Mao Wu Guo

Expression of Fas-Fas ligand in murine testis.

Xu JP, Li X, Mori E, Guo MW, Matsuda I, Takaichi H, Amano T, Mori T. Expression of Fas‐Fas ligand in murine testis. AJRI 1999; 42:381–388

Expression of Fas ligand in murine ovary.

Corresponding to the expression of Fas in the ovarian oocytes as previously reported (Guo et al., Biochem Biophys Res Commun 1994; 203:1438–1446; Mori et al., JSIR 1995; 9:49–50), the expression of Fas ligand (FasL) in the ovarian follicle was found to be restricted in the area of granulosa cells by the indirect immunofluorescence (IIF) test. Reverse transcriptase/polymerase chain reaction (RT/PCR) technique coupled with Southern blot hybridization analysis showed that the highest level of FasL mRNA was demonstrated in murine ovaries and granulosa cells 1 day after the administration of pregnant mares serum gonadotropin (PMSG), while the level of FasL mRNA became very weak on the day 5, respectively. The observed gradual decrease in FasL mRNA could not be attributed to a generalized degradation of cellular RNA during atresia, as evidenced by the presence of constitutive expression of elongation factor 1α (EF‐1α) mRNA in murine ovaries and granulosa cells treated with PMSG. Furthermore, in situ hybridization analysis with a FasL‐specific probe confirmed that FasL was specifically localized in the granulosa cells of most follicles and its expression was regulated by PMSG administration. FasL localized in granulosa cells might possibly play an important role in the formation of the ovarian atretic follicles, most likely depending on PMSG administration.

Expression of Fas-Fas Ligand System Associated with Atresia through Apoptosis in Murine Ovary

We examined the contribution of Fas and its ligand (FasL) in the process of follicular atresia using murine intraovarian follicles and pregnant mares serum gonadotropin (PMSG)-hyperovulated eggs. Reverse transcriptase/polymerase chain reaction-Southern blot hybridization demonstrated positive expression of Fas in both intraovarian oocytes and hyperovulated eggs. In contrast, expression of FasL was only detected in granulosa cells. These findings were histologically confirmed by in situ hybridization using Fas- and FasL-specific probes. A time-course study showed that Fas mRNA was positive in atretic follicles through day 0 and day 2 of PMSG stimulation and negative thereafter. Levels of FasL mRNA were the highest on day 1 and tapered off toward day 5 of PMSG stimulation. Levels of elongation factor 1 alpha mRNA, a constitutive element, were constantly maintained throughout the experimental period. Coculture of ovulated eggs, intact and zona-free, and granulosa cells demonstrated positive TUNEL staining only in zona-free eggs. These findings indicate that follicular atresia is caused by apoptosis, and the apoptosis associated with internucleosomal DNA fragmentation is directly regulated by the Fas/FasL system.

Molecular structure and function of CD4 on murine egg plasma membrane.

In the present study, the expression of the CD4 molecule on murine egg plasma membrane was confirmed by the indirect immunofluorescence (IIF) method. The full-length CD4 cDNA from murine eggs was synthesised by the reverse transcriptase-polymerase chain reaction (RT-PCR) method and its authenticity verified by Southern blot hybridisation using an end-labelled internal oligonucleotide. The results of DNA sequencing showed that the nucleotide sequence of the cDNA of CD4 from murine egg mRNA was identical to that of immune T cells. To demonstrate the direct interaction of CD4 from murine egg with murine sperm cells bearing MHC (major histocompatibility complex) class II molecule, we employed a baculovirus expression system to generate CD4 on the surface of Spodoptera frugiperda (Sf9) cells. Expression of CD4 on Sf9 cells infected with Autographa californica nuclear polyhedrosis virus (AcNPV)-CD4 was demonstrated by IIF and immunoblotting. The CD4-expressing Sf9 cells adhered to MHC class II-bearing sperm cells since the adhesion was specifically blocked by anti-CD4 monoclonal antibody (mAb) or anti-monomorphic region of MHC class II mAb. Taking our previous and present experimental results together, they strongly suggest that intercellular membrane adhesion between two gametes at the fusion step in fertilisation is mediated by the MHC class II molecule located on the posterior region of the sperm head and the CD4 molecule on egg plasma membrane.

Aberrant expression and dysfunction of Fas antigen in MRL/MpJ-lpr/lpr murine ovary

In lpr mice the insertion of an early transposable element (ETn) into intron 2 of the Fas gene, which mediates apoptosis, causes the development of massive lymphadenopathy, splenomegaly and autoimmune disease. In the present study we investigated the influence of this mutation on ovarian development of lpr mice. By means of in situ hybridisation, the expression of Fas mRNA was detected at the same levels in the ovarian cells of MRL/MpJ-lpr/lpr (MRL/lpr) mice as in those of MRL/MpJ-(+)/+ (MRL/+) mice. However, indirect immunofluorescence (IIF) staining with anti-Fas monoclonal antibody (mAb) on the membrane of follicle and egg of MRL/lpr mice was significantly weaker than that of MRL/+ mice. Furthermore, the expression level of Fas protein at the 45 kDa band from ovarian cell lysates of MRL/lpr mice was much lower than that of MRL/+ mice. The co-incubation of Spodoptera frugiperda (Sf9)-Fas lig- and (L) cells with eggs of MRL/+ mice resulted in apoptosis of eggs, as detected by the terminal deoxynucleotide transferase mediated dUPT-nick end labelled (TUNEL) method. In contrast the co-incubation of Sf9-FasL cells with eggs of MRL/lpr mice did not generate apoptosis in eggs. Following intraperitoneal administration of anti-Fas mAb into both types of mice, most oocytes, a proportion of granulosa cells in the ovary and hepatocytes in liver of MRL/+ mice were positively stained by the TUNEL method, corresponding to the appearance of DNA fragmented ladders by DNA fragmentation assay, while negative signals were obtained in those cells of MRL/lpr mice. As the mice aged, the ovarian size of MRL/lpr mice was found to be much larger than that of MRL/+ mice due to the increased number of ovarian follicles. Therefore, the ovarian adenopathy in MRL/lpr mice was strongly suggested to be caused by the dysfunction of Fas antigen in the ovary.

Molecular and immunological approaches to mammalian fertilization

By means of hybridoma technology, we obtained six hydriboma cell lines producing monoclonal antibody (mAb) to porcine zona pellucid (ZP), two of which recognizes the steric structure of common antigens between porcine ZP and humans. Furthermore, we have analyzed all or partial structures of N- and O-linked sugar chains of ZP glycprotein from porcine or murine oocytes. Then, we have clarified that beta-galactose and Le(X) residues on ZP played the binding roles to sperm cells in porcine and murine fertilization. We have also succeeded Sp38 cDNA cloning from cDNA library of porcine testis. We found that Sp38 protein bind to porcine ZP2 and expressed in murine and human sperm cells. Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg plasma membrane was clearly shown by indirect IIF and immunoprecipitation test. Furthermore, the transcriptional expression of CD4/p56(lck) in eggs was confirmed by RT-PCR method. In addition, the p56(lck) associated with CD4 underneath the plasma membrane of eggs was autophosphorylated after cross-linking of CD4 with anti CD4 mAb. The binding between eggs or Sf9-CD4 cells labeled with anti-CD4 mAb and sperm cells labeled with anti-monomorphic region of class II mAb was completely blocked. Considering these findings together with the fact that an interspecies heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we elucidated that one of species specific intercellular adhesions between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4/p56(lck) complex on the plasma membrane of egg.

Expression of CD4/p56lck Complex on Murine Egg Vitelline Membrane and Its Role in Fertilization

Corresponding to the presence of major histocompatibility complex (MHC) class II on murine sperm, CD4 on the murine egg vitelline (plasma) membrane was clearly shown by indirect immunofluorescence (IIF) test with anti CD4 monoclonal antibody (mAb). By means of immunoprecipitation, CD4 from murine eggs was found to have the same size as that of the authentic CD4 from T-cells. Furthermore, the full-length of CD4 cDNA from eggs was synthesized by RT/PCR-Southern blot hybridization. The sequence of CD4 cDNA from eggs was found to be identical with that of T cells. In addition, src-related tyrosine protein kinase (p56lck) was demonstrated to be associated with CD4 under neath the plasma membrane of eggs by IIF with anti-p56lck. The autophosphorylation of p56lck after cross-linking of CD4 with anti CD4 mAb has been shown in eggs as well as in T cells by immunocomplex kinase assay. The transcriptional expression of the signal transducing region of lck gene in eggs was further confirmed by RT-PCR method. The binding between eggs labeled with anti-CD4 mAb and sperm labeled with anti-monomorphic region of class II mAb was completely blocked. In addition, sperm cells adhered extensively to CD4-expressed Spodoptera frugiperda (Sf9) cells and the adhesion between both cells was specifically blocked by labeling Sf9-CD4 cells with anti-CD4 mAb and sperm cells with anti-class II mAb. Considering these findings together with the fact that an interspecies’ heterogeneity is present in CD4 amino acid sequence at the interactive site with class II, we strongly suggested that one of species’ specific intercellular adhesion between two gametes at the fusion step in fertilization is definitely mediated by class II located on the posterior region of sperm head and CD4 on the plasma membrane of egg associated with p56lck.