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Gamete Biology |
Department of Life Science,2 Kwangju Institute of Science and Technology (K-JIST), Kwangju 500-712, Republic of Korea
Gamete Biology Section,3 Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, North Carolina 27709
Department of Biology4
Center for Research on Reproduction and Women's Health and Department of Obstetrics and Gynecology,5 University of Pennsylvania, Philadelphia, Pennsylvania 19104
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, gamete biology, gametogenesis, sperm, spermatogenesis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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A single protamine gene is expressed in most mammals, but a second protamine gene is expressed in a few mammals, including mice and humans [1]. The protein encoded by the second gene (Prm2) constitutes about half of the protamine in human sperm and two thirds of the protamine in mouse sperm. To determine if sperm need both protamines, we disrupted the coding sequence of the Prm1 or Prm2 gene in embryonic stem (ES) cells from 129-strain mice and injected the ES cells into blastocysts from C57BL/6-strain mice to produce chimeras. The male chimeras produced 129-genotype sperm, C57BL/6-genotype sperm, and C57BL/6-genotype offspring but no 129-genotype-bearing offspring. Disruption of one allele of either Prm1 or Prm2 resulted in relatively modest reductions in the amount of PRM1 (by 
16% of total protamine) or PRM2 (by 33% of total protamine), respectively. In addition, the amount of PRM1 was reduced (less than 16% of total protamine) in PRM2-deficient sperm. These findings strongly suggest that sperm require a full complement of both protamines to convey the male genome to the next generation [2]. Although sperm are haploid, all 129-genotype sperm were affected because spermatids develop in syncytial clones, share mRNA and protein through cytoplasmic bridges, and are phenotypically diploid [3, 4]. Thus, inactivation of one copy of either Prm1 or Prm2 caused a protein deficit (haploinsufficiency) in all of the sperm produced by a clone.
We had observed that the nuclei of sperm with reduced amounts of protamine were less resistant to chemical disruption than the nuclei of wild-type sperm [2]. In addition, earlier studies reported that PRM2 deficiency correlates with infertility in men [5, 6]. Furthermore, the frequency of sperm with DNA damage is predictive of failure of development of human embryos after intracytoplasmic sperm injection (ICSI) [7]. The present study examines why a reduction in the amount of either protamine results in failure to transmit the male genome to the next generation. We find that the chimeras are unable to produce 129-genotype-bearing offspring because PRM2 haploinsufficiency results in alterations in the organization and integrity of sperm DNA.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chimeric mice were produced as described previously [2]. In brief, the Prm2 gene was disrupted by homologous recombination in ES cells from 129-strain mice, and the ES cells were injected into blastocysts from C57BL/6-strain mice. Each male chimera was paired with one or two C57BL/6 females for 2–4 mo to test fertility. To obtain sperm without killing the chimeras, they were mated with C57BL/6 female mice primed with pregnant mare's serum gonadotropin and human chorionic gonadotropin. The mice were placed together at 0700 h, and the females were euthanized 1–5 h after a copulation plug was found, the uteri were removed, and sperm were flushed from them with PBS (pH 7.4). These sperm were subjected to two separate polymerase chain reaction (PCR) assays to distinguish 129-genotype sperm from C57BL/6-genotype sperm and to determine the relative amounts of 129-genotype sperm with a copy of the intact allele (129-Prm2+) and the mutant allele (129-Prm2-) in the sample [2]. Animal care was in accordance with U.S. Public Health Service guidelines for use of animals, and the Institutional Animal Care and Use Committee, National Institute of Environmental Health Sciences, reviewed and approved all procedures before use.
Comet Assay for DNA Damage
DNA damage was assessed with the single-cell gel electrophoresis (comet) assay [8]. Sperm from chimeric and wild-type mice were suspended in 0.7% low-temperature-melting agarose, placed on a frosted slide, and chilled on ice. The slides were submerged in lysis buffer (2.5 M NaCl, 100 mM EDTA, 10 mM Tris-base, pH 10, and 1% Triton X-100) at 4°C for 1 h, in lysis buffer with 10 mM dithiothreitol (DTT) at 4°C for 30 min, and finally in lysis buffer with 4 mM lithium diiodosalicylate at room temperature for 90 min. The slides were subjected to electrophoresis in 300 mM NaOH and 1 mM EDTA at 25 V for 10 min, neutralized with 0.4 M Tris-Cl (pH 7.5), and stained with ethidium bromide. The experiments were repeated two times, and approximately 600 sperm from each mouse were examined. The slides were observed using a Leica TCS laser scanning confocal microscope (Leica Microsystems, Mannheim, Germany).
Electron Microscopy
Sperm for scanning electron microscopy were isolated from the epididymis and vas deferens and fixed in 2.5% glutaraldehyde and 2% formaldehyde in 0.15 M sodium phosphate buffer overnight at 4°C. They were washed in buffer, collected on Nuclepore filters, subjected to critical point drying, coated with gold/palladium, and examined in a Cambridge S200 scanning electron microscope at 20 KV. For transmission electron microscopy, cauda epididymides were fixed under the same conditions, postfixed in 2% osmium tetroxide in cacodylate buffer, and embedded in Lowicryl resin. Sections were stained with uranyl acetate and lead citrate and examined in a LEO910 transmission electron microscope at 80 KV.
ICSI and Embryo Genotyping
Sperm from the epididymis and vas deferens were suspended in M16 medium containing 0.01% polyvinyl alcohol, filtered through Kimwipes, and resuspended in NIM medium (123 mM KCl, 2.6 mM NaCl, 7.8 mM NaH2PO4, 1.4 mM KH2PO4, 3 mM EDTA, pH 7.2) containing 1% polyvinyl alcohol and 50% glycerol [9]. Sperm from Prm2 chimeric mice and wild-type C57BL/6 mice were sonicated mildly to remove tails, and randomly selected heads were injected in less than 1 pl of medium into metaphase II-arrested eggs from B6D2 mice (progeny derived from matings of C57BL/6 and DBA mice). Injected eggs were transferred into CZB medium [10] and cultured at 37°C under 5% CO2 in air for 3–5 days. The cultured embryos were observed under a dissecting microscope to determine developmental stages and immediately frozen. For subsequent PCR analysis, each embryo was suspended in 10 µl of embryo lysis buffer (1% Tween-20, 1% Triton X-100, 5 mM EDTA, 10 mM Tris-Cl, pH 7.5, 2 mM DTT, and 4 U/ml of proteinase K) and incubated at 65°C for 10 min and subsequently at 95°C for 10 min. A total of 5 µl of lysate was used in PCR reactions as template. To distinguish embryos produced by injecting 129- or C57BL/6-genotype sperm, PCR was performed (35 cycles) using primers (sense, 5'-GATCTTCCTTTATACACAAGTCATAGC-3'; antisense, 5'-GTGGTACAGAACTTAGGTGTTTAATTG-3') that amplify a microsatellite in the D10Mit180 locus of mouse chromosome 10. The PCR products were 134 base pair (bp) for the C57BL/6 genotype, 206 bp for the 129 genotype, and 156 bp for the DBA genotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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We generated male chimeric mice and selected chimeras with zero or a relatively high percentage of 129-genotype sperm for experimental analyses (2 chimeras with 0% 129-genotype sperm as control and the other 2 chimeras with 70% or 90% of 129-genotype sperm as test). None of the chimeras produced progeny with 129-genotype-derived coat color (agouti). The chimeras with 70% or 90% 129-genotype sperm produced more morphologically abnormal sperm (30% and 37%, respectively) than did the chimeras with 0% 129-genotype sperm (average, 8.1%). In addition, staining of sperm nuclei with acridine orange showed that the percentage of sperm with abnormal staining patterns was much higher in the chimeras with 70% or 90% 129-genotype sperm (70% and 79%, respectively) than the chimeras with 0% 120-genotype sperm (0%). Consistent with a previous study [2], these results indicate alteration of sperm structure and nuclear integrity in 129-genotype sperm from the chimeras.
The DNA of Sperm from Chimeras Is Damaged
The comet assay was used to assess the integrity of DNA in individual sperm from chimeras. This assay has been widely used to measure DNA damage in many different cell types, including sperm from laboratory animals and humans [8, 11]. The assay was performed under alkaline electrophoresis conditions to measure single-stranded DNA breaks and alkali-labile sites of DNA. Approximately 80% of the sperm from a chimera that produced 70% PRM2-deficient sperm had a comet tail, whereas 10% of the sperm from a wild-type mouse had a comet tail (Fig. 1). It is unknown how much of the 10% background level was due to prior DNA damage or was caused by preparative procedures. Although we did not measure comet dimensions, comet tail lengths were variable, suggesting that the degree of DNA damage differed between individual sperm. This is consistent with the differences in sperm head morphology noted herein and the differences in acridine orange staining reported previously [2]. There were no apparent differences between intact and sonicated sperm in the frequency and quality of comet tails, suggesting that sonication did not cause additional DNA damage (Fig. 1).
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Frequency of Abnormal Sperm Correlates with 129 Genotype
Scanning and transmission electron microscopy were used to characterize the differences between PRM2-deficient and wild-type sperm. The shape and external appearance of sperm were examined by scanning electron microscopy. Sperm from a Prm2 chimera producing 90% 129-genotype sperm were compared with sperm from wild-type mice (Fig. 2A). The most common differences seen were a decrease in lateral width and a reduced ventral flexure of the head of most sperm from the chimera (Fig. 2B and C). What appeared initially to be globular sperm heads was found on closer inspection to be the result of the heads being folded back onto and fused with the flagellum (Fig. 2D and E).
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Transmission electron microscopy was used to determine the appearance of the chromatin, the association between the acrosome and the nucleus, and the organization and placement of flagellar components. The chromatin in most sperm from a chimera producing 90% of 129-genotype sperm appeared to be loosely packed and heterogeneous in density (Fig. 3B-D). This appearance is more similar to the incompletely packed chromatin in condensing spermatids (data not shown) than to the tightly packed and homogeneously dense chromatin seen in sperm from wild-type mice (Fig. 3A). In addition, the chromatin of sperm from chimeras was often loosely dispersed in the outer portion of the nucleus, the acrosomes frequently were irregular in contour and appeared to be attached loosely to the nucleus, and flagellar components were seen to be abnormally located in close apposition to the acrosome and underlying nucleus (Fig. 3B-D). These results indicate that a reduction in amount of PRM2 causes distinct changes in chromatin packing, in the shape and organization of the acrosome, and in the overall architecture of the sperm.
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129-Genotype Sperm Fail to Produce Viable Embryos
Embryos in the early stages of development were analyzed to determine if there was a relationship between the frequency of DNA damage and the failure of chimeras to transmit the mutant or intact allele to their progeny. The ICSI procedure was used to minimize the number of variables that might be contributing to the failure. Sperm from a chimera or a wild-type mouse were microinjected into metaphase II-arrested eggs, and the ensuing preimplantation development was monitored in vitro to determine if the embryos developed to the blastocyst stage. The chimera produced a mixed population of C57BL/6-genotype (30%) and 129-genotype (70%) sperm, and oocytes were from B6D2 mice (progeny derived from matings of C57BL/6 and DBA mice). Cultured embryos were analyzed by PCR to identify those developing from eggs injected with 129-genotype sperm or with wild-type sperm from the chimera (Fig. 4A). Sperm from a wild-type mouse were injected as controls, and all six embryos that survived injection developed to the blastocyst stage. A total of 33 eggs survived injection with sperm from the chimera, and 12 developed into blastocysts, 7 developed into morula, 10 developed into two-cell to six-cell stage embryos, and 4 underwent fragmentation. PCR analysis determined that of the 19 embryos that developed to the morula or blastocyst stage, only two (11%) developed from eggs injected with 129-genotype sperm. In addition, 12 (86%) of the 14 embryos that developed only to the two-cell to six-cell stage or underwent fragmentation were derived from 129-genotype sperm (Fig. 4B). The frequency of failures in development that occurred for eggs injected with sperm from the chimera and the genotypes of the failed embryos was comparable to the ratio of 129-genotype to C57BL/6-genotype sperm produced by the chimera. The comet analysis was performed on sperm from the same chimera.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Observations at the light microscope level suggested that some protamine-deficient sperm had abnormally shaped heads [2]. By using scanning electron microscopy, we found the frequency of sperm with small or abnormally shaped heads that were loosely enclosed by the plasma membrane correlated with the percentage of PRM2-deficient sperm. Transmission electron microscopy revealed that the chromatin in PRM2-deficient sperm was less compact than chromatin in sperm from wild-type mice and similar to condensing chromatin in late elongating spermatids. These differences might be due to structural changes that result from the reduced level of PRM2, because the positively charged arginine groups on protamines are believed to neutralize the negatively charged phosphate groups on DNA [15, 16]. Therefore, a reduction in the amount of protamine would change not only the stoichiometry of the major components of the chromatin but also the net charge in the sperm nucleus, thereby affecting chromatin condensation and stability.
Considered along with other studies, our results provide insights into the relationship between PRM2 deficiency and developmental failure after ICSI. To date, the minimal requirement for a functional paternal genome to participate in embryogenesis is the sperm nucleus with a structurally intact nuclear matrix. Sperm nuclei stripped of cytoplasmic components and used for ICSI produced embryos that developed to live offspring [17]. However, treatment with a protease or a disulfide-bond-reducing agent before ICSI resulted in developmental failure [18]. We have found previously that PRM2-deficient sperm are immotile [2]. Impaired sperm motility could be due to loss of sperm viability, leading to chromatin deterioration. In the present study, sperm were mildly sonicated to separate heads and tails before ICSI. It is possible that sonication causes additional damage to altered chromatin integrity of PRM2-deficient sperm. Thus, the developmental failure of some of the eggs injected with PRM2-deficient sperm might be an indirect effect due to damages present in dead sperm and/or induced by sonication. Nonetheless, the aberrant nuclear shape and reduced chromatin density of PRM2-deficient sperm strongly suggest that the structural integrity of these nuclei has been compromised, leading to the failure of embryo development.
Incomplete processing of PRM2 was seen in sperm of mice with heterozygous mutations in Prm1 and Prm2 [2] and is frequently seen where there are changes in the level of PRM1 or PRM2 or of other nuclear components. This was observed in sperm of mice with null mutations in Tp1 [19], Tp2 [20], and Camk4 [21, 22]; with suppression of Prm1 translation in MSY4 gain-of-function transgenic mice [23]; with premature translation of PRM1 in transgenic mice [24]; and in sperm of infertile men [6, 25]. Although infertility occurred in some of these studies [23, 24], it was not seen in others [19–21], suggesting that incomplete processing of PRM-2 is not a major cause of developmental failure that occurs after ICSI with PRM2-deficient sperm.
The defects in condensation of sperm chromatin seen in mice with heterozygous mutations in Prm1 and Prm2 [2] also occurred when a transgene-expressing avian protamine was expressed in spermatids [26]; in null mutations of Tp1 [19], Tp2 [20], and Csnk2a2 [27]; with suppression of Prm1 translation in MSY4 gain-of-function transgenic mice [23]; and in sperm of some infertile men [28]. Since infertility was seen in only some of the studies that observed incomplete DNA condensation [2, 23], defective chromatin condensation alone might not be sufficient to prevent participation of the male genome in development. However, more severe defects in chromatin condensation that include disruption of the nuclear matrix might have an effect on DNA integrity.
PRM2 constitutes from 0% to nearly 80% of the total protamine in sperm from different mammalian species, indicating that PRM1, PRM2, and DNA interact to form complexes in different species with considerably dissimilar stoichiometries [1]. Although the DNA of mammalian sperm is packaged in toroidal subunits substantially larger than nucleosomes [29], protamines do not interact with each other in a consistent manner, and their binding to DNA is sequence independent [1]. However, for a given species, the assembly of protamine and DNA into complexes is likely to have precise stoichiometries. Although spermatids in mice can compensate for overexpression of PRM1 [30] and expression of high levels of avian protamine [26], they fail to compensate for reduction in the amount of PRM1 or PRM2 [2, 21, 23]. This suggests that the set points for expression of PRM1 and PRM2 are near the minimum required for complex formation and DNA protection.
PRM2-deficient sperm have a reduced amount of PRM1, indicating that PRM1 requires the normal amount of PRM2 to become incorporated into sperm chromatin. Although the mechanism responsible for the DNA damage in PRM1/PRM2-deficient sperm remains to be determined, we believe a likely scenario is that haploinsufficiency of PRM2 perturbs the stoichiometries of complex formation by major components of the chromatin. This results in incomplete chromatin condensation, compromising the structural integrity of the nuclear matrix. This condition is exacerbated by a decrease in disulfide bond formation due to the reduced amount of PRM1 and PRM2. As a result of these changes, the DNA is highly susceptible to damage in the reproductive tract by conditions that can be tolerated by sperm with a full complement of nuclear components but not by PRM1/PRM2-deficient sperm.
In summary, we found that 1) the percentage of sperm with damaged DNA seen with the comet assay, 2) the frequency of sperm with abnormally shaped heads seen by electron microscopy, 3) and the percentage of metaphase II-arrested mouse eggs that failed to develop to the blastocyst stage after ICSI were comparable to the percentage of PRM2-deficient sperm in the samples analyzed. Although similar results have been seen individually in other studies, we believe this is the first study to use an animal model to demonstrate that they occur together. These findings strongly suggest that a moderate reduction in protamine levels in sperm results in damage to DNA incompatible with transmission of the male genome to the next generation.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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6 Correspondence: E. M. Eddy, Gamete Biology Section, Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC 27709. FAX: 919 541 3800; eddy{at}niehs.nih.gov
Received: 31 December 2002.
First decision: 23 January 2003.
Accepted: 26 February 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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