Breeding and larval performance of novel hybrids from reciprocal crosses of Asian catfish Pangasianodon hypophthalmus (Sauvage, 1878) and African catfish Clarias gariepinus (Burchell, 1822) were investigated in this study. Spawning was by hormonal injection of brood fish, artificial fertilization, and incubation in triplicate aquarium tanks (0.5 × 0.5 × 0.5 m3) with continuous aeration. Reciprocal crosses (♀C. gariepinus × ♂P. hypophthalmus and ♀P. hypophthalmus × ♂C. gariepinus) had lower hatchability (≤50%) than their pure siblings (≥75%). Fish from all crosses survived until the juvenile stage but survival at 35 days post hatching (dph) was higher for pure C. gariepinus sib. ♀C. gariepinus × ♂P. hypophthalmus was observed to be less resistant to degradation of water quality than the other crosses, however it had higher body weight compared with the other crosses that showed similar performance. Morphological comparison of surviving juvenile at 35 dph, showed that all ♀P. hypophthalmus × ♂C. gariepinus and 13% of the ♀C. gariepinus × ♂P. hypophthalmus exhibited the very same morphology as that of their maternal parent species, while the other portion of the ♀C. gariepinus × ♂P. hypophthalmus cross exhibited morphological traits that were intermediate between those of both parent species. This study been the first successful attempt to hybridize both species and therefore, laid the groundwork for further studies on the aquaculture potentials of the novel hybrids.

The purpose of the present study was to determine whether the plasma membrance of the human oocyte is reorganised following fertlisation and during early cleavage. In order to characterise and localise the major sugar moieties on surface glycoporteins, oocytes and embroys were labelled with a range of flourescent lectins. Regional organisation of plasma membrane microvilli in oocytes and embryos was also studied using scanning electron microscopy (SEM). The plasma membrance of human oocytes, zygotes and early blastomeres stained strongly and homogeneously with concanavalin A and Triticum vulgaris lectin (WGA), indicationg the presence of plasma membrance glycoconjugates with α-D-mannosyl residues, sialic acid and β-NAc-glucosaminyl groups. We did not observe regional domains in oocytes and zygotes, suggesting that the plasma membrane is not topographically reorganised following fertilisation. SEM shows the surface of the human zygote to be organised into short microvilli 0.2–3.0 μm in length and at a density of 5–20/μm2. In early cleavage stages the microvilli are shorter and less frequent (0.2–1.0 μm; 1–5/μm2); however, there is no evidence of polarisation at this level of organisation, at either stage of development. The surface of cell fragments, common in the human embryo in vitro, differs in having few microvilli and numerous cytoplasmic blebs. In conclusion, there are no obvious morphological signs of regionalisation in the plasma membrane of the human embryo before the 8-cell stage.

During mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE–ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE–ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40–100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25–100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i.

Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.

Gene expression at the onset of bovine embryogenesis is developmentally regulated and histone deacetylases (HDACs) have been shown to play a key role in the control of gene expression during this period of development in other species. We determined expression pattern(s) of powerful repressors, namely histone deacetylase-1, -2 and -3, that may in part regulate gene expression during bovine oogenesis and early embryogenesis at the mRNA and protein levels. Detected fragments of the hdac genes were sequenced and comparison of the sequences showed very high homologies between DNA and amino acid sequences of bovine HDACs and those of human and mouse. RPD3, a yeast global regulator of transcription, was also detected in bovine oocytes and embryos. Results suggest that HDACs may be operative in regulation of zygotic/embryonic gene expression in cattle.

Hyaluronan (HA) is glycosaminoglycan that is present from the start of embryonic development and its role and concentration increases with embryo development. The objective of this study was to evaluate if the presence of HA in TCM-199 culture medium had an effect on the development and quality of bovine embryos. There was no effect of HA on the total number of zygotes developing to blastocysts on day 7, however more expanded and hatched blastocyst stages were observed on days 8 and 9 in the group supplemented with HA (p < 0.05). Following freeze/thawing, significantly more (p < 0.05) embryos cultured in medium supplemented with HA hatched than those cultured in TCM-199 alone or those with BSA. Medium supplemented with HA and BSA significantly increased the level of expression of glucose metabolism Glut-1 gene and embryo compaction Cx43 gene (p < 0.05), and had no effect on Glut-5 and IGF-II expression. In addition, HA presence in culture decreased the level of expression of apoptosis Bax and oxidative stress SOX genes (p < 0.05). There was significant difference in total number of nuclei between TCM-199 medium only and the remaining media containing BSA or HA plus BSA, between which there was no difference. In summary, our results indicate that the addition of high molecular weight HA to TCM-199 medium that contains BSA on day 4 of culture improved embryo development to hatching and hatched blastocysts and the quality of produced embryos, which were superior to embryos cultured without HA addition.

In this study our aim was to characterise the presence and the role of DNA alterations during sperm decondensation in the mouse. To visualise the changes during decondensation we investigated for the presence of DNase I hypersensitive sites in situ and for a putative role for topoisomerase II by examining the effect of teniposide, a topoisomerase II inhibitor, during fertilisation. In situ nick translation without the previous addition of DNase I failed to reveal the presence of endogenous nicks in decondensing sperm and pronuclei whereas preincubation of fixed oocytes with DNase I indicated that decondensing sperm were sensitive to this enzyme. Addition of 100 μM teniposide did not completely inhibit pronuclei formation but its addition to the fertilisation medium did lead to the presence of endogenous DNA nicks in decondensing sperm. These observations suggest that DNase I hypersensitivity during sperm decondensation is related to the dramatic conformational changes that the chromatin undergoes during the decondensation process, in which topoisomerase II may be implicated.

Expression of various developmentally regulated markers was screened throughout the preimplantation stages of in vitro-derived bovine embryos. This was done by investigating the distribution of several nuclear, cytoplasmic and extracellular proteins by means of immunofluorescence microscopy. While lamin B appeared as a constitutive component of nuclei of all preimplantation stages, lamins A/C had a stage-related distribution. The early cleavage stage nuclei contained lamins A/C which generally disappeared in the following stages, with the possible exception of a few positive nuclei in the morula and early blastocyst stage. In the expanded blastocyst stage the nuclei of trophectoderm cells became positive while no positivity was observed in the inner cell mass cells. Starting from day 6, the appearance and/or polarised distribution of various cytoskeletal and cytoskeleton-related components such as Factin, α-catenin and E-cadherin gave an insight into the timing of events related to compaction of bovine e bryos. Compaction was correlated with the first differentiation event, i.e. the formation of trophectoderm; this is the first embryonic epithelium, characterised by cytokeratins and desmoplakin. Extracellular fibronectin was first detected in the early blastocyst stage shortly before the morphological differentiation of primitive endoderm, and in the later stages it was localised at the interface between trophectoderm and extraembryonic endoderm. Laminin and collagen IV were expressed by the endoderm cells and contributed to the extracellular matrix underlying the trophectoderm. This study is a first attempt to characterise the cells of in vitro-derived bovine embryos valid for cell line derivation.

The present study aimed to investigate the effect of quercetin as an alternative antioxidant to cysteamine on in vitro maturation. Oocytes were collected from goat ovaries, destined for in vitro maturation and distributed into three groups: CIS group, oocytes were immersed in MIV base medium; in Groups Q4 and Q8, oocytes were immersed in the medium of the CIS group, adding 4 μM or 8 μM of quercetin, respectively, and cultured for 24 h at 38.5°C with 5% CO2. The CIS and Q4 groups presented the same percentage of expanded cumulus cells, but the per cent in the Q8 group was significantly lower than that of the other groups (P<0.05). The oocyte retraction rate in the Q8 group was higher (P<0.05) than in the CIS and Q4 groups. Treatment with 8 μM of quercetin presented a lower proportion of expanded oocytes than the CIS group and 4 μM of quercetin (P<0.05). The percentage of MII oocytes was higher in the Q4 group than in the CIS group (P<0.05), but the percentages in the CIS and Q8 groups were similar. The rate of apoptosis was higher in the CIS group than in the other groups (P<0.05). In addition, oocytes matured with 4 μM quercetin showed higher mitochondrial activity than matured oocytes in the CIS and Q8 groups (P<0.05). In conclusion, 4 μM of quercetin can be used as an alternative to cysteamine in the in vitro maturation of goat oocytes.

The effects of zinc (as ZnCl2) on in vitro production of bovine embryos (IVMFC) and components of the procedure, that is in vitro oocyte maturation (IVM), fertilisation (IVF) and embryo development in culture (IVC), and the effect of added zinc on sperm motility were studied. Immature cumulus oocyte complexes (COCs) were aspirated from ovarian follicles (2-5 mm diameter) at slaughter, and matured, fertilised and cultured in chemically defined conditions. The presence of zinc (10, 100 or 1000 μg added per millilitre) throughout IVMFC inhibited fertilisation. After addition of 10 μg zinc per millilitre separately to media for IVM and IVF, fertilisation was inhibited only when zinc was present for IVM. When present for IVF, 80% of oocytes selected for IVM reached 2- to 4-cell stages by 46 h after insemination whereas 67% of control oocytes (inseminated without added zinc) cleaved. Higher zinc concentrations (100 and 1000 μg added per millilitre) for IVF inhibited fertilisation. Sperm motility was reduced with addition of 10 μg per millilitre of zinc for sperm preparation (i.e. capacitation interval). Addition of 1.0 μg zinc per millilitre to media used through IVMFC, or to the IVC medium alone, resulted in inhibition of development after 2- to 4-cell stages. When added to IVM or to both IVM and IVF media 1.0 μg/ml of zinc compromised development to the morula stage and beyond. Maturing bovine oocytes may be more sensitive to 1.0 μg ml of zinc in vitro than in vivo because a concentration of 3.0 μg/ml has been reported for bovine follicular fluid. Fertilisation was not adversely affected by 10 μg/ml of zinc; however, higher concentrations were inhibitory.

This paper reports on the activation of p90rsk during meiotic maturation and the inactivation of p90rsk after electrical parthenogenetic activation of rat oocytes. In addition, the correlation between p90rsk and MAP kinases after different treatments was studied. We assessed p90rsk activity by examining its electrophoretic mobility shift on SDS-PAGE and evaluated ERK1+2 activity by both mobility shift and a specific antibody against phospho-MAP kinase. The phosphorylation of p90rsk during rat oocyte maturation was a sequential process that may be divided into two stages: the first stage was partial phosphorylation, which was irrelevant with MAP kinases because p90rsk phosphorylation took place prior to activation of MAP kinases. The second stage inferred full activation occurred at the time when MAP kinases began to be activated (3 h after germinal visicle breakdown). Evidence for the involvement of MAP kinases in the p90rsk phosphorylation was further obtained by the following approaches: (1) okadaic acid (OA) accelerated the phosphorylation of both MAP kinases and p90rsk; (2) OA induced phosphorylation of both MAP kinases and p90rsk in the presence of IBMX; (3) when activation of MAP kinases was inhibited by cycloheximide, p90rsk phosphorylation was also abolished; (4) dephosphorylation of p90rsk began to take place at 3 h post-activation, temporally correlated with the completion of MAP kinase inactivation; (5) phosphorylation of both kinases was maintained in oocytes that failed to form pronuclei after stimulation; (6) OA abolished the dephosphorylation of both kinases after parthenogenetic activation. Our data suggest that MAP kinases are not required for early partial activation of p90rsk but are required for full activation of p90rsk during rat oocyte maturation, and that p90rsk dephosphorylation occurs following MAP kinase inactivation after parthenogenetic activation of rat oocytes.

The zona pellucida (ZP) is a transparent envelope that surrounds the mammalian oocyte and mediates species-selective sperm–egg interactions. Porcine and bovine ZPs consist of glycoproteins ZP2, ZP3, and ZP4. In both pig and bovine a heterocomplex consisting of ZP3 and ZP4 binds to sperm, however it is not clarified whether ZP3 or ZP4 in the complex is responsible for the sperm binding. Previously, we have established a baculovirus-Sf9 cell expression system for porcine ZP glycoproteins. A mixture of recombinant ZP3 (rZP3) and rZP4 displayed sperm-binding activity toward bovine sperm but not porcine sperm, probably due to differences in carbohydrate structure between the native and recombinant ZP glycoproteins. In this study, a mixture of porcine rZP3 and native ZP4 (nZP4) inhibited the binding of porcine sperm to the ZP. In contrast, a mixture of porcine nZP3 and rZP4 did not inhibit the binding of porcine sperm, although the mixture inhibited the binding of bovine sperm. The porcine rZP3/nZP4 mixture bound to the acrosomal region of porcine sperm, in a manner similar to that of the nZP3/nZP4 mixture. nZP3 was precipitated with rZP4, and nZP4 was precipitated with rZP3 by utilising the N-terminal tags on the recombinant proteins. These results indicated that nZP4, but not rZP4, is necessary for binding activity of porcine ZP3/ZP4 complex towards porcine sperm and further suggested that the carbohydrate structures of ZP4 in the porcine ZP3/ZP4 complex are responsible for porcine sperm-binding activity of the complex.

Mitogen-activated protein (MAP) kinase in human eggs has been investigated by using immunoblotting with both anti-Active MAPK and anti-ERK2 antibodies. The results showed that the main form of MAP kinase was p42ERK2. It was in a dephosphorylated form in oocytes at the germinal vesicle stage, but fully phosphorylated in unfertilised mature eggs. MAP kinase phosphorylation was significantly decreased when pronuclei were formed after intracytoplasmic sperm injection. Neither MAP kinase expression nor activity was detected in morphologically degenerated eggs. Although MAP kinase still existed in early embryos arrested at the 8-cell or morula stages, little, if any, activity could be detected. These data suggest that MAP kinase may play an important role in the cell cycle regulation of human eggs, as in other mammalian species.

To ascertain whether the Kunming (KM) mouse is an available model for age-related decline in female fertility in human or not, oocytes from young (6–8 weeks), middle-aged (9 months) and aged (12 months) female mice were compared with respect to number of oocytes, frequency of in-vitro maturation (IVM) and in-vitro fertilization (IVF), and meiotic chromosome segregation and alignment. The mean number of pups born per mouse decreased significantly from the young to the middle-aged and the aged mice. The mean number of ovarian follicles, ovarian germinal vesicle oocytes and ovulated MII oocytes decreased significantly with maternal age. The rate of IVM in oocytes from young mice (73.9%) was less significantly than that in oocytes from middle-aged and aged mice (86.1% and 84.4%, respectively). Immunocytochemical analysis showed that ageing caused a significantly higher rate (49.3%) of chromosome misalignment than that (15.7%) of the young mice. The presence of premature chromatids was also significantly higher in MII oocytes of aged mice as compared with young mice (37.8 versus 8.3%). Pronuclear formation was delayed in oocytes of middle-aged and aged females (35.5 and 42.3% respectively in 5 h of IVF) as compared with young mice (88.1%). The study suggests that KM mouse exhibits an age-related decline in female fertility. Significant reduction of germinal vesicle (GV) and MII oocytes and significant increase of metaphase chromosome misalignment and premature chromatid segregation after meiotic maturation of oocytes, similar to human, presumably contribute to the decline in aged KM mice.

Our previous studies have shown that the addition of 100 μM cysteamine to the in vitro maturation (IVM) medium increased the embryo development of prepubertal goat oocytes. The aim of the present study was to evaluate the effect of adding different concentrations of cysteamine to the IVM medium and to the in vitro embryo culture medium (IVC) on the embryo development of prepubertal goat oocytes selected by the brilliant cresyl blue (BCB) test. Oocytes were exposed to BCB and classified as: oocytes with a blue cytoplasm or grown oocytes (BCB+) or oocytes without blue cytoplasm or growing oocytes (BCB−). In Experiment 1, oocytes were matured in a conventional IVM medium supplemented with 100 μM, 200 μM or 400 μM cysteamine. In Experiment 2, oocytes were matured with 400 μM cysteamine and following in vitro fertilization (IVF) were cultured in SOF medium supplemented with 50 μM and 100 μM cysteamine. In Experiment 1, BCB+ oocytes matured with 100 μM and 200 μM cysteamine showed higher normal fertilization and embryo development rates than BCB− oocytes. Oocytes matured with 400 μM cysteamine did not present these differences between BCB+ and BCB− oocytes. In Experiment 2, the addition of 50 μM and 100 μM cysteamine to culture medium did not affect the proportion of total embryos obtained from BCB+ oocytes (35.89% and 38.29%, respectively) but was significantly different in BCB− oocytes (34.23% and 29.04%, respectively, P<0.05). In conclusion, the addition of 400 μM cysteamine to the IVM improved normal fertilization and embryo development of BCB− oocytes at the same rates as those obtained from BCB+ oocytes. The proportions of morulae plus blastocyst development were not affected by the treatments.

The intracellular pH of the mouse egg was measured during fertilisation to determine whether an increase in pH accompanies activation of this mammalian egg. The pH-sensitive dye BCECF [2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein] was introduced into the mouse egg by incubation in BCECF-AM or by microinjection of dextran-conjugated BCECF. The cells were also loaded with the DNA-specific fluorochrome Hoechst 33342 to confirm fertilisation by observation of Hoechst-stained, decondensing sperm heads in the cytoplasm. The ratio of emission intensities for the dye (494/440 nm excitation wavelengths) was monitored continuously with a photon-counting photomultiplier tube. There was no change in pH during or after fertilisation. Control eggs displayed the expected increase in pH when exposed to NH4C1. In other experiments, intracellular pH and intracellular Ca2+ were monitored simultaneously during fertilisation. The eggs were injected with BCECF dextran and Fura dextran. Fluorescence emission was recorded at excitation wavelengths of 495 nm (BCECF, pH-sensitive wavelength) and 385 nm (Fura, Ca2+-sensitive wavelength). A decrease in emission intensity at 385 nm excitation clearly marked the repetitive Ca2+ transients at egg activation. There was no change in the fluorescence emitted at 495 nm excitation, indicating an absence of any change in intracellular pH. These results indicate that intracellular alkalinisation of the cytoplasm does not accompany activation of this vertebrate egg.

Three assays were performed. In assay 1, oocytes harvested during the winter months were subjected to kinetic heat shock by stressing the oocytes at 39.5°C (HS1) or at 40.5°C (HS2) for either 6, 12, 18 or 24 h and then matured at control temperature (38.5°C). The nuclear maturation rates (NMR) of all oocytes were recorded after 24 h. In assay 2, oocytes collected year-round maturated, were implanted via in vitro fertilization (IVF) and developed for 9 days. Gene expression analysis was performed on target genes (Cx43, CDH1, DNMT1, HSPA14) with reference to the two housekeeping genes (GAPDH and SDHA) in embryos. Similarly, in assay 3, genetic analysis was performed on the embryos produced from heat-stressed oocytes (from HS1 and HS2). In assay 1, the duration of heat stress resulted in a significant decline in NMR (P < 0.05) with HS1 for maturated oocytes at 86.4 ± 4.3; 65.5 ± 0.7; 51.3 ± 0.9; 38.1 ± 1.9 and 36.3 ± 0.9, for control, 6 h, 12 h, 18 h and 24 h, respectively. For assays 2 and 3, results demonstrated that DNMT1, Cx43 and HSPA14 were down-regulated in the embryos produced in the warm with respect to the cold months (P < 0.05). A constant up- and down-regulation of DNMT1 and HSPA14 genes were observed in both HS-treated samples. Also, an inconsistent pattern of gene expression was observed in Cx43 and CDH1 genes (P < 0.05). Targeted gene expression was aberrant in embryo development, which can provide evidence on early embryo arrest and slowed embryo development.

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.

Cycle synchronization of donor cells in the G0/G1 stage is a crucial step for successful somatic cell nuclear transfer. In the present report, we evaluated the effects of contact inhibition, serum starvation and the reagents – dimethyl sulphoxide (DMSO), roscovitine and cycloheximide (CHX) – on synchronization of canine fibroblasts at the G0/G1 stage. Ear fibroblast cells were collected from a beagle dog, placed into culture and used for analysis at passages three to eight. The population doubling time was 36.5 h. The proportion of G0/G1 cells was significantly increased by contact inhibition (77.1%) as compared with cycling cells (70.1%); however, extending the duration of culture did not induce further synchronization. After 24 h of serum starvation, cells were effectively synchronized at G0/G1 (77.1%). Although synchronization was further increased gradually after 24 h and even showed significant difference after 72 h (82.8%) of starvation, the proportion of dead cells also significantly increased after 24 h. The percentage of cells at the G0/G1 phase was increased (as compared with controls) after 72 h treatment with DMSO (76.1%) and after 48 h treatment with CHX (73.0%) or roscovitine (72.5%). However, the rate of cell death was increased after 24 and 72 h of treatment with DMSO and CHX, respectively. Thus, we recommend the use of roscovitine for cell cycle synchronization of canine ear fibroblasts as a preparatory step for SCNT.

Localisation of the acrosome reaction inducing activity in egg-jelly was examined in the newt, Cynops pyrrhogaster. The jelly has six layers: the J0, J1, J2, J3, J4 and st layers. Jelly was mechanically dissected and placed on a Millipore filter. When sperm were added from the outer surface side of the jelly, most of them exhibited the acrosome reaction after passing through the jelly. When egg-jelly was divided into four layers, strong activity for the induction of acrosome reaction was detected in the outer layers, J4+st. These findings suggest that the acrosome reaction is induced by a substance in the outer layers of the egg-jelly. Among jelly components separated by SDS-PAGE, a fraction of more than 500 kDa in molecular weight induced the acrosome reaction. Wheat germ agglutinin (WGA), Griffonia simplicifoliar agglutinin 1 (GS-1), Maclura pomifera agglutinin (MPA) and Arachis hypogaea agglutinin (PNA) inhibited the induction of the acrosome reaction by jelly extract, and WGA did so in a dose-dependent manner. Those lectins precipitated some molecules of over 500 kDa. These results suggest that the acrosome reaction is induced by the high molecular-weight components of egg-jelly in C. pyrrhogaster.

There can be little doubt that one of the major technical advances in the discipline of mammalian reproductive physiology in recent years has been the development and refinement of procedures of cell maintenance and culture outside the body. Such in vitro systems have been applied, for example, to questions of (1) ovarian follicular physiology, especially the respective hormonal contributions and interactions of granulosa and theca interna cells, (2) oviduct and uterine activity, especially the novel secretions of endosalpinx and endometrium at different stages of the reproductive cycle, and (3) maturation of gametes, especially endocrine support of the resumption of meiosis in artificially liberated oocytes and the requirements for final maturation (capacitation) of epididymal or ejaculated spermatozoa, either fresh or cryopreserved. Establishment of in vitro systems has been assisted and encouraged by the ready availability of chemically-defined culture media, disposable plastic dishes and tubes, and computer-controlled incubators set to predetermined gas phase, humidity and temperature. Laminair flowhoods for tissue preparation and culture laboratories with filtered air and ultraviolet irradiation have together reduced problems of contamination and infection. Overall, the technical standards are impressive and inspection of modern tissue culture facilities invariably generates confidence in the quality of studies being pursued. In a phrase, such aesthetically pleasing and excellent laboratory facilities must surely be producing excellent science.