DIFFERENTIATION OF FEMALE PRIMORDIAL GERM CELLS IN THE MALE TESTES OF CHICKEN

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1 DIFFERENTIATION OF FEMALE PRIMORDIAL GERM CELLS IN THE MALE TESTES OF CHICKEN (Diferensiasi Primordial Grem Sel dalam Testes Ayam) TAKAHIRO TAGAMI National Institute of Livestock and Grassland Science 2 Ikenodai, Tsukuba, Ibaraki, Japan ABSTRACT We demonstrated that female primordial germ cells (PGCs) have the ability to differentiate into W chromosome-bearing (W-bearing) spermatozoa in male gonads of germline chimeric chickens. In this study, three germline chimeric chickens were generated by injecting female PGCs into the male recipient embryos to investigate the differentiation pattern of female PGCs in male gonads in chickens. After these male chimeras reached sexual maturity, the semen samples were analyzed for detecting W-bearing cells by PCR and in situ hybridization analyses. The results indicated that the female PGCs had settled and differentiated in their testes. A histological analysis of the seminiferous tubule in those chimeras demonstrated that the W- bearing spermatogonia, spermatocytes and round spermatids accounted for 30.8, 32.7 and 28.4%, respectively. However, the W-bearing elongating spermatid was markedly lower (7.7%) as compared to the W-bearing round spermatid. The W-bearing spermatozoa were hardly ever observed (0.2%). We concluded that although female PGCs in male gonads are capable of passing through the first and second myeotic division in adapting themselves to a male environment, they are hardly complete spermiogenesis. Key Words: Chimeras, Primordial Germ Cells, Spermiogenesis ABSTRAK Makalah ini menjelaskan diferensiasi Primordial Germ Cell (PGC) betina menjadi spermatozoa dengan kromosom W-bearing dalam gonad jantan dari ayam Chimeric. Pada penelitian ini tiga ayam germline chimeric diproduksi dengan menyuntikkan PGC betina kedalam resipien yaitu embrio jantan. Setelah ayam Chimeric ini mencapai dewasa kelamin maka dilakukan sampling semen untuk mendeteksi adanya sel W- bearing dengan menggunakan PCR dan dilakukan analisa hibridisasi In Situ. Hasil menunjukan bahwa PGC betina dapat melakukan diferensiasi da berkembang dalam testes. Analisa histopatologi tubulus seminiferus ayam Chimeric menunjukan adanya spermatogonia, spermatosit dan spermatid bulat dengan w-baering sebanyak: 30,8, 32,7, dan 28,4% berturut turut. Akan tetapi spermatid lonjong dengan W-bearing sangat sedikit (7,7%) dibandingkan dengan spermatid bulat dengan W-bearing. Spermatozoa dengan W-bearing sangat sedikit (0,2%). Dari hasil tersebut disimpulkan walaupaun PGC betina dapat melewati tahap pertama dan kedua dari pembelahan myeotik dan menyesuaikan diri pada lingkungan testes, akan tetapi PGC betina tidak dapat mencapai fase spermatogenesis yang sempurna. Kata Kunci: Chimera, Primordial Germ Cell, Spermatogenesis INTRODUCTION Global domestic animal diversity is considered to be under threat, and worldwide, a large number of domestic animal breeds are endangered, in a critical status or already extinct. Cryopreservation of germplasm (sperm or ovum) is considered a very good ex situ strategy for conservation of existing allelic diversity for future use. In avian species, however, the cryopreservation of the ova and fertilized eggs are very difficult because of their huge yolk. The cryopreservation of 15

2 primordial germ cells (PGCs), progenitors of eggs and spermatozoa, provides an alternative way to preserve both female and male genetic materials. PGCs can be specifically harvested from chicken (TAGAMI and KAGAMI, 1998), and a technique for producing germline chimeric chicken by transferring PGCs has been established (YASUDA et al., 1992; TAJIMA et al., 1993). The collected PGCs for cryopreservation usually were not known of their sex. When the thawed PGCs are injected into the opposite sex's recipient embryos for making the germline chimeras, there is no information whether the donor PGCs are able to differentiate into a normal gamete. PGCs normally have the potential to differentiate into either spermatozoa or ova, depending on their sex. After colonization in the gonads, male PGCs in testes differentiate into spermatozoa and female PGCs in ovaries into ova. Although the study of mechanisms underlying germ cell development has been one of the most popular subjects in developmental biology, little is known about the mechanisms of the sexspecific growth and differentiation of germ cells. In this study, germline chimeric chickens were generated with female PGCs and male recipient embryos. The differentiation of female PGCs in the male chicken was clarified by genetic and histological experiments. MATERIALS AND METHODS To analyze the developmental fate of female PGCs in the male gonads, germline chimeric chickens were generated by a novel strategy, as illustrated in Fig. 1. TAGAMI et al. (2007) 16

3 Preparation of donor PGCs The fertilized eggs were cultured until stage 15 (HAMBURGER and HAMILTON, 1951). Embryonic blood cells were collected from the dorsal aorta of the stage 15 embryos (Fig.1 a). After the sex determination of each sample, the female PGCs were then concentrated by Ficoll 400 density gradient centrifugation from the collected blood (Fig.1 b,c). These PGCs were used as donor cells. Preparation of recipient embryos A cell cluster in the center of the area pellucida was removed from a stage X blastoderm using a sharp needle (Fig. 1A) (KAGAMI et al., 1997). These cells were used for sexing of the recipient embryos by PCR. The manipulated embryos were cultured ex vivo by the methods of PERRY (1988) until the injection of donor PGC at stage 15. Production of chimeric chickens The germline chimeric chickens were generated by microinjecting of pooled female donor PGCs into the bloodstream through the dorsal aorta of the treated recipient embryos (Fig. 1 C). The embryos were cultured for an additional 18 days in the incubator at 38.5 C until they hatched (Fig. 1 D, E). Detection of W-bearing spermatogenic cells in semen by fluorescence in situ hybridization To determine whether the transferred female PGCs adhered to the seminiferous tubules of male recipients and could differentiate into spermatogenic cells, the semen samples were fixed with a solution of methanol-acetic acid (3:1), dropped onto slides, and air dried. The procedure of in situ hybridization of sperm nuclei was performed as reported earlier (TAGAMI et al., 1997). For accurate confirmation of the W-bearing spermatozoa, two female-specific DNA probes were cross-hybridized for in situ hybridization (KODAMA et al., 1987; SAITOH et al., 1991). Assessment of germlinet transmission of donor PGC After the chimeras reached sexual maturity, they were mated with female BPR (i/i) by artificial insemination. The distribution of black (i/i) and yellow (I/i) offspring was recorded to estimate the respective contribution of the recipient and donor to the germline. Therefore, test mating should be used to clarify whether or not the female-pgc-derived spermatozoa are fertile. Detection of W-bearing spermatogenic cells in chimeric testes using in situ hybridization analysis The male chimeric chickens were reared to about 12 months of age to ascertain the developmental fate of female-derived PGC in the testes. The gonad was sectioned to a width of 5 µm. The hybridization mixture of W- chromosome specific probes was applied to the samples on slides. After hybridization, the detection of signals was performed according to the manufacturer's instructions with NBT- BCSP staining. Quantitative analysis of the differentiation of the female-derived PGCs in testes was carried out by counting the numbers of W-chromosome positive and negative cells in the section of testes from the chimeric chickens RESULTS AND DISCUSSION Survival and hatching rates of manipulated embryos The chimeric embryos were generated by the injection of female PGCs into male recipient embryos. Survival and hatching rates of the manipulated embryos are shown in Table 1. Among these 13 chicks, 4 (Nos. G19, G21, G37, G46) were raised to sexual maturity. 17

4 Table 1. Survival and hatching rates of chick embryos following injection of PGCs into embryonic bloodstream Donor PGCs Recipient embryo No. of embryos No. (%) of embryos surviving on days a manipulated Hatched (%) WL (F) BP (M) (65.5) 17 (58.6) 16 (55.2) 9 (31.0) 9 (31.0) BP (F) WL (M) (78.6) 7 (50.0) 7 (50.0) 5 (35.7) 4 (28.6) TAGAMI et al. (2007) The round germ cells with two sets of red and white signals were identified as premelotic spermatocytes, while those with one set were indentified as post-meiotic spermatids. Detection of W-bearing spermatogenic cells in semen W-specific DNA fragments were detected in the spermatozoa and/or detached spermatogenic cells (spermatocytes, round and elongating spermatids) from the seminiferous epithelium of all male. Thus, it was shown that female PGCs had settled in the testes and could be differentiated in the seminiferous tubules of these chimeras (Fig. 2, Table 2). The round germ cells with two sets of red and white signals were identified as premeiotic spermatocytes, while those with one set were identified as post-meiotic spermatids. 18

5 Table 2. Frequencies of spermatozoa and round cells in semen detected with female-specific DNA probes by two-color FISH Chimera ID Total no. of spermatozoa No. (%) of w- spermatozoa Total no. of rounc No. (%) of w- cells a round cells b G (0.058) (31 (44.3) G (0.0) 25 3 (12.0) G (0.012) (23.1) Normal male (0.0) 21 0 (0.0) a Round cells include spermatocytes, round spermatids and early clongated spermatids b W-round cells denote W chromosome-bearing round cells TAGAMI et al. (2007) DONOR CONTRIBUTION TO THE GERMLINE The contribution of donor cells to the germline in these chimeras was estimated from the number of black (i/i) and yellow (I/i) offspring (Table 3). All 3 chimeras failed to produce any female donor-derived offspring. Detection of female-derived PGCs in testes In adult male chimeric chickens, the W- specific sequences were detected in spermatogonia (white arrowhead in a ), spermatocytes (white arrow in a ), and round spermatids (black arrow in b ) (steps 1-3; TIBA et al., 1993) (Fig. 3a-c). However, the W- bearing elongating spermatids (black arrowhead in b ) (steps 4-8; TIBA et al., 1993) were markedly fewer compared to the W- bearing round spermatids. c is a HE staining of adjacent sections of a (Fig. 3). W-bearing spermatozoa (black arrow in d ) were hardly ever observed (Fig. 3d). Serial sections in the testes of germline chimeras stained by digoxygenin-labeled W-chromosome specific sequence as well as hematoxylin and eosin illustrated that a cluster of W-bearing spermatogenic cells coincided with a clump indicating disruption spermiogenesis in a number of seminiferous tubules (Fig. 3e, f). That cluster contained scarcely any elongating spermatid or spermatozoa. These figures showed that the W-bearing round spermatids that could hardly extend their flagellum accumulated and formed the clump of W- bearing round spermatids in the seminiferous tubules of chimeras. Table 3. Number of donor- and recipient-derived offspring form germline chimeras produced by injection of female PGCs into the male embryonic bloodstrem Chimera ID Donor Recipient Sex of chimera No. (%) of offspring derived donor recipient G19 WL (F) BP (M) M 0 (0.0) 566 (100) G46 WL (F) BP (M) M 0 (0.0) 279 (100) G37 WL (F) BP (M) M 0 (0.0) 506 (100) 19

6 Fig 3. Histological examination of mature germline chmeric testes TAGAMI et.al. (2007) The extent of W-bearing spermatogenic cells among all stages was analyzed with the chi-square test (Table 4). The female-pgcderived spermatogonia normally differentiated into spermatocytes in the same way as recipient-pgc-derived spermatogonia. There was no significant difference between the occupation rate of female and male-derived spermatocytes and round spermatids. The differentiation of female-derived spermatocytes into round spermatids is not restricted. 20

7 Table 4. Differentiation of W chromosome-bearing spermatogenetic cells in chimeric testes No. of spermatogenic cells Spermatogonia Spermatocyte Round spermatid Elongating spermatid Spermatozoa Total cells observed W-bearing cells Occupation rate of 30.8 a 32.7 a 28.4 a 7.7 b 0.2 c W-bearing cells (%) Spermatogenic cells were counted in 48 seminiferous tubeles from 3 chimeras (G19, G37, G46); abc Values in rows having different superscripts are significantly different (P < 0.001) Mol. Rprod. Dev. 74, (2007) TAGAMI et al. A small number of W-bearing round spermatids could begin to elongate their nuclei; however, it was difficult for them to accomplish spermiogenesis. The very few W- bearing elongating spermatids that survived to this stage could differentiate into W-bearing spermatozoa. Therefore, the female PGCs in male gonads appeared to be capable of passing through the first and second meiotic divisions, adapting themselves to a male environment, but hardly able to undergo morphological differentiation and development into elongating spermatids. IN CONCLUSION This basic research suggested that avian PGCs are difficult to differentiate into gametes that have fertilizing ability in the opposite sex gonads. Therefore the PGCs should be collected by their sex, when they are cryopreserved for saving avian genetic resources. One of the aims in our laboratory is the establishment of more effective preservation method of avian species using PGCs. We are studying not only basic researches but also applied researches for the aim. SETIOKO et al. (2007) selected the suitable cryoprotectant in attempts to preserve chicken PGCs. NAKAMURA et al. (2008) have improved the efficient removal of PGCs from recipient chicken embryos. These researches are expected to lead to establish the efficient system for saving the avian genetic resources. REFERENCES HAMBURGER, V. and H.L. HAMILTON A series of normal stages in the development of the chicken. J. Morphol. 88: KAGAMI, H., T. TAGAMI, Y. MATSUBARA, T. HARUMI, H. HANADA, K. MARUYAMA, M. SAKURAI, T. KUWANA and M. NAITO 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. 48: KODAMA, H., H. SAITOH, M. TONE, S. KUHARA, Y. SAKAKI and S. MIZUNO Nucleotide sequences and unusual electrophoretic behavior of the W chromosome-specific repeating DNA units of the domestic fowl, Gallus gallus domesticus. Chromosoma 96: NAKAMURA, Y., Y. YAMAMOTO, F. USUI, Y. ATSUMI, Y. ITO, T. ONO, K. TAKEDA, K. NIRASAWA, H. KAGAMI and T. TAGAMI Increased Proportion of Donor Primordial Germ Cells in Chimeric Gonads by Sterilization of Recipient Embryos using Busulfan-Sustained-release Emulsion in Chicken. Reprod Fertil Dev. (in press). PERRY, M.M A complete culture system for the chick embryo. Nature 331: SAITOH, Y., H. SAITOH, K. OHTOMO and S. MIZUNO Occupancy of the majority of DNA in the chicken W chromosome by bent-repetitive sequences. Chromosoma 101:

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