Spindles were isolated from deposited eggs of the Mediterranean mealmoth, Ephestia Kuehniella. Their structure and development were studied using anti-tubulin immunofluorescence. The microtubules were labelled with three different monoclonal antibodies. These were directed against β-tubulin, tyrosinated α-tubulin and acetylated α-tubulin. Significant differences in the staining behaviour were not detected with the three antibodies. An unusual mode of spindle formation was observed during the first mitotic division after fusion of the pronuclei. Several of the ensuing embryonic divisions may show the same phenomenon. Prophase of these divisions was characterised by an irregular arrangement of microtubules in the nuclear area. The microtubule mass in the nuclear area increased concomitantly with chromosome condensation. Microtubular foci, comparable to the forming asters of canonical spindles, were not detected. The formation of an orderly pattern in the microtubule mass was signalled by the appearance of minispindles apparently developing around individual chromosomes. Several minispindles subsequently aligned and formed metaphase-like entities within the nuclear area. The metaphase-like entities, in turn, aligned with one another and gave rise to a conventional bipolar metaphase spindle with small asters. The further development of the spindle was conventional. The chromosomes migrated towards the spindle poles and finally daughter nuclei formed. The anaphase and telophase spindles possessed both a prominent array of interzone microtubules and asters. The events in prophase of early embryonic mitosis of E. kuehniella may represent a rare case of chromosomeinduced spindle formation.

After spermatozoa bind to and penetrate the extracellular matrix of the egg, the zona pellucida, they adhere to and fuse with the plasma membrane of the egg. Since sperm–egg fusion may involve membrane glycoproteins and/or carbohydrate binding proteins, we sought to test this hypothesis by challenging sperm–egg fusion in hamster and in mouse with added carbohydrates. In this study, a number of carbohydrate and glycoconjugates were examined for their ability to inhibit sperm–eggfusion. In the hamster, D(+)-glucosamine, D(+)-galactosamine, albumin-bovine-glucosamide and-galactosamide, fucoidan and dextran sulphate inhibited the fusion of spermatozoa with zona-free eggs. The same effects were seen in the mouse, except for the toxic effects of D(+)-galactosamine. These facts suggest a role of carbohydrate binding proteins or glycoproteins in the fertilisation process at the level of binding to and fusing with the oolemma.

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.

As the amphibian oocyte becomes the fertilisation-competent egg an actin-myosin assembles in the cortex which provides for the cortical contraction that accompanies the fertilisation. A number of recent investigations provide data for development of a model detailing the structural changes which should accompany the development of this contractile network as well as the signalling mechanisms which regular assembly and contraction.

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.

Moderate heat treatment increases the penetrability of zonae pellucidae of salt-stored mammalian oocytes by spermatozoa

Ryuzo Yanagimachi, Haruo Katayose, Gary Killian, Chin N. Lee, Douglas T. Carrell and Thomas T. F. Huang

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.

The nuclei of spermatozoa leaving the testies are transcritionally inactivated and are structurally stabilised by extensive association of sperm DNA with protamines, highly basic proteins that replace somatic histones during spermiogenesis. During sperm matureation within teh epididymis, protamine–SH is oxidised to -SS-(Bedford & Calvin, 1974).