Cellular and molecular events during oocyte maturation in mammals: molecules of cumulus-oocyte complex matrix and signalling pathways regulating meiotic progression.

Mammalian oocytes acquire their intrinsic ability in a stepwise manner through ovarian folliculogenesis, ultimately reaching the competence to undergo complete oocyte maturation at the final stage of Graafian follicle development. The fully-grown oocyte is tightly surrounded by compact layers of specialized granulosa cells (cumulus cells) to form a cumulus-oocyte complex (COC). After a preovulatory gonadotrophin surge, the COCs rapidly organize a special muco-elastic extracellular matrix (ECM) consisting of large amounts of hyaluronan (HA) and HA binding matrix glycoproteins. Simultaneously, the oocytes undergo meiotic resumption and cytoplasmic modification and attain the fertilizable metaphase II (MII) stage. These cellular events that immediately occur in COCs in the ovulatory phase are strictly regulated by pituitary hormones, steroids, growth factors and so on. Knowledge of the efficient mechanisms and the downstream cascades of the key molecules controlling oocyte maturation may gradually lead to improvement of the present oocyte/ embryo culture systems and gamete biotechnology. Recent studies by our group imply that i) the interaction of HA-CD44 identified in the porcine COC matrix is likely to participate in gap junctional communication and meiotic progression, and that ii) phosphatidylinositol 3-kinase (P13-K) and Akt contribute to the progress of follicle stimulating hormone (FSH)-induced meiosis in mice. Furthermore, this review focuses on the current understanding of biosynthetic regulation, the presumptive role of COC matrix molecules and the signalling pathways for meiotic modulators, such as the protein kinase A (PKA) pathway, the P13-K/Akt pathway and the mitogen activated protein kinase (MAPK) pathway.

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen.

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.

Induction of pseudopregnancy in the mongolian gerbil (Meriones unguiculatus) by vaginal stimulation.

In rats, pseudopregnancy has been induced by mating with vasectomized males, by mechanical stimulation of the uterine cervix with a glass rod or vibrator, and by stimulation of the vagina with a tampon. On the other hand, no practical data are available in reports on the induction of pseudopregnancy in Mongolian gerbils. Pseudopregnancy of gerbils has been induced by mating with vasectomized males. But this method was uncertain because the incidence of pseudopregnancy was lower than that obtained in rats by other means. In the present study, two experiments were undertaken as follows. 1) Copulatory behavior of gerbils was observed for one hour to determine the most effective stimulation interval. 2) From the results of Experiment 1, female gerbils in estrus were mechanically stimulated to test the effectiveness of inducing pseudopregnancy by vaginal stimulation at various time intervals. The results of these experiments indicated that, although the frequency of copulatory behavior varied among individuals, on average the most effective method for inducing pseudopregnancy was stimulation of 5 min duration and at 20 or 30 min intervals. Because the incidence of pseudopregnancy induced by such mechanical stimulation (83.3%) was higher than that induced by mating with vasectomized males (30.0%), this method might be useful in inducing pseudopregnancy in Mongolian gerbils.

Studies on a chemically defined medium for in vitro culture of in vitro matured and fertilized porcine oocytes.

The present study evaluated the effects of various components in a chemically defined medium on the development of IVM/IVF porcine embryos. The investigated components included energy substrates (lactate, pyruvate or glucose, alone or in various combinations), amino acids (glutamine, glycine or alanine), PVP and HEPES buffer. The effects of each energy substrate were the same as the control. However, a mixture of lactate with either of the other energy substrates increased the development rate. Glutamine tended to decrease rate of the development more than other amino acids, and this inhibition was dose dependent. Both PVP and HEPES buffer did not affect development rate. However, more than 35 mM HEPES buffer induced fragmentation From the above results, a new culture medium was designed (supplemented with 0.276 mM glycine, 0.176 mM alanine, 15 mM HEPES buffer and 1% (wt/vol) PVP in BSA-free Whitten's medium with or without glucose). The new medium resulted in a higher embryo development rate (20.4 and 16.3%) than that obtained with the control medium (10.0%).

Study on the estrous cycle in the Mongolian gerbil (Meriones unguiculatus).

The paucity of information and the unagreed consensus about the estrous cycle of the Mongolian gerbil (Meriones unguiculatus) have rendered such studies rather difficult. The estrous cycle of the Mongolian gerbils in this report were divided into 5 stages: Stage I, proestrus; Stage II, estrus (scattering); Stage III, estrus (gathering); Stage IV, metestrus and Stage V, diestrus. The normal estrous cycle in the Mongolian gerbils was 4-6 days long. In our experimental conditions, 67.9% of virgin females had a 4-6 day cycle, whereas 26.4% had an unsettled cycle and 5.7% assumed pseudopregnancy. The changing pattern of the estrous cycle and copulatory behavior of the Mongolian gerbils after hormone (PMSG: pregnant mare serum gonadotropin, 10 IU; hCG: human chorionic gonadotropin, 10 IU) injection were examined. The change in the estrous stage after hormone injection could be roughly classified into two types. The vaginal smear of virgin females injected with PMSG at Stage I, II or III changed to Stage V the next day and to Stage I or Stage II after 48 hr, but in the case of Stage IV or V, the smear changed to Stage I after 48 hr. The females injected with PMSG at Stage I, II or III copulated at from 13:00 to 23:00, whereas others injected at Stage IV or V scarcely copulated at all between 13:00 and 23:00. Mating of these females with the male midnight was not observed.

Antigenic and genetic characterization of H1 influenza viruses isolated from feral ducks and swine in Japan.

The strains of H 1 N 4 influenza A virus isolated from feral ducks in Japan in 1977-78 were compared to swine-origin H 1 N 1 viruses antigenically and genetically. Homologous characteristics were found among the H 1 N 4 isolates from feral ducks in hemagglutination-inhibition (HI) tests, viral RNA patterns on polyacrylamide gel electrophoresis and oligonucleotide mapping. Although the hemagglutinins of duck-origin viruses employed in this study were identified as H 1, the viruses were distinguishable from A/New Jersey/8/76 (H 1 N 1), A/duck/Alberta/35/76 (H 1 N 1) and the virus isolated from swine in Japan in the cross HI test. Also, the viral RNA patterns of the duck- and swine-origin H 1 viruses were found to be quite different, indicating that genetic reassortment of HA genes between them is unlikely. After H 1 N 4 virus of duck-origin was intranasally inoculated into pigs, a brief period of virus recovery with no serological response was observed; whereas swine-origin H 1 N 1 virus produced seroconversion in the pigs inoculated.

The follow-up study of swine and Hong Kong influenza virus infection among Japanese hogs.

Pigs in Miyagi Prefecture, Japan were examined for swine (Hssw1N1) and Hong Kong (H3N2) influenza virus infection by serological tests. The results obtained revealed that a swine influenza was prevalent with relatively high positive ratios throughout that time, and that the Hong Kong influenza virus closely related to a recent human epidemic strain, A/Yamanashi/2/77, also persisted, corresponding to a human endemic. These epidemiological findings strongly suggested the possibility of direct transmission of Hong Kong virus from humans to pigs and vice versa.