The present study was designed to examine the ability of bovine oocytes, after germinal vesicle breakdown (GVBD), to be activated by sperm penetration and the sequence of sperm nuclear transformation. Bovine oovytes cultured for 8 h in maturation medium (tissue culture medium TCM-199 containing 10% fetal calf serum) were inseminated in Brackett and Oilphant's medium supplemented with bovine serum albumin (10 mg/ml), caffeine (5mM) and heparin (10 μg/ml). When oocytes were transferrred to the maturation medium 8 h after insemination and additionally cultured for 5−40 h at 39°C in 5% CO2 in air, 71−76% of oocytes were penetrated and polyspermy (67–75%) was common. The proportions of penetrated oocytes that were activated significantly increased with the lapse of the additional culture time, reaching 88% and 87% by 25 and 40 h after additional culture, respectively. When compared with unpenetrated oocytes, signifcantly higher proportions of penetrated oocytes, reached metaphase II or beyond 15 and 25 h after additional culture. After penetration, sperm nuclei were transformed into metaphase chromosomes and then to telophase chromomes before the formation of male pronuclei. These results provide evidence that bovine oocytes acquire the ability to respond to sperm-mediated activation soon after GVBD.

Global activation of the embryonic genome occurs at the 4– to 8–cell stage in human embryos and is marked by continuation of early cleavage divisions in the presence of transcriptional inhibitors. Here we demonstrate, using recerse transcripase–polymerase chin reaction (Rt–PCR), the presence of transcripts for wo paternal Y chromosomal genes, ZFY and SRY in human preimplantation embryos. ZFY transcripts were detected as early as the pronucleate stage, 20–24 h post-insemination In vitro and at intermediate stages up to the blastocyst stage. SRY Transcripts were also detected at 2–cell to blastocyos observed in many mammalian species focuses attention on the role of events in six determination prior to gonad differentiation.

Although much information about such processes as cell cycle control, second messenger systems, protein kinases and steroid hormone action has been collected from studies of Xenopus oocyte maturation, we still have very little idea about how the steroid hormone, progesterone, signals the resumption of meiosis from the oocyte plasma membrane. In this review we re-examine the data on second messenger systems in Xenopus oocytes and discuss some of the unresolved questions about hormone signal transduction during maturation. We outline some reasons for the contradictions in the literature and offer some suggestions for avenues of future research.

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.

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)

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.

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.

In most mammals, oocytes initiate meiosis in late fetal life; by the time of birthe they have already entered the diplotene stage of prophase I of meiosis and becaome arrested thereafter at the dictyate state(Baker, 1972). At this stage they became surrounded by a few nonproliferating flat follicle cells forming a unit called the resting or primordial follicle.

Although many validated techniques for oocyte maturation in vitro have been developed and eggs with normal development competence can be produced by these methods, the mechnism triggering oocyte maturation in vivo is still poorly understood. Little doubt exists that meiosis is reinitiated in pre-ovlulatory follicles by peak levels of gonadotrophins and that follicle somatic cells are directly involved since no receptors for gonadotrophins have been found on the oolemma.

Recent Spectacular achievements have enabled the identification of key molecules responsible for mitotic cell cycle progression through the stages of G1, the gap before DNA replication; S, the phase of DNA synthesis; G2, the gap before chromosome segregation; and M, mitosis itself. The last stage has been most intensively studied, where MPE, maturation promotion factor, has been found responsible for the nuclear events associated with chromosomal segregation and the prodcution of two identical daughter cells (see Murray & Hunt, 1993).

Before fertilisation the egg is metabolically quiescent and its nucleus is arrested at metaphase of the second meiosis. After sperm-egg fusion, the arrested nucleus resumes meiosis, and then changes into the female pronucleus. Such a sequence of morphological and biochemical events is called “activation”. The inital responses of the egg to activation by the sperm include cortical granule exocytosis and resumption of meiosis.

The spermatoozoon is an excitable cell that responds to specific effectors by rapidly changing its behaviour. In species with external fertilisation, spermatozoa are stored in a quiescent state in the testis, but within seconds after spawning, dilution into water triggers several activation events such as increases in motility and respiration. In some species, these are followed by the acrosome reaction, an exocytotic process that allows the spermatozoon to penetrate the egg investments and activate the egg (Dale, 1983). The majority of information on sperm activation has come from the sea urchin; secondarily teleosts and starfish have proved to be useful models.

Successful liberation and apposition of gametes are essential components of fertility. Normal fertilisation depends upon the establishment of a pre-ovulatory sperm gradient in the female tract between the site of semen deposition and the site of fertilisation in the Fallopian tubes. As a consequence, sperm: egg ratios may be close to unity at the time of activation of most secondary oocytes under conditions of spontaneous mating. In the absence of a sufficient sperm gradient, newly ovulated eggs would be confronted by an excess of spermatozoa resulting in polyspermic fertilisation. Penetration of the vitellus by more than one spermatozoon is pathological in mammals (Beatty, 1957; Austin, 1963). Accordingly, systems that act to regulate sperm progression and competence before the time of ovulation assume a particular importance. During the 1950s, 1960s and 1970s, there was much controversy as to the rate of sperm transport into the Fallopian tubes. Because observations failed to focus on those spermatozoa that could fertilise eggs, the controversy was largely sterile. Nor were the disagreements well founded since some experiments employed artificial insemination whilst others used natural mating. These two quite distinct approaches to introducing a sperm suspension into the female tract could not reasonably form the basis of disagreements on the physiological events of cellular progression. More recent studies have been set in perspective by Overstreet (1983), Harper (1988), Yanagimachi (1988), Hunter (1988, 1991, 1995) and Drobnis & Overstreet (1992)After mating at the onset of oestrus, ram and bull spermatozoa require a minimum of 6–8 h to reach the Fallopian tubes in sufficient numbers to promote suc.cessful fertilisation (Hunter et al., 1980; Hunter & Wilmut, 1982). Spermatozoa displaced to the tubes in a small number of minutes are moribund or dead, not.

Enjaculated spermatozoa do not have the immediate capacity for ferilisation. Austin (1952) and Chang (1951) first realised that the sperm gain this capacity within the female genital tract. The physiogical changes that make the spermatozoon competent to fertilise were defined as ‘Capacitation’ (Austin, 1952).

Spermatozoa leaving the testis of normal animals are not ready to fertilise oocytes. They gain this ability while passing through the epididymis. It is interesting that spermatozoa of most animals take about 10 days to pass through the epididymis despite huge interspecies variations in the length of the epididymal tubule (e.g. some 30–50 m in large farm animals and about 1 m in the laboratory mouse) (Robaire & Hermo, 1988). The site where the spermatozoa begin to acquire fertilising capacity varies according to species, but it is generally the corpus epididymis or the proximal segment of the cauda epididymis where a large proportion of the spermatozoa become fertilisationcompetent. The distal segment of the cauda epididymis is the principal site for the storage of mature spermatozoa. Prolonged sojourn in the cauda epididymis (and vas deferens), however, could be detrimental to the spermatozoa.

Oocytes are metabolically repressed cells that, in most cases, require a signal from the fertilising spermatozoon to trigger activation. Physiological competence of the activated oocyte, a prerequisite for early embryogenesis, depends, therefore, not only on the quality of the oocyte, but also on the quality of the spermatozoon (Dale, 1983; Tosti & Dale, 1992). There are two current opinions as to how the spermatozoon triggers the oocyte into metabolic activity (Dale & DeFelice, 1990). The first hypothesis is that following gamete fusion the spermatozoon releases a soluble activating factor into the oocyte cytoplasm that is then amplified globally by second messengers in the oocyte. The second is that sperm binding to receptors spanning the oocyte plasma membrane is the signal that is transduced to the second messenger system of the oocyte (Swann, 1993; Foltz & Shilling, 1993).

While the first attempt to cool and preserve stallion semen was done 200 years ago (by Spallazani) and the first pregnancy achieved with frozen-thawes stallion semen almost 40 years ago (Barker & Gandier, 1957), frozen stallion semen has not been used widely, mainly because of its association with low fertility.