Microtubule organization during rabbit fertilization by intracytoplasmic sperm injection with and without sperm centrosome

Abstract

Aim: In most mammalian fertilization, the sperm introduces the centrosome, which acts as a microtubule organizing center (MTOC) and is essential for pronuclear movement. In rabbit fertilization, biparental centrosomal contribution in microtubule organization has been suggested.

Methods: To reveal the function and inheritance of the centrosome during rabbit fertilization, we compared microtubule organization and early embryonal development following intracytoplasmic sperm injection (ICSI) with and without sperm centrosome. Sperm centrosome was removed by sonication, and the isolated sperm head was injected by a Piezo‐driven micromanipulator. Samples were studied by light microscope after immunocytological stain.

Results: The sperm aster formation was observed 2–3 h after ICSI with intact sperm. In contrast, microtubules were organized between the male and female pronucleus without a nucleation site in the eggs after ICSI with an isolated sperm head. In the late pronuclear stage following ICSI with an isolated sperm head, microtubule organization was the same as in late pronuclear stage eggs after intact sperm injection. The first mitotic spindle was organized in eggs following ICSI with an isolated sperm head, as observed in eggs following ICSI with an intact sperm.

Conclusions: These results indicate that the MTOC is in oocyte cytoplasm during fertilization and fulfils the function when the sperm centrosome is absent. (Reprod Med Biol 2005; 4: 169–178)

INTRODUCTION

FERTILIZATION IS THE dynamic motility of the female and male genomes towards their union. ¹ The microtubule, which is one of the cytoskeletal structures, plays important roles in the movement of male and female genomes. The microtubule is a polar structure, one end of which is stabilized by its embedding in a structure called the microtubule organizing center (MTOC). The centrosome is a complex structure made up of a pair of centriole and pericentriolar proteins, and it acts as the MTOC in a cell cycle. ²

In general, a zygote requires one functional centrosome, organizing its microtubules before the first cleavage of fertilization. Except for very few known species, such as in the mouse, ³ the centrosome is provided by the sperm, and it forms a monopolar array of microtubules known as the sperm aster, the function of which is essential for pronuclear movement for the union of the male and female genomes. ⁴ , ⁵ , ⁶ , ⁷ The centrosome is provided by the sperm in primates, ⁸ cows, ⁹ pigs, ¹⁰ and sheep. ¹¹ In mice, the paternal centrosome degenerates during spermiogenesis, ¹² and microtubules assemble in association with the maternal pericentriolar material. ⁶ In human fertilization, the sperm introduces the centrosome, and the incorporated sperm centrosome organizes the sperm aster that is essential for syngamy. ⁶ , ¹³ The pattern of the centrosome inheritance during fertilization is different in each species.

In rabbit fertilization, the centrosome has been considered to follow the paternal pattern of inheritance because of the presence of a monoastral sperm aster during fertilization. ⁴ , ¹⁴ Isolated sperm head without a midpiece failed to nucleate sperm asters in rabbit eggs. ¹⁵ These facts suggest that the male centrosome is required to organize microtubules in rabbit fertilization. In in vivo rabbit fertilization, microtubules are organized into a radial aster from the sperm head, and cytoplasmic microtubules are organized around the male and female pronuclei. In parthenogenetically activated eggs, microtubule arrays were organized around the single female pronucleus. ¹⁶ These facts point to the possibility of biparental centrosomal contribution during rabbit fertilization as opposed to a strictly paternal inheritance pattern, suggested by previous studies. ⁴ , ¹⁴ However, the specific roles of the paternal and maternal centrosome are still unclear in biparental inheritance.

To reveal the role of the sperm centrosome in rabbit fertilization, we prepared a paternal centrosome dysfunction model. The sperm centrosome was removed by sonication from the sperm nucleus. Intracytoplasmic sperm injection (ICSI) with an isolated sperm head by a Piezo‐driven pipette (Piezo‐ICSI) ¹⁷ , ¹⁸ was carried out as a paternal centrosomal dysfunction model. For comparison, rabbit Piezo‐ICSI using intact rabbit sperm was also undertaken. To assess the role of paternal and maternal centrosomal function during rabbit fertilization, we examined microtubule organization in rabbit eggs after Piezo‐ICSI. Microtubule organization and early embryonal development were compared between rabbit zygotes following Piezo‐ICSI with and without sperm centrosome.

MATERIALS AND METHODS

Oocyte collection

ALL ANIMAL PROCEDURES were carried out under Tohoku University procedures and ethical guidelines. Mature New Zealand White rabbits were superovulated with six s.c. injections of 0.3 mg follicle‐stimulating hormone from porcine (Antorin; Denka Pharmacy, Kawasaki, Japan) at 12 h interval, followed by a single i.v. injection of 70 IU human chorionic gonadotropin (hCG) (Gonatotropin; Teikokuzouki Pharmacy, Tokyo, Japan). Fourteen hours post‐hCG, the rabbit was euthanized by 5 mL of 10% KCl i.v. injection. Oocytes were collected by flushing the oviduct. Cumulus cells were removed by a brief incubation 2 mg/mL hyaluronidase (Sigma, St. Louis, MO, USA) in medium 199 with Hanks salts (Gibco, Grand Island, NY, USA) containing 10% fetal bovine serum (FBS).

Sperm preparation

Ejaculated sperm were collected from mature New Zealand White rabbits by using an artificial vagina. Sperm were washed in Brackett and Oliphant medium containing 10% FBS. ¹⁹ Sperm suspensions were centrifuged at 50 g for 3 min. The sperm pellet was resuspended, and the sperm were allowed to swim up for 30 min. The isolated sperm heads were obtained by the modified method reported by the Kuretake et al. ²⁰ For isolation of sperm centrosome from sperm head, the sperm were separated by ultrasound for 3 min at 100% output in a Bransonic sonicator (Emerson Japan, Kanagawa, Japan).

Intracytoplasmic sperm injection by Piezo‐micromanipulator

Prepared sperm were resuspended and then added to EARL's balanced salt solution (Sigma) 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; Primtech, 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 oolemma 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 EARL's balanced salt solution with 10% BSA (v/v) at 37°C with 5% CO₂ in air under mineral oil, and fixed periodically.

Immunocytochemical detection of microtubules and DNA

Rabbit zona pellucidae were removed with 0.75% Pronase (Sigma) in media. After a 30 min recovery at 37°C, zona‐free eggs were extracted for 15 min by Buffer M (25% (v/v) glycerol, 50 mM MgCl₂, 0.1 mM ethylenediaminetetraacetic acid, 1 mM glycoletherdiaminetetraacetic acid (EGTA), 50 mM imidazole hydrochloride and 1 mM 2‐mercaptoethanole, pH 6.8) containing 5% (v/v) methanol and 1% (v/v) Triton‐X‐100 detergent and fixed in −20°C methanol for 10 min, according to the method of Simerly and Schatten. ²¹ Fixed eggs were then permeabilized overnight with 0.1 M phosphate‐buffered saline (PBS) containing 0.1% (v/v) Triton‐X detergent. Microtubules were labeled with a mixture of monoclonal antibody against β‐tubulin (clone 2‐28‐33; diluted 1 : 100; Sigma) and acetylated α‐tubulin (clone 6–11‐B1; diluted 1 : 100; Sigma). Primary antibodies were detected by fluorescein‐conjugated goat antimouse immunoglobulin G (IgG; diluted 1 : 40; Zymed, San Francisco, CA, USA). DNA was detected after labeling with 10 mg/mL of Hoechst 33342. Coverslips were mounted in antifade medium (Vectashield; Vector Laboratories, Burlingame, CA, USA). Samples were studied by a light microscope (Leica DMRXA/HC; Leica Microsystems, Heidelberg, Germany) or scanning confocal microscope (Leica TCS NT). Images were pseudocolored using Adobe Photoshop 4.0 (Adobe Systems, Moutain, CA, USA).

Data about microtubules formation rates, pronucleus formation rates and cleavage rates were compared between the group of ICSI with intact sperm and group of ICSI with sperm head by the χ² test. A P‐value of less than 0.05 was considered to indicate statistical significance.

Immunocytochemical detection of centrosome in sperm

Sperm and isolated sperm heads were extracted by media consisted of 2% paraformaldehyde (v/v), 50 mM piperazine‐N, N′‐bis (2‐ethane sulfonic acid), 5 mM EGTA 5 mM and MgSO₄ (pH 7.4) for 60 min at 37°C. They were fixed in −20°C methanol for 20 min. Fixed samples were then permeabilized overnight with 0.1 M PBS containing 0.1% (v/v) TritonX‐100 detergent. Microtubules were labeled with a mixture of monoclonal antibody against β‐tubulin (clone 2‐28‐33; diluted 1 : 100; Sigma) and acetylated α‐tubulin (clone 6–11‐B1; diluted 1 : 100; Sigma). Primary antibodies were detected by fluorescein‐conjugated goat antimouse immunoglobulin G (IgG; diluted 1 : 40; Zymed). DNA was detected after labeling with 10 mg/mL of Hoechst 33342. Centrosome was labeled with anticentrin‐1 (Sigma) and detected by tetramethyl rhodamine isothiocyanate conjugated antirabbit IgG (Sigma). Coverslips were mounted in antifade medium (Vectashield). Samples were studied by leight microscope (Leica DMRXA/HC). Images were pseudocolored using Adobe Photoshop 4.0 (Adobe Systems).

Transmission electron microscope

Isolated sperm heads were examined by electron microscopy. The fixative for electron microscopy consisted of 2.5% glutaraldehyde, 0.7% paraformaldehyde and 0.5% potassium ferricyanide in 0.075 M phosphate buffer. After osmication in 2% aqueous osmium tetroxide, the specimens were stained in 0.5% aqueous uranyl acetate overnight, dehydrated in an alcohol series with increasing concentrations, and embedded in epoxy resin. Thin sections were studied under the electron microscope (Hitachi H‐7100 FA; HITACHI, Tokyo, Japan).

RESULTS

Microtubule organization and chromatin configuration in rabbit eggs following Piezo‐ICSI with intact sperm or sperm head

MICROTUBULE ORGANIZATION AND chromatin configuration in rabbit eggs after ICSI with an intact sperm or with an isolated sperm head are shown in 1, 2, 3, 4. The unfertilized rabbit egg had an anastral barrel‐shaped meiotic spindle. The only microtubules present within the unfertilized metaphase II‐arrested rabbit egg were those of the metaphase spindle (Fig. 1). At 2 h post‐ICSI with an intact sperm, a radial microtubule array was observed around decondensed sperm nuclei (Fig. 2a). At 3 h post‐ICSI with an intact sperm, microtubules were organized around the male pronuclei and continued to enlarge toward the female pronucleus (Fpn) (Fig. 2b). At 2 h post‐ICSI with an isolated sperm head, a male pronucleus (Mpn) and a Fpn were observed and microtubules were organized between two pronuclei without a distinct nucleation site (Fig. 2d). At 3 h post‐ICSI with an isolated sperm head, microtubules were organized between both pronuclei and enlarged around them (Fig. 2e). Confocal images demonstrate the remarkable difference of microtubule organization (Fig. 4). At 10 h post‐ICSI with an intact sperm or with an isolated sperm head, pronuclear centration was observed, and microtubule arrays without a distinct nucleation site were organized around the Mpn and Fpn (Fig. 2c,f).

Figure 1.

Microtubule (green) and chromatin (blue) configurations in a rabbit unfertilized egg. The only microtubule organization was observed in the anastral second meiotic spindle. Bar = 10 µm.

Figure 2.

Microtubule (green) and chromatin (blue) configurations in rabbit eggs following Piezo‐intracytoplasmic sperm injection (ICSI) with an intact rabbit sperm (a–c), or with an isolated sperm head (d–f). At 2 h post‐ICSI with an intact sperm, astral microtubule organization (sperm aster: arrow) was organized from the base of the sperm head (a). At 3 h post‐ICSI with an intact sperm, the radially extensive microtubules organize around both male pronucleus (Mpn) (arrow: sperm tail) and female pronucleus (b). At 2 h post ICSI with an isolated sperm head, male pronucleus and female pronucleus (Fpn) was observed and microtubles without a distinct nucleation site were organized between both pronuclei (d). Eggs 3 h post‐ICSI with an isolated sperm head, both male and female pronucleus were surrounded by the dense microtubule array without single nucleation site (e). At 10 h post‐ICSI, microtubule organization was the same in the eggs following ICSI with an intact sperm and with an isolated sperm head. (c, f). Bar = 10 µm.

Figure 3.

Microtubule (green) and chromatin (blue) configurations in rabbit eggs 12 h post‐intracytoplasmic sperm injection (ICSI) with an isolated sperm head. First mitotic spindle formation was observed, and the shape of the first mitotic spindle was the same as observed in eggs following ICSI with an intact sperm. (image is not shown) Bar = 10 µm.

Figure 4.

Confocal images of the microtubule (green) in rabbit eggs. (a,b) Rabbit eggs at 2 h and 3 h after intracytoplasmic sperm injection (ICSI) with an intact sperm, respectively. (c,d) Rabbit embryo at 2 h and 3 h after ICSI with an isolated sperm head, respectively. Images show remarkable differences of microtubule organization in fertilization with an intact sperm or with an isolated sperm head. arrow: sperm tail, Bar = 10 µm.

In the mitotic stage at 12 h post‐ICSI with an isolated sperm head, a bipolar mitotic spindle was observed (Fig. 3).

Detachment of centrosome from sperm head

Sperm centrosome isolated by sonication were analyzed by light (Fig. 5) and electron microscopy (Fig. 6) to evaluated the efficacy of the techniques. Detachment of sperm centrosome from sperm midpiece was confirmed from these figures.

Figure 5.

Centrin (red), microtubule (green) and chromatin (blue) configurations in rabbit sperm. (a) Centrin expression is observed in intact sperm midpiece. (b) Centrin expression is absent in isolated sperm head. Bar = 10 µm.

Figure 6.

Electron micrograph in midpiece of intact rabit sperm, (a) and isolated sperm head. (b) Images shows sperm centrosome in sperm midpiece was completely removed from sperm head (arrow).

Pronuclear formation rates and cleavage rates

The pronuclear formation rate and cleavage rate are summarized in Table 1. Pronuclear formation was observed at 4–6 h post‐ICSI, two‐cell cleavage and four‐cell cleavage was observed at 12–16 h and 22–24 h post‐ICSI, respectively. In eggs post‐ICSI with isolated sperm head, pronuculear formation was observed in 140 of 190 eggs (73%), two‐cell cleavage was observed in 29 of 159 eggs (18%) and four‐cell cleavage was observed in eight of 30 eggs (26%). In eggs post‐ICSI with an intact sperm, pronuclear formation was observed in 156 of 177 eggs (88%), two‐cell cleavage was observed in 24 of 147 eggs (16%) and four‐cell cleavage was observed in four of 14 eggs (28%). The pronuclear formation rate was significantly higher in eggs after ICSI with an intact sperm (P < 0.001). The cleavage rates were not significantly different.

Table 1.

Pronucleus formation rate and cleavage rate

	Pronucleus formation	Two‐cell cleavage	Four‐cell cleavage
Sperm head	73% (140/190) a	18% (29/159) b	26% (8/30) c
Intact sperm	88% (156/177)	16% (24/147)	28% (4/14)

Pronucleus formation rate: 4–6 h after intracytoplasmic sperm injection (ICSI). Two‐cell cleavage rate: 12–16 h after ICSI. Four‐cell cleavage rate: 18–20 h after ICSI.

^a P < 0.05, in comparison with intact sperm;

^b,c P > 0.05, in comparison with intact sperm.

Microtubule organization rate

The number of rabbit zygotes examined, and their microtubule organization at 1.5–3 h after ICSI is summarized in Table 2. In eggs post‐ICSI with an isolated sperm head, microtubule organization was observed in one of five eggs (20%) at 1.5 h, 11 of 16 eggs (66%) at 2 h and 16 of 45 eggs (35%) at 3 h. In eggs post‐ICSI with an intact sperm, microtubule organization was observed in two of 41 eggs (4%) at 1.5 h, seven of 43 eggs (16%) at 2 h and 21 of 38 eggs (55%) at 3 h. At 2 h post‐ICSI with an isolated sperm head, the microtubule organization rate was significantly high (P < 0.05).

Table 2.

Microtubule organization rate

Time after ICSI	1.5 h	2 h	3 h
Sperm head	20% (1/5) a	68% (11/16) b	35% (16/45) c
Intact sperm	4% (2/41)	16% (7/43)	55% (21/38)

^b P < 0.05, in comparison with intact sperm;

^a,c P > 0.05, in comparison with intact sperm.

DISCUSSION

IN PIEZO‐ICSI WITH intact sperm, we observed that microtubule organization and chromatin configuration were the same as in in vivo fertilization, as observed previously. ¹⁶ In in vivo rabbit fertilization, microtubules were organized into a radial aster from the sperm head, and cytoplasmic microtubules were organized around the male and female pronuclei in the cytoplasm without a distinct nucleation site. ¹⁶ In mice, the paternal centrosome degenerates during spermiogenesis, ¹² and sperm asters were not observed at the base of the incorporated sperm head. ⁶ In parthenogenetically activated rabbit eggs, de novo formation of centrioles was observed at the stage of morula or early blastocyst, but not in the first cell cycle of fertilization. ²² Based on these facts, MTOC inheritance in rabbit fertilization is regarded as a biparental.

In the present experiment, no aster formation was observed in oocytes following injection with an isolated sperm head, and microtubule organization without a distinct nucleation site between male and female pronuclei was observed. In contrast, sperm aster formation was observed in eggs following Piezo‐ICSI with an intact sperm. After the early pronuclear stage, sperm aster vanished in eggs with an intact sperm, and microtubule organization was the same in embryos with and without centrosome. At 12 h post‐ICSI with isolated sperm head, the first mitotic spindle was organized, as observed in eggs with an intact sperm. These findings indicate that the MTOC existed in oocyte cytoplasm and could complete the microtubules organization that was essential for genomic union and first mitotic spindle formation. There was no difference in cleavage rate. This fact indicated that the function of the first mitotic spindle with and without sperm centrosome was the same. In addition, since microtubule organization without a nucleation site was not observed in oocyte cytoplasm following ICSI with an intact sperm, the maternal centrosome seemed to fulfil the paternal sperm centrosome's function when the sperm centrosome was absent. In rabbit fertilization, the function of the MTOC derived from the paternal centrosome can be replaced by the maternal cytoplasmic centrosome. This fact suggests that normal fertilization in rabbits can progress without a sperm‐derived centrosome.

How is it possible for normal fertilization to occur without a sperm‐derived centrosome in the oocyte? There may be compensating factors in the oocyte, including γ‐tubulin or a self‐organization system of microtubules. γ‐Tubulin is present in the microtubule organization centers that are not associated with a morphological centriole. ²³ During rabbit fertilization, the sperm centriole could attract the oocyte γ‐tubulin. The disappearance of γ‐tubulin in spermiogenesis indicates that sperm aster nucleation is dependent on oocyte γ‐tubulin. ²⁴ Furthermore, in eggs following ICSI without a sperm‐derived centrosome, microtubule organization seems to be promoted by oocyte γ‐tubulin. Experiments have been done to reveal the function of the microtubule. ²⁵ , ²⁶ After a part of the cytoplasm of a fish pigment cell was cut by a needle, a new MTOC without a centriole was observed in the detached cell. ²⁵ Although it was not a gamete cell, the result indicated the possibility of a microtubule self‐organization system as a cytoskeleton. These results support our findings that maternal cytoplasm contributed to the first cell cycle in rabbits.

By observation of a paternal centrosomal dysfunction model, we proved the biparental centrosome participation in rabbit fertilization. In intact sperm injection, the sperm aster vanishes and the MTOC is replaced by oocyte cytoplasm. In isolated sperm head injection, the MTOC is in the oocytoplasm at the beginning of fertilization. Organization of the MTOC in the absence of the sperm aster by the sperm centrosome can produce pronucleus fusion and the first mitotic spindle, which leads to completion of the first cell cycle of fertilization.

We concluded that rabbits have biparental inheritance of centrosomes during the first cell cycle of fertilization, and that maternal cytoplasm could replace the function of sperm‐derived centrosome.