Nonhuman primates, in particular macaques, demonstrate marked similarities to humans in almost all aspects of their anatomy, endocrinology, and physiology. These similarities underlie the value of these primates for studies across a broad range of disciplines. In reproductive biology, nonhuman primates have been used as models to study the mechanisms and processes associated with fertility, infertility, pregnancy, and parturition. This review provides an overview of reproductive studies for which commonly used female macaque species such as rhesus (Macaca mulatta), long-tailed (M. fascicularis), and Japanese macaques (M. fuscata) are appropriate subjects, and a summary of the advantages and problems of using nonhuman primates in such research are described.

Microtubules and microfilaments are elements of the cytoskeleton that are involved in cell motility. Dynamic and proper organization of the cytoskeleton is crucial for completion of oocyte maturation and fertilization. When performing mammalian developmental and reproductive techniques, information concerning the dynamism of the cytoskeleton is necessary and indispensable. Although rodents are widely used for developmental and reproductive technology, there are numerous differences between the cytoskeletal organization of rodent gametogenesis/fertilization and that of primates, including humans. Herein, a review of cytoskeletal organization during oocyte maturation and fertilization involving the differences between rodents, primates and other mammalian species is presented. Furthermore, we also review the function of the centrosome as a microtubule organizing center (MTOC). Proper information on the kinetics of the cytoskeleton is crucial for further expansion of developmental biology.

In mice, basic reproductive technologies, such as oocyte/sperm collection, embryo production, micromanipulation, and embryo transfer, have been established. With these technologies, production of transgenic mice has become routine. The cynomolgus monkey, which is one of the laboratory animals closest to human beings, has been used to obtain vaccine approval and for medical research. Therefore, production of transgenic animals in the monkey is a very significant subject. We describe herein the current state of related studies in addition to current findings regarding reproductive technologies.

The common marmoset (Callithrix jacchus) is a non-endangered New World primate that is native to Brazil. Marmosets offer many advantages compared with other laboratory primates for studying reproductive biology: they are the only anthropoid primates that routinely ovulate multiple oocytes per ovarian cycle, have a short gestation period and reach sexual maturity at around 1 year of age. Moreover, it is possible to synchronize the ovarian cycle, and efficient protocols for superovulation have been developed over the last few decades. As this species is increasingly used in reproductive technology, basic technologies have been established to rival those available in Old World primates. In 2005, common marmoset embryonic stem (ES) cell lines were established and applied to several differentiation studies, which accelerated the development of regenerative therapies using human ES cells, and to the production of transgenic animals for human disease. With the recent development of induced pluripotent stem cells (iPS), non-human primate models using ES cells and iPS cells are needed for elucidation of the safety and efficacy of new technologies in regenerative medicine. In addition to their natural advantages as a model of humans, marmosets are also advantageous as experimental animals, and this should lead to a surge of interest among biological researchers.

Maternal transcripts are indispensable and play an essential role in regulation of the resumption of meiosis, nuclear reprogramming and subsequent development. In this study, we conducted interactive subtraction analysis to investigate the dynamic changes in the gene expression profiles of mouse oocytes after artificial activation. To accomplish this, we compared the cDNA libraries of freshly ovulated metaphase II (MII) oocytes and pronuclear-stage eggs at 8 h after activation, which were obtained by interactive subtraction. After constructing the cDNA libraries, we sequenced 635 subtracted clones of differentially expressed genes and the identities of 515 of these genes were determined by online BLAST analysis. The analysis revealed that genes down-regulated after activation were mainly responsible for enzyme and receptor activities, and also for DNA, chromatin, ATP, protein, and ion binding. On the other hand, genes up-regulated after activation were mainly responsible for enzyme activities, ion binding, and the maintenance of cell structure. For further insight into the functions of the genes, gene network analysis was performed, and in order to validate differential gene expression, the gene expression levels were determined by using real-time quantitative PCR. The present analysis was capable of identifying the genes that changed dynamically following the resumption of meiosis, and these genes may control the developmental programs in oocytes and preimplantation embryos.

In this study, to avoid the virus contamination in the maturation medium by addition of donor follicular fluid (FF) from other in vitro fertilization (IVF) cycles, we examined the outcome of in vitro maturation (IVM) protocol using medium containing patient's own serum. Data were retrospectively collected from 68 irregularly cycling women (31 ± 3.6 years) who were diagnosed as having polycystic ovarian syndrome. Immature oocytes were retrieved transvaginally 36 h after an injection of 10,000 IU hCG. Immature oocytes were matured for 24–26 h in medium supplemented with 10% (v/v) heat-inactivated patient's own serum (group S) or with 20% (v/v) heat-inactivated FF from donors who had undergone IVF cycles (group FF). All mature oocytes were inseminated by intracytoplasmic sperm injection. There were no statistical differences in the rates of maturation (49.2% vs. 45.6%), fertilization (85.0% vs. 84.2%), and pregnancy (16.0% vs. 15.4%) between group S and group FF. The supplementation of patient's own serum to the maturation medium as a substitute for follicular fluid collected from other patients did not affect the outcome of fertility treatment. Thus, we are able to eliminate the risk of transferring infectious diseases by using the patient's own serum.

During in vitro culture, embryo density in culture medium affects the developmental competence of the embryo. However, in many cases, it is necessary to culture human embryos individually or in small groups. A microwell provides a single embryo with a microenvironment suitable for its development (diameter, 500 μm; depth, 300 μm; and volume, 0.04 μl). In the present study, the clinical results of a microwell system were compared with those of a microdroplet system, and the clinical validity of the microwell system was considered for routine clinical use. In the microwell system, the percentage of embryos that reached the blastocyst stage and the subsequent clinical pregnancy rate (61.7% and 41.4%, respectively) were slightly higher than those in the microdroplet system (51.8% and 34.5%, respectively). Moreover, the microwell system was suitable for routine clinical use with respect to safety, convenience and commercial availability. In conclusion, the microwell system not only slightly enhanced the developmental competence of the individual embryos and the subsequent clinical pregnancy rate, but also made it possible to follow the course of development of an individual embryo. It may therefore be beneficial for conducting elective single embryo transfer to avoid the risks of multiple pregnancy.

In this study, we investigated the effect of adding glucose to in vitro maturation (IVM) medium on the nuclear maturation and ATP metabolism of cumulus cell-enclosed porcine oocytes (COs). When various concentrations of glucose (0.00, 2.78, 5.55, 11.10 and 16.65 mM) were added to IVM medium, the nuclear maturation rate up to the metaphase stage of the second meiotic division (M-II stage) in the 2.78 mM group (67.90%) was significantly higher than that of the non-glucose (0.00 mM, 31.66%) group (p<0.05). In addition, when cumulus cell-denuded oocytes (DOs) were matured in medium with or without 2.78 mM glucose, the rate of DOs maturing to the M-II stage with glucose (53.17%) was significantly higher (p<0.05) than that in the group in which glucose was not added (23.53%). However, these values for DOs were significantly lower (p<0.05) than those for COs (with glucose:71.85% and without glucose: 38.10%). Conversely, the ATP content of COs and DOs matured with glucose were almost the same as those matured without glucose. These results indicate that glucose may stimulate nuclear maturation without changing ATP metabolism.

To clarify the cytological characteristics of the porcine oocytes having meiotic competence, the oocytes retrieved from small (1–2 mm in diameter) and large follicles (3–6 mm) were compared. Cumulus-lacking oocytes were also examined to distinguish the characteristics between growing and atretic follicular oocytes. Distributions of cytoskeleton and mitochondria were observed by fluorescence staining in relation to the germinal vesicle (GV) stages, classified as GV 0 and GV I - GV IV. Oocyte size (107.2 ± 0.6 μm vs. 116.9 ± 0.7 μm, p<0.05) and maturation rate (9 vs. 87%, p<0.01) were lower in the small follicular oocytes than the large follicular oocytes. GV 0 oocytes were more frequently found in the small follicles than in the large follicles (67 vs. 9%, p<0.01). Most of the GV 0 oocytes from small follicles were likely forming the cytoplasmic microtubules and microfilaments. In contrast, most of the GV oocytes from large follicles showed an even distribution of fibrous microtubules in the ooplasm. In some of the small follicular oocytes beyond the GV II stage, fluorescence intensities of micro tub ules and mitochondria decreased. These features were similar to those observed in the cumulus-lacking oocytes. The results suggest that most of the GV 0 oocytes may be developing cytoplasmic cytoskeleton, and consequently are unable to complete meiosis. Also, advanced GV stage oocytes with low density of cytoskeleton and mitochondria, especially those from small follicles, may be derived from atretic follicles.

Survivin is a bi-functional protein which has been shown to suppress apoptosis and regulate cell division in mammalian carcinoma cell lines and some somatic cells. In previous studies, we showed that survivin is an essential anti-apoptotic gene which is expressed in mouse pre-implantation embryos, and found that embryos in which survivin has been disrupted develop only to the blastocyst stage. Based on what is known about survivin in mammals and other organisms, we proposed that maternal survivin mRNA may be required for oocyte maturation and early embryonic development, including a role in regulation of chromosome segregation. To test this, we assessed changes in survivin mRNA levels over time in mouse oocytes and embryos, and asked if survivin mRNA levels change in a cell cycle-dependent manner in mouse oocytes and embryos. We found that maternal survivin mRNA levels were higher than zygotically transc ribed surviv in mRNA levels in mouse preimplantation embryos, that zygotic survivin was regulated in a cell-cycle dependent manner, and that survivin mRNA levels were high in young mouse oocytes but decreased as mice aged. Our data suggest that survivin may play a role as a maternal gene in the development of mouse oocytes and pre-implantation embryos.

During ovulation, granulosa cells and cumulus cells synthesize and secrete a wide variety of factors via the exocytosis system. Exocytosis is controlled by the SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) complex consisting of proteins residing in the vesicle membrane and the plasma membrane. SNAP25 localizes on the plasma membrane whereas synaptotagmins (SYT) reside in secretion vesicle membranes. After Ca² -induced binding of SNAP25 and SYT, the release of vesicle-contained factors is triggered. Therefore, we sought to determine if induction of Syt mRNA and binding of SYT to SNAP25 in granulosa and cumulus cells were involved in exocytosis during ovulation. After hCG stimulation, the expression levels of Syt1, Syt2, Syt4 and Syt6 were induced markedly in granulosa cells, whereas the levels of Syt3 and Syt7 mRNAs did not change dramatically. The levels of SYT1 protein were also increased 4 hr after hCG injection and remained increased until 16 hr post-hCG. Immunofluorescence using an SYT1 specific antibody showed increased staining for SYT1 in the granulosa cells and in the cumulus cells of hCG-primed mice. Additionally, the SYT1 antibody coimmunoprecipitated SNAP25 in granulosa cell lysates collected at 8–16 hr post-hCG. From these results, we conclude that the interactions of SYT1 and SNAP25 facilitate the exocytosis system in granulosa cells and cumulus cells during ovulation.