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BOR - Papers in Press, published online ahead of print December 11, 2002.
Biol Reprod 2002, 10.1095/biolreprod.102.011726
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BIOLOGY OF REPRODUCTION 68, 1821–1827 (2003)
DOI: 10.1095/biolreprod.102.011726
© 2003 by the Society for the Study of Reproduction, Inc.

Reproductive Technology

Fertilization of Oocytes and Birth of Normal Pups Following Intracytoplasmic Injection with Spermatids in Mastomys (Praomys coucha)1

Narumi Ogonuki3, Keiji Mochida3, Kimiko Inoue3, Junichiro Matsuda4, Yoshie Yamamoto4, Kaoru Takano4, and Atsuo Ogura2,3

Bioresource Center,3 RIKEN, Tsukuba, Ibaraki 305-0074, Japan Department of Veterinary Science,4 National Institute of Infectious Diseases, Shinjuku, Tokyo 162-8640, Japan


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The mastomys is a small laboratory rodent that is native to Africa. Although it has been used for research concerning reproductive biology, in vitro fertilization (IVF) and intracytoplasmic sperm injection are very difficult in mastomys because of technical problems, such as inadequate sperm capacitation and large sperm heads. The present study was undertaken to examine whether mastomys spermatids could be used to fertilize oocytes in vitro using a microinsemination technique, because spermatids are more easily injected than mature spermatozoa into oocytes. Most mastomys oocytes (80%–90%) survived intracytoplasmic injection with either round or elongated spermatids. Round spermatids had little oocyte-activating capacity, similar to those of mice and rats, and exogenous stimuli were needed for normal fertilization. Treatment with an electric pulse in the presence of 50 µM Ca2+ followed by culture in 10 mM SrCl2 led to successful oocyte activation. After injection of round spermatids into preactivated oocytes, 93% of oocytes were normally fertilized (male and female pronuclei formed), and 100% of cultured oocytes developed to the 2-cell stage. However, none reached term after transfer into recipient females. Elongated spermatids, which correspond to steps 9–11 in rats, activated oocytes on injection without additional activation treatment. After embryo transfer, five offspring (6% per transfer) developed to term. These results indicate that microinsemination with spermatids is a feasible alternative in animal species that are refractory to IVF and sperm injection and that using later-stage spermatids may lead to increased production of viable embryos that can develop into normal offspring.

early development, embryo, gamete biology, in vitro fertilization, spermatid


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Intracytoplasmic sperm injection (ICSI) is a technique used to fertilize oocytes by delivering spermatozoa directly into the ooplasm using micromanipulating devices. Mammalian ICSI was initially designed to examine the fertilization steps in the ooplasm after delivering sperm heads from epididymal or testicular spermatozoa [1, 2]. Early ICSI experiments were conducted with golden hamsters, because these animals provided the best model for studying mammalian fertilization [3–5] and because the injection procedure was well tolerated by hamster oocytes [1]. However, it was very difficult to evaluate the ability of fertilized oocytes to develop into fetuses or offspring in the hamster, because hamster embryo development in vitro is arrested at the 2-, 4-, and 8-cell stages. In 1995, Kimura and Yanagimachi [6] established highly reproducible ICSI in mice and confirmed that, in laboratory species, at least some oocytes fertilized by direct injection with spermatozoa could develop to term. Since then, immature sperm cells (spermatids and spermatocytes) [7–10], sperm with motility defects [11], misshapen sperm [12], and freeze-dried spermatozoa [13] have been used to produce healthy offspring in mice, and the range of ICSI applications has been significantly expanded (for review, see [14]). In general, however, a high degree of skill is needed for successful ICSI in rodents because of technical or biological problems, such as fragile oocytes, large sperm heads (e.g., rats [15]), and arrest of embryo development in vitro (e.g., golden hamsters [16]).

The mastomys (Praomys coucha) is a small rodent that is native to Africa. It has good reproductive performance under conventional breeding conditions and has been used for biomedical research since its introduction to the laboratory in the 1900s [17]. Although the mastomys provides a good experimental model for studying oncology, parasitology, virology, and endocrinology [17], its use for reproductive biology, especially embryology, is limited because of the poor availability of fertilized oocytes for experimentation. Female mastomys respond well to the conventional superovulation regimen for laboratory mice and rats, and 10–30 oocytes per female are usually obtained (unpublished results). However, hormonal treatment of females fails to induce normal estrous behavior for unknown reasons, and fertile mating rarely occurs [18]. In vitro fertilization (IVF) of superovulated oocytes with epididymal spermatozoa is possible, but fertilization efficiency and fertilized oocyte developmental ability are too poor for IVF to be practical [19]. This results, at least in part, from difficulty in capacitating mastomys spermatozoa and in maintaining their motility [19]. For the same reason, artificial insemination in this species is usually unsuccessful (unpublished results). Additionally, ICSI is very difficult in mastomys, because the oocytes are fragile and the sperm have large heads.

An alternative method of in vitro oocyte fertilization is the use of immature sperm cells (spermatogenic cells), such as spermatids. As in most mammalian species, the mastomys spermatid nucleus is smaller and softer than the sperm nucleus and, therefore, should be more safely injected into oocytes using smaller injection pipettes. In some species, including mice [20], rats [21], rabbits [22], and humans [23], it has been demonstrated that the spermatid genome can support full-term development after incorporation into the ooplasm. The present study was undertaken to examine whether microinsemination with spermatids is an efficient strategy for obtaining fertilized mastomys oocytes in vitro. If the technique is practical, it may also be applied to other species in which IVF and ICSI are not feasible because of technical difficulties.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animals

Three inbred strains of mastomys (MCC, RI-7, and RI-M) [18, 24] and hybrid F1 mastomys (RI7 x RI-M) were used in the present study. No strain-related differences were observed in the ability of oocytes and male germ cells to support subsequent embryo development when they were adequately handled [18]. The mastomys were raised under specific-pathogen-free conditions at the National Institute of Infectious Diseases and were provided with water and commercial laboratory mouse chow ad libitum. They were kept under controlled lighting conditions (lights-on 0700–2100 h). All animals were maintained in accordance with the guidelines at the National Institute of Infectious Diseases.

Oocyte Collection

Females were induced to superovulate at 2–4 mo of age by sequential injections of 10 IU of eCG (Sankyo Co., Tokyo, Japan) and 10 IU of hCG (Sankyo) given 48 h apart. Mature oocytes were collected from the oviducts 15 h after hCG injection. The number (mean ± SEM) of oocytes ovulated using this superovulation regime is 25.0 ± 5.4 (n = 40) in our laboratory (unpublished results). They were freed from cumulus cells by treatment with 0.1% bovine testicular hyaluronidase (Sigma Chemical Co., St. Louis, MO) in CZB medium supplemented with 5.6 mM glucose (modified CZB) [25] and incubated in fresh medium at 37°C under 5% CO2 in air.

Collection of Male Germ Cells

Spermatogenic cell suspensions were prepared according to a mechanical method previously described for hamsters [26]. Briefly, testes were removed from 2- to 12-mo-old males and placed in erythrocyte-lysing buffer (155 mM NH4Cl, 10 mM KHCO3, 2 mM EDTA, pH 7.2). The tunica albuginea was removed, and the seminiferous tubule masses were transferred into cold (4°C) Dulbecco PBS supplemented with 5.6 mM glucose, 5.4 mM sodium lactate, and 0.1 mg/ml of polyvinyl alcohol (originally, polyvinylpyrrolidone) (GL-PBS) [26]. The seminiferous tubules were cut into small pieces and gently pipetted to allow spermatogenic cells to be released into the GL-PBS. Then, the cell suspension was filtered through a 50-µm nylon mesh and washed three times by centrifugation (200 x g for 5 min).

Mature spermatozoa were collected from the epididymides of 4- to 12-mo-old males by puncturing the caudal part in silicon oil [19]. The sperm mass was placed on the bottom of a plastic centrifuge tube and overlaid with CZB medium. The spermatozoa that swam to the surface were used for microinsemination.

Microinsemination

Intracytoplasmic injection with spermatozoa and spermatids was performed using a Piezo-driven micromanipulator (Prime Tech Ltd., Ibaraki, Japan) according to a method developed for mice [6]. Unlike the original method, the injection was performed at room temperature without cooling the manipulation stage. Because the mastomys spermatozoon tail is too long to inject along with the head, we isolated the head by homogenizing the sperm suspension in nucleus-isolation medium [27] using a Teflon homogenizer (Wheaton Science Products, Millville, NJ). Unlike mouse spermatozoa, the head and tail of mastomys spermatozoa are very tightly connected and cannot be separated by Piezo-impact.

When round spermatids were injected, mastomys oocytes were artificially activated, because they had no oocyte-activating capacity (see Results). The efficiencies of two activation methods that are commonly used for mouse oocytes, electric-pulse stimulation and strontium treatment, were evaluated for mastomys oocytes. A group of mastomys oocytes was subjected to a single or double activation pulse (3500 V/cm, 10 µsec) in 300 mM mannitol solution containing 50 µM CaCl2 and 100 µM MgSO4 [28]. The second group of oocytes was treated with 10 mM SrCl2 in Ca2+-free CZB medium for 30 min [29]. The third group of oocytes was activated with a combination of the two (a single electric pulse followed by SrCl2 treatment).

Culture and Embryo Transfer

Oocytes fertilized with spermatids were cultured in modified CZB medium at 37°C under 5% CO2 in air. Irrespective of the genetic background of the embryos, CZB medium supports a high rate of 1-cell mastomys embryo development to the blastocyst stage [18]. The embryos were cultured for 24 h, and 2-cell embryos were transferred into pregnant females on the day following mating (Day 1). These females were expected to deliver pups with different coat colors from those of the transferred embryos. Embryos were also transferred into pseudopregnant females mated with vasectomized males.

Statistical Analysis

Data were analyzed using the Fisher exact probability test when appropriate.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Oocyte-Activating Capacity of Mastomys Male Germ Cells

In the first series of experiments, we injected round and elongated spermatids of mastomys into mouse oocytes to examine their oocyte-activating capacity. Heterologous microinsemination of mouse oocytes is a very reliable method for assessing oocyte-activating capacity that has been demonstrated for various species, including humans [30–32]. At 2.0–2.5 h after injection, the oocytes were fixed with 2.5% glutaraldehyde and processed for whole-mount preparation [33], and their stage was then assessed under a phase-contrast microscope. Elongated and round spermatids from mature male mastomys were injected into mouse oocytes to evaluate the stage at which oocyte-activating capacity became active in the male germ cells.

The round spermatids, observed with Nomarski optics, were similar in appearance to those of hamsters and mice. They were 12–14 µm in diameter and were easily distinguished from other small round cells (leukocytes or spermatogonia) by their low nucleus:cytoplasm ratio (Fig. 1). The nucleolar structure in the center of the nucleus was not distinct compared with the mouse nucleolus. Although relatively few elongated spermatids were observed in the testicular cell population, they were more easily identified than round spermatids. As shown in Table 1, mastomys round spermatids failed to activate oocytes and underwent premature chromosome condensation in mouse metaphase II (MII) oocytes (Fig. 2A). Therefore, for further microinsemination experiments with round spermatids, mastomys oocytes were artificially activated before injection. Approximately half the oocytes were activated by elongated spermatid injection. Whole-mount preparations revealed that oocyte and elongated spermatid chromosomes were successfully synchronized by telophase II (Fig. 2B).



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  		FIG. 1. Mastomys male germ cells under a Nomarski microscope. Mastomys spermatozoa (a) have a large, hooked head. Elongated spermatids (b) used for microinsemination correspond to those at steps 9–11 in rats. Round spermatids (c) can be distinguished from other small round cells, such as spermatogonia (inset), by their low nucleus:cytoplasm ratio and distinct nuclear membrane. Because spermatogonia are basally located in the seminiferous epithelium, very few are found among mechanically isolated spermatogenic cells. x900


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  		TABLE 1. Oocyte-activating capacity of mastomys male germ cells.a



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  		FIG. 2. Oocytes injected with mastomys male germ cells. A) A mouse oocyte 2.5 h after injection with a mastomys round spermatid. The oocyte stays at metaphase II (chromosome: F) and the spermatid chromosomes (M) are prematurely condensed. (x1000) B) A mouse oocyte 2.5 h after injection with a mastomys elongated spermatid. The oocyte has proceeded to telophase II and the female (F) and male (M) chromosomes are synchronized. (x1000) C) A mastomys oocyte fertilized with a round spermatid 3 h after activation. The male (M) and female (F) pronuclei are developing synchronously. (x2000) D) Another mastomys oocyte fertilized with a round spermatid 4 h after activation. The spermatid-derived male pronucleus (M) is the original size, whereas the female pronucleus (F) is fully expanded. P, Second polar body. (x2000)

Microinsemination with Round Spermatids

Because round spermatids have little oocyte-activating capacity, we first established a parthenogenetic activation protocol for mastomys oocytes. Mastomys oocytes were submitted to electric pulse, Sr2+ treatment, or a combination of both. The combination treatment successfully activated most mastomys oocytes and induced formation of a female pronucleus and the second polar body within 3 h (Table 2). It was apparent that, when used alone, each activating treatment (the first and second groups) was less efficient (Table 2). Stronger stimuli were not feasible, because higher-voltage electric pulses caused the formation of multiple pronuclei or oocyte lysis and concentrations of more than 10 mM SrCl2 precipitated during culture. The first polar body of mastomys oocytes usually disappeared by the time of pronuclear formation. Telophase II, the stage most suitable for the round spermatid nucleus to develop into the male pronucleus [7, 20, 28], lasted from 60 to 120 min after the activation treatment (data not shown). Thus, the best protocol for microinsemination with mastomys round spermatids was as follows: Oocytes were activated by an electric pulse followed by SrCl2 treatment for 30 min, incubated in CZB medium for a further 30 min (until 60 min postactivation), and then injected with round spermatids.


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  		TABLE 2. Parthenogenic activation of mastomys oocytes by different activating protocols.a

If round spermatids are injected into preactivated oocytes at the appropriate time, spermatid and oocyte chromosomes synchronize and form a well-developed male pronucleus, mimicking the time course of normal fertilization. To evaluate whether mastomys round spermatids could participate in normal fertilization in our microinsemination protocol, we examined whole-mount preparations of injected oocytes for the development of spermatid-derived male pronuclei. At 2–3 h after round spermatid injection (3–4 h after oocyte activation), most oocytes (93%) were successfully activated, as demonstrated by the formation of a female pronucleus and second polar body. Of the activated oocytes, 80% had a well-developed male pronucleus (Table 3 and Fig. 2C). Far fewer oocytes contained a small male pronucleus that remained the same size as the original spermatid nucleus (Table 3 and Fig. 2D). Thus, according to morphological criteria in fertilized oocytes, we confirmed that our microinsemination protocol with mastomys round spermatids led to normal fertilization.


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  		TABLE 3. Pronuclear formation in mastomys oocytes after microinsemination with round spermatids

When oocytes that survived injection with round spermatids were cultured for 24 h, all (n = 89) developed into 2-cell embryos with a normal appearance (Table 4). Sixty-two 2-cell embryos were transferred into the oviducts of six recipient pregnant females, but none developed to term (Day 25) (Table 4). When the remaining 27 embryos derived from round spermatid injection were further cultured until 96 h, 11% (3/27) and 15% (4/27) developed into morulae and blastocysts, respectively, indicating that developmental failure in vivo could not be explained by developmental arrest at the 2-cell stage, which typically occurs in mastomys embryos when in vitro conditions are inadequate [18].


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  		TABLE 4. Development of mastomys embryos following microinsemination with different male germ cells

Microinsemination with Elongated Spermatids

The latest spermatids that could easily be injected into oocytes were those with a condensing nucleus before the head elongated into a hook shape (Fig. 1). These spermatids correspond to rat elongated spermatids at steps 9–11 [34]. As expected from experiments with mouse oocytes, these spermatids activated the majority of homologous oocytes and participated in normal fertilization without any artificial stimuli. All activated oocytes had two pronuclei that were easily discernable under a stereomicroscope, indicating that the female and male chromosomes were successfully synchronized on elongated spermatid injection. After 24 h in culture, 79% of oocytes injected with elongated spermatids developed to the 2-cell stage (Table 4). After transfer into six recipient females, five pups were born from two recipients (Table 4 and Fig. 3). One recipient was subjected to cesarean section, and the other was allowed to deliver young. Three pups (two males and one female) were apparently normal and grew into fertile adults (Fig. 3). One was stillborn, and another was cannibalized shortly after delivery by the recipient female.



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  		FIG. 3. A) A newborn male mastomys and its placenta obtained by microinsemination with an elongated spermatid. The pup was delivered by cesarean section from a pseudopregnant female. B) The newborn (center) was fostered by an ICR lactating mother together with its native pups (albino mice) and grew into a normal adult. (Shown 20 days after birth.) Bar = 5 mm

Microinsemination with Spermatozoa

Because mastomys spermatozoa have large heads, the survivability of oocytes after sperm head injection was relatively poor even when special care was taken to minimize oocyte injury (Table 4). Furthermore, only approximately 25% of fertilized oocytes developed to the 2-cell stage, and none developed beyond the 3-cell stage, probably because of mechanical damage during microsurgical injection (Table 4).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Recent advances in microinsemination techniques have enabled the use of immature sperm cells as substitute gametes, but the common consensus is that mature sperm yield better results than younger cells in terms of fertilization rates, embryo development, and production of normal young [14, 35]. This is true for all species examined so far (i.e., mice, humans, rabbits, and rats). From the biological point of view, spermatozoa are better than younger cells, because they have full oocyte-activating capacity and their cell cycle easily synchronizes with that of oocytes on injection. However, technical factors associated with sperm and oocyte handling in the laboratory may compromise the biological superiority of spermatozoa. In mice, for example, the first microinsemination-derived pups resulted from electrofusion with round spermatids [20], because ICSI into mouse oocytes was extremely difficult before development of the Piezo-assisted microinsemination technique. The present study also demonstrated that in mastomys, spermatids might offer a better chance of fertilization and normal embryo development than mature spermatozoa using the micromanipulation techniques now available.

In general, rodent spermatozoa have a large, hook-shaped head and a long tail. These features make ICSI difficult, although oocyte resistance to injection stimuli may also be a critical factor. Laboratory rat spermatozoa are very similar to those of mastomys in size and shape, and ICSI had been unsuccessful in rats until a recent study by Hirabayashi et al. [15]. According to that study, the technical problems of injecting large rat sperm can be overcome by hanging an isolated sperm head on the tip of smaller injection pipettes (diameter, 2–4 µm) instead of introducing the whole sperm head into larger injection pipettes (diameter, 7–10 µm). However, this modified ICSI technique cannot be employed in mastomys, because the oocytes are highly fragile. This same group also demonstrated that the use of round spermatids for microinsemination yielded high oocyte survival rates (>90%) and fertilization rates (~50%) by conventional injection of nuclei into oocytes. However, subsequent development into offspring was poor (0.5%–1.4% per transfer) [21], as demonstrated for mastomys in the present study.

Although we demonstrated that mastomys oocytes were efficiently fertilized with round or elongated spermatids, only the latter type produced normal full-term offspring. This difference might have resulted from several biological factors that distinguish the two types of spermatids (Table 5). First, round spermatids have a soft, interphase nucleus, whereas elongated spermatids have a condensing nucleus. Thus, in elongated spermatids, histones are largely replaced by protamines, which contribute to the physical and physiological stabilization of the sperm nucleus. Therefore, elongated spermatid nuclei probably are introduced into oocytes more safely and support embryo development better than round spermatid nuclei.


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  		TABLE 5. Comparison of spermatozoa and spermatids as male germ cells for microinsemination

Second, these two types of spermatids have different oocyte-activating abilities. Mastomys round spermatids have little activating capacity; consequently, oocytes have to be activated artificially before microinjection. This is similar to round spermatids in mice [7, 20, 28] and rats [21]. We found that the combination of electric stimulation and strontium treatment yielded the best activation rate for mastomys oocytes. An electric pulse generally induces a single intracellular calcium rise in all mammalian oocytes examined to date [29]. In contrast, in mouse oocytes, strontium treatment induces repetitive rises that resemble those associated with normal fertilization [29], although we did not monitor the intracellular calcium concentration in mastomys oocytes. It is interesting that strontium treatment itself does not induce oocyte activation but seems to prolong the activation status triggered by the electric pulse. Thus, these two types of stimulation probably exert their effects synergistically on oocyte activation machinery. We previously found that the same activation regimen was effective for rabbit oocytes (unpublished results), which also cannot be fully activated by a single electric pulse [36]. Nonetheless, it is apparent that artificial stimulation cannot exactly mimic sperm-induced oocyte activation in terms of efficiency and safety, despite great efforts to elucidate the exact molecular mechanisms of oocyte activation. In at least some mastomys elongated spermatids, the spermborne oocyte-activating factor was already active, as evidenced by the activation of mouse and mastomys oocytes following injection. This might have led to the difference in the ability of round and elongated spermatids to support embryonic development.

Third, the need for precise control of the oocyte cell-cycle stage for injection may also be a limiting factor in the success of round spermatid conception. Only round spermatids injected into oocytes at telophase II develop into a single, swollen male pronucleus [7]. In contrast to round spermatid injection, the elongated spermatid protocol is very simple. The elongated spermatid cell cycle is readily synchronized with the oocyte cell cycle on injection (see Fig. 2B), similar to fertilization by mature spermatozoa. In the mouse, the complicated prerequisite for round spermatid injection can be avoided by injecting round spermatids into MII-arrested oocytes and then activating and restoring the diploid state by enucleation [37]. If this technique can be applied to mastomys oocytes and round spermatids, then the efficiency of embryo development might be improved.

It is possible that the mastomys round spermatid genome has not completed paternal genomic imprinting, which ensures monoallelic expression of paternally imprinted genes during normal embryonic development. However, according to a study in mice [38], imprinted genes were correctly expressed by either the paternal or maternal allele in all spermatid-derived embryos so far examined. Therefore, aberrant expression of imprinted genes is unlikely to be the major cause of development failure in mastomys embryos from round spermatid injection. The birth of normal rats following round spermatid injection also supports this notion [21].

In mastomys, elongated spermatid use is superior to round spermatid use because of the simplicity of the protocol and the viability of the embryos obtained. Elongated spermatids have the technical advantages of round spermatids and the biological advantages of mature spermatozoa (Table 5). In humans, it has also been reported that if round spermatids are used instead of elongated spermatids, then the clinical outcome is often dramatically reduced [35], and treatment of male factor infertility by round spermatid conception remains highly controversial. Detailed analysis using animal models for microinsemination experiments with round and elongated spermatids may provide deeper insight regarding this subject. Another notable feature of elongated spermatids is their high tolerance of freezing and thawing for cryopreservation, although round spermatids can also be cryopreserved if specific technical precautions are taken [8]. For instance, we have obtained normal mouse pups using elongated spermatids that were retrieved from Sl/Sld mice with restored spermatogenesis following gene transfer and then frozen in liquid nitrogen and transported overseas to our laboratory [39]. Elongated spermatids are potentially useful for preserving and propagating invaluable genetic resources, especially for certain Rodentia species in which IVF and ICSI are impractical.


   FOOTNOTES
 
1 Supported by grants from MEXT, Japan; MHLW, Japan; and the Japan Health Sciences Foundation, Japan. Back

2 Correspondence: Atsuo Ogura, RIKEN Bioresource Center, 3-1-1, Koyadai, Tsukuba, Ibaraki 305-0074, Japan. FAX: 81 298 36 9172; ogura{at}rtc.riken.go.jp Back

Received: 24 September 2002.

First decision: 22 October 2002.

Accepted: 9 December 2002.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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