Fertilization and development of quail oocytes ... - CiteSeerX

Fertilization and development of quail oocytes after intracytoplasmic sperm

injection

Anna Hrabia3,4, Soichi Takagi3, Tamao Ono5, Kiyoshi Shimada2,3

Laboratory of Animal Physiology,3 Graduate School of Bioagricultural Sciences,

Nagoya University, Nagoya, Aichi 464-8601, Japan

JSPS Postdoctoral Fellow on leave from Department of Animal Physiology,4 Agricultural

University of Cracow, Al. Mickiewicza 24/28, 30-059 Krakow, Poland

Faculty of Agriculture,5 Shinshiu University, Minamiminowa 399-4598, Japan

1This research was supported by grant-in-aid from the Japan Society for the Promotion of

Science, Postdoctoral Fellowship for Foreign Researchers awarded to A.H. (P 01319) and from

the Japan Society for the Promotion of Science, Basic Research (C2) to K.S. (No. 13660286).

2Correspondence and request for reprints:

Kiyoshi Shimada, Ph.D.

Laboratory of Animal Physiology, Graduate School of Bioagricultural Sciences, Nagoya

University, Nagoya, Aichi 464-8601, Japan.

tel. & fax. 052-789-4065

e-mail:[email protected]

Running title: Fertilization of quail oocytes by ICSI

Key Words: early development, in vitro fertilization, ovum, sperm, spermatid

BOR Papers in Press. Published on July 9, 2003 as DOI:10.1095/biolreprod.103.019315

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ABSTRACT

The present study was conducted to establish the intracytoplasmic sperm injection (ICSI)

method for in vitro fertilization and development in quail. The efficiency of fertilization of

oocytes was compared (1) between spontaneous and premature ovulation and (2) among

testicular round spermatids, elongated spermatids and immature and mature spermatozoa. The

oocytes were injected with a single spermatozoon or spermatid and cultured for 24 h. Cell

division was histologically observed with Hematoxylin-Eosin (HE) and a nuclear specific

fluorescent dye DAPI. Five out of 30 (16.6%) and 4 out of 30 (13.3%) oocytes injected with

mature sperm were fertilized in the spontaneous and induced ovulation group, respectively. Those

embryos showed development at stages II-VII. Half of the number (3/6) of the oocytes injected

with testicular spermatozoa were fertilized and developed to stages IV-VII and 2 out of 5 oocytes

injected with elongated spermatids were fertilized and developed to stage VI. All oocytes injected

with round spermatids were unfertilized. The results demonstrate that intracytoplasmic injection

of a single sperm into quail oocyte can activate the oocyte and lead to fertilization. Oocytes

prematurely ovulated are capable of fertilizing with mature sperm as those spontaneously

ovulated. In addition, the results suggest that the testicular round spermatids may not possess

sufficient oocyte-activating potency but the elongated spermatids and immature spermatozoa are

competent to participate in fertilization and early embryonic development in quail.

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INTRODUCTION

Fertilization in birds takes place in the infundibulum, the most upper region of the oviduct,

soon after ovulation. This must occur before the outer layer of vitelline membrane (outer

perivitelline layer) is laid down since spermatozoa are not able to penetrate this layer [1, 2] and

bind to sperm receptors in the inner layer of the vitelline membrane [3]. Unlike mammals, but

common with reptiles, some amphibians and most urodeles (newts and salamanders), birds

exhibit physiological polyspermy. Many sperm pass through the inner layer of vitelline

membrane into germinal disc of an oocyte, where the nuclei undergo decondensation and form

male pronuclei although only one sperm successfully fertilizes the oocyte [1, 4-7]. The

supernumerary pronuclei degenerate at the very early developmental stages of embryo [5-7].

However, it has been shown that the number of spermatozoa reaching the ovum is positively

correlated with ovum size in variety of birds suggesting that a larger ovum requires more

spermatozoa to ensure fertilization [8, 9]. While the physiological role of polyspermy is not

known, it is unclear whether or not a single sperm is capable of activating an oocyte in birds.

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-

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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, Olszańska 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 still 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 human, (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 to study 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 for

production of transgenic birds. Accordingly, the aims of present experiments were (1) to

establish a method of fertilization of quail ova using ICSI followed by short-term culture system

of embryo, (2) to compare fertilization ability of premature 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

Birds and chemicals

Male and female Japanese quail (Coturnix japonica) were purchased from Tokai Yuki

Company (Toyohashi, Japan) at 6 weeks of age and raised in environmentally controlled room

(room temperature at 24 ± 1ºC with 50 - 60% relative humidity). Birds were caged under a

photoperiod of 14L:10D (lights on at 5:00 and off at 19:00) with free access to food and water.

Total number of 159 quail was used and birds were divided into 3 groups. The first were oocyte

suppliers for ICSI. Female quail individually caged were used at the age of 11-19 weeks. Time of

egg laying was recorded for each quail by video camera-recorder system (camera – ExwaveHAD

SSC-DC430, camera adapter - YS-W170, VHS time lapse – SVT-S 960 ES; Sony, Tokyo,

Japan). They laid eggs consecutively for more than 5 days in a sequence between 14:00-18:00.

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 [14]. The second group was used

to supply testicular spermatids, spermatozoa and semen for ICSI. Adult male quail (12-25 weeks

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 above.

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

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a) Controls

For morphological standardization of early embryo development and examination of the

effect of culture medium and polyvinylpyrrolidone (PVP) on embryo development, the 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 were

collected from the oviduct at different times after ovulation i.e. 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), 4) in vivo

fertilized eggs 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).

b) 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.

c) Premature ovulation

Quail (n=30) were subcutaneously injected with aLHRH ([Gln8]-LHRH; 25 µg/ 0.1 ml of

0.9% NaCl/ quail; Sigma-Aldrich, Tokyo, Japan) 12 h before expected ovulation and sacrificed

9-11 h after the treatment i.e. 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 h-5 h prematurely.

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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 3 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 Petri dish (35 x 10 mm; cat. no. 35 1008; Falcon, Becton Dickinson Labware, Franklin Lakes,

NJ, USA), 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 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 2 parts of PVP solution and kept for up to 5 h on 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 o.d., 0.75 mm i.d., cat.

no. B100-75-10; Sutter Instrument Co., Novato, CA, USA) was drawn with a pipette puller (P-

97/IVF; Sutter Instrument Co.) 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 Instrument Co.), dipped in 70% ethanol and dried. Then the injection

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pipette was connected to micromanipulator (ONM-1; Narishige Instrument), half-filled with

silicon oil and a few µl 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. The round spermatids in

diameter 6-8 µm have centrally located condensed chromatin, and elongated spermatids are easy

to distinguish by visible elongated tail (Fig. 1). The ovum was placed into DMEM in a plastic

dish (35 x 18 mm; multidish 6 wells, Nunclon Delta, Denmark) and 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

stereomicroscope (SZ11; Olympus) (Fig. 1). This manipulation was performed with the aid of an

image-processor system (Image Σ-III, Nippon Avionics, Tokyo, Japan). It took 10-15 min from

sacrifice of female quail (oocyte supplier) to completion of sperm or spermatid injection into

oocyte.

Culture

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The sperm or spermatid injected ova were cultured in plastic dishes (35 x 18 mm; multidish 6

wells) for 24 h at 41°C under 5% CO2 in air, in DMEM containing 3 mg/ ml BSA (fraction V;

Sigma-Aldrich, Tokyo, Japan) and 2 µl/ ml antibiotic-antimycotic solution (with 10,000 units

penicillin, 10 mg streptomycin and 25 µg amphotericin B per ml).

Observation of embryo

Fertilization and development of embryo were staged as described by Eyal-Giladi and Kochav

[15] 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 a fluorescent dye 4,6-diamidino-2-

phenylindole (DAPI) for nuclei in order to examine the cell division by the method of

Waddington et al. [7] and observed under epifluorescent microscope (BX60; Olympus) within 20

h. Additionally, the other half of sections was stained with hematoxylin and eosin (HE) using

standard procedures. Stained sections were imaged using a light microscope.

RESULTS

Controls

Figure 2 shows examples of control blastoderms of in vivo fertilized, developing oocytes.

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

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was observed in the embryo collected from the shell gland 12 h after ovulation (stage V; Fig.

2B). In embryo at stage VIII (Fig. 2C) obtained 16-17 h after ovulation the area pellucida was

under a way of formation. In the blastoderm of the egg collected immediately after oviposition

(stage X) formation of the area pellucida was completed (Fig. 3A). In contrast, unfertilized

oviposited eggs from virgin quail always contained numerous vacuoles at the periphery of

germinal disc and a part of their cytoplasm was often segmented into pseudo cells (Fig. 3B). One

of sham-operated oocytes in which PVP was injected into unfertilized oocyte collected from the

infundibulum had cleaved cytoplasm similar to stage IV (pseudo blastoderm) (Fig. 3C).

Remained four sham-operated oocytes did not show any cleavage and had vacuoles at the

periphery of germinal disc. Naturally (in vivo) fertilized oocytes collected from the infundibulum

developed to stages IX-XI in DMEM and fertilized oocytes in vivo which were injected with

PVP and incubated in DMEM, developed to stages VII-X (data not shown).

DAPI-stained nuclei giving intensive blue fluorescence in fertilized oocytes were

distinguished by their round shape, size and location. The number of nuclei observed on sections

varied depending on stage of embryo development i.e. 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.

2D, E, F and 3D) and HE (Figs. 2G, H, I and 3G) staining. In unfertilized eggs no nuclei were

seen after staining, although a part of their cytoplasm showed cleavage (Figs. 3E and H). Two

out of 5 sham-operated control oocytes showed irregularly fragmented DNA bodies with DAPI

(Fig. 3F) and HE (Fig. 3I) staining, while in 3 other oocytes no fragmented DNA was present.

HE staining showed individual cells with nuclei (Figs. 2H, I and 3G) as well as layers of the

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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 or

exhibited signs of degeneration of nuclei seen as bright irregular spots. They were considered as

unfertilized eggs. Figure 4 depicts example of blastoderm after ICSI into spontaneously ovulated

oocyte. Blastoderms of the embryos developed from the ICSI of premature ovulated oocytes are

not shown. Table 1 presents fertilization and development of quail oocytes spontaneously and

prematurely ovulated followed by ICSI and culture for 24 h in DMEM. Five out of 30 (16.6%)

and 4 out 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.

Intracytoplasmic injection of testicular sperm and round and elongated spermatids

Figure 5 shows a representative blastoderm of the embryos from 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 of the number (3/6) of oocytes injected with

testicular spermatozoa were fertilized and developed to stages IV-VII and 2 out of 5 oocytes

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injected with elongated spermatids were fertilized and developed to stage VI. In contrast, all

oocytes injected with round spermatids were unfertilized.

DISCUSSION

To our knowledge, this is the first paper demonstrating by ICSI that a single sperm is capable

of activating the oocyte for further fertilization process and embryonic development in the quail.

The present study showed that the rate of fertilization after ICSI of spontaneously ovulated

oocytes, 16.6%, is comparable to that of Olszańska et al. [13] who demonstrated 15% of

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). Higher rate of fertilization (75%) was

reported by Nakanishi et al. [6] when the development was observed with DAPI staining only 1-

4 h after culture in the conventional IVF in chicken oocytes. The conventional IVF may retain

more physiological conditions than ICSI, but the present study suggests that ICSI method is valid

to the same degree as the conventional IVF method. The 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

useful means to study mechanisms of fertilization and development in birds as in mammals. As

one of examples in this regard, this study provided evidence, that one sperm is capable of

activating an oocyte for fertilization in quail, although polyspermy naturally occurs in birds.

The present study also showed that oocytes collected from quail of prematurely induced-

ovulation are capable of fertilizing with a single mature sperm as those in spontaneous ovulation,

suggesting that oocytes are matured for fertilization several hours before the destined ovulation

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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 [16, 17]. We propose the fertilizing ability of the oocyte as a new index for

follicle maturation.

Although both the conventional IVF [13] and ICSI in quail oocytes exhibited the formation of

embryonic blastoderms with a typical cleavage pattern of nucleated blastomeres at progressive

stages, the development was somehow delayed when compared to that in vivo conditions. As

demonstrated in the present study and by Stępinska and Olszańska [18], in natural conditions in

the oviduct quail embryos developed to stages III-IV 9-10 h, to stage V 12 h, to stage VI 13-14 h,

to stages VII-VIII 15-17 h and to stages X-XI 24 h after ovulation. Delay of embryo

development in our ICSI experiment may be attributable at least in a part, to high volume of PVP

injection as well as mechanical damage to the germinal disc due to injection. During this

manipulation the germinal disc was exposed to air, which in turn, may cause it to dryness.

However, these points cannot fully explain for the delay, because avian oocytes well tolerated

microsurgery as has been reported in the foreign DNA injection studies in quail [19] and chicken

[20, 21]. In these studies, already fertilized ova were microinjected with DNA, whereas the

unfertilized ova were used in the 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 determined by DAPI

staining method. Olszańska 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 only criterion

14

as a base to estimate the early embryo development in culture. The examination of

presence/absence of the nuclei is important to determine true development of the quail embryo.

Kosin [22] 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 lead to very early degeneration. In a selected line

of turkeys a few percent of parthenogenetically derived embryos hatched and grew into adult

male [23, 24].

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, birth of normal offspring was shown by

direct injections of immature spermatozoa from the testis [25, 26]. The present study

demonstrates that injection of the testicular sperm or elongated spermatid into quail oocyte

resulted in the 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 spermatid did not result in successful

fertilization and development. Kimura and Yanagimachi [25] reported that mouse round

spermatids could not activate the oocytes, unless the oocytes were activated artificially. The rate

of fertilization was improved by activating oocyte by electric current before injection of

spermatid nucleus. In quail, the reason for the failure of round spermatids is not clear but may

attributed to insufficient amounts of the oocyte-activating factor(s). Quail round spermatids may

need preactivation of quail oocytes similar to that in mammals.

To sum up, 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

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maximum stages of VI-VII, (3) spontaneously and prematurely ovulated oocytes possess similar

degree of fertilizing competency and (4) the testicular elongated spermatids and spermatozoa can

activate the oocyte like mature sperm in semen. Accordingly, microfertilization method in birds

has the potential to assist in the production of transgenic birds and protection of endangered

species of birds.

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FIGURE LEGENDS

FIG. 1. ICSI system for identification, isolation and injection of testicular spermatid and sperm

of quail. A - section of quail testis stained with HE. Small and large rectangles indicate areas for

magnification in B. Ba, b - left and right photos are magnification from A, respectively. Sperm

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(sp); elongated spermatid (esd); round spermatid (rsd); spermatocyte (stc). C - quail sperm,

elongated spermatid, round spermatid and spermatocyte in PVP solution under Hoffman

modulation contrast microscope. Legends are the same as B. D - isolation of single sperm from

PVP solution by injection pipette. E - single sperm isolated in the pipette as indicated by arrow.

F - apparatus for sperm injection into oocyte in medium. G - close-up of ICSI into quail oocyte.

H - close-up of ICSI into the center of germinal disc.

FIG. 2. Quail blastoderms of in vivo fertilized, developing oocytes under stereomicroscope (A,

B, C) and after DAPI (D, E, F) and HE (G, H, I) staining. A, D, G - blastoderm at stage III. B, E,

H - blastoderm at stage V. C, F, I - blastoderm at stage VIII. a.o. - area opaca; a.p. - area

pellucida; c – cytoplasm; c.f. - cleavage furrows; y - yolk; arrows indicate the representative

nuclei. Bar = 0.1 mm

FIG. 3. Quail blastoderms of control group under stereomicroscope (A, B, C) and after DAPI (D,

E, F) and HE (G, H, I) staining. A, D, G - blastoderm of in vivo fertilized and developed oocyte

after oviposition at stage X. B, E, H - germinal disc of unfertilized egg after oviposition, with

segmentation of cytoplasm and numerous vacuoles at the periphery, absence of nuclei after

DAPI and HE staining in pseudo cells. C, F, I - sham-operated control, DAPI-stained irregular

fragmented DNA bodies. a.o.- area opaca; a.p. - area pellucida; c – cytoplasm; e – epiblast; h –

primary hypoblast; p.c. - pseudo cells; v – vacuoles; y – yolk; arrows indicate the presence of

nuclei, arrow heads indicate fragmented DNA bodies. Bar = 0.1 mm.

20

FIG. 4. Quail blastoderm at stage V after ICSI into spontaneously ovulated oocyte under

stereomicroscope (A) and after DAPI (B) and HE (C) staining. Arrows indicate representative

nuclei; y-yolk. Bar = 0.1 mm.

FIG. 5. Quail blastoderm at stage VI in oocyte injected with elongated spermatid under

stereomicroscope (A) and after DAPI (B) and HE (C) staining. Other explanation is the same as

in Fig. 4.

21

Table 1. Development of quail oocytes spontaneously and prematurely ovulated after

intracytoplasmic sperm injection and culture.

No. of developing embryos up to

following stages2:

Ovulation of the

oocyte

No. of

injected oocytes1

No. of

fertilized oocytes (%) II III IV V VI

Spontaneous

30

5 (16.6)

1

0

1

2

1

Induced (premature)

30

4 (13.3)

1

1

1

0

1

1Only one oocyte was collected from one female quail

2Developmental stages of embryos were estimated according to those fertilized and developed in vivo.

22

Table 2. Development of quail oocytes after intracytoplasmic injection of spermatogenic cells

and culture.

No. of developing embryos up to

following stages2:

Cell

Injected

No. of

injected oocytes1

No. of

fertilized oocytes (%) IV VI VII

Round spermatid

10

0 (0)

0

0

0

Elongated spermatid

5

2 (40)

0

2

0

Testicular sperm

6

3 (50)

1

1

1

1, 2The same as in Table 1.