| By using distant hybridization and subsequently selective breeding,allotetraploid(4n=200,abbreviated 4nAT)population has been originated from red crucian carp(Carassius auratus red var.,abbreviated RCC)× common carp(Cyprinus carpio L.).The artificial triploid crucian carp(abbreviated as 3nCC)was then obtained by hybridization between the 4nAT(male)and diploid RCC(female).An natural triploid crucian carp(3n=150,abbreviated 3nDTCC)in the Dong Ting lake of China also had been reported,the male and female 3nDTCC were all fertile which mated each other to produce offspring.Those sterile 3nCC and fertile 3nDTCC provided good materials to comparative analyze the meiotic configuration during gametogenesis.In the present study,we studied the cytological characteristics of spermatogenesis in male sterile 3nCC and fertile 3nDTCC through the observation of testis histology,ultrastructure,chromosome spreads of germ cells,and FISH(Fluorescence in situ hybridization)analysis,germ cell apoptosis verification,in which the meiosis characteristics between fertile triploid and sterile triploid had been compared.The results would give new insights why the different triploid fish were fertile or not.The main contents of this study were:1.The testis of 3n DTCC and 3n CC in the second breeding season were selected for histological observation.It was found that there were a large number of spermatogonia,primary spermatocytes and apoptotic spermatocytes in 3nCC,and no mature sperm was found.However,there were a large number of mature sperm and a small number of spermatogonia and primary spermatocytes in 3nDTCC could be found.2.Using transmission electron microscope(TEM)to compare the testes,ultrastructure of 3nDTCC and 3nCC.It was found that there were a large number of spermatogonia,primary spermatocytes and postapoptotic germ cells in 3n CC,in which the development process of secondary spermatocytes,spermatids and mature spermatids after the metaphase of the first meiosis was not observed;In the testis of 3n DTCC,a large number of mature spermatozoa and the flagellum structure of "9 + 2" microtubule arrangement was found.3.Using peripheral blood cells of triploid wild crucian carp as control,the DNA content of mature sperm of 10 3nDTCC was detected by flow cytometry.The results showed that 9 of 10 3n DTCC samples produced gametes with 1.5n DNA content,and 1 sample produced mixed gametes with 1n and 2n DNA content.4.The apoptosis of germ cells in testis of 3nDTCC and 3nCC was compared by TUNEL(TdT-mediated dUTP Nick-End Labeling)method.The results showed that the high incidence of apoptotic spermatocytes in 3nCC,while 3nDTCC testis sometime showing very low levels of apoptosis could be found.5.To observe the difference of cytological characteristics of 3n CC and 3nDTCC in meiosis phase by chromosome spread observation.During prophase of meiosis I pachytene,half chromosomes in pachytene were thick and some others were slim in 3n CC,after they went to metaphase,50 bivalents and other unsynapsed univalents could be found.When it came to 3nDTCC,the chromosomes in pachytene showed uniform thickness,and after they went to metaphase of meiosis?,no obvious bivalent could be found,the chromosome spreads with 150 chromosomes being easily detected,which were looked unpaired.6.The chromosomes of 3nDTCC and 3nCC were analyzed by FISH using 340 bp 5S rDNA repeat as probes.5S rDNA localization in meiosis chromosome spreads of 3nCC showed that two strong signals were detected in a pair of chromosomes with a few weak signals being detected in other chromosomes,while that in 3nDTCC showed there were 3-6 strong signals in the separated-distributing chromosomes,with a few weak signals being detected in other chromosomes,in which no chromosome pairings could be found. |
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