Although progesterone is the maturation inducer in amphibians, it has been demonstrated that in Bufo arenarum oocytes resumed meiosis with no need of exogenous hormonal stimulus if derived of their enveloping, follicle cells. This phenomenon, called spontaneous maturation, is quite rare in amphibians. In B. arenarum, spontaneous maturation took place only in oocytes obtained during the reproductive period (spring-summer). During this period the oocytes also demonstrated a respiratory activity characteristic of mature oocytes. Interestingly, full-grown B. arenarum oocytes always responded to progesterone regardless of the season in which they were obtained and of their respiratory activity. The disposition of oocytes competent or not competent to mature spontaneously provides a useful system for the study of molecular mechanisms involved in the maturation process. The data presented here indicate that the activation of protein kinase C (PKC) induces germinal vesicle breakdown (GVBD) in denuded oocytes unable to mature spontaneously (winter oocytes) and is involved in the in vitro spontaneous maturation of B. arenarum full-grown oocytes. The inhibition of PKC by 1-(5-isoquinolynyl-sulphonyl-2-methyl-piperazine (H-7) impeded spontaneous maturation in a dose-dependent manner, thus supporting the participation of the PKC pathway during this process. Interestingly phorbol 12-myristate-13-acetate (PMA)-induced GVBD is inhibited by the incubation of the oocytes in dibutyryl cAMP (dbcAMP), indicating that both pathways, PKC and protein kinase A (PKA), are related at a certain point. However, spontaneous GVBD is less sensitive than PMA-induced GVBD to dbcAMP. This fact would support the suggestion that in spontaneous GVBD mechanisms different from activation of PKC are at work.

The influence of unprotected freezing of mammalian spermatozoa on their oocyte activating capacity and chromosome integrity is unknown. However, this type of sperm treatment has been used in assisted reproduction by intracytoplasmic sperm injection in cattle and humans. The mouse oocyte injection test was used to analyse the influence of unprotected freezing of human spermatozoa on their reproductive characteristics. Mouse oocytes were microinjected with intact human spermatozoa or spermatozoa treated with two cycles of unprotected freeze-thawing. Oocytes surviving the injection were either cultured without further treatment or exposed to ethanol solution to induce parthenogenetic activation. Both injected and activated oocytes were used for sperm chromosome analysis. The results revealed a significant reduction in oocyte activating capacity and a tenfold increase in the incidence of structural chromosomal abnormalities in human spermatozoa treated by unprotected freezing. We conclude that unprotected freezing of human spermatozoa has a detrimental effect on their reproductive characteristics. Our data also provide a new perspective on the stability of mammalian spermatozoa to physical factors and demonstrate the importance of detailed analysis of the stability of sperm structures for successful development of new approaches in assisted reproduction.

In the mouse, mature oocytes injected with prespermatozoal cell nuclei remain unactivated. Additional stimulation is needed to trigger oocyte activation leading to embryo development. We compared various electrical stimulations, treatment with cycloheximide alone or in combination with electrical stimulation, and injection of sperm-borne oocyte-activating factor (oscillogen) in terms of their oocyte activation and embryo development rates. Of all the treatments tested, a single electrical pulse (1.0 kV / cm, 128 μs) was the simplest, yet very effective, in allowing the development of the oocytes injected with spermatid nuclei.

The tolerance of the oocyte plasma membrane (oolemma) to electrical pulses (TEP) was investigated for oocytes, zygotes, and embryos at the early and late 2-cell stage. The oocyte survival rate after two electrical pulses (1.4KV/cm for 640μs each) was used as an indicator of the TEP of the oolemma. Survival rate of mid-pronuclear zygotes (94.3%±2.3%) was significantly (p < 0.05) higher than that of recently ovulated (2.1%±1.9%) and in vivo aged (25.1%±2.6%) oocytes; survival rate of in vivo aged oocytes was also significantly higher than that of recently ovulated oocytes. Soon after fertilisation, the survival rate of the oocytes markedly increased, up to 94% at the mid-pronuclear stage. Survival ratedropped thereafter. These results suggest that the characteristics of the oocyte plasma membrane (oolemma) change after fertilisation.

The recently reported human pregnancies and births after fertilising oocytes with round spermatids recovered from the ejaculate of men with non-obstructive azoospermia have underscored the need for a more accurate evaluation of the nuclear and cytoplasmic maturation status of ejaculated germ cells. In this study we describe our first experience with a method combining the immunocytochemical visualisation of proacrosin with autosomal DNA fluorescence in situ hybridisation (FISH) to assess ejaculated germ cells from patients with a spermiogenesis defect. The proacrosin immunoreactivity, analysed with the use of the monoclonal antibody 4D4, has been detected in cells of round spermatid size presenting a haploid FISH figure as well as in larger cells whose ploidy corresponds to primary and secondary spermatocytes. These observations are in agreement with previously published results obtained, with the use of the same antibody, by immunocytochemical analysis of histological sections of testicular tissue. All the cells of round spermatid size possessing proacrosin immunoreactivity were found to be haploid by FISH. On the other hand, some of the haploid cells of round spermatid size did not possess proacrosin immunoreactivity. The structural pattern of proacrosin immunoreactivity was highly variable both in spermatids and in younger spermatogenic cells. These data show that cell size is the main criterion to be used for the identification of ejaculated round spermatids, whereas the presence of the developing acrosome represents only an auxiliary criterion. The scoring of acrosomal development in ejaculated spermatids may be useful as part of pre-treatment diagnosis before the inclusion of infertile couples in a spermatid conception programme.

This paper addresses the proposition, first advanced by Wilson (1925), that successful embryogenesis depends on an ordered series of events in oogenesis. It is at the completion of this varied set of intracellular changes that the oocyte finally acquires its full capacity to support fertilisation and development. Amongst the earliest nuclear events are those associated with chromosome pairing and meiotic recombination. During the growth phase cell volume increases 300-fold and the cytoplasm becomes the storage site for RNA and protein which will be mobilised during early development. Finally, a short phase of intracellular reprogramming, or maturation, completes the series of events during oogenesis that confer developmental competence upon the oocyte. Follicle cell support is an indispensable requirement for ordered oocyte development and provides the early germline cell with many of the essential nutrients and growth regulators required to ensure progression through the protracted growth phase (see contributions by Cecconi & Rosella and De Felici et al. this issue). Although different, the interactions between the full-grown oocyte and the antral follicle are no less crucial to the acquisition of competence than those involved in the earlier stages of oogenesis.

The presence of intra-ovarian nerves was reported more than a century ago and successive investigations have demonstrated that the mammalian ovary is innervated by both sympathetic and sensory fibres distributed to the different compartments of the gland, such as blood vessels, ovarian stroma and follicle wall. Despite the extensive ovarian innervation and the experimental evidence indicating that neuromediators can influence sex steroid production, the role of the nervous system in the control of ovarian activity is still largely unknown.

In each oestrous cycle only a limited number of follicles are selected for ovulation whereas the remaining majority undergo atresia. The earliest and most prominent feature of atresia is the death of granulosa cells. Recent biochemical evidence has demonstrated that granulosa cell death during follicular atresia in swine (Tilly et al., 1992), bovine (Jolly et al., 1994) and rodent (Tilly et al., 1991) ovaries occurs by apoptosis, a process whereby cells die in a controlled manner. A biochemical event considered to be characteristic of apoptotic cell death is the intranucleosomal cleavage of genomic DNA into fragments 180–200 bp in size, which separate into a distinctive ladder-like pattern on agarose gel electrophoresis. Detection of this pattern of oligonucleosomes in DNA provides a marker of apoptotic cell death.

In mammals the ability of an oocyte to become fertilised is the result of a complex process occurring within the ovarian follicle which depends on the stagespecific expression of oocyte genes and the presence of granulosa cells (for a review see Buccione et al., 1990a). The coordinated development of germinal and somatic components of the follicle is regulated by two principal systems of interaction, based on the presence of gap junctions and on the production of paracrine factors. Gap junctions link granulosa cells to each other and to the oocyte (Anderson & Albertini, 1976), and represent a major route for the transfer of small molecules involved in oocyte metabolism (for a review see Mangia et al., 1992) and regulation of the arrest and resumption of meiosis (for a review see Eppig, 1993). The production of paracrine factors by granulosa cells has been suggested by the findings that these cells express the production of the Steel locus, the Steel factor (SLF) or kit ligand (KL; Motro et al., 1991; Manova et al., 1993), and that this factor promotes oocyte growth in vitro when used at high concentrations (Packer et al., 1994). Since KL is too large to be transmitted through gap junctions, it must necessarily be released in the extracellular environment before binding to the c-kit receptor present on oocyte membrane (Manova et al., 1990; Horie et al., 1991).

Nerve growth factor (NGF) belongs to a family of related target-derived proteins required for the survival and development of discrete neuronal populations in the central and peripheral nervous systems (Levi-Montalcini, 1987; Snider, 1994). Although initial observations led to the conclusion that the biological actions of neurotrophins are restricted to the nervous system (Thoenen, 1991; Raffioni et al. 1993), new evidence suggests that they, and in particular NGF, can also affect non-neuronal cells.

In a Graafian follicle, granulosa cells are classified into two principal cell subpopulations: cumulus cells, which are closely associated with the oocyte to form the cumulus cell-oocyte complex (COC), and mural granulosa cells, which are organised as a stratified epithelium at the periphery of the follicle. Following the preovulatory gonadotropin surge, cumulus cells lose contact with mural granulosa cells and start to synthesise and secrete a large amount of hyaluronan (HA), a glycosaminoglycan with high molecular weight and large hydrodynamic domains (Salustri et al., 1992). Proteins derived from serum (Chen et al., 1992, 1994) and synthesised by cumulus cells (Camaioni et al., 1993, 1996) organise the strands of HA into an intercellular elastic network that traps the cumulus cells and the oocyte in a unit which can not be mechanically dissociated – a process also referred to as cumulus expansion. At ovulation, the expanded COC is released through the ruptured follicle wall and transferred to the oviduct. The matrix in the expanded COC facilitates its extrusion from the follicle and its capture by oviductal fimbria, and provides, together with the cumulus cells, a suitable microenvironment for sperm penetration and fertilisation (for references see Salustri et al., 1993).

Investigations of strains of mice defective in germ cell development have revealed the importance of oocytes for the initial stages of folliculogenesis (Pellas et al., 1991; Huang et al., 1993). Various aspects of follicular development are dependent upon and/or influenced by the presence of oocytes, including granulosa cell proliferation (Vanderhyden et al., 1990, 1992) and cumulus expansion (Buccione et al., 1990; Salustri et al., 1990; Vanderhyden et al., 1990; Vanderhyden, 1993). We are investigating the possibility that oocytes influence one of the primary functions of granulosa cells: steroidogenesis. In many species, granulosa cells removed from preovulatory follicles luteinise in vitro (Channing et al., 1982), presumably due to loss of contact with follicular luteinisation inhibitory factor(s). Indeed, follicular fluid can prevent granulosa cell luteinisation in vitro (Ledwitz-Rigby et al., 1977). Follicular fluid, however, may simply be the medium for transport of factors secreted by oocytes to regulate granulosa cell activities.

Each oestrous cycle the ovary produces one or more oocytes, depending on the species considered, which are fully competent to sustain the development of a new individual and are defined as matured. Ovulation is the terminal phase of a lengthy and complex selection process which enables only a minute proportion of the oocytes present in the ovary to be ovulated and possibly fertilised. The use of exogenous gonadotrophins for the pharmacological stimulation of the ovaries has clearly indicated that oocytes naturally excluded by the selection process can also be matured and, if fertilised in vivo or in vitro, can generate normal offspring.

The ovulatory follicle has the dual role of releasing a viable oocyte capable of fertilisation and the production of key endocrine signals that result in mating behaviour, the induction of the preovulatory LH surge and ovulation. Further, following ovulation the ovulatory follicle must be capable of forming a viable corpus luteum if pregnancy is to be maintained. Follicle growth is therefore a developmental process during which the follicle progressively acquires a number of properties, each of which is an essential prerequisite for further development. Failure to acquire these properties at the correct time and in an exact sequence will lead to failure of the developmental process and to the deterioration of the follicle through atresia and degeneration of the oocyte.

As meiosis is initiated and the oogonium is transformed into a primary oocyte, the female germ cell becomes intimately invested by a single squamous layer of sex cord epithelium. As the follicle cell population expands during the initial stages of the ovarian cycle, oocyte and follicle cells become increasingly connected to one another by one of the most extensive populations of gap junctions documented in any epithelium (reviewed in Larsen & Wert, 1988).

Much effort has been focused on establishing optimal conditions for obtaining in vitro maturation of oocytes from different species with results comparable to those achieved after in vivo development (reviewed by Brackett, 1992). However, even though extraordinary progress has been made, the in vitro technology for oocyte maturation lags far behind that in vivo and improvements are needed to increase the quantity and quality of the embryos produced from these matured oocytes.