森 千里

Our recent study clearly shows that fetuses are exposed to multiple chemicals including endocrine disruptors in Japan. Although the embryo and fetus stages are the most sensitive period to chemicals in humans' life cycle, the health effects of the chemicals such as endocrine disruptors to them are largely unknown. The conventional risk assessment method cannot assess the risk to fetuses precisely. Now we need a new risk assessment, in which the target is fetuses and not the adults, in addition to the conventional risk assessment. At the same time, we also need a new strategy to practically eliminate the risk for the future generations. To make the strategy effective, we suggest a new approach to reduce the risk and avoid the possible adverse health effects, using primary, secondary and tertiary preventions as they are used in public health. We also suggest a new concept of "pre-primary prevention" to reduce the risk for fetuses. Furthermore, to make this method even more practical, we suggest a new risk communication method. In this paper, we present a framework of risk avoidance of multiple chemical exposure to fetuses.

We investigated the long-term estrogenic influence of genistein on the male reproductive system in mice. Newborn ICR male mice were treated with genistein (10, 100, or 1000 mug/mouse) for neonatal 5 days. As positive control, administration of diethylstilbestrol (0.5-50 mug/mouse) was carried out. In mice exposed to genistein,we examined weight of testes, sperm counts, sperm motility, and mRNA expression levels of estrogen receptor alpha (ER alpha) and androgen receptor (AR) at 4, 8 or 12 weeks after birth. Moreover, at 12 weeks of age, we evaluated protein level of ER alpha. In our conventional reproductive-toxicological study (weight of testes, sperm counts and sperm motility), neonatal transient exposure to genistein did not show adverse effects on the male reproductive system in 4, 8 or 12 week old mice. However, in mice treated with genistein mRNA expression levels of ER alpha and AR were reduced at 8 weeks. This reduction was recovered at 12 weeks in mice treated with a lower dose (10 mug) of genistein but not in those with higher doses (100 mug and 1000 mug). In addition, ER alpha protein levels tended to decrease in 12 weeks of adulthood. Our results exhibited that the disruption of gene expression continued for long term such as 3 months after administration of genistein, even if no effect was found at conventional reproductive-toxicological level. We have shown that neonatal administration of weak estrogenic compound (genistein) affects male reproductive organs at molecular levels in adulthood.

Testicular heat shock was used to characterize cellular and molecular mechanisms involved in male fertility. This model is relevant because heat shock proteins (HSPs) are required for spermatogenesis and also protect cells from environmental hazards such as heat, radiation, and chemicals. Cellular and molecular methods were used to characterize effects of testicular heat shock (43 degreesC for 20 min) at different times posttreatment. Mating studies confirmed conclusions, based on histopathology, that spermatocytes are the most susceptible cell type. Apoptosis in spermatocytes was confirmed by TUNEL, and was temporally correlated with the expression of stress-inducible Hsp70-1 and Hsp70-3 proteins in spermatocytes. To further characterize gene expression networks associated with heat shock-induced effects, we used DNA microarrays to interrogate the expression of 2208 genes and thousands more expression sequence tags expressed in mouse testis. Of these genes, 27 were up-regulated and 151 were down-regulated after heat shock. Array data were concordant with the disruption of meiotic spermatogenesis, the heat-induced expression of HSPs, and an increase in apoptotic spermatocytes. Furthermore, array data indicated increased expression of four additional non-HSP stress response genes, and eight cell-adhesion, signaling, and signal-transduction genes. Decreased expression was recorded for 10 DNA repair and recombination genes; 9 protein synthesis, folding, and targeting genes; 9 cell cycle genes; 5 apoptosis genes; and 4 glutathione metabolism genes. Thus, the array data identify numerous candidate genes for further analysis in the heat-shocked testis model, and suggest multiple possible mechanisms for heat shock-induced infertility.

近年,環境中に放出された化学物質の多くにホルモン様あるいは抗ホルモン様作用があることが見出され,化学物質の内分泌撹乱作用による生態系への悪影響が,社会問題となっている。その理由は,内分泌撹乱物質の曝露が野生動物の生殖腺や生殖腺付属器官の発生や機能分化に異常をきたし,ホルモン代謝に悪影響を及ぼしていることが判明してきたためである。諸外国より発表された論文では,ヒトにおける「精子数の減少」や「男性生殖器の発生異常の増加」などの生殖異変の原因として,内分泌撹乱物質をあげている。さらに,ヒトの疫学調査から内分泌撹乱物質の悪影響として,精巣ガン,乳ガン,子宮内膜症,女性の思春期の早期化,免疫系・神経系への影響,さらに次世代への影響が懸念されている。ただし,内分泌撹乱物質のヒトに対する影響は,現時点では明白でない。よって,本稿では,「内分泌撹乱物質とその健康影響」と題し,内分泌撹乱物質の定義や種類および生体作用の特徴,作用メカニズム,検出法等について概説し,内分泌撹乱物質のヒトヘの影響の可能性について整理する。特に,ヒトヘの影響に関しては不明な点も多いため,当方が行っている調査研究結果の一部も紹介しながら「男性生殖能の異変」に関する報告を中心に概説する。

Some chemicals are known to induce limb malformations in mice. The occurrence of limb abnormality induced by chemical reagents is due to changes in the programmed cell death (PCD), 5-Bromodeoxyuridine (BrdU) is known as a potent teratogen and has been reported to induce polydactyly and other limb malformations in rodents (DiPaolo, Science 145, 501-503, 1964). Here, we undertook the morphological and genetic analyses of fetuses with limb malformations in BrdU-treated mice, in order to investigate an alteration of gene expression that resembles that of mutant mice with similar limb malformations. The fetuses of the BrdU-treated mice exhibited preaxial polydactyly and preaxial triphalangism of the hindlimb at a high incidence. Our observations showed that the PCD in the preaxial necrotic zone was found to be delayed or absent on day 11 of pregnancy. Histological analyses of these fetuses showed that the preaxial apical ectodermal ridge (AER) of the hindlimb was hyperplastic and consisted of several irregular layers. In observation of the whole-mount in situ hybridization, we detected the anterior-extended overexpression of Hoxd-11 and Hoxd-13 genes in the mesenchyme cells and the overexpression of Fgf4 and Fgf8 genes in the anterior region of the AER of hindlimbs of BrdU-treated fetuses. Our study shows that the injection of BrdU changed the PCD and gene expression during limb development and induced time-specific limb malformations during fetal development. This examination of the changes of the PCD and gene expression will be useful markers for the investigation of toxicities and teratologieties of other chemicals now present in the world environment. (C) 2000 Academic Press.

Spermatogenesis is a developmental process that occurs in several phases and is regulated by a large number of gene products. An insertional transgenic mouse mutant (termed kisimo mouse) has been isolated that results in abnormal germ-cell development, showing: abnormal elongated spermatids in the lumina of seminiferous tubules. We cloned the disrupted locus of kisimo and identified a novel testis-specific gene, THEG, which is specifically expressed in spermatids and was disrupted in the transgenic mouse. The yeast two-hybrid screening method revealed that THEG protein strongly interacts with chaperonin containing t-complex polypeptide-le, suggesting that THEG protein functions as a regulatory factor in protein assembly. Our findings indicate that the kisimo locus is essential for the maintenance of spermiogenesis and that a gene expression disorder may be involved in male infertility.

Although the process of glycolysis is highly conserved in eukaryotes, several glycolytic enzymes have unique structural or functional features in spermatogenic cells. We previously identified and characterized the mouse complementary DNA (cDNA) and a gene for 1 of these enzymes, glyceraldehyde 3-phosphate dehydrogenase-s (Gapds). This gene is expressed only in spermatids. The enzyme appears to have an essential role in energy production required for fertilization, and it is reported to be susceptible to inhibition by certain environmental chemicals. We have now cloned and sequenced the cDNA for the human homologue of glyceraldehyde 3-phosphate dehydrogenase (GAPD2) and determined the structure of the gene. The messenger RNA (mRNA) was detected in testis, but not in 15 other human tissues analyzed by Northern blot technique. The deduced GAPD2 protein contains 408 amino acids and is 68% identical with somatic cell GAPD. GAPD2 has a 72-amino acid segment at the amino terminal end that is not present in somatic cell GAPD. This segment is proline-rich but contains smaller stretches of polyproline and is 30 amino acids shorter than the comparable segment of mouse GAPDS. The structure of the human GAPD2 gene was determined by polymerase chain reaction (PGR) to identify exon-intron junctions in a genomic clone and in total genomic DNA. The locations of these junctions in the GAPD2 gene corresponded precisely to those of the 11 exon-intron junctions in the mouse Gapds gene. Immunohistochemical studies found that GAPD2 is located in the principal piece of the flagellum of human spermatozoa, as are GAPDS in mouse and rat spermatozoa. GAPD2 extracted from human spermatozoa and analyzed by Western blot technique migrated with an apparent molecular weight of similar to 56 000, although the calculated molecular weight is 44 501. The conserved nature of the mouse, rat. and human enzymes suggests that they serve similar roles in these and other mammalian species.

To investigate the incorporation of oligonucleotides (ODNs) into the tissues of cultured fetal mouse palates and their effects on in vitro palatogenesis, we cultured day-12.5 fetal mouse palates in a chemically defined serumless medium supplemented with either antisense or sense ODNs to epidermal growth factor receptor (EGF-r). The EGF-r ODNs were found to be incorporated into the palatal tissue and remained detectable for at least 72 hr. Immunohistochemical and immunoblot analyses revealed that the treatment with 5μM EGF-r antisense ODN suppressed the production of EGF-r protein. No pathological change was observed in the explanted palates when they were treated with 5 μM EGF-r antisense or sense ODNs, but the treatment with 10 or 20 μM ODN caused pyknotic changes in the palatal epithelium, probably due to the ODN toxicity. The present results indicate that under optimal conditions, antisense ODNs to EGF-r can be incorporated into fetal organs cultured in vitro and specifically inhibit the production of EGF-r protein. Since the suppression of the production of EGF-r protein did not prevent the palate fusion, EGF and/or EGF-r alone may not play a critical role in palatogenesis, as suggested by previous studies. The antisense ODN technique could be of potential use for analyzing the roles of specific molecules in normal and abnormal morphogenesis.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase, However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Retroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd, In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a "promoter-trap" fashion, One such promoter ("promoter B" at the Mintb locus) was found in a CPG island, associated with an upstream enhancer ("enhancer B"), Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit ("R" for reverse), which apparently interfered with transcription from promoter B, Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U), Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.

The present study investigated the occurrence of apoptotic cell death in the mouse testis at various intervals following the administration of hydroxyurea (HU). The presence of apoptosis was assessed by the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) method and by DNA fragmentation assay using ligation-mediated polymerase chain reaction, Both the incidence of apoptotic cells and the level of DNA fragmentation in the testis increased depending on the HU dose, and they were most apparent at the highest dose (400 mg/kg). The incidence of apoptotic cells in the HU-treated group increased continuously and peaked at 12 h, but then decreased gradually, reaching control levels by 48 h. After HU treatment, TUNEL-positive apoptotic cells increased in the seminiferous epithelium of the tubules, and affected cells were found synchronously in the tubules of animals treated with HU. Spermatogonia and spermatocytes were found to be affected selectively. TUNEL-positive cells were found to be stage-specific and were primarily in stage IV-VI tubules. It has been shown that in vivo HU exposure induced testicular germ cell apoptosis dose dependently in a time- and stage-specific manner, and damaged cells appeared to be eliminated by phagocytosis by neighboring cells. Apoptosis of damaged testicular germ cells is apparently a common response to various testicular toxicants therefore protecting the next generations of germ cells from the damaged cell population. (C) 1999 Academic Press.

Tissue distribution and cellular localization of rhophilin, a 71 kDa Rho-binding protein, were examined in mice. Rhophilin mRNA was highly expressed in adult testis, but was absent in the testis of WIWV mice deficient in germ cells.,An anti-rhophilin antibody detected a band of an expected size in sperm extracts, which was enriched in the tail fraction. Immunofluorescence analysis revealed two lines of striated staining running in parallel in the principal piece of the sperm tail, These results suggest that rhophilin is expressed in germ cells and localized in the fibrous sheath of the sperm tail. (C) 1999 Federation of European Biochemical Societies.

Retroviral genes are not usually expressed in mouse embryonal carcinoma (EC) cells, but they are readily expressed upon differentiation of these cells. We previously reported the isolation of EC cell lines that express a neomycin resistance (neo) gene introduced by a recombinant transducing Moloney murine leukemia virus from specific integration sites, Minta, Mintb, Mintc, or Mintd. In some of these clones, the entire 5' long terminal repeat (LTR) was deleted, and the neo gene was expressed by read-through transcription from upstream cellular promoters in a 'promoter-trap' fashion. One such promoter ('promoter B' at the Mintb locus) was found in a CpG island, associated with an upstream enhancer ('enhancer B'). Although enhancer B caused expression of the neo gene in the transductant EC cell line, no endogenous transcription from promoter B was detected in the parental EC or NIH3T3 cells. In contrast, we found a strong counter-flow endogenous transcription unit ('R' for reverse), which apparently interfered with transcription from promoter B. Promoter R turned out to have a bidirectional activity in transfection assays. In normal tissues, promoter R activates gene R, which encodes an 800-residue protein that is highly homologous to the rat and human heterogeneous nuclear ribonucleoprotein U (hnRNP U). Northern and in situ hybridization analyses revealed that gene R was abundantly expressed in the testis, especially in the pachytene spermatocytes and round spermatids.

Hsp70-2 is a unique member of the mouse 70-kDa heat shock protein family that is synthesized during meiosis in spermatogenic cells. Germ cells in male mice homozygous for a targeted mutation in the Hsp70-2 gene (Hsp70-2(-/-)) arrest in development and undergo apoptosis at the end of the pachytene spermatocyte stage of meiotic prophase. However, cells with a putative acrosome were present occasionally in histological sections of the testes of juvenile and adult Hsp70-2(-/-) mice. This study verified that acrosomes were present and investigated the relationship between acrosome formation and the process of meiosis. Histochemistry with the periodic acid-Schiff procedure and immunostaining with monoclonal antibody MN7 verified that acrosomes were present in Hsp70-2(-/-) mice, and electron microscopy showed that some of these cells had condensing nuclei characteristic of step 8-9 spermatids. The frequency of acrosome-containing cells in Hsp70-2(-/-) mice was less than 0.01% of that in wild-type mice. Propidium iodide staining and cytophotometry indicated that the average DNA content of nuclei in MN7-positive cells in Hsp70-2(-/-) mice was usually about twice, or occasionally the same as, that of nuclei in round spermatids of wild-type mice. Meiotic metaphase I and II chromosome spreads were observed in spermatogenic cells from Hsp70-2(-/-) mice but at a much lower frequency than in wild-type mice. These results indicate that not all pachytene spermatocytes in Hsp70-2(-/-) mice arrest in meiosis, but they may divide once or sometimes twice and begin acrosome formation and nuclear condensation. This demonstrates that some aspects of spermatid development can occur without the completion of meiosis in mice, as has been reported recently for Drosophila.

Unique type 1 hexokinase (HK1) mRNAs are present in mouse spermatogenic cells (mHk1-s). They encode a spermatogenic cell-specific sequence region (SSR) but not the porin-binding domain (PBD) necessary for HK1 binding to porin on the outer mitochondrial membrane. This study determined the origin of the multiple Hk1-s transcripts in mouse spermatogenic cells and verified that they are translated in mouse spermatogenic cells. It also showed that a single mHk1 gene encodes the mHk1 transcripts of somatic cells and the mHk1-sa and mHk1-sb transcripts of spermatogenic cells, that alternative exons are used during mHk1 gene expression in mouse spermatogenic cells, and that mHK1-S is translated in mouse spermatogenic cells and is localized mainly with the fibrous sheath in the tail region, not with the mitochondria in the midpiece of mouse sperm. (C) 1998 Wiley-Liss, Inc.

Spermatogenic cell degeneration in the mature mammalian testis occurs both spontaneously during normal spermatogenesis and in response to cytotoxic agents. Mitomycin C (MC) is an antibiotic that affects DNA synthesis. In the present study, we examined the induction of mouse spermatogenic cell apoptosis by MC, using TdT-mediated dUTP-biotin nick end labeling (TUNEL) to detect high levels of DNA fragmentation in situ, transmission electron microscopy (TEM) to observe nuclear chromatin condensation, and molecular methods to detect DNA ladders. This study shows that in the testis of MC-treated mice: (i) apoptotic cell death with fragmentation of nuclear DNA is induced by MC dose-dependently, (ii) apoptotic cell death is most commonly found in the spermatogonia and less frequently in spermatocytes, and (iii) apoptotic cell death induced by MC is not specific for the seminiferous stage of the tubules. The present study suggests that the spermatogenic cell apoptosis induced by MC might be involved in its testicular toxicity. (C) 1997 Academic Press.

Cisplatin and VP-16 were used to study the induction of apoptosis in Panc-1 cells. Cisplatin and VP-16 inhibited the growth of Panc-1 cells. After 2 hours exposure to cisplatin or VP-16, attached and detached cells were subjected to TUNEL staining to calculate the ratio of apoptosis. In detached cells TUNEL positive ratios increased in a time-and dose-dependent manner In Western blotting, Bm expression was obviously upregulated but Bcl-2 remained almost constant. The results suggested that in Panc-1 cells cisplatin and VP-16 induced apoptotic cell death which was mediated through the interaction of Bax expression in the presence of mutated p53.

An iron chelate, ferric nitrilotriacetate (Fe-NTA), induces renal proximal tubular necrosis, a consequence of free radical-associated damage, that ultimately leads to a polycystic change of the renal cortex and a high incidence of renal cell carcinoma (RCC) in rodents. The differential display technique was used to search for inducible genes in the kidney of male Wistar rats treated with Fe-NTA and in the induced RCCs. Six fragments were selected that showed specific quantitative changes in mRNA. Two of them exhibited similar patterns in northern blots as well. One fragment showed a high homology (89%) to murine integrin-associated protein (IAP; CD47). We thus cloned rat IAP cDNA including the entire coding region for use in further analysis. Rat IAP cDNA showed a 21-amino-acid deletion that was also observed in human, but not in mouse. Northern blots revealed that IAP was consistently overexpressed in non-tumorous parts of the kidney (2.4-fold increase, n = 9, P<0.0001) as compared with matched controls 1 to 2 years after Fe-NTA treatment. IAP overexpression of more than 2.9-fold was found in 25% (2/8) of RCCs studied, and was limited to cases of a high histological grade and lung metastasis. Unexpectedly, IAP expression was higher in the non-tumorous part of the kidney after Fe-NTA treatment (2.8-fold) than in RCC (1.5-fold) in each case (n = 4, P < 0.05). Abundant expression of IAP mRNA in the renal tubular epithelium after Fe-NTA treatment and RCC cells was observed by in situ hybridization. The results suggest that IAP overexpression may be associated with Fe-NTA-induced renal cortical tubular damage and regeneration that lead to a polycystic state, and with tumor progression and metastasis of the induced RCCs.

It has been recognized in mammals that after pancreatic duct obstruction, acinar cells progressively disappear and pancreatic islets are preserved. Previous studies by electron microscopy have suggested the involvement of apoptosis in acinar cell deletion. In the present study, we employed molecular biological methods and investigated whether acinar cell deletion is due to apoptosis. In male Sprague-Dawley rats, pancreatic duct Ligation was performed through a left paramedian incision. Pancreatic tissue was studied at each of the following intervals after ligation: 3, 6, 12, 18, and 24 h and 2, 3, 5, and 7 days. DNA fragmentation was determined by in situ labeling of DNA strand breaks on tissue sections [fluorescein-labeled terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling method (TUNEL)] and by electrophoretic detection of the fragments of extracted DNA. Tissue sections were also examined by hematoxylin/eosin staining and immunohistochemical staining of insulin. Pancreatic duct ligation induced acinar cell deletion by day 5. Pancreatic tissue from control rats demonstrated no TUNEL-positive nuclei. In contrast, acinar cells from rats 12 h to 5 days after duct ligation showed TUNEL-positive nuclei. The number of TUNEL-positive nuclei was maximum 2 days after duct ligation. Electrophoresis showed DNA fragmentation after duct ligation. Control rats showed a genomic DNA pattern. Islets were preserved throughout the experimental period in duct-ligated rats and control rats. The results suggest that apoptosis may be the dominant form of acinar cell death in the rat pancreatic duct ligation model.

The present study examined the occurrence of apoptotic cell death in the testis of wild-type mice from postnatal days 3 to 28 and in juvenile Hsp70-2 knockout mice. Adult Hsp70-2 knockout males are infertile and lack spermatids and spermatozoa (Dir et al. [1996a] Proc. Natl. Acad. Sci. U.S.A. 93:3264-3268). To identify the cell types undergoing apoptosis, we also examined the relationship between the occurrence of apoptotic cell death and the expression pattern of the Hsp70-2 gene product (heat-shock protein 70-2 [Hsp70-2]; marker for spermatocytes and spermatids), germ cell nuclear antigen 1 (GCNA1; marker for spermatogonia and spermatocytes), and vimentin (marker for Sertoli cells). This study shows that during postnatal development of the wildtype mouse testis (1) the percentage of apoptotic cell death detected by the TdT-mediated dUTP-biotin nick end labeling (TUNEL) method is higher in mice from days 8 to 22 than in younger or older mice, (2) the majority of apoptotic cells are spermatogonia and less frequently are spermatocytes, and (3) the degenerative cell death of spermatogonia and primary spermatocytes involves apoptosis with fragmentation of DNA. The analysis of apoptotic cell death in the testes of juvenile Hsp70-2 knockout mice showed an additional increased level of apoptosis at day 17, during the first wave of spermatogenesis, in pachytene spermatocytes. (C) 1997 Wiley-Liss, Inc.

Several enzymes in the glycolytic pathway are reported to have spermatogenic cell-specific isozymes. We reported recently the cloning of cDNAs representing three unique type 1 hexokinase mRNAs (mHk1-sa, mHk1-sb, and mHk1-sc) present only in mouse spermatogenic cells and the patterns of expression of these mRNAs (Mori et al., 1993: Biol Reprod 49:191-203). The mRNAs contain a spermatogenic cell-specific sequence, but lack the sequence for the porin-binding domain that somatic cell hexokinases use to bind to a pore-forming protein in the outer mitochondrial membrane. We now report the cloning of cDNAs representing three unique human type 1 hexokinase mRNAs (hHK1-ta, hHK1-tb, and hHK1-tc) expressed in testis, but not detected by Northern analysis in other human tissues. These mRNAs also contain a testis-specific sequence not present in somatic cell type 1 hexokinase, but lack the sequence for the porin-binding domain. The hHK1-tb and hHK1-tc mRNAs each contain an additional unique sequence. The testis-specific sequence of the human mRNAs is similar to the spermatogenic cell-specific sequence of the mouse mRNAs. Furthermore, Northern analysis of RNA from mouse, hamster, guinea pig, rabbit, ram, human, and rat demonstrated expression of type 1 hexokinase mRNAs lacking the porin-binding domain in the testes of these mammals. These results suggest that hexokinase may have unique structural or functional features in spermatogenic cells and support a model proposed by others for hexokinase gene evolution in mammals. (C) 1996 Wiley-Liss, Inc.

In addition to the five 70-kDa heat shock proteins (HSP70) common to germ cells and somatic tissues of mammals, spermatogenic cells synthesize HSP70-2 during meiosis, To determine if this unique stress protein has a critical role in meiosis, we used gene-targeting techniques to disrupt Hsp70-2 in mice. Male mice homozygous for the mutant allele (Hsp70-2(-/-)) did not synthesize HSP70-2, lacked postmeiotic spermatids and mature sperm, and were infertile, However, neither meiosis nor fertility was affected in female Hsp70-2(-/-) mice, We previously found that HSP70-2 is associated with synaptonemal complexes in the nucleus of meiotic spermatocytes from mice and hamsters. While synaptonemal complexes assembled in Hsp70-2(-/-) spermatocytes, structural abnormalities became apparent in these cells by late prophase, and development rarely progressed to the meiotic divisions, Furthermore, analysis of nuclei and genomic DNA indicated that the failure of meiosis in Hsp70-2(-/-) mice was coincident with a dramatic increase in spermatocyte apoptosis. These results suggest that HSP70-2 participates in synaptonemal complex function during meiosis in male germ cells and is linked to mechanisms that inhibit apoptosis.