臺灣博碩士論文加值系統

動物之射出精子內含有信使RNA（messenger RNA, mRNA）之事實，已於人、鼠與牛等物種被證明；推論此等mRNA之出現，其功能可能涉及受精卵或胚之早期發育、訊息傳遞，甚或癌症發生等基因表現之調控。鑑於台灣地處亞熱帶，夏季高溫多濕，遂公豬在夏季時射出之精液品質常有低落之情事；惟存在於公豬精子內之mRNAs，其質與量是否隨著冬季與夏季而有顯著之不同，進而影響精子於完成受精作用後，受精卵早期發育所需重要基因之表現，則迄未有明。
本論文旨在針對臺灣地區飼養之公豬，探討其射出精子中含有mRNAs之質與量，是否係隨著季節（冬季，11月~翌年2月；夏季，6月~ 8月）之不同而有顯著性之差異。試驗之進行包括：1. 首先將源自性成熟公豬收集獲得之新鮮射出精子（freshly ejaculated spermatozoa），分別完成其cDNA基因庫之構築，並透過5′端定序與身份註解；2. 續而依據業經建立之表現序列標記株系（expressed sequence tags, ESTs）資料庫，完成豬精子特異性表現探針（probe）之建構於寡核苷酸微陣列晶片（oligonucleotide microarray）；3. 最後再針對冬、夏兩季各公豬射出精子之mRNAs標的物（target）進行雜合試驗，並進一步詳實分析比較各公豬於冬、夏兩季射出精子中基因表現之差異性。
試驗結果證明：1. 公豬之射出精子中確有mRNAs存在之事實；2. 將獲得之4,562條序列，進一步完成序列比對與群集分析（cluster analysis）；結果獲得獨特序列（unique sequences）合計514條，其中包括：188條序列為組合片段（contigs；36.58％）者，與326條為單一序列（singletons；63.42％）者；3. 為謀先行推論前述完成構築之公豬射出精子的cDNA基因庫中，各表現序列之生理角色，本研究乃進一步透過基因功能分類體系（Gene Ontology, GO）資料庫，完成各表現序列可能扮演生理角色之功能性分類預測分析；分析結果顯示，前述表現序列之可被歸納於參與分子功能（molecular function）、細胞組成（cellular component）與生物作用（biological process）等各分項功能者，其百分率分別為39.1、40.3，與38.7％。4. 另外，就公豬於冬季與夏季之射出精子中含有寡核苷酸微陣列晶片之差異性比較分析試驗而言，結果證明在公豬射出精子所測得之表現序列中，特別於冬季與夏季彼此間，具有顯著性差異表現之序列者，合計多達67個之譜；其中有33個基因，經進一步透過基因功能分類體系，完成第四層級生物作用功能之分項分析描述，並確認多數係屬於涉及調控細胞代謝相關功能者。
綜合上述，本研究證明在亞熱帶地區之臺灣，公豬射出之精子中確實含有眾多mRNAs，且其mRNAs之表現圖譜可因季節（冬季 vs 夏季）之不同而有顯著性之差異；未來設若能進一步針對彼等在冬季與夏季呈現顯著性差異表現之候選基因，分別詳加探討並釐清其在調控雄性動物生育力相關作用中，可能扮演之功能角色，則可望提供做為選拔優異種用公豬之良好分子標幟。

While the existence of specific messenger RNA (mRNA) remnants contained within freshly ejaculated spermatozoa were described in several species, most of these studies were conducted by the use of high-throughput techniques for screening the population of transcripts from the ejaculated spermatozoa, and these studies frequently confronted a major hindrance by the limitation of probes which mostly derived from nucleic acids of either human or mice testicular tissues. In the present study, attempts were made to investigate mRNA remnants from swine ejaculated spermatozoa harvested during both winter and summer seasons. It is anticipated that an available technique platform and a valuable model would be established not only for comparison the global variety of mRNA remnants expressed during both winter and summer seasons but also for meeting those purposes of biomedical research requested.
In the initial studies, a non-redundant 5´-end complementary DNA library was generated from swine ejaculated spermatozoa. After sequence quality verification, 4,562 clones remained. These clones were then clustered and assembled into 514 unique sequences including 188 contigs (36.58%) and 326 singletons (63.42%), representing those clusters containing at least two clones and those clusters without having enough similarity with other clones. These unique gene sequences were annotated in gene ontology (GO); they included biological processes (38.7%), molecular functions (39.1%) and cellular components (40.3%). Based on the analysis, a broad spectrum of messenger RNAs existed in swine ejaculated spermatozoa and was closely correlated with nucleic acid binding, structural modifications, and transcriptional regulation. In the microarray analysis, 67 transcripts were differentially expressed with statistical differences between seasons of sperm samples collected, including forty-nine in winter (49/67) and eighteen in summer (18/67), respectively. There were only 33 of these transcripts could be annotated to gene ontology hierarchy with the database of Homo sapiens and their functions mostly were involved in variety of metabolic processes. Moreover, these studies also confirmed that significant differences of gene expression profiles were found in swine spermatozoa when comparisons were made between ejaculates collected during the winter and the summer season under the subtropical area such as Taiwan.
The conclusion came to these present studies was that the spermatozoal RNAs provides a useful molecular tool in functional genomic studies of the male reproductive system, in evaluating the testis function and in developing new selection makers for swine industry.

口試委員會審定書............................................................................................ I
誌謝....................................................................................................................... II
摘要........................................................................................................................ III
ABSTRACT............................................................................................................ V
LIST OF FIGURES............................................................................................... XI
LIST OF TABLES.................................................................................................. XIII
CHAPTERS 1 INTRODUCTION ..................................................................... 1
CHAPTERS 2 REVIEW OF LITERATURES.................................................... 4
2.1 The Spermatogenesis.............................................................................. 4
2.1.1 Spermatocytogenesis Phase........................................................ 6
2.1.2 Spermiogenesis Phase............................................................... 8

2.1.3 Gene Expression Profiling during Mammalian Spermatogenesis……………………….…............................... 9

2.1.4 Recent Evidences for Spermatozoal RNAs............................... 13
2.2 Methods for Profiling Gene Expression at the Transcriptome Level..... 17
2.2.1 Direct Sampling of Sequences.................................................. 18
2.2.2 DNA Microarray..................................................................... 21
2.2.2.1 Oligonucleotide Microarray.................................. 23
Page

2.2.2.2 Spotted Microarray..................................................... 26
2.2.2.3 Platform Comparison.................................................. 30
CHAPTERS 3 SEQUENCING AND IDENTIFICATION OF REMNANT MESSENGER RNAS FOUND IN FRESHLY EJACULATED SPERMATOZOA OF DOMESTIC SWINE (Sus Scrofa) ............................................................................ 31

3.1 Objective...................................................................................................... 31
3.2 Materials and Methods................................................................................ 32
3.2.1 Spermatozoa Collection................................................................. 32
3.2.2 Total RNA Extraction and Quality Control.................................... 33
3.2.3 Expressed Sequence Tag Library Construction.............................. 34
3.2.4 DNA Sequencing............................................................................ 36
3.2.5 Sequence Analysis and Functional Annotation.............................. 36
3.2.6 Northern Blot Analysis................................................................... 38
3.2.7 Reverse Transcription-Polymerase Chain Reaction....................... 38
3.3 Results......................................................................................................... 39
3.3.1 Evidence for RNA Existence in Spermatozoa................................ 39

3.3.2 Construction and Characterization of a Swine Spermatozoal
Expressed Sequence Tag Library................................................... 39
3.4 Discussion...................................................................................................... 45
Page

3.4.1 Transcriptional and Translational Characteristics of Spermatozoa.................................................................................... 45

3.4.2 The Function of Spermatozoal mRNA Transcripts Related to
Early Embryo Development........................................................... 49

3.4.3 Variation in Spermatozoal Protamine Transcripts........................... 51
3.4.3.1 Protamine P1 (PRM1).................................................. 51
3.4.3.2 Protamine P2 (PRM2).................................................. 52 3.5 Summary..................................................................................................... 54

CHAPTERS 4 SEASONAL EFFECT ON SPERMATOZOAL MESSENGER RNA PROFILE OF DOMESTIC SWINE (Sus scrofa) .......................................................................... 55

4.1 Objective..................................................................................................... 55
4.2 Materials and Methods................................................................................ 56
4.2.1 Spermatozoa Collection.................................................................. 56
4.2.2 Target RNA Preparation for Microarray......................................... 58
4.2.3 Microarray Analyses of Gene Expression Profiles......................... 60

4.2.4 Reverse Transcription Real-Time Polymerase Chain Reaction Analyses......................................................................................... 61

4.3 Results......................................................................................................... 63
4.3.1 Seasonal Variation in Gene Expression........................................... 63
4.3.2 Validation of Microarray Data........................................................ 63
Page

4.4 Discussion................................................................................................... 66

4.4.1 Utilization of Spermatozoal mRNA Profile as a Potential
Investigating Tool............................................................................ 66

4.4.2 Effects of Elevated Environment Temperature on Reproduction
Efficiency....................................................................................... 68

4.4.3 Serine/Threonine Protein Kinase Essential for Male Fertility........ 70
4.4.3.1 Testis-Specific Serine Kinase 6 (TSSK6) .................... 71
4.4.3.2 Testis-Specific Protein Kinase 1 (TESK1).................... 72
4.5 Summary.............................. ...................................................................... 73
CONCLUSION AND FUTURE WORK.................................................................. 74
REFERENCES........................................................................................................... 75
APPENDICIES......................................................................................................... 102

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