Molecular and biological aspects of early germ cell development in interspecies hybrids between chickens and pheasants

Available online at www.sciencedirect.com

Theriogenology 75 (2011) 696 –706 www.theriojournal.com

Molecular and biological aspects of early germ cell development in interspecies hybrids between chickens and pheasants Seok Jin Kanga,b, Sea Hwan Sohnc, Kyung Soo Kanga, Hyung Chul Leea, Seul Ki Leed, Jin Won Choia,b, Jae Yong Hana,* a

WCU Biomodulation Major, Department of Agricultural Biotechnology, Seoul National University, Seoul 151-921, Korea b Research Institute for Agriculture and Life Sciences, Seoul National University, Seoul 151-921, Korea c Department of Animal Science and Biotechnology, Jinju National University, Jinju 660-758, Korea d Avicore Biotechnology Institute, Optifarm Solution Inc., Hanlim Human Tower #707, Gyeonggi-do, 435-050, Korea Received 2 July 2010; received in revised form 11 October 2010; accepted 11 October 2010

Abstract Interspecific hybrids provide insights into fundamental genetic principles, and may prove useful for biotechnological applications and as tools for the conservation of endangered species. In the present study, interspecies hybrids were generated between the Korean ring-necked pheasant (Phasianus colchicus) and the White Leghorn chicken (Gallus gallus domesticus). We determined whether these hybrids were good recipients for the production of germline chimeric birds. PCR-based species-specific amplification and karyotype analyses showed that the hybrids inherited genetic material from both parents. Evaluation of biological function indicated that the growth rates of hybrids during the exponential phase (body weight/week) were similar to those of the pheasant but not the chicken, and that the incubation period for hatching was significantly different from that of both parents. Primordial germ cells (PGCs) of hybrids reacted with a pheasant PGC-specific antibody and circulated normally in blood vessels. The peak time of hybrid PGC migration was equivalent to that of the pheasant. In late embryonic stages, germ cells were detected by the QCR1 antibody on 15 d male gonads and were normally localized in the seminiferous cords. We examined the migration ability and developmental localization of exogenous PGCs transferred into the blood vessels of 63 h hybrid embryos. Donor-derived PGCs reacted with a donor-specific antibody were detected on 7 d gonads and the seminiferous tubules of hatchlings. Therefore, germ cell transfer into developing embryos of an interspecies hybrid can be efficiently used for the conservation of threatened animals and endangered species, and many biotechnological applications. © 2011 Elsevier Inc. All rights reserved. Keywords: Interspecies hybrid; Primordial germ cell (PGC); Pheasant; Chicken; Germline Chimera

1. Introduction Interspecific hybrids are bred by mating two species: they typically display intermediate traits and character-

* Corresponding author. Tel.: ⫹82-2-880-4810; fax: ⫹82-2-8744811. E-mail address: [email protected] (J.Y. Han). 0093-691X/$ – see front matter © 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.theriogenology.2010.10.010

istics of both parent species. In domestic poultry, a variety of hybrids have been obtained between chickens and other types of fowl, such as turkeys [1], quails [2] and pheasants [3,4]. The hybrids have been used for comparative studies between parental species and their hybrid progenies, and to investigate the mechanisms underlying the development and differentiation of germ cells. The production of fertile interspecific hybrids

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

may be limited by differences in chromosome number, which can make female hybrids poorly fertile and male hybrids sterile. In these cases, normal gametogenesis (production of spermatozoa and oocytes/eggs) cannot occur in the germline tissues due to chromosome dissimilarity [3,5], hormonal imbalance [4] and other biological and physiochemical differences such as sperm abundance, motility and morphology [6]. In previous studies, we successfully produced germline chimeras by intraspecific transfer of primordial germ cells (PGCs) in chickens [7,8] and quails [9,10], and interspecific transfer of PGCs between pheasants and chickens [11]. Technical progress has been made to improve the rates of germline transmission, but transmission efficiency still remains low. Several approaches have been taken to enhance the germline transmission rate including enrichment of gonadal migration of donor-derived PGCs, depletion of endogenous germ cells in the recipient, and the use of sterile recipients [12–16]. In transgenic mice, the germline transmission rate was elevated by the use of sterile recipients depleted of endogenous germ cells [17]. Likewise, in an avian system, interspecific hybrids could enhance the germline transmission due to their sterility; however, little is known about the biological characteristics of recipient hybrids and the development of transferred donor-derived germ cells. In the present study, we examined the genetic and physiological traits of interspecific hybrids and evaluated germ cell development in hybrid embryos. Interspecific hybrids exhibited genotypic and phenotypic characteristics from both parents, and their germ cells showed a normal pattern of development in the embryo. The study of hybrids as sterile hosts in germ cellmediated technology will help increase the production efficiency of donor-derived progeny in transgenesis and species conservation. It will also contribute to the un-

697

derstanding of the mechanisms of germ cell and early developmental biology. 2. Materials and methods 2.1. Experimental animals and animal care Korean ring-necked pheasants (Phasianus colchicus) and White Leghorn chickens (Gallus gallus domesticus) were maintained under the standard management program at the University Animal Farm, Seoul National University, Korea. The procedures for animal management, reproduction and embryo manipulation followed the standard operating protocols of our laboratory. 2.2. Production of interspecific hybrids To generate chicken-pheasant hybrids, semen collected from sexually mature male chickens was used for the artificial insemination of female pheasants. Hybrid hatchlings from pheasant eggs were maintained under our standard management program. 2.3. Hybrid detection and characterization using genetic markers Species-specific primers were developed to characterize the genotype of the hybrids (Table 1). Pheasantspecific primers were designed based on sequence differences in CYTB, TAPBP, and IGLC between both pheasants and chickens [18]; IGLC was cloned via 3=-rapid amplification of cDNA ends using two chicken variable domain framework region-specific primers [gene specific primer 1 (GSP1), 5=-CCT GGC AGT GCC CCT GTC A C-3=; GSP2, 5=-CAC ATT AAC CAT CAC TGG GGT CC-3=]. The sequences at the 3=-end matched the pheasant sequences, but not those of the chicken. The chicken-specific primers were CSP#1 [19] and AS3554-I9 [20].

Table 1 Sequences of primers used for PCR. Gene CYTB IGLC TAPBP CSP#1 AS3554-I9

Sequence (5=-3=): forward and reverse

Product size (bp)

GenBank accession no.

CAC ACA TGT CGA AAT GTG CAG CTC ATG GAA GGA CAT ATC CTA CG ACC ATC AAA GGA GGA GCT GGA A GGT GCT GTG GTC TCG CCA CT GGG ACA CAG TGA TGG ACA GC GTA GAG CCA ACG GAT GAG GC GAG TGT AGA CAG TAG TGT ATC CTC AGG GCA CCA TTT TCA CTG AGC AGC GGC GAT GAG CGG TG CTG CCT CAA CGT CTC GTT GGC

205

AY368060.1 AF354171 Cloned in our lab.

120 230 363

AL004999 AJ972781 D85614

222

AY636126

698

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

2.4. Karyotype analysis Hybrid blood samples were collected for karotyping. Whole blood cells were cultivated in RPMI-1640 medium (Invitrogen, Carlsbad, CA, 90%) supplemented with penicillin and streptomycin (Invitrogen, 0.1%), phytohemagglutinin (Invitrogen, 2%) and fetal bovine serum (Hyclone, Logan, UT, 10%) for 72 h; 0.1 mL colcemid (Sigma-Aldrich, St. Louis, MO, 10 ␮g/mL) was added and cells were incubated for 45 to 50 min. Cultured-blood cells were harvested, treated with 0.06 M KCl (hypotonic solution) for 15 min at room temperature, and fixed with acetic acid and methanol (1:3). The fixed cells were dropped onto a slide, dried on a plate warmer for 1 to 2 h, and stained with 4% Giemsa solution for 5 min. 2.5. Immunochemical analysis of endogenous germ cells To estimate the number of circulating PGCs (cPGCs) in hybrid embryos, blood was collected at three time points during incubation (58, 63, and 68 h); blood samples from embryos of the parent species were collected as controls (48, 53, 58, and 63 h of incubation for chickens; 58, 63, 68, and 73 h of incubation for pheasants). Technically, it is very difficult to collect blood from 48 and 53 h-incubated hybrid embryos and pheasant embryos because embryonic blood vessel is not fully developed at these stages. Then 1 ⫻ 105 blood cells were seeded on 96-well culture dishes, incubated for 3 h, and fixed in 4% paraformaldehyde (PFA). Immunocytochemical analysis with QCR1 and antiSSEA-1 antibodies was used to identify hybrid and pheasant, and chicken-derived cells, respectively; the numbers of antibody-positive cells were counted. In addition, embryonic gonads from hybrid, pheasant and chicken were immunostained with the PGC-specific antibodies. Briefly, whole gonads retrieved from 7 d embryos were fixed in PFA and incubated with QCR1 [mouse IgG isotype, diluted 1:100 in 1⫻ phosphatebuffered saline (PBS), 0.1% Triton X, 5% bovine serum albumin] or anti-SSEA-1 (Santa Cruz Biotechnology, Santa Cruz, CA, mouse IgM isotype, diluted 1:200) antibodies overnight. Samples were then incubated with fluorescein isothiocyanate (FITC)-conjugated goat anti-mouse IgG (Santa Cruz Biotechnology, diluted 1:100) for QCR1 or IgM (Santa Cruz Biotechnology, diluted 1:100) for anti-SSEA-1, and examined under a fluorescence microscope (IX70; Olympus, Tokyo, Japan). In addition, 15 d hybrid germline tissues were sectioned (10 ␮m thickness) with a cryotome (HM 505 E; Microm, Walldorf, Germany) and evalu-

ated with a peroxidase LSAB1 kit (Dako, Carpinteria, CA). 2.6. Gonadal migration and localization of transferred PGCs To monitor gonadal migration and developmental localization of exogenous PGCs after transfer, cPGCs retrieved from the blood of 2.5 d Korean Oge (KO) embryos were cultured in vitro as previously described [21]. The cultured PGCs were labeled with PKH26 fluorescent dye (Sigma-Aldrich) or targeted with green fluorescence protein (GFP) gene before transplantation and subsequently 1 ⫻ 104 PGCs were transferred into the blood vessels of hybrid embryos. Migration of PKH-labeled PGCs and localization of GFP-positive PGCs were monitored in 7 d embryonic gonads and 1 d testes after hatching, respectively. 2.7. Statistical analysis Incubation times, body weights, and cPGCs in the bloodstream of chickens, pheasants, and hybrids were compared. Values were analyzed using the general linear model (PROC-GLM) with the Statistical Analysis System (SAS Institute, Cary, NC) software. When the model effects were significant, treatment effects were compared by the least-squares method. Significance was reached at ⬍ 0.05. 3. Results Interspecies hybrids were generated by crosses between female pheasants and male chickens (Fig. 1A). The fertilization rate and hatchability of hybrids were 25.9% (50/193) and 36.0% (18/50), respectively (Table 2). Of 18 hatchlings, ten were male (55.6%) and eight were female (44.4%). 3.1. Genotype and phenotype of hybrids The genotypes of hybrids generated from crosses between female pheasants and male chickens were evaluated using species-specific markers. The pheasant-specific markers, CYTB, IGLC, and TAPBP, and the chicken-specific markers, CSP#1 and AS3554-I9 (Fig. 1B), were detected in the genomic DNA of five offspring (Hybrid#1 to Hybrid#5). These results indicated that chicken–pheasant hybrids were intermediate birds and their genotypes consist of each parental genome. Chromosome karyotyping was conducted in female and male hybrids (Fig. 2). The hybrid genome exhibited a different chromosome number and a different centromeric index between paired chromosomes. Diploid

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

699

Fig. 1. Phenotype and genotype of chicken–pheasant hybrids. Male White Leghorn chicken (A) and female Korean ring-necked pheasant (B) were used to produce interspecies hybrids. Male and female hybrids are the dominant white color (C). The presence of parental genotypes was identified by polymerase chain reaction (D). Five hybrids (lanes 2– 6) were analyzed with pheasant-specific (CYTB, IGLC, TAPBP) and chicken-specific (CSP#1, AS3554-I9) DNA markers. Chicken (lane 7) and pheasant genomic DNAs (lane 8) were used as positive controls and no template was used as the negative control (lane 9); 200 ng genomic DNA was used in every lane. Lane 1: molecular size marker; lane 2: Hybrid #1; lane 3: Hybrid #2; lane 4: Hybrid #3; lane 5: Hybrid #4; lane 6: Hybrid #5; lane 7: chicken; lane 8: pheasant; and lane 9: no template (ddH2O).

chromosomes were counted in metaphase spreads; the total number was 80 (2n) and was a mixture of chromosomes from the chicken (n ⫽ 39) and the pheasant (n ⫽ 41) (Fig. 2A,B). Chromosome banding and the centromeric index were evaluated for the sex chromosomes (Z and W) and macrochromosomes (chromosomes 1 to 7); the microchromosomes could not be analyzed because of their very short length. The chromosome sets of chicken and pheasant origin were paired by Giemsa banding (Fig. 2C,D). Evaluation of the centromeric index of the sex and macrochromosomes of the hybrids showed that three chromosomes (chromosomes 1, 2 and 4) were different from those of chicken and pheasant origin (Fig. 2E). For example, hybrid chromosome 1 was paired of submetacentric originated from pheasant and metacentric from chicken, and submetacentic of pheasant was longer than that of chicken origin. Briefly, different centromeric index between each paired chromosomes and different chromosome number were observed on hybrid genome. Table 2 The rate of fertilization, hatchability, and the sex ratio of hybrids generated by crosses between male chickens and female pheasants. No. of eggs incubated 193 a b c

No. of eggs fertilized (%)a 50 (25.9)

No. of hatchlings (%)b 18 (36.0)

Percentage of the number of eggs incubated. Percentage of the number of eggs fertilized. Ratios of male to female and female to male.

Sex ratio (%)c of Male

Female

55.6

44.4

The mean hatching time of the hybrid progenies generated from the crossbreeding of male chickens and female pheasants was 21.6 d, which was closer to that of chickens than pheasants (chicken, 20.7 d vs pheasant, 24.4 d; Table 3). The body weights of the hatchlings were significantly different from those of chickens (P ⬍ 0.0001; Table 3). Body weights of pheasants, chickens, and hybrids were analyzed according to sex and age (Table 4). The body weights of male and female hybrids were significantly different from those of pheasants and chickens. The weights of the males and females of the three species were significantly different at 12 and 24 wks (P ⬍ 0.05); the body weights of the 12- and 24-wk hybrid males were nearly double those of the hybrid females (12 wks, 687.7 vs 368.3 g; 24 wks, 1395.8 vs 676.7 g; male vs female). 3.2. Primordial germ cells in developing embryos The reactivity of the circulatory PGCs (cPGCs) and gonadal PGCs (gPGCs) was investigated by immunostaining with the germ cell-specific antibodies antiSSEA-1 for chickens (Fig. 3 A,D) and QCR1 for pheasants (Fig. 3C,G). Hybrid PGCs reacted specifically with the QCR1 antibody (Fig. 3B,F), but not the antiSSEA-1 antibody (Fig. 3E). The numbers of hybrid cPGCs were compared with those of the parent species (Fig. 4). Blood was collected at different times during incubation (48, 53, 58, and 63 h for chickens; 58, 63, and 68 h for hybrids; 58, 63, 68, and 73 h for pheasants). The numbers of hybrid cPGCs were 7.9, 12.4, and 6.4 per 1 ⫻ 105 blood cells at 58, 63, and 68 h,

700

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

Fig. 2. Chromosomal karyotyping and analysis of female and male interspecies hybrids. Metaphase chromosome spreads of male and female hybrids (A,B). Chromosomes for sex chromosomes (Z and W) and macrochromosomes (chromosomes 1 to 7) were banded by Giemsa staining (C,D). Diploid chromosomes and the centromeric index of the hybrids were evaluated (E). Centromeric index (%), 50.0 - 37.0; metacentric (M), 37.0 - 25.0; submetacentric (S), 25.0 - 0.0; acrocentric (A).

respectively. The ratio of cPGCs increased significantly at 63 h (0.0124%; P ⬍ 0.0001). Due to the limited number of hybrid embryos, we could not measure the cPGC number at 73 h. However, the cPGC number dramatically decreased after 63 h and it could be expected that the cPGC number of hybrid embryos at 73 h

is lower than that at 68 h. In pheasants at similar developmental stages, the numbers of PGCs were 4.3, 10.4, 3.9, and 1.1 (58, 63, 68, and 73 h, respectively). The numbers of chicken PGCs peaked at 53 h and were 2.9, 11.5, 5.3, and 0.7 at 48, 53, 58, and 63 h, respectively.

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

701

Table 3 Comparison of incubation times for hatching and body weights among pheasants, chickens, and hybrids. Species

Eggs

1d

No. of eggs analyzed (n)

Weight (g)

No. of eggs analyzed (n)

Incubation time for hatching (d)

Body weight (g)

30 30 N/A

26.9b 53.3c —

21 20 20

24.4d 20.7b 21.6c

16.5b 38.0c 17.6b

Pheasant Chicken Hybrida

Model effect of each parameter was P ⬍ 0.0001. a Hybrids were produced from pheasant eggs mated with male chickens. b– d Different superscripts within each parameter are significantly different, P ⬍ 0.05.

To investigate endogenous germ cells at a later stage of embryonic development, immunohistochemical staining of male germline tissues was performed at 15 d. Germ cells that reacted specifically with the QCR1 antibody were detected on the seminiferous cords of hybrids (Fig. 5). 3.3. Exogenous germ cell migration and developmental localization in germline tissues of hybrids To monitor migration of donor-derived PGCs to the gonadal tissues, exogenous cPGCs were retrieved from the blood of 2.5 d embryonic chickens, cultured in vitro, labeled with PKH26 fluorescent dye, and transferred into the blood vessels of 63 h hybrids. From the 81 manipulated eggs, 25 hybrids were hatched (30.9%). PKH fluorescence was detected on the left and right gonads of 7 d hybrids (Fig. 6A to D). The colonized, fluorescent cells on the hybrid gonads were identified as chicken PGCs by whole-mount immunohistochemical staining with anti-SSEA-1; this chicken PGC-specific antibody did not react with hybrid gPGCs (Fig. 6E,F). To investigate localization of exogenous germ cells after hatching, cPGCs targeted with the GFP gene were

Table 4 Body weight of pheasants, chickens and hybrids were evaluated in 12- and 24-week males and females. Sex Male

a

Femaleb

a,b

c– e

Species Pheasant Chicken Hybrid Pheasant Chicken Hybrid

No. of stocks analyzed (n) 9 15 13 11 15 6

Weight (g) at 12 wk c

531.7 948.0e 687.7d 441.4d 810.3e 368.3c

24 wk 1055.1c 1509.7e 1395.8d 729.1c 1289.2d 676.7c

Significant differences between the weights of males and females were observed in each species, P ⬍ 0.05. Different superscripts within each species of the same sex and age are significantly different, P ⬍ 0.05.

transferred. Green fluorescent cells were detected on the testes of 1 d hybrids (Fig. 7A); subsequently these cells were located on the seminiferous tubules together with endogenous germ cells (Fig. 7B to D). These results indicated that germ cells transferred into hybrids show normal development on the recipient gonads. These data provide new possibilities for the use of interspecific hybrids as an efficient recipient model for production of transgenic birds and preservation of endangered species. 4. Discussion In the present study, chicken–pheasant hybrids exhibited genotypic and phenotypic characteristics from both parents. In addition, the germ cells of developing hybrids showed normal patterns of migration during the embryonic stages. Exogenous germ cells transferred into the blood vessels of early developing hybrids were detected on both gonads of 7 d embryos, and on the testes of 1 d hatchlings. The understanding of the genetic and phenotypic traits of hybrids and the migratory ability of transferred germ cells in hybrid gonads during embryo development is essential. It may identify hybrids as potential recipient models in germ cell-mediated reproductive technologies, and be applied to the generation of germline chimeras and transgenic species for the conservation of endangered birds. During our investigations, 32 embryos (64%) from 50 fertilized eggs generated by crosses between female pheasants and male chickens ceased their development in the early embryonic stages. However, 18 hatchlings (36%) were successfully obtained. Purohit et al reported that malformations such as exencephaly were frequently present in chicken–pheasant hybrid progenies [22]. We observed that eight of 18 hatched chicks (44.4%) showed exencephaly. The high mortality of early embryos and the incidence of exencephaly may result from the dissimilarity of chromosome numbers and the incompatibility of maternal cytoplasm and pa-

702

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

Fig. 3. Detection of primordial germ cells (PGCs) by immunostaining. Circulating PGCs (cPGCs) collected from blood vessels and gonadal PGCs (gPGCs) retrieved from the gonads of 7-d embryos were reacted with anti-SSEA-1 specific for chicken (A,D), and QCR1 antibody specific for pheasant (C,G). In hybrid, cPGCs and gPGCs were reacted with only QCR1 (B,F), not anti-SSEA-1 (E). cPGCs positive for species-specific antibodies are indicated by arrows (A to C). Detection of gPGCs was conducted by whole-mount immunostaining (D to G). All squares in (A) to (G) were magnified around twofold (A= to G=). Scale bars; (A to C) ⫽ 100 ␮m and (D to G) ⫽ 400 ␮m.

ternal chromosomes [3,23]. In these hybrids, the mechanisms involved in the pairing of the homologous chromosomes during cell division and the trace and

Fig. 4. Estimation of primordial germ cells (PGCs) during early embryonic development in chickens, pheasants, and hybrids. Blood was collected from chickens, hybrids, and pheasants at different incubation times. The number of cPGCs reacting with anti-SSEA-1 antibody specific for chicken, and QCR1 antibody specific for the hybrid and pheasant, were counted in 1 ⫻ 105 blood cells. Three replicates for each incubation time in all three species were independently evaluated to estimate the number of PGCs. A significant effect (P ⬍ 0.0001) was found at each observation time in each species. More PGCs were detected in circulating blood at 53 h in chickens (11.5 PGCs), and at 63 h in hybrids (12.4 PGCs) and pheasants (10.4 PGCs). Different superscripts indicate significant differences (P ⬍ 0.05).

distribution of the additional chromosomes originating from the pheasant are unclear. More karyological studies are needed to clarify cytogenetic patterns and gametogenesis in hybrids. Generally, interspecies hybrids are infertile and unable to produce normal gametes. In our preliminary study, we also observed abnormal spermatogenesis and oogenesis in chicken–pheasant hybrids (data not shown). The chromosomal dissimilarity between parental karyotypes may result in an interruption of meiosis (the process of cell division that produces gametes). In chicken– quail hybrids, chickens and quails and their progenies had the same chromosome number (2n ⫽ 78). However, no spermatogenesis in the testes was observed [24] due to chromosomal incompatibility and failure of synapsis during meiosis. Guinea fowl hybrids generated from crosses between guinea fowl and male vulturine guinea fowl are sterile regardless of the morphological similarity of the parental chromosomes [5]. These results indicated that a difference in gene arrangement may be the reason for hybrid sterility and lethality even if the number and size of chromosomes are similar between parents. Besides chromosomal dissimilarity, the effect of hormone abnormalities in pheasant– chicken hybrids has been observed. Purohit et al reported that the plasma of these hybrids contained low levels of testos-

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

703

Fig. 5. Detection of germ cells during late embryonic development. Germ cells of 15-d testicular tissues reacted with the QCR1 antibody (A,B). Counterstaining was with hematoxylin. Samples were 10 ␮m thick. Scale bars; (A) ⫽ 200 ␮m and (B) ⫽ 50 ␮m.

Fig. 6. Migration of exogenous primordial germ cells (PGCs) in the embryonic gonads. PGCs retrieved from the blood of 2.5-d chicken embryos were cultured in vitro, labeled with PKH26 fluorescent dye, and transferred into 63-h hybrid embryos. Gonadal migration was observed 7 d after transfer. PKH signals were detected on the left gonad (A,B) and right gonad (C,D). The presence of migrated PGCs on the left gonad of the hybrid was confirmed with anti-SSEA-1 antibody, the chicken PGC-specific antibody (E,F). (F) is a magnification of (E). Scale bars; (A to E) ⫽ 200 ␮m and (F) ⫽ 100 ␮m.

704

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

Fig. 7. Localization of chicken primordial germ cells (PGCs) in the hybrid embryo. Chicken germ cells targeted with the green fluorescent protein (GFP) gene regulated by the chicken DAZL promoter were detected on 1-d intact testes (A). The testes were sectioned and treated with QCR1 antibody (red) to detect endogenous germ cells (B) and GFP (green) to detect exogenous germ cells (C). GFP-positive cells (arrowhead) were located in the seminiferous tubules with endogenous germ cells (arrow) (D). (D) is a magnification of squares from (B) and (C). Counterstaining was with DAPI (blue). Scale bars; (A to C) ⫽ 400 ␮m and (D) ⫽ 50 ␮m.

terone and that spermatogenesis in the seminiferous tubules did not progress beyond the primary spermatocyte stage [4]. Testosterone is secreted by Leydig cells in the testes and is required for the development of Sertoli cells, the supporter cells for spermatogenesis. Therefore, low levels of testosterone may result in the absence of Sertoli cells and sterilization of the testes. Purohit and Basrur administrated testosterone to hybrids, but this resulted in the degeneration of most of the germ cells in the seminiferous tubules [25]. The localization of 3-beta-hybroxysteroid dehydrogenase (HSD)-positive cells in the testes of chicken–pheasant hybrids is known to be different from that of chickens; the steroid generated by these 3-beta-HSD positive cells may not be able to maintain normal spermatogenesis in the hybrids [26]. Alternatively, the hybrids may be deficient in trophic hormones such as follicle-stimulating hormone (FSH) and luteinizing hormone (LH). Investigations into the interactions between these hormones are

important for understanding the microenvironment in hybrid testes. The hatching time of the hybrids was 21.6 d, which was close to the 20.7 d hatching time of chickens (Table 3). Although the offspring hatched from pheasant eggs, the incubation time for hatching was not close to that of pheasants (24.4 d). Incubation periods are influenced by egg size, body weight, and egg constituents [27,28]. However, the incubation times of the hybrids differed from that of pheasants despite having the same egg volume, hatchling sizes, and egg compositions. In preliminary studies, we attempted to produce hybrids from chicken eggs by artificial insemination using pheasant semen. Because of the technical difficulties involved in obtaining sufficient pheasant semen, only one hybrid was successfully generated (data not shown); hatching time was 21 d, similar to chickens. The incubation period for hatching is likely determined by unknown genetic factors derived from the chicken rather than the pheasant.

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706

The body weight of male hybrids were nearly double that of the female. It may result from chromosomal dissimilarity between two species; however, little was known about this mechanism in bird. In mammals, there are evidences that genomic imprinting genes were disrupted in interspecies hybrid. Loss of Peg1, paternal imprint gene, was occurred and correlated with body growth in interspecies mouse hybrid [29]. PGCs in the hybrid embryos showed normal patterns of migration, circulation in the blood vessels, settlement on the gonads, and location on the seminiferous cords. PGCs in intact gonads isolated from 7 d embryos were investigated for reactivity with QCR1 and antiSSEA-1 antibodies (Fig. 3). Hybrid PGCs reacted with the QCR1 antibody, but not with the chicken-specific anti-SSEA-1 antibody, which could have been due to the presence of maternal cytoplasmic factors in developing germ cells. In mammals, specific inherited factors from oocytes during fertilization support early embryonic development [22,30]. In birds, the detection of cPGCs in the bloodstream of the early embryo is important for determining the appropriate time point of germ cell transplantation, and for improving the transmission rate of donor-derived gametes in germline chimeras. In chicken–pheasant hybrid embryos, the number of cPGCs peaked at 63 h of incubation, and then dwindled (Fig. 4). This profile was close to that of the pheasant in which cPGC migration peaked at 64 h, and then gradually decreased [31]. In chickens, the number of cPGCs in the bloodstream began to decline at stage 16. Based on these data, 63 h of incubation was selected as an optimal time point for the transplantation of donor-derived cPGCs into the embryonic blood vessels of hybrids. Consequently, a majority of donor-derived PGCs migrated to both gonads of the hybrids (Fig. 6), which suggests that chicken–pheasant female hybrids could be used as recipient animals in germ cell-mediated technology. In this study, an infertile hybrid was used as a recipient during the production of avian germline chimeras. The genetic and physiological characteristics of the hybrid species as a host animal may allow optimization of germ cell-mediated technology, conservation of endangered avian species, and generation of transgenic birds.

Acknowledgments We thank Dr. H. Aoyama (Hiroshima University, Japan) and Dr. T. Ono (Shinshu University, Japan) for kindly providing the QCR1 antibody. This study was

705

supported by World Class University (WCU) program (R31-10056) through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology. The authors declare no conflict of interest. References [1] Olsen MW. Turkey-chicken hybrids. J Hered 1960;51:69 –73. [2] McFarquhar AM, Lake PE. Artificial insemination in quail and the production of chicken-quail hybrids. J Reprod Fertil 1964; 8:261–3. [3] Yamashina Y. Studies on sterility in hybrid birds. IV. Cytological researches on hybrids in the family Phasianidae. J Fac Sci Hokkaido Univ Ser 1943;8:307– 86. [4] Purohit VD, Basrur PK, Smith VG. Testosterone levels in the blood plasma of male chicken-pheasant hybrids. Poult Sci 1978; 57:513–7. [5] Takahashi E, Kurihara Y, Kondo N, Makino S. Karyological studies on hybrids between the guinea fowl (乆) and the Vulturine Guinea fowl (么). Proc Japan Acad 1975;51:593–7. [6] Malone JH, Chrzanowski TH, Michalak P. Sterility and gene expression in hybrid males of Xenopus laevis and X. muelleri. PLoS ONE 2007;2:e781. [7] Park TS, Jeong DK, Kim JN, Song GH, Hong YH, Lim JM, Han JY. Improved germline transmission in chicken chimeras produced by transplantation of gonadal primordial germ cells into recipient embryos. Biol Reprod 2003;68:1657– 62. [8] Han JY, Park TS, Hong YH, Jeong DK, Kim JN, Kim KD, Lim JM. Production of germline chimeras by transfer of chicken gonadal primordial germ cells maintained in vitro for an extended period. Theriogenology 2002;58:1531–9. [9] Kim MA, Park TS, Kim JN, Park HJ, Lee YM, Ono T, Lim JM, Han JY. Production of quail (Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos. Theriogenology 2005;63:774 – 82. [10] Park TS, Kim MA, Lim JM, Han JY. Production of quail (Coturnix japonica) germline chimeras derived from in vitrocultured gonadal primordial germ cells. Mol Reprod Dev 2008; 75:274 – 81. [11] Kang SJ, Choi JW, Kim SY, Park KJ, Kim TM, Lee YM, Kim H, Lim JM, Han JY. Reproduction of wild birds via interspecies germ cell transplantation. Biol Reprod 2008;79:931–7. [12] Yasuda Y, Tajima A, Fujimoto T, Kuwana T. A method to obtain avian germ-line chimaeras using isolated primordial germ cells. J Reprod Fertil 1992;96:521– 8. [13] Kim JN, Kim MA, Park TS, Kim DK, Park HJ, Ono T, Lim JM, Han JY. Enriched gonadal migration of donor-derived gonadal primordial germ cells by immunomagnetic cell sorting in birds. Mol Reprod Dev 2004;68:81–7. [14] Vick L, Luke G, Simkiss K. Germ-line chimaeras can produce both strains of fowl with high efficiency after partial sterilization. J Reprod Fertil 1993;98:637– 41. [15] Kagami H, Tagami T, Matsubara Y, Harumi T, Hanada H, Maruyama K, Sakurai M, Kuwana T, Naito M. The developmental origin of primordial germ cells and the transmission of the donor-derived gametes in mixed-sex germline chimeras to the offspring in the chicken. Mol Reprod Dev 1997;48:501–10. [16] Carsience RS, Clark ME, Verrinder Gibbins AM, Etches RJ. Germline chimeric chickens from dispersed donor blastodermal

706

[17]

[18]

[19]

[20]

[21]

[22] [23]

S.J. Kang et al. / Theriogenology 75 (2011) 696 –706 cells and compromised recipient embryos. Development 1993; 117:669 –75. Zhang Z, Shao S, Meistrich ML. Irradiated mouse testes efficiently support spermatogenesis derived from donor germ cells of mice and rats. J Androl 2006;27:365–75. Choi JW, Kang SJ, Park MS, Kim JK, Han JY. Paractical Use of DNA Polymorphisms in the Avian Immunoglobulin Light Chain Constant Domain for Sepcies-specific PCR. J Anim Sci & Technol (Kor.) 2008;50:9 –18. Li HC, Zhao L, Kagami H, Matsui K, Ono T. Identification of transferred chicken germ cells in quail gonad and semen by amplification of chicken-specific PCR products. J Poult Sci 2001;38:308 –16. Choi JW, Lee EY, Shin JH, Zheng Y, Cho BW, Kim JK, Kim H, Han JY. Identification of breed-specific DNA polymorphisms for a simple and unambiguous screening system in germline chimeric chickens. J Exp Zool Part A Ecol Genet Physiol 2007;307:241– 8. Choi JW, Kim S, Kim TM, Kim YM, Seo HW, Park TS, Jeong JW, Song G, Han JY. Basic Fibroblast Growth Factor Activates MEK/ERK Cell Signaling Pathway and Stimulates the Proliferation of Chicken Primordial Germ Cells. PLoS ONE 2010;5:e12968. Purohit VD, Basrur PK, Reinhart BS. Exencephaly in chickenpheasant hybrids. Br Poult Sci 1977;18:143– 6. Wakasugi N. A genetically determined incompatibility system between spermatozoa and eggs leading to embryonic death the mouse. J Reprod Fertil 1974;41:85–96.

[24] Takashima Y, Mizuma Y. The testes of chicken-quail hybrids. Tohoku Journal of Agricultural Research 1981;Vol. 32. No 4. [25] Purohit VD, Basrur PK. Effects of testosterone on the testes of sexually quiescent pheasants: evidence for a negative feedback mechanism. Br Poult Sci 1978;19:709 –12. [26] Purohit VD, Basrur PK, Bhatnagar MK. Histochemical localization of 3 beta-hydroxysteroid dehydrogenase in the testes of chicken-pheasant hybrids. Histochem J 1977;9:293–9. [27] Mashiko K. Relationships between egg size and incubation time among the populations of two freshwater prawns. Ecol Res 1987;2:97–9. [28] Badzinski SS, Ankney CD, Leafloor JO, Abraham KF. Egg size as a predictor of nutrient composition of eggs and neonates of Canada geese (Branta canadensis interior) and lesser snow geese (Chen caerulescens caerulescens). Canadian J Zool 2002; 80:333– 41. [29] Shi W, Lefebvre L, Yu Y, Otto S, Krella A, Orth A, Fundele R. Loss-of-imprinting of Peg1 in mouse interspecies hybrids is correlated with altered growth. Genesis 2004;39:65–72. [30] Renard J-P, Baldacci P, Richoux-Duranthon V, Pournin S, Babinet C. A maternal factor affecting mouse blastocyst formation. Development 1994;120:797– 802. [31] Kim JN, Lee YM, Park TS, Jung JK, Cho BW, Lim JM, Han JY. Detection and characterization of primordial germ cells in pheasant (Phasianus colchicus) embryos. Theriogenology 2005; 63:1038 – 49.