The objective of this study was to determine developmental pattern, total cell number, apoptosis and apoptosis-related gene expression in haploid and diploid embryos following parthenogenetic activation. In vitro-matured porcine oocytes were activated by electrical pulses and cultured in the absence or presence of cytochalasin B for 3 h. Zygotes with two polar bodies (haploid) and one polar body (diploid) were carefully selected and were further cultured in NCSU 23 medium containing 0.4% bovine serum albumin (BSA) for 7 days. The percentage of development to blastocyst stage was higher (p<0.01) in the diploid than in the haploid parthenotes. In haploid blastocysts, average total cell number was significantly reduced (p<0.05) and apoptosis was increased at day 7. The relative abundance of Bcl-xL and Bak mRNA in the diploid blastocysts was similar to that of in vivo-fertilized embryos. However, Bcl-xL was significantly decreased, and Bak mRNA was significantly increased (p<0.05) in haploid parthenotes compared with the diploid parthenotes. These results suggest that the haploid state affects apoptosis-related gene expression which results in increased apoptosis and decreased developmental competence of haploid parthenotes.

While the fertilising spermatozoon supplies the active centre directing the human zygote's first mitotic division, the relative contributions of the sperm head and tail (as well as the importance of the sperm's general structural integrity) to subsequent developmental processes remain incompletely studied. The sperm nucleus contains paternal chromatin necessary for restoration of a diploid genome, but the functional role of the sperm tail (either attached or dissected) in early human embryonic growth is not known. In this investigation using oocytes donated by in vitro fertilisation patients, human oocytes were injected with isolated sperm heads (n = 73), isolated sperm flagella (n = 11) or both (dissected sperm heads + free sperm tails, n = 26). The formation of bipronucleate zygotes was recorded for each method. Among oocytes surviving injection with isolated sperm heads, 44 of 66 (67%) formed two pronuclei. Of oocytes receiving only sperm tails, 2 of 11 (18%) displayed two pronuclei, but a single polar body was evident in both cases. When dissected spermatozoa parts (head + tail) were jointly injected, 12 of 26 (46%) developed two pronuclei. From embryos resulting from each of these three fertilisation regimes, blastomere biopsies were obtained and subjected to multiprobe fluorescent in situ hybridisation (FISH) analysis to detect mosaicism or aneuploidy arising from these experimental treatments. Only embryos with growth sufficient to permit sampling of at least two blastomeres were evaluated, and FISH analysis was successful in 25 of 29 (86%) embryos tested. Of 12 embryos derived from injection of an isolated sperm head, only one was normal diploid; the remaining 11 were mosaic. Both embryos resulting from injection of an unattached sperm tail were mosaic. Of 11 embryos generated from oocyte injection with sperm head + tail segments, 10 (91%) were mosaic and only one was normal diploid. Results from this study show that injection of isolated sperm segments can permit oocyte activation and bipronuclear formation. However, a high rate of mosaicism in human embryos originating from disrupted sperm or sperm components suggests that more than a ‘sum of parts’ is needed for later development. The structural integrity of the intact fertilising spermatozoon appears to contribute to normal human early embryogenesis.

The amelogenin (AMEL) gene exists on both sex chromosomes of various mammalian species and the length and sequence of the noncoding regions differ between the two chromosome-specific alleles. Because both forms can be amplified using a single primer set, the use of AMEL in polymerase chain reaction (PCR)-based methods has facilitated sex identification in various mammalian species, including cattle, sheep and humans. In this study, we designed PCR primers to yield different-sized products from the AMEL genes on the X (AMELX) and Y (AMELY) chromosomes of pigs. PCR amplification of genomic DNA samples collected from various breeds of pigs (European breeds: Landrace, Large White, Duroc and Berkshire; Chinese breeds: Meishan and Jinhua and their crossbreeds) yielded the expected products. For all breeds, DNA from male pigs produced two bands (520 and 350 bp; AMELX and AMELY, respectively), whereas samples from female pigs generated only the 520 bp product. We then tested the use of PCR of AMEL for sex identification of in vitro-produced (IVP) porcine embryos sampled at 2 or 5 to 6 days after fertilization; germinal vesicle (GV)-stage oocytes and electroactivated embryos were used as controls. More than 88% of the GV-stage oocytes and electroactivated embryos yielded a single 520 bp single band and about 50% of the IVP embryos tested produced both bands. Our findings show that PCR analysis of the AMEL gene is reliable for sex identification of pigs and porcine embryos.

In sea urchin embryos, blastula formation occurs between the seventh and tenth cleavage and is associated with changes in the permeability properties of the epithelium although the structures responsible for mediating these changes are not known. Tight junctions regulate the barrier to paracellular permeability in chordate epithelia; however, the sea urchin blastula epithelium lacks tight junctions and instead possesses septate junctions. Septate junctions are unique to non-chordate invertebrate cell layers and have a characteristic ladder-like appearance whereby adjacent cells are connected by septa. To determine the function of septate junctions in sea urchin embryos, the permeability characteristics of the embryonic sea urchin epithelia were assessed. First, the developmental stage at which a barrier to paracellular permeability arises was examined and found to be in place after the eighth cleavage division. The mature blastula epithelium is impermeable to macromolecules; however, brief depletion of divalent cations renders the epithelium permeable. The ability of the blastula epithelium to recover from depletion of divalent cations and re-establish a barrier to paracellular permeability using fluorescently labelled lectins was also examined. Finally, septate junction structure was examined in embryos in which the permeability status of the epithelium was known. The results provide evidence that septate junctions mediate the barrier to paracellular permeability in sea urchin embryos.

In cattle early gastrulation-stage embryos (Stage 5), four tissues can be discerned: (i) the top layer of the embryonic disc consisting of embryonic ectoderm (EmE); (ii) the bottom layer of the disc consisting of mesoderm, endoderm and visceral hypoblast (MEH); (iii) the trophoblast (TB); and (iv) the parietal hypoblast. We performed microsurgery followed by RNA-seq to analyse the transcriptome of these four tissues as well as a developmentally earlier pre-gastrulation embryonic disc. The cattle EmE transcriptome was similar at Stages 4 and 5, characterised by the OCT4/SOX2/NANOG pluripotency network. Expression of genes associated with primordial germ cells suggest their presence in the EmE tissue at these stages. Anterior visceral hypoblast genes were transcribed in the Stage 4 disc, but no longer by Stage 5. The Stage 5 MEH layer was equally similar to mouse embryonic and extraembryonic visceral endoderm. Our data suggest that the first mesoderm to invaginate in cattle embryos is fated to become extraembryonic. TGFβ, FGF, VEGF, PDGFA, IGF2, IHH and WNT signals and receptors were expressed, however the representative members of the FGF families differed from that seen in equivalent tissues of mouse embryos. The TB transcriptome was unique and differed significantly from that of mice. FGF signalling in the TB may be autocrine with both FGFR2 and FGF2 expressed. Our data revealed a range of potential inter-tissue interactions, highlighted significant differences in early development between mice and cattle and yielded insight into the developmental events occurring at the start of gastrulation.

Preimplantation mammalian development culminates in the formation of a fluid-filled cavity, the blastocoele, which is a prerequisite for successful implantation and further development. The blastocoele is enclosed by a single layer of polarised cells, the trophectoderm, which is the first epithelium formed in development. In embryos of the mouse and the rabbit, a basolaterally located Na+/K+-ATPase hydrolyses ATP to drive the vectorial transport of ions, which is responsible for the accumulation of blastocoele fluid. Using non-invasive assays of energy substrate consumption and blastocoele fluid accumulation, experiments were carried out on single preimplantation rat embryos, to establish: (1) the roles of the Na+/K+-ATPase and exogenous energy substrates, and (2) the relationship between the consumption and metabolism of energy substrates and fluid accumulation, during blastocoele cavity formation in this species. Ouabain 0.5 mM and energy-substrate-free medium both caused an inhibition in the number of embryos forming a blastocoele in culture, and also reduced the rate of fluid accumulation by day 5 blastocysts collapsed in cytochalasin-D and allowed to re-expand. Ouabain also reduced the consumption of glucose (but not pyruvate) and the production of lactate by re-expanding blastocysts. In the absence of the inhibitor, a direct relationship was seen between fluid accumulation and both glucose (but not pyruvate) consumption and lactate production. However, ouabain had no effect on intact, expanded blastocysts. These results suggest that (1) a basolaterally located, ouabain-inhibitable Na+/K+-ATPase is involved in rat blastocoele formation, (2) this process is dependent on exogenous energy substrates, and (3) there may be a direct relationship between the metabolism of glucose via glycolysis, and blastocoele fluid accumulation.

The aim of the present study was to investigate whether growth hormone (GH) has any effect on the development of cultured rat pre-antral follicles. Pre-antral follicles with a diameter between 120 μm and 160 μm were mechanically isolated from 10-day-old rat ovaries and cultured in groups for 6 days in serum-free medium without GH or with GH supplemented at concentrations of 1, 10 and 100 ng/ml, respectively. DNA content of the follicles before and after culture was measured to determine whether possible growth is due to proliferation of follicular cells. To investigate the quality of follicles cultured under different conditions, the ultrastructure of the cultured follicles was studied with transmission electron microscopy. Furthermore, reverse transcriptase polymerase chain reaction (RT-PCR) was used to assess the expression of growth hormone receptor (GHR) in pre-antral follicles. GH, regardless of the concentration, stimulated the growth of pre-antral follicles. However, follicles cultured in medium supplemented with high-dose GH (100 ng/ml) showed a significantly lower survival rate compared with the other groups. Follicles cultured in GH-containing medium showed a better ultrastructure in comparison with those cultured in medium without GH. Remarkably, scattered cortical granules were observed in oocytes of follicles cultured in the presence of GH. With RT-PCR, the presence of the mRNA of GHR was demonstrated in pre-antral follicles. It can be concluded that GH promotes rat pre-antral follicle development in vitro and better supports the morphology of cultured pre-antral follicles. The gene expression of GHR suggests that the action of GH could be mediated by its receptors present in pre-antral follicles.

With the development of embryo technologies, such as in vitro fertilization, cloning and transgenesis, cryopreservation of mammalian gametes and embryos has acquired a particular interest. Despite a certain success, various cryopreservation techniques often cause significant morphological and biochemical alterations, which lead to the disruption of cell organelles, cytoskeleton damages, cell death and loss of embryo viability. Ultrastructural studies confirm high sensitivity of the cell membrane and organelle membrane to freezing and thawing. It was found that many substances with low molecular weights have a protective action against cold-induced damage. In this concern, an anti-freeze protein (AFP) and anti-freeze glycoproteins (AFGPs), which occur at extremely high concentrations in fish that live in Arctic waters and protect them against freezing, may be of potential interest for cryostorage of animal embryos at ultra-low temperatures. This mini-review briefly describes several models of AFP/AFGP action to preserve cells against chilling-induced damages and indicates several ways to improve post-thaw developmental potential of the embryo.

Medaka (Oryzias latipes) eggs microinjected with the Ca2+-mobilising messenger cyclic adenosine diphosphate ribose (cADPR) underwent a wave of exocytosis of cortical alveoli and were thus activated. The number of eggs activated was sharply dependent on the concentration of cADPR in the pipette, the threshold concentration was approximately 60 nM. After injection, a pronounced latency preceded the onset of cortical alveoli exocytosis; this latency was independent of the concentration of cADPR but decreased markedly with increasing temperature. Heat-treated cADPR, which yields the inert non-cyclised product ADP-ribose, was ineffective in activating eggs. When cADPR was injected into aequorin-loaded eggs, a wave of luminescence arose at the site of cADPR injection and then swept out across the egg with a mean velocity of approximately 13 μm/s; the velocity was independent of the concentration of injected cADPR. In such a large cell (diameter of around 1 mm), this is considerably faster than that possible by simple diffusion of cADPR, which unambiguously demonstrates that cADPR must activate a regenerative process. cADPR has been demonstrated to modulate Ca2+-induced Ca2+ release (CICR) via ryanodine receptors (RyRs) in many cell types, and consistent with this was the finding that microinjection of the pharmacological RyR modulator, ryanodine, also activated medaka eggs. These results suggest that a cADPR-sensitive Ca2+ release mechanism is present in the medaka egg, that cADPR is the most potent activator of medaka eggs described to date, and that it activates eggs by triggering a wave of CICR from internal stores that in turn stimulates a wave of exocytosis.

Somatic cell nuclear transfer (SCNT) is an important technique for life science research. However, most SCNT embryos fail to develop to term due to undefined reprogramming defects. Here, we show that abnormal Xi occurs in somatic cell NT blastocysts, whereas in female blastocysts derived from cumulus cell nuclear transfer, both X chromosomes were inactive. H3K27me3 removal by Kdm6a mRNA overexpression could significantly improve preimplantation development of NT embryos, and even reached a 70.2% blastocyst rate of cleaved embryos compared with the 38.5% rate of the control. H3K27me3 levels were significantly reduced in blastomeres from cloned blastocysts after overexpression of Kdm6a. qPCR indicated that rDNA transcription increased in both NT embryos and 293T cells after overexpression of Kdm6a. Our findings demonstrate that overexpression of Kdm6a improved the development of cloned mouse embryos by reducing H3K27me3 and increasing rDNA transcription.

Infertility is an important reproductive health problem, and male infertility is especially important in more than half of infertility cases. Due to the importance of genetic factors in this condition, analysis of semen alone is not enough to recognize men with idiopathic infertility. A molecular non-invasive investigation is necessary to gain valuable information. Currently, microRNAs (miRNAs) are being used as non-invasive diagnostic biomarkers. miRNAs, single-stranded non-coding RNA molecules, act as post-transcriptional gene silencing regulators either by inhibition or repression of translation. Changes in the regulation of miRNAs have been investigated in several different types of male infertility, therefore the biological role of miRNA and gene targets has been defined. The purpose of this study was to review recent research on the altered expression of miRNA in semen, sperm, and testicular biopsy samples in infertile males with different types of unexplained infertility. Changes in miRNA regulation were investigated using microarray and the miRNA levels were confirmed by real-time qRT-PCR. This review explains why creating a non-invasive diagnostic method for male infertility is necessary and how changes in miRNA expression can be used as new diagnostic biomarkers in patients with differing spermatogenic and histopathologic injury.

The aim of this study was to evaluate the effect of adding a combination of insulin, transferrin and selenium (ITS) and l-ascorbic acid (AA) during in vitro maturation (IVM) and in vitro culture (IVC) on in vitro embryo production. To verify the effect of the supplements, cleavage and blastocyst rates, embryo size and total cell number were performed. Embryonic development data, embryo size categorization and kinetics of maturation were analyzed by chi-squared test, while the total cell number was analyzed by a Kruskal–Wallis test (P < 0.05). When ITS was present during IVM, IVC or the entire culture, all treatments had a cleavage and blastocyst rates and embryo quality, similar to those of the control group (P < 0.05). Supplementation of IVM medium with ITS and AA for 12 h or 24 h showed that the last 12 h increased embryo production (51.6%; n = 220) on D7 compared with the control (39.5%; n = 213). However, no improvement was observed in blastocyst rate when less competent oocytes, obtained from 1–3 mm follicles, were exposed to ITS + AA for the last 12 h of IVM, with a blastocyst rate of 14.9% (n = 47) compared with 61.0% (n = 141) in the control group. The results suggest that the addition of ITS alone did not affect embryo production; however, when combined with AA in the last 12 h of maturation, there was improvement in the quantity and quality of embryos produced. Furthermore, the use of ITS and AA during IVM did not improve the competence of oocytes obtained from small follicles.

Mast cells of connective tissue type are scattered in the interstitial compartment of the lizard Podarcis s. sicula. Their number varies during the year, showing peaks in spring and in winter, respectively.Thermal manipulation affects mast cell number (MCN): high temperature decreased MCN in both January and May, while low temperature increased MCN only in January. Ethane dimethane sulphonate, a toxin which specifically destroys Leydig cells, induced an increase in MCN on days 3 and 7 of treatment. Oestradiol treatment provoked a strong increase in MCN that was blocked by tamoxifen. Blocking androgen receptors with cyproterone acetate resulted in an increase in MCN, while testosterone injection provoked a strong decrease. These results suggest a relationship between the presence of mature Leydig cells and mast cell proliferation and/or differentiation.

Heat shock may affect different aspects of oocyte maturation and its subsequent development to the blastocyst stage. A series of in vitro experiments was performed to determine whether physiologically heat shock (41°C) disrupts the progression of the ovine oocytes through meiosis, activation and blastocyst formation. Cumulus–oocyte complexes (COCs) were aspirated from 2–6-mm follicles and cultured at 38.5°C (control) or 41°C (heat shock) for the first 12 h of maturation. The oocytes were incubated at 38.5°C during the last 10 h of maturation and 8 days after activation. Results showed that most of the oocytes matured under heat-shock conditions remained at the germinal vesicle breakdown (GVBD) stage and they showed an aberrant chromatin configuration. After heat shock, oocyte diameter and time spent for zona pellucida dissolution increased (P < 0.05). The heat-shocked group had a higher percentage of oocytes with incomplete migration of cortical granules (P < 0.05). The heat-shock condition decreased (P < 0.05) cleavage rates (56.19 versus 89.28%) and morula formation (26.85 versus 37.81%). However, there was no significant difference in blastocyst formation and percentage of hatched blastocysts. At 12 h, heat shock had an adverse effect on embryo quality and reduced inner cell mass number (P < 0.05). Quantitative gene expression analysis showed greater transcripts (P < 0.05) for Na/K-ATPase mRNA in heat-shocked oocytes. To sum up, heat shock has disruptive effects on ovine oocyte maturation and can impair cellular and molecular factors that are important for embryo development.

The purpose of this study was to investigate the possible molecular pathways through which ghrelin accelerates in vitro oocyte maturation. Bovine cumulus–oocyte complexes (COCs), after 18 or 24 h maturation in the absence or the presence of 800 pg ml–1 of acylated ghrelin were either assessed for nuclear maturation or underwent in vitro fertilization in standard media and putative zygotes were cultured in vitro for 8 days. In a subset of COCs the levels of phosphorylated Akt1 and ERK1/2 (MAPK1/3) were assessed at the 0th, 6th, 10th, 18th and 24th hours of in vitro maturation (IVM). At 18 and 24 h no difference existed in the proportion of matured oocytes in the ghrelin-treated group, while in the control group more (P < 0.05) matured oocyte were found at 24 h. Oocyte maturation for 24 h in the presence of ghrelin resulted in substantially reduced (P < 0.05) blastocyst yield(16.3%) in comparison with that obtained after 18 h (30.0%) or to both control groups (29.3% and 26.9%, for 18 and 24 h in maturation, respectively). Ghrelin-treated oocytes expressed lower Akt1 phosphorylation rate at the 10th hour of IVM, and higher ERK1/2 at the 6th and 10th hours of IVM compared with controls. In cumulus cells, at the 18th and 24th hours of IVM Akt1 phosphorylation rate was higher in ghrelin-treated oocytes. Our results imply that ghrelin acts in a different time-dependent manner on bovine oocytes and cumulus cells modulating Akt1 and ERK1/2 phosphorylation, which brings about acceleration of the oocyte maturation process.

DNA stability and thiol-disulphide status of rat sperm nuclei was observed in vivo during maturation in the epididymis and penetration of oocytes. When spermatids and spermatozoa were stained with acridine orange after fixation with acetic alcohol, the red/green fluorescence ratio observed under a confocal microscope was not different between spermatids (3.81 ± 0.16) and testicular spermatozoa (4.03 ± 0.34), and then decreased sharply (p < 0.01) as the spermatozoa descended the epidymis to the caput epididymis (1.13 ± 0.03). However, the ratio was not different among corpus (0.69 ± 0.01), cauda epididymis (0.68 ± 0.11) and ejaculated spermatozoa (0.63 ± 0.01). On the other hand, when spermatozoa were labelled with monobromobimane (mBBr), the fluorescence intensities gradually decreased (p < 0.01) during passage of spermatozoa from testis (4.74 ± 0.16) through epididymis (caput, 2.72 ± 0.08; corpus, 1.07 ± 0.03; cauda, −0.05 ± 0.05; ejaculated, 0.08 ± 0.03). The acridine orange red/green fluorescence ratio increased (p < 0.01) during zona penetration (binding sperm, 0.52 ± 0.09; perivitelline sperm, 0.64 ± 0.16) and sperm decondensation (decondensed sperm, 0.69 ± 0.12). When spermatozoa in the perivitelline space were labelled with mBBr, the fluorescence was detected. These results demonstrate that DNA stability against acid appears to be ahead of the oxidation of protamine during sperm maturation in the epididymis and is an initial event of the unpackaging process in rat genome occurring during or just after zona penetration but before ooplasm penetration.

This study compared the developmental competence of somatic cell nuclear transfer (SCNT) embryos reconstructed with different donor cells and analysed gene expression in the resulting embryos. Bovine fetal/adult ear fibroblasts and cumulus cells were used as donor cells and the developmental competence of the reconstructed embryos was monitored. The cell number and allocation in blastocysts were determined by differential staining. The Bax, E-cad, IF-tau, Hsp (heat shock protein) 70, Igf2r (insulin-like growth factor 2 receptor), DNMT (DNA methyltransferase) 1 and Mash (mammalian achaete-scute homologue) 2 genes were selected for gene expression analysis. The relative abundance (ratio to GAPDH mRNA) of gene transcripts in blastocysts was measured by semiquantitative reverse transcription-polymerase chain reaction. In experiment 1, development of SCNT preimplantation embryos and the cell numbers of inner cell masses and trophoblasts were not different among SCNT embryos derived from different cell types. In experiment 2, the relative expression of GAPDH and Hsp 70 transcripts was similar in all embryos. The expression of Bax, Igf2r and Mash2 transcripts was significantly increased in SCNT embryos reconstructed with adult fibroblasts. The E-cad transcript levels were reduced in SCNT embryos reconstructed with fetal fibroblasts. Relative abundance of DNMT1 in SCNT embryos derived from fetal fibroblasts was increased, and IF-tau expression in SCNT embryos derived from cumulus cells was increased. In conclusion, depending on the type of donor cells, preimplantation SCNT embryos displayed marked differences in gene expression. This may affect the developmental competence of SCNT embryos reconstructed with different cell types after implantation or during fetal growth in vivo.

Studies with intact preimplantation mouse embryos and some types of chimaeric aggregates have shown that the most advanced cells are preferentially allocated to the inner cell mass (ICM) rather than the trophectoderm. Thus, differences between 4-cell and 8-cell stage embryos could contribute to the tendency for tetraploid cells to colonise the trophectoderm more readily than the ICM in 4-cell tetraploid[harr ]8 cell diploid chimaeras. The aim of the present study was to test whether 4-cell stage embryos in 4-cell diploid[harr ]8-cell diploid aggregates contributed equally to all lineages present in the E12.5 conceptus. These chimaeras were compared with those produced from standard aggregates of two whole 8-cell embryos and aggregates of half an 8-cell embryo with a whole 8-cell embryo. As expected, the overall contribution of 4-cell embryos was lower than that of 8-cell embryos and similar to that of half 8-cell stage embryos. In the 4-cell[harr ]8-cell chimaeras the 4-cell stage embryos did not contribute more to the trophectoderm than the ICM derivatives. Thus, differences between 4-cell and 8-cell embryos cannot explain the restricted tissue distribution of tetraploid cells previously reported for 4-cell tetraploid[harr ]8-cell diploid chimaeras. It is suggested that cells from the more advanced embryo are more likely to contribute to the ICM but, for technical reasons, are prevented from doing so in simple aggregates of equal numbers of whole 4-cell and whole 8-cell stage embryos.

The aim of this study was to describe a system for embryo morphology scoring at the morula stage and to determine the efficiency of this model in selecting viable embryos for transfer. In total, 519 embryos from 122 patients undergoing intracytoplasmic sperm injection (ICSI) were scored retrospectively on day 4 according to the grading system proposed in this article. Two separate quality scores were assigned to each embryo in relation to the grade of compaction and fragmentation and their developmental fate was then observed on days 5 and 6. Secondly, the prediction value of this scoring system was compared with the prediction value of the traditional scoring system adopted on day 3. Morulas classified as grade A showed a significant higher blastocyst formation rate (87.2%) compared with grades B, C and D (63.8, 41.3 and 15.0%, respectively), (P < 0.001). Furthermore, the ability to form top quality blastocysts was significantly higher for grade A morulas with respect to grades B, and C and D (37.8% vs. 22.4% vs. 11.1%), (P < 0.001). Finally, the morula scoring system showed more prediction power with respect to the embryo scoring a value of 1 [Akaike information criterion (AIC) index 16.4 vs. 635.3 and Bayesian information criterion (BIC) index −68.8 vs. −30.0 for morulas and embryos respectively]. In conclusion, results demonstrated that the presented scoring system allows for the evaluation of eligible embryos for transfer as a significant correlation between the grade of morula, blastulation rate and blastocyst quality was observed. Furthermore, the morula scoring system was shown to be the best predictive model when compared with the traditional scoring system performed on day 3.

The aim of our study was to analyse the cytoskeletal organization of prepubertal goat oocytes. Microtubule and microfilament organization during in vitro maturation of prepubertal and adult goat oocytes and presumptive zygotes of in vitro matured–in vitro fertilized (IVM-IVF) prepubertal goat oocytes were analysed. Oocytes were matured in M-199 with hormones and serum and inseminated with frozen-thawed spermatozoa. Oocytes and presumptive zygotes were treated with anti-α-tubulin antibody and fluorescein isothiocyanate (FITC)-labelled goat anti-mouse antibody to stain the microtubules. Microfilaments were localized by means of phalloidin 5 μg/ml conjugated with fluorescein isothiocyanate (FITC-phalloidin). DNA was stained with propidium iodide. Stained oocytes were observed under a confocal laser scanning microscope. At the germinal vesicle nuclear stage, microfilaments were distributed at the cortex of the oocytes. After in vitro maturation, 91.7% of metaphase II (MII) oocytes from adult goats displayed microfilaments in the cortex and within the polar body and were characterized by the presence of a microfilament thickening at the cortical region over the meiotic spindle. In prepubertal goat MII oocytes only 5.7% of oocytes displayed microfilaments at the cortex and within the polar body. After insemination, most of the zygotes displayed microfilaments distributed at the cortex. An undefined microtubular network was observed in adult and prepubertal goat oocytes at the germinal vesicle stage. After in vitro maturation, 100% of MII oocytes from adult goats displayed microtubules on the meiotic spindle and within the polar body. This pattern of distribution was observed in 71.6% of prepubertal goat oocytes. Undefined microtubule networks were present in most of the zygotes analysed. In conclusion, cytoskeletal differences were found between prepubertal and adult goat MII oocytes. Furthermore, most of the zygotes from IVM-IVF prepubertal goat oocytes displayed cytoskeletal anomalies.