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BOR - Papers in Press, published online ahead of print November 27, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011304
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BIOLOGY OF REPRODUCTION 68, 1470–1476 (2003)
DOI: 10.1095/biolreprod.102.011304
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Development to Blastocyst Is Impaired When Intracytoplasmic Sperm Injection Is Performed with Abnormal Sperm from Infertile Mice Harboring a Mutation in the Protein Phosphatase 1c{gamma} Gene1

Tyler Daviesa, and Susannah Varmuza2,a

a Department of Zoology, University of Toronto, Toronto, Ontario, Canada M5S 3G5


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Idiopathic azoospermia, characterized by abnormal spermatogenesis, is commonly treated by performing intracytoplasmic sperm injection (ICSI) with sperm retrieved from testicular biopsies. However, no controlled experiments have been performed using an animal model to assess the efficacy or safety of the procedure. We have performed ICSI with testicular sperm obtained in a similar manner from testes of male mice homozygous for a null mutation in the protein phosphatase 1c{gamma} gene (PP1c{gamma}) or those of their wild-type littermates. PP1c{gamma} mutant testicular sperm are less resistant to sonication than are wild-type sperm and display a range of morphological abnormalities, similar to those reported for testicular sperm from idiopathic azoospermic men. PP1c{gamma} mutant sperm are unable to support development to the blastocyst stage, resulting in arrested development either before or just after compaction. A comparison of testicular and epididymal sperm from wild-type males revealed that the epididymal sperm caused embryos to fragment at an elevated rate. These results suggest that ICSI with any kind of testicular sperm carries an increased risk of embryo fragmentation and that abnormal testicular sperm has an added risk of embryo wastage at later preimplantation stages.

assisted reproductive technology, early development, embryo, phosphatases, sperm


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Infertility affects 10–15% of human couples and thus represents a significant health problem in terms of quality of life. The development of assisted reproductive technologies over the past 25 yr has provided many infertile couples with the hope of having their own babies. The demand for these services has in part fueled rapid progress in the efficacy of the technologies involved. The speed of progress in our understanding of the basic biology of human reproduction has lagged somewhat behind introduction of the technologies to clinical practice, however. This lag has raised concerns in both the clinical/scientific and general communities about the safety of some procedures.

Approximately half of human infertility can be attributed to male sterility. Causes of male infertility are varied; however, the single largest cause is classified as idiopathic nonobstructive infertility [1, 2]. Men with this syndrome display impaired spermatogenesis, suggesting a fundamental biological defect in testicular function. The majority of these defects are thought to have a genetic basis. Although some limited evidence supports this view [3, 4], only a few mutations, comprising microdeletions of the Y chromosome, have been positively correlated with infertility in about 10% of cases [5–8].

Significantly more is known about the genetics of male fertility in mice. More than 100 genes have been identified, mainly through targeted mutagenesis, as playing a role in spermatogenesis [9]. Mutations in many of these genes affect only males and have no additional phenotypic consequences, a profile similar to idiopathic nonobstructive azoospermia in humans. These genes are therefore good candidates for mutant alleles in the human population.

One such gene is the protein phosphatase 1c{gamma} (PP1c{gamma}) gene. The only phenotype observed so far for a null allele made by targeted mutagenesis is male infertility [10]. Homozygous PP1c{gamma} -/- males display a number of phenotypes strikingly similar to those of men suffering from idiopathic nonobstructive azoospermia: 1) variable testicular histopathology, with tubules displaying oligozoospermia, maturation arrest, and Sertoli cell only [10] (unpublished); 2) elevated serum FSH [11]; 3) elevated levels of DNA fragmentation in testicular or epididymal sperm and spermatids [12]; 4) teratozoospermia and asthenozoospermia [12]; and 5) elevated levels of aneuploidy in haploid gametes [13]. The similiarity of phenotypic profile suggests that either many azoospermic men have mutations in their PP1c{gamma} genes or these phenotypes represent a suite of defects common to males with impaired spermatogenesis.

The treatment option of choice for infertile men is intracytoplasmic sperm injection (ICSI) [14, 15]. Many ICSI babies have been born worldwide since the introduction of this technology. However, there is controversy surrounding the safety of ICSI. Some investigators claim that the frequency of birth defects among ICSI babies is indistinguishable from that in the general population [16–20]. Others, however, believe that significant risks of either birth defects or low birth weight are associated to ICSI conceptions [21–23].

One confusing aspect to many studies is the lack of distinction between ICSI performed with sperm from obstructive azoospermic patients (normal spermatogenesis) and ICSI performed with sperm from nonobstructive azoospermic patients (abnormal spermatogenesis). Moreover, it is difficult to perform controlled experiments with humans. To assess the developmental consequences of ICSI using abnormal sperm from a known source and under controlled conditions, we performed ICSI with testicular sperm from PP1c{gamma} -/- male mice and their wild-type littermates. In a separate series of experiments, we compared the developmental competence following ICSI of testicular and epididymal sperm. PP1c{gamma} -/- sperm had a profoundly negative impact on preimplantation development. In addition, epididymal sperm was more successful than testicular sperm in promoting development to the blastocyst stage.


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

Mice were bred using standard animal husbandry. Individual males from the Pp1c{gamma} colony were genotyped using a polymerase chain reaction-based method previously described [10]. In our laboratory, the Pp1c{gamma} mutant allele has been propagated in a CD-1 background (Charles River Laboratories, St.-Constant, PQ, Canada). In all cases, the term wild type refers to littermates genotyped as +/+, and mutant refers to animals genotyped as -/-.

The C57BL/6 x DBA/2 F1 (B6D2 F1) females used in these experiments were derived from crossing C57BL/6 females with DBA/2 males. All inorganic and organic reagents were purchased from Sigma Chemical Company (St. Louis, MO) unless otherwise stated. B6D2 F1 females, 4–12 wk of age, were induced to superovulate with an i.p. injection of 5 IU of eCG (Vetrepharm Canada, London, ON, Canada) and 5 IU of hCG 46–48 h apart.

All procedures involving laboratory animals were approved by the Canadian Council on Animal Care.

Oocyte Retrieval

Fourteen to 18 h after hCG injection, females were killed by cervical dislocation, and cumulus masses were dissected free from oviducts and treated briefly with bovine testicular hyaluronidase at a final concentration of 0.5 µg/µl to release individual oocytes. In some cases, oocytes with polar bodies of abnormal size or location were discarded. Oocytes were kept until use in kSOM medium [24] supplemented with 5.56 mM D-glucose and 1% essential amino acids and 0.5% nonessential amino acids (both from Gibco-BRL Life Technonogies, Burlington, ON, Canada) in a 37°C incubator inside a humidified microincubator (Billups-Rothenberg, Del Mar, CA) equilibrated with 5% CO2, 5% O2, and 90% N2. One hundred thirty-seven females were used over the couse of this study, with an average yield of 16.8 oocytes/female.

Sperm Preparation

Sperm from the cauda epididymides or testes were isolated by dissection of the organs from freshly killed males. The tubules of each organ were cut using a sterile razor, teased apart, and suspended in 1.5 ml of nucleus isolation medium (NIM) [25] consisting of 121.6 mM KCl, 7.8 mM NaH2PO4, 1.4 mM KH2PO4, 10 mM EDTA disodium salt, and 0.01% polyvinal alcohol (PVA, cold water soluable) in place of BSA. Medium pH was adjusted to 7.2 by the addition of a small quantity of 1 M KOH in double-distilled water. Cell suspensions were centrifuged for 10 min at 2000 rpm in a Canlab Biofuge A microcentrifuge, and each 50- to 400-µl cell pellet was resuspended in 1 ml of fresh NIM and stored on ice until use. For ICSI, 1–3 µl of mutant or wild-type sperm suspension was diluted in a 50-µl microdrop of NIM under poly(dimethylsiloxane) fluid with a viscosity of 50 centiStokes (Aldrich Chemical Company, Milwaukee, WI). To ensure the highest morphological quality, both mutant and wild-type sperm were individually selected prior to oocyte injection. One male of each genotype was used for each experiment. We performed 45 experiments comparing wild-type and mutant testicular sperm and 23 experiments with epididymal sperm. The epididymal and testicular sperm were obtained from different mice, in most cases.

Intracytoplasmic Sperm Injection

ICSI was performed using a micropipette needle etched in 20% hydrofluoric acid, siliconized, and attached to a PMM-01 piezomicropipette-driving unit (Prima Meat Packers, Tsuchiura City, Ibaraki-ken, Japan) as previously described [26], with the modification that Chatot-Tasca-Ziomek (CZB) medium was replaced by kSOM medium containing 0.01% PVA in place of BSA. For comparisons of wild-type and mutant testicular sperm, each batch of oocytes was divided into two groups, and one oocyte each was injected with either mutant or wild-type sperm. In this way, minor experimental fluctuations in oocyte quality or medium were internally controlled. Embryos were activated by 6-h treatment with 5 mM SrCl2 in Ca2+-free kSOM and cultured in kSOM for 5 days at 37°C in a humidified microincubation chamber equilibrated with 5% CO2, 5% O2, and 90% N2. Development was monitored at the same time each day, and images were captured with a Camedia C-2020 Z digital camera attached to an IX70 inverted microscope (Olympus, Tokyo, Japan) fitted with phase contrast optics.

Sonication of Sperm

Sonication was used to assess the fragility of mutant sperm following the methodology described previously [25]. Sperm from mutant or wild-type males was isolated by first dissecting out the testes and then cutting the tubules using a sterile razor. Tubules were teased apart and suspended in 3 ml of NIM on ice. Each sperm suspension was divided into two samples of equal volume (1.5 ml); one sample was stored immediatly on ice, and the second sample was prepared for sonication. Sperm sample volume was increased to 3 ml, and samples were sonicated for 5 sec at 60% output with a model 250 sonifier (Branson Ultrasonics Corp., Danbury, CT) fitted with a 12-mm-diameter horn. Sonicated and unsonicated samples were centrifuged to pellet cells and then resuspended in 1 ml cold NIM on ice. Sperm counts were performed by introducion of 10 µl of each suspension into a Bright-Line hemacytometer (American Optical, Buffalo, NY).

Statistical Analysis

Data were subjected to standard 2 x 2 chi-square analysis, using categories outlined in Table 4.


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  		TABLE 4. Chi-square comparisons of mouse oocyte/embryo survival at different stages of preimplantation development after ICSI with wild-type (wt) or mutant testicular sperm or epididymal sperm


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
PP1c{gamma} mutant males are severely oligospermic, and most do not shed any sperm into the epididymis [10, 12]. Thus, the only source of mature sperm is the testis. Testicular sperm from mutants are typically abnormal in appearance, with misshapen heads (Fig. 1). There may often be multiple tails. We performed our experiments with the best sperm we could find in any given testicular suspension, using head shape and size as the criteria. Mutant sperm heads were less resilient than wild-type sperm heads and burst more easily in the injection pipette. This fragility was confirmed by counting sonication-resistant sperm heads (Table 1). Wild-type testicular sperm were relatively resistant to sonication, as shown by the negligible change in sperm counts following sonication. Sonication appeared to increase sperm count, probably by releasing clumps. Mutant testicular sperm, however, were highly sensitive to sonication; there was an average of 65% reduction in numbers after treatment. Numbers of sperm for each preparation varied widely, probably because of variation in the efficiency of mincing testes.



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  		FIG. 1. PP1c{gamma} mutant sperm are abnormal in appearance. Sperm retrieved from wild-type (+/+) or mutant (-/-) mouse testes were chosen for ICSI based on appearance. Mutant sperm often appeared abnormal, with misshapen heads and tail abnormalities (not shown). Magnification x1000


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  		TABLE 1. Mutant testicular sperm heads are sensitive to sonication. Testes from four males of each genotype were minced, and cell suspensions were divided into equal portions, one of which was sonicated. Sperm heads in each portion were counted in a hemacytometer. Numbers are sperm heads per milliliter suspension

We injected testicular sperm from PP1c{gamma} mutant males and their wild-type littermates into paired batches of oocytes retrieved from superovulated B6D2 F1 females and followed development of the resulting embryos for 5 days. In a separate series of experiments performed over the same time period but with different wild-type males, epididymal sperm were injected into oocytes, and development of resulting zygotes was monitored. Oocytes were scored for survival through the initial activation stage (Table 2), and the resulting two-cell embryos were scored for developmental progress after 5 days in culture (Table 3).


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  		TABLE 2. Survival of mouse oocytes injected with wild-type or mutant testicular sperm or epididymal sperm


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  		TABLE 3. Development of two-cell ICSI mouse embryos after 5 days in culture.

Chi-square analysis was performed on the data presented in Tables 2 and 3 to determine which aspects of the ICSI procedure were susceptible to developmental failure (summarized in Table 4). Comparisons were made between oocytes fertilized with wild-type and mutant testicular sperm and between oocytes fertilized with wild-type testicular and epididymal sperm. Comparisons were made only between oocytes/embryos that were still alive or developmentally competent with respect to the stage/parameter being tested. For example, embryos that had arrested at the two-cell stage were not included in the comparison of embryos undergoing compaction to avoid obfuscation of the results. Thus, we were able to gain some insight into stage specificity of the paternal effects under investigation. All three classes of oocytes were equally susceptible to lysis (P = 0.28 and 0.11) and activation failure (P = 0.25 and 0.32). Cleavage arrest at the two-cell and four-cell stages was only marginally significantly different in the comparison of wild-type testicular and epididymal sperm (P = 0.031 and 0.046) and was not different when comparing wild-type and mutant testicular sperm (P = 0.169 and 0.985). A highly significant difference in fragmentation rates was observed between wild-type testicular and epididymal sperm (P = 0.00012), but the difference between wild-type and mutant testicular sperm was only marginally significant (P = 0.03). The highly significant difference in fragmentation rate of oocytes fertilized with epididymal and testicular sperm accounts for almost all of the increased rate of development of embryos to blastocysts when epididymal sperm as opposed to testicular sperm was used. In contrast, mutant testicular sperm embryos were not different from wild-type testicular sperm embryos until the eight-cell stage, at which point those fertilized with mutant sperm began to fail. Failure was not confined to arrest of embryos at the eight-cell stage (P = 0.0000116) but was also evident among embryos that began compaction/cavitation but apparently could not complete the process (P = 0.000017).

Embryos fertilized with mutant testicular sperm (Fig. 2) often appeared to have stalled during the compaction process, with some blastomeres remaining uncompacted. Those few that proceeded to form blastoceol cavities did so poorly, often making misshapen cavities reminiscent of parthenogenetic embryos. However, because none of the embryos fertilized with wild-type sperm had this appearance, these dysmorphic embryos probably were not parthenotes, because a similar subset should have then appeared among the controls. Moreover, in a subset of later experiments, oocytes with two pronuclei (2PN) were segregated just after activation. None of the oocytes with one or no pronuclei developed beyond the one-cell stage. However, development of 2PN oocytes fertilized with mutant sperm remained relatively poor, and those few embryos that proceeded to the blastocyst stage displayed the parthenogenetic embryo-like appearance. This finding supports the idea that the mutant paternal genome was defective in its activity rather than in its ability to form a pronucleus. Karyotype analysis would confirm this interpretation.



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  		FIG. 2. Oocytes fertilized with mutant sperm display abnormal morphology. Wild-type B6D2 F1 oocytes injected with wild-type (A, +/+) or mutant (B–F, -/-) sperm were cultured for 5 days until blastocyst formation was maximized. Blastocysts derived from wild-type sperm (A) typically displayed normal morphology, whereas those derived from mutant sperm displayed incomplete (B) or abnormal (C) cavitation. Many embryos derived from mutant sperm arrested at the compacted morula stage, with extruded blastomeres (D). Fragmentation (E) was indistinguishable between the embryos produced with wild-type or mutant testicular sperm but was greatly reduced in embryos produced with epididymal sperm. Some fragmented embryos produced with mutant sperm had fragments of equal size, as if blastomeres had completely failed to compact but had continued to cleave (F). These embryos were nevertheless classified as fragmented. Magnification x300


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We performed strictly controlled experiments in which wild-type and mutant testicular sperm were compared for their ability to support mouse embryo development following ICSI. This is the first study using an animal model of male infertility to test the efficacy of ICSI. Although the ICSI procedure itself negatively affected the overall rate of development, the nature of the experimental design allowed us to discount experimental effects and assess biological effects. The PP1c{gamma} mutant sperm performed poorly compared with wild-type sperm, suggesting an underlying biological defect in the ability of abnormal sperm to support development. The 2 x 2 chi-square tables consisted of focused data, in which only those oocytes/embryos informative for a particular process were analyzed. By looking at the data this way, we were able to gain some insight into the stage specificity of developmental defects caused by the use of abnormal sperm in ICSI. The developmental block first appeared at the eight-cell stage, where increased numbers of embryos fertilized with mutant sperm arrested. However, there were also significantly larger numbers of embryos fertilized with mutant sperm that failed to progress from compaction to cavitation. Moreover, both compacted and cavitated embryos fertilized with mutant sperm displayed abnormalities in morphology (uncompacted blastomeres or irregular blastocoel cavities). The defects in these embryos reflect poor trophoblast formation and or function.

Testicular sperm were less potent than epididymal sperm in generating blastocysts. The defect exhibited by testicular sperm when compared with epididymal sperm was different from the defects exhibited by mutant testicular sperm. Testicular sperm appeared to cause an increased rate of embryo fragmentation when compared with epididymal sperm. The reduction in fragmentation in the group of embryos fertilized with epididymal sperm accounts for almost all of the increased rate of development to the blastocyst stage. (statistical comparisons, Table 4). The only comparison between testicular and epididymal sperm that produced a dramatic difference was fragmentation rate. All other comparisons were either nonsignificant or only marginally significant. This observation suggests that the response of oocytes to sperm from different sources may depend on the type of sperm used, i.e., the problem lies with the sperm rather than the egg. This observation challenges the notion that sperm are mere genome packages.

Other investigators have suggested that there may be a paternal effect when using sperm from sources other than ejaculate [27–30]. However, there is some controversy regarding this issue, with not all investigators reporting negative effects with abnormal sperm in ICSI or in vitro fertilization [31]. Moreover, each study seems to produce a different outcome with respect to developmental stages affected by the use of abnormal sperm. This variation in results from one study to another may reflect variation in underlying causes of infertility. Comparisons of the effect of performing ICSI with sperm from different mouse mutants should determine whether blocks at compaction are common or specific to certain mutations. There also may be species differences in sperm function with respect to development of embryos. For example, mouse sperm does not contribute a centrosome to the oocyte, which contains several endogenous microtubule-organizing centers. Some species differences in sperm genome condensation during passage through the epididymis may also exist [reviewed in 32]. Because the mouse reproductive tract is much smaller than that of the human, mouse sperm have a shorter distance to travel and probably complete the journey much more quickly.

Increased fragmentation of oocytes following activation has been linked with DNA fragmentation [33]. Testicular sperm may still contain DNA strand breaks that have not been sealed, a process that is completed as the sperm progresses through the epididymis [34]. Such an explanation would account for the increased fragmentation observed in embryos injected with testicular sperm compared with those injected with epididymal sperm but would not account for the failure of embryos fertilized with mutant testicular sperm, which displayed a level of fragmentation indistinguishable from that of wild-type sperm. PP1c{gamma} mutant sperm and spermatids display elevated levels of DNA breakage compared with wild-type sperm and spermatids, as measured by TUNEL [12]. This finding suggests that severe DNA strand breakage detectable by TUNEL may have the same effect on embryo fragmentation as the mild, undetectable levels of DNA strand breakage that exist in normal testicular sperm. Comet assays of embryos fertilized with testicular or epididymal sperm may shed some light on these questions.

Although embryo fragmentation is indistinguishable when comparing PP1c{gamma} mutant and wild-type testicular sperm, development to the blastocyst stage is impaired; transitions from uncompacted to compacted embryo and from compacted morula to blastocyst are severely affected. These developmental transitions are coincident with the appearance of the trophoblast. Polarization and adhesion of blastomeres (compaction) may be a prerequisite for trophoblast formation [35], and formation of the blastocoel is certainly dependent on trophoblast function through the action of the NA+K+-ATPase present in trophoblast epithelium. These observations suggest that in addition to defects associated with having been obtained from the testis (i.e., increased embryo fragmentation compared with epididymal sperm), PP1c{gamma} mutant sperm are also impaired in their ability to support the first differentiation event in mammalian embryos, i.e., formation of trophoblast. Thus, either some component of the sperm that is missing from PP1c{gamma} mutants affects trophoblast formation and function or the activity of the embryonic genome is adversely affected by the presence of a PP1c{gamma} mutant paternal genome. Pronuclear transplants could be used to help resolve this issue.

ICSI is widely used to treat male factor infertility. Approximately one-third of infertile men suffer from idiopathic nonobstructive azoospermia, which is characterized by impaired spermatogenesis. The etiology of idiopathic azoospermia is unknown but thought to be genetic [2–8, 36–39]. Limited evidence suggests that there may be some forms of familial male factor infertility [3, 4]. However, apart from de novo microdeletions of the Y chromosome, no other genetic lesions have been found. This lack of progress may in part reflect the nature of the defect, which requires invasive testing to reveal, unlike many other genetic diseases, which produce readily recognizable defects, often affecting children.

There are numerous reports of babies born following ICSI with testicular sperm from idiopathic azoospermic men. There are also some reports of reduced rates of preimplantation embryo development or pregnancy following ICSI with abnormal sperm [27–30]. Given that many clinics transfer embryos at the two-cell stage, embryo wastage due to losses later in preimplantation development would account for reduced pregnancy rates. What is unclear is whether these results can be extrapolated in a blanket fashion to describe the risk factors for all idiopathic azoospermic men.

Testicular histopathology is one way to classify idiopathic azoospermic men as (progressively) normozoospermic, hypospermatogenic with some areas of normal spermatogenesis, maturation arrest, or Sertoli cell only. The various mouse mutations that affect spermatogenesis can be similarly categorized. PP1c{gamma} falls into the hypospermatogenic category, whereas the various meiotic arrest mutations (mlh-1, hsp70-2) fall into the maturation arrest category. Problems with insufficient sampling of testis tissues in humans notwithstanding [40–44], it seems reasonable to assume that mutations in at least several genes cause idiopathic azoospermia in humans. Do they all have the same effect on embryo development, i.e., do they all carry the same risk of embryo wastage?

There are two ways to address this question. One way would be to test other mouse mutations for ICSI success rates. This approach would address the question of whether ICSI with abnormal sperm in general is highly risky or whether problems are confined to a subset of mutations affecting spermatogenesis. A second approach would be to identify those mutations in the human population that cause male infertility and to test their murine counterparts for embryo wastage following ICSI. These experiments may well be worth the effort required to establish the safety of ICSI. Recent reports of increased rates of birth defects [21], increased rates of low birth weight [23], and increased rates of maternal hypertension and preeclampsia [45] following ICSI suggest that some caution should be applied to the use of this treatment for male infertility.


   ACKNOWLEDGMENTS
 
We thank Keith Latham for technical advice on use of the piezo drill and the culture of embryos and for many helpful discussions. We also thank two anonymous reviewers for many insightful comments on our manuscript.


   FOOTNOTES
 
1 Financial support for this work was provided by the Canadian Institutes of Health Research (to S.V.). Back

2 Correspondence: Susannah Varmuza, Department of Zoology, University of Toronto, 25 Harboard St., Toronto, ON, Canada M5S 3G5. FAX: 416 978 8532; svarmuza{at}zoo.utoronto.ca Back

Received: 11 September 2002.

First decision: 20 October 2002.

Accepted: 1 November 2002.


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TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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