The acrosome reaction (AR) is a fundamental event for fertilization, which is induced in concert with acrosome reaction-inducing substance (ARIS) and asterosap, both of which are components of starfish egg jelly (EJ). During the AR, a spermatozoon undergoes a series of physiological changes, such as in intracellular cGMP concentration ([cGMP]i), pHi and intracellular Ca2+ concentration ([Ca2+]i). Affinity purification of cGMP-binding protein resulted in the isolation of a regulatory subunit of the cAMP-dependent protein kinase A (PKA), suggesting the involvement of a cAMP-dependent pathway in the AR. By using a cAMP enzyme immunoassay, [cAMP]i was found to increase in starfish spermatozoa when stimulated with ARIS and asterosap. ARIS could also increase the [cAMP]i in the presence of high pH seawater. Pretreatment of spermatozoa with two specific and cell-permeable PKA inhibitors, H89 and KT5720, prevented the induction of the AR in a concentration-dependent manner. These results suggest that PKA activity participates in the induction of the AR with ARIS and asterosap. To investigate this, we have cloned a gene that encodes a regulatory subunit of PKA that had been identified in starfish spermatozoa.

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.

Asterosap, a group of equally active isoforms of sperm-activating peptides from the egg jelly of the starfish Asterias amurensis, functions as a chemotactic factor for sperm. It transiently increases the intracellular cGMP level of sperm, which in turn induces a transient elevation of intracellular Ca2+ concentration ([Ca2+]i). Using a fluorescent Ca2+-sensitive dye, Fluo-4 AM, we measured the changes in sperm [Ca2+]i in response to asterosap. KB-R7943 (KB), a selective inhibitor of Na+/Ca2+ exchanger (NCX), significantly inhibited the asterosap-induced transient elevation of [Ca2+]i, suggesting that asterosap influences [Ca2+]i through activation of a K+-dependent NCX (NCKX). An NCKX activity of starfish sperm also shows K+ dependency like other NCKXs. Therefore, we cloned an NCKX from the starfish testes and predicted that it codes for a 616 amino acid protein that is a member of the NCKX family. Pharmacological evidence suggests that this exchanger participates in the asterosap-induced Ca2+ entry into sperm.

Advanced female age and extended in vitro culture have both been implicated in zona pellucida (ZP) hardening and thickening. This study aimed to determine the influence of (i) the woman's age and (ii) prolonged in vitro culture of embryos on ZP thickness and density using non-invasive polarized light (LC-PolScope) microscopy. ZP thickness and density (measured as retardance) were determined in oocytes, embryos and blastocysts in women undergoing intracytoplasmic sperm injection (ICSI) in two age groups (older, >38 years; younger, ≤38 years). A total of 193 oocytes from 29 patients were studied. The younger group contained 100 oocytes and the older group 93 oocytes. The ZP was significantly thicker in metaphase II oocytes in the older group compared with the younger group (mean ± SD: 24.1 ± 2.5 μm vs 23.1 ± 3.3 μm; p = 0.01) but ZP density was equal (2.8 ± 0.7 nm). By day 2 of culture, embryos from the two groups had similar ZP thickness (22.2 ± 2.2 μm vs 21.7 ± 1.6 μm; p = 0.28) and density (2.9 ± 0.7 nm vs 2.8 ± 0.8 nm; p = 0.57). For the embryos cultured to blastocyst (older: n = 20; younger: n = 18) ZP thickness was similar in the two groups (19.2 ± 2.7 μm vs 19.1 ± 5.0 μm; p = 0.8) but thinner than on day 2. The older group had significantly denser ZP than the younger group (4.2 ± 0.5 nm vs 3.3 ± 1.0 nm, p < 0.01). Blastocysts from both groups had significantly denser ZP than their corresponding day 2 embryos (older: 4.2 ± 0.5 nm vs 2.9 ± 0.7 nm, p < 0.001; younger: 3.3 ± 1.0 nm vs 2.8 ± 0.8 nm, p = 0.013). It is concluded that there is little relationship between ZP thickness and its density as measured by polarized light microscopy. While ZP thickness decreases with extended embryo culturing, the density of the ZP increases. ZP density increases in both age groups with extended culture and, interestingly, more in embryos from older compared with younger women.

Using an interspecies microinsemination assay with bovine oocytes, it was examined whether centrosomes of Antarctic minke whale spermatozoa function as the microtubule-organizing centre (MTOC). Bull and rat spermatozoa were used as positive and negative controls, respectively. Vitrified-warmed bovine mature oocytes were subjected to immunostaining against α-tubulin 4–6 h after intracytoplasmic injection (ICSI) of 5 mM dithiothreitol-treated spermatozoa. Aster formation occurred from whale spermatozoa (33%) and bull spermatozoa (33%), but very little from rat spermatozoa (3%). Activation treatment for the microinseminated oocytes with 7% ethanol + 2 mM 6-dimethylaminopurine resulted in a similar proportion of oocytes forming a whale sperm aster (35% vs 27% in the non-treated group; 4 h after ICSI) but a significantly larger aster (ratio of aster diameter to oocyte diameter, 0.57 vs 0.30 in the non-treated group). These results indicate that the centrosome introduced into bovine oocytes by whale spermatozoa contributes to the MTOC and that assembly of the microtubule network is promoted by oocyte activation.

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.

We report that the first localized Ca2+ transient visualized in the blastodisc cortex of post-mitotic zebrafish zygotes has unique features. We confirm that this initial ‘furrow positioning’ Ca2+ transient precedes the physical appearance of the first cleavage furrow at the blastodisc surface and that it has unique dynamics, which distinguish it from the subsequent furrow propagation transients that develop from it. This initial transient displays a distinct rising phase that peaks prior to the initiation of the two linear, subsurface, self-propagating Ca2+ waves that constitute the subsequent furrow propagation transient. Through the carefully timed introduction of the Ca2+ buffer, dibromo-BAPTA, we also demonstrate the absolute requirement of this initial rising phase Ca2+ transient in positioning the furrow at the blastodisc surface: no rising phase transient, no cleavage furrow. Likewise, the introduction of the inositol 1,4,5-trisphosphate receptor (IP3R) antagonist, 2-aminoethoxydiphenyl borate, eliminates both the rising phase transient and the appearance of the furrow at the cell surface. On the other hand, antagonists of the ryanodine receptor and NAADP-sensitive channels, or simply bathing the zygote in Ca2+-free medium, have no effect on the generation of the rising phase positioning transient or the appearance of the furrow at the surface. This suggests that like the subsequent propagation and deepening/zipping Ca2+ transients, the rising phase furrow positioning transient is also generated specifically by Ca2+ released via IP3Rs. We propose, however, that despite being generated by a similar Ca2+ release mechanism, the unique features of this initial transient suggest that it might be a distinct signal with a specific function associated with positioning the cleavage furrow at the blastodisc surface.

A major challenge in the widespread application of hES (human embryonic stem) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of less than 5% as reported by previous studies. This study characterized cell death in frozen-thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (approximately 98%), as assessed by the trypan blue exclusion test. However, when the freshly thawed hES colonies were placed in a 37 °C incubator, there was a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed down by keeping the freshly thawed hES colonies at 4 °C, with more than 90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperatures. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Hence, our observations would strongly suggest involvement of a self-induced apoptotic mechanism, as opposed to cellular necrosis arising from cryoinjury.

We investigated the relationship between the morphology of oocytes collected from small antral follicles and their developmental capacity. Immature oocytes were classified into seven groups and cultured in vitro for maturation (IVM), fertilization (IVF) and development to blastocysts (IVC). After IVF, sperm penetration and normal fertilization rates were higher in the oocytes whose cytoplasm appeared brown. The rate of polyspermy was highest in the oocytes whose cytoplasm was black. After IVC, the rates of cleavage and of development to the blastocyst stage were also higher in the brown oocytes. Although the oocytes with dark clusters in a pale cytoplasm showed lower cleavage rates, cleaved zygotes had high developmental rates the same as the oocytes with a brown cytoplasm. Transmission electron microscopy showed that the oocytes with a pale or black cytoplasm had organelles arranged differently from other oocytes before IVM. Most of the oocytes with a brown and homogeneous cytoplasm or small diameter had the characteristics of immature cytoplasm (large clusters of cortical granules) even after IVM. On the other hand, the brown oocytes with a dark zone at the periphery or with dark clusters showed the same arrangement of organelles as in vivo matured oocytes. The oocytes with a pale or black cytoplasm appeared to be degenerating and/or ageing. In conclusion, a dark ooplasm indicates an accumulation of lipids and good developmental potential, while a light-coloured ooplasm indicates a low density of organelles and poor developmental potential. A black ooplasm indicates ageing and low developmental potential.

We have shown that the assembly of lamin-associated polypeptide (LAP) 2β was detected surrounding the chromatin mass around the time of extrusion of the second polar body (PB) in some fertilized oocytes, but not in most activated oocytes, by using A23187 and cycloheximide (CaA + CH). Here, we immunohistologically analysed the correlation between LAP2β assembly and chromatin condensation in fertilized and activated oocytes during the second meiosis. In bovine cumulus cells, the onset of LAP2β assembly was observed around anaphase chromosomes with strongly phosphorylated histone H3. No LAP2β assembled around the chromosomes in the first and second polar bodies and the alternative oocyte chromatin (oCh) if histone H3 was phosphorylated. Only histone H3 of oCh was completely dephosphorylated during the telophase II/G1 transition (Tel II/G1), and then LAP2β assembled around only the oCh without phosphorylated histone H3. In the oocytes activated by CaA + CH, LAP2β did not assemble around the condensed oCh during the Tel II/G1 transition, although their histone H3 dephosphorylation occurred rather rapidly compared with that of the fertilized oocytes. The patterns of histone H3 dephosphorylation and LAP2β assembly in oocytes activated by CaA alone showed greater similarity to those in fertilized oocytes than to those in oocytes activated by CaA + CH. These results show that LAP2β assembles around only oCh after complete dephosphorylation of histone H3 after fertilization and activation using CaA alone, and that the timing of histone H3 dephosphorylation and LAP2β assembly in these oocytes is different from that of somatic cells. The results also indicate that CH treatment inhibits LAP2β assembly around oCh but not histone H3 dephosphorylation.

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.

Testicular cell-to-cell interactions play a key role in the regulation of spermatogenesis. In the testis, cell contacts are mediated through several mechanisms, including paracrine and direct contacts depending on gap junctional pathways. Gap junctions require connexin (Cx) channels and connexin-43 (Cx43) represent the most abundant Cx found in mammalian testis. Little is known about Cx expression in non-mammalian testis. Here we report the partial cloning of a Cx43 transcript of 381 bp from Rana esculenta testis. We also demonstrate that, in the frog testis, Cx43 transcript and protein show a parallel temporal and spatial pattern of expression throughout the reproductive annual cycle, with higher levels from September to January (when spermatogenesis is at a maximum level). In situ hybridization, carried out on testis collected in October, indicated that Leydig cells (LC) and Sertoli cells express Cx43 transcript, while the hybridization signal was less intense in germ cells. To obtain more information on Cx43 expression in the frog testis, we have used ethane-dimethane sulphonate (EDS), a toxin that specifically destroys LC. RT-PCR analysis shows a progressive decrease in Cx43 expression in EDS-treated testis from day 1 to day 4 after the injection, associated with LC destruction. Moreover, Cx43 expression returns to normal on day 28, when a new population of LC reappear in the interstitium, indicating that Cx43 is mainly expressed by LC. Taken together our data provide evidence that Cx43 is present in the frog testis with an important role in spermatogenesis.

The phenomenon of the developmental arrest at the 2-cell stage of 1-cell embryos from some mouse strains during in vitro culture is known as the 2-cell block. We investigated the specific factors involved in the 2-cell block of AKR embryos by means of a modified culture system, the production of reconstructed embryos by pronuclear exchange and a cross experiment. In a culture medium with phosphate, 94.6% of 1-cell embryos from the C57BL mouse strain developed to the blastocyst stage, but 95.7% of embryos from the AKR mouse strain showed 2-cell block. Phosphate-free culture medium rescued the 2-cell block of AKR embryos and accelerated the first cell cycle of the embryos. Co-culture with BRL cells and a BRL-conditioned medium fractionated below 30 kDa also rescued the 2-cell block of AKR embryos. Examinations of in vitro development of reconstructed embryos and of embryos from F1 females between AKR and C57BL strains clearly demonstrated that the AKR cytoplast caused the 2-cell block. In the backcrossed female progeny between (AKR × C57BL) F1 males and AKR females, about three-quarters of the embryos were of the 2-cell blocking phenotype and about one-quarter were of the non-blocking phenotype. These results suggest that two genes are responsible for the 2-cell block of AKR embryos.

The aim of the work was to study a potential relationship between acrosome response characteristics of bovine spermatozoa and their ability to fertilize oocytes and produce in vitro embryos. Sperm of artificial insemination bulls with a high rate (22.0 ± 4.1%, group A, n = 7) or a low rate (10.3 ± 4.1%, group B, n = 8) of embryos were used. For acrosome assessment, motile spermatozoa from a Percoll gradient were incubated with or without heparin and examined by the fix-vital sperm assay (FVSA). The differences between the heparin-treated (H+) and the non-treated (H−) spermatozoa were significant (p < 0.01) in all bulls at all tested intervals. According to the kinetics of the heparin response, the bulls fell into three categories: fast (FR, n = 7), moderate (MR, n = 5) or slow (SR, n = 3) acrosome responses (p < 0.01). Five MR bulls were found in group A in comparison with two MR bulls in group B (57.1 vs 12.5%; p < 0.05). Intensity of the acrosome response (response index) was significantly higher in bull group A compared with bull group B (7.0 vs 4.6, p < 0.01). A positive correlation was recorded between response index and embryo rate (r = 0.668, p < 0.01). In conclusion (a) the kinetics of spermatozoa response to heparin may be important for in vitro fertilization, bulls with a moderate response appearing to be most suitable for embryo production; (b) greater spermatozoa response to heparin was related to more effective embryo production.

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.

There is very little information available on stage-specific gene expression during early embryo development, particularly in the pig. Here, we accurately identified the genes that are specifically or prominently expressed in parthenogenetic porcine blastocysts as compared with 2-cell stage embryos. We accomplished this by using a PCR technology regulated by annealing control primers (ACPs). By utilizing 120 ACPs, a total of 46 expressed sequence tags (ESTs) of genes that are differentially expressed in blastocysts as compared with 2-cell stage embryos were cloned and sequenced. The cloned genes or ESTs all exhibited significant sequence similarity with known genes or ESTs of other species. Of the known genes, six genes [renin-binding protein (RNBP), BMDP, solute carrier family 25 (SLC25A6), MTHFD1, TRK-fused gene (TFG), spermidine synthase (SRM)] were selected and their stage-specific expression levels in porcine parthenotes were determined by real-time quantitative polymerase chain reaction at the 1-, 2-, 4-cell, morula and blastocyst stages. While RNBP, BMDP, SLC25A6, MTGFD1 and SRM were highly expressed only at the blastocyst stage, TFG was highly expressed at the 1-cell stage, then declined after genomic activation, high levels of expression being again detected at the morula and blastocyst stages. This analysis suggests that the ACP system is an effective tool for use in the identification of stage-specific genes in small numbers of porcine parthenotes. Examination of the genes differentially expressed in the blastocyst, which we have identified here, will provide insight into the molecular basis of preimplantation development.

It is well known that the preimplantation culture environment to which embryos are exposed influences the expression of developmentally important genes. Recently, it has been reported that MEMα, a culture medium commonly used for somatic cells, allows high rates of preimplantation development and development to term of mouse somatic cell nuclear transfer (SCNT) embryos. The objective of this study was to compare the differential effects of this medium and of the nuclear transfer procedure on the relative mRNA abundance of several genes with key roles during preimplantation. The relative mRNA levels of nine genes (Glut 1, Glut 5, G6PDH, Bax, Survivin, Gpx 1, Oct4, mTert and IGF2bp1) were quantified at blastocyst stage on cumulus cell cloned embryos cultured in MEMα, as well as on in vivo cultured and MEMα cultured controls. Only three of the nine transcripts analysed (Glut 5, Gpx 1 and Igf2bp1) were significantly down-regulated at blastocyst stage in in vitro produced controls. However, most genes analysed in our MEMα cultured cloned embryos showed altered transcription levels. Interestingly, between cloned and in vitro produced controls only the transcription levels measured for Glut 1 were significantly different. This result suggests that Glut 1 may be a good marker for embryo quality after cumulus cell nuclear transfer.