人類BOLL基因屬於DAZ 基因家族中的祖先基因，在人類造精過程中的減數分裂過程扮演重要的角色。BOLL的蛋白質結構中在胺基酸端含有一段RNA-recognizing motif，所以可能具有穩定mRNA或者是調控轉譯的功能。果蠅中的Twine基因，屬於CDC25磷酸酶的家族，表現量和細胞內的Boule有相關性，而在Boule 突變種 ，Twine的含量會有意義地下降，因此在果蠅的實驗中，Boule被認為可以藉由和Twine mRNA直接結合，影響Twine的表現。這次的研究目的主要是要探討人類造精過程中，CDC25A這個基因的表現並且將BOLL和CDC25A兩基因之間的關係闡明。
我們分別以免疫組織染色技術和real time-PCR技術來達成40位無精蟲症病人的睪丸中CDC25A蛋白質和mRNA的表現量偵測。人類精蟲的CDC25A表現則以西方墨點技術和免疫螢光染色來觀察。BOLL蛋白質的表現也是用免疫組織染色技術，而以I.O.D (integrated optical density) 為單位來表示訊號的強度。BOLL蛋白質表現量和CDC25A mRNA量兩者的相關性則是以計算Pearson correlation coefficient來表示。我們對造精功能正常男人的睪丸切片做雙免疫螢光染色，來探求BOLL和CDC25A是否有colocolization的現象。另外，利用HeLa細胞中過表現 BOLL並接下去做RNA-免疫沉澱實驗來探求BOLL protein和CDC25A mRNA是否同時存在。
CDC25A蛋白質主要表現在spermatocyte, spermatid甚至連正常男性精液的精蟲中也有豐富表現。在造精功能障礙病人中，CDC25A的轉錄量會有意義的降低。 另外，CDC25A轉錄量愈高，後續接受取精手術成功率也愈高。BOLL蛋白質的表現量同樣的在造精功能障礙組顯著降低。經過統計的計算，BOLL蛋白質的表現量和CDC25A轉錄量存在顯著的正相關。( r=0.5858, P=0.0013) 雙免疫螢光染色發現BOLL和CDC25A同時表現在人類睪丸切片中primary spermatocyte的細胞質中。當在人類HeLa cell過度表現BOLL，利用RNA免疫沉澱技術可以證實CDC25A mRNA和BOLL protein有同時連結存在。
在這個實驗中，我們證實了不孕症男性造精功能障礙的病患中睪丸切片CDC25A的表現量會顯著下降。我們也直接的證實在人類細胞中，BOLL蛋白質和CDC25A mRNA會聯結在一起。因此，我們認為人類造精過程中CDC25A扮演BOLL的下游受質，BOLL調控CDC25A得以使細胞進入減數分裂。

Human BOLL is the ancestor gene of DAZ gene family and crucial for meiosis completion in human spermatogenesis. The BOLL protein contains a RNA-recognizing motif (RRM) at its amino terminus, suggesting a functional role in mRNA stability or translation regulation. In Drosophila, protein expression of Twine, which is a Cdc25-type phosphatase, correlates with the intracellular accumulation of Boule and is significantly reduced by Boule mutants, indicating that Boule could influence Twine expression through direct binding to the twine mRNA in Drosophila. This study was performed to investigate the CDC25A expression and unravel the relationships between BOLL and CDC25A in human spermatogenesis.

The protein expressions and mRNA transcript levels of CDC25A in the testes of 40 azoospermic men were determined by immunohistochemical staining and quantitative real time-polymerase chain reaction. The protein expression of CDC25A in human spermatozoa was investigated by Western blotting and immnunoflorescence staining. BOLL expressions were acquired by immunohistochemical staining and the signals were interpreted as I.O.D. Pearson product moment correlation coefficient was used to determine the correlation between the protein levels of BOLL and transcript level of CDC25A. The colocolization of BOLL and CDC25A protein in the testis of men with normal spermatogenesis was investigated by double immunofluorescence staining. Over-expression of BOLL and RNA immunoprecipitation (IP) in HeLa cells were also applied to evaluate the co-existence of BOLL protein and CDC25A mRNA.

The CDC25A protein was expressed mainly in spermatocyte, spermatid as well as spermatozoa. CDC25A transcript levels were significantly decreased in patients with spermatogenic failure. Significantly higher CDC25A transcript levels were detected in patients with successful sperm retrieval than in patients with failed sperm retrieval. BOLL expression also decreased significantly in the testis of spermatogenetic failure. With statistical calculation, significant positive correlation was found between BOLL protein and CDC25A transcript levels. (r = 0.5858, P = 0.0013). Double fluorescence labeling micrographs indicated the co-localization of BOLL and CDC25A in the cytoplasma of primary spermatocyte in human testis slices. After overexpression of BOLL in human HeLa cells, co-existence of CDC25A mRNA and BOLL protein was confirmed by RNA IP assay.

In the present study, we demonstrated that decreased CDC25A and BOLL expressions were associated with spermatogenic failure in infertile men. Moreover, we confirmed the direct evidence that BOLL would bind with CDC25A mRNA in human cells. These results suggest that CDC25A act as downstream substrate of BOLL required for meiotic entry in human spermatogenesis.

References

Blomberg I and Hoffmann I. Ectopic expression of Cdc25A accelerates the G(1)/S transition and leads to premature activation of cyclin E- and cyclin A-dependent kinases. Mol Cell Biol 19, 6183-6194 (1999).

Castrillon DH, Gonczy P, Alexander S, Rawson R, Eberhart CG, Viswanathan S, Di Nardo S and Wasserman SA. Toward a molecular genetic analysis of spermatogenesis in Drosophila melanogaster: characterization of male-sterile mutants generated by single P element mutagenesis. Genetics 135, 489-505 (1993).

Eberhart CG, Maines JZ and Wasserman SA. Meiotic cell cycle requirement for a fly homologue of human Deleted in AZoospermia. Nature 381, 783-785 (1996).

Ferlin A, Moro E, Garolla A and Foresta C. Human male infertility and Y chromosome deletions: role of the AZF-candidate genes DAZ, RBM and DFFRY. Hum Reprod 14, 1710-1716 (1999).
Fox M, Urano J and Reijo Pera RA. Identification and characterization of RNA sequences to which human PUMILIO-2 (PUM2) and deleted in Azoospermia-like (DAZL) bind. Genomics 85, 92-105 (2005).

Galaktionov K and Beach D. Specific activation of CDC25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell 67, 1181-1194 (1991).

Galaktionov K, Chen X and Beach D. Cdc25 cell-cycle phosphatase as a target of c-myc. Nature 382, 511-517(1996).

Jiao X, Trifillis P and Kiledjian M. Identification of target messenger RNA substrates for the murine deleted in azoospermia-like RNA-binding protein. Biol Reprod 66, 475-485 (2002).

Jinno S, Suto K, Nagata A, Igarashi M, Kanaoka Y, Nojima H and Okayama H. CDC25A is a novel phosphatase functioning early in the cell cycle. EMBO 13, 1549-1556 (1994).

Kuo PL, Wang ST, Lin YM, Lin YH, Teng YN and Hsu CC. Expression profiles of the DAZ gene family in human testis with and without spermatogenic failure. Fertil Steril 81, 1034-1040 (2004).

Lee TH, Solomon MJ, Mumby MC, and Kirschner MW. INH, a negative regulator of MPF, is a form of protein phosphatase 2A. Cell 64, 415-423 (1991).

Lin YM, Kuo PL, Lin YH, Teng YN and Lin JSN. Messenger RNA transcripts of the meiotic regulator BOULE in the testis of azoospermic men and their application in predicting the success of sperm retrieval. Hum Reprod 20, 782-788 (2005).

Lin YM, Lin YH, Teng YN, Hsu CC, Lin JSN and Kuo PL. Gene-based screening for Y chromosome deletions in Taiwanese men presenting with spermatogenic failure. Fertil Steril 77, 897-903 (2002).

Luetjens CM, Xu EY, Reijo RA, Kamischke A, Nieschlag E and Gromoll J. Association of meiotic arrest with lack of BOULE protein expression in infertile men. J Clin Endocrinol Metab 89, 1926-1933 (2004).
Lundgren K, Walworth N, Booher R, Dembski M, Kirschner M and Beach D. mik1 and wee1 cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64, 1111-1122 (1991).

Maines JZ and Wasserman SA. Post-transcriptional regulation of the meiotic CDC25 protein Twine by the DAZL orthologue BOULE. Nat Cell Biol 1, 171-174 (1999).

Masui Y and Markert CL. Cytoplasmic control of nuclear behavior during meiotic maturation of frog oocytes. J Exp Zool 177, 129-145 (1971).

Paul J. Turek Male infertility. Smith’s general urology 16th edition, 678-712 (2004).

Reynolds N, Collier B, Maratou K, Bingham V, Speed RM, Taggart M, Semple CA, Gray NK and Cooke HJ. Dazl binds in vivo to specific transcripts and can regulate the pre-meiotic translation of Mvh in germ cells. Hum Mol Genet 14, 3899-3909 (2005).

Schrader M, Muller-Tidow C, Ravnik S, Muller M, Schulze W, Diederichs S, Serve H and Miller K. Cyclin A1 and gametogenesis in fertile and infertile patients: a potential new molecular diagnostic marker. Hum Reprod 17, 2338-2343 (2002).

Silber SJ, Alagappan R, Brown LG and Page DC. Y chromosome deletions in azoospermic and severely oligozoospermic men undergoing intracytoplasmic sperm injection after testicular sperm extraction. Hum Reprod 13, 3332-3337 (1998).

Skendzel LP and Youden WJ. Systematic versus random error in laboratory surveys. Am J Clin Pathol 54, 448-450 (1970).

Steger K, Fink L, Failing K, Bohle RM, Kliesch S, Weidner W and Bergmann M. Decreased protamine-1 transcript levels in testes from infertile men. Mol Hum Reprod 9, 331-336 (2003).

Teng YN, Lin YM, Lin YH, Tsao SY, Hsu CC, Lin SJ, Tsai WC and Kuo PL. Association of a single-nucleotide polymorphism of the deleted-in-azoospermia-like gene with susceptibility to spermatogenic failure. J Clin Endocrinol Metab 87, 5258-5264 (2002).

Venables JP, Ruggiu M and Cooke HJ. The RNA-binding specificity of the mouse DAZL protein. Nucleic Acids Res 29, 2479-2483 (2001).

Vigo E, Muller H, Prosperini E, Hateboer G, Cartwright P, Moroni MC and Helin K. CDC25A phosphatase is a target of E2F and is required for efficient E2F-induced S phase. Mol Cell Biol 19, 6379-6395 (1999).

Wickramasinghe D, Becker S, Ernst MK, Resnick JL, Centanni JM, Tessarollo L, Grabel LB and Donovan PJ. Two CDC25 homologues are differentially expressed during mouse development. Development 121, 2047-2056 (1995).

Wolgemuth DJ, Lele KM, Jobanputra V and Salazar G. The A-type cyclins and the meiotic cell cycle in mammalian male germ cells. Int J Androl 27, 192-199 (2004).

Wu S and Wolgemuth DJ. The distinct and developmentally regulated patterns of expression of members of the mouse CDC25 gene family suggest differential functions during gametogenesis. Dev Biol 170, 195-206 (1995).

Xu EY, Moore FL and Reijo RA. A gene family required for human germ cell development evolved from an ancient meiotic gene conserved in metazoans. Proc Natl Acad Sci USA 98, 7414-7419 (2001).