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Embryo |
Laboratory of Animal Physiology,3 Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan
Faculty of Agriculture,4 Shinshiu University, Minamiminowa 399-4598, Japan
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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early development, in vitro fertilization, ovum, sperm, spermatid
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Compared to mammals, information about in vitro fertilization is very limited in birds. Howarth [10] first demonstrated by Delafield's hematoxylin staining of nuclei that the ovum immediately after ovulation could be fertilized in vitro when the germinal disc of the ovum was introduced to millions of sperm in medium. More recently, Nakanishi et al. [6] provided direct evidence of in vitro fertilization (IVF) of chicken oocytes by showing male and female pronuclei formation. They also compared details of early nuclear events between in vivo and in vitro fertilization during 1–4 h of culture and concluded that the process of IVF is comparable to that in vivo. Furthermore, Nakanishi et al. [11] demonstrated the fertility competency of multiple-ovulated eggs in the chicken after IVF. Tanaka et al. [12] produced viable chicks by implanting in vitro-fertilized ova into the oviduct of recipient hens followed by incubation of the eggs. Recently, Olsza
ska et al. [13] investigated development of quail embryos after IVF using in vitro-ovulated oocytes with a special emphasis on the link between the steps of ovulation and fertilization and with the early cleavage stages under in vitro conditions. They demonstrated for the first time that cytoplasmic segmentation can occur in the absence of nuclear divisions in the germinal disc of the quail and showed the existence and significance of ooplasmic maternal information in birds. These conventional IVF techniques gave an insight to the early nuclear events to some extent in chicken and quail, but questions remain unanswered.
The methods of the direct injection of a sperm into an oocyte called intracytoplasmic sperm injection (ICSI) have been well developed in mammals and contributed to 1) the basic studies of fertilization, 2) clinical practice in humans, 3) production of normal offspring using morphologically normal and abnormal spermatozoa and spermatogenic cells, and 4) production of transgenic animals. However, in birds no ICSI has been reported.
Establishment of the ICSI method in birds is advantageous for studying the mechanism of fertilization and the role of gametes in this process and to protect endangered species of birds. In addition, the use of sperm as a vector to carry foreign DNA to the oocyte may help production of transgenic birds. Accordingly, the aims of the present experiments were 1) to establish a method of fertilization of quail ova using ICSI followed by short-term culture of the embryo; 2) to compare fertilizing ability of prematurely ovulated oocytes to those spontaneously ovulated; and 3) to test the competency of testicular round spermatids, elongated spermatids, and immature spermatozoa in fertilization and embryonic development.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Male and female Japanese quail (Coturnix japonica) were purchased from Tokai Yuki Company (Toyohashi, Japan) at 6 wk of age and raised in an environmentally controlled room (room temperature at 24 ± 1°C with 50–60% relative humidity). Birds were caged under a photoperiod of 14L:10D (lights-on at 0500 h and off at 1900 h) with free access to food and water. A total of 159 quail were used, and birds were divided into three groups. The first were oocyte suppliers for ICSI. Female quail individually caged were used at the age of 11–19 wk. Time of egg laying was recorded for each quail by a video camera recorder system (camera—ExwaveHAD SSC-DC430, camera adapter—YS-W170, VHS time lapse—SVT-S 960 ES; Sony, Tokyo, Japan) [14]. They laid eggs consecutively for more than 5 days in a sequence between 1400 and 1800 h. Time of ovulation was estimated by the time of oviposition. Ovulation was considered to occur within 30 min after oviposition of the previous egg in the series [15]. The second group was used to supply testicular spermatids, spermatozoa, and semen for ICSI. Adult male quail (12–25 wk old; n = 10) were kept individually. The third group supplied fertilized oocytes. Each female was coupled with a male in a cage. The time of egg laying was recorded as described previously.
All chemicals were obtained from Sigma-Aldrich (Tokyo, Japan) except ethanol, glacial acetic acid, and eosin, which were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan).
Collection of Oocytes
Controls For morphological standardization of early embryo development and examination of the effect of culture medium and polyvinylpyrrolidone (PVP) on embryo development, positive and negative control ova were prepared. The positive control was as follows: 1) in vivo-fertilized ova collected from the oviduct within 50 min after ovulation and cultured in the same conditions as those after ICSI (n = 5); 2) in vivo-fertilized ova collected from the oviduct within 50 min after ovulation and PVP was injected, followed by culture (n = 5); 3) in vivo-fertilized ova collected from the oviduct at different times after ovulation, that is, 3.5, 4–5, 6, 7, 9, 10, 12, 13, 15, 16, 17, and 20 h, which reflect different stages of embryonic development (n = 33); and 4) in vivo-fertilized eggs collected immediately after spontaneous oviposition (n = 10). As a negative control, unfertilized ova were collected within 50 min after ovulation and PVP was injected, followed by culture (sham-operated control; n = 5). Unfertilized eggs were also collected from virgin quail after oviposition (n = 10).
Spontaneous ovulation To obtain ova after spontaneous ovulation, birds (n = 51) were killed 50–60 min after oviposition, and oocytes were recovered from the infundibulum or the upper part of the magnum.
Premature ovulation Quail (n = 30) were subcutaneously injected with aLHRH ([Gln8]-LHRH; 25 µg/0.1 ml of 0.9% NaCl/quail; Sigma-Aldrich) 12 h before expected ovulation and killed 9–11 h after the treatment, that is, about 1 h after premature oviposition. Oocytes were collected from the infundibulum or the upper part of the magnum. Any oocytes present in the other region of the oviduct were not used. Induced ovulation occurred 1.5–5 h prematurely.
Collection of Semen Sperm, and Testicular Spermatids and Spermatozoa
The isolated male was introduced to the isolated female for mating. Semen was collected with swab from cloaca of female quail immediately after mating and washed three times by centrifugation at 1000 x g for 5 min in 1 ml of 0.9% NaCl solution. The washed sperm were diluted with 12% PVP in Dulbecco Modified Eagle Medium (DMEM). About 100 µl of this solution was placed on a Petri dish (35 x 10 mm; cat. no. 35 1008; Falcon, Becton Dickinson Labware, Franklin Lakes, NJ), covered with mineral oil, and kept until injection at room temperature (25–26°C).
To collect round and elongated spermatids and testicular spermatozoa, male quail (n = 4) were killed by dislocation of the atlas bone, and the testes were excised. They were cut in pieces, and a piece of testis was placed in 0.9% NaCl and minced by scissors. One part of this suspension was mixed thoroughly with two parts of PVP solution and kept for up to 5 h on a Petri dish before spermatozoa or spermatids were selected for injection into spontaneously ovulated oocytes.
Preparation of Injection Pipettes
To prepare pipettes for ICSI, borosilicate glass capillary tubing (1-mm outer diameter, 0.75-mm inner diameter, cat. no. B100-75-10; Sutter Instrument Co., Novato, CA) was drawn with a pipette puller (P-97/IVF; Sutter), and the tip of the pipette was broken with a microforge (MF-900; Narishige Instrument, Tokyo, Japan) such that the inner diameter at and near the tip was approximately 10 µm. Next, the tip of the pipette was beveled at a 30° angle with a micropipette beveler (BV-10; Sutter), dipped in 70% ethanol, and dried. Then the injection pipette was connected to a micromanipulator (ONM-1; Narishige Instrument) and half-filled with silicon oil, and a few microliters of 0.9% NaCl were sucked into the pipette. The injection pipettes for round and elongated spermatids were prepared in the same way except beveling.
Intracytoplasmic Injection of Sperm and Round and Elongated Spermatids
Immediately before injection, a single sperm or spermatid was isolated from PVP-sperm or PVP-testicular tissue solution, respectively, into the injection pipette under the Hoffman modulation contrast microscope (IX70; Olympus, Tokyo, Japan). The round and elongated spermatids were recognized from other cells by their size and shape. Round spermatids, 6–8 µm in diameter, have centrally located condensed chromatin, and elongated spermatids are easy to distinguish by a visible elongated tail (Fig. 1). The ovum was placed into DMEM in a plastic dish (35 x 18 mm; multidish six wells, Nunclon Delta, Roskilde, Denmark), and the sperm or spermatid was injected into the central area of the germinal disc using a micromanipulator connected to the injector (IM-9B; Narishige Instrument) with silicon tubing filled with silicon oil, under a stereomicroscope (SZ11; Olympus) (Fig. 1). This manipulation was performed with the aid of an image-processor system (Image 
-III, Nippon Avionics, Tokyo, Japan). It took 10–15 min from the time the female quail (oocyte supplier) was killed to completion of sperm or spermatid injection into oocyte.
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Culture
The sperm- or spermatid-injected ova were cultured in plastic dishes (35 x 18 mm; multidish six wells) in DMEM containing 3 mg/ml BSA (fraction V; Sigma-Aldrich) and 2 µl/ml antibiotic-antimycotic solution (with 10 000 units penicillin, 10 mg streptomycin, and 25 µg amphotericin B/ml) for 24 h at 41°C under 5% CO2 in air.
Observation of Embryo
Fertilization and development of the embryo were staged as described by Eyal-Giladi and Kochav [16] under stereomicroscope (SZ11; Olympus). The blastoderm was cut and, in conformity with the method of Nakanishi et al. [6], fixed in ethanol/glacial acetic acid (3:1), embedded in paraffin, and sectioned vertically. Half the sections were stained with 4,6-diamidino-2-phenylindole (DAPI) a nuclei-staining fluorescent dye, in order to examine the cell division by the method of Waddington et al. [7] and observed under an epifluorescent microscope (BX60; Olympus) within 20 h. Additionally, the other half of the sections were stained with hematoxylin and eosin (HE) using standard procedures. Stained sections were imaged using a light microscope.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Figure 2 shows examples of control blastoderms of in vivo fertilized, developing oocytes. The quail embryo collected from the shell gland 9 h after ovulation was at stage III, in which individual blastomeres and cleavage furrows were seen (Fig. 2A). Much more advanced cleavage was observed in the embryo collected from the shell gland 12 h after ovulation (stage V; Fig. 2B). In the embryo at stage VIII (Fig. 2C), obtained 16–17 h after ovulation, pellucida formation was under way. In the blastoderm of the egg collected immediately after oviposition (stage X), formation of the pellucida was completed (Fig. 3A). In contrast, unfertilized oviposited eggs from virgin quail always contained numerous vacuoles at the periphery of the germinal disc, and a part of their cytoplasm was often segmented into pseudocells (Fig. 3B). One of the sham-operated oocytes in which PVP was injected into an unfertilized oocyte collected from the infundibulum had cleaved cytoplasm similar to stage IV (pseudoblastoderm) (Fig. 3C). The remaining four sham-operated oocytes did not show any cleavage and had vacuoles at the periphery of the germinal disc. Naturally (in vivo) fertilized oocytes collected from the infundibulum developed to stages IX–XI in DMEM, and oocytes fertilized in vivo that were injected with PVP and incubated in DMEM developed to stages VII–X (data not shown).
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DAPI-stained nuclei emitting intense blue fluorescence in fertilized oocytes were distinguished by their round shape, size, and location. The number of nuclei observed on sections varied depending on the stage of embryo development, that is, several at very early stages (Fig. 2D) and numerous at advanced stages (Figs. 2F and 3D). Control embryos of in vivo-fertilized oocytes developed in the oviduct or in culture for 24 h always showed nuclei identified by DAPI (Figs. 2, D, E, and F and 3D) and HE (Figs. 2, G, H, and I and 3G) staining. In unfertilized eggs, no nuclei were seen after staining, although a part of their cytoplasm showed cleavage (Fig. 3, E and H). Two of five sham-operated control oocytes showed irregularly fragmented DNA bodies with DAPI (Fig. 3F) and HE (Fig. 3I) staining, while in three other oocytes no fragmented DNA was present. HE staining showed individual cells with nuclei (Figs. 2, H and I and 3G) as well as layers of the germinal disc, such as yolk, cytoplasm (Figs. 2G and 3I), epiblast, and primary hypoblast (Fig. 3G). DAPI staining showed more distinguishable nuclei.
Spontaneously and Prematurely Ovulated Oocytes
Several embryos after ICSI showed "normal" cleavage but contained no fluorescent nuclei nor exhibited signs of degeneration of nuclei seen as bright irregular spots. These embryos were considered unfertilized eggs. Figure 4 depicts an example of blastoderm after ICSI into a spontaneously ovulated oocyte. Blastoderms of the embryos developed from the ICSI of prematurely ovulated oocytes are not shown. Table 1 presents data on the fertilization and development of quail oocytes spontaneously and prematurely ovulated followed by ICSI and culture for 24 h in DMEM. Five of 30 (16.6%) and 4 of 30 (13.3%) oocytes injected with mature sperm were fertilized in the spontaneous and induced ovulation groups, respectively. There was no clear difference in stages of embryo development after ICSI between the two groups. Both groups showed maximum development at stage VI.
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Intracytoplasmic Injection of Testicular Sperm and Round and Elongated Spermatids
Figure 5 shows a representative blastoderm of the embryos from an experiment in which a single testicular sperm or spermatid was injected into an individual oocyte followed by culture for 24 h in DMEM. All data are summarized in Table 2. Half the number (three of six) of oocytes injected with testicular spermatozoa were fertilized and developed to stages IV–VII, and two of five oocytes injected with elongated spermatids were fertilized and developed to stage VI. In contrast, all oocytes injected with round spermatids were unfertilized.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ska et al. [13], who demonstrated 15% efficiency after conventional IVF using 1–2 x 104 semen sperm of quail. The embryonic development is also comparable between the two studies (stages II–VI 24 h after culture in the present study vs. stages IV–VI 18 h after culture in theirs). A higher rate of fertilization (75%) was reported by Nakanishi et al. [6] when development was observed with DAPI staining only 1–4 h after culture in conventional IVF in chicken oocytes. Conventional IVF may retain more physiological conditions than ICSI, but the present study suggests that the ICSI method is valid to the same degree as the conventional IVF method. ICSI omits some physiological steps preceding the fertilization required in IVF, such as sperm maturation or interaction of sperm membrane with oocyte. Thus, this method simplifies the experimental conditions and provides a useful means to study the mechanisms of fertilization and development in birds, as in mammals. As one example in this regard, this study provided evidence in quail that one sperm is capable of activating an oocyte for fertilization, although polyspermy naturally occurs in birds. The present study also showed that oocytes collected from quail after prematurely induced ovulation are as capable of fertilizing with a single mature sperm as those spontaneously ovulated, suggesting that oocytes are mature for fertilization several hours before the destined ovulation time. Maturity of the ovarian follicle in birds has been characterized in terms of ovulability in response to exogenous progesterone, luteinizing hormone, or luteinizing hormone-releasing hormone injections [17, 18]. We propose the fertilizing ability of the oocyte as a new index for follicle maturation.
Quail oocytes exhibited the formation of embryonic blastoderms with a typical cleavage pattern of enucleated blastomeres at progressive stages with both conventional IVF [13] and ICSI; development was somewhat delayed compared to that achieved in vivo. As demonstrated in the present study and by St
pinska and Olszaska [19], in natural conditions in the oviduct quail embryos developed to stages III–IV at 9–10 h, to stage V at 12 h, to stage VI at 13–14 h, to stages VII–VIII at 15–17 h, and to stages X–XI at 24 h after ovulation. Delay of embryo development in our ICSI experiment may be attributable at least in part to the high volume of PVP injected as well as mechanical damage to the germinal disc due to injection. During this manipulation, the germinal disc is exposed to air, which may cause it to dry. However, these points cannot fully explain the delay because avian oocytes tolerated microsurgery well, as has been reported in the foreign DNA injection studies in quail [20] and chicken [21, 22]. In these studies, already fertilized ova were microinjected with DNA, whereas unfertilized ova were used in conventional IVF and in the present ICSI. Nevertheless, there is room to improve the culture system for avian IVF.
Several embryos after ICSI showed a similar cleavage pattern to a "normal" one, but these embryos did not contain any nuclei or fragmented DNA bodies when examined by the DAPI staining method. Olszaska et al. [13] also reported this phenomenon in quail oocytes after IVF and suggested that experimental procedures may have activated the egg, but fertilization did not go to completion. The appearance of the external features of the embryo cannot be the only criterion to estimate early embryo development in culture. The examination of the presence/absence of the nuclei is important to determine the true development of the quail embryo. Kosin [23] observed degenerated nuclei in the germinal disc of the unfertilized eggs of chicken. Some breeds of turkeys and strains of chicken exhibit limited parthenogenesis. In most cases this spontaneous development without fertilization led to very early degeneration. In a selected line of turkeys, a small percentage of parthenogenetically derived embryos hatched and grew into adult males [24, 25].
We also provided a new finding that a single elongated spermatid and immature sperm in the testis can fertilize the oocyte in quail. In mammals, the birth of normal offspring was shown by direct injections of immature spermatozoa from the testis [26, 27]. The present study demonstrated that injection of testicular sperm or elongated spermatid into a quail oocyte resulted in fertilization and the embryo development to stages VI–VII. The results clearly indicate that these cells are competent to participate in fertilization and early embryonic development. In contrast, the injection of round spermatids did not result in successful fertilization and development. Kimura and Yanagimachi [26] reported that in mice, round spermatids could not activate the oocytes unless the oocytes were activated artificially. The rate of fertilization was improved by activating the oocyte by electric current before injection of spermatid nucleus. In quail, the reason for the failure of round spermatids is not clear but may be attributed to insufficient amounts of the oocyte-activating factor(s). Quail round spermatids may need preactivation of quail oocytes similar to that in mammals.
In summary, the present study demonstrates for the first time that 1) intracytoplasmic injection of a single sperm isolated from semen into quail oocyte can activate the oocyte and lead to fertilization; 2) using the employed ICSI and culture conditions, embryos can develop to maximum stages of VI–VII; 3) spontaneously and prematurely ovulated oocytes possess a similar degree of fertilizing competency; and 4) the testicular elongated spermatids and spermatozoa can activate the oocyte as do mature sperm in semen. Accordingly, the microfertilization method has the potential to assist in the production of transgenic birds and protection of endangered species of birds.
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FOOTNOTES |
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2 Correspondence: Kiyoshi Shimada, Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, Aichi 464-8601, Japan. FAX: 81 52 789 4065; kshimada{at}agr.nagoya-u.ac.jp
Received: 13 May 2003.
First decision: 6 June 2003.
Accepted: 1 July 2003.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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ska B, Stpiska U, Perry M. Development of embryos from in vitro ovulated and fertilized oocytes of the quail (Coturnix coturnix japonica). J Exp Zool 2002 292:580-586[CrossRef][Medline]piska U, Olszaska B. Cell multiplication and blastoderm development in relation to egg envelope formation during uterine development of quail (Coturnix coturnix japonica) embryo. J Exp Zool 1983 228:505-510[CrossRef]This article has been cited by other articles:
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  S. Takagi, T. Ono, A. Tsukada, Y. Atsumi, S. Mizushima, N. Saito, and K. Shimada Fertilization and Blastoderm Development of Quail Oocytes After Intracytoplasmic Injection of Chicken Sperm Bearing the W Chromosome Poult. Sci., May 1, 2007; 86(5): 937 - 943. [Abstract] [Full Text] [PDF]
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S. Takagi, A. Tsukada, N. Saito, and K. Shimada Fertilizing Ability of Chicken Sperm Bearing the W Chromosome Poult. Sci., April 1, 2007; 86(4): 731 - 738. [Abstract] [Full Text] [PDF]
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