Cumulus-enclosed or cumulus-free pig oocytes were cultured for 48 h in a serum-free maturation medium, TCM-199B containing 1 mg/ml polyvinylalcohol, supplemented with gonadotropins (PMSG + hCG) and/or epidermal growth factor (EGF), and inseminated with frozen-thawed ejaculated spermatozoa. Fourteen hours after insemination, the ability of oocytes to form male pronucleus (MPN) was examined. EGF significantly increased the ability of cumulus-enclosed oocytes to form MPN irrespective of the concentrations (1–100 ng/ml) tested. Gonadotropins had a greater effect than EGF on MPN formation in cumulus-enclosed oocytes, and a synergetic effect was observed when EGF and gonadotropins were added together. However, neither EGF nor gonadotropins exhibited a stimulatory effect on MPN formation in cumulus-free oocytes. These results indicate that the role(s) of both EGF and gonadotropins in stimulating cytoplasmic maturation of pig oocytes is mediated by cumulus.

Sperm capacitation is crucial for fertilization. However, debate continues on exactly how, where and when capacitation is elicited in the bovine female genital tract. In this study we used merocyanine-540 and the chlortetracycline (CTC) assay to test how capacitation of bull spermatozoa is affected in vitro by exposure to oviductal fluid (ODF) collected in vivo, various glycosaminoglycans (GAGs) or bicarbonate. Following different durations of exposure, spermatozoa were stained with CTC or merocyanine-540, and evaluated with epifluorescent light microscopy or flow cytometry, respectively. Incubation time did not significantly affect capacitation. Exposure (30–120 min) to ODF capacitated (p < 0.05) bull spermatozoa as measured by either merocyanine-540 or CTC. Hyaluronan was the only GAG that induced a significant increase in B-pattern spermatozoa (capacitated; p = 0.012) compared with controls. Dermatan sulphate also induced capacitation (merocyanine-540 high fluorescence; p = 0.035). Exposure to bicarbonate-enriched media also yielded an increase in merocyanine-540 high fluorescence (p < 0.0001). When bicarbonate was added to the other treatments (ODF or GAGs) an equal increase in merocyanine-540 high fluorescence was noted (p < 0.0001), compared with before addition of bicarbonate and independent of the treatment before exposure. There was no significant difference in the number of B-pattern spermatozoa when bicarbonate was added, but an significant increase in spermatozoa with an acrosome-reacted (AR)-pattern (p < 0.0001) was observed. Exposure of spermatozoa to solubilized zonae pellucidae significantly increased the AR-pattern spermatozoa (p = 0.016). In conclusion, ODF was more potent in inducing capacitation of bull spermatozoa than the individual GAGs. Our results also indicate that bicarbonate is an effector of bull sperm capacitation.

This study was aimed to establish embryonic stem (ES)-like cells from blastocysts derived from somatic cell nuclear transfer (SCNT) in pig. Somatic cells isolated from both day-30 fetus and neonatal cloned piglet were used for donor cells. A total of 60 blastocysts (46 and 14 derived from fetal and neonatal fibroblast donor cells, respectively) were seeded onto a mitotically inactive mouse embryonic fibroblast (MEF) monolayer and two ES-like cell lines, one from each donor cell type, were established. They remained undifferentiated over more than 52 (fetal fibroblast-derived) and 48 (neonatal fibroblast-derived) passages, while retaining alkaline phosphatase activity and reactivity with ES specific markers Oct-4, stage-specific embryonic antigen-1 (SSEA-1), SSEA-4, TRA-1–60 and TRA-1–81. These ES-like cells maintained normal diploid karyotype throughout subculture and successfully differentiated into embryoid bodies that expressed three germ layer-specific genes (ectoderm: β-III tubulin; endoderm: amylase; and mesoderm: enolase) after culture in leukemia inhibitory factor-free medium. Microsatellite analysis confirmed that they were genetically identical to its donor cells. Combined with gene targeting, our results may contribute to developing an efficient method for producing transgenic pigs for various purposes.

The present study was designed to examine whether in vitro produced porcine embryos can be used to establish an embryonic stem (ES) cell line. Porcine embryos were produced by in vitro maturation and in vitro fertilization. Embryos at the 4-cell to blastocyst stages were cultured in an ES medium containing 16% fetal bovine serum with mouse embryonic fibroblasts as a feeder layer. It was found that ES-like colonies were derived only from blastocysts. When these ES-like colonies were separated in 0.25% trypsin–0.02% EDTA solution and cultured again, ES-like colonies were further observed in the subsequent culture until the fourth passage. The cells from ES-like colonies showed positive alkaline phosphatase activity. Some cells from the colonies differentiated into several types of cells in vitro when they were cultured in the medium without feeder layers and leukemin inhibitory factor. Embryoid bodies were also formed when the cells were cultured in a suspension status. These results indicate that porcine ES-like cells can be derived from in vitro produced porcine blastocysts and these ES-like cells are pluripotent. The culture system used in the present study is useful to isolate and culture ES cells from in vitro produced porcine embryos.

Incomplete reprogramming of the donor cell nucleus after nuclear transfer (NT) probably leads to the abnormal expression of developmentally important genes. This may be responsible for the low efficiency of cloned animal production. Insulin-like growth factor 2 (IGF2) and IGF2 receptor (IGF2R) are imprinted genes that play important roles in preimplantation development. To obtain an insight into abnormal gene expression after nuclear transfer, we assessed the transcription patterns of IGF2-IGF2R in single in vitro fertilised and cloned embryos by reverse-transcription polymerase chain reaction (RT-PCR). IGF2R expression did not differ significantly but IGF2 was more highly expressed in cloned embryos than in IVF embryos (p < 0.05). This was confirmed by a quantitative RT-PCR method. Thus, incomplete reprogramming may induce abnormal transcription of IGF2 in cloned embryos.

It is now accepted that sperm trigger deuterostome egg activation by causing an increase in egg cytoplasmic Ca2+ levels (Jaffe, 1983; Whitaker & Swann, 1993). This increase generally takes the form of a single wave of Ca2+ release that crosses the egg from the point of sperm-egg interaction (Jaffe, 1983)eggs Ca2+ waves occur repetitively and soon turn into synchronous pulses, or homogeneous oscillations, that last for hours after sperm-egg fusion (Miyazaki et al., 1993b; Homa et al., 1993). Despite their extensive characterisation it is still not established how sperm trigger these Ca2+ changes in eggs. The signal transduction mechanism is missing. There is a proliferation of overlapping and complex schemes for how the sperm may initiate Ca2+ release (Miyazaki et al., 1993b;Whitaker & Swann, 1993). Here, my aim is to present one simple scheme in its generic form. The brevity of this essay restricts citations and necessitates using reviews to reference original work.

We have studied the developmental activation of the metaphase checkpoint, and the consequences of activating this checkpoint, in the zebrafish embryo. (1) Treatment with nocodazole (a microtubule destabiliser) before mid-blastula transition (MBT) produces complete destruction of all nuclei in the deep cell layer of the embryo. In contrast, nocodazole treatment after MBT efficiently produces metaphase arrest in this cell layer. Thus, the metaphase checkpoint becomes activated at MBT. (2) Although a metaphase arrest is induced by nocodazole, it is not induced by paclitaxel (a microtubule stabiliser). Thus the metaphase checkpoint appears to sense a destabilisation, but not a stabilisation, of spindle microtubules. (3) Metaphase-arrested cells (in nocodazole) can be driven into the next interphase by adding the Ca2+-specific ionophore A23187. Thus, a Ca2+-signalling pathway lies downstream of, or parallel to, the metaphase checkpoint. (4) After mid-gastrula stage, treatment with nocodazole produces DNA fragmentation in all three cell layers. In the enveloping epithelial monolayer (EVL), this is associated with a classical apoptotic phenotype. In the deep layer, it is associated with an unusual, highly condensed nuclear state that is entered directly from metaphase arrest. Thus, after the mid-gastrula stage, the embryo responds to nocodazle by undergoing apoptosis. (5) Nocodazole-induced apoptosis in the deep cell layer can be blocked by the caspase-1,4,5 inhibitors Ac-YVAD-CHO and Ac-YVAD-CMK. This suggests that a homologue of the C. elegans ced-9—ced-4—ced-3 pathway is involved in control over apoptosis in the early zebrafish embryo.

Endometrial injury is an important cause of intrauterine adhesion (IUA), amenorrhea and infertility in women, with limited effective therapies. Recently, stem cells have been used in animal experiments to repair and improve injured endometrium. To date, our understanding of adipose-derived stem cells (ADSCs) in endometrial injury repair and their further therapeutic mechanisms is incomplete. Here, we examined the benefit of ADSCs in restoration of injured endometrium by applying a rat endometrial injury model. The results revealed by immunofluorescence showed that green fluorescent protein (GFP)-labelled ADSCs can differentiate into endometrial epithelial cells in vivo. At 30 days after ADSCs transplantation, injured endometrium was significantly improved, with increased microvessel density, endometrial thickness and glands when compared with the model group. Furthermore, the fertility of rats with injured endometrium in ADSCs group was improved and had a higher conception rate (60% vs 20%, P = 0.014) compared with the control phosphate-buffered saline (PBS) group. However, there was no difference in the control group compared with the sham group. In addition, expression levels of the oestrogen receptor Eα/β (ERα, ERβ) and progesterone receptor (PR) detected by western blot and enzyme-linked immunosorbent assay (ELISA) were higher in the ADSCs group than in the PBS group. Taken together, these results suggested that ADSC transplantation could improve endometrial injury as a novel therapy for IUA.

Egg activation at fertilisation is composed of a complex, choreographed series of events, the initiation of which still is not understood. Two major hypotheses have emerged as explanations for the mechanism of egg activation (reviewed by Nuccitelli, 1991; Whitaker & Swann, 1993). The first holds that the sperm delivers an ‘activating factor’ (e.g. inositol trisphosphate, calcium, or a protein) that diffuses into the egg cytoplasm after gamete fusion. The second hypothesis holds that sperm bind to receptors spanning the egg plasma membrane which then transduce a signal to second messenger enzymes. We present here the evidence for receptor-mediated signal transduction in egg activation at fertilisation. By ‘receptor’ we mean only molecules that bind an extra-membrane ligand and which transduce a signal to molecules residing on the cytoplasmic side of the egg membrane. It is critical to be aware that several alternative activating mechanisms are not mutually exclusive and that species differences may exist. In fact, as more has been learnt about the molecules involved in gamete recognition and binding and of the signalling pathways in the egg, it seems likely that multiple pathways exist to trigger complete egg activation.

In the dominant model, monozygotic (MZ) twinning is universally accepted as a post-fertilization event resulting from splitting of the embryo along its first 2 weeks of development. The stage at which splitting occurs determines chorionicity and amnionicity. A short history on how the model was built is presented, stressing the role played by some embryologists, in particular George Corner, in its completion and final success. Strikingly, for more than 60 years no deep criticisms have been raised against the model, which, in virtue of its rational and plausible character, enjoys the status of undisputed truth. At close examination, the embryological support of the model shows some important weak points, particularly when dealing with late splitting. In the author's view, the model not only has contributed to ‘suspend’ our knowledge on the timing of MZ twinning, but seems indefensible and claims to be substituted. That factor could imply relevant consequences for embryology and bioethics. As an alternative to the model, a new theory to explain the timing of MZ twinning is proposed. It is based on two premises. First, MZ twinning would be a fertilization event. In that case, due to an alteration of the zygote–blastomere transition, the first zygotic division, instead of producing two blastomeres, generates twin zygotes. Second, monochorionicity and monoamnionicity would not depend on embryo splitting, but on fusion of membranes. Some support for this theory can be found in recent embryological advances and also in some explanations of old.

Improper activation and swelling of in vitro produced eggs of European eel, Anguilla anguilla, has been shown to negatively affect embryonic development and hatching. We investigated this phenomenon by examining the effects of salinity and sea salt type on egg dimensions, cell cleavage patterns and egg buoyancy. Egg diameter after activation, using natural seawater adjusted to different salinities, varied among female eels, but no consistent pattern emerged. Activation salinities between 30–40 practical salinity unit (psu) produced higher quality eggs and generally larger egg diameters. Chorion diameters reached maximal values of 1642 ± 8 μm at 35 psu. A positive relationship was found between egg neutral buoyancy and activation salinity. Nine salt types were investigated as activation and incubation media. Five of these types induced a substantial perivitelline space (PVS), leading to large egg sizes, while the remaining four salt types resulted in smaller eggs. All salt types except NaCl treatments led to high fertilization rates and had no effect on fertilization success as well as egg neutral buoyancies at 7 h post-fertilization. The study points to the importance of considering ionic composition of the media when rearing fish eggs and further studies are encouraged.

Ovarian folliculogenesis in mammals is a complex process. Several compounds have been tested during in vitro culture of follicular cells for a better understanding of the mechanisms and factors related to ovarian folliculogenesis in mammals. From these compounds, vascular endothelial growth factor (VEGF) can be highlighted, as it is strongly associated with angiogenesis and, in recent years, its presence in ovarian cells has been investigated extensively. Previous studies have shown that the presence of VEGF protein, as well as mRNA expression of its receptor 2 (VEGFR-2) increases during follicular development. Therefore, it is likely that the interaction between VEGF and VEGFR-2 is crucial to promote follicular development. However, few studies on the influence of this factor on follicular development have been reported. This review addresses aspects related to the structural characterization and mechanism of action of VEGF and its receptors, and their biological importance in the ovary of mammals.

The acrosome reaction is a fundamental event in the biology of the sperm and is a prerequisite to fertilization of the egg. Members of the Rho family of GTPases and their effectors are present in the cytoplasm and/or plasma membrane overlying the acrosome of porcine sperm. We have implicated the Rho family of GTPases and the Rho-activated kinase, ROCK-1, in mediating the zona-pellucida-induced acrosome reaction. Others have implicated the Rho GTPase in regulating the ionophore-induced acrosome reaction in the sperm of several mammalian species as well as in motility of bovine sperm. In this study, the localization of the Rho GTPases (RhoA, RhoB, Rac1 and Cdc42) as well as the effectors RhoGDI, PI(4)P5K and ROCK-1, was determined in boar, human, rat, ram, bull and elephant sperm. The four GTPases were each present in the sperm head of all species examined. RhoGDI was expressed in the head and tail of sperm from all species except pig, where it was present only in the head. PI(4)P5K was expressed in both head and tail of sperm from all species, but expression was typically weaker in the tail. Finally, ROCK-1 was expressed in the heads and tails of all sperm except that of the boar, where it was present only in the acrosomal region. These observations taken together suggest that the expression of Rho GTPases in sperm has been conserved throughout mammalian evolution, most likely due to the role of these GTPases in regulating acrosomal exocytosis.

We previously demonstrated that the development of cultured rat pre-antral follicles is stimulated by growth hormone (GH) and insulin-like growth factor-I (IGF-I) and that the mRNA of IGF-I and type I IGF receptor (IGFR) is present in the oocyte and wall of these follicles. To gain a closer insight into the regulation of early folliculogenesis by GH and IGF-I, the present study investigated the gene expression of GH and GHR mRNA in isolated oocytes and follicular wall cells of pre-antral follicles, using reverse transcriptase polymerase chain reaction, and the localisation of immunoreactive IGF-I, IGFR, GH and GHR proteins in ovarian sections of 10-day-old rats. GH was detected in oocytes and follicular wall tissue of pre-antral follicles, whereas expression of the GH mRNA was absent. The GHR mRNA was present in follicular wall tissue and not in the oocyte, while positive immunostaining for GHR was observed in all cells of the pre-antral follicles. Immunoreactive IGF-I and IGFR was also visible in the pre-antral follicles, especially in the oocytes. In conclusion, the data show that the previously demonstrated local gene expression of IGF-I and IGFR in oocytes and their enveloping follicular cells also leads to translation, which points to the involvement of intrafollicular IGF-I in early follicular development. The presence of the GHR mRNA and the GHR and GH proteins in pre-antral follicles in the absence of ovarian GH mRNA suggest a direct effect of systemic GH on early follicular development.

Nuclear and cytoplasmic maturation of porcine oocytes collected from different sizes of follicles were examined. Oocyte-cumulus complexes were collected from small (1-2 mm in diameter), medium (3-6 in diameter) and large (7-8 mm in diameter) follicles and cultured in a modified tissue culture medium 199 for 44 h. Nuclear maturation was evaluated after orcein staining, and cytoplasmic maturation was evaluated by intracellular glutathione (GSH) assay. Oocyte diameter, cumulus morphology, steroid hormones and glutathione in the follicular fluid (FF), were also examined. Significantly higher proportions of oocytes collected from large and medium follicles reached metaphase II than did oocytes from small follicles. Oocytes from small follicles also had a smaller size. GSH content was significantly higher (p < 0.05) in oocytes from large (14.24 ± 2.1 pmol/oocyte) and medium (13.69 ± 1.5 pmol/oocyte) follicles than in oocytes from small (9.44 ± 1.28 pmol/oocyte) follicles just after collection. After maturation, oocytes from medium follicles had a higher GSH concentration than oocytes from small follicles. It was found that between 49.7 ± 5.18 nM and 52.25 ± 0.78 nM GSH was present in FF but there was no statistical difference between follicle sizes. A significantly higher (p < 0.001) estradiol level was present in FF from large follicles (299.2 ± 68.6 ng/ml) than from medium (40.0 ± 6.4 ng/ml) and small (41.2 ± 3.7 ng/ml) follicles. Progesterone concentrations in FF from large (281.6 ± 45.9 ng/ml) and medium (267.5 ± 38.6 ng/ml) follicles were significantly higher than that (174.7 ± 22.0 ng/ml) from small follicles. These results indicate that the oocyte's ability to accumulate intracellular GSH during maturation, and extracellular steroid hormones and cumulus cells, affect the competence of porcine oocytes to undergo nuclear and cytoplasmic maturation.

The aim of this study was to evaluate the influence of two-dimensional (2D) and three-dimensional (3D) alginate culture systems on in vitro development of pre-antral caprine follicles. In addition, the influence of the reproductive age of the ovary donor on the in vitro culture success was investigated. Pre-antral follicles from pre-pubertal or adult goats were isolated and cultured directly on a plastic surface (2D) or encapsulated in an alginate-based matrix (3D). After 18 days, the oocytes underwent in vitro maturation (IVM) and in vitro fertilization (IVF) to produce embryos. The 3D system showed higher rates of follicle survival, lower rates of oocyte extrusion, and a greater number of recovered oocytes for IVM and IVF (P < 0.05). Only pre-antral follicles from adult animals produced MII oocytes and embryos. The estradiol concentrations increased from day 2 to day 12 of culture in all groups tested (P < 0.05). Conversely, progesterone concentrations were lower in 3D-cultured follicles than in 2D-cultured follicles, with differences on days 2 and 6 of culture (P < 0.05). We provide compelling evidence that a 2D or 3D alginate in vitro culture system offers a promising approach to achieving full in vitro development of caprine pre-antral follicles to produce mature oocytes that are capable of fertilization and viable embryos.

In this study we imaged integral changes in microfilament assembly and cortical granule distribution, and examined effects of microfilament inhibitor on the cortical granule distribution during oocyte maturation, parthenogenetic activation and in vitro fertilisation in the pig. The microfilament assembly and cortical granule distribution were imaged with fluorescent-labelled lectin and rhodamine-labelled phalloidin under laser scanning confocal microscopy. At the germinal vesicle stage, cortical granule organelles were located around the cell cortex and were present as a relatively wide area on the oolemma. Microfilaments were also observed in a wide uniform area around the cell cortex. Following germinal vesicle breakdown, microfilaments concentrated in the condensed chromatin and cortical granules were observed in the cortex. Treatment with cytochalasin B inhibited microfilament polymerisation and prevented movement of cortical granules to the cortex. Cortical granule exudation following sperm penetration was evenly distributed in the entire perivitelline space. These results suggest that the microfilament assembly is involved in the distribution, movement and exocytosis of cortical granules during maturation and fertilisation.

The selection of competent oocytes for in vitro maturation is still a major problem during bovine in vitro embryo production. Markers for in vitro cytoplasmic maturation, based on the organization of cortical granule and mitochondria, are lacking. We examined the pre-selection of immature bovine oocytes by brilliant cresyl blue stain (BCB test) based on glucose-6-phosphate dehydrogenase (G6PDH) activity during oocyte development. Oocytes were recovered from ovarian follicles exposed to 26 μM BCB stain and classified according to the aspect of their cytoplasm: BCB+ (oocytes with blue cytoplasm) and BCB− (unstained cytoplasm) and then in vitro matured into a conventional in vitro maturation (IVM) medium and standard procedure. In Experiment 1, nuclear maturation was determined by polar body identification, while cytoplasmic maturation was based on cortical granule (CG) migration (peripheral) and mitochondria distribution (central). Evidence of polar body, cortical granule migration and of centrally located mitochondria was significantly (p < 0.05) higher in BCB+ oocytes than in BCB− (polar body present: 65% vs 20%; peripheral CG: 72% vs. 14%; and central mitochondria: 85% vs. 19%, respectively). In Experiment 2, the efficiency pre-selection of bovine oocytes by BCB on embryo development in vitro was assessed. Cleavage rates were similar (75%) among control, BCB+ and BCB− groups, while blastocyst rates on D7 were (p < 0.05) higher (35%) in BCB+ vs BCB− (10%) or control (28%). We showed that the BCB test is efficient to identify competent immature bovine oocytes to undergo synchronous nuclear and cytoplasmic in vitro maturation thus yielding higher in vitro embryo development to blastocyst stage.

In this report we demonstrate the successful in vitro culture of fertilised embryos from 1-cell to blastocyst stage, albeit in a strain-dependent fashion. We report procedures for the enucleation of rat oocytes; nuclear transfer by injection of nuclei (NT) from adult rat cumulus cells, rat primary embryonic fibroblasts and genetically modified rat fibroblasts; and activation resulting in advanced preimplantation development. Blastocyst stage rat embryos were obtained after in vitro culture of nuclear transfer zygotes at similar frequencies with each of these nuclear donor cell types. Transfer of NT embryos to surrogate mothers leads to implantation of 24% of the zygotes. These results suggest that the nuclei of cultured rat cells, even following genetic modification, can be reprogrammed to support early embryonic development, which is a prerequisite to cloning the rat.

Controlled ovarian stimulation was induced in 19 women for in vitro fertilisation/embryo transfer. After ovum pick-up, haptoglobin titres were determined by ELISA in sera and homologous follicular fluids. The haptoglobin phenotype of each subject was assessed and the penetration of the protein forms through the blood-follicle barrier was predicted on the basis of their molecular weight. The penetration threshold compatible with the barrier integrity was calculated as 92%, 73% and 57% of the blood level of phenotypes Hpt 1-1, Hpt 1-2 and Hpt 2-2 respectively. Penetration values comparable/lower or higher than threshold were found associated with 46 of 49 and 3 of 49 fertilised oocytes, respectively. Complexes of haptoglobin with apolipoprotein A-1 were isolated from follicular fluids by affinity chromatography with haemoglobin. The haptoglobin β chain, after Western blotting and incubation with apolipoprotein A-1, was found to be involved in the protein–protein interaction as detected by anti-apolipoprotein A-1 antibodies. Complexes from separate fluids were analysed by electrophoresis and densitometry: the plain β chain/apolipoprotein A-1 stoichiometric ratio was 0.75 and 1.40 in fluids associated with fertilised and unfertilised oocytes respectively. The results suggest that haptoglobin transport in the follicle depends on the integrity of the blood–follicle barrier and might be associated with oocyte quality, possibly by interfering with the role of apoliprotein A-1 in cholesterol or vitamin E exchange between high-density lipoproteins and granulosa cells.