Golgi matrix protein gene, Golga3/Mea2, rearranged and re-expressed in pachytene spermatocytes restores spermatogenesis in the mouse

In a transgenic mouse, Golga3/Mea2 gene (human homolog: GOLGA3/golgin-160) was disrupted by a translocation at the site of the transgene integration. Exons 8-24 of the disrupted gene remained intact and formed a fusion gene (DeltaMea2) with the antisense strand of E. coli-derived transgene by means of a cryptic splice signal in there. The protein product of DeltaMea2, virtually a form truncated to 2/3 of the normal size, localized to Golgi apparatus of pachytene spermatocytes and round spermatids. DeltaMea2 expression was specific to the testis, but varied among separate seminiferous tubules. It also showed variation among homozygous individuals from 0.5 to 4.3% of the wild type (wt) level. At the lowest levels, neither spermatids nor spermatozoa were present in the homozygous testes, but when the expression of DeltaMea2 increased to 4.3% of the wt level, high sperm production was restored and a sporadic (1/22) fertile homozygous male was obtained. The earliest apoptotic degeneration of pachytene spermatocytes evidenced at 17 dpp in homozygous testes in some discrete seminiferous tubules was preceded by DeltaMea2 expression in a variegated fashion at 16 dpp. These results consistently indicated that in homozygous testes, the pachytene spermatocytes which failed to express DeltaMea2 may undergo apoptotic degeneration. Golga3/Mea2, and DeltaMea2 in homozygotes, in a certain excessive amount may be important for survival of pachytene spermatocytes in the mouse.     

Regulation of acrosome formation in mice expressing green fluorescent protein as a marker

Transgenic mice expressing enhanced green fluorescent protein under acrosin promoter were used to study the role of the Golgi complex and of the cytoskeleton during early development of the acrosomic system in exactly defined stages of the seminiferous epithelial cycle during in vitro differentiation. First acrosin expression was found uniformly in the cytoplasm of stage IV pachytene spermatocytes. The steady-state level increased up to stage X pachytene spermatocytes, and in diakinetic primary spermatocytes, acrosin started to accumulate into the Golgi complex. During step 2 of spermiogenesis, several small fluorescent proacrosomic granules were seen in various parts of the Golgi complex, and they fused to a solid acrosomic system at step 3. In cultured stage I-III seminiferous tubule segments, nocodazole slowed down acrosin incorporation and increased the distance of the acrosomic system from the nucleus. Follicle stimulating hormone had an opposite effect by increasing density of the acrosomic system together with activation of the surrounding microtubule network. The observations suggest that microtubules have an important function during the early differentiation of the acrosomic system.     

Identification of the mouse beta'-COP Golgi component as a spermatocyte autoantigen in scleroderma and mapping of its gene Copb2 to mouse chromosome 9

In an immunological screening of a mouse testicular cDNA library with a human CREST serum we isolated five overlapping cDNA clones encoding the mouse homolog of a Golgi coatomer complex protein (accession number AF043120), designated beta'-COP in bovine and p102 in humans. We generated antibodies against this protein which specifically recognize the Golgi apparatus of mouse spermatocytes. FISH analyses assigned the beta'-COP gene Copb2 to mouse Chromosome 9, region E3-F1. Our results demonstrate that CREST sera can contain antibody components against Golgi proteins as well as against nuclear proteins.     

Phosphorylation of high-mobility group protein A2 by Nek2 kinase during the first meiotic division in mouse spermatocytes

The mitogen-activated protein kinase (MAPK) pathway is required for maintaining the chromatin condensed during the two meiotic divisions and to avoid a second round of DNA duplication. However, molecular targets of the MAPK pathway on chromatin have not yet been identified. Here, we show that the architectural chromatin protein HMGA2 is highly expressed in male meiotic cells. Furthermore, Nek2, a serine-threonine kinase activated by the MAPK pathway in mouse pachytene spermatocytes, directly interacts with HMGA2 in vitro and in mouse spermatocytes. The interaction does not depend on the activity of Nek2 and seems constitutive. On progression from pachytene to metaphase, Nek2 is activated and HMGA2 is phosphorylated in an MAPK-dependent manner. We also show that Nek2 phosphorylates in vitro HMGA2 and that this phosphorylation decreases the affinity of HMGA2 for DNA and might favor its release from the chromatin. Indeed, we find that most HMGA2 associates with chromatin in mouse pachytene spermatocytes, whereas it is excluded from the chromatin upon the G2/M progression. Because hmga2-/- mice are sterile and show a dramatic impairment of spermatogenesis, it is possible that the functional interaction between HMGA2 and Nek2 plays a crucial role in the correct process of chromatin condensation in meiosis.     

Molecular cloning and characterization of SRG-L, a novel mouse gene developmentally expressed in spermatogenic cells

Full-length cDNA of a novel mouse gene upregulated in late stages of spermatogenic cells was cloned from mouse testis using overlapping RT-PCR and RACE. The mRNA of the gene was expressed mainly in diplotene/pachytene spermatocytes, round and elongating spermatids. We named this gene as SRG-L (Spermatogenesis Related Gene expressed in late stages of spermatogenic cells, GenBank Accession No. AY352586). The tissue-specific analysis showed a higher expression level in testis and spleen. The gene is mapped on chromosome 8q33.1 and contains 18 exons. The full-length of cDNA is 2,843 bp with an open reading frame (ORF) of 2,625 bp that encodes a 104 kDa protein (874 amino acids) with a putative transmembrane region. The bioinformatics analysis revealed that the SRG-L has two conserved regions, transglutaminase-like homologues domain and D-serine dehydratase domain, rich phosphorylation sites and methylation sites. The SRG-L protein was detected in diplotene/pachytene spermatocytes and spermatids by immunohistochemical staining and Western blot. The results suggest that SRG-L may play definite roles regulating differentiation of germ cells during spermatogenesis, particularly during meiosis and spermiogenesis.     

X-Y chromosome univalency in the testes of hyperthermic mice: II. Light and electron microscopic analysis of synaptonemal complexes

Hyperthermia-induced X-Y dissociation has been observed in diakinesis-metaphase I sper-matocytes but not in pachytene spermatocytes, which have been implicated as highly susceptible to heat stress. To determine X-Y dissociation in pachytene spermatocytes and to compare levels of dissociation between pachytene and diakinesis-metaphase I spermatocytes male ICR mice were exposed to 35°C ± 0.07°C and 65% ± 0.14% relative humidity for 2 or 4 days. Control mice were housed at 24°C ± 0.04°C and 43% ± 0.58% relative humidity. Mice were killed 0, 3, 5, 8, or 10 days after stress and the testes processed for meiotic chromosome display at diakinesis-metaphase I and synaptonemal complex display at pachynema. Twenty-five to thirty cells per mouse at both stages of meiosis were observed with light microscopy, and pachytene spreads with transmission electron microscopy to determine heat-stress effects on synaptonemal complex structure. Statistical analyses revealed significant linear increases in X-Y dissociation with prolonged exposure to heat at pachynema and diakinesis-metaphase I. Levels of pachytene dissociation were one-half the levels of dissociation at diakinesis-metaphase I. The resolvable structure of the lateral elements of the synaptonemal complex was not affected by heat stress.     

Expression of a mouse zinc finger protein gene in both spermatocytes and oocytes during meiosis.

In order to identify genes regulating meiosis, a mouse spermatocyte cDNA library was screened for sequences encoding proteins with C2H2-type zinc finger motifs which are typically expressed by the Drosophila Krüppel gene. Three new cDNAs were isolated, and they were designated CTfin33, CTfin51, and CTfin92. Among them, CTfin51 was selected for further study. The deduced amino acid sequence revealed seven zinc finger motifs in its C-terminal region. Northern blot and in situ hybridization showed CTfin51 mRNA expression in spermatocytes after the pachytene stage and in early stage round spermatids of prepuberal and adult males. Immunocytochemical staining with an antiserum against beta-gal-CTfin51 fusion protein was localized within nuclei of spermatocytes and spermatids. Oocyte nuclei after the pachytene stage also were immunoreactive for CTfin51 protein. Immunoblots revealed a band at M(r) 75,000 in protein extracts from the testis and the ovary. These results suggest that the CTfin51 gene encodes a DNA-binding regulatory protein functionally associated with meiosis in both male and female gametogenesis.     

2P1, a novel male mouse cDNA specifically expressed during meiosis

We screened a mouse germinal cell expression library with a probe derived from Sob1, a human testis-specific cDNA, and identified 2P1, a new mouse cDNA. A database search revealed that 2P1 was 91% identical to ORF1 of E3-3, a rat gene probably involved in the regulation of alternative splicing. Sequencing showed that 2P1 has a destabilization motif in its 3'-untranslated region. Northern blotting showed strong gene expression in the testis and weak expression in the epididymis, with no signal detected in other tissues. RT-PCR analysis confirmed testis and epididymis expression. In situ hybridization revealed that 2P1 mRNA was absent in spermatogonia but expressed in spermatocytes. This last result was confirmed by RT-PCR of FACS isolated primary spermatocytes (pachytene stage). Using RT-PCR, purified spermatids were also shown to express 2P1.     

Localization of lysosomal acid phosphatase mRNA in mouse tissues.

We studied the expression of lysosomal acid phosphatase (LAP) in mouse by hybridizing Northern blots and tissue sections with the mouse LAP cDNA. Three mRNA species of 2.3, 3.2 and 5.2 KB were identified, which differ in the length of their 3' untranslated region (UTR). The 3.2 KB mRNA is expressed in equal amounts in all tissues and represents the major species in most tissues, whereas the amounts of the 2.3 and 5.2 KB species differ. In situ hybridization of different tissues of adult mice showed a uniform expression of LAP, as expected for a housekeeping gene, except in testis and brain. In testis we found an increase in the LAP mRNA level in spermatocytes. By Northern blot analysis of young mouse testis, this increase could be attributed to late pachytene primary spermatocytes or secondary spermatocytes. In brain tissue the neurons were predominantly labeled, especially the Purkinje and pyramidal cells, whereas glial cells expressed only low amounts of LAP mRNA. Very high LAP expression was also found in the epithelial cells of the choroid plexus. Analysis of LAP expression during mouse embryonic development between Days 9.5 and 17.5 revealed a prominent expression relative to other tissues in the neural tube from Day 9.5 to Day 13.5.     

Mouse testes contain two size classes of actin mRNA that are differentially expressed during spermatogenesis.

Using several actin isotype-specific cDNA probes, we found actin mRNA of two size classes, 2.1 and 1.5 kilobases (kb), in extracts of polyadenylated and nonpolyadenylated RNA from sexually mature CD-1 mouse testes. Although the 2.1-kb sequence was present in both meiotic and postmeiotic testicular cell types, it decreased manyfold in late haploid cells. The 1.5-kb actin sequence was not detectable in meiotic pachytene spermatocytes (or in liver or kidney cells), but was present in round and elongating spermatids and residual bodies. To differentiate between the beta- and gamma-actin mRNAs, we isolated a cDNA, pMGA, containing the 3' untranslated region of a mouse cytoplasmic actin that has homology to the 3' untranslated region of a human gamma-actin cDNA but not to the 3' untranslated regions of human alpha-, beta-, or cardiac actins. Dot blot hybridizations with pMGA detected high levels of presumptive gamma-actin mRNA in pachytene spermatocytes and round spermatids, with lower amounts found in elongating spermatids. Hybridization with the 3' untranslated region of a rat beta-actin probe revealed that round spermatids contained higher levels of beta-actin mRNA than did pachytene spermatocytes or residual bodies. Both probes hybridized to the 2.1-kb actin mRNA but failed to hybridize to the 1.5-kb mRNA.     

DNA synthesis at selective sites during pachytene in mouse spermatocytes

DNA repair replication has been previously demonstrated to occur in mouse spermatocytes during the pachytene stage. The results reported in this study provide a more detailed characterization of pachytene repair by focusing upon specific properties of the sites of replication. Our data demonstrate that single-strand breaks persist within replicated sequences throughout a period which corresponds to a defined interval of the pachytene stage. A large fraction of the sites may be nicked more than once within the same DNA strand, allowing the selective release of replicated DNA sequences from gently denatured spermatocyte DNA. DNA fragments thus prepared from pachytene spermatocytes are not of random sequence composition, but are derived from a specific subset of the mouse genome. Sites of replication are also associated with chromatin of distinctive structure in pachytene spermatocytes, as evidenced by the sensitivity of replicated chromatin to DNase II, and its solubility in the presence of Mg²⁺. In each of these respects, sequences replicated in pachytene spermatocytes closely resemble their counterparts in the LiHum genome.     

Oxidative stress in mature rat testis and its developmental changes

Active oxygen causes various problems including male infertility through the oxidation of DNA, proteins, and lipids. In the present study, we examined the immunohistochemical localization of molecules involved in oxidative stress including 8-hydroxy-2-deoxyguanosine (8-OHdG), superoxide dismutase (SOD), and protein disulfide isomerase (PDI) in mature and developing rat testes. In mature rat testes, 8-OHdG was detected in leptotene, zygotene, and early pachytene spermatocytes, while its expression was weak in late pachytene stage spermatocytes. On the other hand, SOD was detected in late pachytene spermatocytes but not in early pachytene and former spermatocytes, suggesting the efficient removal of active oxygen by SOD in late pachytene spermatocytes. In developing rat testes, 8-OHdG expression peaked at 4 weeks when spermatocytes started to differentiate to the late pachytene stage, while SOD started to be expressed at 4 weeks after birth. These findings suggest that the defense system against oxidative stress by SOD is developed in late pachytene stage spermatocytes at 4 weeks after birth. The present findings aid our understanding of the defensive mechanism against oxidative stress in developing and mature testes.