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Full-Term Development of Golden Hamster Oocytes Following Intracytoplasmic Sperm Head Injection

^a Graduate School of Applied Biosciences ^b Department of Bioresources, Hiroshima Prefectural University, Hiroshima 727-0023, Japan ^c Institute for Biogenesis Research, University of Hawaii School of Medicine, Honolulu, Hawaii 96822

	ABSTRACT

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The golden hamster is the mammalian species in which intracytoplasmic sperm injection (ICSI) was first tried to produce fertilized oocytes. Thus far, however, there are no reports of full-term development of hamster oocytes fertilized by ICSI. Here we report the birth of hamster offspring following ICSI. Keys to success were 1) performing ICSI in a dark room with a small incandescent lamp and manipulating both oocytes and fertilized eggs under a microscope with a red light source and 2) injecting sperm heads without acrosomes. All oocytes injected with acrosome-intact sperm heads died within 3 h after injection, while those oocytes injected with acrosomeless sperm heads survived injection. Under illumination with red light in a dark room, the majority of the oocytes injected with acrosomeless sperm heads were fertilized normally (77%), cleaved (91%), and developed into morulae (49%). Of the 47 morulae transferred to five recipient females, nine (19%) developed to live offspring.

assisted reproductive technology, embryo, fertilization, sperm

	INTRODUCTION

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Intracytoplasmic sperm injection (ICSI) has been widely used in human infertility clinics as the means for overcoming male infertility. ICSI is a powerful tool to overcome certain types of male infertility [1], but it can be valuable in determining what is essential and what is not essential for fertilization [2]. The species in which ICSI yielded live offspring include rabbits [3, 4], cattle [5], humans [1], mice [6], sheep [7], horses [8], cats [9], monkeys [10], and pigs [11]. The golden hamster was the first mammal in which ICSI was performed [12]. Injection of intact hamster spermatozoa into eggs is technically difficult due to large acrosomes and long tails of the spermatozoa. Therefore, Uehara and Yanagimachi [12] injected isolated sperm heads into oocytes to obtain fertilized eggs. Although many ICSI experiments were carried out in the hamster to analyze the processes and mechanisms of sperm head transformation into pronuclei [12–14], all the previous attempts to produce live hamster offspring by ICSI were not successful, mainly due to the difficulty in culturing ICSI-fertilized eggs to the embryonic stage suitable for transfer to surrogate mothers. The problem of culturing hamster embryos in vitro has been overcome largely by endeavors by Bavister and his associates [15–19]. We now report the birth of live offspring following intracytoplasmic sperm head injection.

	MATERIALS AND METHODS

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Reagents

Inorganic salts were purchased from either Sigma Chemical Co. (St. Louis, MO) or Nacalai Tesque Inc. (Kyoto, Japan). All organic reagents were purchased from Sigma unless otherwise stated.

Culture Media

The medium used for collection, micromanipulation of hamster oocytes, and culturing embryos from one-cell to two-cell stages was TCM 199 (with Earle salt, 26 mM sodium bicarbonate, and 25 mM Hepes; Gibco BRL, Grand Island, NY) supplemented with 5% heat-inactivated fetal bovine serum (ICN Biomedicals Inc., Aurora, OH), 5 mM taurine, and 25 µM EDTA (ethylenediaminetetraacetic acid). This medium was called M199TE. Sperm-injected oocytes were cultured for 24 h in the above medium under the gas phase of 5% CO₂, 10% O₂, and 85% N₂. During the next 2 days, they were cultured in Hamster Embryo Culture Medium-9 (HECM-9) [18] under the same gas phase.

Preparation of Oocytes

Golden hamsters (Mesocricetus auratus) were raised and maintained in an air-conditioned room with a 14L:10D light cycle (light from 0600 h). Mature females, 2–3 mo after birth, were induced to superovulate by i.m. injection of 30 IU eCG on the day of the postestrus discharge [20] followed by an i.m. injection of 30 IU hCG 56 h later. Mature unfertilized oocytes were collected from oviducts approximately 15 h after hCG injection [21]. They were freed from the cumulus cells by a 1-min treatment with 0.1% bovine testicular hyaluronidase in M2 medium [22]. The oocytes were rinsed and kept in M199TE at 37.5°C under 5% CO₂, 10% O₂, and 85% N₂ for up to 60 min before sperm head injection. Initially, manipulation of oocytes and embryos was performed in a windowless room with ordinary, cool-white fluorescent ceiling lamps (100 V, 40 watts). Light intensity at desk height, measured with an illuminometer, was 200 lux. A dissecting microscope had a regular incandescent lamp (6 V, 15 watts). Light intensity at the specimen stage was 1300 lux. Subsequently, the same room was illuminated with a single small incandescent lamp (100 V, 20 watts). Light intensity at desk height was 8 lux. A red cellophane filter was placed in front of the microscope lamp. Light intensity at the specimen was 250 lux. These measures were taken to avoid short-wave visible light harmful to hamster oocytes and embryos [23, 24].

Microinjection of Sperm Heads into Oocytes (ICSI)

A dense sperm mass was collected from the cauda epididymis and a small drop (about 5 µl) was placed at the bottom of a 1.5-ml centrifuge tube containing 300 µl of M2 medium (37°C) [25]. Spermatozoa were allowed to swim up into this medium for 5–10 min at 37°C before collection of the upper 100 µl of the medium. Virtually all spermatozoa in the medium were motile. Intracytoplasmic injection with sperm heads was carried out, with some modifications, according to the method of Kimura and Yanagimachi [6, 26, 27]. In a series of experiments, one drop (6 µl) of sperm suspension was mixed thoroughly with twice the volume of M2 medium containing 12% (w/v) polyvinylpyrrolidone (molecular weight 360 000). This mixture was transferred to the micromanipulation chamber on the microscope stage. A single live spermatozoon was drawn tail first into the injection pipette and moved back and forth until the sperm-tail junction was at the opening of the injection pipette. The head was separated from the tail by applying one or more piezo pulses [26]. After discarding the tail, the head with acrosome was redrawn into the pipette and injected into an oocyte (Fig. 1, A and B). Other series of experiments were performed in the same way except that spermatozoa were killed as follows prior to injection. A polypropylen microcentrifuge tube containing 100 µl of fresh sperm suspension was plunged into liquid nitrogen for 1 min. It was then thawed in a water bath (37°C). By this freeze-thawing procedure, all spermatozoa were immobilized (dead) and lost acrosomes prior to injection. Again, only sperm heads were injected into oocytes.

View larger version (130K): [in this window] [in a new window]   FIG. 1. Hamster ICSI and in vitro embryo development. A) A single sperm head (arrow) inside of injection pipette. Injection pipette had already penetrated the zona pellucida by a few piezo pulses. B) Sperm head was introduced into the ooplasm after the injection pipette penetrated deep into the vitellus. C) A pronuclear egg with two pronuclei and two polar bodies (Pb 1, Pb 2) 5 h after ICSI. D) ICSI embryos at the two-cell stage. E) ICSI embryos at the morula stage. F) Hoechst-stained nuclei of morula

Oocytes that were micromanipulated in the same way but without injection of sperm heads (sham injection) were manipulated to assess the effect of the injection procedure on development.

Embryo Culture

The ICSI oocytes were incubated in 35-µl droplets of M199TE under mineral oil at 37.5°C under 5% CO₂, 10% O₂, and 85% N₂ for about 5 h, then examined using an inverted microscope (Diaphot TMD, Diaphot 300; Nikon, Tokyo, Japan) equipped with Hoffman modulation contrast optics. An oocyte with two distinct pronuclei and the second polar body (2PN + 2Pb, Fig. 1C) was considered normally fertilized. Fertilized eggs were cultured in 35-µl droplets of M199TE. The eggs that reached the two-cell stage (Fig. 1D) by 24 h after ICSI were selected and transferred into 35-µl droplets of HECM-9 [18] under mineral oil. They were cultured for 48 h at 37.5°C under 5% CO₂, 10% O₂, and 85% N₂. Some morulae (Fig. 1E) were fixed and stained with Hoechst 33342 to determine cell numbers. Others were transferred to pseudopregnant surrogate females to see if they were capable of developing into live offspring.

Embryo Transfer to Foster Mothers

About 72 h after sperm head injection, morulae were selected randomly and transferred to the uteri of surrogate mothers on Day 2 of pseudopregnancy, which was induced by mating an estrus female with a vasectomized male of proven infertility. Oocytes and spermatozoa were obtained from wild-type (golden) hamsters with brown coats and black eyes. Surrogate mothers were albino (white coat and red eyes). The albino colony had not produced anything but albino offspring during the preceding 4 years. Transfer of wild-type embryos to albino surrogate mothers would result in the delivery of wild-type offspring only, but we cotransferred albino embryos cultured from the two-cell stage to improve pregnancy maintenance [28]. We transferred 3–5 wild-type ICSI-derived morulae into each uterus of surrogate mothers on Day 2 of pseudopregnancy, together with 5–6 morulae/blastocysts developed from normally (in vivo) fertilized albino eggs.

Statistical Analysis

Comparison of fertilization and embryonic development rates following sperm head injection and sham operation was made using the chi-square test.

	RESULTS

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Effect of Light on Fertilization and Cleavage Following Sperm Head Injection

The effect of light on fertilization and cleavage is shown in Figure 2. Although ordinary light illumination did not affect fertilization rate (84% vs. 84%), it lowered cleavage rate considerably (16% vs. 92%). None of cleaved embryos manipulated under regular lighting conditions developed beyond the two-cell stage. Apparently the use of a dark room and red filters for illumination during and after sperm head injection protected oocytes and zygotes from detrimental effects of white or short wavelength light on subsequent embryo development.

View larger version (23K): [in this window] [in a new window]   FIG. 2. Effect of visible light on ICSI fertilization and cleavage. Fertilized eggs examined 5 h after ICSI; cleaved eggs were examined 24 h and 48 h after ICSI. Forty-five oocytes/embryos in three experiments in each group. a, b) P < 0.05 (chi-square test).

Detrimental Effect of Acrosome-Intact Sperm Head Injection on the Oocytes

The effect of the presence or absence of acrosomes during sperm head injection on the survival of oocytes is shown in Table 1. All oocytes (n = 33) injected with acrosome-intact sperm heads shrunk within 3 h after injection (Fig. 3A), and none developed pronuclei within the ooplasm. On the other hand, all oocytes (n = 34) injected with acrosomeless sperm heads survived injection (Fig. 3B), and 79% reached the pronuclear stage by 5 h after injection.

View larger version (100K): [in this window] [in a new window]   FIG. 3. Hamster eggs 3 h after injection of acrosome-intact or acrosomeless sperm heads. A) Deformed (wrinkled and shrunken) eggs after injection of acrosome-intact sperm heads. B) Normally fertilized eggs after injection of acrosomeless sperm heads

Pronuclear Formation and In Vitro Development of Eggs after Injection of Acrosomeless Sperm Heads or Sham Operation under Red Light

Tables 2 and 3 summarize the rates of fertilization and development of eggs after sperm head injection. A total of 325 oocytes were injected with acrosomeless sperm heads and 150 oocytes were sham operated. About 77% of the oocytes were fertilized normally by sperm head injection. Of those, 49% developed into morulae by 72 h after injection. The average cell number (±SEM) in each morula was 15.8 ± 0.3 (Table 3 and Fig. 1F). Sham-operated oocytes cleaved at a high rate (88%), but the vast majority of them failed to develop into morulae.

Full-Term Development of Embryos Fertilized by Sperm Head Injection

Of a total of 47 embryos transferred to surrogate mothers, 9 (19%) developed into live young (Table 4). Figure 4 shows surrogate mothers that gave birth to three pups (black fur) developed from ICSI-fertilized oocytes.

View larger version (183K): [in this window] [in a new window]   FIG. 4. Three pups (colored coat, arrow) developed from ICSI-fertilized eggs. Albino pups were derived from normally fertilized embryos cotransferred with ICSI-derived embryos to a surrogate mother

	DISCUSSION

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This study showed that hamster oocytes fertilized by sperm head injection could develop into live offspring. At present, ICSI embryos are far less development competent than in vivo-fertilized embryos (Table 4). Obviously, there is room for technical improvements. The golden hamster is an excellent animal model for the study of fertilization and postfertilization events [2, 29], but the difficulty of culturing fertilized eggs to a transferable embryonic stage has hampered embryologic studies using this species.

Thanks to the endeavors by Bavister and his associates [15–19], the culture of in vitro-fertilized hamster eggs to blastocysts is now possible. In the present study, we cultured ICSI-fertilized eggs in TCM 199 with 5% serum, 5 mM taurine, and 25 µM EDTA. TCM 199 supplemented with 5% serum has been used successfully to culture zona-free hamster eggs for the analysis of human sperm chromosomes [30]. We used this medium for ICSI as well as a culture of ICSI-fertilized eggs up to the two-cell stage. Because two-cell hamster embryos could not develop further in this medium, we transferred them to HECM-9 for further development. For unknown reasons, all embryos failed to reach the blastocyst stage. Therefore, we transferred morulae to surrogate mothers. Even though we could obtain live offspring this way, the method of culturing ICSI embryos must be improved through further study. We reported here that lighting conditions of the room and microscopes greatly affect in vitro development of hamster embryos. Hirao and Yanagimachi previously reported that short wavelength visible light (<470–480 nm) emitted from ordinary fluorescent light sources was detrimental to the meiosis of hamster oocytes during activation [23]. The reason for harmful effects of light on oocytes and embryos is not clear, but generation of radical oxygen species may damage genomic and mitochondrial DNA in oocytes and embryos. Although golden hamster oocytes/embryos may be exceptionally sensitive to light [23, 24], possible detrimental effects of light on mammal oocytes and embryos [31–33] should be taken into consideration during their manipulation.

Spermatozoa of most laboratory rodents have very long tails, which makes the injection of a whole spermatozoon difficult, if not impossible. Although live mouse offspring can be obtained by injecting whole spermatozoa into oocytes [6], injection of isolated heads is technically much easier [34]. Because the sperm-borne factor that activates the oocyte is within the head, not in the tail [34, 35], injection of sperm tails is not necessary when oocyte activation is the concern.

In most mammals, the centriole within the sperm tail connecting piece serves as a microtuble-organizing center during fertilization [36], and therefore the injection of a whole spermatozoon is essential for successful ICSI. Laboratory rodents are exceptional in this regard. It is not the sperm centrosome but the centrosome of maternal origin that serves as the microtuble-organizing center during fertilization [36]. This is the reason why we are able to obtain normal mouse and hamster offspring by injecting tailless sperm heads into oocytes ([34], this study).

Kimura et al. [35] previously reported that mouse oocytes injected with acrosome-intact rabbit and hamster spermatozoa deformed and did not develop into two-cell embryos. When the same sperm heads were freed from acrosomes prior to injection, oocytes did not deform. Introduction of the contents of large acrosomes of hamster and rabbit spermatozoa appeared to be detrimental to the cytoplasm of mouse oocytes. In the present study, we found that hamster oocytes injected with acrosome-intact sperm heads died, while those injected with acrosomeless heads not only survived injection but developed into live offspring. The cytoplasm of hamster oocytes seems to be very sensitive to acrosomal content (including hydrolyzing enzymes) that normally does not enter the ooplasm. Unlike hamster oocytes, human and mouse oocytes can survive injection of acrosome-intact spermatozoa [1, 6]. Human sperm acrosome is very small; mouse sperm acrosome is by no means small, but its principal segment with hydrolyzing enzymes is rather small [2]. Perhaps this is one of the reasons why mouse and human oocytes can survive the injection of acrosome-intact spermatozoa.

Producing living offspring by ICSI is now possible in the golden hamster. With further improvement of embryo culture and ICSI technique, the hamster may become another animal model for embryological and biotechnological research.

	FOOTNOTES

 
First decision: 6 November 2001.

¹ Correspondence: Toshitaka Horiuchi, Department of Bioresources, Hiroshima Prefectural University, Shoubara, Hiroshima 727-0023, Japan. FAX: 81 8247 4 1750; toshi{at}bio.hiroshima-pu.ac.jp

Accepted: March 4, 2002.

Received: October 4, 2001.

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This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal My Folders Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Yamauchi, Y. Articles by Horiuchi, T. Search for Related Content PubMed PubMed Citation Articles by Yamauchi, Y. Articles by Horiuchi, T. Agricola Articles by Yamauchi, Y. Articles by Horiuchi, T.