1 Boston College, Chestnut Hill, MA 02167 and 2 Boston IVF, Beth Israel Hospital, Brookline, MA 02146, USA
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Abstract |
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Key words: fertilization failure/Hoechst 33342 dye/human and mouse IVF
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Introduction |
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In light of the moral, ethical, and legal issues involved in working with human gametes, as well as their limited availability, it would be very useful to have an appropriate animal model in which to study the reasons for human fertilization failures. The murine IVF system and embryo culture is widely used as a model for human IVF (Martin-DeLeon, 1989; Alsalili et al., 1997
; Janssenswillen et al., 1997
; Matson et al., 1997
). However, differences may exist between the two species that would make the mouse system a poor model, specifically with regard to methods of centrosome inheritance. During human fertilization the spermatozoon restores the zygotic centrosome (Simerly et al., 1995
), whereas the mouse follows a maternal method of centrosome inheritance (Schatten et al., 1985
, 1986
). This study was undertaken to characterize and describe the distribution of DNA in human oocytes that have failed to undergo normal fertilization and to attempt to evaluate the mouse IVF system as a model to gain insight into reasons for human fertilization failures.
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Materials and methods |
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A total of 63 patients, all having signed informed consent, with 237 failed fertilization oocytes were included in this study. All IVF procedures with oocytes that had failed to undergo normal fertilization were included in this study, and no selection of oocytes based on type of infertility was made. All forms of infertility treated at Boston IVF were part of this study, including male factor, tubal factor, secondary infertility and some unexplained infertility. No oocytes from intracytoplasmic sperm injection (ICSI) procedures were included in this study.
B6SJLF1/J mice, purchased from Jackson Laboratory, Bar Harbor, ME, USA, were used for all experiments. Ovulated eggs were collected from 612 week old females. Ovulation was induced by i.p. injection of 7.5 IU of pregnant mare's serum (PMS, Sigma, St Louis, MO, USA) and 7.5 IU of human chorionic gonadotrophin (HCG, Sigma), 48 h apart. Cumulus-cell complexes were collected from the ampullar oviduct, 1517 h post-HCG into an organ culture dish (Falcon; Allegiance, McGraw Park, IL, USA) containing non-bicarbonated HTF supplemented with 10% plasmanate.
Males were caged individually for at least 3 weeks prior to removing intact caudal epididymides. Spermatozoa were manually squeezed from the epididymis using a 30G needle and tweezers. The spermatozoa were kept in an organ culture dish containing 1 ml of HTF medium at 37°C in an atmosphere of 5% CO2 for 1 h, to allow capacitation to take place. Cumulus enclosed oocytes were inseminated with 1x106 spermatozoa/ml, in 100 µl drops under mineral oil. Insemination took place 11
h post-removal of the oocytes from the oviduct. The gametes were incubated at 37°C and 5% CO2.
Oocytes were incubated for 10 min at 37°C, in human tubal fluid medium supplemented with 0.5% bovine serum albumin and 10 µg Hoechst-33342 dye (bisbenzimide trihydrochloride, Sigma). Oocytes were then placed on a coverslip for viewing. Oocytes were examined using a Zeiss ICM 405 fluorescent microscope with a 100 W arc bulb for epifluorescence. The DNA + H-33342 complex was excited with a 355 nm UV light and epifluorescence emission of 465 nm was viewed and photographed. A G 365 excitation filter, an FT 395 dichromatic beam splitter and an LP 420 barrier filter were used. Both epifluorescent and brightfield photographs were taken using a Sony video graphic printer (UP-870MD), or the video frame grabber function of the IPLab Spectrum Software (Scanalytics Inc., Fairfax, VA, USA). Analysis of chromatin was performed either manually on the Sony photographs or using NIH Imaging software (Research Service Branch of the National Institute of Mental Health, Bethesda, MD, USA) on the video frame grabbed computer images. The chromatin organization of the maternal and paternal genomes were determined from analysis of the fluorescent images.
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Results |
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Discussion |
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In both populations, the majority of failed fertilization oocytes were mature, 80.6 and 85.7% for human and mouse, respectively. At least in this strain of mice (B6SJLF1/J), first polar body degeneration appears to be more prevalent than in human oocytes.
In comparison with mouse, human failed fertilization oocytes show a higher rate of sperm penetration of the plasma membrane, followed by developmental arrest prior to pronuclear development. Human failed fertilization oocytes from patients with greater than 50% normal fertilization rates have a 35.7% sperm penetration rate with subsequent developmental arrest prior to pronuclear formation. Human failed fertilization oocytes have been assessed in several cytogenetic studies, and it was shown that 8.2% (Wall et al., 1996) and 32.3% (Edirisinghe et al., 1997
) of human failed fertilization oocytes contained sperm chromosomes. In contrast, if penetration occurs in failed fertilization mouse oocytes there is only a 1.4% rate of the spermatozoa arresting in development prior to 2-cell formation. These observations suggest that mouse failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte, whereas human failed fertilization oocytes arise from an inability of the cytoplasm to support pronuclear development despite a higher rate of sperm penetration. This could suggest that human spermatozoa more easily penetrate the zona pellucida, but that there is an additional block present in human oocyte cytoplasm that prevents some spermatozoa from developing into functional pronuclei.
Dozortsev et al. (1997) suggested that in some cases, failure of oocyte activation after human ICSI could be due to the relative deficiency of sperm-associated oocyte activating factor (SAOAF) in the selected spermatozoa. It is possible that some human failed fertilization oocytes are the result of a deficiency of activating factors in the spermatozoa; however, this was not specifically investigated in our study. It has also been postulated that failure of fertilization after human ICSI may be due, in part, to poor chromatin packaging and/or damaged DNA of the penetrated spermatozoa (Sakkas et al., 1996).
It is unlikely that the block to pronuclear development in our system is due to cytoplasmic immaturity, since this appears to be associated with an increase in premature chromosome condensation (PCC) after IVF (Calafell et al., 1991). PCC was not observed in the human failed fertilization oocytes. Furthermore, evidence has been provided that the gonadotrophin-releasing hormone analogue/human menopausal gonadotrophin (GnRHa/HMG) stimulation results in an increase in the number of mature failed fertilization oocytes observed over the use of other stimulation protocols (Pieters et al., 1991
).
A potential bias in the results of this study could be the use of epididymal spermatozoa in the mouse IVF system and the use of ejaculated spermatozoa in the human system. Normal fertile mouse offspring have been obtained after ICSI of not only mature (epididymal) and immature (testicular) but also round spermatids, suggesting that genomic imprinting of the male germ cell is complete before spermiogenesis in the mouse (Yanagimachi, 1998). Furthermore, the high fertilization rates using mouse epididymal spermatozoa in IVF (control 2-cell rate of 73.6%) would also suggest that these spermatozoa are mature and can be compared to IVF results using ejaculated spermatozoa.
The observations presented here suggest that in mice, the failed fertilization oocytes arise from an inability of the spermatozoa to penetrate the oocyte, whereas in humans, the failed fertilization oocytes have a higher incidence of sperm penetration but the cytoplasm fails to support pronuclear development. This study highlights observable differences between failed fertilization eggs from human and mouse IVF systems and suggests that the mouse is not a clinically relevant model for human fertilization failures.
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Acknowledgments |
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Notes |
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References |
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Submitted on June 2, 1999; accepted on September 29, 1999.