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Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection
FERTILITY Copyright
AND STERILITY~ ‘. 1997 Amencan
Society
Vol. 67, No. 2, February for Reproductive
Medicme
Printed
on acid-free
paper
1997
in U. S. A.
Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection*
Dong Ryul Lee, M.S.: Jeoung Eun Lee, M.S. Hyun Soo Yoon, Ph.D. Sung 11 Roh, M.D. Infertility
Research
Center,
Jeil
Women’s
Hospital,
Seoul,
Korea
Objective: To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). Design: Prospective study. Setting: Infertility Research Center, Jeil Women’s Hospital. Main Outcome Measure(s): Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF: and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light micro-. scope. Result(s): Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF., pentoxifylline + FF, and PAF treated groups were 10.5% i- 2.6%, 10.3% 2 1.7%, 16.4% 2 1.8%., 24.8% z 5.6%, 28.4% ? 3.8%, and 33.3% ? 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6%( 3/54), 19.0% (1X8), and 18.9% (10153) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. Conclusion(s): Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI. Fertil Steril@ 1997;67:315-20 Key Words: Acrosome reaction, tion, pronucleus formation
human
sperm, hamster
oocyte, intracytoplasmic
sperm injec-
Mammalian spermatozoa having matured in the male reproductive organ, epididymis, undergo capacitation during exposure to the female reproductive tract, resulting in the physiological alteration
of the plasma membrane (11, and they rneet the oocyte in the ampullary portion of the fallopian tube. Penetration of spermatozoa through the cumulus oophorus could result in an acute exposure to P and
Received January 22, 1996; revised and accepted October 15, 1996. * Presented at the 9th World Congress on In Vitro Fertilization and Assisted Reproduction, Vienna, Austria, April 3 to 7, 1995, where it was granted a poster award.
f Reprint requests: Dong Ryul Lee, M.S., Infertility Research Center, Jeil Women’s Hospital, 1021-4 Daechi-Dong, KangnamKu, Seoul 135-280, Korea (FAX: 82-2-552-7964; e-mail: [email protected]).
Vol. 67, No. 2, February 1997
Lee
et al.
Acrosome
reaction
and ICSI
315
initiation of acrosome reaction (AR), and then interaction with the zona pellucida (ZP) would trigger its completion (2). After penetration, the plasma membrane of acrosome-reacted spermatozoon fuses with oolemma (3). A recent introduction of intracytoplasmic sperm injection (ICSI), the breakthrough for severe male factor infertility and repetitive failure or low percentage of fertilization in the IVF-ET program, showed a higher rate of fertilization and pregnancy than the other microsurgical fertilization techniques, such as partial zona dissection or subzonal sperm insertion (SUZI) (4-6). The mechanism of fertilization in ICSI may be different from that in conventional IVF. It is known that the AR in most of the spermatozoa introduced and penetration of the ZP and plasma membrane of oocytes on the ICSI were bypassed. In SUZI, the AR of spermatozoa appears to be prerequisite for fertilization (7). Until recently, it has not been reported whether AR of spermatozoa has any effects on the process of fertilization in the ICSI. To obtain excellent results in ICSI, it was suggested that pretreatment of spermatozoa by follicular fluid or electroporation (5) and elevated Ca2+ concentration in the injection medium for parthenogenetic activation of oocytes were necessary (8). But, Liu et al. (9) reported that the high fertilization rate after the ICSI was not influenced by the different procedures used to enhance acrosoma1 loss in the spermatozoa or by incubation of the spermatozoa in medium with metabolic stimulants such as pentoxifylline and 2-deoxyadenosine. Even though it is known that the fertilization mechanism by ICSI is different from the that of conventional IVF, it has not been studied whether induction of the AR in spermatozoa influences developments of injected eggs. Therefore, we are to evaluate the relationship between the AR rate of spermatozoon that is introduced and the time of pronucleus (PN) formation after ICSI. MATERIALS Preparation
of Human
AND
METHODS
Spermatozoa
This study was approved by the Institutional Review Board on the use of human subjects in research at the Infertility Research Center, Jeil Women’s Hospital. Human semen samples were obtained from the Infertility Research Center’s donor program at the Jeil Women’s Hospital. Good quality semen (count, >lOO X lo6 spermatozoa/ml; motility, >50%; normal morphology, >50%) was used for this study. Human semen preservation medium (10) containing 15% (vol:vol) glycerol (G-9012; Sigma Chemi316
Lee
et al.
Acrosome
reaction
and ICSl
cal Co., St. Louis, MO) and 0.47~ bovine serum albumin (BSA; GIBCO BRL, Grand Island, NY) was used for semen cryopreservation. The semen was diluted 1:l with human semen preservation medium and placed in 2 mL cryovials (Corning L,aboratory Science Co., Corning, NY). The cryovials were cooled in a programmed freezer (Kryo 10 series III; Planer, Sunbury, Middlesex, United Kingdom.) using the following protocols: -0.5”C/min from 20°C to 4°C and -1O”CYmin from 4°C to -90°C. The cryovials were removed and then plunged into liquid nitrogen. For thawing, the cryovials were placed in a 37°C incubator for 1 hour and semen was evaluated using a Hamilton-Thorn Motility Analyzer ( HTM-C, Danvers, MA). Human tubal fluid medium (HTF) (11) supplemented with 0.5% BSA was used for sperm preparation. The semen sample was divided into six groups. Washed control group: the semen aliquot was washed by centrifugation at 300 x g for 10 minutes twice. Percoll-treated group: the semen aliquot was laid on the top of discontinuous six-layer Percoll (Pharmacia, Uppsala, Sweden) gradient (loo%, 90%, 80%, 70%, 6010, and 50%) and centrifuged at 600 x g for 20 minutes. The pellet was washed by centrifugation at 300 x g for 10 minutes twice and then the sample was incubated in a CO:! incubator (BB6220; Heraeus, Hanau, Germany) at 37°C for 6 hours. Pentoxifylline-treated group: After Percoll gradient treatment, the sample was treated with 3 mM pentoxifylline (P-1784; Sigma Chemical Co.) in HTF medium for 30 minutes. Follicular fluidl-treated group (FF): After Percoll gradient treatment, the sample was treated with 50% human FF in HTF medium for 6 hours. Pentoxifylline and FF treated group (pentoxifylline + FF): After Percoll gradient treatment, the sample was treated with 3 .mM pentoxifylline for 30 minutes and then 50% human FF for 6 hours. Platelet activating factor-treated group (PAF): After Percoll gradient treatment, the sample was incubated for 1 hour and then treated with a 1 x 10 -’ M PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, P-9515; Sigma Chemical Co.) in HTF medium for 1 hour. Each group was divided into two aliquots. One was used for detection of AR, the other was used for ICSI. Evaluation
of Acrosome
Reaction
Acrosome reaction was assessed lay using fluorescein isothiocyanate (FITC)-conjugated Pisum satiuum agglutinin (Vector Laboratories, Burlingame, CA) (12) or FITC-Arachis hypogea (peanut) agglutinin (Vector Laboratories) (13) as follows: aliquots of sperm suspension were washed in phosphate-buffered saline (PBS; GIBCO BRL) and smeared on the Fertility
and
SterilityB
clean slides. After air drying, sperm smears were fixed in the methanol for 1 minute at room temperature (RT) and stained with either FITC-Pisum satiuum agglutinin or FITC-Arc&is hypogea agglutinin at 50 pg/mL concentration in PBS for 30 minutes at RT. The slides were rinsed in distilled water and, on each slide, 2200 spermatozoa were examined under a fluorescence microscope (Optiphot-2; Nikon, Tokyo, Japan) with x400 magnification. Spermatozoa were classified as acrosome intact, partial acrosome reacted, or complete acrosome reacted according to the status of the acrosome. Preparation
of Hamster
Oocyte
Female golden hamsters (8 to 10 weeks old) were injected intraperitoneally with 30 IU of pregnant mare serum gonadotropin (PMSG, G-4877; Sigma Chemical Co.). Germinal vesicle-stage oocytes were collected from ovaries at 52 to 56 hours after PMSG injection and cultured in HTF medium supplemented with 0.5% BSA for in vitro maturation. After 20 hours, metaphase II-stage oocytes were selected after ascertaining the extrusion of the first polar body and no sign of activation and used for micromanipulation. Micromanipulation
Techniques
A microinjection procedure was carried out on a heated stage of an inverted microscope (Diaphot 300; Nikon) at ~400 magnification under differential interference contrast optics (Nikon). The micropipettes were fitted to a tool holder controlled by two IM-6 micromanipulators (Narishige Co., Tokyo, Japan). Holding and injection pipettes were made from glass capillaries (Drummond Scientific Co., Broomall, PA) using a puller (P-97; Sutter, Novato, CA), microgrinder (EG-6; Narishige Co.), and microforge (MF-9; Narishige Co.). The outer and inner diameters of holding pipettes were 100 and 20 pm, respectively. The outer diameter of the injection pipette was 5pm. The sperm of washed control, pentoxifylline + FF, and PAF-treated group, with a higher AR rate, were used for ICSI. The microinjection procedures were carried out by the method of Palermo et al. (5); sperm suspension was mixed with 10% polyvinylpyrrolidone (PVP-360; Sigma Chemical Co.) solution in the ratio of 1:4. A single, immobilized, living sperm was aspirated into the injection pipette and the oocyte was held by the holding pipette and the injection pipette containing the sperm was introduced deeply into the cytoplasm, and the sperm was deposited together with the smallest possible amount of medium. After ICSI, the oocytes were washed and culVol.
67, No. 2, February
1997
tured in preincubated HTF media supplemented with 0.5% BSA at 37°C for overnight. Assessment
of Pronucleus
Formation
Hamster oocytes were fixed with 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.2, C-0250; Sigma Chemical Co.) at 6,9,12, and 15 hours after ICSI. For examination of PN formation, fixed oocytes were stained with 0.1% Toluidine blue (Merck) in 0.5% boric acid (B6768; Sigma Chemical Co.). They were observed under a light microscope (Optiphot-2; Nikon) and degenerated oocytes were discarded. Cases of oocytes with two or more PNs were regarded as a PN formation because the hamster oocyte was activated easily, although we injected one sperm into the oocyte. Cases of oocytes with no or only one PN were regarded as a failed PN formation. If an intact sperm head existed in cytoplasm of oocyte, it was regarded as failed PN formation even if the oocyte had two or more PNs. Fluorescence
In Situ
Hybridization
Hamster oocytes with two or more PNs by ICSI and spontaneous activation during in vitro maturation were used for fluorescence in situ h;ybridization analysis. The ZP of oocytes were removed by treatment with 0.5% pronase E (P-5147; Sigma Chemical Co.) in HTF medium. These zona-free oocytes were placed in a hypotonic solution (6 mg/mL BSA and 0.5% sodium citrate [S-4641; Sigma Chemical Co.]) for 2 to 5 minutes and fixed to preclleaned glass slides. The position of the nucleus then was marked. Slides were dehydrated at 70%, 85%, 95%, and 100% ethanol (Merck) for 2 minutes each. The probes used for this study were directly labeled DNA probes (Vysis Inc, Framingham, MA): CEP 15-Spectrum Orange and CEP 18-Spectrum Green. Fluorescence in situ hybridization was done according to the basic technique of Munnle et al. (14); the 10 PL of the hybridization solution (1 yL of each probe was added to 8 PL of hybridization buffer) was dropped onto a glass slide containing fixed fertilized oocytes and covered with a coverslip. Nuclear and probe DNA were denatured simultaneo-usly at 80°C for 8 minutes. The slide was incubated in a dark, moist chamber at 40°C for 6 hours to allow hybridization of the DNA probes. The slides were washed with 50% formamide/2x SSC at 42°C for 15 minutes and 2~ SSC at 42°C for 10 minutes twice. After air drying, the 8 PL of 4’,6-diamino-2-phenylindole in antifade solution (0.5 mg/mL; Vysis Inc.) was used as a counterstain and the slide was observed under a fluorescence microscope (Optiphot-2; Nikon) using specific filter (B, G, and UV filter) sets. Lee et al.
Acrosome
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317
Table 1 Partial or Complete Acrosome Reaction Rate in Washed Control Group, Pentoxifylline + FF-Treated and PAF-Treated Group Assessed by Pisum sativum and Arachis hypogea Lectin Staining*
satiuum
FITC-Pisum agglutinin
Group
(n = 9)
Washed control Pentoxifylline + FF PAF
Complete AR
11.4 31.5 34.1
Partial AR
2 1.8 z 5.9f 5 5.4”i
*Values are means x SEM. i Significantly different from
Statistical
16.0 39.7 41.3
washed
-t 4.2 2 6.9; t 5.0t
control
Group,
FITC-Arachis agglutinin
hypogea
Complete m
Partial AFL
16.2 40.1 44.4
group,
z 3.0 k 5.0t ? 5.4t
19.9 48.1 54.6
+ 3.9 2 5.1; x 6.4+
P < 0.01.
Analysis
Differences in AR rates of spermatozoa in various treatment groups were analyzed by a Student’s t-test. The results were expressed as means 2 SEM. Differences in the PN formation rate of oocytes that were injected with sperm of various treatment groups were analyzed by a x2 test. A P value < 0.05 was defined to be statistically significant. RESULTS
The AR rates of spermatozoa in various treatment groups were assessedby FITC-Pisum sativum agglutinin staining. There were no significant differences in the AR rate between washed control (10.5% t 2.6%) and Percoll gradient (10.3% i 1.7%). But, the AR rate of sperm in pentoxifylline, FF, pentoxifylline + FF, and PAF groups was higher than that of washed control (16.4% ? 1.8%, 24.8% -C 5.6%, 28.4% 5 3.8%, 33.3% 2 5.2% versus 10.5% -C 2.6%; P < 0.01). The AR rate is different according to the different methods of monitoring the acrosome status. Because the probe of FITC-Arc&is hypogea agglutinin binds to the acrosomal outer membrane, which disappeared before the acrosomal content of the spermatozoa was released, AR is detected earlier than FITCPisum sativum agglutinin, which binds to the acrosomal contents (15). Therefore, the spermatozoa of the washed control, pentoxifylline + FF, and PAF groups, with a higher AR rate than others, were assessed a partial and complete AR by FITC-Pisum sativum agglutinin and FITC-Arachis hypogea agglutinin (Table 1). Metaphase II stage hamster oocytes were injected with the sperm of the washed control, pentoxifylline + FF, and PAF groups, and then PN formation was observed at 6,9, 12, and 15 hours after ICSI (Table 2). At 6 hours after ICSI, the PN formation rate was significantly higher in the pentoxifylline + FF or 318
Lee
et al.
Acrosome
reaction
and ICSI
PAF groups than in the washed control (19.0% [ll/ 581 and 18.9% [lo/531 versus 5.6% 131541;P < 0.05). At 9 hours after ICSI, the PN formation rate of the pentoxifylline + FF or PAF groups was significantly higher than that of the washed control (51.8% [29/ 561 and 57.4% 1311541 versus 32.7% [18/55]; P < 0.05). But, there was no significant difference in the PN formation rate between the washed control, pentoxifylline + FF, and PAF groups at 12 hours (53.6% [30/561 and 50.0% [27/541 versus 36.1% [22/ 611; 0.05 < P < 0.1) and 15 hours (64.8% [35/541 and 53.6% [30/561 versus 47.2% [25/531; P > 0.051, although the PN formation rate of the pentoxifylline + FF and PAF groups was higher than that of the washed control (Table 2). To determine whether one of the PNs originated from human spermatozoa or oocyte p-roper, 10 fertilized hamster oocytes with two or more PNs (2PN [n = 91 + 3PN [n = 11)and 10 activated hamster oocytes were analyzed by fluorescence in situ hybridization using human chromosome specific DNA probes. Eight fertilized oocytes had two signals for chromosome 15 and 18 at one of the PNs, anfd one fertilized oocyte had one signal for chromosome 18. One fertilized oocyte had two signals for chromosome 15 and 18 in each PN. Activated oocytes had no fluorescence in situ hybridization signals (Fig. 1). DISCUSSION
In conventional IVF, AR is induced by P in cumulus complexes during penetration of the sperm. After binding to ZP, AR is completed by mechanisms of signal transduction pathways (2). Penetrated spermatozoon lost all of their acrosomal. contents and membrane. Putative fusogenic molecu.les in an equatorial segment of sperm head may be activated by acrosins during AR and mediated fusion with the cytoplasm of the oocyte (16). Consequently, the
Table 2 Pronucleus Formation Rat.es in Hamster Oocytes of Human Spermatozoa Among Washed Control Group, Pentoxifylline + FF-Treated Group and PAF-Treated Group After ICSI*
Hours+ 6 9 12 15
Washed control 3/54 (5.6) 18/55 (32.71 22/61 (36.11 25153 (47.21
Pentoxifylline i- FF 11/58 29/56 30/56 35154
PAF
(19.013: (51.8)$ (53.6)Q: (64.8)
10/53 31154 27/54 30156
(18.9 I$ (57.4)$ (50.0 19 (53.6~
* Values are No. of two or more PN hamster oocytes/no. of hamster oocytes injected with human sperm with percentages in parentheses. t Time after ICSI. $ Significantly different from washed control group, P r: 0.05. 9: 0.05 < P < 0.1. Fertility
and
Sterility*
Figure 1 In situ hybridization of fluorochrome-labeled 15 (red) and 18 (green) chromosome-specific DNA probes. (a and b), Fertilized hamster oocyte. (c and d), Activated hamster oocyte. a and c show position of 4’,6-diamino-Z-phenylindole-labeled PN under ultraviolet illumination. b shows two hybridization signals observed at one of the PNs. d shows no observed hybridization signal.
sperm head is incorporated into the oocyte. After incorporating, the nucleus of spermatozoon is decondensed and eventually changes to male PN. The frequency with which spermatozoa from a given sample undergo the AR represents an important parameter in clinical evaluation of sperm function (17). Acrosome reaction in humans is well investigated, owing to microfertilization techniques that were introduced in order to overcome male factor infertility, particularly the development of SUZI, which required acrosomal loss of spermatozoa. It is known that the induction of AR of human spermatozoa is very difficult, unlike that of other animals. Acrosome reaction was induced artificially by treatment of chemicals such as pentoxifylline (18), P (2), FF, PAF (19), or calcium ionophore (20), and a mechanical stimulus such as electroporation (5). Nevertheless, the AR rate of human sperm does not exceed 50% in any of the aforementioned treatments. Edwards and Van Steirteghem (8) suggested that the higher fertilization rate in ICSI depended on activation, which is brought about by a parthenogenetic mechanism. The injection of a spermatozoon into the ooplasm seems to be a sufficient stimulus to activate the oocyte. And, the Ca” concentration of the injection medium was not related to the fertilization rate (9). It was demonstrated recently that introduction of the spermatozoon into the oocyte may be necessary for human oocyte activation (21). When cytosolic factors of spermatozoa were injected into oocytes, a higher fertilization rate was acquired Vol.
67, No. 2, February
1997
than in mimic ICSI (22). Therefore, it is known that breakage in the outer plasma membrane and acrosoma1 membranes of spermatozoa is necessary to complete oocyte activation and fertilization in ICSI. Bypassing the AR and fusion process in ICSI, most injected spermatozoa still may contain contents of the acrosome. It is important to know that the effects of the acrosomal contents of spermatozoa in oocytes are evaluated and to know how its membrane effects PN formation after ICSI. Generally, PN was formed in the injected oocytes within 8 to 12 hours after ICSI (23). However, in some cases, delayed PN formation could be observed. This could be caused by incomplete maturation of the oocyte and functional disorders of the spermatozoa. Indeed, intact sperm were observed inside the cytoplasm of some oocytes that failed to fertilize (24). Also, in some patients with male factor infertility who had a functional disorder of the spermatozoa, enhancement of the spermatozoa1 func:tion by pretreatment, such as pentoxifylline or 2-deoxyadenosine, increased the fertilization rate and promoted embryonic development in IVF-ET programs. And it was reported that an ET of faster developed embryos obtained a higher pregnancy rate than the delayed ones in IVF-ET programs (25). In this experiment, we observed that treatment of sperm with pentoxifylline + FF and PAF induced a higher rate of AR compared with washed control. After injection of the sperm of these groups into hamster oocytes, we observed a faster PN formation than that of the washed control group after 6 or 9 hours. But, PN formation rate was not different between the groups after 12 or 15 hours. From these results, we suggest that acrosome-reacted sperm injection does not induce a higher fertilization rate but facilitates the time of PN formation in ICSI. To determine whether one of the PNs originated from human spermatozoa or oocyte activation, fertilized hamster eggs were analyzed by fluorescence in situ hybridization using human chromosome specific DNA probes. All fertilized eggs had signals of human chromosomes, but activated oocytes did not. We ascertained that human spermatozoon decondensed and formed male PN within hamster oocytes after ICSI. It appeared that injection of acrosome-reacted human spermatozoon with no intact plasma or outer acrosomal membrane into hamster oocytes induced faster PN formation than that of acrosome-intact spermatozoon. Therefore, we suggest that induction of AR of spermatozoa before ICSI accelerates the time of PN formation of the embryo. Whether inducing the AR in human sperm advances PN formation in injected human oocytes needs to be examined. Lee
et al.
Acrosome
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and ICSI
319
Acknowledgment. We are grateful ful comments on the manuscript.
to Sug Oh, M.D.,
for his help13.
REFERENCES 1. Fraser LR, Harrison RAP, Herod JE. Characterization of a decapacitation factor associated with epididymal mouse spermatozoa. J Reprod Fert 1990;89:135-48. 2. Roldan RES, Murase T, Shi Q-X. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science 1994;266:1578-81. 3. Yanagimachi R. Sperm-egg association in mammals. In: Moscona AA, Monroy A, editors. Current topics in developmental biology. Vol. 12. New York: Academic Press, 1978:83-105. 4. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-8. 5. Palermo G, Joris H, Derde M-P, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril1993; 59:826-35. 6. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;7:1055-60. 7. Lacham 0, Trounson AO, Holden C, Mann J, Sathananthan H. Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induced the acrosome reaction. Gamete Res 1989;23:233-43. 8. Edwards RG, Van Steirteghem AC. Intracytoplasmic sperm injection: does calcium hold the key to success? Hum Reprod 1993;8:988-9. 9. Liu J, Nagy Z, Joris H, Tournaye H, Devroey P, Van Steirteghem AC. Intracytoplasmic sperm injection does not require special treatment of the spermatozoa. Hum Reprod 1994;6:1127-30. 10. Mahadevan M, Trounson AO. Cryopreservation of human semen. In: Artificial insemination by donor. Melbourne: Brown Prior Anderson, 1980:19-32. 11. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8. 12. Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss
320
Lee
et al.
Acrosome
reaction
and ZCSZ
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
by a one-step staining method using Pisum sativum agglutinin. J Reprod Fert 1992;95:755-63. Mortimer D, Curtis EF, Miller RG. Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J Reprod Fert 1987;81:127-35. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993;8:2185-91. Aitken RJ, Brindle JP. Analysis of the ability of three probes targeting the outer acrosomal membrane or acrosomal contents to detect the acrosome reaction in human spermatozoa. Hum Reprod 1993;8:1663-9. Takano H, Yanagimachi R, Urch U. Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with oolemma: inhibitor studies using the golden hamster. Zygote 1993; 1:79-91. Tesarik J, Testart J. Human sperm-egg interactions and their disorders: implications in the management of infertility. Hum Reprod 1989;4:729-41. Tesarik J, Mendoza C. Sperm treatment w-ith pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil Steril 1993;60:141-8. Sengoku K, Tamate K, Takaoka Y, Ishikawa M. Effects of platelet activating factor on human sperm function in vitro. Hum Reprod 1993;8:1443-7. Pampiglione JS, Tan S-L, Campbell S. The use of the stimulated acrosome reaction test as a test of fertilizing ability in human spermatozoa. Fertil Steril 1993;59: 1280-4. Tesarik J, Sousa M, Testart J. Human 0ocyt.e activation after intracytoplasmic sperm injection. Hum Reprod 1994;9:5118. Dozortsev D, Rybouchkin A, De Sutter P, Qian C, Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod 1995; 10:404-7. Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem A. Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod 1994;9:1743--8. Dozortsev D, De Sutter P, Dhont M. Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum Reprod 1994;9:213944. Edwards RG, Fishel S, Cohen J, Fehilly CB, Purdy JM, Slater JM, et al. Factors influencing the success of IVF for alleviation of human infertility. J In Vitro Fert Embryo Transf 1984; 1:3-23.
Fertility
and
Sterility@
AND STERILITY~ ‘. 1997 Amencan
Society
Vol. 67, No. 2, February for Reproductive
Medicme
Printed
on acid-free
paper
1997
in U. S. A.
Induction of acrosome reaction in human spermatozoa accelerates the time of pronucleus formation of hamster oocytes after intracytoplasmic sperm injection*
Dong Ryul Lee, M.S.: Jeoung Eun Lee, M.S. Hyun Soo Yoon, Ph.D. Sung 11 Roh, M.D. Infertility
Research
Center,
Jeil
Women’s
Hospital,
Seoul,
Korea
Objective: To assess the relationship between the incidence of acrosome reaction (AR) and the timing of pronucleus (PN) formation after intracytoplasmic sperm injection (ICSI). Design: Prospective study. Setting: Infertility Research Center, Jeil Women’s Hospital. Main Outcome Measure(s): Human semen obtained from fertile donors was prepared by one of the following methods: washing only (washed control); Percoll gradient; pentoxifylline; human follicular fluid (FF); pentoxifylline + FF; or platelet-activating factor (PAF) treatment. The AR of each group was assessed by fluorescein isothiocyanate-conjugated Pisum sativum agglutinin or Arachis hypogea agglutinin. Spermatozoa of washed control, pentoxifylline + FF: and PAF treated groups, with significantly higher AR rate than others, were injected into mature hamster oocytes. Spermatozoon-injected oocytes were cultured for 6, 9, 12, or 15 hours. Then they were stained with Toluidine blue for PN formation examination under a light micro-. scope. Result(s): Acrosome reaction rates of washed control, Percoll gradient, pentoxifylline, FF., pentoxifylline + FF, and PAF treated groups were 10.5% i- 2.6%, 10.3% 2 1.7%, 16.4% 2 1.8%., 24.8% z 5.6%, 28.4% ? 3.8%, and 33.3% ? 5.2%, respectively. Pronuclear formation rate in washed control, pentoxifylline + FF, and PAF treated groups were 5.6%( 3/54), 19.0% (1X8), and 18.9% (10153) at 6 hours; 32.7% (18/55), 51.8% (29/56), and 57.4% (31/54) at 9 hours; 36.1% (22/61), 53.6% (30/56), and 50.0% (27/54) at 12 hours; and 47.2% (25/53), 64.8% (35/54), 53.6% (30/56) at 15 hours after ICSI. Pronuclear formation rate was significantly higher in pentoxifylline + FF, and PAF treated groups than that in the washed control group at 6 and 9 hours after ICSI. Conclusion(s): Pronuclear formation of oocytes takes place faster on those that were injected with acrosome-reacted spermatozoon than those injected with acrosome-intact spermatozoon. It could be concluded that induction of the AR of spermatozoa accelerates the time of PN formation and early development of the embryo in ICSI. Fertil Steril@ 1997;67:315-20 Key Words: Acrosome reaction, tion, pronucleus formation
human
sperm, hamster
oocyte, intracytoplasmic
sperm injec-
Mammalian spermatozoa having matured in the male reproductive organ, epididymis, undergo capacitation during exposure to the female reproductive tract, resulting in the physiological alteration
of the plasma membrane (11, and they rneet the oocyte in the ampullary portion of the fallopian tube. Penetration of spermatozoa through the cumulus oophorus could result in an acute exposure to P and
Received January 22, 1996; revised and accepted October 15, 1996. * Presented at the 9th World Congress on In Vitro Fertilization and Assisted Reproduction, Vienna, Austria, April 3 to 7, 1995, where it was granted a poster award.
f Reprint requests: Dong Ryul Lee, M.S., Infertility Research Center, Jeil Women’s Hospital, 1021-4 Daechi-Dong, KangnamKu, Seoul 135-280, Korea (FAX: 82-2-552-7964; e-mail: [email protected]).
Vol. 67, No. 2, February 1997
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initiation of acrosome reaction (AR), and then interaction with the zona pellucida (ZP) would trigger its completion (2). After penetration, the plasma membrane of acrosome-reacted spermatozoon fuses with oolemma (3). A recent introduction of intracytoplasmic sperm injection (ICSI), the breakthrough for severe male factor infertility and repetitive failure or low percentage of fertilization in the IVF-ET program, showed a higher rate of fertilization and pregnancy than the other microsurgical fertilization techniques, such as partial zona dissection or subzonal sperm insertion (SUZI) (4-6). The mechanism of fertilization in ICSI may be different from that in conventional IVF. It is known that the AR in most of the spermatozoa introduced and penetration of the ZP and plasma membrane of oocytes on the ICSI were bypassed. In SUZI, the AR of spermatozoa appears to be prerequisite for fertilization (7). Until recently, it has not been reported whether AR of spermatozoa has any effects on the process of fertilization in the ICSI. To obtain excellent results in ICSI, it was suggested that pretreatment of spermatozoa by follicular fluid or electroporation (5) and elevated Ca2+ concentration in the injection medium for parthenogenetic activation of oocytes were necessary (8). But, Liu et al. (9) reported that the high fertilization rate after the ICSI was not influenced by the different procedures used to enhance acrosoma1 loss in the spermatozoa or by incubation of the spermatozoa in medium with metabolic stimulants such as pentoxifylline and 2-deoxyadenosine. Even though it is known that the fertilization mechanism by ICSI is different from the that of conventional IVF, it has not been studied whether induction of the AR in spermatozoa influences developments of injected eggs. Therefore, we are to evaluate the relationship between the AR rate of spermatozoon that is introduced and the time of pronucleus (PN) formation after ICSI. MATERIALS Preparation
of Human
AND
METHODS
Spermatozoa
This study was approved by the Institutional Review Board on the use of human subjects in research at the Infertility Research Center, Jeil Women’s Hospital. Human semen samples were obtained from the Infertility Research Center’s donor program at the Jeil Women’s Hospital. Good quality semen (count, >lOO X lo6 spermatozoa/ml; motility, >50%; normal morphology, >50%) was used for this study. Human semen preservation medium (10) containing 15% (vol:vol) glycerol (G-9012; Sigma Chemi316
Lee
et al.
Acrosome
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and ICSl
cal Co., St. Louis, MO) and 0.47~ bovine serum albumin (BSA; GIBCO BRL, Grand Island, NY) was used for semen cryopreservation. The semen was diluted 1:l with human semen preservation medium and placed in 2 mL cryovials (Corning L,aboratory Science Co., Corning, NY). The cryovials were cooled in a programmed freezer (Kryo 10 series III; Planer, Sunbury, Middlesex, United Kingdom.) using the following protocols: -0.5”C/min from 20°C to 4°C and -1O”CYmin from 4°C to -90°C. The cryovials were removed and then plunged into liquid nitrogen. For thawing, the cryovials were placed in a 37°C incubator for 1 hour and semen was evaluated using a Hamilton-Thorn Motility Analyzer ( HTM-C, Danvers, MA). Human tubal fluid medium (HTF) (11) supplemented with 0.5% BSA was used for sperm preparation. The semen sample was divided into six groups. Washed control group: the semen aliquot was washed by centrifugation at 300 x g for 10 minutes twice. Percoll-treated group: the semen aliquot was laid on the top of discontinuous six-layer Percoll (Pharmacia, Uppsala, Sweden) gradient (loo%, 90%, 80%, 70%, 6010, and 50%) and centrifuged at 600 x g for 20 minutes. The pellet was washed by centrifugation at 300 x g for 10 minutes twice and then the sample was incubated in a CO:! incubator (BB6220; Heraeus, Hanau, Germany) at 37°C for 6 hours. Pentoxifylline-treated group: After Percoll gradient treatment, the sample was treated with 3 mM pentoxifylline (P-1784; Sigma Chemical Co.) in HTF medium for 30 minutes. Follicular fluidl-treated group (FF): After Percoll gradient treatment, the sample was treated with 50% human FF in HTF medium for 6 hours. Pentoxifylline and FF treated group (pentoxifylline + FF): After Percoll gradient treatment, the sample was treated with 3 .mM pentoxifylline for 30 minutes and then 50% human FF for 6 hours. Platelet activating factor-treated group (PAF): After Percoll gradient treatment, the sample was incubated for 1 hour and then treated with a 1 x 10 -’ M PAF (1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylcholine, P-9515; Sigma Chemical Co.) in HTF medium for 1 hour. Each group was divided into two aliquots. One was used for detection of AR, the other was used for ICSI. Evaluation
of Acrosome
Reaction
Acrosome reaction was assessed lay using fluorescein isothiocyanate (FITC)-conjugated Pisum satiuum agglutinin (Vector Laboratories, Burlingame, CA) (12) or FITC-Arachis hypogea (peanut) agglutinin (Vector Laboratories) (13) as follows: aliquots of sperm suspension were washed in phosphate-buffered saline (PBS; GIBCO BRL) and smeared on the Fertility
and
SterilityB
clean slides. After air drying, sperm smears were fixed in the methanol for 1 minute at room temperature (RT) and stained with either FITC-Pisum satiuum agglutinin or FITC-Arc&is hypogea agglutinin at 50 pg/mL concentration in PBS for 30 minutes at RT. The slides were rinsed in distilled water and, on each slide, 2200 spermatozoa were examined under a fluorescence microscope (Optiphot-2; Nikon, Tokyo, Japan) with x400 magnification. Spermatozoa were classified as acrosome intact, partial acrosome reacted, or complete acrosome reacted according to the status of the acrosome. Preparation
of Hamster
Oocyte
Female golden hamsters (8 to 10 weeks old) were injected intraperitoneally with 30 IU of pregnant mare serum gonadotropin (PMSG, G-4877; Sigma Chemical Co.). Germinal vesicle-stage oocytes were collected from ovaries at 52 to 56 hours after PMSG injection and cultured in HTF medium supplemented with 0.5% BSA for in vitro maturation. After 20 hours, metaphase II-stage oocytes were selected after ascertaining the extrusion of the first polar body and no sign of activation and used for micromanipulation. Micromanipulation
Techniques
A microinjection procedure was carried out on a heated stage of an inverted microscope (Diaphot 300; Nikon) at ~400 magnification under differential interference contrast optics (Nikon). The micropipettes were fitted to a tool holder controlled by two IM-6 micromanipulators (Narishige Co., Tokyo, Japan). Holding and injection pipettes were made from glass capillaries (Drummond Scientific Co., Broomall, PA) using a puller (P-97; Sutter, Novato, CA), microgrinder (EG-6; Narishige Co.), and microforge (MF-9; Narishige Co.). The outer and inner diameters of holding pipettes were 100 and 20 pm, respectively. The outer diameter of the injection pipette was 5pm. The sperm of washed control, pentoxifylline + FF, and PAF-treated group, with a higher AR rate, were used for ICSI. The microinjection procedures were carried out by the method of Palermo et al. (5); sperm suspension was mixed with 10% polyvinylpyrrolidone (PVP-360; Sigma Chemical Co.) solution in the ratio of 1:4. A single, immobilized, living sperm was aspirated into the injection pipette and the oocyte was held by the holding pipette and the injection pipette containing the sperm was introduced deeply into the cytoplasm, and the sperm was deposited together with the smallest possible amount of medium. After ICSI, the oocytes were washed and culVol.
67, No. 2, February
1997
tured in preincubated HTF media supplemented with 0.5% BSA at 37°C for overnight. Assessment
of Pronucleus
Formation
Hamster oocytes were fixed with 2.5% glutaraldehyde (Merck, Darmstadt, Germany) in 0.1 M cacodylate buffer (pH 7.2, C-0250; Sigma Chemical Co.) at 6,9,12, and 15 hours after ICSI. For examination of PN formation, fixed oocytes were stained with 0.1% Toluidine blue (Merck) in 0.5% boric acid (B6768; Sigma Chemical Co.). They were observed under a light microscope (Optiphot-2; Nikon) and degenerated oocytes were discarded. Cases of oocytes with two or more PNs were regarded as a PN formation because the hamster oocyte was activated easily, although we injected one sperm into the oocyte. Cases of oocytes with no or only one PN were regarded as a failed PN formation. If an intact sperm head existed in cytoplasm of oocyte, it was regarded as failed PN formation even if the oocyte had two or more PNs. Fluorescence
In Situ
Hybridization
Hamster oocytes with two or more PNs by ICSI and spontaneous activation during in vitro maturation were used for fluorescence in situ h;ybridization analysis. The ZP of oocytes were removed by treatment with 0.5% pronase E (P-5147; Sigma Chemical Co.) in HTF medium. These zona-free oocytes were placed in a hypotonic solution (6 mg/mL BSA and 0.5% sodium citrate [S-4641; Sigma Chemical Co.]) for 2 to 5 minutes and fixed to preclleaned glass slides. The position of the nucleus then was marked. Slides were dehydrated at 70%, 85%, 95%, and 100% ethanol (Merck) for 2 minutes each. The probes used for this study were directly labeled DNA probes (Vysis Inc, Framingham, MA): CEP 15-Spectrum Orange and CEP 18-Spectrum Green. Fluorescence in situ hybridization was done according to the basic technique of Munnle et al. (14); the 10 PL of the hybridization solution (1 yL of each probe was added to 8 PL of hybridization buffer) was dropped onto a glass slide containing fixed fertilized oocytes and covered with a coverslip. Nuclear and probe DNA were denatured simultaneo-usly at 80°C for 8 minutes. The slide was incubated in a dark, moist chamber at 40°C for 6 hours to allow hybridization of the DNA probes. The slides were washed with 50% formamide/2x SSC at 42°C for 15 minutes and 2~ SSC at 42°C for 10 minutes twice. After air drying, the 8 PL of 4’,6-diamino-2-phenylindole in antifade solution (0.5 mg/mL; Vysis Inc.) was used as a counterstain and the slide was observed under a fluorescence microscope (Optiphot-2; Nikon) using specific filter (B, G, and UV filter) sets. Lee et al.
Acrosome
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317
Table 1 Partial or Complete Acrosome Reaction Rate in Washed Control Group, Pentoxifylline + FF-Treated and PAF-Treated Group Assessed by Pisum sativum and Arachis hypogea Lectin Staining*
satiuum
FITC-Pisum agglutinin
Group
(n = 9)
Washed control Pentoxifylline + FF PAF
Complete AR
11.4 31.5 34.1
Partial AR
2 1.8 z 5.9f 5 5.4”i
*Values are means x SEM. i Significantly different from
Statistical
16.0 39.7 41.3
washed
-t 4.2 2 6.9; t 5.0t
control
Group,
FITC-Arachis agglutinin
hypogea
Complete m
Partial AFL
16.2 40.1 44.4
group,
z 3.0 k 5.0t ? 5.4t
19.9 48.1 54.6
+ 3.9 2 5.1; x 6.4+
P < 0.01.
Analysis
Differences in AR rates of spermatozoa in various treatment groups were analyzed by a Student’s t-test. The results were expressed as means 2 SEM. Differences in the PN formation rate of oocytes that were injected with sperm of various treatment groups were analyzed by a x2 test. A P value < 0.05 was defined to be statistically significant. RESULTS
The AR rates of spermatozoa in various treatment groups were assessedby FITC-Pisum sativum agglutinin staining. There were no significant differences in the AR rate between washed control (10.5% t 2.6%) and Percoll gradient (10.3% i 1.7%). But, the AR rate of sperm in pentoxifylline, FF, pentoxifylline + FF, and PAF groups was higher than that of washed control (16.4% ? 1.8%, 24.8% -C 5.6%, 28.4% 5 3.8%, 33.3% 2 5.2% versus 10.5% -C 2.6%; P < 0.01). The AR rate is different according to the different methods of monitoring the acrosome status. Because the probe of FITC-Arc&is hypogea agglutinin binds to the acrosomal outer membrane, which disappeared before the acrosomal content of the spermatozoa was released, AR is detected earlier than FITCPisum sativum agglutinin, which binds to the acrosomal contents (15). Therefore, the spermatozoa of the washed control, pentoxifylline + FF, and PAF groups, with a higher AR rate than others, were assessed a partial and complete AR by FITC-Pisum sativum agglutinin and FITC-Arachis hypogea agglutinin (Table 1). Metaphase II stage hamster oocytes were injected with the sperm of the washed control, pentoxifylline + FF, and PAF groups, and then PN formation was observed at 6,9, 12, and 15 hours after ICSI (Table 2). At 6 hours after ICSI, the PN formation rate was significantly higher in the pentoxifylline + FF or 318
Lee
et al.
Acrosome
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and ICSI
PAF groups than in the washed control (19.0% [ll/ 581 and 18.9% [lo/531 versus 5.6% 131541;P < 0.05). At 9 hours after ICSI, the PN formation rate of the pentoxifylline + FF or PAF groups was significantly higher than that of the washed control (51.8% [29/ 561 and 57.4% 1311541 versus 32.7% [18/55]; P < 0.05). But, there was no significant difference in the PN formation rate between the washed control, pentoxifylline + FF, and PAF groups at 12 hours (53.6% [30/561 and 50.0% [27/541 versus 36.1% [22/ 611; 0.05 < P < 0.1) and 15 hours (64.8% [35/541 and 53.6% [30/561 versus 47.2% [25/531; P > 0.051, although the PN formation rate of the pentoxifylline + FF and PAF groups was higher than that of the washed control (Table 2). To determine whether one of the PNs originated from human spermatozoa or oocyte p-roper, 10 fertilized hamster oocytes with two or more PNs (2PN [n = 91 + 3PN [n = 11)and 10 activated hamster oocytes were analyzed by fluorescence in situ hybridization using human chromosome specific DNA probes. Eight fertilized oocytes had two signals for chromosome 15 and 18 at one of the PNs, anfd one fertilized oocyte had one signal for chromosome 18. One fertilized oocyte had two signals for chromosome 15 and 18 in each PN. Activated oocytes had no fluorescence in situ hybridization signals (Fig. 1). DISCUSSION
In conventional IVF, AR is induced by P in cumulus complexes during penetration of the sperm. After binding to ZP, AR is completed by mechanisms of signal transduction pathways (2). Penetrated spermatozoon lost all of their acrosomal. contents and membrane. Putative fusogenic molecu.les in an equatorial segment of sperm head may be activated by acrosins during AR and mediated fusion with the cytoplasm of the oocyte (16). Consequently, the
Table 2 Pronucleus Formation Rat.es in Hamster Oocytes of Human Spermatozoa Among Washed Control Group, Pentoxifylline + FF-Treated Group and PAF-Treated Group After ICSI*
Hours+ 6 9 12 15
Washed control 3/54 (5.6) 18/55 (32.71 22/61 (36.11 25153 (47.21
Pentoxifylline i- FF 11/58 29/56 30/56 35154
PAF
(19.013: (51.8)$ (53.6)Q: (64.8)
10/53 31154 27/54 30156
(18.9 I$ (57.4)$ (50.0 19 (53.6~
* Values are No. of two or more PN hamster oocytes/no. of hamster oocytes injected with human sperm with percentages in parentheses. t Time after ICSI. $ Significantly different from washed control group, P r: 0.05. 9: 0.05 < P < 0.1. Fertility
and
Sterility*
Figure 1 In situ hybridization of fluorochrome-labeled 15 (red) and 18 (green) chromosome-specific DNA probes. (a and b), Fertilized hamster oocyte. (c and d), Activated hamster oocyte. a and c show position of 4’,6-diamino-Z-phenylindole-labeled PN under ultraviolet illumination. b shows two hybridization signals observed at one of the PNs. d shows no observed hybridization signal.
sperm head is incorporated into the oocyte. After incorporating, the nucleus of spermatozoon is decondensed and eventually changes to male PN. The frequency with which spermatozoa from a given sample undergo the AR represents an important parameter in clinical evaluation of sperm function (17). Acrosome reaction in humans is well investigated, owing to microfertilization techniques that were introduced in order to overcome male factor infertility, particularly the development of SUZI, which required acrosomal loss of spermatozoa. It is known that the induction of AR of human spermatozoa is very difficult, unlike that of other animals. Acrosome reaction was induced artificially by treatment of chemicals such as pentoxifylline (18), P (2), FF, PAF (19), or calcium ionophore (20), and a mechanical stimulus such as electroporation (5). Nevertheless, the AR rate of human sperm does not exceed 50% in any of the aforementioned treatments. Edwards and Van Steirteghem (8) suggested that the higher fertilization rate in ICSI depended on activation, which is brought about by a parthenogenetic mechanism. The injection of a spermatozoon into the ooplasm seems to be a sufficient stimulus to activate the oocyte. And, the Ca” concentration of the injection medium was not related to the fertilization rate (9). It was demonstrated recently that introduction of the spermatozoon into the oocyte may be necessary for human oocyte activation (21). When cytosolic factors of spermatozoa were injected into oocytes, a higher fertilization rate was acquired Vol.
67, No. 2, February
1997
than in mimic ICSI (22). Therefore, it is known that breakage in the outer plasma membrane and acrosoma1 membranes of spermatozoa is necessary to complete oocyte activation and fertilization in ICSI. Bypassing the AR and fusion process in ICSI, most injected spermatozoa still may contain contents of the acrosome. It is important to know that the effects of the acrosomal contents of spermatozoa in oocytes are evaluated and to know how its membrane effects PN formation after ICSI. Generally, PN was formed in the injected oocytes within 8 to 12 hours after ICSI (23). However, in some cases, delayed PN formation could be observed. This could be caused by incomplete maturation of the oocyte and functional disorders of the spermatozoa. Indeed, intact sperm were observed inside the cytoplasm of some oocytes that failed to fertilize (24). Also, in some patients with male factor infertility who had a functional disorder of the spermatozoa, enhancement of the spermatozoa1 func:tion by pretreatment, such as pentoxifylline or 2-deoxyadenosine, increased the fertilization rate and promoted embryonic development in IVF-ET programs. And it was reported that an ET of faster developed embryos obtained a higher pregnancy rate than the delayed ones in IVF-ET programs (25). In this experiment, we observed that treatment of sperm with pentoxifylline + FF and PAF induced a higher rate of AR compared with washed control. After injection of the sperm of these groups into hamster oocytes, we observed a faster PN formation than that of the washed control group after 6 or 9 hours. But, PN formation rate was not different between the groups after 12 or 15 hours. From these results, we suggest that acrosome-reacted sperm injection does not induce a higher fertilization rate but facilitates the time of PN formation in ICSI. To determine whether one of the PNs originated from human spermatozoa or oocyte activation, fertilized hamster eggs were analyzed by fluorescence in situ hybridization using human chromosome specific DNA probes. All fertilized eggs had signals of human chromosomes, but activated oocytes did not. We ascertained that human spermatozoon decondensed and formed male PN within hamster oocytes after ICSI. It appeared that injection of acrosome-reacted human spermatozoon with no intact plasma or outer acrosomal membrane into hamster oocytes induced faster PN formation than that of acrosome-intact spermatozoon. Therefore, we suggest that induction of AR of spermatozoa before ICSI accelerates the time of PN formation of the embryo. Whether inducing the AR in human sperm advances PN formation in injected human oocytes needs to be examined. Lee
et al.
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Acknowledgment. We are grateful ful comments on the manuscript.
to Sug Oh, M.D.,
for his help13.
REFERENCES 1. Fraser LR, Harrison RAP, Herod JE. Characterization of a decapacitation factor associated with epididymal mouse spermatozoa. J Reprod Fert 1990;89:135-48. 2. Roldan RES, Murase T, Shi Q-X. Exocytosis in spermatozoa in response to progesterone and zona pellucida. Science 1994;266:1578-81. 3. Yanagimachi R. Sperm-egg association in mammals. In: Moscona AA, Monroy A, editors. Current topics in developmental biology. Vol. 12. New York: Academic Press, 1978:83-105. 4. Palermo G, Joris H, Devroey P, Van Steirteghem AC. Pregnancies after intracytoplasmic injection of single spermatozoon into an oocyte. Lancet 1992;340:17-8. 5. Palermo G, Joris H, Derde M-P, Camus M, Devroey P, Van Steirteghem AC. Sperm characteristics and outcome of human assisted fertilization by subzonal insemination and intracytoplasmic sperm injection. Fertil Steril1993; 59:826-35. 6. Van Steirteghem AC, Liu J, Joris H, Nagy Z, Janssenswillen C, Tournaye H, et al. Higher success rate by intracytoplasmic sperm injection than by subzonal insemination. Report of a second series of 300 consecutive treatment cycles. Hum Reprod 1993;7:1055-60. 7. Lacham 0, Trounson AO, Holden C, Mann J, Sathananthan H. Fertilization and development of mouse eggs injected under the zona pellucida with single spermatozoa treated to induced the acrosome reaction. Gamete Res 1989;23:233-43. 8. Edwards RG, Van Steirteghem AC. Intracytoplasmic sperm injection: does calcium hold the key to success? Hum Reprod 1993;8:988-9. 9. Liu J, Nagy Z, Joris H, Tournaye H, Devroey P, Van Steirteghem AC. Intracytoplasmic sperm injection does not require special treatment of the spermatozoa. Hum Reprod 1994;6:1127-30. 10. Mahadevan M, Trounson AO. Cryopreservation of human semen. In: Artificial insemination by donor. Melbourne: Brown Prior Anderson, 1980:19-32. 11. Quinn P, Kerin JF, Warnes GM. Improved pregnancy rate in human in vitro fertilization with the use of a medium based on the composition of human tubal fluid. Fertil Steril 1985;44:493-8. 12. Mendoza C, Carreras A, Moos J, Tesarik J. Distinction between true acrosome reaction and degenerative acrosome loss
320
Lee
et al.
Acrosome
reaction
and ZCSZ
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
by a one-step staining method using Pisum sativum agglutinin. J Reprod Fert 1992;95:755-63. Mortimer D, Curtis EF, Miller RG. Specific labelling by peanut agglutinin of the outer acrosomal membrane of the human spermatozoon. J Reprod Fert 1987;81:127-35. Munne S, Lee A, Rosenwaks Z, Grifo J, Cohen J. Diagnosis of major chromosome aneuploidies in human preimplantation embryos. Hum Reprod 1993;8:2185-91. Aitken RJ, Brindle JP. Analysis of the ability of three probes targeting the outer acrosomal membrane or acrosomal contents to detect the acrosome reaction in human spermatozoa. Hum Reprod 1993;8:1663-9. Takano H, Yanagimachi R, Urch U. Evidence that acrosin activity is important for the development of fusibility of mammalian spermatozoa with oolemma: inhibitor studies using the golden hamster. Zygote 1993; 1:79-91. Tesarik J, Testart J. Human sperm-egg interactions and their disorders: implications in the management of infertility. Hum Reprod 1989;4:729-41. Tesarik J, Mendoza C. Sperm treatment w-ith pentoxifylline improves the fertilizing ability in patients with acrosome reaction insufficiency. Fertil Steril 1993;60:141-8. Sengoku K, Tamate K, Takaoka Y, Ishikawa M. Effects of platelet activating factor on human sperm function in vitro. Hum Reprod 1993;8:1443-7. Pampiglione JS, Tan S-L, Campbell S. The use of the stimulated acrosome reaction test as a test of fertilizing ability in human spermatozoa. Fertil Steril 1993;59: 1280-4. Tesarik J, Sousa M, Testart J. Human 0ocyt.e activation after intracytoplasmic sperm injection. Hum Reprod 1994;9:5118. Dozortsev D, Rybouchkin A, De Sutter P, Qian C, Dhont M. Human oocyte activation following intracytoplasmic injection: the role of the sperm cell. Hum Reprod 1995; 10:404-7. Nagy ZP, Liu J, Joris H, Devroey P, Van Steirteghem A. Time-course of oocyte activation, pronucleus formation and cleavage in human oocytes fertilized by intracytoplasmic sperm injection. Hum Reprod 1994;9:1743--8. Dozortsev D, De Sutter P, Dhont M. Behaviour of spermatozoa in human oocytes displaying no or one pronucleus after intracytoplasmic sperm injection. Hum Reprod 1994;9:213944. Edwards RG, Fishel S, Cohen J, Fehilly CB, Purdy JM, Slater JM, et al. Factors influencing the success of IVF for alleviation of human infertility. J In Vitro Fert Embryo Transf 1984; 1:3-23.
Fertility
and
Sterility@