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Biology of Reproduction 61, 813-822 (1999)
©Copyright 1999 Society for the Study of Reproduction, Inc.

Articles

Completion of Meiosis Is Not Always Required for Acrosome Formation in HSP70-2 Null Mice1

Chisato Mori2,a,c, James W. Allend, David J. Dixe, Noriko Nakamuraa, Makio Fujiokab, Kiyotaka Toshimorif, and Edward M. Eddyc

a Department of Anatomy and Developmental Biology and Central Laboratory for Electron Microscopy, b Faculty of Medicine, Kyoto University, Kyoto 606-8501, Japan c Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709 d Environmental Carcinogenesis Division, Reproductive Toxicology Division, e National Health and Environmental Effects Research Laboratory, US Environmental Protection Agency, Research Triangle Park, North Carolina 27711 f Department of Anatomy, Miyazaki Medical College, Miyazaki 889-16, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The developmental program of spermatogenesis in mammals occurs in tightly coordinated mitotic, meiotic, and postmeiotic phases. Spermatogonia proliferate by mitosis to expand the spermatogenic cell population; spermatocytes undergo chromosomal synapsis and genetic recombination before completing the two meiotic divisions; and spermatids undergo profound remodeling during the postmeiotic phase to become spermatozoa. These processes involve dramatic shifts in patterns of gene expression, including activation of genes that encode unique proteins, that are regulated developmentally, and that are either transcribed only in male germ cells or produce mRNAs specific to these cells [1, 2].

Hsp70-2 is a member of the mouse 70-kDa heat shock protein (HSP70) family. These proteins serve as chaperones to assist other proteins in folding, transport through the cytoplasm, assembly into complexes, and refolding after cellular stress [3].

Hsp70-2 gene expression begins in meiotic prophase in leptotene spermatocytes [4, 5], and spermatogenesis is disrupted in mice homozygous for a targeted mutation of the Hsp70-2 gene [6]. Late pachytene and diplotene spermatocytes undergo developmental arrest and apoptosis in Hsp70-2-/- mice, resulting in severe depletion of postmeiotic germ cells and infertility. Hsp70-2 is associated with the synaptonemal complex of the paired homologous chromosomes in spermatocytes [7]. Although synaptonemal complexes form in spermatocytes of Hsp70-2-/- mice, desynapsis of the paired chromosomes is defective in diplotene spermatocytes at the end of meiotic prophase [8].

Failure of meiosis in Hsp70-2-/- mice is associated with a lack of CDC2 (p32cdc2) protein kinase activity in spermatocytes [9]. Hsp70-2 serves as a chaperone for CDC2, with their interaction required before CDC2 can form a heterodimer with cyclin B1 to become an active protein kinase [9]. Spermatocytes of Drosophila that are mutant for either cdc2 [10, 11] or twine, a cdc25 homologue [12, 13], are able to skip crucial events of meiotic division and carry out spermatid differentiation. Both flagellar elongation and nuclear condensation and shaping proceed, despite the failure to complete meiotic chromosome segregation and cytokinesis [13, 14], indicating that completion of meiotic cell cycle progression is not obligate for initiation of the spermatid differentiation program. Flagellar formation without progression through the second meiotic division has also been reported for secondary spermatocytes of the newt that have been cultured in the presence of cycloheximide [15].

This study examined spermatogenic cells containing an acrosome-like structure seen occasionally in sections of testis from Hsp70-2-/- mice. Histochemistry with the periodic acid-Schiff (PAS) procedure and immunostaining with a monoclonal antibody specific for the acrosome [16, 17] and electron microscopy confirmed that acrosomes were present. In addition, the nucleus and cell body of most acrosome-containing cells in Hsp70-2-/- mice appeared larger than those of acrosome-containing cells in wild-type mice. The nuclei of these cells in Hsp70-2-/- mice often contained DNA similar in amount to that in diploid cells and occasionally the amount in haploid spermatids in wild-type mice. Furthermore, meiotic metaphase I and II nuclei were found in chromosome spreads of spermatogenic cells from Hsp70-2-/- mice. These results indicate that initiation of spermatid differentiation and acrosome formation can occur without completion of meiosis in Hsp70-2-/- mice. Although these findings are similar to what has been reported recently in Drosophila, there are also significant differences when these events occur during meiosis that may relate to differences in spermatogenesis in the two species.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Mice with a targeted mutation in the Hsp70-2 gene were produced with E14TG2a embryonic stem cells derived from strain 129/SvOla mouse blastocysts as described previously [6]. All studies were performed according to protocols approved by National Institute of Environmental Health Sciences and United States Environmental Protection Agency Institutional Animal Care and Use Committees. Most studies were performed with juvenile and adult mice homozygous for a mutation in the Hsp70-2 gene (Hsp70-2-/-) and their wild-type littermates with a predominantly C57BL/6N genetic background. They were in the third or fourth generation produced by crossing a male chimera with a wild-type female C57BL/6N mouse and then mating their progeny. In addition, Hsp70-2-/- mice produced by crossing a male chimera with a 129SvEv female and then mating their progeny were used for some histological preparations and meiotic chromosome spreads.

Tissue Preparation for Histochemical and Immunocytochemical Staining

Testes analyzed in this study were from Hsp70-2-/- and wild-type mice that were 24 or 28 postnatal days of age (day of birth designated as Day 0) or adults. Testes were immersion fixed for at least 16 h at 4°C in freshly prepared 4% paraformaldehyde buffered with 0.1 M sodium phosphate (pH 7.4), dehydrated with ethanol, and embedded in paraffin according to standard procedures. Paraffin sections (5 µm thick) were placed on slides pretreated with 3-amino-propyltriethoxysilane (Matsunami, Osaka, Japan) and stored at room temperature until further processing.

Sections were deparaffinized and either stained with PAS and counterstained with hematoxylin to identify the acrosome and nucleus [18, 19], or immunostained with monoclonal antibody MN7 specific for a constituent of the anterior acrosome of spermatozoa from several mammalian species [16, 17]. Monoclonal antibody MN7 (1:200) was used either with a Vectastain ABC kit (Vector Laboratories, Burlingame, CA) according to the manufacturer's instructions, or with an fluorescein isothiocyanate-conjugated goat anti-mouse IgG second antibody (Cappel, West Chester, PA). Slides were prepared for immunofluorescence using antifade mounting medium [20] containing 1 µg/ml of propidium iodide (PI).

Digital imaging techniques were used to measure DNA content in PI-stained nuclei and the cross-sectional area of nuclei. The DNA content of individual nuclei was determined using cytophotometry to measure fluorescence resulting from intercalation and stoichiometric binding of PI to DNA [21]. Fluorescence intensity and the cross-sectional area of the nuclei were measured using a fluorescence microscope with a silicon intensifier tube camera (Hamamatsu Photonics, Hamamatsu, Shizuoka, Japan) and a digitized image processor (Argus-50; Hamamatsu Photonics) [22]. The stages of the cycle of the seminiferous epithelium were identified on sections stained with PAS and hematoxylin by the criteria of Oakberg [23] and Russell et al. [19].

Transmission Electron Microscopy (TEM)

The testes of Hsp70-2-/- and wild-type mice (postnatal Day 24 and 28, and adult) were fixed with 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.2), postfixed in 1% phosphate-buffered OsO4 with 0.1 M sucrose, dehydrated with ethanol, and embedded in epoxy resin. Sections 70–90 nm thick were placed on 150-mesh copper grids, stained with uranyl acetate followed by lead citrate, and examined using an electron microscope (Hitachi H7000, Tokyo, Japan).

Preparation of Meiotic Chromosome Spreads

Spermatocytes were processed for meiotic chromosome analysis using methods described earlier [24]. Briefly, cell suspensions from seminiferous tubules were centrifuged, and the pelleted contents were treated with hypotonic (1%) sodium citrate for 10 min and fixed in 3:1 methanol:acetic acid. Chromosome spreads were prepared by applying droplets of fixed cell suspensions onto cold, wet slides and staining with 4% Giemsa (Biomedical Specialties, Santa Monica, CA). Brightfield microscopy was performed with a Nikon (Garden City, NY) FXA microscope and x63 Leitz (Leitz Wetzlar GBH, Wetzlar, Germany) objective.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Spermatogenic Cells with an Acrosome Were Present in Hsp70-2-/- Mice

We reported previously that pachytene spermatocytes undergo meiotic arrest and apoptosis in Hsp70-2-/- mice [6, 8, 25]. However, an occasional spermatogenic cell in Hsp70-2-/- mice contained an acrosome-like structure, a feature characteristic of postmeiotic germ cells. The acrosome-like structures were seen in sections of testes from postnatal Days 24 and 28 and adult Hsp70-2-/- mice (Figs. 1–4). It was confirmed that these structures were acrosomes by staining with PAS (Fig. 1), immunostaining with monoclonal antibody MN7 (Figs. 1 and 3), and TEM (Figs. 2 and 4). An acrosome was found in 1–5% of the spermatogenic cells in 2 or 3 of the approximately 50 seminiferous tubules per testis cross section in Hsp70-2-/- mice. This is less than 0.01% of the frequency of cells with an acrosome in wild-type mice. Only a few spermatogenic cells in postnatal Day 24 Hsp70-2-/- mice had an MN7-positive acrosome (data not shown). Spermatogenic cells with an acrosome and a condensed nucleus, features typically found in step 8–9 condensing spermatids, were seen infrequently in adult Hsp70-2-/- mice (Figs. 1C and 4C).



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  		FIG. 1. Spermatogenic cells with acrosomes were detected by PAS staining (A–D) or by immunohistochemistry with monoclonal antibody MN7 (E, F) in Hsp70-2-/- (A-–C-, E-) and wild-type mice (D+, F+). In Hsp70-2-/- mice, cells containing PAS-stained (magenta color) acrosomes are shown that appeared to be at approximately A-) step 3 (round nucleus with adjacent Golgi complex), B-) step 6–7 (round nucleus with cap-phase acrosome), and C-) step 8–9 (condensed nucleus) of spermatid development (arrows). In a wild-type mouse (D+), step 3 spermatids (round nucleus with Golgi-phase acrosomes) were present in the tubule on the left (arrowheads), and step 6–7 spermatids were present in the tubule on the right (arrowheads). With antibody MN7, acrosomes (brown reaction product) are shown in cells of Hsp70-2-/- mice (E-) at approximately step 6–7 of spermatid development (arrows), and in wild-type mice (F+) at step 5 of spermatid development (arrowheads). Sections were counterstained with hematoxylin (dark purple color; A–D) or methyl green (light green color; E, F). Bar = 5 µm (A–D), 4 µm (E, F).



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  		FIG. 2. TEM of spermatogenic cells with a chromatoid body (A, B; arrows) or an acrosome (C, D, arrowheads) in Hsp70-2-/- (A-, C-) and wild-type mice (B+, D+). Pachytene spermatocytes (P) are identifiable by the presence of synaptonemal complexes (small arrowhead) in the nucleus of A-) Hsp70-2-/- and B+) wild-type mice. The presence of these cells indicated that the adjacent cells containing a chromatoid body were probably early spermatids. Step 6–7 spermatids with acrosomes (arrowheads) were seen in C-) Hsp70-2-/- and D+) wild-type mice. Bar = 1 µm.

Nucleus and Cell Body of Spermatogenic Cells with an Acrosome Were Larger in Hsp70-2-/- Mice Than in Wild-Type Mice

Comparison of spermatogenic cells with PAS-stained acrosomes suggested that the nucleus and cell body often were larger in Hsp70-2-/- mice (Fig. 1A, approximately step 3 spermatid with Golgi-phase acrosome; Fig. 1B, step 6–7 spermatid with cap-phase acrosome) than in wild-type mice (Fig. 1D, step 3 Golgi-phase and step 6–7 cap-phase spermatids). This was also seen for spermatogenic cells immunostained with MN7 in Hsp70-2-/- mice (Fig. 1E) and wild-type mice (Fig. 1F). The same relationship was seen at the TEM level upon comparison of cells with similar structural features (chromatoid bodies or cap-phase acrosomes) in Hsp70-2-/- mice (Fig. 2A, large arrows; Fig. 2C, arrowhead) and wild-type mice (Fig. 2B, large arrow; Fig. 2D, arrowheads). This was confirmed on sections labeled with PI to identify nuclei and immunostained with MN7 to identify acrosomes. The PI-labeled nuclei of most cells with MN7-positive acrosomes were larger in both postnatal Day 28 and adult Hsp70-2-/- mice (Fig. 3, A–C and E; arrows) than in wild-type mice (Fig. 3, D and F; arrows). In contrast, the nuclei of pachytene spermatocytes containing synaptonemal complexes appeared to be approximately the same size in Hsp70-2-/- (Fig. 2A) and wild-type mice (Fig. 2B). However, the difference between the size of nuclei of pachytene spermatocytes and that of cells with chromatoid bodies or cap-phase acrosomes in Hsp70-2-/- mice (Fig. 2, A and C) usually appeared to be less than that between comparable cells in wild-type mice (Fig. 2, B and D).



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  		FIG. 3. Sections of mouse testis observed using MN7 antibody to identify acrosomes by immunofluorescence (greenish yellow color; arrows) and nuclei by fluorescence with PI (orange) in adult Hsp70-2-/- (A-–C-) and wild-type (D+) mice, and in postnatal Day 28 Hsp70-2-/- (E-) and wild-type (F+) mice. In adult Hsp70-2-/- mice, spermatids were seen at approximately A-) step 3, B-) step 5, and C-) step 6–7 of development. Spermatids were seen at approximately step 5 of development in D+) adult wild-type, E-) Day 28 Hsp70-2-/-, and F+) Day 28 wild-type mice. Bar = 10 µm.

Because of the subjective nature of these observations, digital imaging techniques were used to measure the size of nuclei on sections of testes from juvenile and adult Hsp70-2-/- and wild-type mice that were stained with PI and immunostained with MN7. Pachytene spermatocytes from Hsp70-2-/- and wild-type mice were confirmed to be similar in size by this approach (Table 1). In addition, the PI-stained nuclei in pachytene spermatocytes were larger than PI-stained nuclei of MN7-positive cells in both Hsp70-2-/- and wild-type mice (Table 1). However, there usually was less difference between the sizes of these nuclei in pachytene spermatocytes and acrosome-containing cells in Hsp70-2-/- mice than in wild-type mice (Table 1). These results verified that nuclei in cells containing acrosomes are significantly (p < 0.01) larger in Hsp70-2-/- mice than nuclei in spermatids of wild-type mice (Table 1).


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  		TABLE 1. Area of nuclei of spermatogenic cells in Hsp70-2-/- and wild-type mice (mean ± SD).a

DNA Content of Spermatogenic Cell Nuclei in Hsp70-2-/- and Wild-Type Mice

The difference in size of nuclei of acrosome-containing cells in Hsp70-2-/- and wild-type mice suggested that their DNA content would also differ. This was examined by using digital cytophotometry to determine the relative fluorescence intensity of PI-stained nuclei of pachytene spermatocytes and MN7-positive cells in 28-day-old and adult Hsp70-2-/- and wild-type mice (Fig. 3). The fluorescence intensity of nuclei in MN7-positive cells of Hsp70-2-/- mice was usually twice that of nuclei in spermatids of wild-type mice (Table 2). The fluorescence intensity of most nuclei of MN7-positive cells in Hsp70-2-/- mice was comparable to that of Leydig cells (Table 2) and of most spermatogonia (data not shown). These results suggest that the nuclei of most MN7-positive cells in Hsp70-2-/- mice contain DNA comparable in amount to that found in diploid cells, but that a few contain the amount of DNA found in haploid spermatids. Only a few nuclei of the spermatids in Hsp70-2-/- mice were approximately the same size as those in wild-type mice (Fig. 4).


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  		TABLE 2. DNA content of spermatogenic cells stained by PI in Hsp70-2-/- and wild-type mice (mean ± SD).a



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  		FIG. 4. TEM of spermatogenic cells with an acrosome in Hsp70-2-/- (A-, C-) and wild-type (B+, D+) mice. The spermatids were at approximately A-) step 7, B+) step 5–6, and C-, D+) step 9 of development. In these examples, the nuclei of the spermatids in Hsp70-2-/- mice (A-, C-) were approximately the same size as those in wild-type mice (B+, D+). Bar = 0.5 µm.

Meiotic Metaphase I and II Chromosome Spreads

Further evidence that meiotic divisions occur came from examining chromosome spreads prepared from spermatogenic cells from Hsp70-2-/- mice of predominantly C57BL/6N and 129SvEv genetic backgrounds. Although infrequent, nuclei were seen that contained bivalent chromosomes with chiasmata characteristic of diakinesis-meiotic metaphase I cells (Fig. 5A) or the haploid complement of chromosomes characteristic of meiotic metaphase II cells (Fig. 5B). The disruption of spermatogenesis prevented accurate comparisons of meiotic metaphase indices between mice heterozygous (Hsp70-2+/-) and homozygous (Hsp70-2-/-) for the mutant allele. Consequently, relative frequencies of mitotic and meiotic (either diakinesis-meiotic metaphase I or meiotic metaphase II) stages were determined for mice with each genotype from both genetic backgrounds. In 100 dividing cells from a C57BL/6N and in 100 dividing cells from each of two 129SvEv heterozygous mice, about 91% of the nuclei with condensed chromosomes were identified as meiotic, and the remaining as mitotic (putative spermatogonia). In 130 dividing cells from two C57BL/6N Hsp70-2-/- mice, this ratio was reversed, and only about 10% of the chromosome spreads were from meiotic nuclei. Metaphase II cells were rarely observed (1 per 130 dividing cells). Interestingly, two 129SvEv Hsp70-2-/- mice gave intermediate and variable frequencies of meiotic metaphase I divisions, although, again, metaphase II cells were infrequently seen (5 per 200 dividing cells). In 100 cells from these mice, meiotic nuclei accounted for 28% and 70% of chromosome spreads. Not only were meiotic nuclei from 129SvEv Hsp70-2-/- mice more prevalent, but the chromosomes were more distinct than in spreads from C57BL/6N Hsp70-2-/- mice. In the latter animals, meiotic chromosomes appeared to be diffuse and indistinct, and normal spreads were rarely observed.



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  		FIG. 5. Chromosome spreads of spermatogenic cells from the testis of Hsp70-2-/- mice. A) Nuclei were present that contained bivalent chromosomes with chiasmata (arrows) characteristic of diakinesis-metaphase I of primary spermatocytes. B) Nuclei were also present with the haploid complement of metaphase II chromosomes characteristic of secondary spermatocytes.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Spermatid Development Can Occur Without Completion of Meiosis in Hsp70-2-/- Mice

Germ cells in male Hsp70-2-/- mice were reported previously to arrest in development and to undergo apoptosis in the late pachytene and diplotene stages of meiotic prophase I [6, 25]. However, the present study demonstrates that 1) cells with acrosomes stained with PAS or monoclonal antibody MN7 occasionally are present in sections of the testis of Hsp70-2-/- mice; 2) some of these cells have a condensing nucleus typical of step 8–9 spermatids; 3) well-developed spermatozoa are not observed; 4) the nucleus and cell body of acrosome-containing cells in Hsp70-2-/- mice are often larger than those of spermatids in wild-type mice; 5) the average DNA content of MN7-positive spermatogenic cells in Hsp70-2-/- mice is usually twice, but sometimes the same as, that of round spermatids in wild-type mice; and 6) meiotic metaphase I and II chromosome spreads are observed at low frequency in germ cells from Hsp70-2-/- mice.

Although the earlier reports are correct for most spermatocytes in Hsp70-2-/- mice, we conclude that a few spermatocytes escape the block to development, undergo one and sometime two meiotic divisions, begin acrosome formation, and initiate nuclear condensation. Our conclusions are based on several lines of evidence. The PAS procedure stains the carbohydrate moieties in the acrosome and is used commonly to define the steps of spermiogenesis in postmeiotic germ cells [18, 19]. Together with the results from MN7 monoclonal antibody immunostaining [16, 17] and electron microscopy, this provides strong evidence that acrosome development occurs in some germ cells of male Hsp70-2-/- mice. Furthermore, we observed that the nucleus and cell body of cells with acrosomes are often larger in Hsp70-2-/- mice than acrosome-positive cells in wild-type mice. Secondary spermatocytes are about one third larger than spermatids in wild-type mice [19], suggesting that the larger acrosome-positive cells from Hsp70-2-/- mice may not have completed both meiotic divisions. Consistent with this was the finding that most cells with acrosomes in Hsp70-2-/- mice have higher DNA content than comparable cells in wild-type mice. Diploid spermatids approximately twice the normal size also have been observed in mice after treatment with doxorubicin or radiation [26, 27]. Finally, meiotic metaphase I and II chromosomes were seen in spermatogenic cells from Hsp70-2-/- mice. The importance of genetic background was suggested in the observed strain-specific reductions in spermatocyte progression to the metaphase I stage. In both strains, negative selection against meiotic cells resulted in few spermatocytes reaching metaphase II stage. Taken together, these findings provide compelling evidence that some germ cells undergo one or both meiotic divisions in these mice. They also indicate that it is more common for germ cells to complete the first meiotic division than both divisions in Hsp70-2-/- mice, and that initiation of acrosome formation and other aspects of spermatid development do not require completion of both meiotic divisions.

Role of CDC2 in Completion of Meiosis in Male Hsp70-2-/- Mice

Cell cycle checkpoints control the order and timing of transitions through various phases of the cell cycle and ensure that critical events such as DNA replication and chromosome segregation are completed with high fidelity [28]. One of these checkpoints is present at the G2/M-phase transition and regulates entry into cell division. MPF (maturation-promoting factor, metaphase-promoting factor, or M-phase factor) is a protein kinase that has a pivotal role in this process [29]. It consists of a heterodimer of CDC2 catalytic and cyclin B1 regulatory subunits. Most pachytene spermatocytes fail to complete the G2/M-phase transition in Hsp70-2-/- mice [6] and lack assembled CDC2-cyclin B1 heterodimers and protein kinase activity [9]. However, addition of recombinant Hsp70-2 protein to homogenates of testis from Hsp70-2-/- mice results in assembly of the CDC2-cyclin B1 heterodimer and protein kinase activity. Furthermore, Hsp70-2 is bound to CDC2 but not to cyclin B1 or to the MPF complex in homogenates of testis from wild-type mice. These findings strongly suggest that Hsp70-2 is a chaperone for CDC2 and serves to establish or maintain CDC2 in a conformation competent to form a heterodimer with cyclin B1 and thereby gain protein kinase activity [9].

Because of the evidence that CDC2 has an essential role in G2/M checkpoint function in oocytes and somatic cells, it is surprising that spermatogenic cells in Hsp70-2-/- mice occasionally are able to begin spermatid development. This suggests either that the activation of additional kinases is involved in the G2/M transition in spermatocytes (e.g., [30]), or that some CDC2 activity is present in Hsp70-2-/- mice. Because CDC2 can dimerize with A or B cyclins in somatic cells [31], it might dimerize with other cyclins in spermatocytes of Hsp70-2-/- mice to become an active kinase. Mouse spermatocytes contain cyclin A1 that is unique to germ cells [32], and cyclins A2 [33] and B2 [34] present in most other cells. However, little if any cyclin A2 is present in late pachytene spermatocytes [33], and male cyclin B2-null mice are fertile [35], indicating that cyclin B2 is not essential for spermatogenesis. On the other hand, cyclin A1 mRNA is present in late pachytene spermatocytes, and CDC2-cyclin A1 heterodimers are present in mouse testis lysates [32]. This suggests that activation of CDC2 by cyclin A1 in spermatocytes could occur in Hsp70-2-/- mice. However, significant CDC2 kinase activity was not detected in homogenates of testis from Hsp70-2-/- mice [9]. In addition, intercellular bridges allow transcripts and proteins to move freely between spermatogenic cells [36], and it is not clear how cell-autonomous CDC2 activation might occur in this situation.

Meiosis and Spermatid Development in Drosophila and Mice

There are strong parallels between spermatogenesis in Hsp70-2-/- mice and in Drosophila with mutations in cdc2 [10, 11] and twine, a CDC25 homologue [12, 13]. CDC25 is a cell cycle protein phosphatase that participates in activation of the CDC2 kinase at the G2/M transition [29]. Meiosis is blocked in flies with mutations in cdc2 or twine [12, 37], but spermatogenic cells undergo flagellar elongation and changes in nuclear shape and condensation typical of spermatid differentiation. As in Hsp70-2-/- mice, completion of spermiogenesis fails in flies with these mutations and results in sterility. However, there are also significant differences between what occurs in mice and flies. Acrosome formation in Hsp70-2-/- mice occurs after one or sometimes two meiotic divisions, whereas spermatid differentiation occurs without a meiotic division in the mutant flies [13, 14]. In addition, chromosomal recombination occurs during spermatogenesis in mice but not in Drosophila. Checkpoint mechanisms that monitor DNA integrity during meiotic recombination in mice may prevent activation of the spermatid developmental program until after the first meiotic division. However, Drosophila apparently are free of this constraint and able to begin spermatid development prior to meiotic division.

Another significant difference between these species occurs in their patterns of gene transcription during spermatogenesis. Genes required for spermatid differentiation in Drosophila are transcribed mainly in primary spermatocytes [37–39], but transcription is quite active during the postmeiotic phase of spermatogenesis in mice [40]. Many of the mouse genes expressed in spermatids encode proteins required for the form and function of spermatozoa. However, some transcripts utilized in spermatids are also present in pachytene spermatocytes of the mouse. These include the acrosomal components proacrosin [41, 42] and acrogranin [43]. Although transcription of genes for spermatid components begins before completion of meiosis in both mice and flies, the present study indicates that the progression of spermatid development is more tightly coupled to completion of the first meiotic division in mice than in flies.

Gene Knockouts with Effects on Meiosis in Mice

Knockouts for nearly 50 genes in mice have been reported to disrupt spermatogenesis either directly or indirectly [44], with meiosis being affected by several encoding proteins involved in DNA repair and recombination. Failure of homologue pairing occurs in mice with disruption of genes for Pms2 [45], Dmc1 [46, 47], or Atm [48–50]. Chromosomal chiasma formation and recombination failed to occur in Mlh1 knockouts [51, 52]. In addition, in Ccna1 knockout mice, middiplotene nuclei were not observed in sections, meiotic metaphase chromosomes were not seen in cytogenetic preparations, and spermatogenesis did not progress beyond the diplotene stage [53]. The effects on meiosis appear to occur earlier in these knockouts than in Hsp70-2-/- mice [4, 8, 25]. Although spermatids or abnormal sperm were seen occasionally in mice with disruption of the Hr6b [54], Mlh1 [51], and Pms2 [45] genes, meiotic divisions were not reported. Partial rescue of the prophase I defects of ATM-deficient mice occurred when these mice were also made deficient for p53 or p21, but germ cell development progressed only to the pachytene stage of spermatogenesis [55]. However, the knockout of the gene for basegin caused most spermatogenic cells to arrest and to degenerate at metaphase of meiosis I, with a small number of germ cells differentiating to step 1 spermatids [56]. Basegin is a transmembrane glycoprotein that participates in intercellular recognition processes in multiple tissues, but its role in germ cell development or meiosis is unknown.

In conclusion, these results indicate that initiation of acrosome formation and spermatid development can occur without completion of meiosis in Hsp70-2-/- mice. Although similar findings have been reported recently for Drosophila with mutations in cdc2 or twine, there are significant differences between the two species. These data suggest that the developmental program for acrosome formation and other events usually associated with the postmeiotic phase of spermatogenesis in mice can begin after the first meiotic division (secondary spermatocytes) in Hsp70-2-/- mice (Fig. 6). In contrast, flagellar elongation and changes in nuclear shape and chromatin organization typical of postmeiotic germ cell development can occur before the first meiotic division (pachytene spermatocytes) in Drosophila (Fig. 6). Completion of meiosis can be bypassed and spermiogenesis initiated in both species when CDC2 activity is disrupted, although this is infrequent in Hsp70-2-/- mice. These findings suggest that significant differences may exist in the G2/M checkpoint of meiosis in germ cells of males compared to other cell types, and that the genetic program for spermatid development is initiated before completion of the meiotic phase of spermatogenesis.



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  		FIG. 6. Relationship between completion of meiosis and initiation of acrosome formation and spermiogenesis in mice and Drosophila. The present study indicates that the developmental program for acrosome formation and other events of spermiogenesis in mice can begin in secondary spermatocytes, and does not require the second meiotic division and completion of the meiotic phase of spermatogenesis. Prior studies in the mouse indicate that transcription for some acrosome constituents begins in late prophase of meiosis I. In Drosophila with mutations in cdc2, twine, or genes that regulate their expression or activity, spermatogenic cells are able to skip the meiotic divisions and undergo flagellar elongation and changes in nuclear shape and chromatin organization. However, unlike the situation in Drosophila, many genes required for acrosome formation and spermiogenesis probably are transcribed in the postmeiotic phase of spermatogenesis in the mouse. Although spermatids as late as step 8–9 of development were seen in Hsp70-2-/- mice, they apparently failed to complete development, possibly because genes required for the latter phases of spermiogenesis are not expressed.


   ACKNOWLEDGMENTS
 
The authors would like to thank N. Kenmotsu (Kyoto University) and Barbara Collins (U.S. EPA) for expert technical assistance. The research described in this article has been reviewed by the National Health and Environmental Effects Research Laboratory and approved for publication. Approval does not signify that the contents necessarily reflect the views of the U.S. EPA, nor does mention of trade names or commercial products constitute endorsement or recommendation for use.


   FOOTNOTES
 
1 This work was supported by grants from the Japanese Ministry of Education, Science and Culture, and the Toyota Foundation. Back

2 Correspondence. FAX: 81 75 751 7529; mori{at}med.kyoto-u.ac.jp Back

Accepted: May 6, 1999.

Received: March 9, 1999.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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