From antiquity humans have tried to understand how a new individual is generated after sexual intercourse. A popular notion was that a woman's blood and a man's semen were involved. Aristotle, based on studies of chick embryology, proposed that the mixture produced an egg from which the foetus developed in the uterus. This idea lasted for nearly two thousand years. William Harvey disproved the old notion by finding an empty uterus in a variety of animals soon after mating. Although he did not find any eggs he insightfully proposed “ex ovo omnia”, implying that all creatures arise from eggs. This lead to an intensive search for the source of eggs in mammals and resulted in the discovery of the role of the ovaries and fallopian tubes. With the introduction of the microscope in the 17th century sperm were discovered in the semen. Studies involving impregnation of amphibian eggs revealed that single sperm could produce a foetus. Despite this information the mammalian egg remained undiscovered for another 200 years. Finally, Karl Ernst von Baer discovered the ovum by microscopic examination of the ovarian follicle contents of the dog. Studies on fertilization and embryo development in mammals were initiated. With the advent of the cell theory it was recognized that the ovum was a cell, its nucleus was discovered, meiosis was described and the role of the chromosomes in heredity was revealed. Egg and embryo transfer in animals developed quickly and was facilitated by the introduction of tissue culture. The establishment of egg and embryo culture techniques encouraged John Rock and Miriam Menkin to attempt the first studies on human in vitro fertilization and embryo culture. Subsequent refinement of chemically defined culture media set the stage for successful in vitro fertilization, embryo culture and transfer for treating infertility.

Following the maturation of follicular cells, the zona pellucida, the oocyte nucleus and cytoplasm, oocytes are ready for fertilization and further embryonic development. The mechanism of nuclear maturation has been investigated previously, however, cytoplasmic maturation has not been described clearly. From our ultrastructural observation, we defined cytoplasmic maturation as development of smooth endoplasmic reticulum, aggregation and development of mitochondria, and extension and ramification of microvilli on the surface of human oocytes. Furthermore, metaphase I oocytes, which show no specific features under light microscopy, have been found to undergo important changes in the whole maturation process. In metaphase I oocytes, organella such as mitochondria and smooth endoplasmic reticulum develop well and the communication between follicular cells is initiated. Thus, ultrastructural study may contribute to the elucidation of the complicated maturation process of human oocytes during in vitro maturation (IVM).

Within growing follicles oocytes gradually acquire developmental competence to further support early embryonic development. This process is intrinsically linked to folliculogenesis. Thus, the fate of the germ cell depends on the health of the developing follicle, e.g. its vascularization, oxygen content and cumulus cell characteristics. Consequently, gametes of varying quality are harvested after follicular puncture. This review focuses on extracytoplasmic anomalies that should clearly be distinguished from intracytoplasmic ones. In detail, all relevant dysmorphisms related to the zona pellucida and perivitelline space will be discussed. Interestingly, some correlation can be found between extracytoplasmic dysmorphisms and fertilization and preimplantation development. Since genetic constitution is not affected by anomalies of the outer shell, it is postulated that it occurs in the late stages of maturation.

In human oocytes, many types of abnormal phenotypes have been observed both within the cytoplasm and outside of the cytoplasm. The morphological evaluation of oocytes and its impact on embryo quality has been controversial. However, abnormal oocyte phenotypes can be directly influenced by the follicular environment, ovarian function and the effects of ovarian stimulation used in assisted reproductive technology. This review focuses on four critical intracytoplasmic anomalies; fluid-filled vacuoles, smooth endoplasmic reticulum clusters (sERC), refractile bodies / lipofuscin bodies and centrally located cytoplasmic granularity (CLCG). The fine structures, morphological and immunochemical characteristics and the possible mechanism giving rise to each phenotype are discussed.

Scanning electrochemical microscopy (SECM) is a non-invasive and sensitive technique for measuring cellular respiration. In this paper, we review the SECM technique, to establish it as an accurate method for measuring the respiratory activity of single cumulus-oocyte complexes (COCs) and oocytes in animals as well as in humans. Oxygen consumption rates of COCs are influenced by the surrounding cumulus volume and the mitochondrial activity of the cumulus cells. An increase in the oxygen consumption rate was found in bovine oocytes, whereas the oxygen consumption of human oocytes tends to decrease during in vitro maturation (IVM). To analyze the metabolic activity of mitochondrial respiration, ATP content and mitochondrial distribution in bovine oocytes have been examined. An electron microscopic study confirmed mitochondrial reorganization in bovine oocytes during oocyte maturation. These results show that the respiratory activity of oocytes changes with maturation status during IVM and mitochondrial reorganization may partly influence respiratory activity. The SECM procedure is therefore a useful technique for evaluating the metabolic activity and quality of oocytes and cumulus cells in the IVM process.

Bovine in vitro fertilized (IVF) embryos were cultured in either CR1aa (CR1) or TCM-199 (TCM) medium and compared for their daily development based on the number of cleavage divisions (CDN), as calculated from the total cell numbers of the embryos. Embryos with advanced developmental stage and higher morphological quality were selected for use in the experiment. The relation between the CDN and the number of embryonic days after IVF in both groups showed a linear correlation; no significant difference (P > 0.05) was found between the two groups. However, CDN on days 3 and 8 after IVF in the CR1 group did not increase, which suggests that transient developmental arrests occurred at these stages. In contrast, embryos in the TCM group showed a transient evelopmental arrest 3 d after IVF, but CDN increased regularly with age in days at later stages of culture with 100 µM βmercaptoethanol. A significant difference was found between the regression lines of the two groups during 5–9 d after IVF (P < 0.001). Consequently, numerical analysis of embryonic development in terms of CDN enabled objective evaluation of the developmental progress of bovine IVF embryos.

The distribution of lysosomes during porcine oocyte maturation and fertilization in vitro was revealed by using acridine orange staining. The lysosomal distribution was classified into three types: generally even distribution throughout the ooplasm (Type I), less distribution in the peripheral ooplasm (Type II), and less distribution in the peripheral and inner ooplasm (Type III). All oocytes examined at 0 and 8 h after maturation culture showed the Type I lysosome distribution and 97% were at the germinal vesicle (GV) stage. When cultured for 22 h, the relative abundance of Type I oocytes decreased to 58%, while Type II and Type III oocytes appeared at relative abundances of 35 and 8%, respectively. After 32 and 44 h of culture, 35 and 80% of the oocytes, respectively, were Type III. When cultured with olomoucine, IBMX or dbcAMP for 22 h, 100, 79 and 94% of the oocytes, respectively, showed the Type I distribution of lysosomes, and their nuclei were almost all at the GV stage (100, 93 and 100%). The results of the present study suggest that there may be a close relationship between nuclear maturation and the distribution of lysosomes in the cytoplasm, and that the distribution of lysosomes may be one of the criteria of cytoplasmic maturation of porcine oocytes.

The aim of this study was to confirm the effect of dithiothreitol (DTT)-treated spermatozoa on oocyte activation following intracytoplasmic sperm injection (ICSI). Boar spermatozoa with or without DTT treatment (5 mM, 30 min) were injected into in vitro matured porcine oocytes, and the nuclear phase in presumptive zygotes was observed at 3 h intervals up to 12 h after ICSI. Furthermore, developmental competence of embryos produced by DTT-treated or non-treated spermatozoa was monitored after cultivation in vitro for 144 h. Male and female pronuclear formation rates in the oocytes injected with DTT-treated spermatozoa were significantly (P < 0.05) higher than those in the oocytes injected with non-treated spermatozoa. Additionally, we observed that female pronuclear formation was linked to male pronuclear formation. Sperm treatment with DTT improved (P < 0.05) subsequent development up to the blastocyst stage. These findings confirm the efficiency of DTT in in vitro porcine embryo production mediated by ICSI. We conclude that DTT treatment improves the formation of not only male pronuclei but also female pronuclei in porcine ICSI.

The effect of cryopreservation and subsequent xenotransplantation on the follicular reserve of the canine ovary by using non obese diabetic-severe combined immunodeficient (NOD-SCID) mice was examined. Vitrified-warmed canine ovarian tissues were placed into the ovarian bursa of mice, and then were removed and subjected to histological examination at 4 weeks after the transplantation. Over 30% of primordial follicles and 65% of early primary follicles survived after cryopreservation. However, regardless of breed or age, percentages of survived primordial follicles and early primary follicles after the transplantation ranged from 0–7% and from 0–15%, respectively. These results indicate that the majority of primordial follicles and early primary follicles in vitrified-warmed canine ovarian tissues disappear after xenotransplantation. Further studies will be required to be able to enhance the survival of transplanted cryopreserved ovarian follicles in canine.