Glycine is the most concentrated amino acid in the female genital tract. In this study, we report its conversion and incorporation into proteins in the presence or absence of methionine, in borth 1-cell and blastocyst mouse embryos. The uptake, incorporation and conversion of radiolabelled glycine were studied in the presence or absence of unlabelled methionine. For control purposes, the reciprocal experiment was performed with labelled methionine in the presence or absence of unlabelled glycine. At the 1-cell stage neither glycine uptake nor its incorporation into proteins is inhibited by methionine. Glycine is, however, highly used as an oxidisable energy substrate, via glycolate. At the blastocyst stage, glycine conversion into other amino acids is high and mainly utilised in the formation of glutamic acid. Glycine is highly incorporated into proteins, resulting in a poor exchange of glycine from the preloaded embryos. Methionine competes for glycine uptake and consequently reduces its overall incorporation into proteins. For methionine, neither its uptake nor its incorporation into proteins is reduced in the presence of glycine for the two embryonic stages tested here. The embryo has different mechanisms for incorporation and utilisation of methionine and glycine. Glycine, which has an important function in the embryo, has an inefficient transport system compared with methionine. We were unable to demonstrate the presence of methyiglycine since SAM-glycine-methyltransferase (EC 2.1.1.20) was not detected. The same results were obtained when exogenous methionine was added. We therefore concluded that glycine does not compete in transmethylation within the embryo.

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.

Dorsal–ventral patterning in the Xenopus egg becomes established midway through the first cell cycle during a 30° rotation of the subcortical yolk mass relative to the egg cortex. This rotation of symmetrisation is microtubule dependent, and its direction is thought to be cued by the usually eccentric sperm centrosome. The fact that parthenogenetically activated eggs also undergo a directed rotation, despite the absence of a sperm centrosome, suggests that an endogenous asymmetry in the unfertilised egg supports the directed polymerisation of microtubules in the vegetal cortex, in the way that an eccentric sperm centrosome would in fertilised eggs. Consistent with this idea, we noticed that the maturation spot is usually located an average of more than 15° from the geometric centre of the pigmented animal hemisphere. In parthenogenetically activated eggs, this eccentric maturation spot can be used to predict the direction of rotation. Although in most fertilised eggs the yolk mass rotates toward the sperm entry point (SEP) meridian, occasionally this relationship is perturbed significantly; in such eggs, the maturation spot is never on the same side of the egg as the SEP. In oocytes tilted 90° from upright during maturation in vitro, the maturation spot developed 15° or more from the centre of the pigmented hemisphere, always displaced towards the point on the equator that was up during maturation. This experimentally demonstrated lability is consistent with an off-axis oocyte orientation during oogenesis determining its eccentric maturation spot position, and, in turn, its endogenous rotational bias.

DNA methylation is one of the proposed biochemical mechanisms involved in cell differentiation and in genomic imprinting, and DNA methyltransferase (DMT) is a key enzyme in the embryo since mutation of its gene is lethal early in development. In order to verify that non-viability of uniparental embryos was not due to a defect in the regulation of DMT activity, we compared the metabolism of methylation in parthenogenetic embryos (maternal genome) and in fertilised embryos (maternal and paternal genomes). As regards total methylation, estimated by a measure of S-adenosyl methionine (SAM) and S-adenosyl homocysteine (SAH) formation, no significant difference was found between the two kinds of embryos during preimplantation development. Mean values were 4.5 ± 0.6 fmol (SAM +SAH)/h per 2-cell embryo and 0.40 ± 0.05 fmol SAH/h per 2-cell embryo, i.e. a SAH/(SAM + SAH) ratio of 9%; there was no detectable SAH formation in blastocysts. The same observation can be made for DMT activity, with mean values of: 7.8 fmol/h per oocyte, 8.5 fmol/h per 2-cell embryo, 6.1 fmol/h per 4-cell embryo, 4.1 fmol/h per morula, and no detectable activity in blastocysts. Total methylation as well as DNA methylation is characterised by a progressive drop in activity during preimplantation development.

Production of embryos that are free of tough outer coats facilitates studies that are not possible with embryos surrounded by impenetrable envelopes. This report describes a new procedure for preventing formation of fertilisation membranes in the sea urchin (Lytechinus pictus) model. This procedure involves treating unfertilised eggs with the enzyme alpha-amylase, which cleaves alpha-1,4 glucosidic bonds in the vitelline layer. A major advantage of this method is that it is very well defined and completely controllable with alpha-amylase inhibitor. The results suggest that intact alpha-1,4 glucosidic bonds are essential for vitelline layer integrity required for formation of the fertilisation membrane. Eggs treated with alpha-amylase possessed the same surface lectin receptors as untreated eggs and, as shown by light and transmission electron microscopy, produced healthy, cleaving embryos that were free of fertilisation envelopes.

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.

Xenopus ooctye cytoplasmic poly(A)+ RNA has been shown to include two major complex classes: mRNA and interspersed RNA. the former is defined by its translatalility, the latter consists of non–translatable repeat–containing transcripts with unknown functions. In this study we compared the accumulation patterns of total mRNA and a subfamily of interspersed RNA, the XR family (McGrew&Richter, 1989, Dev. Biot. 134, 267–70)

We previously showed in organ culture of testes fragments from Cynops pyrrhogaster that mammalian folicle-stimulating hormone (FSH) stimulates secondary spermatogonia to differentiate into primary spermatocytes. In this report, we demonstrate in organ culture that FSH stimulates DNA synthesis in secondary spermatogonia and Sertoli cells: the numbers of secondary spermatogonia and Sertoli cells incorporating 5-bromo-2′ -deoxyuridine (BrdU)throughout the culture period in the presence of FSH were 3-5 times those incorporating BrdU in the absence of FSH. Moreover, addition of FSH, induced after a day a remarkable increase in the number of spermatogonia and Sertoli cells incorporating BrdU. The above results indicate that FSH stimulates and induces DNA synthesis in spermatogonia and Sertoli cells. Most of the spermatogonia within a cyst were labelled simultaneously and at the same density, indicating that they underwent synchronous DNA synthesis, whereas all the Sertoli cells within a cyst were not labelled simultaneously, indicating that they synthesised DNA asynchronously. When testes fragments pulse-labelled with Brdu were cultured in FSH for 14 days, the secondary spermatogonia differentiated into primary spermatocytes, whereas in the absence of FSH they failed to differentiate and most died by the 7th day. The above results together show that FSH is required for the proliferation of both secondary spermatogonia and Sertoli cells as well as the differentiation of secondary spermatogonia into primary spermatocytes.

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 site of gamete interaction of electrophysiologically recorded Lytechinus variegatus eggs, fixed with osmium tetroxide (O5O4) and/or glutaraldehyde (GTA) at varying intervals after the onset of the increase in membrane conductance induced by an attached sperm, has been examined by high-voltage and conventional transmission electron microscopy. Although GTA and a GTA-O5O4 mixture iduced different electrical responses, specimens prepared with the two fixatives were ultrastructurally similar. In specimens observed within 5 s of the change in conductance, the acrosomal process projected through the vitelline layer and abutted the egg plasma membrane. A conspicuous layer of bindin surrounded the acrosomal process and connected the sperm to the egg's vitelline layer. In a fortuitous specimen fixed within 4 s following the change in conductance, the area of contact between the gamete plasma membranes possessed a trilaminar structure that separated the egg's and sperm's cytoplasms. The morphology of this area of contact was consistent with previously proposed intermediates of membrane fusion. Five to six seconds after the change in conductance, the sperm was connected to the egg via a narrow cytoplasmic bridge that consisted of the former acrosomal process and a projection of the egg cortex. The region of the bridge midway between the fused gametes was encircled by dense material that marked the site of sperm-egg fusion. Gamete interactions in which the activation potential was recorded (unclamped egg) were comparable in time and ultrastructure to events taking place in voltage-clamped eggs except for one major difference. Intact cortical granules (one to three) were observed beneath the tip of the incorporating sperm in unclamped eggs fixed following the onset of the activation potential, whereas all cortical granules dehisced in clamped eggs.

Using monoclonal anti-calmodulin antibodies in conjunction with confocal scanning laser microscopy we have analysed the spatial variations in the distribution pattern of calmodulin (CaM) during the sequential events of pollen hydration, germination and tube growth in Nicotiana tabacum. These immunocytochemical observations have been complemented by immunochemical studies wherein the anti-calmodulin antibody raised against pea CaM recognises a polypeptide of c. 18 kDa in the pollen extracts. Digitisation of confocally acquired optical sections of immunofluorescence images reveals that in hydrated pollen a high level of CaM is consistently present in the region of the germinal apertures. Subsequently, with the onset of germination a high CaM concentration was found associated with the plasma membrane of the germination bubble and in the cytoplasm in its vicinity, while in the vegetative cytoplasm a weak diffuse and intense punctate signal was registered. CaM immunostain was also detected in association with the plasma membrane of the tube tips in both short and long pollen tubes. Furthermore, the cytosol of the tubes invariably manifested an apically focused CaM gradient. We were, however, unable to detect any vacuolar association of CaM in the older regions of the pollen tubes. Although punctate immunostain was obvious across the pollen tube numerous punctate structures were invariably present in the extreme tip. The possible implications of these findings in development of cell polarity, polarised growth, maintenance of calcium homeostasis and CaM interactions with other mechanochemical motor proteins in effecting propulsion of organelles during pollen hydration, germination and pollen tube growth are discussed.

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.

A study was conducted to evaluate the ability of mammalian oocytes to develop to the pronuclear stage and beyond if injected with freeze-dried sperm nuclei. The rate of development of hamster eggs to the pronuclear stage after microinjection of freeze-dried hamster sperm nuclei was 90%. The pronuclear formation rate of hamster eggs injected with freeze-dried human sperm nuclei was 85%. On the other hand, the rates for eggs injected with untreated sperm nuclei were 84% and 89% respectively. The survival rate of rabbit eggs microinjected with freeze-dried rabbit sperm nuclei was 64%, the fertilisation rate 56% and the cleavage rate 38%. The survival, fertilisation and cleavage rates of eggs injected with non-freeze-dried sperm nuclei were 78%, 45% and 34%, respectively. There was no difference between eggs injected with freeze-dried sperm nuclei and those injected with sperm nuclei that had not been freeze-dried. Of the viable rabbit eggs injected with freeze-dried rabbit sperm nuclei, 6% developed to the 6- to 8-cell stage 62 h after injection.

To further our Knowledge on the mechanisms and molecules involved in mouse sperm–oocyte plasma membrane interaciton, exteraction, experiments were carried out to determine the stage during oogenesis at which an oocte acquires the capacity ot fuse with acrosome-reacted sperm. Zona-ferr oocytes 10 μm in diametes do not fuse with sperm. Oolemma fusibility is first acquired when the oocyte reaches about 20μm in diameter. Fusibility is maniatained even after fertilisation has accurred and is lost completely by the 4–cell stage.

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.

Isolated seminiferous tubules of rat testis contain considerable urokinase-inhibiting activity. An immunohistological analysis revealed the presence of plasminogen activator inhibitor type 1 (PAI-1) in the basement membrane as well as in the interior of the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the tubules. Distribution and intensity of the intratubular immunoreactivity depends on the stage of the seminiferous cycle. A relatively weak signal is present around elongated nuclei of spermatids at the beginning of chromatin condensation. The signal intensity increases in the course of differentiation until a maximum is reached at stages VII-VIII. In these stages PAI-1 immunoreactivity is localised around the nuclei of the late spermatids as well as along their tails. Spermatozoa in the ductus epididymis also strongly react with the PAI-1-specific antiserum, suggesting that the inhibitor remains associated with the germ cells after spermiation and during maturation in the epididymis. In intact mature spermatozoa isolated from epididymis cauda by ‘swimming-up’ in non-capacitation medium, PAI-1 antigen is localised on the plasma membrane surrounding the head. In addition, in fixed and permeabilised cells the immunoreactivity is detectable in the acrosome and in the tail. Possible functions of PAI-1 in spermatogenesis, sperm motility and sperm-egg interaction are discussed.

The purpose of this investigation was to determine the parental origin og the pronucleus furthest from the second polar body (the distal pronucleus) in dispermic human zygotes. Infact dispermic embryos (n = 53) and those from which the distal pronucles (n =50) was removed at the zygote stage were biopsied after cleavage. Blastomeres were sexed using either coamplification of X and Y probes using a duplex polymerase chain reaction (PCR), or simultaneous fluorescence in situ hybridisation (FISH) with directly fluorochrome-labelled probes for chromosomes X, Y and 18. The ratio X/Y was determined in both groups of embryos by assessing a minimum of two blastomeres. If the pronuclei in dispermic zygotes are topographcially in a fixed position, the X/Y ratio should change from 1:3 in dispermic embryos to 1:1 in enucleated ones. The ratio of embryos containing only an X chromosome and those with X as well as Y chromosomes in the intact dispermic zygotes was 1.0:2.3 which is similar to the theoretical ratio of 1:3. This ratio was 1.0:1.3 in dispermic zygotes from which the distal pronuclei were removed. This ratio is not significantly different from the 1:1 ratio based on a statistical analysis with a sample size of 50. These sex ratios would have been considered different if more than 200 enucleations had been performed. Although the ratio X/Y was altered following removal of distal pronuclei, suggesting frequent targeting of male pronuclei, accidental removal of the female pronucleus could not be excluded. This indicates that enucleation of dispermic zygotes could produce high yields of gynogenetic and androgenetic embryos for research purposes. Clinical application aimed at producing biparental zygotes may be hazardous, since mosaicism was common among enucleated embryos.