Expression of recombinant mouse sperm protein sp56 and assessment of its potential for use as an antigen in an immunocontraceptive vaccine

Recombinant mouse sp56 protein was produced for testing as an antigen in an immunocontraceptive vaccine. The coding sequence for the mature sp56 protein was cloned into the bacterial expression system pFLAG using a PCR‐based method on mouse testis cDNA. Polyclonal antisera were raised in mice against affinity purified recombinant sp56 fusion protein (sp56FLAG) or an artificial sp56 peptide fused to a carrier protein (KLH) and shown to cross‐react to a protein band of 75 kD in detergent extracts of mouse sperm by Western immunoblot analysis under reducing conditions. The antisera to sp56FLAG also immunolocalized over the entire acrosome of mouse sperm. Female BALB/c mice were immunized intraperitoneally with sp56FLAG in a fertility trial with 20 μg sp56FLAG in Freund's Complete Adjuvant and boosted three to five times with 20 μg sp56FLAG in Freund's Incomplete Adjuvant. Litter sizes of sp56FLAG‐treated mice were significantly smaller than control‐treated animals after five boosts. Mol. Reprod. Dev. 52:216–224, 1999. © 1999 Wiley‐Liss, Inc.     

Molecular cloning and characterization of avian N-methyl-d-aspartate receptor type1 (NMDA-R1) gene

Birds have several advantages in the study of memory formation, as imprinting and passive avoidance behaviors in chick are often used as model systems. However, the primary structure of the bird N-methyl-d-aspartate (NMDA) responsive glutamate receptor, which is assumed to play a critical role in memory formation, has not been determined. In this report we describe the cDNA cloning of a subunit of NMDA receptors (NMDA-R1) from duck and analysis of its structure and distribution in the brain. The N-terminal 898 amino acids of the NMDA-R1 were well conserved between duck and mammals, but the homology was completely lost in the C-terminus. In situ hybridization showed that the duck NMDA-R1 gene was expressed throughout the brain as it is in mammals.Special issue dedicated to Dr. Bernard W. Agranoff.     

Molecular cloning and gene expression of Foxl2 in the Nile tilapia, Oreochromis niloticus

A Foxl2 cDNA was cloned from the Nile tilapia ovary by RT-PCR and subsequent RACE. Alignment of known Foxl2 sequences from vertebrates confirmed the conservation of the Foxl2 open reading frame and protein sequences, especially the forkhead domain and C-terminal region, while some homopolymeric runs of amino acids are found only in mammals but not in non-mammalian vertebrates. RT-PCR revealed that Foxl2 is expressed in the tilapia brain (B), pituitary (P), gill, and gonads (G), with the highest level of expression in the ovary, reflecting the involvement of Foxl2 in B-P-G axis. Northern blotting and in situ hybridization also revealed an evident sexual dimorphic expression pattern in the gonads. Foxl2 mRNA was mainly detected in the granulosa cells surrounding the oocytes. The ovarian expression of Foxl2 in tilapia begins early during the differentiation of the gonads and persists until adulthood, implying the involvement of Foxl2 in fish gonad differentiation and the maintenance of ovarian function.     

Cloning and sequencing of cDNA encoding for a human sperm antigen involved in fertilization

A cDNA encoding for a sperm antigen, designated NZ-2, was cloned and sequenced from human testis cDNA-λgt11 expression library by using antibodies to human sperm surface antigens belonging to 14–18 kD molecular regions. These sperm antigens are involved in binding to zona pellucida of the human oocyte. Computer generated translation analysis of 963-bp cDNA yielded an open reading frame (ORF) of 163 amino acids (aa) with first ATG, Met start codon at nucleotide (nt) 335 and the stop codon TAA at nt 824. The NZ-2 cDNA has 335-bp 5′ and 139-bp 3′ noncoding regions. The translated protein has a calculated molecular weight of ∼19 kD, and has two casein kinase II (CK-2) sites at aa 94–97 and 149–152, respectively. Extensive computer search in the GenBank, National Biomedical Research Foundation (NBRF), and Swiss database indicates it to be a novel protein, having 99.5% nt sequence similarity, except for the first 40-bp, only with the human bacterial artificial chromosome (BAC) containing cloned human sperm DNA, at position 76935–76009. The in vitro translated product of T3 RNA polymerase by using NZ-2 cDNA digested with XhoI yielded a protein band of ∼20 kD, indicating it to be sense strand. The in vitro translated product of T7 RNA polymerase by using NZ-2 cDNA digested with NotI did not yield any protein band, indicating it to be antisense strand. The ∼20 kD protein was recognized specifically by the antisperm IgG, not by the control IgG in the Western blot procedure. Neither antisperm IgG nor control IgG recognized any protein band in the in vitro translation products of the antisense strand. The human genomic DNAs from three different cells/tissues namely, sperm, kidney, and testis when cut by HindIII, and then hybridized with the NZ-2 cDNA probe in the Southern blot procedure, showed restriction fragment length polymorphism (RFLP). The recombinant human sperm NZ-2 antigen may find applications in the development of a contraceptive vaccine, and diagnosis and treatment of infertility in humans. Mol. Reprod. Dev. 51:176–183, 1998. © 1998 Wiley-Liss, Inc.     

Post-meiotic expression of the mouse dihydropyrimidinase-related protein 3 (DRP-3) gene during spermiogenesis

The dihydropyrimidinase-related protein (DRP) family, originally identified in humans by their homology to dihydropyrimidinase, contains at least four members. Genes of this family, and their counterparts in other mammals and chickens, are expressed mainly in fetal and neonatal brain, suggesting that the encoded proteins have a physiological role in the development of the central nervous system. In addition, the DRP-3 gene is expressed in testis as a shorter mRNA than the brain form. As a first step in understanding the extra-neuronal function of DRP-3, the structure and expression of testis DRP-3 were examined. Testis DRP-3 cDNA showed the same sequence as brain DRP-3 cDNA, except for the 5′-terminal end, which encodes a 5′-untranslated region and the 11 N-terminal amino acid residues, indicating that the two forms of DRP-3 mRNA were transcribed from a single copy gene. Northern blotting analysis detected DRP-3 mRNA in 30-, 40- and 70-day-old, but not in 10- and 20-day-old testes. In situ hybridization analysis indicated that the expression of DRP-3 in testis is restricted to post-meiotic round spermatids. This is the first report of the expression of DRP genes in extra-neuronal cells. Mol. Reprod. Dev. 51:105–111, 1998. © 1998 Wiley-Liss, Inc.     

Acrocentric centromere organization within the chromocenter of the human sperm nucleus.

It has recently been reported that in human sperm cells, the centromeres are clustered in a chromocenter in the interior region of the nucleus. The aim of the present study was to determine the intra-chromocenter organization of the five centromeres of the acrocentric chromosomes responsible for the biosynthesis of rRNA. The acrocentric centromeres were labeled by fluorescence in situ hybridization (FISH) after mild decondensation of the sperm nuclei to preserve the tail structure. The tail was used as a topographical marker for the orientation of the nucleus. The following results were obtained: (a) the association among the five centromeres was higher than expected from random distribution; (b) all the centromeres observed were randomly located within the chromocenter, occupying about 87% of the total area of the internal nucleus; (c) a major subpopulation of centromeres was located in a preferred area occupying 8.3% of the total nuclear area, with a peak 0.6 microm on the lateral axis and 1.0 microm on the apical side of the longitudinal axis; and (d) The dispersion of the centromeres was not influenced by the degree of the nuclear decondensation. We conclude that in human sperm nuclei, the acrocentric centromeres are organized within a nonlocalized structural element in the chromocenter. The chromocenter can range from an expanded size of 87% of the whole nucleus to a preferred size of 8.3% independent of the degree of nuclear decondensation. These findings have important implications for nuclear function (rRNA) that is not directly related to sperm cell function or early embryo development.     

The murine Spam1 gene: RNA expression pattern and lower steady-state levels associated with the Rb(6.16) translocation

Recently we mapped the murine Spam1 gene to the proximal region of chromosome 6 (MMU 6). Based on the map location and physiological characteristics of its encoded sperm antigen, the gene is an attractive candidate for the sperm dysfunction seen in Rb(6.16) translocation heterozygotes and the reduced fertility of homozygotes. We have analyzed the expression of Spam1 mRNA in normal and Rb(6.16) mice. The expression of Spam1 mRNA was found to be: 1) tissue specific; it is expressed exclusively in testis; and 2) developmentally regulated, with a haploid expression. Notably, the steady-state mRNA level of Spam1 in Rb(6.16) homozygotes was 25–30% of that in chromosomally normal consomic mice and those homozygous for Rb(2.8) (7.18). In Rb(6.16) and Rb(6.15) heterozygotes the levels were 61% and 66% of the normal. Studies are currently under way to determine the protein levels and gene structure of Spam1, to detect the underlying cause of the mRNA reduction associated with these translocations. Mol. Reprod. Dev. 46:252–257, 1997. © 1997 Wiley-Liss, Inc.     

Identification and characterization of a novel cancer/testis antigen gene CAGE

We applied serological analysis of cDNA expression library technique to identify cancer-associated genes. We screened cDNA expression libraries of human testis and gastric cancer cell lines with sera of patients with gastric cancers. We identified a gene whose expression is testis-specific among normal tissues. We cloned and characterized this novel gene. It contains D-E-A-D box domain and encodes a putative protein of 630 amino acids with possible helicase activity. It showed wide expression in various cancer tissues and cancer cell lines. The corresponding gene was named cancer-associated gene (CAGE). PCR of human x hamster Radiation Hybrids showed localization of CAGE on the human chromosome Xp22. Transient transfection of CAGE showed predominantly nuclear localization. Both Western blot and plaque assay indicated seroreactivity of CAGE protein. We found that demethylation played a role in the activation of CAGE in some cancer cell lines that do not express it. Cell synchronization experiments showed that the expression of CAGE was related with cell cycle. This suggests that CAGE might play a role in cellular proliferation. Because CAGE is expressed in a variety of cancers but not in normal tissues except testis, this gene can be a target of antitumor immunotherapy.