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Independent Spatial and Temporal Functions of Human Sperm Centrosomes After Dispermic Microinjection Into Bovine Oocytes

From the Department of Obstetrics and Gynecology, Tohoku University School of Medicine, Sendai, Japan.

Correspondence to: Dr Yukihiro Terada, Associate Professor, Department of Obstetrics and Gynecology, Tohoku University School of Medicine, Seiryo-machi, Aoba-ku, Sendai, Miyagi, 980-8574, Japan (e-mail: terada{at}mail.tains.tohoku.ac.jp).

Received for publication December 27, 2008; accepted for publication April 1, 2009.

Abstract

During mammalian fertilization, a centrosome is introduced by the sperm during the first cell cycle to organize a radial array of microtubules known as the sperm aster. In nature, multiple human sperm centrosomes may exist in the same egg cytoplasm during polyspermy. However, critical information concerning individual sperm centrosomal function with regards to the latter case remains unknown. We subsequently examined the sperm aster formation after injection of multiple human sperm into a bovine egg. When 2 fertile human sperm were simultaneously microinjected into different regions of the same bovine egg cytoplasm, no difference in sperm aster formation rate was observed compared to cases in which a single sperm was injected. Two human sperm were also microinjected into bovine eggs 30-, 60- and 120-minute intervals apart from one another, and no difference in sperm aster formation rates were observed. Among eggs in which 1 sperm aster was organized, there was no observable bias towards the first or second injected sperm. These findings indicated that when multiple human sperm are present in a single egg cytoplasm, each centrosome can function independently from the other. This fact suggests the possibility of transplanting a normal sperm centrosome into an egg with a sperm known to have centrosomal dysfunction.

     Key words: Fertilization, sperm aster, polyspermy, microtubules

We recently reported that heterologous ICSI of human sperm into bovine eggs can be used to assess human sperm centrosomal function (Nakamura et al, 2001). Using this assay, we reported several examples of human infertility with poor sperm centrosomal function (Nakamura et al, 2002; Rawe et al, 2002). Developing the treatment for this type of infertility caused by poor human sperm centrosomal function is a novel strategy of assisted reproductive technology (ART).

In a previous study, we presented gamete manipulation for activation of the sperm centrosome and centrosomal transplantation as a possible treatment for poor sperm centrosomal function. We endeavored to restore defective sperm centrosomal function in human sperm with known dysplasia of fibrous sheath (DFS). Prior to heterologous ICSI of human sperm into bovine egg, sperm were treated with dithiothreitol. On the other hand, oocytes were treated with the cytoskeletal stabilizer paclitaxel after ICSI. However, this cytoskeletal manipulation of gametes did not improve the sperm aster formation rates in the bovine egg in DFS sperm (Nakamura et al, 2005b).

The other candidate for treating infertility caused by sperm centrosomal dysfunction is sperm centrosomal transplantation. From this, we reported the occurrence of microtubule organization during egg cleavage in the absence of the sperm centrosome (Morita et al, 2005; Morito et al, 2005). In bovine parthenogenesis, the egg cytoplasm has been observed to reorganize microtubules into a cytoplasmic aster to move the female pronucleus to the cell center. This report suggested that the egg cytoplasm functioned as a microtubule organizing center (MTOC) when the sperm centrosome was absent (Morito et al, 2005). Moreover, sperm aster formation without cytoplasmic aster formation indicated that egg centrosomes were suppressed during normal fertilization (Simerly et al, 1995; Nakamura et al, 2001). In addition, the function of the paternal centrosome was selectively shut off during fertilization of Spisula solidissima (Wu and Palazzo, 1999). These reports suggested that a functional relationship between centrosomes existed.

Upon the transplantation of a normally functioning centrosome to a sperm with centrosomal dysfunction, the interaction between each individual centrosome (paternal vs maternal, paternal vs paternal) must be considered. If paternal centrosomes are able to act independently from one another, the possibility of assisted fertilization in such cases, through transplantation of a normal paternal centrosome, in highly likely.

In this study, we examined sperm aster formation after heterologous ICSI with multiple human sperm into a bovine egg. The timing and position of each injection were varied and the interaction between multiple sperm centrosomes within the same egg cytoplasm was analyzed.

Consequently, the function of each sperm centrosome was noted to be independent of the other, which suggested that the presence of multiple sperm centrosomes had no determinable interactive effects.

Materials and Methods

All procedures were performed under the approval of an internal review board at the Tohoku University School of Medicine.

In Vitro Maturation

Bovine ovaries were obtained at a local slaughterhouse and eggs were recovered by aspiration from 2–8-mm follicles. Eggs were matured for 22–24 hours in Medium 199 (M199; Gibco, Grand Island, New York) supplemented with 10% (vol/vol) fetal calf serum (FCS; Gibco), 0.12 IU/mL FSH (Antrin; Denka Pharmaceutical, Kanagawa, Japan), and 50 ng/mL recombinant human epidermal growth factor (Upstate; Lake Placid, New York) at 38.5°C with 5% CO₂ in air. Cumulus cells were removed and briefly incubated in 1 mg/mL collagenase (Sigma, St Louis, Missouri) and 2 mg/mL hyaluronidase (Sigma) in M2 culture medium (Sigma). Eggs that paused at the second meiotic metaphase were selected for ICSI.

ICSI With Human Sperm Using a Piezo-Micromanipulator

Surplus sperm from 3 fertile donors were obtained with informed consent and frozen in TEST-yolk buffer (Irvine Scientific Co, Santa Ana, California). The sperm samples were thawed at room temperature and washed with modified human tubal fluid medium (Irvine Scientific) supplemented with 10% serum substitute supplement (Irvine Scientific) by centrifugation at 500 x g for 5 minutes. The sperm pellet was resuspended in M2 culture medium. Half of the sperm samples were incubated in M2 culture medium containing 0.5 µM MitoTracker orange CMTMRos (Molecular Probes, Eugene, Oregon) for 10 minutes at 37°C. The stained sperm were then washed with M2 culture medium. 10% polyvinyl pyrrolidone was added to the sperm samples before use. Only motile and normal-shaped sperm were used. After being immobilized with a piezo-micromanipulator (MB-U; Prim Tech, Tsuchiura, Japan), sperm were loaded into the pipette. The zona pellucida was penetrated using piezo-pulses. A piece of the zona pellucida in the pipette was expelled, and the immobilized sperm were moved to the tip of the injection pipette. The pipette was inserted into the ooplasm as the oolemma was punctured by applying 1 piezo-pulse. The sperm on the tip of the pipette was injected into the ooplasm simultaneously with a minimum amount of sperm suspension medium.

Study Design

Experimental design is summarized in Figure 1. ICSI was performed in the following 5 groups: Control: One sperm was injected into the bovine egg cytoplasm (group A). Two sperm at the same time: Two sperm stained by MitoTracker orange were injected into either the same region (group B) or into different regions (group C) of respective bovine egg cytoplasm simultaneously. Two sperm with injection interval: Two sperm were injected at 30-minute (group D), 60-minute (group E), or 120-minute (group F) intervals into the same bovine egg cytoplasm. All later-injected sperm were stained by MitoTracker orange. After injection, eggs were cultured in M199 supplemented with 10% FCS at 38.5°C with 5% CO₂ in air. Eggs were fixed 6 hours after their first injections.

View larger version (34K): [in this window] [in a new window]   Figure 1. Experimental design. MII indicates metaphase II oocyte.

Statistics

The sperm aster formation rates of these groups were compared using the ² test. P values less than .05 were considered to be statistically significant.

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 a single human sperm (group A) is shown in Figure 2a. At 6 hours post-ICSI, the sperm aster was organized, with microtubules elongating throughout the cytoplasm until coming into contact with the developing female pronucleus, whereas the male pronucleus decondensed. When 2 human sperm were injected into the same place and time (group B), it was impossible to identify the number of MTOCs even by confocal microscopic observation (image not shown), probably because the 2 sperm centrosomes were too close to one another to detect. The microtubule organization and chromatin configuration in bovine eggs after ICSI with 2 human sperm at the same time and in different locations (group C) are shown in Figure 2b and c. At 6 hours post-ICSI, 2 sperm asters (Figure 2b) or 1 (Figure 2c) sperm aster was organized in bovine eggs. The microtubule organization and chromatin configuration in bovine eggs after ICSI with 2 human sperm injected 30 minutes apart from one another (group D) are shown in Figure 2d through f. At 6 hours post-ICSI, 2 sperm asters (Figure 2d) or 1 (Figure 2e: prior-injected sperm organize a sperm aster; Figure 2f: later-injected sperm organize a sperm aster) sperm aster was organized in bovine eggs. Similar results were obtained for the 60-minute (group E) and 120-minute (group F) injection intervals. For all double sperm injections, Mitotracker was used to stain the sperm mitochondria of the second sperm.

View larger version (64K): [in this window] [in a new window]   Figure 2. Microtubule (green), chromatin (blue), and sperm mitochondria (orange) configurations in bovine eggs after intracytoplasmic injection by a piezo-driven pipette of human sperm into bovine egg. In group C, both sperm were stained by MitoTracker orange. In groups D, E, and F, the later-injected sperm was stained by MitoTracker orange. At 6 hours post-ICSI, eggs were fixed and microtubule and chromatin were stained by immunofluorescein. (a) In the case of single human sperm injection (group A), a radial array of microtubules (arrow, sperm aster) was organized from a single sperm centrosome. (b) Two human sperm were microinjected at the same time and at different locations (group C). Two sperm asters were organized in bovine eggs. (c) Two human sperm were microinjected at the same time and at different locations (group C). One sperm aster was organized in bovine eggs. (d) Two human sperm were microinjected 30 minutes apart from one another (group D). Two radial arrays of microtubules were organized from 2 sperm centrosomes. (e) Two human sperm were microinjected 30 minutes apart from one another (group D). A single radial array of microtubules was organized from the centrosome of the first sperm injected. (f) Two human sperm were microinjected 30 minutes apart from one another (group D). A single radial array of microtubules was organized from the centrosome of the second sperm injected. Fpn indicates female pronucleus; Mpn, male pronucleus. Bar = 20 µm.

Sperm Aster Formation Rates

The results for sperm aster formation are summarized in Tables 1, 2, 3. Sperm aster formation rates of 1 or 2 human sperm in bovine eggs at 6 hours post–first ICSI are summarized in Table 1. In group A (single sperm), the sperm aster was organized in 39 out of 56 injected eggs (69.6%). As compared to group A, there was no difference between sperm aster formation rate in each condition when compared to 1 sperm (Table 1).

Table 2 shows the number of the eggs that formed 2 sperm asters or 1 sperm aster after the injection of 2 human sperm at 6 hours post–first ICSI. We counted only eggs with 2 sperm in groups C–F. In group C (simultaneous double injection in 2 different locations in the egg), 2 sperm asters were observed in 15 out of 35 injected eggs (42.9%). One sperm aster was organized in 9 eggs (25.7%). In group D (30-minutes injection interval), 2 sperm asters were observed in 14 injected eggs (50.0%). One sperm aster was organized in 9 of the eggs (32.1%). In group E (60-minute intervals), 2 sperm asters were organized in 10 injected eggs (40.0%). One sperm aster was organized in 11 eggs (44.0%). In group F (120-minute intervals), 2 sperm asters were organized in 7 injected eggs (36.8%). One sperm aster was organized in 8 eggs (42.1%). In comparison to sperm aster formation rates when 2 sperm were injected simultaneously, there was no difference in sperm aster formation rate for each condition when evaluated by injection duration (Table 2). Table 3 shows the number of eggs that organized single sperm asters after 2 sperm injections at different times. In group D, the first sperm injected organized an aster in 4 eggs and the second sperm injected organized an aster in 5 eggs. In group E, the first sperm injected organized an aster in 2 eggs and the second sperm injected organized an aster in 9 eggs. In group F, the first sperm injected organized an aster in 5 eggs and the second sperm injected organized an aster in 3 eggs.

Discussion

According to a study by Rawe et al (2000), involving microtubule organization and DNA configuration of fertilization failure human eggs after ICSI, 20% of oocytes will be arrested during pronuclei apposition with lack of a sperm aster. This report indicated that sperm centrosomal function (sperm aster formation) was an essential event toward the goal of fertilization, the union of male and female genome inside of an egg (Terada, 2007).

We invented a novel assay for human sperm centrosomal function by using heterologous ICSI of human sperm into bovine eggs. Sperm aster formation rates of fertile human sperm inside of bovine eggs were around 60.0% (Nakamura et al, 2001). Previously, we reported 2 cases of infertility with defective sperm centrosomal function: 1 was a case of globozoospermia (Nakamura et al, 2002; Dam et al, 2007) and the other was a case of DFS (Rawe et al, 2002). The development of a treatment for such examples of sperm centrosomal dysfunction would greatly improve clinical ART.

Here, we have proposed gamete manipulation for activation of sperm centrosome and centrosomal transplantation as treatment for sperm centrosomal dysfunction. However, molecular mechanisms for the function of sperm centrosomes during fertilization remain elusive (Schatten, 1994). Abnormal alignments of the head-tail junction were observed in the DFS sperm with a disorganized centrosome. Furthermore, the sperm aster formation rate of DFS sperm inside of bovine eggs was significantly lower than that of normal fertile sperm (Rawe et al, 2002). We tried to restore defective sperm centrosomal function in human sperm with DFS by chemical manipulation of gametes, as mentioned in the introduction. However, complications in chemical manipulation of gametes did not work for DFS sperm, which congenitally lacked normal sperm centrosome (Nakamura et al, 2005b).

With the possible development of techniques to inject normally functioning sperm centrosomes during ICSI with defective sperm centrosomes, treating sperm centrosomal dysfunction will be achievable. In human somatic cell studies, isolated centrosomes (Mitchison and Kirshner, 1984, 1986), transplanted into the oocyte were found to be functioning normally (Picard et al, 1987).

One important factor in the development of exogenous sperm centrosomal transplantation is understanding the functional relation of sperm centrosomes, especially in this situation, in which 1 sperm contains a dysfunctional centrosome and the other is an isolated exogenous centrosome. Moreover, because our observation of microtubule organization during bovine parthenogenesis (Morito et al, 2005) and rabbit ICSI using sperm without a sperm centrosome (Morita et al, 2005) indicated that the egg cytoplasmic centrosomes can be functional, we also have to examine the functional relationship between paternal centrosomes and maternal centrosomes. As for maternal MTOC, these are technically too difficult to capture and operate functionally at this point in time. For our study, we simply tested how multiple centrosomes in the same oocyte cytoplasm interacted by microinjecting 2 human sperm into the same bovine oocyte cytoplasm.

When 2 sperm were injected simultaneously but in different locations in the egg (Figure 2b and c), the overall sperm aster formation rate was the same as when a single sperm was injected (Table 1). Sperm asters were organized from about 40% of the sperm injected, and only a single sperm formed an aster in 30% of eggs. Egg activation was confirmed in all injected eggs using a piezo-ICSI system. These results indicated that each human sperm centrosome was functional even when multiple human sperm centrosomes were in the same egg cytoplasm. Moreover, the material to organize 2 sperm asters (eg, tubulin and centrosomal proteins) existed in bovine eggs.

Though little has been known about the microtubule organization during human polyspermic fertilization (Asch et al, 1995), the function of sperm centrosome during polyspermic fertilization has been well studied in amphibians. During amphibian fertilization, the distribution of tubulin and functional proteins necessary for sperm aster formation in the ovum cytoplasm is not uniform (Nakamura et al, 2005a). A sperm that is in a suitable position can organize an aster and fuse its pronucleus with the female pronucleus, but sperm that are not at the appropriate position disappear in the egg cytoplasm (Iwao et al, 1997). In contrast, during porcine polyspermic fertilization, multiple male pronuclei are separated by microtubule domains, and if 2 male pronuclei are distant from one another, pronuclear apposition and fusion do not occur. Furthermore, with a high degree of polyspermy, pronuclear apposition did not proceed and microtubule assembly was delayed (Suzuki et al, 2003). Our results showing the formation of 2 human sperm asters in the same bovine ovum suggested that both human sperm centrosomes can function in the same egg cytoplasm. Also, recent findings suggested that mammalian eggs may be at risk for abnormal development as compared to amphibian eggs, because when 2 sperm fuse, each may form a spindle.

In general, cells cannot tolerate multiple active centrosomes because of the subsequent introduction of multipolar spindles (Brinkley and Goepfert, 1998). Fertilization in S. solidissima eggs results in cells with 3 active centrosomes, 2 maternal and 1 paternal, but the function of the paternal centrosome is selectively shut off, possibly because the embryos can identify and control the function of centrosomes (Wu and Palazzo, 1999). When we injected 2 human sperm into bovine eggs 30 minutes apart from one another, aster formation from only 1 sperm was observed in 25% of eggs, and there was no bias towards either the first or the second sperm injected (Table 3). This suggests that a human sperm centrosome that is already present in the egg does not influence the function of the centrosome of the later-injected sperm.

The maturation-promoting factor (MPF) activity after bovine ICSI was previously shown to fluctuate, decreasing to 30% of the value of metaphase II oocytes post-ICSI, decreasing further, increasing temporarily, then decreasing again at 8 hours post-ICSI (Fujinami et al, 2004). We observed that sperm aster formation with a 60- or 120-minute sperm injection interval was similar to results obtained with a 30-minute injection interval (Table 2), suggesting that MPF does not significantly influence human sperm centrosomal function.

In conclusion, these results indicate that human sperm centrosomes can function independently of one another. This fact suggests that when a normal sperm centrosome is injected into an egg simultaneously with sperm with centrosomal dysfunction, the latter normal centrosome can work independently.

Acknowledgments

We are grateful to Ms Aiko Takahashi for her technical support and to Dr Clarissa Velayo for preparation of the manuscript.

Footnotes

Supported by a Grant-in Aid for Scientific Research from Japan Society for the Promotion of Science (Y.T., N.Y., and K.O.).

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This Article Abstract Full Text (PDF) All Versions of this Article: 30/5/559    most recent Author Manuscript (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Terada, Y. Articles by Okamura, K. Search for Related Content PubMed PubMed Citation Articles by Terada, Y. Articles by Okamura, K.