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Testis |
Department of Experimental Radiation Oncology,4 The University of Texas M.D. Anderson Cancer Center, Houston, Texas 77030
Institute for Biogenesis Research,5 University of Hawaii Medical School, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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embryo, fertilization, sperm, spermatogenesis, testis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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To investigate whether sperm production could continue in the absence of either TP, two lines of transgenic mice were generated, each carrying a null mutation for either Tnp1 or Tnp2 [1, 2]. Epididymal sperm were recovered from male mutants homozygous for either the Tnp1- or Tnp2-null allele. The sperm counts were not reduced, and the males were fertile; however, their fertility was lower than normal. Chromatin condensation was abnormal during spermiogenesis in both mutants. Morphological abnormalities were also evident. The requirement of TPs for the production of functional sperm was demonstrated in Tnp1- and Tnp2-null double mutant males, which had major defects in sperm production [3].
Single-knockout mutants had only subtle changes in phenotype, which suggested the TPs had functional redundancy, i.e., one TP could functionally compensate for the absence of the other. However, in those studies, determination of whether phenotypic changes were due to the absence of a specific TP or to an overall decrease in total TP protein levels was not possible. Further, it was not known if the morphological abnormalities explained the decrease in male fertility and whether abnormalities were associated with a specific TP. To investigate whether TPs fulfilled unique roles or if their functions were entirely redundant required the characterization of sperm from mice with different levels of both of the Tnp genes and the TP proteins and a comparison of such results to those from single- and double-knockout mutants. Because TP1 is more highly expressed than TP2 (approximately 2.5-fold), we can obtain mice with many different levels of total TP protein from different combinations of null Tnp1 and Tnp2 genes. Examination of the phenotypes of sperm from single Tnp heterozygotes, single Tnp nulls, double heterozygotes (+/–)(+/–), mice heterozygous for one Tnp and null for the other (+/–)(–/–) and (–/–)(+/–), and the double null should allow us to relate the specificity and severity of the effects to the differing amounts of protein and the presence of specific TPs. Changes in sperm morphology could also be assigned to specific changes in expression and protein levels.
Here we report the function and morphology as well as reproductive potential of epididymal sperm from all Tnp-null mutant genotypes and present evidence that suggests each TP fulfills some unique function during spermiogenesis even though sperm phenotypes strongly indicate defects are largely attributable to an overall gene dosage effect.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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All procedures were approved by the M.D. Anderson Cancer Center institutional animal care and use committee. Mice carrying both mutations were generated through the mating of 129S-Tnp1tm1Mlm [1] homozygous null females with 129S-Tnp2tm1Mzh [2] heterozygous males. Resultant double heterozygous males (Tnp1(±) Tnp2(±)) were initially crossed with females of the same genotype to produce eight mutant genotypes and wild-type controls; further production of mice continued by breeding various pairs of genotypes to achieve a nearly equal production rate of the nine genotypes. Overall, about 15% of all male mice exhibited testicular abnormalities, mainly unilateral atrophy and tumors. Data from these affected animals were not included in the final analysis.
Fertility Testing
Mutant males 8–9 wk of age were housed with 6- to 8-wk-old virgin C57BL/6 females for 8 wk. The numbers and sizes of litters were recorded. Single heterozygotes, previously reported to have normal fertility, were used as concurrent, positive controls. Values from the concurrent group did not differ from those of the historic groups [1, 2] and all data were combined. Females paired with males that never produced litters were subsequently placed with males of proven fertility and, if they produced a litter, were considered to be fertile and included in the study; otherwise, they were excluded.
Sperm Counts
For epididymal sperm counts and motility and viability assessments, cauda epididymides were isolated from males ranging in age from 8.6 to 34.9 wk and placed in a 250-µl drop of HEPES-buffered, calcium-free, modified Krebs-Ringer sperm medium (mKR, 300–310 mOsmolar) [4, 5] supplemented with 0.5% fatty acid-free bovine serum albumin. Epididymides were dissected and sperm were recovered in the medium under oil at 37°C. Clumps of sperm that occurred in double-mutant animals were counted as one.
Assessment of Motility and Viability
An aliquot of sperm sample in the above medium was loaded onto a Cell-Vu (Fertility Technologies, Inc., Natick, MA) counting chamber prewarmed to 37°C. More than 100 cells were examined in duplicate samples and judged to be progressively motile, motile without progression, or nonmotile. Viability was determined by assessing the permeability of the plasma membrane with a commercially available dual fluorescent stain (Sperm Viability Stain, Molecular Probes, Inc., Eugene, OR) using a protocol modified from that given by the manufacturer [6]. A total of >200 cells per sample were counted using an epifluorescent microscope equipped with 415- to 490-nm excitation and 515-nm long-pass emission filters. Sperm displaying green fluorescence were considered viable and those with red fluorescence nonviable.
Sperm Morphology
Air-dried smears of sperm isolated in mKR without BSA were stained with hematoxylin solution, and were viewed at 1000x magnification. Sperm head or tail morphology was determined independently for each mouse by counting more than 100 cells when judging head abnormalities and more than 200 cells when judging tail abnormalities. The percentage of sperm nuclei with noticeably less staining was also determined on more than 100 sperm (range 112–404 cells) from 2–4 animals of each genotype.
To assess whether tail abnormalities were a result of altered membrane permeability or more permanent structural changes, sperm isolated in BSA-free mKR medium were exposed to either 1% Triton X-100 or 1% sodium dodecyl sulfate (SDS). Between 100 and 400 sperm were assessed for all morphological characteristics in wet, coverslipped preparations.
Nuclear Staining with DNA-Specific Probes
Air-dried smears of caput and cauda epididymal sperm in calcium- and magnesium-free PBS were prepared, and DNA was stained by applying a coverslip using commercially available antifade mounting medium containing either propidium iodide (PI, 1.5 µg/ml) or 4',6-diamidino-2-phenylindole (DAPI, 1.5 µg/ml) (Vectashield, Molecular Probes, Inc.). The fluorescence was scored as abnormal if the sperm head was visibly less bright than the majority of others on the slide.
Recovery of Basic Nuclear Proteins and Separation by Electrophoresis
Cauda epididymides from 3–5 mice (age range 18–67 wk) were minced and sperm recovered in calcium- and magnesium-free PBS. Contaminating epididymal tissue was removed by filtering through an 80-µm screen. Basic nuclear proteins were recovered, modified with ethyleneimine, and analyzed by acid-urea polyacrylamide gel electrophoresis as previously described [2, 7, 8]
Electron Microscopy
Samples were prepared for electron microscopy as previously described [1, 2]. Areas of embedded tissue were chosen based on light microscopy of thick sections, and thin sections displaying silver-gold interference colors were cut from those areas and viewed using the electron microscope.
Intracytoplasmic Sperm Injection (ICSI)
Sperm and oocyte recovery and sperm injection into the oocyte were performed as previously reported [9]. Five to six hours after sperm injection, the oocytes were examined with a dissecting microscope and those with two distinct pronuclei and the second polar body (distinguishable from the first polar body by its clear contour) were considered normally fertilized. Normally fertilized oocytes were cultured 1–3 days more and their development assessed each day. Separate experiments of fertilization and embryo culture were done using contralateral epididymides from each mouse.
Assessments of Postimplantation Loss
Embryo survival after implantation was assessed both with oocytes fertilized in vivo by natural matings or in vitro by ICSI. Males with genotypes previously shown to produce functional sperm capable of in vivo fertilization (single Tnp1- or Tnp2-knockout mutants) were housed with B6D2F1 females. Females were checked daily for a copulation plug, and those that had them (always within 7 days of pairing) were held until Day 14 or 17 postcoitus when they were killed and the uterus inspected for nonviable or viable offspring. Males were housed alone for 2–4 days before a new female was introduced. A total of 2–4 females were mated with each male. For males with genotypes that rendered them infertile, as well as Tnp1-null mutants and wild-type controls, oocytes were fertilized by ICSI (as described above) and resultant embryos were transferred to pseudopregnant females. Recipients were killed on Day 19 and Cesarean sections performed. Total numbers of implantation sites were recorded and live fetuses obtained were raised by lactating CD-1 foster mothers.
Statistical Analysis
Results were expressed as mean values ± SEM. Statistical significance of values in the mutant mice vs. the wild type was analyzed with one-way ANOVA, and differences between mutant genotypes were compared by Tukey honestly significant difference (HSD) adjustment for multiple comparisons. Significant differences in the percentage of mice that were fertile among the genotypes were determined using Fisher exact test and, because of multiple comparisons (n = number of comparisons), normally acceptable P-values (
= 0.05) were adjusted using the Bonferroni correction (n = 8; = 0.05/8 = 0.006). Significance of differences between fluorescence of caput sperm from different genotypes were determined using the one-sample t-test with values compared with zero. Significance of differences between fluorescence of caput and cauda sperm from the same genotype were determined using the paired t-test (PI stain) or unpaired t-test (DAPI) and normally acceptable P values (0.05) used. Significance of differences in fertilization rates, in vitro development of embryos, and postimplantation development resulting from ICSI were determined using 
2 analysis or Fisher exact test. Bonferroni corrections were used to adjust the levels of significance as follows: n = 10, 0.05/10 = 0.005 when five genotypes were compared or n = 6, = 0.05/6 = 0.008 when four genotypes were compared.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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To determine and rank the overall fertility of males that had reduced numbers of Tnp genes, the average number of total pups produced per mouse was calculated for each genotype and called the fertility index (Table 1). Four genotypes homozygous for null alleles of at least one of the Tnp genes were significantly less fertile than wild-type mice. Additionally, the fertility index demonstrated that the (–/–)(+/+)-mutant males were significantly less fertile than the (+/+)(–/–)-mutant males (P < 0.02). Mice with one or zero copies of a Tnp gene (severe genotypes) were sterile.
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Impairment of Male Fertility Associated with Poor Epididymal Sperm Quality
Of all the genotypes, only sperm counts from (–/–)(+/–) and (–/–)(–/–)-mutants (12 ± 1 million and 0.2 ± 0.1 million, respectively) were significantly lower than counts from age-matched wild-type controls (25 ± 2 million) (Fig. 1A). The percentage of sperm that displayed forward progression was reduced in Tnp2-single knockouts and the percentages of motile sperm, sperm with forward progression, and viable sperm were significantly lower in mice with (–/–)(+/+), (+/–)(–/–), (–/–)(+/–), and (–/–)(–/–) genotypes than in wild-type mice (Fig. 1, B–D). Additionally, viability and overall motility of sperm from (–/–)(+/–) and (–/–)(–/–) mutants were significantly lower than those of sperm recovered from (+/–)(–/–) mutants. Thus, disruption of Tnp1 appeared to be more effective than that of Tnp2 at producing loss of both motility and integrity of the plasma membranes.
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These results indicate that the impaired fertility observed during breeding trials is most likely a result of poor progressive motility and poor viability of epididymal sperm. The viability and motility of sperm from mutants lacking only either TP1 or TP2 were sufficient to allow some sperm to fertilize oocytes, but none of the sperm from the more severe genotypes did.
Prominent Tail Abnormalities in Sperm from Tnp Mutants
Sperm tail abnormalities evident in the Tnp mutants were grouped into five major categories (Fig. 2): sperm with the head bent back onto the midpiece, sperm with the midpiece bent and bridged to itself, sperm tails showing evidence of unraveling, sperm with tails coiled around their heads, and sperm that were clumped together with cellular remnants along their heads and/or midpieces. All others were classified as straight. The percentage of sperm with heads bent back was 3% or less in wild-type or single or double heterozygotes and (+/+)(–/–) mice but was elevated to 19% in (–/–)(+/+) mice (Fig. 3). In (+/–)(–/ –) and (–/–)(+/–) mutants, each possessing only one copy of either Tnp1 or Tnp2, the percentage of such sperm was even higher, about 30%. The percentage of epididymal sperm with the midpiece bent and bridged to itself was about 29% in both single knockouts compared with 6% in wild-type controls. The numbers of sperm with bridged midpieces declined in the more severe genotypes probably because of the increase in more severe defects, such as coiled sperm or clumps of sperm. The percentage of sperm with portions of the flagella unraveled was increased (14%) only in Tnp2-single knockouts and in double knockouts (14%), whereas it was about 3% in all other genotypes. The percentage of epididymal sperm with their tails coiled around their heads was elevated only in the more severe genotypes, increasing to 12% in mutants expressing one copy of Tnp1 ((+/–)(–/–)) and then to 27% in (–/–)(+/–) mutants and double knockouts. Sperm that apparently failed to individualize at spermiation were recovered as clumps from the epididymides. The percentage of sperm that were in clumps, which ranged from 0% to 1% in wild-type, heterozygotes, and single knockouts, reached 10% in (+/–)(–/ –) and about 18% in (–/–)(+/–) and (–/–)(–/–) mutants. Among the severe genotypes, approximately 70% of clumps contained two or three sperm. The percentage of clumps in which attachment involved heads was 87% ± 2% in double knockouts, a significantly higher percentage (P < 0.01) than that in (+/–)(–/–) (63% ± 2%) (P < 0.01). Nearly all sperm produced in the double Tnp null mutants had some form of morphological abnormality.
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In addition, more sperm from Tnp1-single knockouts (22% ± 2%) but not Tnp2-single knockouts (6% ± 1%) had a bent or blunted shape at the normally sharply pointed apex of the sperm nucleus than did wild-type or heterozygotes (range 4–5%) [1, 2]. This same head malformation was even more frequent in (+/–)(–/–)-, (–/–)(+/–)-, and (–/–)(–/–)-double mutant animals, with 49% ± 5%, 65% ± 4%, 45% ± 1% of sperm having blunt tips, respectively.
Tail Abnormalities Unaffected by Detergent Treatment
To determine whether tail abnormalities were due to membrane attachments, osmotic effects [10], volume regulatory mechanisms [11], or more permanent structural changes during development, mutant sperm were exposed to either 1% Triton or 1% SDS. Most bending was not alleviated by detergent treatment, and few significant changes were seen in the tail angularity (Fig. 4). However, SDS treatment produced head-tail dissociation in 80% to 85% of wild-type and single- and double-heterozygote sperm, but in the single knockouts, the rate was significantly lower (60% in Tnp1-null mice) and still lower in the more severe genotypes: (+/–)(–/–), (–/–)(+/–), (–/–)(–/–), with 42%, 22%, and 14%, respectively. Because only sperm with attached heads and tails were scored, the stronger attachment of the heads to tails in sperm with abnormal tail morphology could account for the apparent rise with SDS treatment in the percentage of sperm with heads bent back seen in samples from the Tnp1 knockout. The fact that the percentage of sperm from (+/–)(–/–), (–/–)(+/–), and (–/–)(–/–) mutants that had heads bent back or were clumped or coiled (coiled data not shown) was not alleviated by detergent treatment indicates that sperm were held together by structural elements that develop in mutant sperm in response to decreased levels of TPs.
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Nuclear Proteins in Epididymal Sperm
Protamine precursors were absent in the wild-type sperm samples, but incompletely processed protamine 2 precursors [12] were present in epididymal sperm from single-knockout mutants [1, 2]. The partially processed precursors represented 30% of protamines in (+/+)(–/–) mutants and 39% in (–/–)(+/+) mutants. Only low levels were observed in some heterozygotes (e.g., 8% in double heterozygotes) (Fig. 5). In samples from the severe genotypes, the full-length protamine 2 precursor was present at high levels (32% (±)(–/–); 39% (–/–)(+/–)) with partially processed forms of the precursor constituting 24% and 25% of protamines, respectively. Despite the defects in protamine 2 processing, the ratio of total protamine 2 to protamine 1 remained between 1.9 and 2.6 and therefore appeared to be independent of the Tnp mutations.
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No data on distribution of protamine types could be obtained from double-knockout epididymal sperm because of low protein yields. Only about 0.1 pg protein per sperm head was isolated from double-knockout sperm compared with 2.1–6.7 pg/head from all other genotypes and 2.8 pg per head from data in the literature on wild-type epididymal sperm [13]. Whereas no histones were detected in the epididymal sperm of wild-type, single heterozygotes, and single knockouts, 2% of proteins isolated from (+/–)(+/–) mutants and about 9% of that recovered from (+/–)(–/–) and (–/–)(+/–) mutants were histones.
Ultrastructure of Nuclei and Tails of Mutant Epididymal Sperm
To determine effects of Tnp mutations on chromatin condensation, sperm within the cauda epididymides were examined by electron microscopy. In wild-type controls, sperm chromatin was uniformly dense (Fig. 6A). From both single-knockout mutants, chromatin was relatively highly condensed (Fig. 6, B and C, white arrowheads); however, the translucent areas in some sperm (asterisks) suggested incomplete or abnormal condensation. In (+–)(–/–) (Fig. 6D) and (–/–)(+/–) (Fig. 6E) mutants, relatively smoothly condensed chromatin was occasionally identified (white arrowhead); however, translucent areas within many nuclei were larger and more numerous (asterisk), suggesting that chromatin was less condensed than in single-knockout mutants. In double-knockout mutants (Fig. 6F), chromatin was packaged into distinct focal units evenly spaced throughout the nucleus (white asterisk, Fig. 6F). In some sperm, nuclear membranes were disrupted and what appear to be units of chromatin were found outside the boundaries of the nucleus (black arrowheads, Fig. 6F). The inner acrosomal membrane was pulled away from the sperm head in epididymal sperm from (–/–)(+/+) and more severe mutants (Fig. 6, black arrows in C, D, E, and F); however, acrosomes of sperm from (+/+)(–/–) mutants (Fig. 6B) appeared unaffected.
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Morphological abnormalities in the configuration of the tail, initially identified at the light microscopic level, were further characterized by electron microscopy. Electron microscopy of sperm in which the head was bent back (Fig. 7B) or the midpiece was bent (Fig. 7C) revealed a single outer membrane that enclosed the abnormalities within a common cytoplasm. Also, outer dense fibers lay just beneath the sperm plasma membrane in an area of missing mitochondria (Fig. 7D); the resulting spread of these fibers may account for the unraveling observed by light microscopy. Additionally, the normal pattern of repeated gyres of the highly compact mitochondrial sheath was abnormal and disorganized (Fig. 7C), as suggested by light microscopy (see Fig. 2F). In contrast, the ultrastructure and position of microtubules, outer dense fibers (except where unraveled in the midpiece), and fibrous sheath appeared normal in all the mutants (not shown).
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Irregular Staining of Sperm Heads from Tnp Mutants
The initial study of TP1-knockout mutants reported that some sperm stained abnormally darkly with hematoxylin. However, we now believe that, at least on the 129 genetic background, the presence of abnormally light or poorly stained nuclei of epididymal sperm is more common and reproducible. In smears, cauda epididymal sperm from wild-type, single and double heterozygotes, and Tnp2-null males, 100% of the nuclei stained well with hematoxylin and the staining intensity was uniform (data not shown). However, only 87% of sperm from Tnp1-null and (+/–)(–/–) mutants appeared to have normal nuclear staining. The percentage of normally stained sperm further decreased in (–/–)(+/–) and (–/–)(–/–) mutants (
77%).
Nearly all epididymal sperm treated with either PI (Fig. 8) or DAPI (data not shown) from wild-type and Tnp2-knockout mutants fluoresced normally (100% and 99%, respectively). The percentages fell to about 85% in (–/–)(+/+) and (+/–)(–/–) mutants (the differences from controls were statistically significant in the case of DAPI) and to 55–59% or 68–84% in (–/–)(+/–) and (–/–)(–/–) mutants, respectively.
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To determine if the decline in nuclear DNA fluorescence occurred in earlier stages of sperm development or developed during epididymal transit, caput sperm were also assessed for fluorescence intensity. The percentages of caput sperm in Tnp1-null, (+/–)(–/–), and (–/–)(+/–) mutants that showed normal fluorescence intensities were about 96% (significantly different from wild-type for DAPI), 93% (significantly different from wild-type for PI), and 81% (significantly different from control for both dyes). Most importantly, for Tnp1-null, (+/–)(–/–), and (–/–)(+/–) sperm, the percentages of cauda sperm showing normal PI fluorescence were significantly lower than for caput sperm. Similar results were obtained with DAPI.
Fertilization and Embryo Development Impaired Following Injection of Mutant Sperm
To determine whether mutant sperm could normally fertilize oocytes, ICSI was performed using cauda epididymal sperm (Table 2). Whereas sperm from (–/–)(+/+) and (+/–)(–/–) mutants fertilized normally, fertilization rates decreased by about 10% when sperm from (–/–)(+/–) mutants were injected and drastically when sperm from double knockouts were used.
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Zygotes produced using epididymal sperm from the two genotypes demonstrating decreased fertilization also showed decreased cleavage rates and in the development of the two-cell embryos remaining in culture to morula/ blastocyst stage, although the limited number of embryos from double-knockout mutants precluded statistical analysis with much power. Thus, fertilization and embryo development were not blocked at one particular step; rather the Tnp-null mutations had a continuous effect on each step of development.
To further assess the developmental potential of embryos derived from ICSI, embryos at the morula/blastocyst stage were transferred into pseudopregnant females (Table 3). The pregnancy percentage for recipients as well as the percentage of implants producing viable offspring was lower for embryos produced with Tnp1-null sperm than wild-type. Although the percentages of implantation and survival of implants were lower than controls for (+/–)(–/–) and (–/–)(+/–), the differences were not significant.
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Postimplantation loss was also evaluated in studies in which wild-type and single-knockout males were paired with females (Table 4). As expected, significantly fewer females became pregnant following mating with Tnp1-null males, but the small differences in postimplantation loss among the three genotypes tested were not statistically significant.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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Because TP1 is expressed at levels nearly 2.5 times that of TP2, each wild-type Tnp1 allele expresses 35% of the total amount of TP and each Tnp2 allele expresses 15%. Furthermore, no changes in RNA levels from the unaltered Tnp alleles were detected in testes of single heterozygotes and single-knockout mutants, the levels of the TP protein corresponding to the null allele were half that of wild-type in single heterozygotes, and the levels of the remaining TP in step-12 and -13 spermatids appeared to be the same in single-knockout mutants as in wild-type (see Note Added in Proof). Though not all genotypes have been tested, we believe that it is valid to calculate the levels of TP proteins in step-12 and -13 spermatids in Tnp mutants of all genotypes based on the gene copy number and the relative levels of TP1 and TP2 in wild-type mice (Table 5).
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Although, the severity of the defects appeared in general to increase with decreasing levels of total TP protein in the step-12 and -13 spermatids, evidence for a unique function(s) of TP2 emerged when phenotypes of specific mutant genotypes were compared. For example, in (+/–)(–/–) and (–/–)(+/+) mutants, the comparable levels of either TP1 or TP2 (35% and 30%, respectively), expressed on the null background of the other had different effects on the phenotype: Mutants with only one copy of Tnp1 but no Tnp2 had severe sperm defects and were completely infertile, whereas those with two copies of Tnp2 but no Tnp1 had lower levels of sperm defects and remained moderately fertile (Table 5). As another example, double heterozygous mutants (50% of protein levels) were normally fertile, and sperm from these mice appeared to be morphologically normal with normal motility and viability. In contrast, TP2 single-knockout mice (70% of protein levels) were less motile and had more abnormalities than those from double heterozygous mutants and the mice were subfertile. That sperm with one copy of each Tnp were superior to those with two copies of Tnp1 and no Tnp2 further supports a function preferentially fulfilled by TP2. Furthermore, the observation of a specific morphological defect associated with the absence of a given TP, such as unraveled sperm midpiece fibers in (+/+)(–/–) mutants, supports a preferential role for TP2. The results presented here provide evidence for some level of redundancy between the TPs so that, in the absence of one TP, the other can compensate to some extent to preserve fertility. However, although individual function(s) of each transition protein remains unclear, TP1 and TP2 are not sufficiently redundant to fully compensate for one another and each likely fulfills some unique roles.
Additionally, this study presents data demonstrating which phenotypic characteristics of Tnp mutants are most closely associated with the impaired fertility of Tnp mutants. Sperm counts did not appear to be a factor, but reduction in progressive motility and sperm tail abnormalities, which were significantly altered in the Tnp2-knockout mice, the first genotype to show a reduction in fertility, did appear to be related to fertility. Sperm nuclear morphology, nuclear condensation, acrosomal integrity, and DNA integrity, which were first significantly altered in Tnp1-knockout mice, the first genotype that shows a drastic decline in fertility, may also have been involved. The increased severities of all of these defects in the double-mutant mice likely contribute to the complete disappearance of in vivo fertility of those mice. Although the amount of the mature protamine 2 decreased with increasing infertility, this was an unlikely direct cause of the sterility because normal fertilization and development occurred following ICSI with testicular spermatids from (–/–)(–/–) mice [3]; however, the defects in protamine 2 processing could have led to some of the other defects described above.
We were surprised that some Tnp-mutant sperm were lightly stained or unstained with DNA-specific dyes, as had been the case in mice with abnormalities in protamines [14, 15]. Here, we studied 3 stains—hematoxylin, PI, and DAPI, which bind by different mechanisms. Hematoxylin binds to DNA phosphoryl groups within the nucleus [16], PI intercalates among DNA strands, and DAPI preferentially binds to the minor groove [17, 18]. Interestingly, the loss of staining appears to occur during transport because sperm recovered from the testis of double-null mutants stain normally (unpublished observations). There are slight reductions in the percentages of poorly stained sperm in the caput, but the major decline occurs between the caput and the cauda epididymis.
The poor staining of some of the Tnp-mutant sperm with DNA probes is unlikely to be a result of interference with the intercalation or binding of the dye due to abnormal DNA condensation in mutant sperm. Flow cytometry experiments actually demonstrated an increase in PI fluorescence in sperm from TP1-knockout mutants [19], the genotype in which an increase in the number of nuclei that do not retain the dye is first elevated. Important, though, is that the population of sperm that fluoresce poorly with PI were not detected in the flow cytometry analysis because these cells were excluded by the threshold set to eliminate background noise. We conclude that the poor staining of individual mutant sperm heads reflects a reduced DNA content as a result of degradation of the DNA. In most of the mutant sperm, DNA was only nicked and chromatin was incompletely condensed, accounting for some elevation in PI staining and DNA degradation had begun, the epididymal sperm that fluoresced poorly were more advanced in this degradative process.
The failure of fertilization and subsequent development after ICSI may also be related to the DNA damage. Embryos derived from ISCI using sperm from (–/–)(+/+) were less likely to implant and produce live young than wild-type. In contrast, postimplantation loss of embryos produced through natural matings with Tnp1-null males was unchanged from wild-type. These results are consistent with the idea that the sperm from these mutants that have suboptimal DNA integrity are selected against during the processes imposed by the female reproductive tract and natural penetration of the oocyte. Further support of the relationship between DNA degradation and failure of development comes from the fact that, when sperm from (–/–)(+/–) mutants, which have the highest incidence of poorly staining sperm of all genotypes, are injected into oocytes, fertilization and preimplantation development are impaired. Finally, the much higher rates of fertilization and embryo viability using testicular sperm rather than epididymal sperm from (–/–)(–/–) mutants [3], combined with the observation that all testicular sperm fluoresced well with DNA dyes (unpublished observations) but many epididymal sperm fluoresced poorly, strengthens the conclusion that DNA degradation was primarily responsible for reduced reproductive potential of epididymal sperm of Tnp mutants when ICSI is used.
Interestingly, the increase in the percentage of sperm with tail abnormalities parallels the rising percentage of poorly staining sperm nuclei. Many of the tail abnormalities occur in the midpiece, where the mitochondria are located. Increased amounts of strand breaks during spermatid development, as evidenced by increased TUNEL staining in the developing spermatids of double-null mutants and inferred by the increase in DNA denaturability in single-knockout mutants, may initiate steps in the apoptotic signaling pathway that induce changes in the mitochondria [3, 19]. Thus, these tail abnormalities may be secondary to the degradation of DNA within the nucleus. The mechanism by which DNA degradation occurs in Tnp-mutant sperm is not known; perhaps the incompletely condensed DNA is not fully protected by the nuclear proteins and so is exposed and susceptible to nucleases within the sperm nuclei [20, 21].
The results from the use of Tnp-mutant sperm for fertilization are clinically applicable. Male-factor infertility is most commonly treated by the use of ICSI. Usually, the etiology of poor sperm in these cases is never identified, but the sperm are characterized by poor motility, incomplete protamine 2 processing, loss of membrane integrity, and DNA fragmentation [22–25]. The suitability of such sperm for use in ICSI has been questioned, and DNA fragmentation in particular was shown to positively correlate with poor fertilization following ICSI [25]. The similarities of sperm defects found in both infertile patients and Tnp-mutant mice show that the multiple defects observed can be caused by the elimination of one or two specific genes. The sperm from these mice provide a valuable tool with which to study the effects on embryo development and pregnancy outcome following fertilization using sperm with poor DNA integrity and may provide valuable information on possible health risks in children conceived by ICSI using sperm with severely compromised DNA.
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NOTE ADDED IN PROOF |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 Correspondence and current address: Cynthia R. Shirley, California National Primate Research Center, University of California, Davis, CA 95616. FAX: 530 752 2880; crshirley{at}primate.ucdavis.edu
3 Current address: Department of Obstetrics and Gynecology, Fukushima Medical University, Fukushima, 960-1295, Japan
Received: 10 March 2004.
First decision: 3 April 2004.
Accepted: 1 June 2004.
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONNOTE ADDED IN PROOFREFERENCES |
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R. Suganuma, R. Yanagimachi, and M. L. Meistrich Decline in fertility of mouse sperm with abnormal chromatin during epididymal passage as revealed by ICSI Hum. Reprod., November 1, 2005; 20(11): 3101 - 3108. [Abstract] [Full Text] [PDF]
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R.-M. Laberge and G. Boissonneault On the Nature and Origin of DNA Strand Breaks in Elongating Spermatids Biol Reprod, August 1, 2005; 73(2): 289 - 296. [Abstract] [Full Text] [PDF]
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