Tomoko Nakanishi

Green mice' as a source of ubiquitous green cells

The green fluorescent protein (GFP) is responsible for the green bioluminescence of the jellyfish Aequorea victoria. Many classes of GFP mutants exist that display modified fluorescence spectra and an increased extinction coefficient. We produced transgenic mouse lines with an ‘enhanced’ GFP (EGFP) cDNA under the control of a chicken beta‐actin promoter and cytomegalovirus enhancer. All of the tissues from these transgenic lines, with the exception of erythrocytes and hair, were green under excitation light. The fluorescent nature of the cells from these transgenic mouse lines would facilitate their use in many kinds of cell transplantation experiments.

Real‐time observation of acrosomal dispersal from mouse sperm using GFP as a marker protein

We produced transgenic mouse lines that accumulate mutated green fluorescent protein (EGFP) in sperm acrosome, a membrane limited organelle overlying the nucleus. The sperm showed normal fertilizing ability and the integrity of their acrosome was easily examined in a non‐invasive manner by tracing the GFP in individual ‘live’ sperm with fluorescent microscopy. The time required for the dispersal of acrosomal contents was demonstrated to be approximately 3 s after the onset of acrosome reaction.

Acrosin Accelerates the Dispersal of Sperm Acrosomal Proteins during Acrosome Reaction

Using homologous recombination, we have previously produced male mice carrying a disruptive mutation (Acr −/−) in the acrosin gene. AlthoughAcr −/− mouse sperm lacking the acrosin protease activity still penetrated the zona pellucida and fertilized the egg, the mutant sperm exhibited a delay in penetration of the zona pellucida solely at the early stages after insemination. To further elucidate the role of acrosin in fertilization, we have examined the involvement of acrosin in the acrosome reaction of sperm using theAcr −/− mutant mice. When the ability of sperm to adhere (attach) and bind to the zona pellucida of cumulus-free eggs was assessed in vitro, no significant difference was observed among Acr +/+,Acr +/−, and Acr −/−mouse sperm. Immunocytochemical analysis demonstrated that the release of several acrosomal proteins from the acrosome ofAcr −/− mouse sperm was significantly delayed during the calcium ionophore- and solubilized zona pellucida-induced acrosome reaction, despite normal membrane vesiculation. These data indicate that the delayed sperm penetration of the zona pellucida in the Acr −/− mouse results from the altered rate of protein dispersal from the acrosome and provide the first evidence that the major role of acrosin is to accelerate the dispersal of acrosomal components during acrosome reaction.

Comparison of gene expression in male and female mouse blastocysts revealed imprinting of the X-linked gene, Rhox5/Pem, at preimplantation stages.

Mammalian male preimplantation embryos develop more quickly than females . Using enhanced green fluorescent protein (EGFP)-tagged X chromosomes to identify the sex of the embryos, we compared gene expression patterns between male and female mouse blastocysts by DNA microarray. We detected nearly 600 genes with statistically significant sex-linked expression; most differed by 2-fold or less. Of 11 genes showing greater than 2.5-fold differences, four were expressed exclusively or nearly exclusively sex dependently. Two genes (Dby and Eif2s3y) were mapped to the Y chromosome and were expressed in male blastocysts. The remaining two (Rhox5/Pem and Xist) were mapped to the X chromosome and were predominantly expressed in female blastocysts. Moreover, Rhox5/Pem was expressed predominantly from the paternally inherited X chromosome, indicating sex differences in early epigenetic gene regulation.

Disruption of Mouse CD46 Causes an Accelerated Spontaneous Acrosome Reaction in Sperm

ABSTRACT Human membrane cofactor protein (MCP, CD46) is a ubiquitously expressed protein known to protect cells from complement attack. Interestingly, when we examined the expression of mouse CD46, which we recently cloned, the message was found only in testis and the protein was found on the inner acrosomal membrane of sperm. In order to elucidate the function of CD46, we produced mice carrying a null mutation in the CD46 gene by using homologous recombination. Despite the absence of CD46, the mice were healthy and both sexes were fertile. However, to our surprise, the fertilizing ability of males appeared to be facilitated by disruption of the CD46 gene, as the average number of pups born from CD46−/− males was significantly greater than that of wild-type males. It was also revealed that the incidence of the spontaneous acrosome reaction doubled in CD46−/− sperm compared to that in wild-type sperm. It was assumed that this increase caused the heightened fertilizing ability found in CD46−/− sperm. These data suggest that CD46 may have some role in regulating sperm acrosome reaction.

Identification and characterization of a haploid germ cell-specific nuclear protein kinase (Haspin) in spermatid nuclei and its effects on somatic cells.

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I–III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (forhaploid germ cell-specific nuclearprotein kinase). Western blot analysis showed that specific antibodies recognized a protein ofM r 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

1 Green Fluorescent Protein (GFP) as a Vital Marker in Mammals

Publisher Summary It is reported that the green fluorescent protein (GFP), derived from the jellyfish Aequoreu victoria, can serve as a useful marker of gene expression without substrate loading or any other pretreatment. GFP has opened the door for the use of intact cells and organisms as experimental systems. This chapter presents the application of GFP and its variants as vital markers in mammals both in vitro and in vivo . Various GFPs emitting blue (BFP) or yellow (YFP) light have been made commercially. It has been reported that the mutations affect not only the spectra but also other characteristics of GFP such as magnitude and solubility. The most striking and novel nature of GFP is that one can observe the expression in real time in a noninvasive manner. In this context, GFPs possess an exclusive advantage. By using GFP variants, several groups of scientists succeeded in expressing them and detecting fluorescence in mouse embryonic stem (ES) cells. Green-ES cells were easily traced during chimerization and embryogenesis. These results suggest that GFP can be a reporter for knock-in, gene trap, or negative selection in ES-cell-mediated transgenesis.

Mouse Sperm Lacking ADAM1b/ADAM2 Fertilin Can Fuse with the Egg Plasma Membrane and Effect Fertilization

Fertilin, a heterodimeric protein complex composed of α (ADAM1) and β (ADAM2) subunits on the sperm surface, is believed to mediate adhesion and fusion between the sperm and egg plasma membranes. Here we have shown that mutant male mice lacking ADAM1b are fertile and that the loss of ADAM1b results in no significant defect in sperm functions such as migration from the uterus into oviduct, binding to egg zona pellucida, and fusion with zona pellucida-free eggs. ADAM1b-deficient epididymal sperm showed a severe reduction of ADAM2 on the cell surface, despite the normal presence of ADAM2 in testicular germ cells. The appearance of ADAM1b and ADAM2 on the sperm surface depended on formation and abundance of ADAM1b/ADAM2 fertilin in testicular germ cells. These results suggest that mouse ADAM1b/ADAM2 fertilin may play a crucial role not in the sperm/egg fusion but in the appearance of these two ADAMs on the sperm surface.

Functional Roles of Mouse Sperm Hyaluronidases, HYAL5 and SPAM1, in Fertilization

Although sperm entry into the oocyte-cumulus complex and subsequent sperm penetration through the cumulus matrix to reach the oocyte zona pellucida are essential for mammalian fertilization, the molecular mechanism remains controversial. Previously, we have shown that mouse sperm lacking SPAM1 are capable of penetrating the cumulus matrix despite a delayed dispersal of cumulus cells. We also have identified another sperm hyaluronidase, HYAL5, as a candidate enzyme involved in sperm penetration through the cumulus. In the present study, we produced HYAL5-deficient mice to uncover the functional roles of HYAL5 and SPAM1 in fertilization. The HYAL5-deficient mice were fully fertile and yielded normal litter sizes. In vitro fertilization assays demonstrated that HYAL5-deficient epididymal sperm is functionally normal. We thus conclude that HYAL5 may be dispensable for fertilization. Comparative analysis among wild-type, HYAL5-deficient, and SPAM1-deficient epididymal sperm revealed that only SPAM1 is probably involved in sperm penetration through the cumulus matrix. Notably, the loss of SPAM1 resulted in a remarkably increased accumulation of sperm on the surface or outer edge of the cumulus. These data suggest that SPAM1 may function in sperm entry into the cumulus and sperm penetration through the cumulus matrix.

Selective Passage Through the Uterotubal Junction of Sperm from a Mixed Population Produced by Chimeras of Calmegin-Knockout and Wild-Type Male Mice

Abstract Loss of calmegin, a testis-specific putative chaperone protein of the endoplasmic reticulum, leads to male sterility because the sperm show defects in migration into the oviduct and do not bind to the zona pellucida. To clarify the mechanism of defective migration, XY ⟷ XY chimeras were produced by aggregating wild-type embryos with embryos of transgenic mice lacking functional calmegin genes and expressing enhanced green fluorescent protein (EGFP) in their acrosomes. Chimeric ejaculates contained wild-type, nonfluorescent sperm as well as sperm with EGFP-tagged acrosomes and the defective calmegin gene. Transgenic, wild-type, and chimeric males were mated to wild-type females; however, only wild-type sperm were ever found within the oviducts. Calmegin-knockout sperm, even when they were combined in chimeric ejaculates with wild-type sperm, remained outside of the uterotubal junction. These findings indicate that the presence of wild-type sperm cannot compensate for the inability of calmegin-knockout sperm to enter the oviduct and that successful ascent into the oviduct depends on the capabilities of individual sperm.