage of preimplantation development.

The use of somatic cells for coculture with embryos has been amply investigated to study embryo maternal interactions. The use of bovine oviduct epithelial cells (BOEC) has been shown to improve the blastocyst rate and quality, affecting their gene expression profile. In this study, we evaluated different timings of BOEC coculture for the development of in-vitro-produced embryos and their effects on blastocysts rate and mRNA abundance of some genes that are important for embryo development. Our results confirmed the positive effects of BOEC on early development of bovine embryos. The presence of the cells during the first four days or during the last four days of development was enough to produce the full BOEC effect. When the presence of BOEC was restricted to the four first days, the kinetics of blastocyst development was accelerated, with significantly more blastocysts at Days 6 and 7 than when the cells were present all along the culture or only during the last four days. Older cells used at early stage were not active anymore. Using young cells at late stage did not improve the cell effect, compared with the older ones. Therefore, the lower effect of BOEC at late stage, compared with early period, may not be explained by cell aging. In addition, the presence of BOEC, at early or late stages, induced changes in the embryos expression profile of genes known to be related to embryo quality, suggesting reduced apoptosis and increased capacity to struggle against oxidative stress after coculture. In conclusion, we confirmed the effect of BOEC on the rate and quality of bovine IVP embryos development. We found for the first time that the presence of BOEC during the four first days of the 8-days development is enough to produce these effects. These first four days represent the period of the presence of the embryos in the oviduct in vivo, highlighting the physiological relevance of this in vitro model of coculture. In addition, we found that the presence of BOEC at early stages of development induced modification of transcription profile in the blastocyst, four days later, suggesting an epigenetic regulation induced by BOEC in growing embryos.

Effect of different culture systems on adipocyte differentiation-related protein (ADRP) in bovine embryos.

Bovine embryos cultured in serum-containing media abnormally accumulate lipid droplets, compared to their in vivo counterparts. The objective of this study was to investigate the effect of different culture systems on the mRNA expression and on the quantification and localisation of adipocyte differentiation-related protein (ADRP), a protein associated with lipid accumulation in bovine blastocysts. Two experiments were independently performed for ADRP mRNA expression analysis. In experiment A, blastocysts were produced in modified synthetic oviduct fluid (mSOF)+10% foetal calf serum (FCS), in coculture (bovine oviduct epithelial cells, Boec) and in ewe oviducts, whereas in experiment B, they were produced in mSOF+10µM docosahexaenoic acid (DHA) and in vivo. Control groups were also performed. ADRP mRNA expression was downregulated in the Boec, ewe oviduct and in vivo groups compared to the 10% FCS or DHA groups, respectively. Moreover, the expression of this protein was downregulated in the Boec group compared to the control group (P<0.05). A third experiment (experiment C) was performed to quantify and localise ADRP protein. Boec, in vivo and control groups were tested. After immunofluorescence staining followed by confocal microscopy analysis, embryonic ADRP was clearly localised around lipid droplets, indicating that ADRP is also a lipid droplet coat protein in bovine embryos. In conclusion, our results demonstrate that bovine embryos at the blastocyst stage expressed ADRP mRNA and protein, and that the embryonic culture system modified this expression.

[Use of an in vitro model in bovine to evidence a functional and molecular dialogue between preimplantation embryo and oviduct epithelial cells]. / Mise en évidence d'un dialogue moléculaire et fonctionnel entre l'embryon préimplantatoire bovin et l'oviducte grâce à un modèle in vitro.

Beyond being a pipe between ovary and uterus, the oviduct is an active player in different aspects of early reproductive processes, in particular in the transport of embryos to the site of implantation and the regulation of its early development. Different studies evidenced a communication between oviduct and early embryo at the molecular and functional levels. Since the study of these interactions is difficult in vivo, different in vitro systems have been developed to mimic the maternal milieu during early development. These systems allowed to confirm the action of the cells on the quality of early development (blastocyst rate and viability). In turn, the embryos are producing signals that are able to modify and adapt the activity of maternal cells.

Breast-cancer anti-estrogen resistance 4 (BCAR4) encodes a novel maternal-effect protein in bovine and is expressed in the oocyte of humans and other non-rodent mammals.

STUDY QUESTION: Does BCAR4 have a role in mammalian embryo development? SUMMARY ANSWER: Expression, localization and functional data support that BCAR4 is a maternal-effect protein in non-rodent mammals. WHAT IS KNOWN ALREADY: BCAR4 was previously identified as an oocyte-specific gene in cattle, and as a marker of certain breast tumors in humans. STUDY DESIGN, SIZE, DURATION: Human oocytes were obtained from patients undergoing IVF, but had failed to mature after ovarian stimulation. Dog oocytes were obtained from ovariectomized bitches. Pig, horse and bovine ovaries were obtained from commercial slaughterhouses for extraction of immature oocyte-cumulus complexes. In vivo matured bovine matured oocytes were obtained after ovulation induction and ovulation inducing treatment of Montbeliard heifers. MATERIALS, SETTING AND METHODS: Expression at the RNA level was analyzed by reverse transcription coupled to polymerase chain reaction. Western blot and immunolabeling coupled to confocal or electronic microscopy were used to analyze bovine protein expression and intracellular localization. For the functional approach, short-interfering RNA were microinjected into mature bovine oocytes, followed by IVF; cleavage and embryo development were recorded. MAIN RESULTS AND THE ROLE OF CHANCE: The BCAR4 gene is conserved in mammalian species from various orders and has been lost in rodents after divergence with lagomorphs. The transcript is expressed in the oocytes of humans and domestic species. We bring the first experimental evidence of the BCAR4 protein in mammals. In cattle, the protein is not detected in immature oocytes but starts to be synthesized during maturation, increases in the zygote and persists until the morula stage. The protein is detected throughout the cytoplasm in mature oocytes, concentrates in and around the pronuclei in the zygote, and appears to shuttle in and out of the nuclei starting in the 2-cell embryo; BCAR4 is also present at the junctions between blastomeres from 2-cell to morula. In our functional approach, targeting the BCAR4 transcript by small-interfering RNA significantly compromised development to the morula or/and blastocyst stages (P < 0.05, logistic regression). LIMITATIONS, REASONS FOR CAUTION: As indicated above, protein expression and function were investigated in cattle and mostly in vitro matured oocytes were used. WIDER IMPLICATIONS OF THE FINDINGS: This study provides a novel candidate gene whose mutation or deregulation may underlie certain cases of unexplained female infertility.

Sex and PRNP genotype determination in preimplantation caprine embryos.

The objective of this study was to test the accuracy of genotype diagnosis after whole amplification of DNA extracted from biopsies obtained by trimming goat embryos and to evaluate the viability of biopsied embryos after vitrification/warming and transfer. Whole genome amplification (WGA) was performed using Multiple Displacement Amplification (MDA). Sex and prion protein (PRNP) genotypes were determined. Sex diagnosis was carried out by PCR amplification of ZFX/ZFY and Y chromosome-specific sequences. Prion protein genotype determination was performed on codons 142, 154, 211, 222 and 240. Embryos were collected at day 7 after oestrus and biopsied either immediately after collection (blastocysts and expanded blastocysts) or after 24 h of in vitro culture (compacted morulae). Biopsied embryos were frozen by vitrification. Vitrified whole embryos were kept as control. DNA of biopsies was extracted and amplified using MDA. Sex diagnosis was efficient for 97.4% of biopsies and PRNP genotyping was determined in 78.7% of biopsies. After embryo transfer, no significant difference was observed in kidding rate between biopsied and vitrified control embryos, whereas embryo survival rate was different between biopsied and whole vitrified embryos (p = 0.032). At birth, 100% of diagnosed sex and 98.2% of predetermined codons were correct. Offspring PRNP profiles were in agreement with parental genotype. Whole genome amplification with MDA kit coupled with sex diagnosis and PRNP genotype predetermination are very accurate techniques to genotype goat embryos before transfer. These novel results allow us to plan selection of scrapie-resistant genotypes and kid sex before transfer of cryopreserved embryo.

Kinetics of gene expression and signaling in bovine cumulus cells throughout IVM in different mediums in relation to oocyte developmental competence, cumulus apoptosis and progesterone secretion.

In vitro maturation of oocytes is a crucial step in assisted reproductive technologies in cattle; however, the molecular mechanisms of cumulus contribution to oocyte developmental potential require more investigation. Based on transcriptomic data, we studied by using real-time RT-PCR and western blot in bovine cumulus cells, the kinetics of expression of several candidate genes involved in oxidative stress response, apoptosis, steroid metabolism and signal transmission throughout IVM. Phosphorylations of the components of the main signaling pathways were also analyzed. In addition, IVM was performed in different maturation mediums which influenced the cumulus apoptosis, progesterone secretion and oocyte developmental competence. Glutathione-S-transferase A1 (GSTA1) transcript and protein abundance significantly decreased throughout IVM progression. Similarly, transcript levels of FSH receptor and aromatase (CYP19A1) and protein levels of three steroidogenic enzymes (steroidogenic acute regulatory protein, cytochrome P450scc and 3-beta-hydroxysteroid dehydrogenase) decreased along with progression of maturation and especially since 10 hours of IVM. Expression of progesterone receptor (PGR) and clusterin (CLU) mRNA and phosphorylations of protein kinases AKT, MAPK P38 and SMAD2 were particularly increased at 10 hours of IVM. This expression pattern supposed the role of these factors during oocyte metaphase-I check point of meiosis. Levels of CLU, GSTA1 and FSHR transcripts were higher in 199 basic hormone-free medium as compared to the medium 199EM, enriched in gonadotropins and growth factors, in which we recorded the higher developmental rate and progesterone secretion. Higher phosphorylation levels of SMAD2, AKT and MAP kinase JNK1, but not of MAP kinases ERK1/ERK2 or P38, was positively correlated with oocyte developmental competence and progesterone secretion and negatively correlated with cumulus apoptosis rate. These factors and signaling pathways in cumulus cells are potentially involved in controlling different stages of oocyte nuclear maturation and acquirement of its developmental potential.

Expression of maternal transcripts during bovine oocyte in vitro maturation is affected by donor age.

The primary objective of this study was to compare expression of maternal transcripts in bovine oocyte populations with differential developmental competence: oocytes from prepubertal and pubertal animals; and oocytes from small (3-4 mm) and large (6-10 mm) follicles from pubertal animals. All transcripts were examined in oocytes prior to and after in vitro maturation (IVM). Genes were selected based on their known maternal effect in mouse (ZAR1, STELLA, HSF1, MATER/NLRP5 and its paralogue NLRP9), or their identification as markers of oocyte maturation, either involved in redox metabolism (PRDX1, PRDX2) or meiotic progression (AURKA). Total or polyadenylated forms of the transcripts were followed by reverse transcription coupled to real-time PCR. Six polyadenylated transcripts were found significantly reduced after maturation irrespective of donor age or follicle diameter (p<0.05). Within these six polyadenylated transcripts, ZAR1, NLRP9, HSF1, PRDX1 and PRDX2 were significantly reduced in oocytes from prepubertal animals compared to adult animals (p<0.05). A younger age was also associated with lower abundance (total form) of PRDX2/PRDX1 irrespective of maturation. Total HSF1, PRDX1 and polyadenylated NLRP9 showed a tendency (p values from 0.053 to 0.08) for a higher detection in oocytes from small follicles, thus encouraging further investigation of the follicle diameter model. However, at the present time, follicle size did not significantly affect expression of transcripts examined. In conclusion, this study demonstrates differences in the maternal store of RNA and its regulation during IVM which is dependent on donor age.

Factors affecting oocyte quality: who is driving the follicle?

Mammalian ovaries contain a large stock of oocytes enclosed in primordial follicles. Ovarian cyclic activity induces some of these follicles to initiate growth towards a possible ovulation. However, most of these follicles terminate their growth at any moment and degenerate through atresia. In growing follicles, only a subset of oocytes are capable to support meiosis, fertilization and early embryo development to the blastocyst stage, as shown through embryo in vitro production experiments. This proportion of competent oocytes is increasing along with follicular size. Growing lines of evidence suggest that oocyte competence relies on the storage of gene products (messenger RNA or protein) that will be determinant to support early stages of embryo development, before full activation of embryonic genome. Given these facts, the question is: are these gene products stored in oocytes during late folliculogenesis, allowing an increasing proportion of them to become competent? Alternatively, these transcripts may be stored during early folliculogenesis as the oocyte grows and displays high transcription activity. Several arguments support this latter hypothesis and are discussed in this review: (i) many attempts at prolonged culture of oocytes from antral follicles have failed to increase developmental competence, suggesting that developmental competence may be acquired before antral formation; (ii) the recent discovery of oocyte secreted factors and of their ability to regulate many parameters of surrounding somatic cells, possibly influencing the fate of follicles between ovulation or atresia, suggests a central role of oocyte quality in the success of folliculogenesis. Finally, in addition to their role in interfollicular regulation of ovulation rate, late folliculogenesis regulation and atresia could also be seen as a selective process aimed at the elimination through follicular atresia of oocytes that did not succeed to store proper gene products set during their growth.

Expression of lumican, a small leucine-rich proteoglycan with antitumour activity, in human malignant melanoma.

BACKGROUND: The family of small leucine-rich proteoglycans (SLRPs), which includes decorin, lumican, biglycan and fibromodulin, constitutes an abundant component of the skin extracellular matrix. We previously demonstrated that human lumican inhibits melanoma growth and progression in a mouse experimental model, by regulating cell migration, proliferation and apoptosis. AIM: The aim of this study was to investigate the expression of lumican and decorin in human malignant melanoma and adjacent peritumoral tissue, to understand better their role in the control of growth and invasion of human melanoma. METHODS: Expression of both proteoglycans was studied by immunohistochemistry using specific antibodies in 34 malignant melanomas, 12 Hutchinson's melanotic freckles and 4 cutaneous metastatic melanomas. RESULTS: We showed that lumican and decorin are located in the dermis and in the peritumoral stroma of malignant melanoma, but are not found in melanoma cells or dense tumour tissue. In the healthy dermis, distant from the tumour, the increasing ratio of lumican to decorin was inversely correlated with the proliferation of the tumour cells (P = 0.035). The comparison of the level of expression of lumican protein in superficial vs. nodular subtypes of malignant melanomas showed a decrease of lumican but not decorin in the peritumoral stroma of nodular subtypes. In the peritumoral stroma, the level of expression of lumican but not decorin decreased significantly (P = 0.016) with increasing Clark levels. In addition, immunocytochemical and reverse transcription PCR analyses of malignant melanoma cell lines (A-375, HT-144) and of MRC-5 and dermal fibroblasts from healthy donors in vitro confirmed that dermal fibroblasts are responsible for lumican and decorin synthesis in skin. CONCLUSIONS. Lumican may regulate vertical progression of human malignant melanoma, but further study is necessary to clarify the antitumour mechanism and the downstream signal transduction pathways involved.

Comparison of cell proliferation index in equine and caprine embryos using a modified BrdU incorporation assay.

The measurement of cell proliferation and cell viability using 5'bromo-2'deoxy-uridine (BrdU) labelling has been described in several cell types and species. The aim of this study was to adapt this technique to equine embryos and to compare the index of DNA replication (S-phase) between equine and caprine embryos. Seventeen equine embryos were recovered at day 6.5 post-ovulation and 20 caprine embryos were recovered at day 7 after the onset of estrus. Equine embryos were incubated during 1h at 39 degrees C in PBS containing 1mM of BrdU. Embryos were then treated in 0.05% trypsin during 15 min at 39 degrees C to permeabilise the capsule, and then embryos were rinsed in PBS containing 10% of foetal calf serum. After washing, embryos were immediately fixed in 2.5% paraformaldehyde with 0.3M NaOH during 15 min at ambient temperature. The S-phase was detected by immunocytochemistry technique. In caprine embryos, BrdU was visualised by the same technique but without the trypsin treatment. The percentage of cells (+/-S.E.M.) with BrdU incorporated into newly synthesised DNA strands was significantly higher in equine embryos (74+/-1) than in caprine (38+/-2). Our results demonstrated that BrdU incorporation assay can be used in equine embryos. This assay allows the determination of the proliferation index of live cells and could be used as an additional tool for evaluating the viability of embryos. The high percentage of cells incorporating BrdU during 1h of incubation with BrdU suggests that in comparison with the caprine embryos the cellular activity of proliferation is more intense in equine embryos and suggests that the cellular cycle is shorter in equine embryos.

Effect of roscovitine, a cdk1 inhibitor, and of the presence of oocyte on bovine cumulus cell expansion and cyclooxygenase-2 expression.

Oocyte maturation is accompanied by differentiation of surrounding cumulus cells. These cells produce hyaluronic acid (HA) and its storage in intercellular spaces results in expansion of the cells. The cumulus cells also accumulate cyclooxygenase-2 (cox-2) during maturation. Both expansion and cox-2 storage are regulated by FSH and EGF. The aim of this study was to determine whether oocyte meiotic resumption is involved in the regulation of cumulus differentiation or not. We investigated the effects of roscovitine, a reversible inhibitor of meiosis resumption of cattle oocytes on EGF induced expansion and cox-2 expression at the transcript and protein levels respectively (RT-PCR and Western blot), in cumulus oocyte complexes (COCs) and cumulus complexes alone (CCs). EGF induced expansion and cox-2 expression in both COCs and CCs. These effects were prevented by roscovitine, whether in the presence or in the absence of oocyte. However, the oocyte was essential for the reversibility of inhibition by roscovitine. In conclusion, our results indicate that i) oocyte secreted-factors are not essential for cumulus expansion, and ii) roscovitine mediated inhibition of meiotic resumption also respects the functionality of the surrounding somatic cells.

Effect of follicular size on meiotic and developmental competence of porcine oocytes.

In several species, the developmental competence of the oocyte is acquired progressively during late follicular growth, after the acquisition of the competence to resume and complete meiosis. In the pig, full meiotic competence of the oocyte is reached in ovarian follicles with a diameter of 3 mm or more. However, there is no information about developmental competence acquisition. We analyzed the ability of oocytes from three foll icular size classes to resume and complete meiosis, to be fertilized, and to develop in vitro to the blastocyst stage. A total of 941 follicles were dissected from slaughterhouse gilt ovaries and classified as small (<3 mm, n = 330), medium (3-5 mm, n = 373), or large (>5 mm, n = 238). The cumulus-oocyte complexes recovered from these follicles were submitted to in vitro maturation for 44 h in TCM199 supplemented with 10 ng/ml EGF, 400 ng/ml pFSH and 570 microM cysteamine; in vitro fertilized for 18 h in mTBM with 10(5) frozen-thawed percoll-selected sperms/ml; and developed for 7 days in mSOF. Samples of oocytes or presumptive zygotes were fixed and stained at the end of maturation and fertilization. Groups of oocytes were cultured for 3 h in the presence of 35S-methionine before or after maturation for SDS-PAGE analysis of protein neosynthesis. More oocytes originating from medium and large follicles were competent for maturation than oocytes from small follicles (77 and 86% of metaphase II, respectively, versus 44%, P < 0.05). More oocytes from medium and large follicles werepenetratedby spermatozoa during in vitro fertilization, resulting in significantly more oocytes presenting two or more pronuclei at the end of fertilization (73 and 77% for medium and large follicles, respectively, versus 53% for small follicles, P < 0.05). More oocytes from medium and large follicles developed to the blastocyst stage (14 and 23%, respectively) than those from small follicles (3%, P < 0.05), even if the development rates were corrected by the maturation or fertilization rates. It is concluded that a high proportion of oocytes harvested from follicles of less than 3 mm in the pig are not fully competent for meiosis and are cytoplasmically deficient for development.

Birth of piglets after OPS vitrification and transfer of compacted morula stage embryos with intact zona pellucida.

In swine, five to six days post-insemination, morulae and blastocysts are collected together after uterine flushing. The purpose of this study was to vitrify zona pellucida-intact morulae with Open Pulled Straw (OPS) technology and obtain piglets after transfer. Morulae (200) were vitrified after a two-step equilibration in ethylene glycol, dimethyl sulfoxide and sucrose in Hepes-buffered TCM199 + 20% NBCS medium (TCM). 2-6 morulae were loaded into OPS and plunged into liquid nitrogen. At embryo warming, a three-step dilution with decreasing concentrations of sucrose was applied. In each of 10 recipients, 20 morulae were transferred surgically. Day 25, gestation rate and the farrowing rate were 80% and 70%, respectively. The pregnant recipients farrowed from 1 to 8 piglets and the survival of total transferred embryos was 13%. Although survival rates are still compromised, OPS technology is therefore appropriate to cryopreserve porcine morulae with intact zona pellucida.

Meiotic and developmental competence of prepubertal and adult swine oocytes.

The present study was conducted to compare meiotic and cytoplasmic competence of prepubertal and adult porcine oocytes, and the effects of EGF (0 to 100 ng/mL), FSH (0 to 400 ng/mL) and prepubertal pFF (0 to 10%) on nuclear maturation. Prepubertal oocytes were less responsive to FSH and pFF than were adult oocytes in terms of stimulation of nuclear maturation. The best nuclear maturation rates for prepubertal oocytes were obtained with 10 ng/mL EGF and 400 ng/mL FSH, whereas for adult oocytes no additional effect of EGF was seen in the presence of 400 ng/mL FSH. Supplementation with pFF had no additional effect on MII yield over that obtained with EGF plus FSH. After maturation in the presence of EGF, FSH and cysteamine, fertilization rates were not different between adult and prepubertal oocytes, but polyspermy was more frequent in prepubertal oocytes (31 +/- 17% vs. 17 +/- 7% in prepubertal and adult oocytes, respectively, P < 0.05). The addition of pFF to maturation medium decreased oocyte fertilization of adult oocytes and polyspermic fertilization in prepubertal oocytes. Blastocyst yield and developmental competence were significantly reduced in prepubertal oocytes compared to adult oocytes. The mean cell numbers in blastocysts cultured for 7 days ranged from 61 to 74, and did not differ among groups. Finally, the viability of the 2- to 4-cell embryos and blastocysts produced was assessed by embryo transfer experiments. One offspring was obtained after transfer of 2- to 4-cell embryos, and one after transfer of in vitro-produced blastocysts. In conclusion, although prepubertal gilt oocytes appeared less meiotically and developmentally competent than their adult counterparts, they can be used to produce blastocysts able to develop to term.

Piglets born after vitrification of embryos using the open pulled straw method.

Morulae and unhatched blastocysts from Large White hyperprolific (LWh) and Meishan (MS) gilts were selected to test an ultrarapid open pulled straw (OPS) vitrification method with two media. The viability of vitrified/warmed embryos was estimated by the percentage of embryos that developed to the hatched blastocyst stage in vitro or by birth after transfer. In Experiment 1, two cryoprotectant dilution media were compared for cryopreservation of MS and LWh blastocysts: TCM was a standard Hepes-buffered TCM199 + 20% NBCS medium and PBS was a PBS + 20% NBCS medium. After a two-step equilibration in ethylene glycol, dimethyl sulfoxide, and sucrose, 2-5 blastocysts were loaded into OPS and plunged into liquid nitrogen. Embryos were warmed; a four-step dilution with decreasing concentrations of sucrose was applied. In PBS, LWh blastocysts (27%) had a lower viability in vitro than MS blastocysts (67%; P = 0.001). In TCM, no significant difference was observed between genotypes (41% for LWh and 43% for MS blastocysts) and both viability rates were lower than that of the control groups. In Experiment 2, morula-stage LWh and MS embryos were vitrified and warmed using PBS. The viability rate was low and did not differ between LWh (11%) and MS (14%). In Experiment 3, 200 MS and 200 LWh blastocysts were vitrified/warmed as described in Experiment 1 (PBS). In each of 20 MS recipients, 20 embryos were transferred. The farrowing rate was 55% and recipients farrowed four and five piglets (median) for MS and LWh blastocysts, respectively. The OPS method is therefore appropriate for cryopreservation of unhatched porcine blastocysts.

Modulation of sulfated glycosaminoglycan and small proteoglycan synthesis by the extracellular matrix.

Cell culture in collagen lattice is known to be a more physiological model than monolayer for studying the regulation of extracellular matrix protein deposition. The synthesis of sulfated glycosaminoglycans (GAG) and dermatan sulfate (DS) proteoglycans by 3 cell strains were studied in confluent monolayers grown on plastic surface, in comparison to fully retracted collagen lattices. Cells were labelled with 35S-sulfate, followed by GAG and proteoglycan analysis by cellulose acetate and SDS-polyacrylamide gel electrophoresis, respectively. The 3 cell strains contracted the lattice in a similar way. In monolayer cultures, the major part of GAG was secreted into culture medium whereas in lattice cultures of dermal fibroblasts and osteosarcoma MG-63 cells but not fibrosarcoma HT-1080 cells, a higher proportion of GAGs, including dermatan sulfate, was retained within the lattices. Small DS proteoglycans, decorin and biglycan, were detected in fibroblasts and MG-63 cultures. They were preferentially trapped within the collagen gel. In retracted lattices, decorin had a higher Mr than in monolayer. Biglycan was detected in monolayer and lattice cultures of MG-63 cells but in lattice cultures only in the case of fibroblasts. In this last case, an up regulation of biglycan mRNA steady state level and down regulation of decorin mRNA was observed, in comparison to monolayers, indicating that collagen can modulate the phenotypical expression of small proteoglycan genes.