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Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds
General and Comparative Endocrinology 196 (2014) 100–105
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Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds Kiyoshi Shimada a,⇑, Tamao Ono b, Shusei Mizushima c a
WCU, Major in Biomodulation, College of Agriculture and Life Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, South Korea Faculty of Agriculture, Shinshu University, Kamiina, Nagano 399-4598, Japan c Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan b
a r t i c l e
i n f o
Article history: Received 29 October 2012 Revised 22 October 2013 Accepted 1 November 2013 Available online 13 November 2013 Keywords: GFP-expressing blastoderm ICSI Inositol trisphosphate PLCzeta Quail oocyte Transgene
a b s t r a c t Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24 h at 41 °C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12–18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37–50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37 °C after the 24 h incubation at 41 °C under 5% CO2 in air. It survived up to 2 days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24 h incubation at 41 °C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Intracytoplasmic sperm injection (ICSI) has now been well recognized as the excellent assisted-reproductive technology in mammals and made a great contribution to better understanding of fundamental gamete biology as well as application in animal biotechnology. It is also employed for protection of endangered species of animals and for clinical medicine in humans. Nevertheless, in birds, ICSI has not been attempted until recently (Hrabia et al., 2003a,b; Mizushima, 2012). Since artificial insemination has been employed in poultry industries widely, it has been thought the ICSI might not have much advantage in practical term. Furthermore, opacity of the avian oocyte is a big barrier for sperm injection into the oocyte. However, once this methodology is established, it seems promising to contribute to studies of fertility mechanism as well as biotechnology application in birds. In physiological states, polyspermic fertilization takes place at the infundibulum soon after ovulation, although only one sperm ⇑ Corresponding author. Fax: +82 52 804 7257. E-mail address: [email protected] (K. Shimada). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.11.001
successfully fertilizes oocyte. The yolk including oocyte moves along the magnum where albumen is secreted, and the isthmus where shell-membrane is formed, and stayed in the shell gland where shell is formed for about 20 h. Meanwhile, the first cell division takes place when the egg enters at the shell gland, and subsequent cell divisions follow. By the time of oviposition the oocyte consists of more than 4–6 104 cells. Polyspermy is unique phenomenon in birds as observed in some reptiles, some amphibians and most urodetes (newts and salamanders). The significance of polyspermy remains unknown, but application of the ICSI method revealed that even a single sperm is capable of fertilizing oocyte in quail (Hrabia et al., 2003a,b), although more sperm can cause higher rate of fertility (Mizushima et al., 2008). Conventional artificial insemination (AI) has been conducted in poultry industry or research farms in many domestic fowls. Usually the semen is collected by massage of the abdominal area of males, dilutes it and is injected into vagina. The AI using cryo-preserved semen is not popular due to inconsistent results of fertility rate and hatchability. Experimentally, in vitro fertilization has been conducted in some studies, culture of the embryo is limited within a day. We have developed ICSI technology in quail and provided
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several new findings on oocyte activation mechanism, and a new trial of gene transfer. Here, we describe our ICSI methodology and our outputs. 2. Method of development of ICSI-assisted fertilization Male and female Japanese quail (Coturnix japonica) were kept in an environmentally controlled room. Female quail laying eggs consecutively for more than 5 days in a relatively regular sequence were used for experiments. Ovulation time was estimated by oviposition time. Oviposition time was recorded by video camera and video recorder. Ova were recovered from the infundibulum or the upper magnum. Unlike mammals (i.e. mouse), only a single oocyte can be recovered from each female quail because of the difficulty of inducing multiple ovulation (Hrabia et al., 2003b). Under the inverted microscope a single sperm was isolated by a micro capillary pipette and was injected into the central area of the germinal disc using a micromanipulator connected to the injector under the stereomicroscope. The sperm-injected ova were cultured in plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) for 24 h at 41 °C under 5% CO2 in air (Fig. 1). The blastoderm was excised and development of the embryo were staged by Eyal-Giladi and Kochav (Eyal-Giladi and Kochav, 1976) under stereomicroscope. The blastoderm was fixed in ethanol/glacial acetic acid, embedded in paraffin, and sectioned vertically. Some eggs were assessed by whole mount tissue. The sections were stained either with 40 6 diamidino-2-phenylindole (DAPI) or with the Hematoxylin and Eosin (HE). DAPI-stained nuclei emitting intense blue fluorescence in fertilized oocytes were distinguishable by their round shape, size and location. Table 1 shows data on the fertilization and development of quail oocytes by ICSI and culture for 24 h in DMEM. Five out of 30 (16.6%) oocytes injected with mature sperm were fertilized and they developed at stage VI. The rate (16.6%) is very low, as was found in vitro fertilization with semen reported by Olszanska et al. (2002).
3. Improvement of fertility rate Recently, phospholipase Czeta (PLCzeta) has been isolated from spermatid as a novel isoform of PLC family has been cloned and its protein has been suggested as sperm-derived oocyte activation factor (Saunders et al., 2002). Accordingly, we studied effects of quail sperm extract (SE) and PLCzeta cRNA on ICSI-assisted fertilization in terms of pronuclear formation, and cytoplasmic segmentation or embryonic development. 3.1. Effects of SE and PLCzeta on pronuclear formation The sperm concentration was adjusted to 1 108 sperm/ml and the suspension was lysed by one freeze–thaw cycle in liquid nitrogen. Subsequently, the supernatant was recovered as quail SE and the protein concentration was adjusted to 2.0 mg protein/ml. Oocytes were placed in DMEM in a 6-well dish and injected into their germinal discs with about 3 nl of SE solution of increasing protein concentration (0.02, 0.07, 0.13, 0.2 and 2 mg/ml, equivalent to 3, 10, 20, 30, and 300 sperm/3 nl, respectively) diluted with Dulbecco’s phosphate-buffered saline (DPBS). The incubation procedure, which was a modification of System Q1a described by Ono (2001), was performed in a 20 ml plastic cup. Fig. 2 shows an example of oocytes with (A) and without pronuclear formation (metaphase II chromatin, B) after intracytoplasmic injection of 0.1 M calcium and buffer solution, respectively (Mizushima et al., 2009). Table 2 summarized the data. Between 0 (buffer) and 0.13 mg protein/ml of SE the rate of pronuclear formation increased in a dose-dependent manner (5.7–61.5%) and at the higher doses beyond 0.13 mg protein/ml, the rate remained substantially the same as with 0.13 mg protein/ml (59.3% and 63.6%). The injection more than 0.13 mg protein/ml increased rates of pronuclear formation significantly compared to those injected with 0.02 mg protein/ml, respectively. Treatment of SE at 90 °C for 10 min before microinjection completely blocked pronuclear formation in quail oocytes. On
Fig. 1. Isolation of a single sperm with a capillary under inverted microscopy (top 3) and under stereomicroscopy a sperm is injected into oocyte. Adopted from Hrabia et al. (2003a) (Biol Reprod).
Table 1 Development of quail oocytes spontaneously ovulated after ICSI and culture. No. of injected oocytes
30
No. of fertilized oocytes (%)
5 (16.6)
Adopted from Hrabia et al. (2003a) (Biol. Reprod.).
No. of embryos developing up to following stages: below I
II
III
IV
V
VI
0
1
0
1
2
1
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K. Shimada et al. / General and Comparative Endocrinology 196 (2014) 100–105
Fig. 2. Photographs of quail oocytes showing pronuclear formation (A) and metaphase II chromatin (B) after DAPI after microinjection of 0.1 M calcium and buffer solution, respectively, Scale bar = 20 lm. Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
Table 2 Pronuclear formation of quail oocytes 3 h after injection with quail sperm extract (SE) at different concentrations. Concentration of SE injected (mg/ml) /conditions
No. of oocytes injected
No. of oocytes showing pronuclear formation (%)*
Buffer 0.02 0.07 0.13 0.2 2.0 0.13/Heat** 0.13/BAPTA***
35 37 35 39 39 33 10 22
2 (5.7)a 8 (21.6)a,b 12 (34.3)b 24 (61.5)c 23 (59.0)c 21 (63.6)c 0 (0)a 1 (4.5)a
*
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009) ** Quail oocytes were injected with 0.13 mg protein/ml SE pre-heated at 90 °C for 10 min. *** Quail oocytes were pre-injected with BAPTA 15 min before microinjection of 0.13 mg protein/ml SE.
Table 3 Pronuclear formation of quail oocytes 3 h after injection with quail PLCzeta cRNA at different concentrations. Concentration of PLCzeta cRNA injected (lg/ml)/conditions
No. of oocytes injected
No. of oocytes showing pronuclear formation (%)*
Buffer 2.7 60 200 60/cycloheximide** 60/BAPTA***
30 25 23 21 20 12
1 (3.3)a 7 (28.0)b 15 (65.2)c 14 (66.7)c 0 (0)a 0 (0)a
*
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009) ** Quail oocytes were pre-loaded with 10 lM cycloheximide 30 min before microinjection of PLCzeta cRNA. *** Quail oocytes were pre-injected with BAPTA 15 min before microinjection of 60 lg/ml PLCzeta cRNA.
the other hand, the treatment with the Ca2+ chelator, 1,2-bis (o-aminophenoxy ethane-N,N,N0 ,N0 -tetraacetic acid, BAPTA), before injection of SE blocked pronuclear formation that could be induced at concentration of 0.13 mg protein/ml). Consequently, only one of 20 oocytes showed pronuclear formation. On the other hand, pronuclear formation was not induced by extracts from brain and liver. Table 3. The quail PLCzeta cDNA encoding the ORF was cloned and PLCzeta cRNA synthesized (Mizushima et al., 2009). PLCzeta cRNA solution of increasing RNA concentration (2.7, 60 and
200 lg/ml) was diluted with DPBS, or of tissue extract solution at concentration of 0.13 mg protein/ml diluted with DPBS. The injected 3 nl volume consisted approximately 0.3% of a germinal disc volume. The injection of PLCzeta cRNA increased the rate of pronuclear formation in a dose-dependent manner (3.3–65.2%, Table 3) and at the higher dose (200 lg/ml) the rate remained essentially the same as that observed at 60 lg/ml (66.7%). To further investigate whether pronuclear formation is dependent upon endogenous PLCzeta as the protein according to PLCzeta cRNA, quail oocytes were incubated with the protein synthesis inhibitor cycloheximide before microinjection of PLCzeta cRNA (at concentration of 60 lg/ml). The pre-incubation of 10 lM cycloheximide completely blocked pronuclear formation in quail oocytes after the microinjection of intact PLCzeta cRNA (at concentration of 60 lg/ml). To further evaluate whether PLCzeta cRNA-induced oocyte activation is related to a Ca2+ increase in the ooplasm, the oocytes were treated by BAPTA. Consequently, no oocytes showed pronuclear formation. 3.2. Cytoplasmic segmentation of quail oocytes injected with SE and PLCzeta cRNA 24 h after incubation Whether or not oocyte activation will continue further development after injection of SE and PLCzeta cRNA, parthenogenetic cytoplasmic segmentation was evaluated 24 h after injection in the different treatment groups as described previously (Hrabia et al., 2003a,b; Olszanska et al., 2002). Fig. 3 shows examples of cytoplasmic segmentation after injection of SE (at concentration of 0.13 mg protein/ml) and PLCzeta cRNA (at concentration of 60 lg/ml). In the activated oocytes, their cytoplasm showed normal cleavage assessed by stereomicroscopic observation (Fig. 3A–D), although no nuclei were seen after DAPI staining as demonstrated in the previous report (Olszanska et al., 2002). Oocytes that did not develop after injection of SE (at concentration of 0.13 mg protein/ml) were shown in Fig. 3E and F. The results were summarized in Table 4. When oocytes were injected with buffer solution, very few oocytes developed (5.0%). In contrast, when oocytes were injected with SE (at concentration of 0.13 mg protein/ml) or PLCzeta cRNA (at concentration of 60 lg/ ml), 25.0% or 36.4% of oocytes developed. The oocytes showed pseudoblastodermal development from stage II to V among these groups. 3.3. Effect of microinjection of round spermatid and PLCzeta cRNA When oocytes were injected with a round spermatid together with PLCzeta cRNA (at concentration of 60 lg/ml), blastodermal development with numerous distinct nuclei was evident. These developing oocytes showed a maximum of stage VI and the fertilization rate was 46.7% (Table 5). In contrast, when oocytes were injected with a round spermatid alone, no oocytes developed. Previously we demonstrated that elongated spermatid itself is capable of fertilizing oocyte as does sperm but round spermatid is not (Hrabia et al., 2003a,b). The RT-PCR assay showed that mRNA of PLCzeta is expressed in elongated spermatid but not in round spermatid (Mizushima et al., 2009). Our preliminary experiments repeatedly confirmed that the inositol trisphosphate (IP3) injection augmented the rate of ICSIassisted fertilization by two folds over that by PLCzeta (Mizushima et al., 2012). Overall results of our previous report indicate a possible pathway of oocyte activation in relation fertilization in quail oocyte as follows (Fig. 4). Namely, sperm binds to the oocyte membrane and diffuses PLCzeta which in turn catalyzes phosphatidylinositol biphosphate into IP3 and diacyl glyceride
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Fig. 4. Possible molecular pathway in the quail egg in relation to oocyte activation.
4. Gene transfer trial in quail 4.1. Novel method toward gene transfer in birds: intracytoplasmic sperm injection (ICSI) for green fluorescent protein (GFP) expression in quail blastoderms
Fig. 3. Cytoplasmic segmentation of oocytes 24 h after microinjection of 0.13 mg protein/ml SE (pseudoblastoderm stage III in A,B and undeveloped blastodisc in E,F) and of oocytes injected with PLCzeta cRNA (pseudoblastoderm stage IV in C,D). (A,C,E) under stereomicroscope and (B,D,F) after DAPI staining. V, vacuoles; arrow indicates fragmented DNA body; arrowheads indicate pseudoblastomere. Scale bar = 1.0 mm (A,C,E); 20 lm (B,D,F). Adopted from Mol. Reprod. Dev. (Mizushima et al., 2009).
and then IP3 binds to its receptor (IP3R) in the endoplasmic reticulum triggering Ca2+ release. Ca2+ release is involved in resumption of meiosis.
Among different methods of gene transfer in birds, the major includes direct DNA injection, viral transfection, testis-mediated system and chimeric approaches. A direct injection of DNA is conducted by injected DNA into the germinal disc of the fertilized oocyte at the single-cell stage (Love et al., 1994; Naito et al., 1994; Ono et al., 1994; Sherman et al., 1998). However, injection of DNA into male pronucleus is difficult due to the opaque cytoplasm of the egg yolk, resulting low efficiency of integration of foreign DNA into chromosomes. Viral infection method is meritorious for relatively high efficiency for incorporation of the transgene, but it may be risky to introduce a potentially hazardous viral infection (Han, 2009; Naito, 2003). Testis-mediated system using spermatogonial stem cells is performed by the transplantation of testicular cells into the seminiferous tubules of heterologous recipients, which yielded around 8% chimeric efficiency (Han, 2009; Lee et al., 2006). And storage of ejaculated semen and spermatogonial cells is developing (Blesbois, 2012; Trefil et al., 2012), but these techniques have not yet applied for transgenic chicken production. Chimeric approach is to introduce a foreign gene into blastodermal cells (Etches et al., 1997), primordial germ cells (PGCs) (Vick et al., 1993; Zhu et al., 2005) or embryonic stem cells (Vick et al., 1993) and these cells are transplanted into another recipient eggs at early embryonic stage. Recently, successful production of germline chimeara using PGCs has been reported, but the efficiency of gene transduction is highly variable (van de Lavoir et al., 2006). Here the
Table 4 Parthenogenetic cytoplasmic segmentation of quail oocytes 24 h after injection with either 0.13 mg/ml SE or 60 lg/ml PLCf cRNA. Sample
SE Buffer 0.13 mg/ml SE RNA buffer 60 lg/ml PLCf cRNA
No. of oocytes injected
20 24 21 22
No. of oocytes developed Parthenogenetically (%)
1 6 1 8
No. of oocytes developing up to following stages
(5.0) (25.0) (4.8) (36.4)
II
III
IV
V
0 0 0 1
0 2 0 0
1 3 1 5
0 1 0 2
Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
Table 5 Blastodermal development of quail oocytes 24 h after microinjection of a round spermatid with PLCf cRNA. Concentration of RNA (lg/ml)
No. of oocytes injected
No. of oocytes developed (%)
0 60
17 15
0 (0)a 7 (46.7)b
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
No. of oocytes developing up to following stages IV
V
VI
0 1
0 3
0 3
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Table 6 Blastoderm development of quail oocytes 24 h after simultaneous injection of PLCf cRNA and sperm pretreated with TX-100. Sperm treatment
No. of oocytes
NIM (control) TX-100
No. of embryos developed to the stage of
Injected
Developed (%)
III
IV
V
VI
14 23
6 (42.9) 10 (43.5)
0 1
2 3
1 2
3 4
NIM, nuclear isolation medium. Adopted from Mizushima et al. (2010) (Biol Reprod).
Table 7 Blastoderm development of quail oocytes at 24 h after microinjection of a sperm incubated with GFP vector and PLCf cRNA simultaneously. Sperm treatment
No. of oocytes Injected
NIM (control) TX-100
10 16
No. of embryos Developed (%)
4 (40.0) 7 (43.8)
Expressing GFP (%)
0 (0) 6 (85.7)
Developed to the stage of III
IV
V
VI
0 1
2 3
1 2
3 4
NIM, nuclear isolation medium. Adopted from Mizushima et al. (2010) (Biol Reprod).
ICSI method is employed for a new approach for gene transduction assuming the sperm injected fertilizes and the DNA is incorporated chromosomally. When sperm is treated with Triton X-100 (TX-100), the sperm membranes damaged. TX-100-treated sperm was injected into a quail oocyte. It was subsequently cultured for 24 h, but no oocytes developed to blastodermal stages. In contrast, microinjection of TX-100-treated sperm together with PLCzeta cRNA induced blastoderm development at stage V. Numerous nuclei were observed in the blastoderm, indicating active cell division. When a single sperm was injected into oocytes either without or with TX-100 pretreatment, 43% of blastoderm developed up to stage VI after 24 h of culture (Table 6) (Mizushima et al., 2010). For evaluation of ICSI-produced GFP-expressing embryos, blastoderm development 24 h after multiple injections of EGFP vector, a TX-100 treated-sperm and PLCzeta cRNA is observed under a stereomicroscope by fluorescent stereomicroscope and after DAPI staining. Emission of green fluorescent light was detected in numerous blastomeres. As shown in Table 7, when oocytes were injected with a single sperm incubated with EGFP vector without TX-100 pretreatment, 4 out of 10 oocytes (40.0%) showed blastoderm development between stages from IV to VI, but none of them expressed the fluorescent green protein. In contrast, when oocytes were injected with a single sperm incubated with EGFP vector with TX-100 pretreatment, 7 out of 16 oocytes (43.8%) showed blastoderm development between stages III to VI, and 6 of those 7 blastoderms expressed the fluorescent green protein (85.7%). Furthermore, the PCR result shows EGFP fragments obtained from GFP-expressing blastoderms and the bands of EGFP at the expected size (about 730 bp) in the DNA of 3 of 6 quail blastoderms. It is worthy of noting that disruption of quail sperm membrane prior to incubation of sperm with an exogenous gene and before co-injection of PLCzeta cRNA results in high concentration of the transgene in the oocytes and PLCzeta is highly effective in assisting the fertilizability of TX-100-treated sperm. Chromosomal integration of the genes and development beyond the blastodermal stages up to hatching, are always important for production of transgenesis. More recently, cryopreservation and in vitro culture techniques of PGCs in blastmderm (Glover and McGrew, 2012) and a more simplified ex vivo culture protocol for avian embryos from the blastoderm stage through to hatching using a single surrogate eggshell have been well developed (Kato et al., 2013a,b). These techniques could provide helpful toward solution for this subject in future.
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General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen
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Application of intracytoplasmic sperm injection (ICSI) for fertilization and development in birds Kiyoshi Shimada a,⇑, Tamao Ono b, Shusei Mizushima c a
WCU, Major in Biomodulation, College of Agriculture and Life Sciences, Seoul National University, 599 Gwanak-ro, Gwanak-gu, Seoul 151-921, South Korea Faculty of Agriculture, Shinshu University, Kamiina, Nagano 399-4598, Japan c Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Ohya, Shizuoka 422-8529, Japan b
a r t i c l e
i n f o
Article history: Received 29 October 2012 Revised 22 October 2013 Accepted 1 November 2013 Available online 13 November 2013 Keywords: GFP-expressing blastoderm ICSI Inositol trisphosphate PLCzeta Quail oocyte Transgene
a b s t r a c t Intracytoplasmic sperm injection (ICSI) technology in birds has been hampered due to opacity of oocyte. We developed ICSI-assisted fertilization and gene transfer in quail. This paper reviews recent advances of our ICSI experiments. The oocyte retrieved from the oviduct and a quail sperm was injected into the oocyte under a stereomicroscope. The oocyte was cultured for 24 h at 41 °C under 5% CO2 in air. The fertilization and development was assessed by microscopic observation. The fertility rate ranged 12–18% and development varied from stage II to V in trials. To improve the fertility rate, phospholipase C (PLC) zeta was injected with a sperm. It was increased to 37–50%. Furthermore, injection of inositol trisphosphate increased to over 85%. Quail oocyte can be fertilized with chicken sperm and so can testicular elongated spermatid. To extend embryonic development, chicken eggshell was used as a surrogate culture at 37 °C after the 24 h incubation at 41 °C under 5% CO2 in air. It survived up to 2 days thereafter. Finally, gene transfer was attempted in quail egg. The sperm membrane was disrupted with Triton X-100 (TX100) and was injected with PLCzeta cRNA and enhanced green fluorescent protein (EGFP) gene in oocyte. The GFP expression was evaluated at 24 h incubation at 41 °C under 5% CO2 in air in the embryos. While the expression was not detected in the control oocytes, the experimental treatment induced blastoderm development (44%) of the oocytes and 86% of blastoderm showed fluorescent emission. In addition, PCR analysis detected EGFP fragments in 50% of GFP-expressing blastoderm. Our ICSI method may be the first step toward the production of transgenic birds. Ó 2013 Elsevier Inc. All rights reserved.
1. Introduction Intracytoplasmic sperm injection (ICSI) has now been well recognized as the excellent assisted-reproductive technology in mammals and made a great contribution to better understanding of fundamental gamete biology as well as application in animal biotechnology. It is also employed for protection of endangered species of animals and for clinical medicine in humans. Nevertheless, in birds, ICSI has not been attempted until recently (Hrabia et al., 2003a,b; Mizushima, 2012). Since artificial insemination has been employed in poultry industries widely, it has been thought the ICSI might not have much advantage in practical term. Furthermore, opacity of the avian oocyte is a big barrier for sperm injection into the oocyte. However, once this methodology is established, it seems promising to contribute to studies of fertility mechanism as well as biotechnology application in birds. In physiological states, polyspermic fertilization takes place at the infundibulum soon after ovulation, although only one sperm ⇑ Corresponding author. Fax: +82 52 804 7257. E-mail address: [email protected] (K. Shimada). 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.11.001
successfully fertilizes oocyte. The yolk including oocyte moves along the magnum where albumen is secreted, and the isthmus where shell-membrane is formed, and stayed in the shell gland where shell is formed for about 20 h. Meanwhile, the first cell division takes place when the egg enters at the shell gland, and subsequent cell divisions follow. By the time of oviposition the oocyte consists of more than 4–6 104 cells. Polyspermy is unique phenomenon in birds as observed in some reptiles, some amphibians and most urodetes (newts and salamanders). The significance of polyspermy remains unknown, but application of the ICSI method revealed that even a single sperm is capable of fertilizing oocyte in quail (Hrabia et al., 2003a,b), although more sperm can cause higher rate of fertility (Mizushima et al., 2008). Conventional artificial insemination (AI) has been conducted in poultry industry or research farms in many domestic fowls. Usually the semen is collected by massage of the abdominal area of males, dilutes it and is injected into vagina. The AI using cryo-preserved semen is not popular due to inconsistent results of fertility rate and hatchability. Experimentally, in vitro fertilization has been conducted in some studies, culture of the embryo is limited within a day. We have developed ICSI technology in quail and provided
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several new findings on oocyte activation mechanism, and a new trial of gene transfer. Here, we describe our ICSI methodology and our outputs. 2. Method of development of ICSI-assisted fertilization Male and female Japanese quail (Coturnix japonica) were kept in an environmentally controlled room. Female quail laying eggs consecutively for more than 5 days in a relatively regular sequence were used for experiments. Ovulation time was estimated by oviposition time. Oviposition time was recorded by video camera and video recorder. Ova were recovered from the infundibulum or the upper magnum. Unlike mammals (i.e. mouse), only a single oocyte can be recovered from each female quail because of the difficulty of inducing multiple ovulation (Hrabia et al., 2003b). Under the inverted microscope a single sperm was isolated by a micro capillary pipette and was injected into the central area of the germinal disc using a micromanipulator connected to the injector under the stereomicroscope. The sperm-injected ova were cultured in plastic dishes in Dulbecco’s modified Eagle’s medium (DMEM) for 24 h at 41 °C under 5% CO2 in air (Fig. 1). The blastoderm was excised and development of the embryo were staged by Eyal-Giladi and Kochav (Eyal-Giladi and Kochav, 1976) under stereomicroscope. The blastoderm was fixed in ethanol/glacial acetic acid, embedded in paraffin, and sectioned vertically. Some eggs were assessed by whole mount tissue. The sections were stained either with 40 6 diamidino-2-phenylindole (DAPI) or with the Hematoxylin and Eosin (HE). DAPI-stained nuclei emitting intense blue fluorescence in fertilized oocytes were distinguishable by their round shape, size and location. Table 1 shows data on the fertilization and development of quail oocytes by ICSI and culture for 24 h in DMEM. Five out of 30 (16.6%) oocytes injected with mature sperm were fertilized and they developed at stage VI. The rate (16.6%) is very low, as was found in vitro fertilization with semen reported by Olszanska et al. (2002).
3. Improvement of fertility rate Recently, phospholipase Czeta (PLCzeta) has been isolated from spermatid as a novel isoform of PLC family has been cloned and its protein has been suggested as sperm-derived oocyte activation factor (Saunders et al., 2002). Accordingly, we studied effects of quail sperm extract (SE) and PLCzeta cRNA on ICSI-assisted fertilization in terms of pronuclear formation, and cytoplasmic segmentation or embryonic development. 3.1. Effects of SE and PLCzeta on pronuclear formation The sperm concentration was adjusted to 1 108 sperm/ml and the suspension was lysed by one freeze–thaw cycle in liquid nitrogen. Subsequently, the supernatant was recovered as quail SE and the protein concentration was adjusted to 2.0 mg protein/ml. Oocytes were placed in DMEM in a 6-well dish and injected into their germinal discs with about 3 nl of SE solution of increasing protein concentration (0.02, 0.07, 0.13, 0.2 and 2 mg/ml, equivalent to 3, 10, 20, 30, and 300 sperm/3 nl, respectively) diluted with Dulbecco’s phosphate-buffered saline (DPBS). The incubation procedure, which was a modification of System Q1a described by Ono (2001), was performed in a 20 ml plastic cup. Fig. 2 shows an example of oocytes with (A) and without pronuclear formation (metaphase II chromatin, B) after intracytoplasmic injection of 0.1 M calcium and buffer solution, respectively (Mizushima et al., 2009). Table 2 summarized the data. Between 0 (buffer) and 0.13 mg protein/ml of SE the rate of pronuclear formation increased in a dose-dependent manner (5.7–61.5%) and at the higher doses beyond 0.13 mg protein/ml, the rate remained substantially the same as with 0.13 mg protein/ml (59.3% and 63.6%). The injection more than 0.13 mg protein/ml increased rates of pronuclear formation significantly compared to those injected with 0.02 mg protein/ml, respectively. Treatment of SE at 90 °C for 10 min before microinjection completely blocked pronuclear formation in quail oocytes. On
Fig. 1. Isolation of a single sperm with a capillary under inverted microscopy (top 3) and under stereomicroscopy a sperm is injected into oocyte. Adopted from Hrabia et al. (2003a) (Biol Reprod).
Table 1 Development of quail oocytes spontaneously ovulated after ICSI and culture. No. of injected oocytes
30
No. of fertilized oocytes (%)
5 (16.6)
Adopted from Hrabia et al. (2003a) (Biol. Reprod.).
No. of embryos developing up to following stages: below I
II
III
IV
V
VI
0
1
0
1
2
1
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Fig. 2. Photographs of quail oocytes showing pronuclear formation (A) and metaphase II chromatin (B) after DAPI after microinjection of 0.1 M calcium and buffer solution, respectively, Scale bar = 20 lm. Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
Table 2 Pronuclear formation of quail oocytes 3 h after injection with quail sperm extract (SE) at different concentrations. Concentration of SE injected (mg/ml) /conditions
No. of oocytes injected
No. of oocytes showing pronuclear formation (%)*
Buffer 0.02 0.07 0.13 0.2 2.0 0.13/Heat** 0.13/BAPTA***
35 37 35 39 39 33 10 22
2 (5.7)a 8 (21.6)a,b 12 (34.3)b 24 (61.5)c 23 (59.0)c 21 (63.6)c 0 (0)a 1 (4.5)a
*
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009) ** Quail oocytes were injected with 0.13 mg protein/ml SE pre-heated at 90 °C for 10 min. *** Quail oocytes were pre-injected with BAPTA 15 min before microinjection of 0.13 mg protein/ml SE.
Table 3 Pronuclear formation of quail oocytes 3 h after injection with quail PLCzeta cRNA at different concentrations. Concentration of PLCzeta cRNA injected (lg/ml)/conditions
No. of oocytes injected
No. of oocytes showing pronuclear formation (%)*
Buffer 2.7 60 200 60/cycloheximide** 60/BAPTA***
30 25 23 21 20 12
1 (3.3)a 7 (28.0)b 15 (65.2)c 14 (66.7)c 0 (0)a 0 (0)a
*
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009) ** Quail oocytes were pre-loaded with 10 lM cycloheximide 30 min before microinjection of PLCzeta cRNA. *** Quail oocytes were pre-injected with BAPTA 15 min before microinjection of 60 lg/ml PLCzeta cRNA.
the other hand, the treatment with the Ca2+ chelator, 1,2-bis (o-aminophenoxy ethane-N,N,N0 ,N0 -tetraacetic acid, BAPTA), before injection of SE blocked pronuclear formation that could be induced at concentration of 0.13 mg protein/ml). Consequently, only one of 20 oocytes showed pronuclear formation. On the other hand, pronuclear formation was not induced by extracts from brain and liver. Table 3. The quail PLCzeta cDNA encoding the ORF was cloned and PLCzeta cRNA synthesized (Mizushima et al., 2009). PLCzeta cRNA solution of increasing RNA concentration (2.7, 60 and
200 lg/ml) was diluted with DPBS, or of tissue extract solution at concentration of 0.13 mg protein/ml diluted with DPBS. The injected 3 nl volume consisted approximately 0.3% of a germinal disc volume. The injection of PLCzeta cRNA increased the rate of pronuclear formation in a dose-dependent manner (3.3–65.2%, Table 3) and at the higher dose (200 lg/ml) the rate remained essentially the same as that observed at 60 lg/ml (66.7%). To further investigate whether pronuclear formation is dependent upon endogenous PLCzeta as the protein according to PLCzeta cRNA, quail oocytes were incubated with the protein synthesis inhibitor cycloheximide before microinjection of PLCzeta cRNA (at concentration of 60 lg/ml). The pre-incubation of 10 lM cycloheximide completely blocked pronuclear formation in quail oocytes after the microinjection of intact PLCzeta cRNA (at concentration of 60 lg/ml). To further evaluate whether PLCzeta cRNA-induced oocyte activation is related to a Ca2+ increase in the ooplasm, the oocytes were treated by BAPTA. Consequently, no oocytes showed pronuclear formation. 3.2. Cytoplasmic segmentation of quail oocytes injected with SE and PLCzeta cRNA 24 h after incubation Whether or not oocyte activation will continue further development after injection of SE and PLCzeta cRNA, parthenogenetic cytoplasmic segmentation was evaluated 24 h after injection in the different treatment groups as described previously (Hrabia et al., 2003a,b; Olszanska et al., 2002). Fig. 3 shows examples of cytoplasmic segmentation after injection of SE (at concentration of 0.13 mg protein/ml) and PLCzeta cRNA (at concentration of 60 lg/ml). In the activated oocytes, their cytoplasm showed normal cleavage assessed by stereomicroscopic observation (Fig. 3A–D), although no nuclei were seen after DAPI staining as demonstrated in the previous report (Olszanska et al., 2002). Oocytes that did not develop after injection of SE (at concentration of 0.13 mg protein/ml) were shown in Fig. 3E and F. The results were summarized in Table 4. When oocytes were injected with buffer solution, very few oocytes developed (5.0%). In contrast, when oocytes were injected with SE (at concentration of 0.13 mg protein/ml) or PLCzeta cRNA (at concentration of 60 lg/ ml), 25.0% or 36.4% of oocytes developed. The oocytes showed pseudoblastodermal development from stage II to V among these groups. 3.3. Effect of microinjection of round spermatid and PLCzeta cRNA When oocytes were injected with a round spermatid together with PLCzeta cRNA (at concentration of 60 lg/ml), blastodermal development with numerous distinct nuclei was evident. These developing oocytes showed a maximum of stage VI and the fertilization rate was 46.7% (Table 5). In contrast, when oocytes were injected with a round spermatid alone, no oocytes developed. Previously we demonstrated that elongated spermatid itself is capable of fertilizing oocyte as does sperm but round spermatid is not (Hrabia et al., 2003a,b). The RT-PCR assay showed that mRNA of PLCzeta is expressed in elongated spermatid but not in round spermatid (Mizushima et al., 2009). Our preliminary experiments repeatedly confirmed that the inositol trisphosphate (IP3) injection augmented the rate of ICSIassisted fertilization by two folds over that by PLCzeta (Mizushima et al., 2012). Overall results of our previous report indicate a possible pathway of oocyte activation in relation fertilization in quail oocyte as follows (Fig. 4). Namely, sperm binds to the oocyte membrane and diffuses PLCzeta which in turn catalyzes phosphatidylinositol biphosphate into IP3 and diacyl glyceride
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Fig. 4. Possible molecular pathway in the quail egg in relation to oocyte activation.
4. Gene transfer trial in quail 4.1. Novel method toward gene transfer in birds: intracytoplasmic sperm injection (ICSI) for green fluorescent protein (GFP) expression in quail blastoderms
Fig. 3. Cytoplasmic segmentation of oocytes 24 h after microinjection of 0.13 mg protein/ml SE (pseudoblastoderm stage III in A,B and undeveloped blastodisc in E,F) and of oocytes injected with PLCzeta cRNA (pseudoblastoderm stage IV in C,D). (A,C,E) under stereomicroscope and (B,D,F) after DAPI staining. V, vacuoles; arrow indicates fragmented DNA body; arrowheads indicate pseudoblastomere. Scale bar = 1.0 mm (A,C,E); 20 lm (B,D,F). Adopted from Mol. Reprod. Dev. (Mizushima et al., 2009).
and then IP3 binds to its receptor (IP3R) in the endoplasmic reticulum triggering Ca2+ release. Ca2+ release is involved in resumption of meiosis.
Among different methods of gene transfer in birds, the major includes direct DNA injection, viral transfection, testis-mediated system and chimeric approaches. A direct injection of DNA is conducted by injected DNA into the germinal disc of the fertilized oocyte at the single-cell stage (Love et al., 1994; Naito et al., 1994; Ono et al., 1994; Sherman et al., 1998). However, injection of DNA into male pronucleus is difficult due to the opaque cytoplasm of the egg yolk, resulting low efficiency of integration of foreign DNA into chromosomes. Viral infection method is meritorious for relatively high efficiency for incorporation of the transgene, but it may be risky to introduce a potentially hazardous viral infection (Han, 2009; Naito, 2003). Testis-mediated system using spermatogonial stem cells is performed by the transplantation of testicular cells into the seminiferous tubules of heterologous recipients, which yielded around 8% chimeric efficiency (Han, 2009; Lee et al., 2006). And storage of ejaculated semen and spermatogonial cells is developing (Blesbois, 2012; Trefil et al., 2012), but these techniques have not yet applied for transgenic chicken production. Chimeric approach is to introduce a foreign gene into blastodermal cells (Etches et al., 1997), primordial germ cells (PGCs) (Vick et al., 1993; Zhu et al., 2005) or embryonic stem cells (Vick et al., 1993) and these cells are transplanted into another recipient eggs at early embryonic stage. Recently, successful production of germline chimeara using PGCs has been reported, but the efficiency of gene transduction is highly variable (van de Lavoir et al., 2006). Here the
Table 4 Parthenogenetic cytoplasmic segmentation of quail oocytes 24 h after injection with either 0.13 mg/ml SE or 60 lg/ml PLCf cRNA. Sample
SE Buffer 0.13 mg/ml SE RNA buffer 60 lg/ml PLCf cRNA
No. of oocytes injected
20 24 21 22
No. of oocytes developed Parthenogenetically (%)
1 6 1 8
No. of oocytes developing up to following stages
(5.0) (25.0) (4.8) (36.4)
II
III
IV
V
0 0 0 1
0 2 0 0
1 3 1 5
0 1 0 2
Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
Table 5 Blastodermal development of quail oocytes 24 h after microinjection of a round spermatid with PLCf cRNA. Concentration of RNA (lg/ml)
No. of oocytes injected
No. of oocytes developed (%)
0 60
17 15
0 (0)a 7 (46.7)b
Different superscript letters within column denote significant difference (P < 0.05). Taken from Mol. Reprod. Dev. (Mizushima et al., 2009).
No. of oocytes developing up to following stages IV
V
VI
0 1
0 3
0 3
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Table 6 Blastoderm development of quail oocytes 24 h after simultaneous injection of PLCf cRNA and sperm pretreated with TX-100. Sperm treatment
No. of oocytes
NIM (control) TX-100
No. of embryos developed to the stage of
Injected
Developed (%)
III
IV
V
VI
14 23
6 (42.9) 10 (43.5)
0 1
2 3
1 2
3 4
NIM, nuclear isolation medium. Adopted from Mizushima et al. (2010) (Biol Reprod).
Table 7 Blastoderm development of quail oocytes at 24 h after microinjection of a sperm incubated with GFP vector and PLCf cRNA simultaneously. Sperm treatment
No. of oocytes Injected
NIM (control) TX-100
10 16
No. of embryos Developed (%)
4 (40.0) 7 (43.8)
Expressing GFP (%)
0 (0) 6 (85.7)
Developed to the stage of III
IV
V
VI
0 1
2 3
1 2
3 4
NIM, nuclear isolation medium. Adopted from Mizushima et al. (2010) (Biol Reprod).
ICSI method is employed for a new approach for gene transduction assuming the sperm injected fertilizes and the DNA is incorporated chromosomally. When sperm is treated with Triton X-100 (TX-100), the sperm membranes damaged. TX-100-treated sperm was injected into a quail oocyte. It was subsequently cultured for 24 h, but no oocytes developed to blastodermal stages. In contrast, microinjection of TX-100-treated sperm together with PLCzeta cRNA induced blastoderm development at stage V. Numerous nuclei were observed in the blastoderm, indicating active cell division. When a single sperm was injected into oocytes either without or with TX-100 pretreatment, 43% of blastoderm developed up to stage VI after 24 h of culture (Table 6) (Mizushima et al., 2010). For evaluation of ICSI-produced GFP-expressing embryos, blastoderm development 24 h after multiple injections of EGFP vector, a TX-100 treated-sperm and PLCzeta cRNA is observed under a stereomicroscope by fluorescent stereomicroscope and after DAPI staining. Emission of green fluorescent light was detected in numerous blastomeres. As shown in Table 7, when oocytes were injected with a single sperm incubated with EGFP vector without TX-100 pretreatment, 4 out of 10 oocytes (40.0%) showed blastoderm development between stages from IV to VI, but none of them expressed the fluorescent green protein. In contrast, when oocytes were injected with a single sperm incubated with EGFP vector with TX-100 pretreatment, 7 out of 16 oocytes (43.8%) showed blastoderm development between stages III to VI, and 6 of those 7 blastoderms expressed the fluorescent green protein (85.7%). Furthermore, the PCR result shows EGFP fragments obtained from GFP-expressing blastoderms and the bands of EGFP at the expected size (about 730 bp) in the DNA of 3 of 6 quail blastoderms. It is worthy of noting that disruption of quail sperm membrane prior to incubation of sperm with an exogenous gene and before co-injection of PLCzeta cRNA results in high concentration of the transgene in the oocytes and PLCzeta is highly effective in assisting the fertilizability of TX-100-treated sperm. Chromosomal integration of the genes and development beyond the blastodermal stages up to hatching, are always important for production of transgenesis. More recently, cryopreservation and in vitro culture techniques of PGCs in blastmderm (Glover and McGrew, 2012) and a more simplified ex vivo culture protocol for avian embryos from the blastoderm stage through to hatching using a single surrogate eggshell have been well developed (Kato et al., 2013a,b). These techniques could provide helpful toward solution for this subject in future.
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