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Reproductive Technology |
Graduate School of Applied Biosciences,2 Hiroshima Prefectural University, Hiroshima, 727-0023 Japan
The Institute for Biogenesis Research,3 University of Hawaii School of Medicine, Honolulu, Hawaii 96822
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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assisted reproductive technology, early development, embryo, fertilization, spermatid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Round spermatids are spermatogenic cells that have just completed meiosis. They have a haploid set of chromosomes. After preliminary experiments [10, 11], Ogura et al. [12] obtained normal live mouse offspring after electrofusion of round spermatids with mature oocytes. Intracytoplasmic injection of round spermatids (ROSI) also produced normal offspring [13]. Today, animal species in which live offspring were obtained by ROSI include mice [12, 13], mastomys [14], rats [15], rabbits [16], monkeys [17], and humans [18]. We report here the birth of hamster offspring following ROSI.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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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.
Collection 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 (brown coats and black eyes), 2–4 mo of age, were induced to superovulate by an i.m. injection of 30 IU eCG (Teikoku-Zoki Pharmaceuticals, Tokyo, Japan) on the morning of the day of postestorous discharge [19], followed by an i.m. injection of 30 IU hCG (Sankyo Co., Tokyo, Japan) 56 h later [20]. Mature unfertilized oocytes were collected from oviducts approximately 15 h after hCG injection [20]. They were freed from cumulus cells by a 1-min treatment with 0.1% bovine testicular hyaluronidase in M2 medium [21]. The oocytes were rinsed and kept in TCM199 (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 Biomedical Inc., Aurora, OH), 5 mM taurine, and 25 µM EDTA. This medium, called M199TE, was maintained at 37.5°C under 5% CO2, 10% O2, and 85% N2 for up to 60 min before use for microinjection. All experiments were performed in a dark room with a small incandescent lamp, and red filters were used on the microscope light source, as described previously [9].
Collection of Male Germ Cells
Spermatogenic cell suspensions were prepared according to the mechanical method described for the hamster [10]. In brief, testes were isolated from mature wild-type males (brown coat and black eyes; 2–3 mo old) and placed in the erythrocyte-lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 2 mM EDTA, pH 7.2). The tunica albuginea was removed and dissected seminiferous tubules were transferred to a cold (4°C) Dulbecco phosphate-buffered saline (PBS) supplemented with 5.6 mM glucose and 5.4 mM sodium lactate (GL-PBS). Seminiferous tubules were further cut into small pieces with fine scissors and gently pipetted to release spermatogenic cells into GL-PBS. The cell suspension was filtered through a 40-µm nylon mesh and washed three times in GL-PBS by centrifugation (200 x g for 5 min each).
Mature spermatozoa were prepared as previously described [9]. A dense sperm mass was collected from the cauda epididymis of mature male hamsters (4–5 mo of age). A small drop of sperm mass was placed at the bottom of a 1.5-ml centrifuge tube containing 300 µl of M2 medium. 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. This 100 µl of medium, containing actively motile spermatozoa, was placed in a polypropylene microcentrifuge tube, which was then plunged into liquid nitrogen for 1 min. The tube was then thawed in a water bath (37°C). By this freeze-thawing, almost all spermatozoa were immobilized and lost their acrosomes.
Microinjection of Round Spermatids (ROSI) or Sperm Heads (ICSI) into Hamster Oocytes
Microinjection was carried out, with some modifications, according to the methods for mouse ROSI [13] and hamster ICSI [9]. Spermatids and spermatozoa were from wild-type hamster males.
Round spermatids were easily recognized by their small size and a centrally located chromatin mass within the nucleus [10] (Fig. 1A). In a series of experiments, a single spermatid (about 10 µm in diameter) was sucked into an injection pipette (7 µm i.d.) and injected into an oocyte (intact round spermatid injection: Fig. 1B). In another series of experiments, a single spermatid was drawn in and out of an injection pipette repeatedly until its plasma membrane was completely broken and the nucleus became almost completely separated from the cytoplasm [13]. The nucleus and the bulk of the cytoplasm were then injected together into an oocyte (membrane-broken round spermatid injection; Fig. 1C).
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For sperm-head injection, a single 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. After discarding the tail, the head was redrawn, tip of the head first, into the pipette and injected into an oocyte.
Oocytes micromanipulated in the same way but without injection of spermatids and sperm heads (sham operation) served as a control to assess the effects of injection procedure on oocyte activation and early parthenogenetic cleavage (control I). The volume of medium injected into each oocyte was about 1 pl, which was roughly equivalent to the volume of medium injected with a round spermatid or sperm head. In addition, some unfertilized oocytes were cultured under the same culture conditions but without any operation (culture only). This was to see whether oocytes activated spontaneously in the culture medium (control II).
Embryo Culture
Sperm-injected and control 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 a clearly visible second polar body was considered normally fertilized. Some ROSI and ICSI oocytes were fixed and stained for observation of cytological details of fertilization status [3]. Fertilized eggs, 10–15 in number, were selected and cultured further in a 35-µl droplet of M199TE. The eggs that reached the two-cell stage by 24 h after ROSI or ICSI were transferred into 35-µl droplets of Hamster embryo culture medium-9 (HECM-9) [8] supplemented with 0.5 mg/ml human serum albumin (HSA, Cohn Fraction V, A-1653), which had been previously placed under mineral oil. They were cultured for 52 h at 37.5°C under 5% CO2, 10% O2, and 85% N2. The pH value of the medium was approximately 7.3.
Embryo Transfer
About 76 h after microinjection, 4–7 morulae and blastocysts were selected randomly and transferred into each uterus of recipient albino females, which were naturally mated with albino males 3 days previously. The embryos of these females were at the eight-cell stage. Mothers were allowed to deliver and raise their own pups as well as foster pups (brown coat and black eyes). The reason why we used naturally mated surrogate mothers was based on reports that in vitro-fertilized and developed hamster preimplantation embryos failed to develop to term when transferred to pseudopregnant surrogate females [22].
Statistical Analysis
Comparison of fertilization and embryo development rates following round spermatid and sperm-head injections or sham operation was made using the chi-square test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Table 1 summarizes the status of fertilization by ROSI and ICSI. Over 90% of ROSI and ICSI oocytes were activated. Examination of unactivated ROSI oocytes after fixation and staining revealed that they were arrested at metaphase II and some contained spermatid nuclei with premature chromosome condensation (Fig. 2A). Some had one large female pronucleus plus one decondensing sperm head (Fig. 2B) or one small male pronucleus (Fig. 2C). The incidence of normal fertilization (2Pb+2PN; Fig. 2D) was the highest after ICSI with isolated sperm head. It is important to note that round spermatids with a broken plasma membrane fertilized normally (2Pb+2PN; Fig. 2D) and far better than those with an intact plasma membrane. Most oocytes injected with medium only (Sham operation: control I) were activated. Only 20% of oocytes were activated when they were left in the medium without any operation (control II).
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In Vitro Development of ROSI or ICSI Oocytes
Table 2 summarizes development of ROSI and ICSI oocytes. The proportion of normally fertilized oocytes that developed to morulae and blastocysts was significantly lower after ROSI than after ICSI. None of control oocytes developed beyond the eight-cell stage.
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Full-Term Development of ROSI or ICSI Embryos
Of a total of 57 ROSI-derived embryos (31 morulae and 26 blastocysts) transferred to recipients (9–13 embryos per recipient), three (5%) developed into live young (Fig. 3). Of 65 ICSI-derived embryos (37 morulae and 28 blastocysts) transferred to recipients (12–14 embryos per recipient), four (6%) developed into live young (Table 3). All three young (one male and two females) born after ROSI and four young (three males and one female) born after ICSI grew into healthy, fertile adults.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Hamster round spermatids, unlike mouse spermatids, are able to activate oocytes. According to Yazawa et al. [24], 70% of mouse oocytes injected with hamster round spermatids were activated. It seems to be the cytoplasm of the hamster spermatid, not the nucleus, that is largely responsible for oocyte activation by ROSI. In the present study, we injected both the cytoplasm and nucleus of hamster spermatids after disruption of their plasma membranes. One should be aware that hamster oocytes are easily activated by pricking with a glass needle [25]. Injecting a bolus of medium could have activated the majority of hamster oocytes (see Table 1). Although a temporal influx of extracellular Ca2+ and Na+ through the punctured point of the plasma membrane may trigger oocyte activation, such activation could be incomplete. In fact, Yazawa et al. [24] noted that the vast majority (91%) of mouse oocytes injected with hamster round spermatids display abnormal or only temporal intracellular Ca2+ oscillation even though 70% of the oocytes are activated. Elongated spermatids or spermatozoa (of the hamster), on the other hand, induce typical Ca2+ oscillations, resulting in activation of 74–93% of mouse oocytes. In the future, we should be able to increase the proportion of normally fertilized oocytes (with well-developed male and female pronuclei) and improve pre- and postimplantation development of ROSI embryos by stimulating ROSI oocytes with proper oocyte-activating reagents.
The proportion of transferred hamster ROSI-derived morulae/blastocysts that developed into live offspring was 5% in this study. This rate was similar to that for ICSI-derived morulae/blastocysts in this study (6%) but somewhat lower than our previous ICSI results of 19% [19] or for in vitro fertilization-derived embryos (17%) [22] and in vivo-fertilized embryos (48%) [26]. Again, appropriate stimulation to ROSI oocytes may largely increase proportions of ROSI oocytes capable of developing into normal offspring. This must be the subject of our next study.
The hamster has been an excellent model animal for studies of gametogenesis, fertilization, reproductive, endocrinological, and behavioral studies. Assisted fertilization like in vitro fertilization, ICSI, and ROSI will make this animal more valuable for reproduction studies.
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FOOTNOTES |
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Received: 21 January 2004.
First decision: 11 February 2004.
Accepted: 2 March 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  M. Kanatsu-Shinohara, T. Muneto, J. Lee, M. Takenaka, S. Chuma, N. Nakatsuji, T. Horiuchi, and T. Shinohara Long-Term Culture of Male Germline Stem Cells From Hamster Testes Biol Reprod, April 1, 2008; 78(4): 611 - 617. [Abstract] [Full Text] [PDF]
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