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1 J. Exp. Biomed. Sci. 10 (2004) Seminiferous Epithelium Cycle in the Korea Squirrel, Tamias sibiricus Tae-Dong Jung and Jung-Hun Lee Division of Biology and Chemistry, College of Natural Scineces, Kyungnam University, Kyungnam , Korea The annual changes in testis weight and diameter of seminiferous tubules, and the seminiferous epithelium cycle of Tamias sibiricus were studied by light microscope. Testis weight and diameter of seminiferous tubule are significantly increased from January to July, and decreased rapidly to the size from August to December. Spermatogenesis occurs from January to July, and spermatocytogenesis are produced from August to December. The cycle of the seminiferous epithelium was divided into 12 stages during the development of spermatids as a changes of the nucleus and acrosomal structure, presence and/or absence of residual body, appearance and/or absence of sperm tail and meiotic figure and spermiation. The dark type spermatogonia (Ad) are appeared in all stages (I~XII), and the spermatids of step 10 are observed at I, II, X and XII stages. The spermatids of step 11 are appeared in III and IV stages, only the step 12 spermatid observed in V stage. Key Words: Seminiferous epithelium cycle, Sperm, Spermatogenesis, Tamias sibiricus 서 다람쥐는설치목 (Order Rodentia), 청설모과 (Family Sciuridae), 다람쥐속 (Genus Tamias) 에속하며, 세계적으로아시아중 북부와북미에서식분포하고있다. 한반도에는청설모속 (Genus Sciurus) 청설모 (Sciurus vulgaris), 다람쥐속 (Genus Tamias) 다람쥐 (Tamias sibiricus), 하늘다람쥐속 (Genus Pteromys) 하늘다람쥐 (Pteromys volans) 3속 3종이자연적으로분포하고있는것으로보고되어져있다 (Corbet & Hill, 1991). 청설모과다람쥐는다른동면동물들과같이늦가을부터초봄까지동면하며, 봄부터초가을에걸쳐활동을한다 (Bushberg & Holmes, 1985). 다람쥐는동면동안에는정상적인체온을벗어나변동하는특성을보이는데, 이기간동안주기적으로둔마상태 (torpor) 를겪게되고, 이러한둔마상태는개체마다다르게나타나며, 정자형성의분화시기에많은영향을미친다 (Barnes et al., 1986). 정소의성장은세정관상피의분화에밀접한관련을가지며, 정소의성장이활발한시기에는정자형성과정의전단계들이세정관내에관찰된다. 한편, 야생의경우정소의성 * 논문접수 : 2004 년 7 월 14 일수정재접수 : 2004 년 8 월 16 일 Corresponding autho: Jung-Hun Lee, Division of Biology and Chemistry, College of Natural Scineces, Kyungnam University, Kyungnam , Korea. Tel: , Fax: jhlee@kyungnam.ac.kr 론 장은개체마다차이를나타날뿐만아니라사육의경우에도조금씩변화를보인다 (Foreman, 1997). 세정관상피의주기는연속하는상피에나타나는성숙변화의과정을의미하는것으로, 포유동물에서세정관상피의주기를구분하기위하여일반적인두가지방법을사용한다. 첫번째로는정자세포핵의형태, 성숙정자위치, 감수분열과정자변태등을기초로하여구분하는것과, 두번째방법은정자세포핵과첨체 (acrosome) 의형태적변화에기초를두고구분하는것이있는데, 특히정자세포첨체의발달변화는세정관상피의주기를구분하는데많이사용된다 (Adachi et al., 1992; Kurohmaru et al., 1998). 한편, 우리나라에서식하고있는청설모과에속하는종들의생식에관한연구는아직보고된바없으므로본연구에서는다람쥐 (Tamias sibiricus) 의월별변화에따른정소와세정관의크기변화및세정관상피의주기를알아보기위하여시도하였다. 재료및방법본연구는 2002년 9월부터 2003년 8월까지강원도고성군일대에서서식하고있는다람쥐 (Tamias sibiricus) 성체수컷들로서, 월별로 2개체씩총 24마리를실험재료로하였다. 월별로채집된다람쥐들은 ethyl ether로마취시킨후각개체의정소조직을적출하여 3% glutaraldehyde (4, ph 7.4, Millong's buffer) 로 2시간전고정한후동일한완충액 (ph

weight. 7.4, Millong's buffer) 으로 3회세척한뒤 1.33% OsO 4 (ph 7.4, Millong's buffer) 2시간후고정하였다. 고정이끝난조직편들은동일한완충액으로세척한다음 acetone 농도상승순 (60~100%) 으로탈수하였고, 탈수가끝난조직은 Epon 812 혼합액으로포매하였다.

2 Table 1. Annual changes of testis weight and seminiferous tubule diameter in Tarmias sibiricus Left y-axis indicates the size of diameter of the seminiferous tubule, and right y-axis indicates the testis weight. 7.4, Millong's buffer) 으로 3회세척한뒤 1.33% OsO 4 (ph 7.4, Millong's buffer) 2시간후고정하였다. 고정이끝난조직편들은동일한완충액으로세척한다음 acetone 농도상승순 (60~100%) 으로탈수하였고, 탈수가끝난조직은 Epon 812 혼합액으로포매하였다. 포매가끝난조직편을 ultramicrotome (MT-6000, Sorvall, Dupont) 을사용하여정소와세정관의크기및세정관상피의분화와주기를알아보기위하여이들조직편들을 400 nm의두께로절편을제작한다음 0.5% toluidine용액으로염색후광학현미경으로관찰하였다. 결과 1. 월별에따른정소무게와세정관크기월별에따른정소무게는 3월의실험군 (0.25 g) 을제외하고는 1월부터 7월까지는 (1월: 0.8 g; 2월 : 0.84 g; 4월 : 0.5 g; 5월 : 0.81 g; 6월 : 0.68 g; 7월 : 0.67 g) 높은수치를나타내었고, 8월에서 12월까지는 (8월: 0.17 g; 9월 : 0.11 g; 10월 : 0.09 g; 11월 : 0.1 g; 12월 : 0.3 g) 낮은수치를나타내었다. 또한세정관직경의크기변화도 1월부터 7월까지는 (1월: 142~172 µm; 2월 ; 146~178 µm; 3월 ; 108~140 µm; 4월 : 105~140 µm; 5월 : 140~ 65 µm; 6월 : 135~160 µm; 7월 : 110~140 µm) 높은수치를보이는반면에, 8월부터 12월까지 (8월: 55~70 µm; 9월 : 60~64 µm; 10월 : 60~64 µm; 11월 : 60~65 µm; 12월 : 70~100 µm) 낮은수치를나타내었다 (Table 1). 2. 월별에따른세정관정상피의분화다람쥐의세정관의변화와정상피의분화는 1월부터 7월까지세정관의내강은열려져있었으며정자형성과정의전단계가관찰된반면에, 8월부터 12월까지는정모세포발생단계까지만관찰되어졌다 (Table 2). 3. 세정관정상피주기세정관정상피의분화가활발한시기는 1월부터 7월까지이며, 본연구에서 5월실험군을토대로하여세정관정상피주기를 XII단계로나눌수있었다 (Figs. 1-13). 1) 단계 I 정원세포 ( 어두운정원세포 : Ad형, dark type; 밝은정원세포 : Ap형, pale type) 가기저막에인접하고있었으며, 제1정모세포의후사기 (pachytene) 를비롯하여, 골지기의초기단계인정자세포 (St 1 ) 와성숙중인정자세포 (St 10 ) 들이관찰되었다 (Fig. 1). 2) 단계 II 세정관내에는 I단계와마찬가지로 Ad형과 Ap형의정원세포들이기저막에가까이위치하고있었고, 제1정모세포의후사기및다수의골지기의중기단계인정자세포 (St 2 ) 와성숙중의정자세포 (St 10 ) 들이관찰되었다 (Fig. 2). 3) 단계 III 중간정원세포 (In형정원세포 : Intermediate spermatogonia)

3 Table 2. Degree of appearance of spermatogeneic cell types in the seminiferous tubule of Tarmias sibiricus by monthly Ad, Ap, In and B, dark, pale, intermediate and B types of spermatogonia. 가기저막인접부위에존재하고있었으며, 제1정모세포의후사기, 그리고다수의골지기의후기단계인정자세포 (St 3 ) 와성숙기의후기단계인정자세포 (St 11 ) 들이관찰되었다 (Fig. 3). 4) 단계 IV 세정관내에는 B형정원세포 (B type), 제1정모세포단계의후사기, 두모기의초기단계인정자세포 (St 4 ) 와성숙기의후기단계인정자세포 (St 11 ) 들이관찰되었다 (Fig. 4). 5) 단계 V B형정원세포와제1정모세포의후사기를비롯하여두모기의중기단계인정자세포 (St 5 ) 들과이탈단계의정자세포 (St 12 ) 들이관찰되었다 (Fig. 5). 6) 단계 VI 기저막가까이에는 B형정원세포와제1정모세포의후사기를비롯하여두모기의후기단계인정자세포 (St 6 ) 가다수관찰되었다 (Fig. 6). 7) 단계 VII 제1정모세포의세사기 (leptotene) 를비롯하여, 후사기및첨체기의초기단계인정자세포 (St 7 ) 들이관찰되었다 (Fig. 7). 8) 단계 VIII 제1정모세포의세사기와후사기의세포를비롯하여첨체기의중기단계인정자세포 (St 8 ) 들이관찰되었다 (Fig. 8). 9) 단계 IX 극소수의제1정모세포의세사기를비롯하여후사기그리고첨체기의후기단계인정자세포 (St 9 ) 들이다수존재하고있었다 (Fig. 9). 10) 단계 X 제1정모세포의세사기, 후사기, 그리고성숙기의초기단계인정자세포 (St 10 ) 들이다수관찰되었다 (Fig. 10). 11) 단계 XI 세정관내에는극소수의제1정모세포의접합기와이동기 (diakinesis) 및성숙기의초기단계인정자세포 (St 10 ) 들이관찰되었다 (Fig. 11). 12) 단계 XII 기저막인접부위에제1정모세포의접합기, 제1감수분열단계의중기상들이다수관찰되었고, 성숙기의초기단계인정자세포 (St 10 ) 들도다수관찰되었다 (Fig. 12). 고찰포유동물에있어서세정관상피의주기는정자세포핵의형태, 성숙정자위치, 감수분열과정자이탈등을기초로하여구분하는방법 (Foote et al., 1972; Grocock & Clarkel., 975; Tait & Johnson, 1982), 다른하나는정자세포의첨체형태변화에기초를두어구분하는방법이있다 (Kurohmaru et al.,

4 Figs Light micrographs showing successive stages of spermatogenic cells in the seminiferous tubule of Tamias sibiricus. All scale bars = 20 µm Ad, Ap, In and B, dark, pale, intermediate and B types of spermatogonia; L, leptotene spermatocyte; Z, zygotene spermatocyte; P, pachytene spermatocyte; D, diakinesis spermatocyte; M1, metaphase of meiosis 1; St 1, St 2 and St 3, round spermatids of Golgi phases; St 4, St 5 and St 6, spermatids of cap phases; St 7, St 8 and St 9, spermatids of acrosomal phases; St 10 and St 11 ; spermatids of maturation phases; St 12, sperm of the spermiation phase. Fig. 1. Light micrograph of the stage I of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing spermatogonia (Ad and Ap), pachytene spermatocytes (P) and early round spermatid (St 1 ) and mature spermatids (St 10 ). Fig. 2. Light micrograph of the stage II of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing spermatogonia (Ad and Ap), pachytene spermatocytes (P) and early round spermatid (St 2 ) and mature spermatids (St 10 ). Fig. 3. Light micrograph of the stage III of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) and intermediate (In) types of spermatogonia, pachytene spermatocytes (P) and numerous early round spermatid (St 3 ) and mature spermatids (St 11 ). Fig. 4. Light micrograph of the stage IV of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) and B types of spermatogonia, pachytene spermatocytes (P) and numerous early round (St 4 ) and mature spermatids (St 11 )

5 Fig. 5. Light micrograph of the stage V of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) and B types of spermatogonia, pachytene spermatocytes (P) and cap step (St 5 ) and spermatids of spermiation (St 12 ). Fig. 6. Light micrograph of the stage VI of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) and B types of spermatogonia, pachytene spermatocytes (P) and spermatid of acrosomal step (St 6 ). Fig. 7. Light micrograph of the stage VII of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, leptotene (L) and pachytene spermatocytes (P) and spermatid of acrosomal step (St 7 ). Fig. 8. Light micrograph of the stage VIII of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, leptotene (L) and pachytene spermatocytes (P) and elongating spermatids (St 8 ). 고 찰

6 Fig. 9. Light micrograph of the stage IX of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, leptotene (L) and pachytene spermatocytes (P), and elongating spermatids (St 9 ). Fig. 10. Light micrograph of the stage X of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, leptotene (L) and pachytene spermatocytes (P) and nemerous maturating spermatids (St 10 ). Fig. 11. Light micrograph of the stage XI of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, zygotene (Z) and pachytene spermatocytes (P) and nemerous maturating spermatids (St 10 ). Fig. 12. Light micrograph of the stage XII of the squirrel seminiferous cycle. Squirrel seminiferous epithelium showing dark (Ad) type of spermatogonia, zygotene (Z) and metaphase of meiosis I and nemerous maturating spermatids (St 10 )

7 Fig. 13. Schematic illustration of 12 stages in the squirrel seminiferous cycle. Roman numerals indicate each stage. Ad and Ap, dark and pale type of spermatogonia; In and B, Intermediate and B type of spermatogonia; L, Z, P and D, leptotene, zygotene, pachytene and diakinesis spermatocyte; MI, primary spermatocytes in metaphase of meiosis 1; St( 1-12 ), spermatids during spermiogenesis. 1988; Adachi et al., 1992; Paula et al., 1999; Mizukami et al., 2001; Segatelli et al., 2002). 본연구에서는다람쥐 (Tamias sibiricus) 의세정관주기를첨체의형태적변화와정자세포핵의형태, 감수분열, 정자세포와정자의위치에기초를두고조사한결과 12단계로구분되어졌다 (Fig. 13). 세정관주기에관한연구에서볼때, rat (Leblond & Clermont, 1952) 는 14단계로, musk shew (Kurohmaru et al., 1994), cotton rat (Kurohmaru et al., 1988) 그리고 wild squirrel (Patil & Saidapur, 1991) 는각각 13단계로, watase's shrew (Adachi et al., 1992), greater Japanese shrew mole (Mizukami et al., 2001),

8 chinese hamster (Oud & Rooij, 1977) 그리고 Mongolian gerbil (Segatelli et al., 2002) 는각각 12단계로, prairie dog (Foreman, 1997) 와 mole (Sanchez et al., 1995) 는각각 10단계로, vole (Grocock & Clarke, 1975), capybaras (Paula et al., 1999) 와 grey squirrel (Tait & Johnson, 1982) 는각각 8단계로보고되었다. 이러한단계적차이는종특이성에기인하는것으로여겨진다. 본연구에서정소의크기변화와세정관직경의크기는 3월을제외하고 1월부터 7월까지는 (0.5~0.8 g, 121~162 µm) 높은수치를나타내었고, 8월부터 12월까지는 (0.09~0.3 g, 61~86.5 µm) 낮은수치를나타냈다. 이는정소크기와세정관직경크기는비례관계가성립하며이러한사실은정자형성세포의분화과정과밀접한관련성이있음을시사한다. 실험개체군의몸무게는 60~105 g ( 평균 : 90 g) 정도로나타났으나, 3월의실험군몸무게는최저인 60 g을나타내고, 본실험결과에서세정관직경의크기와정소의무게가낮은수치를나타내었다. 이러한결과는체중은정자형성과는무관함을알수있다. Ground squirrel (Bushberg & Holmes, 1985) 는동면기간의체중은번식시기에성적성숙에중요하다고시사하고있는데, 본연구결과를미루어보아체중이정자형성을직접적인영향을미치지는않는것으로사료된다. 또한동면기간동안체온이 (3~4 ) 떨어지는둔마상태는주기적인중단상태에서때로는평상수준 (35 ) 으로되돌아오는데, 이러한낮은체온은유지는동면을위한에너지절약과먹이의결핍에대한적응전략이라여겨진다. 성적활성의경우, prairie dog (Foreman, 1997) 와 Grey squirrel (Tait & Johnson, 1982) 는겨울철에정소내의정상피분화의활성이높게나타나는반면에, Ground squirrel (Barnes et al., 1986; Foreman, 1998) 는봄철에그활성이높게나타난다. Ground squirrel의경우, 동면시기에세정관상피의생식세포들은변화가없고정소의크기는서서히증가하고, 정소의성장은동면이끝나는시기에성장한다 (Barnes et al., 1996). 그러나본연구에서다람쥐 (Tamias sibiricus) 는 prairie dog와마찬가지로정소의크기가동면전까지는큰차이를보이지않았으나, 동면시기에는정소의성장이증가하였고, 세정관상피의조직변화는활동기에는다수의정원세포와감수분열전기의 1차정모단계세사기 (Leptotene), 접합기 (Zygotene), 후사기 (Pachytene) 세포들이관찰되었다. 이상실험결과를종합해볼때, 월별에따른다람쥐의정소무게와세정관직경의크기변화그리고세정관상피주기에대한소견에서정소무게와세정관직경의크기변화는상관성이있었다. 또한, 1년을통한세정관정상피의분화의경우에는동면기인 1월부터 3월까지그리고활동기인 4월부터 7월까지는정자형성과정의전단계를확인할수있었다. 특히암컷은이기간동안 2회에걸쳐새끼를분만 (5월 과 8월 ) 하는데, 이러한사실로미루어보아한국산다람쥐 (Tamias sibiricus) 수컷의생식세포의분화조절은새끼분만과양육의최적조건을만족시키기위한일종의번식메커니즘이라고여겨진다. 감사의글본연구는 2003학년도경남대학교학술논문게재연구비지원에의해서이루어졌음. REFERENCES Adachi Y, Kurohmaru MA, Hattori S, Hayashi Y. Spermatogenesis in the watase's Shrew (Crocidura watasei): A light and electron microscopic study. Exp Anim : Barnes BM, Kretzmann M, Licht P, Zucker I. The Influence of hibernation on testis growth and spermatogenesis in the golden mantled ground squirrel (Spermophilus lateralis). Biol Reprod : Bushberg DM, Holmes WG. Sexual maturation in male belding's ground squirrel: Influence of body weight. Biol Reprod : Corbet GB, Hill JE. A world list of mammalian species: In Genus Sciurus pp Oxford University Press. Ames. Dubock AC. Male grey squirrel (Sciurus carolinensis) reproductive cycles in Britain. J Zool Lond : Foote RH, Swierstra EE, Hunt WL. Spermatogenesis in the dog. J Anat : Foreman D. Seminiferous tubule stages in the prairie dog (Cynomys ludovicianus) during the annual breeding cycle. Anat Record : Foreman D. Effects of exogenous hormones on spermatogenesis in the male prairie dog (Cynomys ludovicianus). Anat Record : Grocock CA, Clarke JR. Spermatogenesis in mature and regressed testes of the vole (Microtus agrestis). J Reprod Fert : Kurohmaru M, Kobayashi H, Hattori S, Nishida T, Hayashi Y. Spermatogenesis and ultrastructure of a peculiar acrosomal formation in the musk shrew (Suncus murinus). J Anat : Kurohmaru M, Tiba T, Nishida N, Hayashi Y. Spermatogenesis and ultrastructural changes of spermatids during spermiogenesis in the cotton rat (Sigmodon hispidus). Okajimas Folia Anat Jpn : Leblond CP, Clermont Y. Definition of the stage of the cycle of

9 the seminiferous epithelium in the rat. Ann New York Acad Sci : Mizukami T, Kuwahara S, Ohmura M, Iinuma Y, Izumikubo J, Hagiwara M, Kurohmaru M, Hayashi Y, Nishida T. The cycle of the seminiferous epithelium in the greater Japanese shrew mole (Urotrichus talpoides). J Vet Med Sci : Oud JL, Rooij DE. Spermatogenesis in the chinese hamster. Anat Rec : Patil SB, Saidapur SK. Kinetics of spermatogenesis in the wild squirrel (Funambulus palmarum Linnaeus). Acta Anat : Paula TAR, Chiarini-Garcia H, Franca LR. Seminiferous epithe- lium cycle and its duration in capybaras (Hydrochoerus hydrochaeris). Tissue & Cell. 1999: 31: Sanchez A, Stamatopoulos C, Redi CA. Descriptive kinetics of the seminiferous epithelium cycle and genome size in the mole (Talpa occidentalis, Insectivora). J Exp Zool : Segatelli TM, Almedia CCD, Pinheiro PFF, Martinez M, Padovani CR, Martinez FE. Kinetics of spermatogenesis in the Mongolian gerbil (Meriones unguiculatus). Tissue & Cell : Tait AJ, Johnson E. Spermatogenesis in the grey squirrel (Sciurus carolinensis) and changes during sexual regression. J Reprod Fert :

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