The effects of medium supplementation with oestrous goat serum and glycoprotein hormones on caprine oocyte maturation in vitro (IVM) were evidenced by proportions of resulting ova completing in vitro fertilisation (IVF) and development to the morula stage. Oocyte-cumulus complexes (OCCs) were harvested in follicular fluid from 2–5 mm diameter follicles. Oocyte maturation took place during 27 h in TCM-199 supplemented with 20% oestrous goat serum, oestradiol-17β (1.0 μg/ml), and either (a) 0.5 μg FSH/ml, (b) 100 μg LH/ml, (c) 100 μg LH + 0.5 μg FSH/ml, (d) 100 μg hCG + 0.5 μg FSH/ml, (e) 0.5 μg TSH/ml or (f) no added glycoprotein hormone (control). Of 353 immature oocytes cultured in seven experiments, 311 (88.1%) exhibited cumulus expansion at the end of the IVM interval; all normalappearing OCCs were inseminated. In vitro insemination was with ejaculated sperm treated with heparin (10 μg/ml) and caffeine (0.4 μg/ml). Proportions (%) of inseminated ova that were fertilised (cleaved) and that reached the morula stage after IVM with (a) FSH, (b) LH, (c) LH + FSH, (d) hCG + FSH, (e) TSH and (f) no added glycoprotein hormone were (a) 22/52 (42.3%) and 9/52 (17.3%), (b) 25/54 (46.3%) and 14/54 (25.9%), (c) 52/65 (80.0%) and 26/65 (40.0%), (d) 48/78 (61.5%) and 22/78 (28.2%), (e) 14/54 (25.9%) and 4/54 (7.4%), and (f) 11/50 (22.0%) and 1/50 (2.0%), respectively. All treatments yielded better results than IVM with no added glycoprotein hormone. After IVM with added LH + FSH higher proportions of oocytes were fertilised (p<0.05), and higher proportions reached the morula stage (p<0.05) when compared with other treatments.

During fertilisation in starfish oocytes, the fertilisation cone develops temporarily beneath the penetrating sperm. The role of the fertilisation cone in sperm incorporation in the starfish Asterias amurensis was examined using cytochalasin B (CB). CB (2 μM) allowed sperm acrosomal process–egg plasma membrane fusion and egg activation, but inhibited the development of the fertilisation cone containing actin microfilaments. When sperm were added to intact oocytes (with the jelly coat and vitelline coat) in seawater containing CB, the sperm head did not penetrate the fertilisation membrane. Although the acrosomal process fused with egg plasma membrane, the sperm head remained outside the fertilisation membrane. On the other hand, denuded oocytes without the jelly coat and vitelline coat allowed sperm penetration even in the presence of 2 μM CB. Electron microscopy revealed that sperm organelles, including the acrosomal process, nucleus, mitochondrion and tail, were incorporated into the slightly electron-dense cytoplasm, which was similar to the cytoplasm of the fertilisation cone. These results show that the development of the fertilisation cone/actin filament complex is not essential for incorporation of the sperm, since incorporation can occur in denuded oocytes. However, the cone is required for fertilisation of intact oocytes, suggesting that this actin-filament-containing structure is necessary for getting the sperm through the outer egg coats.

We have used rhodamine-conjugated phalloidin and confocal microscopy to examine the organisation of filamentous actin (F-actin) during oogenesis in Xenopus laevis. F-actin was restricted to a thin shell in the cortex of oogonia and post-mitotic oocytes less than 35 μm in diameter. In oocytes with diameters of 35–50 μm F-actin was observed in three cellular domains: in the cortex, in the germinal vesicle (GV) and in a network of cytoplasmic cables. Initially, actin cables were sparsely distributed in the cytoplasm, with no evidence of discrete organising centres. In larger stage I oocytes, a dense network of actin cables extended throughout the cytoplasm, linking the GV and mitochondrial mass to the cortical actin shell. Apart from the F-actin associated with the mitochondrial mass, no evidence of a polarised distribution of F-actin was apparent in stage I oocytes. F-actin was observed also in the cortex and the GV of stage VI oocytes, and a network of cytoplasmic cables surrounded the GV. Cytoplasmic actin cables extended from the GV to the animal cortex, and formed a three-dimensional network surrounding clusters of yolk platelets in the vegetal cytoplasm. Finally, disruption of F-actin in stage VI oocytes with cytochalasin resulted in distortion and apparent rotation of the GV in the animal hemisphere, suggesting that actin plays a role in maintaining the polarised organisation of amphibian oocytes.

We present evidence for the fusion of isolated single maize egg and sperm cell protoplasts in a mannitol solution (400–430 mosmol/kg H2O) containing 0.05 M CaCl2 at pH 11.0, followed by cell division of the fusion products. These findings allow the performance of in vitro fertilisation of higher plants by combining single gametes as in lower plant and animal systems. Further, our findings open new avenues for investigating the basic mechanisms of adhesion and fusion of higher plant gametes and eventually for examining processes that inhibit polyspermy in higher plants.

We have investigated the oolemma block to polyspermy in the mouse. Zona-free and zona-intact eggs were fertilised and subsequently re-inseminated (the latter following zona pellucida removal). The ‘perivitelline’ block to polyspermy in zona-intact eggs renders motile sperm in the perivitelline space unable to bind to the oolemma. This is not connected with irreversible changes in the egg plasma membrane, because freshly added sperm can still fuse with such eggs freed from the zona. Fertilised eggs eventually lose the ability to fuse with sperm within 1 h, while still being able to bind many sperm.

Various researchers describe the morphology of cumulus cells (CC) in vitro, but few have investigated their behaviour on plastic. Knowledge concerning the behaviour of human CC could be useful in improving the success of in vitro fertilisation procedures. This study aimed to describe the morphology and behaviour of CC in vitro and to investigate movement on a collagen-coated substrate. Following collection some cumulus were mechanically dissected from those surrounding the oocyte. Cumulus aggregates were cultured over 24 h using Earle's medium supplemented with 8% albumin. Substrata were plastic coverslips coated with collagens I, IV, or mixed collagens. Cumulus cultured over corresponding time periods on uncoated coverslips served as controls. Specimens were fixed and prepared for scanning electron microscopy. Over 24 h the controls began exhibiting the morphological features associated with cell movement: cell surface protrusions changed from blebs to microridges, lamellipodia and leading lamellae; cell shape altered from rounded and upright, to flattened. Extracellularmatrix (ECM) transformed from a thick, sheet-like substance to a thin, fibrous material. By 24 h, cells contacting ECM remained rounded showing few features of movement. Collagens enhanced attachment of CC as a monolayer on the substrate. Cell morphology varied according to the collagen type used. On mixed collagens, cells attached rapidly, appearing to be predominantly non-motile. On collagen type I there was less attachment of cells but increased motility. On collagen type IV there was decreased attachment and the cells remained spherical. In conclusion, collagens enhance the settling of cumulus cells on a plastic substrate and the cells exhibit some specificity in attaching to collagens.

Magnesium ions as well as calcium ions are required for successful fertilisation in sea urchins. In the absence of Mg2+ spermatozoa attached to the egg plasma membrane, their acrosomal processes passing through the vitelline envelope, but could not enter the egg cytoplasm (Sano et al, Dev. Growth Differ. 22, 531–41,1980). Such an individual spermatozoon was observed microscopically to resume entry into the egg immediately after the addition of a sufficient amount of Mg2+ to the surrounding medium. Neither any change in membrane potential nor an increase in intracellular Ca2+ concentration of the egg was observed after insemination in the absence of Mg2+, although both could be observed after the addition of Mg2+. The sperm heads did not show fluorescence when attached to the surface of an egg previously microinjected with mithramycin A in Mg-free seawater, indicating that there was no connection between the sperm and the egg. Therefore, occurrence of fertilisation potential must be a post-fusional event. These results suggest that Mg2+ are indispensable for fusion between the sperm acrosomal membrane and the egg plasma membrane.

Specific mRNAs coding for a 63 kDa sperm membrane protein (63-SMP) were localised in Strongylocentrotus purpuratus testis sections using in situ hybridisation techniques. 35S-labelled antisense RNA probes transcribed from a 766 base pair fragment of the gene coding for the 63-SMP hybridised to all spermatogenic cells in the basal germinal epithelia of testicular acini, except the most peripherally located (least differentiated) spermatogonia. No hybridisation to the luminally located mature spermatozoa or somatic cells of the testis was observed. Using monoclonal antibody J17/30 and indirect immunofluorescence techniques, the 63-SMP was localised to the same subset of spermatogenic cells that contain the 63-SMP mRNA, suggesting that expression of this gene is transcriptionally controlled. In combination with previous studies on the expression of sperm histones and sperm bindin, these results show that multiple, perhaps sequential, classes of gene activity contribute to the differentiation of sea urchin sperm.

The protein pattern of the follicular fluid (FF) and the ultrastructure of the inner cumulus–oocyte complex (COC) has been analysed in single antral follicles (n = 146) of buffalo B. Bubalis ovaries. The protein population of FF was fractionated by SDS-PAGE; the resulting pattern was Coomassie stained and processed for densitometry. Comparartive analysis of sera and autologous FFs showed a marked difference in the level (measured as the percentage of total proteins) of one 21 kDa polypeptide band, called ‘L’. Concentration of L, which was mainly higher in the serum (2.05 ± 1.5%) than in the surrounding FF(0.98 ± 0.94%), fluctuated widely in fluids from the in the same ovary. On gel filtration of FF and SDS-PAGE of ther factions collected, the L polypeptide was found and eluted together with a 36 kDa polypeptide, called ‘H’, with an exclusion volume lower than that of albumin. The levels of both polypeptides in the eluted fractions were measured by gel densitometry and the same ratio H/L was found (2:1). These data suggest that H and L are subunits of a complex high-molecular-weight protein. The presence of L levels in male sera comparable to those detected in females indicates that this putative protein does not originate in the ovary but is transported from the blood. Moreover, a correlation between the increase in the percentage of Lf (calculated as %L in FF/%L in serum) and atresia was observed. COCs (n = 86) obtained during the collection of the single FF samples were processed for transmission electron microscopy. The ultrastructure of each COC was compared with the SDS-PAGE data of the associated FF. Healthy COCs were found to be related to very low levels of Lf (between 0 and 14% of those measured in serum). COCs with an early atretic ultrastructure undetectable at the dissection microscope, were associated with FFs having Lf levels between 24% and 60%; advanced atresia was associated with Lf values up to 70%. Finally, the acrosome reaction of buffalo precapacitated spermatozoa in vitro was monitored by adding one volume of FF with high (FF +; Lf = 80%) or undetectable (FF - ) values of Lf to the sperm suspension. It was found that 96.2% of spermatozoa treated with FF - showed a normal acrosome reaction, without any change in their motility pattern; spermatozoa treated with FF +, in contrast, exhibited poor motility, and only 66.4% of them underwent the acrosome reaction. The ionophore A23187, in a control experiment, resulted in lower percentages of acrosome reactions (38%). In conclusion, our results suggest that enhanced influx of L from serum into the follicle antrum causes an increase in the polypeptide level in FF. This might be due to a loss of selectivity in the blood-follicle barrier of early atretic follicles. The use of the L polypeptide as a biochemical marker for identifying the earlier stage of follicular atresia is proposed.