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Human Sperm Aster Formation and Pronuclear Decondensation in Bovine Eggs Following Intracytoplasmic Sperm Injection Using a Piezo-Driven Pipette: A Novel Assay for Human Sperm Centrosomal Function

^a Department of Obstetrics and Gynecology, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi 980-8574, Japan ^b Laboratory of Animal Reproduction, Department of Bioresources, Hiroshima Prefectural University, 562, Nanatsuka, Shoubara, Hiroshima 727-0023, Japan

ABSTRACT

In human fertilization, the sperm introduces the centrosome; the microtubule-organizing center and microtubules are organized within the inseminated egg from the sperm centrosome. These microtubules form a radial array, called the sperm aster, the functioning of which is essential to pronuclear movement for union of male and female genome. The sperm centrosomal function is considered to be necessary for the normal human fertilization process. Therefore, the dysfunction of sperm centrosome is a possible cause of human fertilization failure. However, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. To assess the human sperm centrosomal function, we examined sperm aster formation and pronuclear decondensation following intracytoplasmic sperm injection (ICSI) with human sperm into the bovine egg using a Piezo-driven pipette and ethanol activation of eggs. After human sperm incorporation into bovine egg, we observed that the sperm aster was organized from sperm centrosome, and that the sperm aster was enlarged as the sperm nuclei underwent pronuclear formation. The sperm aster formation rate at 6 h post-ICSI and the male pronuclear formation rate at 8–12 h post-ICSI were 60.0% and 83.3%, respectively. No difference of the sperm aster formation rate and the male pronuclear formation rate was observed between eggs activated with ethanol and eggs without artificial activation. We concluded that this heterologous Piezo-ICSI system into bovine egg can be a novel assay for human sperm centrosomal function, and it is possible to explicate a course of fertilization failure that was unknown until now.

early development, fertilization, gamete biology, reproductive technology, sperm

INTRODUCTION

By intracytoplasmic sperm injection (ICSI), humans overcome some types of male infertility caused by problems of sperm entry into the egg [1]. However, fertilization is a "choreography" toward the union of male and female genome, and sperm entry is only the start of this movement. To improve the efficacy and safety of assisted reproductive technology, we have to pay more attention to the process of fertilization after sperm entry.

In human fertilization, sperm introduces the centrosome, and incorporated sperm centrosome organizes the radially arrayed microtubules, or sperm aster, that are essential to pronuclear movement for union of male and female genome [2, 3]. Aberrant microtubule organization in "fertilization failure eggs" during human in vitro fertilization (IVF) suggested that centrosomal dysfunction may be a cause of fertilization arrest [4]. Furthermore, the level of functioning of the zygotic centrosome varies among bulls during IVF, and this variation affects male fertility [5]. These data suggested that sperm centrosomal function plays important roles in the process of fertilization after sperm entry, and that sperm centrosomal dysfunction is a cause of human fertilization failure. By assessing the sperm centrosomal function, we may understand another aspect of a cause of infertility and the treatment for it. However, to our knowledge, little information is available regarding human sperm centrosomal function during fertilization in clinically assisted reproductive technology. Until now, one simple reason for this has been the difficulty of assaying the human sperm centrosomal function.

In bovine fertilization, the centrosome is paternally derived into the egg, and the sperm centrosome organizes the sperm aster, as in human fertilization [6]. Interspecies ICSI using bovine eggs and human sperm might be a relevant model to understand possible sperm centrosomal function. However, until now, it has been difficult to achieve fertilization by performing ICSI with bovine gamete [7]. High fertilization and embryo development rates have recently been reported in bovine ICSI using a Piezo-driven system and ethanol activation after ICSI [8, 9]. In the present study, we examined whether bovine egg can be a good tool for assessing human sperm centrosomal function. We employed a Piezo-ICSI system and artificial activation after ICSI for successful heterologous ICSI using bovine eggs and human sperm. We examined human sperm aster formation and chromatin configuration in bovine eggs for assessing the human sperm centrosomal function. Furthermore, we examined the sperm aster formation rate and the male pronuclear formation rate of the eggs without ethanol activation and compared these data to those of eggs with ethanol activation.

MATERIALS AND METHODS

In Vitro Maturation of Bovine Oocytes

Bovine ovaries were obtained at a local slaughterhouse, and oocytes were recovered by aspiration from 2- to 8-mm follicles. Oocytes were matured for 22–24 h in Hepes-buffered TCM-199 supplemented with 10% (v/v) fetal calf serum (FCS), 0.12 IU/ml of FSH (Antrin, Denka Pharmaceutical, Kanagawa, Japan), and 50 ng/ml of recombinant human epidermal growth factor (Tokyo, Japan) at 38.5°C with 5% CO₂ in air. Cumulus cells were removed by a brief incubation in 2 mg/ml of hyaluronidase (Sigma, St. Louis, MO) in M2 culture medium, and eggs that had arrested at the second meiotic metaphase were used for ICSI.

ICSI with Human Sperm Using a Piezo-Micromanipulator

Human sperm samples were obtained from three fertile donors under informed consent, and the sperm samples were frozen and thawed in a water bath at 37°C. Then, sperm were washed with modified HTF Medium (Irvine Scientific Co., Santa Ana, CA), supplemented with 10% serum substitute (Irvine Scientific), by centrifugation at 500 x g for 5 min. The sperm pellet was resuspended and then added to M2 culture medium with 10% (w/v) polyvinylpyrrolidone. After immobilization by touching the sperm tail with the injection pipette, the sperm was injected using a Piezo-micromanipulator (MB-U; Prim Tech, Tsuchiura, Japan). In Piezo-ICSI, the zona pellucida was penetrated using several Piezo-pulses. After a cylindrical piece of the zona in the pipette was expelled, an immobilized sperm was positioned at the tip of the pipette. The pipette was then inserted deeply into the ooplasm without applying Piezo-pulses. Next, the oolemna was punctured by application of one Piezo-pulse, and the entire sperm was expelled into the ooplasm with a minimum amount of sperm suspension medium.

After injection, eggs were cultured in Hepes-buffered TCM-199 supplemented with 10% (v/v) FCS at 38.5°C with 5% CO₂ in air under mineral oil. At 4 h post-ICSI, some groups of eggs were activated by 5-min exposure to 7% (v/v) ethanol in TCM-199. Eggs were fixed and stained at 6 and 8–12 h after ICSI on the activated group. The eggs without ethanol activation after ICSI were fixed and stained at 3–4, 6, and 8–12 h after ICSI. These examinations were performed at each time of fixation in three repetitions.

Immunocytochemical Detection of Microtubules and DNA

Zonae pellucidae were removed with M2 culture medium supplemented with 0.75% Protease (Sigma). After a 30-min recovery at 38.5°C, zona-free eggs were extracted for 15 min by buffer M (25% [v/v] glycerol, 50 mM KCl, 0.5 mM MgCl₂, 0.1 mM EDTA, 1 mM EGTA, 50 mM imidazole hydrochloride, and 1 mM 2-mercaptoethanol, pH 6.8) containing 5% (v/v) methanol and 1% (v/v) Triton X-100 detergent and fixed in cold methanol for 10 min according to the method of Simerly and Schatten [10]. Fixed eggs were then permeabilized overnight with 0.1 M PBS containing 0.1% (v/v) Triton X-100 detergent.

Microtubules were labeled with a mixture of monoclonal antibody against ß-tubulin (clone 2-28-33; diluted 1:100; Sigma) and acetylated -tubulin (clone 6-11-B1; diluted 1:100; Sigma). The primary antibodies were detected by fluorescein-conjugated goat anti-mouse immunoglobulin G (IgG; diluted 1:40; Zymed, San Francisco, CA). The DNA was detected after labeling with 10 mg/ml of Hoechst 33342.

Coverslips were mounted in antifade medium (Vectashield; Vector Laboratories, Burlingame, CA) and examined using conventional epifluorescence microscopy (Optiphot-2; Nikon, Tokyo, Japan). The images were recorded digitally and archived on magnet optical disks processed using Adobe Photoshop software (Adobe Systems, Inc., Mountain View, CA). Data were compared between the group with artificial activation and the group without artificial activation regarding sperm aster formation rate at 6 h post-ICSI and male pronuclear formation rate at 8–12 h post-ICSI by the chi-square test. A P value of less than 0.05 was considered to indicate statistical significance.

RESULTS

Microtubule Organization and Chromatin Configuration in Bovine Eggs Following ICSI with Human Sperm

Microtubule organization and chromatin configuration in bovine eggs after ICSI with human sperm are shown in Figure 1. The unfertilized bovine egg had an anastral, barrel-shaped meiotic spindle. The only microtubules present within the unfertilized metaphase II-arrest bovine egg were those of the metaphase spindle (Fig. 1A; microtubules, green; DNA, blue). At 3–4 h post-ICSI, microtubules were present in the meiotic midbody between the second polar body and the decondensing female chromosomes. Sperm nuclei were decondensed, but the sperm aster was not observed around the sperm centrosome at this time (Fig. 1B). At 6 h post-ICSI, the sperm aster, a radial microtubule array extending from the sperm centrosome, was organized. No microtubule was organized around female pronucleus (Fig. 1C). Microtubules elongated throughout the cytoplasm until they came into contact with the developing female pronucleus as the male pronucleus decondensed (Fig. 1D). During pronuclear movement to the center of the egg, both the male and female pronuclei were surrounded by a microtubule array (Fig. 1E), and the male and female pronuclei were adjacent (Fig. 1F).

View larger version (88K): [in this window] [in a new window]   FIG. 1. Microtubule (green) and chromatin (blue) configurations in bovine eggs after intracytoplasmic injection with human sperm by a Piezo-driven pipette. A) The unfertilized bovine egg has microtubules only at the second meiotic spindle. B) At 3–4 h post-ICSI, the midbody structure (arrow) connecting the decondensing female chromosomes was observed. Sperm nucleus (arrowhead) was decondensed, but no microtubules were organized around the sperm centrosome. C) At 6 h post-ICSI, a radial array of microtubules (the sperm aster; arrow) was organized from the sperm centrosome. Microtubules were not organized around the female pronucleus. D) As the male and female pronuclei decondensed, the sperm aster enlarged toward the female pronucleus. E) During pronuclear centration, male and female pronuclei were surrounded by a microtubule array (12 h post-ICSI). F) Male and female pronuclei were adjacent, and the extensive microtubules were organized around both pronuclei. Fpn, Female pronucleus; Mpn, male pronucleus. Bar = 25 µm

Sperm Aster Formation Rate and Male Pronuclear Formation Rate

Bovine eggs were scored for sperm aster formation at 6 h post-ICSI (Table 1). The sperm aster was organized in 39 (62.9%) of 62 injected eggs with artificial activation by ethanol at 4 h post-ICSI, whereas the sperm aster was organized in 15 (60.0%) of 25 eggs without artificial activation after ICSI.

Male pronuclear formation was examined at 8–12 h post-ICSI (Table 2). Male pronuclear formation was observed in 67 (79.8%) of 84 eggs with artificial activation by ethanol at 4 h post-ICSI. Also, male pronuclear formation was observed in 45 (83.3%) of 54 injected eggs without artificial activation. Differences in the sperm aster formation rate and the male pronuclear formation rate between groups were not significant. All eggs, which had male pronucleus, had female pronucleus. Premature chromosome condensation of the sperm nucleus (Fig. 2) was observed in only a few eggs in both groups.

View larger version (27K): [in this window] [in a new window]   FIG. 2. Microtubule (green) organization and chromatin (blue) configuration associated with a defect in chromatin decondensation. Sperm nucleus had undergone premature chromosome condensation (arrow, sperm nucleus; arrowheads, sperm tail). The egg nucleus was arrested in metaphase II. Bar = 10 µm

DISCUSSION

The human sperm centrosomal dysfunction is supposed to relate to fertilization failure, but to our knowledge, few studies have been reported. One promising method for the assessment of human sperm centrosomal function is an in vivo system using mammalian eggs. Mouse and hamster are common experimental animals. However, centrosome was maternally derived in mouse fertilization [11]. Furthermore, sperm aster was absent from human sperm centrosome in hamster egg [12]. These reports suggested that mouse and hamster eggs cannot be a tool for the assessment of human sperm centrosomal function.

Paternal centrosomal inheritance during fertilization has been demonstrated in the bovine [6], rabbit [13], and rhesus monkey [14]. Human sperm aster formation following ICSI into rabbit egg has been demonstrated by Terada et al. [13], who found that a heterologous ICSI system using eggs of a species with paternal centrosomal inheritance can be a tool for the assessment of sperm centrosomal function. However, the sperm aster formation rate in rabbit eggs after ICSI using fertile donor sperm was low (30–40%). Eggs from nonhuman primates might be good models, but they remain special experimental animals. Thus, another relevant system to assess human sperm centrosomal function was needed.

In bovine fertilization, the centrosome is paternally derived into the egg, and the sperm centrosome organizes the sperm aster, as in human fertilization [6]. The present study suggests that the bovine egg can be a tool for assessment of human sperm centrosomal function. However, bovine egg activation rates after conventional ICSI were very low [7]. Several reports showing a "breakthrough" of ICSI and micromanipulation technology using a Piezo-driven injection system have appeared [15–17]. Also, in bovine ICSI, high fertilization and embryo development rates have recently been reported using the Piezo-ICSI procedure [8, 9]. We employed this technique for a heterologous ICSI system with human sperm into bovine egg. In this investigation, the male pronuclear formation rate in bovine eggs after Piezo-ICSI with human sperm was 83.3% without artificial activation by ethanol, and female pronuclei were found in all eggs that formed male pronuclei. The male and female pronuclear formation rate in this study was similar to that of the bovine ICSI system using the Piezo-driven pipette as previously reported [8, 9]. Why does ICSI using the Piezo-driven pipette work better than the conventional ICSI? In conventional ICSI, the oolemna is injured severely when the injection pipette penetrates. As a result, cytolysis of the oocyte occurs. In Piezo-ICSI, however, the zona is penetrated smoothly, without deformation of zona and cytoplasm. Furthermore, the oolemna is broken by only single Piezo-pulse, and sperm is expelled into cytoplasm. Therefore, injury to the oolemna is minimal, and a small volume of injection media with polyvinylpyrrolidone is left in the cytoplasm [18, 19]. Human sperm aster formation rate in bovine eggs after Piezo-ICSI was almost 60% in either eggs with artificial activation by ethanol or eggs without artificial activation. As compared with heterologous ICSI using rabbit egg and human sperm, human sperm aster formation rate in bovine eggs was higher. These data indicate that this heterologous ICSI system may be a more relevant way to assess the human centrosomal function.

In bovine ICSI, the biggest problem is low cleavage rate [20, 21]. This problem may be related to insufficient oocyte activation. Both Horiuchi and Numabe [9] and Horiuchi et al. [22] reported high blastocyst formation rate in bovine ICSI by ethanol activation. Artificial activation by ethanol after ICSI improved the cleavage rates (33% without activation, 72% with activation) and blastocyst formation rates (14% without activation, 29% with activation) [9]. Nevertheless, in the present study, artificial activation by ethanol after ICSI with human sperm and bovine eggs did not affect human sperm decondensation or sperm aster formation, which is an interesting result. We speculate that artificial activation after ICSI might have some influence on the events after sperm aster formation, including pronuclear formation.

We conclude that this heterologous ICSI into bovine eggs with human sperm can be a relevant assay for human sperm centrosomal function, which contributes to the diagnosis and treatment of some types of male infertility. In addition, it was indicated that the Piezo-micromanipulator was useful for this heterologous ICSI system.

ACKNOWLEDGMENTS

All procedures were undertaken with the approval of the internal review board of the Tohoku University School of Medicine. We are grateful to Mr. Yasuhiro Yamauchi (Hiroshima Prefectural University) for technical support and Prof. Gerald Schatten (Oregon Regional Primate Research Center) for helpful comments and continuous encouragement.

FOOTNOTES

First decision: 13 March 2001.

¹ Correspondence. FAX: 81 22 717 7258; terada{at}ob-gy.med.tohoku.ac.jp

Accepted: June 13, 2001.

Received: February 15, 2001.

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