This study evaluated the final output and quality of in vitro produced blastocysts derived from in vivo recovered sheep embryos cultured at various early developmental stages to blastocyst. A total of 270 embryos were recovered from the oviduct, at different days of the early luteal phase, and were classified into three different developmental stages: 2- to 4-cell (n = 93); 5- to 8-cell (n = 92) and 9- to 12-cell (n = 85). The effect of culture conditions was studied, at the same time, by randomly allocating the embryos to one of four groups: three groups of culture with fresh oviduct monolayers (2, 4 and 5 days old) and a fourth group with 2-day monolayers derived from frozen-thawed oviduct cells. Two control groups were established: first, embryos cultured in semi-defined medium (n = 29) and, second, blastocysts obtained in vivo and cryopreserved (n = 43). Influence on blastocyst yield of embryo developmental stage at the start of culture was statistically significant (p < 0.001). Two- to four-cell embryos showed a significantly lower developmental rate (67.7%) than the 5- to 8-cell (83.6%; p < 0.001) and 9- to 12-cell groups (90.5%; p < 0.0001) and lower quality in terms of blastocyst cryotolerance (56.0 vs. 83.7%; p < 0.005). There were no detected effects relating to the age or handling of the monolayer on the embryo developmental rate, but the day of blastocyst appearance was different between embryos cultured on monolayers derived from fresh or frozen-thawed cells (p < 0.0001); the main influence was on the group of 9- to 12-cell embryos (p < 0.0001). Current results confirm the temporal sensitivities of sheep embryos to in vitro culture, regardless of the culture conditions.

Follicular fluid meiosis-activating sterol (FF-MAS) has been isolated from the follicular fluid (FF) of several species including man. FF-MAS increases the quality of in vitro oocyte maturation, and thus the developmental potential of oocytes exposed to FF-MAS during in vitro maturation is improved. The aim of the present study was to investigate the effects of FF-MAS on porcine oocyte maturation and pronucleus formation in vitro. Porcine cumulus–oocyte complexes (COCs) were isolated from abattoir ovaries and in vitro matured for 48 h in NCSU 37 medium supplemented with 1 mg/l cysteine, 10 ng/ml epidermal growth factor and 50 μM 2-mercaptoethanol with or without 10% porcine follicular fluid (pFF). For the first 22 h, 1 mM db-cAMP and 10 I.E PMSG/hCG was added. The medium was supplemented with 1 μM, 3 μM, 10 μM, 30 μM or 100 μM FF-MAS dissolved in ethanol. After maturation the COCs were denuded mechanically using a fine glass pipette under constant pH and in vitro fertilized with fresh semen (5 × 105 spermatozoa/ml). The presumptive zygotes were evaluated 18 h after fertilization. The addition of pFF increased the monospermic as well as the polyspermic penetration of oocytes. In the absence of pFF, the addition of FF-MAS decreased the polyspermic penetration rate, whereas FF-MAS in combination with pFF decreased monospermic and increased polyspermic penetration. The degeneration rate of ova decreased in the presence of FF-MAS irrespective of the presence or absence of pFF. In the absence of pFF, FF-MAS at 3–10 μM increased the number of zygotes with advanced maternal pronuclear stages. In supraphysiological doses, i.e. 30–100 μM, FF-MAS dose-dependently and reversibly inhibited nuclear maturation in the absence of pFF.

TEX101, a glycoprotein we recently identified, is primarily characterized as a unique germ-cell-specific marker protein that shows sexually dimorphic expression during mouse gonad development. Based on data obtained from molecular biological as well as immuno-morphological studies, we believe this molecule may play a role in the process underlying germ cell formation. However, many points remain unclear as the molecular characteristics and its physiological functions are far from being completely understood. To clarify the molecular basis of TEX101, we herein report a further biochemical characterization of the molecule using testicular Triton X-100 extracts from mice. Deglycosylation studies using endoglycohydrolases that delete N-linked oligosaccharides (OS) from the molecule show that TEX101 is highly (approximately 47%) N-glycosylated. All potential N-glycosylation sites within TEX101 are glycosylated and most of these sites are occupied by endoglycosidase F2-sensitive biantennary complex type OS units. In addition, an extremely low population among TEX101 possesses only endoglycosidase H-sensitive hybrid type OS units. In studies using phosphatidylinositol-specific phospholipase C against native testicular cells or TEX101 transfectant, the enzyme treatment caused major reduction of the TEX101 expression on the cell, suggesting that TEX101, at least in part, is expressed as a glycosylphosphatidylinositol-anchored protein. Taken together, these findings will help elucidate the molecular nature of TEX101, a marker molecule that appeared on germ cells during gametogenesis.

TATA binding protein (TBP) associated factor 1 (TAF1) is a member of the general transcription machinery. Interference in the function of TAF1 causes a broad transcriptional defect in early development. To explore possible roles of TAF1 in embryonic transcriptional silence and zygotic genome activation, we examined the expression of TAF1 in 1-cell mouse embryos. Using an immunofluorescence assay, TAF1 was not detected in embryos in the first few hours after fertilization. TAF1 appeared in pronuclei 6 h post-fertilization and reached a relatively high level before zygotic genome activation. These data show that besides TBP, another critical member of the general transcription machinery such as TAF1 is also absent or at an extremely low level at the outset of development. Combined deficiency in critical members of the general transcription machinery may account for embryonic transcriptional silence.

This survey was performed to characterize the embryogenesis of Prochilodus lineatus. Seven stages of embryo development were identified – zygote, cleavage, blastula, gastrula, segmentation, larval and hatching – after a period of incubation of 22 h (24 °C) or 14 h (28 °C). The following cleavage pattern was identified: the first plane was vertical (2 blastomeres); the second was vertical and perpendicular to the first (4 blastomeres); the third was vertical and parallel to the first (4 × 2); the fourth cleavage was vertical and parallel to the second (4 × 4); the fifth was vertical and parallel to the first (4 × 8); and the sixth cleavage was horizontal (64 blastomeres). At the blastula stage (3.0–4.0 h (24 °C); 1.66–2.0 h (28 °C)) irregular spaces were detected and periblast structuring was initiated. At the gastrula stage (4.0–8.0 h (24 °C); 3.0–6.0 h (28 °C)) the epiboly, convergence and cell movements, as well as the formation of embryonic layers, had begun. The segmentation stage (10.0–15.0 h (24 °C); 7.0–10.0 h (28 °C)) was characterized by a rudimentary formation of organs and systems (somites, optic vesicle and intestinal delimitation). The embryo at the larval stage (16.0–21.0 h (24 °C); 11.0–13.0 h (28 °C)) showed a free tail, more than 25 somites, an optic vesicle and a ready-to-hatch larval shape. The blastomeres at cleavage stage had disorganized nuclei indicating high mitotic activity. At gastrula, the blastomeres and the periblast had euchromatic nuclei and a large number of mitochondria and vesicles. The yolk was organized into globose sacs, which were dispersed into small pieces prior to absorption.

This report presents data from ultrastructural and morphometric studies on the germinal-body-like structures, nuage, nuage–mitochondrial clusters and chromatoid bodies in 4.5-day embryo cells and spermatogenic cells of the laboratory mouse Mus musculus. In the 4.5-day embryo cells the germinal-body-like structures that, according to previous data, arise by condensation of mitochondria in Graafian oocytes, were found not to undergo any ultrastructural alterations. In spermatogonia the germinal-body-like structures presumably were transformed into nuage that functioned as ‘intermitochondrial cement’ binding the mitochondrial clusters. In primary spermatocytes mitochondria aggregated by nuage were found with large vacuoles containing membraneous conglomerates that were obviously excreted by organelles into the cytoplasm. The chromatoid bodies that arose in spermatocytes and finally disintegrated in the posterior part of late spermatids seemed not to be implicated in the pathway of the germinal-body-like structure. The dispersion of chromatoid bodies was noted to be accompanied by excretion of membraneous conglomerates by late spermatid mitochondria. The spermatozoa were not found to contain either the germinal-body-like structures or any other germ-plasm-related structures.

The present study was designed to evaluate the feasibility of producing pig transgenic blastocysts expressing enhanced green fluorescent protein (GFP) and to examine the effects of shape and preparation methods of donor cells on in vitro developmental ability of pig nuclear transferred embryos (NTEs). In experiment 1, the effect of GFP transfection on development of pig NTEs was evaluated. The cleavage and blastocyst rates showed no significant difference between NTEs derived from transfected and non-transfected donors. In experiment 2, the effect of different nuclear donor preparation methods on in vitro development of NTEs was examined. The cleavage rate showed no statistically significant differences among three preparation methods. The blastocyst rates of donor cells treated once at −4 °C and those of freshly digested cells were similar to each other (26.3% vs 17.9%). The lowest blastocyst rates (5.88%) were observed when cells cryopreserved at −196 °C were used as donors. In experiment 3, the effect of different cell cycle synchronization methods on the in vitro development potential of pig NTEs was evaluated. The cleavage rate of NTEs derived from cycling cells was much better than that of NTEs derived from serum-starved cells (64.4% vs 50.5%, p < 0.05), but no significant difference was observed between the the blastocyst rates of the two groups. In experiment 4, the effect of different shapes of cultured fibroblast cells on the in vitro development of pig NTEs was examined. The fusion rate for couplets derived from rough cells was poorer than that observed in couplets derived from round smooth cells (47.8% vs 76.8%, p < 0.05). However, there were no significant differences observed in the cleavage rate and blastocyst rate. In conclusion, the present study indicated that (i) refrigerated pig GFP-transfected cells could be used as donors in nuclear transfer and these NTEs could be effectively developed to blastocyst stage; (ii) serum starvation of GFP-transfected cells is not required for preimplantation development of pig NTEs; and (iii) a rough surface of GFP-transfected donor cells affects fusion rate negatively but has no influence on the cleavage rate or blastocyst rate of pig NTEs.

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.

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.