, Takahiro Tagami, Hiroshi Kagami and Tamao Ono

Transcription

1 The Journal of Poultry Science,./:,3,,31,,**2 Copyright,**2, Japan Poultry Science Association. doi: +*.,+.+/jpsa../.,3, + Yusuke Atsumi, Takahiro Tagami, Hiroshi Kagami and Tamao Ono +,, Faculty of Agriculture, Shinshu University, 2-*. Minamiminowa, Kamiina, Nagano -33./32, Japan, Interdisciplinary Graduate School of Science and Technology, Shinshu University, 2-*. Minamiminowa, Kamiina, Nagano -33./32, Japan - National Institute of Livestock and Grassland Science, Tsukuba, Ibaraki -*/ *3*+, Japan Primordial germ cells (PGCs) are the progenitor cells of gametes. Avian PGCs are located in the central region of the area pellucida at the blastoderm stage. PGCs enter the circulation soon after the formation of blood vessels in incubating eggs and eventually settle in the gonadal primordium. We have now examined exposure of chicken embryos to soft (low-energy) x-rays as a means of depleting endogenous PGCs and thereby improving the e$ ciency of chimera production. The blastoderm of White Leghorn eggs was exposed to soft x-rays for *,,*,.* or 0* s before incubation. The irradiated embryos manifested delayed development at 0* h of incubation. They also showed reduced numbers of circulating PGCs at stages +. and +/ and of gonadal PGCs at stage -*. The hatchability of irradiated embryos was lower than that of nonirradiated controls. Irradiation for,* s was found to provide the best outcome taking into consideration both the restriction of PGC proliferation and hatchability. Dispersed blastoderm cells of quail (black plumage) embryos were introduced into the blastoderm of chicken embryos irradiated for,* s or of nonirradiated embryos. The number of donor-derived PGCs was higher in the irradiated embryos than in the nonirradiated controls at stage -*. These results suggest that soft x-irradiation of chicken embryos is a feasible approach to depletion of endogenous germ cells and consequent improvement in the e$ ciency of incorporation of donor PGCs. Key words: blastoderm, chimera, embryo culture, primordial germ cells, soft x-ray J. Poult. Sci.,./:,3,,31,,**2 Introduction germline transmission. The production of germline chimeras requires incorporation of donor PGCs into the Primordial germ cells (PGCs) are the first identifiable endogenous gonadal tissue of recipient embryos. The progenitors of gametes and provide the only cellular con- proportion of donor-derived gametes, however, is detertinuity between generations. Avian PGCs are located in mined by the relative numbers of the donor- and recipientthe central region of the area pellucida at the blastoderm derived germ cells in the chimeric gonad. This proportion stage, and they migrate to the extraembryonic germinal would be expected to be increased if the PGCs of the crescent shortly after the onset of incubation. As soon as recipient could be removed, depleted, or inactivated. The blood vessels form, the PGCs enter the circulation and ideal recipient, therefore, would be a healthy animal that finally settle in the gonadal primordium (Gilbert,,**0). has normal reproductive organs but which is sterile as a Attempts have been made to produce avian germline result of the absence only of the germ cells themselves (Li chimeras and donor-derived o# spring by the transfer of et al.,,**+). Exposure to soft (low-energy) x-rays has blastoderm cells or PGCs into host embryos (Petitte et al., been shown to be e# ective in restricting the proliferation +33* ; Kagami et al.,,**,; Naito,,**- a, b; Kim et al., of quail and chicken PGCs (Li et al.,,**+; Lim et al.,,**/). Such procedures will likely play an important role,**0; Nakamichi et al.,,**0). We have now examined in the preservation of foundation stocks and endangered the e# ect of such x-irradiation on the number of PGCs in species (Tajima et al., +33-). Development of a more chicken embryos, and we have evaluated the utility of this e# ective technology for the expression of donor-derived approach for the preparation of recipient chicken embryos germ cells will require improvement in the e$ ciency of for the production of germline chimeras. Received: May 1,,**2, Accepted: May,3,,**2 Correspondence: Dr. T. Ono, Faculty of Agriculture, Shinshu University, 2-*. Minamiminowa, Kamiina, Nagano -33./32, Japan. ( tamaoon@shinshu-u.ac.jp) Materials and Methods Animals The study was performed in accordance with the policies on animal care developed by the Animal Care and

2 Atsumi et al. : Restriction of Germline Proliferation by X-irradiation 293 Management Committee of Shinshu University. Ferti- embryo that had been irradiated for,* s or not prior to the lized White Leghorn ( Gallus domesticus) eggs were ob- injection. Donor-derived PGCs in chimeric embryos at tained from Goto Hatchery (Gifu, Japan). Japanese quail stage -* were identified and counted by immuno- ( Coturnix japonica) with black plumage were maintained histochemical analysis with the QCR+ antibody, which in our laboratory, and fertilized eggs were collected daily. recognizes a protein specific to quail PGCs (Aoyama et The developmental stage of embryos was determined on al., +33, ; Ono et al., +332), as described above. the basis of the normal tables of Eyal-Giladi and Kochav Statistical Analysis ( +310 ) or Hamburger and Hamilton ( +3/+ ) for stages Data were analyzed by Student s t test, Welch s t test, before incubation (expressed in Roman numerals) or after the Mann-Whitney s Utest, Sche# e s Ftest, or Spearman s incubation (expressed in Arabic numerals), respectively. correlation coe$ cient by rank test with the use of the Irradiation Statcel add-in program for Microsoft Excel (Yanai,,**.) A fertilized chicken egg was cracked open and trans- or by Fisher s exact probability test ( ferred to a dish. The surrounding thick albumen was gunma-u.ac.jp/javascript/fisherexacttest.html). A P removed, and the blastoderm on the naked egg yolk was value of **/. was considered statistically significant. positioned upward. The egg was then placed on a turntable in the chamber of a soft x-ray apparatus (model B-.; Results Softex, Tokyo, Japan). The table was rotated at 0 rpm, Embryonic Development and Hatchability and the blastoderm was placed at a distance of,+ cm from Embryos exposed to soft x-rays and cultured according the soft x-ray source ( +2 kv,, ma). Embryos were to system II showed delayed development compared with irradiated for *,*.*,, or 0* s and were designated X- *, X- control embryos (Fig. + ). After 0* h of incubation, the,*, X-.* and X-0* accordingly. The radiation dosage was median developmental stage of normal (noncultured, nonestimated as,*. Gy/min on the basis of data supplied by irradiated) embryos was stage +1 whereas that of cultured Softex showing the dosage to be,. 30 or *. 0+ Gy/min at (X-*) embryos was stage +0 ( P *.*/ versus normal) and distances of +2/ and -.- mm, respectively, and at settings that of irradiated (X-,*, X-.* or X- 0* ) embryos was stage of,* kv and, ma. +/ ( P *.*/ versus X- *). Hatching rates were -. (,2/ Embryo Culture 2- ), +2 (,,/ +,. ), 3 ( 3 / +*/ ) and - (// +0-) for the Manipulated embryos were cultured for 0./ days until X- *, X-,*, X-.* and X-0* embryos, respectively, comthey reached stage -* or until hatching using a previously pared with a hatchability of 2, (-1/./) for normal described method (systems II and III; Perry, +322 ; Naito embryos (Fig.,). The hatchability of cultured (X-*) et al., +33* ). embryos was significantly lower than that of normal Counting of Circulating PGCs embryos ( P **/. ) and it was decreased by x-irradiation Cultured embryos at stages +. to +1 were transferred to in a dose-dependent manner ( P *.*/). new dishes for collection of, ml of blood from the dorsal Population of Circulating and Gonadal PGCs aorta. The blood was diluted with +2 ml of Dulbecco s We next determined the densities of circulating PGCs modified Eagle s medium (Gibco BRL, Grand Island, and erythrocytes in embryos at stages +. to +1 that had NY, USA) supplemented with +* fetal bovine serum been exposed to soft x-rays before culture. The density of (Nippon Bio-test Laboratories, Tokyo, Japan), and a well- PGCs in the bloodstream of nonirradiated (normal or X- dispersed sample was transferred to a hemocytometer for *) embryos was decreased in a developmental stageenumeration of PGCs and erythrocytes. dependent manner ( P **/., Spearman s correlation coe$ Population of Gonadal PGCs cient by rank test), with that for cultured (X-*) Embryos at stage -* were removed from the yolk and embryos not di# ering from that for normal embryos at fixed in Bouin s fluid. The gonadal tissue was serially each stage (Fig. - A). At stage +., the PGC density in X- sectioned at a thickness of 1 mm, and the sections were processed for immunohistochemical detection of gonadal.* or X-0* embryos was significantly lower than that in X- * embryos ( P *.*/); at stage +/, that in X-,*, X-.* or X- PGCs with antibodies to the chicken vasa homolog 0* embryos was significantly lower than that in X-* (CVH) protein (Nakamura et al.,,**1) as described embryos ( P **/. ). The density of erythrocytes did not previously (Ono et al., +332). The population of PGCs di# er significantly between the nonirradiated and irradiated was enumerated by counting the number of stained PGCs in +* sections (every fourth serial section) in the middle region of the left gonad. Production of Chimera groups at any stage examined (Fig. -B). Gonadal PGCs were visualized immunohistochemically with antibodies to CVH at stage -* (Fig..). The number of PGCs in cultured (X-*) embryos (,,+,3, mean The area pellucida of quail blastoderm was isolated SE) was significantly reduced compared with that in from the yolk and suspended in Dulbecco s modified normal embryos (.*. +,, P *.*/). The number of Eagle s medium supplemented with +* chicken serum PGCs was reduced by x-irradiation in a dose-dependent (Nippon Bio-test Laboratories). A portion ( + ml) of the manner ( P **/. ), with the values for X-,*, X-.* and X- blastoderm cell suspension (containing -*** cells) was injected into the area pellucida of an unincubated chick 0* embryos being +/- +3, +/... and 31,/, respectively.

3 294 J. Poult. Sci.,./ (.) Fig.,. ect of soft x-irradiation of the hatchability of chicken embryos. Embryos were exposed to soft x-rays for * s (X-*, n 2- ),,* s (X-,*, n +,. ),.* s (X-.*, n +*/ ) or 0* s (X- 0*, n +0-) before incubation. The hatchability of normal embryos ( n./ ) is shown for comparison. Hatchabilities of normal versus cultured (X-*) embryos and X-* versus irradiated (X-,*, X-.* or X- 0* ) embryos di# ered significantly as indicated by the di# erent letters ( P *.*/, Fisher s exact probability test). Hatchability was also decreased by soft x- irradiation in a dose-dependent manner ( P **/., Spearman s correlation coe$ cient by rank test). marker for tracing the origin of the germline. Its homolog in the chicken, cvh, has been isolated and applied to trace the origin and movement of PGCs (Tsunekawa et al., Fig. +. ect of soft x-irradiation of the distribution of developmental stages of chicken embryos at 0* h of,***), resulting in the detection of -* CVH-expressing culture. Embryos were exposed to soft x-rays for * s PGCs in the central zone of the area pellucida of White (X-*),,* s (X-,*),.* s (X-.*) or 0* s (X- 0* ) before Leghorn embryos at stage X. The population of CVHexpressing incubation. The distribution for normal embryos is PGCs was recently shown to increase gradually shown for comparison. Each group comprised +1 to,3 from stage X ( +-* cells) to stage +* (..* cells) in embryos. Lowercase letters (a, b, c) indicate the median Rhode Island Red embryos (Nakamura et al.,,**1). The values as well as significant di# erences ( P *.*/) di# erence in the size of the PGC population at stage X between the medians of normal and X-* groups and between these two previous studies may be due to the between those of X-* and irradiated (X-,*, X-.* or X- strain di# erence (Nakamura et al.,,**1). The proliferation of avian PGCs occurs during their migratory period 0* ) groups (Mann-Whitney s U test). before they settle in the germinal ridge (Nakamichi et al.,,**0). The population of chicken PGCs in the circulation Chimera Production around stage +. is several hundred or more, although it For chimera production, chick embryos were exposed varies among eggs (Tajima et al., +333 ; Zhao et al.,,**-). to soft x-rays for * s (X-*) or,* s (X-,*), injected with Chicken embryos exposed to soft x-rays in the present quail blastoderm cells, and incubated until stage -*. study manifested delayed development at 0* h of culture, Transferred quail PGCs were positively stained with and this developmental delay persisted until hatching. QCR+ antibody in the chick embryonic gonads (Fig. / A, The irradiated embryos thus hatched one or more days / B). The number of donor-derived quail PGCs in left later than the X-* embryos (data not shown). The gonadal tissue of the chick embryos was then determined hatchability of embryos also decreased in a manner deimmunohistochemically with the QCR+ antibody (Fig. pendent on the dose of radiation. In addition, the / C). The population of quail PGCs in X-,* embryos hatchability of cultured (X-*) embryos was markedly (,0./ -. 0 ) was greater than that in X-* embryos ( +/., reduced compared with that of intact eggs, suggesting a,1., P **/. ). Chimera hatchlings with quail-derived need for improvement in culture techniques. Culture of black plumage were obtained from both X-* and X-,* chick embryos from stage X with the use of embryo embryos (Fig. 0). culture systems II and III was previously shown to result Discussion in a hatching rate of /*.* to 0,./ (Naito and Perry, +323 ; Naito et al., +33* ). The vasa gene is thought to be a reliable molecular Soft x-irradiation of chicken embryos in the present

4 Atsumi et al. : Restriction of Germline Proliferation by X-irradiation 295 Fig. -. ect of soft x-irradiation on the density of PGCs or erythrocytes in the bloodstream of chicken embryos. Embryos were exposed to soft x-rays for * s Fig... ect of soft x-irradiation on the population of (X-*),,* s (X-,*),.* s (X-.*) or 0* s (X- 0* ) before PGCs in the left gonad of chicken embryos. (A) incubation, and the density of PGCs (A) or ery- Embryos were exposed to soft x-rays for * s (X-*),,* s throcytes (B) in the circulation was determined at (X-,*),.* s (X-.*) or 0* s (X- 0* ) before incubation stages +. to +1. The values for normal embryos are until stage -*, when the number of PGCs in the left shown for comparison and did not di# er from those for gonad was determined by immunohistochemistry with cultured (X-*) embryos at each stage (Student s t test). antibodies to CVH. Each dot indicates the value for Lowercase letters (a, b, c) indicate significant di# erone embryo ( n 2 for all groups). Values for normal ences based on Sche# e s F test ( P *.*/). Data are embryos are shown for comparison. The PGC popumeans SE from / to +* embryos. lation of cultured (X-*) embryos was significantly smaller than that of normal embryos ( P **/., Mann- Whitney s U test), and it was decreased by x- study resulted in depletion of endogenous PGCs. The irradiation in a dose-dependent manner ( P **/., number of circulating PGCs at stage +. or +/ in embryos Spearman s correlation coe$ cient by rank test). (B) Representative immunohistochemistry images for irradiated at the blastoderm stage was thus markedly embryos treated as in (A). Arrowheads indicate reduced compared with that in cultured (X-*) embryos. PGCs. Scale bars, /* mm. Exposure to soft x-rays had previously been found to reduce the number of circulating PGCs in quail (Li et al.,,**+), White Leghorn (Lim et al.,,**0), and Barred Plymouth Rock (Nakamichi et al.,,**0) embryos. The The population of gonadal PGCs in the present study was extent of the reduction in the number of circulating PGCs also reduced by soft x-irradiation in a dose-dependent di# ered among these studies, likely because of di# erences manner. X-irradiation induces DNA damage, cell cycle in the apparatus for x-irradiation and in the avian strains. arrest, and apoptosis, with such treatment having been

5 296 J. Poult. Sci.,./ (.) Fig. 0. Chimera hatchlings born from X- * (left) and X-,* (right) chick embryos. The black plumage is derived from the donor quail cells of chimeric chick Fig. /. ect of soft x-irradiation on the population of embryos produced as in Fig. /. donor-derived (quail) PGCs in the left gonad of chicken embryos. Chicken embryos were exposed to soft x-rays for * s (X-*) or,* s (X-,*), injected with quail (Brinster and Avarbock, +33. ; Brinster and blastoderm cells, and incubated until stage -*, when the Zimmermann, +33. ; Ogawa et al., +331). Similarly transplanted number of donor-derived PGCs in left gonadal tissue rat testis cells also underwent spermatogenesis in was determined immunohistochemically with the QCR+ the testis of immunodeficient mice (Clouthier et al., +330; antibody. (A, B) Representative immunohistochemistry images for X-* and X-,* embryos, respectively. derived quail PGCs had settled in the gonads of chicken Shinohara et al.,,**0). In the present study, donor- Scale bars, /* mm. (C) Population of donor-derived embryos at stage -*. It was also shown that the population of donor PGCs was increased when the proliferation PGCs in left gonadal tissue. Data are means SE for +- embryos and di# ered significantly between X-* and of PGCs of the recipient was compromised by x- X-,* embryos ( P *.*/, Welch s ttest), as indicated by the lowercase letters (a, b). irradiation. It remains to be determined whether these donor cells are able to di# erentiate into functional gametes and to produce o# spring. Quail-chick chimeras with quail plumage were obtained shown to delay or arrest mitosis in rat spermatogonia but in the present study, but these animals did not survive not to a# ect the proliferation of somatic cells (West and beyond - to. months of age. A small proportion of such Lähdetie,,**+). Our results suggest that x-irradiation chimeras also showed loss of quail plumage and its reinhibits the mitotic activity of PGCs, possibly by inducing placement with chicken plumage. The growth rate of apoptosis, without a# ecting that of somatic cells including these latter chimeras was reduced while they had quail erythrocytes and gonadal cells. Although the proliferation plumage but increased after its substitution with chicken of blastodermal PGCs appeared to be impaired by x- plumage (data not shown). irradiation, the susceptibility of these cells to this treat- In conclusion, exposure of chicken embryos to soft ment varied markedly among embryos (Fig.. A). x-rays is a feasible approach to depletion of endogenous Depletion of endogenous PGCs would be expected to germ cells and consequent improvement in the e$ ciency increase the chance of incorporation of donor PGCs and of incorporation of donor PGCs. Further optimization of therefore to increase the proportion of the donor cells in this approach may result in a greater extent of endogenous the gonads of chimeric embryos. Such depletion of endog- PGC depletion and donor cell incorporation in recipient enous PGCs, however, is associated with a reduction in embryos. hatchability. Taking into account these conflicting e# ects of x-irradiation, we adopted an irradiation time of,* s (a Acknowledgments radiation dose of *1. Gy) for chimera production in the Part of this study was presented at the XXIII World present study. The ratio of the number of endogenous Poultry Congress in Brisbane, Australia (,**2), with gonadal PGCs in X-,* embryos to that in X-* embryos travel support to Y.A. from the Japan Poultry Science was *1. at stage -*, which resulted in a +1. -fold increase in Association. This study was supported in part by Grantsthe number of donor-derived PGCs in X-,* embryos in-aid for Scientific Research from the Japan Society for relative to that in X-* embryos. the Promotion of Science to T.O. Donor testis cells isolated from a fertile male mouse were able to undergo spermatogenesis when transplanted into the seminiferous tubules of an infertile male mouse

6 Atsumi et al. : Restriction of Germline Proliferation by X-irradiation 297 References Aoyama H, Asamoto K, Nojyo Y and Kinutani M. Monoclonal antibodies specific to quail embryo tissues: Their epitopes in the developing quail embryo and their application to identi- fication of quail cells in quail-chick chimeras. Journal of Histochemistry and Cytochemistry,.*: ,. Brinster RL and Avarbock MR. Germline transmission of donor haplotype following spermatogonial transplantation. Proceedings of the National Academy of Sciences of the United States of America, 3+ : ++-*- ++-* Brinster RL and Zimmermann JW. Spermatogenesis following male germ-cell transplantation. Proceedings of the National Academy of Sciences of the United States of America, 3+ : ++,32 ++-*, Clouthier DE, Avarbock MR, Maika SD, Hammer RE and Brinster RL. Rat spermatogenesis in mouse testis. Nature, -2+:.+2., Eyal-Giladi H and Kochav S. From cleavage to primitive streak formation: a complementary normal table and a new look at the first stages of the development of the chick. I. General morphology. Developmental Biology,.3: -, Gilbert SF. Developmental Biology. 2th ed. Sinauer Associates, Inc. Publishers. Sunderland, MA.,**0. Hamburger V and Hamilton HL. A series of normal stages in the development of chick embryos. Journal of Morphology, 22:.3 3,. +3/+. Kagami H. Developmental genetic analysis of the avian primor- dial germ cells and the applications for production of chimeric chickens. Journal of Poultry Science, -3: ,**,. Kim MA, Park TS, Kim JN, Park HJ, Lee YM, Ono T, Lim JM and Han JY. Production of quail ( Coturnix japonica) germline chimeras by transfer of gonadal primordial germ cells into recipient embryos. Theriogenology, 0-: ,.,**/. Li HC, Kagami H, Matsui K and Ono T. Restriction of prolifera- tion of primordial germ cells by the irradiation of Japanese quail embryos with soft X-rays. Comparative Biochemistry and Physiology. Part A: Molecular and Integrative Physiology, +-* : *.,**+. Lim JM, Kwon HM, Kim DK, Kim JN, Park TS, Ono T and Han JY. Selective decrease of chick embryonic primordial germ cells in vivo and in vitro by soft X-ray irradiation. Animal Reproduction Science, 3/ : ,**0. Naito M and Perry MM. Development in culture of the chick embryo from cleavage to hatch. British Poultry Science, -*:,/+,/ Naito M, Nirasawa K and Oishi T. Development in culture of the chick embryo from fertilized ovum to hatching. Journal of Experimental Zoology,,/.: -,, -,0. +33*. Naito M. Development of avian embryo manipulation techniques and their application to germ cell manipulation. Animal Science Journal, 1. : +/1 +02.,**-a. Naito M. Genetic manipulation in chickens. World s Poultry Science Journal, /3: ,**-b. Nakamichi H, Sano A, Harumi T, Matsubara Y, Tajima A, Kosugiyama M and Naito M. ects of soft X-ray irradia- tion to stage X blastoderm on restriction of proliferation of primordial germ cells in early chicken embryos. Journal of Poultry Science,.-: -3..**.,**0. Nakamura Y, Yamamoto Y, Usui F, Mushika T, Ono T, Setioko AR, Takeda K, Nirasawa K, Kagami H and Tagami T. Migration and proliferation of primordial germ cells in the early chicken embryo. Poultry Science, 20 :,+2,,+3-.,**1. Ogawa T, Aréchaga JM, Avarbock MR and Brinster RL. Transplantation of testis germinal cells into mouse seminiferous tubules. International Journal of Developmental Biology,.+: +++ +,, Ono T, Yokoi R, Maeda S, Nishida T and Aoyama H. Settlement of quail primordial germ cells in chicken gonads. Animal Science and Technology (Jpn.), 03 : /.0 /// Perry MM. A complete culture system for the chick embryo. Nature, --+: 1* 1, Petitte JN, Clark ME, Liu G, Verrinder Gibbins AM and Etches RJ. Production of somatic and germline chimeras in the chicken by transfer of early blastodermal cells. Development, +*2 : +2/ *. Shinohara T, Kato M, Takehashi M, Lee J, Chuma S, Nakatsuji N, Kanatsu-Shinohara M and Hirabayashi M. Rats produced by interspecies spermatogonial transplantation in mice and in vitro microinsemination. Proceedings of the National Academy of Sciences of the United States of America, +*- : +-0,. +-0,2.,**0. Tajima A, Naito M, Yasuda Y and Kuwana T. Production of germ line chimera by transfer of primordial germ cells in the domestic chicken ( Gallus domesticus). Theriogenology,.*: /*3 / Tajima A, Hayashi H, Kamizumi A, Ogura J, Kuwana T and Chikamune T. Study on the concentration of circulating primordial germ cells (cpgcs) in early chick embryos. Journal of Experimental Zoology,,2.: 1/ Tsunekawa N, Naito M, Sakai Y, Nishida T and Noce T. Isolation of chicken vasa homolog gene and tracing the origin of primordial germ cells. Development, +,1 :,1.+,1/*.,***. West A and Lähdetie J. X-irradiation-induced changes in the progression of type B spermatogonia and preleptotene spermatocytes. Molecular Reproduction and Development, /2: ,**+. Yanai H. Statcel The Useful Addin Forms on Excel., nd ed. OMS Publications, Tokorozawa, Japan.,**.. Zhao DF, Yamashita H, Matsuzaki M, Takano T, Abe S, Naito M and Kuwana T. Genetic factors a# ect the number of circulating primordial germ cells in early chick embryos. Journal of Poultry Science,.*: +*+ ++-.,**-.