行政院國家科學委員會專題研究計畫 成果報告
DAZL 在人類黃體細胞扮演調節細胞凋亡的角色研究
計畫類別： 個別型計畫 計畫編號： NSC94-2314-B-006-081- 執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日 執行單位： 國立成功大學醫學系婦產科 計畫主持人： 潘咸安 共同主持人： 郭保麟 計畫參與人員： 廖睿雯 李于青 報告類型： 精簡報告 報告附件： 出席國際會議研究心得報告及發表論文 處理方式： 本計畫可公開查詢中 華 民 國 95 年 10 月 31 日
行政院國家科學委員會補助專題研究計畫
5 成 果 報 告
□期中進度報告
DAZL 在人類黃體細胞扮演調節細胞淍亡的角色研究
計畫類別：5 個別型計畫 □ 整合型計畫
計畫編號：NSC 94－2314 －B －006 －081
執行期間： 94 年 08 月 01 日至 95 年 07 月 31 日
計畫主持人：潘咸安
共同主持人：郭保麟
計畫參與人員： 廖睿雯．李于青
成果報告類型(依經費核定清單規定繳交)：5精簡報告 □完整報告
本成果報告包括以下應繳交之附件：
□赴國外出差或研習心得報告一份
□赴大陸地區出差或研習心得報告一份
5出席國際學術會議心得報告及發表之論文各一份
□國際合作研究計畫國外研究報告書一份
處理方式：除產學合作研究計畫、提升產業技術及人才培育研究計畫、
列管計畫及下列情形者外，得立即公開查詢
□涉及專利或其他智慧財產權，□一年□二年後可公開查詢
執行單位：國立成功大學醫學系婦產科
中 華 民 國 95 年 10 月 30 日
中文摘要及關鍵詞(keywords)
關鍵詞：DAZL 基因, 黃體細胞, 細胞凋亡 黃體分泌黃體素是維持早期懷孕的重要因素, 而藉由以前的文獻報告, 吾人已知黃體 素的產生和黃體細胞的凋亡有直接的相關性. 由先前的研究知道 DAZL 在黃體化顆粒 細胞及黃體細胞都有表現, 也建立了體外細胞株 DAZL 表現和黃體素分泌的初步關係, 於是推測 DAZL 應可藉由調控黃體細胞的分泌黃體素的功能, 進而調控黃體細胞的凋 亡 . 本 研 究 以 人 類 黃 體 化 顆 粒 細 胞 為 模 組 , 先 將 DAZL 基 因 轉 植 到 此 細 胞 株 上 , 以 RT-PCR 定量 DAZL RNA 的量, 接著以 ShRNA 阻斷體外培養之黃體化顆粒細胞 DAZL 蛋白合成，以觀察在沒有 DAZL 蛋白分泌之情況下黃體化顆粒細胞分泌黃體素的功能是 否受到影響。並測試 DAZL 表現量和細胞凋亡指數的相關性.本實驗初步得到的結果是 DAZL 的 RNA 表現量多寡和黃體化顆粒細胞 apoptosis 無關, 但是此結論需要進一步藉由 DAZL 蛋白質(Western blot)的表現量和 TUNAL 染色濃度 表現並作關聯性研究才能確定.
Abstract and keywords
Keywords: DAZL gene, apoptosis, corpus luteum, progesterone
During normal luteolysis, the cells of the corpus luteum undergo apoptosis during structural luteolysis. And the progesterone produced by luteum copora has been proven as a suppressor of apoptosis in bovine luteal cell. According to our preliminary data, we had established an
expression curve between DAZL and progesterone secretion in vitro. And already published the data that DAZL transcripts are expressed in the CL, but the concentrations decreased with
advancing luteal stage . Theoretically, we hypothesize that the distinct expression of DAZL in the human CL will regulate the secretion of the progesterone level and play an important role in regulation of the apoptosis of the CL.
established human granulosa cell line (HO-23) and primary granulose cell culture to test the relationship between the DAZL and apoptosis. First, due to the low DAZL RNA level in primary granulose cell culture, we transfected the DAZL by lipofectamin 2000 into gra nulosa cell then knockdown the DAZL by ShRNA and correlate the DAZL RNA level to apoptosis figure. However, the data showed no positive correlation between the DAZL and apoptosis. But before reaching the conclusion there still need a conformation of the protein level of DAZL by Western blot method and correlated to the TUNAL assay (apoptotic figure) again.
前言
在不同物種中, DAZL基因和它的同源基因的表現為男性或女性生殖細胞發育上所必需。 在 Caenorhabditis elegans，daz-1 是女性生殖細胞進行減數分裂的一個十分重要的因素， 但是對男性減數分裂( 1 ) 則是不必要的。 在Xenopus , Xdazl 為女性原始生殖細胞
(primordial germ cells, PGC ) ( 2 ) 的早期分化和遷移 (migration )所需要。 在Drosophiala， DAZL 同源基因boule的缺失, 會阻斷男性生殖細胞進行減數分裂 ( 3 ) 。 在老鼠中，Dazl 基因的缺失導致公鼠和母鼠無法產生生殖細胞，顯示 Dazl 基因表現對於兩性在生殖細胞 形成過程的重要性( 4 ) 。 在人類女性生殖系統中，卵子和濾泡中的顆粒細胞可以表現 DAZL 基因 ( 5,6,7 )。 此外我們亦發現DAZL可在黃體表現 ( 8 )。 黃體化是維持早期懷 孕的必要過程，顆粒細胞可以保護卵子並且在gonadotropin的作用下形成黃體，分泌 progesterone ( 9 )。 利用細胞培養可以了解更多人類的、豬的、和老鼠的steroidogenesis 控 制因子( 10,11,12 )。 DAZL 蛋白質在女性生殖過程的不同的時期表現，顯示其調節女性生殖的重要功能。 黃體在排卵以後大約 5 天達到成熟並且迅速在 9 - 14 天後崩解。 在正常的黃體化過程 中二個密切相關的因素會形成。 首先會失去 progesterone 和 estradiol 的合成及分泌能力 ( 13 ) ，接著黃體結構內的細胞產生降解而消失 ( 14 )。 progesterone 已被證實可以抑制牛 的黃體細胞進行細胞凋亡( 15 ) 。 根據我們的初步體外研究，DAZL 的表現量和
progesterone 的分泌有關，而且 DAZL 可以在早期的黃體被偵測到，隨著黃體的崩解其表 現量也愈低 ( 16) 。因此我們假設 DAZL 蛋白質的表現在黃體可以抑制其細胞凋亡的產 生，以維持早期黃體的功能。藉著測定 DAZL 及 progesterone 的表現量，並利用 TUNEL 分析比較細胞凋亡的比率，以釐清 DAZL 與細胞凋亡之間的關係。
研究方法
Establishment of human granulosa cell culture and induction of DAZL expression
Human granulosa cell was collected from follicular fluid and was culturedin phenolred – free
DMEM/F12 (1:1) containing 10% FCS, at 37 C on 5% CO2 in ahumidified incubator. The
steroidogenic response of the cell wasmaintained by addition of HCG (1 iu/ ml) in culture medium. DAZL expression was induced by transfection with pcDNA3.1-DAZL vector which contains the full length of DAZL open reading frame. Transfection was carried out using
Lipofectamine 2000 according to manufacturer’s protocol (Invitrogen Co.). Cells were harvested for TUNEL and RT-PCR analysis
TdT-mediated dUTP Nick-End Labeling (TUNEL) Assay
Cell apoptosis were analyzed by TUNEL according DeadEnd fluorometric TUNEL system ( Promega Co.) Cells culture on 3 cm dish were harvested and wash once with PBS by
centrifugation (300xg) at 4℃. Fix the cells by adding 0.1 ml of 1% paraformaldehyde / PBS on ice for 20 minutes. Add 0.9 ml PBS and centrifuge at 300xg for 10 minutes at 4℃. Remove the supernatant and resuspend cells in 0.1 ml 70% ethanol, then stored at -20℃. Before taking experiment, remove 70% ethanol by adding 0.9 ml PBS and centrifuge at 300xg for 10 minutes at 4℃. Descant the supernatant, then equilibrate cells with 50μl equilibration buffer and incubate at room temperature for 5 minutes. Add another 50μl labeling mixture containing recombinant terminal deoxynucleotidyl transferase and fluorescein-12-dUTP and incubate in 37
℃ for 1 hour, protecting from direct light exposure. Remove the reaction mixture by wash with PBS. Resuspend cells in 1ml PBS and store at 4℃ in the dark until flow cytometric analysis.
Total RNA isolation and cDNA synthesis
Total cellular RNA was extracted using TRIZOL Reagent according to manufacturer’s protocol (Invitrogen Co.). For synthesis of complementary DNA (cDNA), 12µl RNA mixture containing 2 µg of RNA, 1 µl of oligo(dT)l2-18 primer (500 ng/µl) (Gibco/BRL, Grand Island, NY, USA) were preheated to 70°C for 10 min and then chilled on ice. Reverse transcription was performed in 25-µl aliquots master mixture containing 1x first strand synthesis buffer, 0.1 M DTT, 10 mM of each dNTP, 40 U RNase inhibitor and 200 U of RNase H minus reverse transcriptase
(SuperscriptTM II, Gibco/BRL, Grand Island, NY, USA). The RT reaction was carried out at 42°C for 2 hr, then inactivated reverse transcriptase activity by heat at 75°C for 15 min. The cDNA was aliquoted and stored at –20°C until use.
Polymerase chain reaction (PCR)
PCR for DAZL, GAPDH, Caspase3 and StAR were carried out using the specific primers : DAZL-F : 5’- GGAGCTATGTTGTACCTCC - 3’ DAZL-RP-R : 5’- TTTAAGCATTGCCCGAC - 3’ GAPDH-F : 5’- TGAAGGTCGGAGTCAACGGATT- 3’ GAPDH-R : 5’- CCTGGAAGATGGTGATGGGATT- 3’ Caspase3-F : 5’- TGGAATTGATGCGTGATGTT- 3’ Caspase3-R : 5’- GGCAGGCCTGAATAATGAAA- 3’ StAR-F : 5’- GACTTTGTGAGCGTGC - 3’ StAR-R : 5’- TGATGACACCCTTCTGC- 3’
Four microlitres of cDNA was used as the template in a 25μl reaction mixture comprising of 1.25U AmpliTaq DNA polymerase （Perkin-Eelmer）, 1X PCR buffer [10 mM Tris-HCl, pH 8.3
（at 25℃）, 50 mM KCl, 1.5 mM magnesium chloride, 0.001%（w/v）gelatin], 200 nM each primers and 200μM of each dNTP （Promega）. PCR was carried out in a HYBAID
PCR Express machine using the following cycling profile: 30 cycles of 60 sec at 95℃,
90 sec at 64℃ and 60 sec at 72℃, and a final extension for 5 min at 72℃. The PCR fragments were analyzed by 2.5% agarose gel electrophoresis.
Suppression of DAZL expression in human granulosa cell
The pSM2c vector carry a sequence to generate human DAZL shRNA (termed shRNA-DAZL, from Open Biosystems) was transfected into human granulose cell to suppress DAZL gene expression. The knockdown efficiency was established by RT-PCR.
結果
由於人類黃體細胞的培養尚未成功，而卵子周圍的顆粒細胞是形成黃體的前身，所以分離 濾泡液中的顆粒細胞進行培養，以分析其DAZL之表現與細胞凋亡的關係。但是培養後的 顆粒細胞DAZL表現量高，試圖尋找可以誘導DAZL表現量提高的方法。在培養的顆粒細胞 中加入spermidine、 HCG (1 iu/ml)、 PMSG (1 iu/ml)、 E2 (0.05 mM)、 progesterone (0.05 mM)、或利用lipofectamine 2000 進行基因轉殖將可以表現DAZL蛋白質的載體
pcDNA3.1-DAZL送入細胞內進行表現。結果顯示只有基因轉殖可以提高DAZL RNA的表 現。(圖1)
將顆粒細胞已經表現的DAZL RNA再利用基因轉殖的方法送入DAZL的shRNA (short hairpin RNA)以抑制其表現，由結果可知抑制效果的存在且可維持至少三天，但效果並未 達到顯著的水準(圖2)。進一歩調整pcDNA3.1-DAZL與shRNA-DAZL轉殖DNA量的比率， 發現1比1.5以上的比率可有效抑制DAZL RNA的表現。(圖3)
利用本條件進行顆粒細胞DAZL表現與細胞凋亡的關係。實驗設計分成三組，A組不作任何 處理，B組只進行DAZL基因的轉殖，C組進行DAZL基因的轉殖後再利用shRNA-DAZL抑 制DAZL RNA的表現。利用RT-PCR分析三組之間DAZL(圖4)、 Caspase 3、 StAR RNA(圖 5)的表現量，並利用TUNEL分析細胞凋亡的程度(圖6, 7)。結果顯示基因轉殖確實可以增加 或抑制DAZL基因的表現，但是其與Caspase 3、 StAR RNA的表現量以及細胞凋亡之間並 沒有顯著的相關性。 圖1 1. spermidine (1000x dilution) 2. HCG (1 iu/ml) 3. PMSG 4. Control 5. E2 (0.05 mM) 6. progesterone (0.05 mM) 2.5% agarose gel 7. lipofectamine 2000/ pcDNA3.1-DAZL 8. Calcium phosphate/ pcDNA3.1-DAZL 9. genomic DNA 10. 100 bp DNA ladder -500 bp -300 bp -200 bp -100 bp -400 bp 1 2 3 4 5 6 7 8 9 10 -500 bp -300 bp -200 bp -100 bp -400 bp -500 bp -300 bp -200 bp -100 bp -400 bp 1 2 3 4 5 6 7 8 9 10 圖2 1. 100 bp DNA ladder 2. day 1 control
3. day 1 lipofectamine 2000/ pcDNA3.1-DAZL 4. day 1 lipofectamine 2000/ shRNA-DAZL
5. day 1 lipofectamine 2000/ pcDNA3.1-DAZL/ shRNA-DAZL 6. day 3 lipofectamine 2000/ shRNA-DAZL
7. day 3 lipofectamine 2000/ pcDNA3.1-DAZL/ shRNA-DAZL
8. day 3 lipofectamine 2000/ pcDNA3.1-DAZL/ shRNA-DAZL (day 1 treatment) 9. blank DAZL GAPDH 100 200 300 400 500 bp-1 2 3 4 5 6 7 8 9 bp-1 2 3 4 5 6 7 8 9 DAZL GAPDH 100 200 300 400 500 100 200 300 400 500 bp-1 2 3 4 5 6 7 8 9 bp-1 2 3 4 5 6 7 8 9 2.5% agarose gel
圖3 DAZL : shRNA_DAZL = 1 : 1.5 DAZL : shRNA_DAZL = 1 : 0.5 DAZL : shRNA_DAZL = 1 : 0 0.8 + 1.2 0.6 + 0.9 0.4 + 0.6 0.2 + 0.3 3 0.8 + 0.4 0.6 + 0.3 0.4 + 0.2 0.2 + 0.1 2 0.8 + 0 0.6 + 0 0.4 + 0 0.2 + 0 1 2 μg 1.5 μg 1 μg 0.5 μg Max. amount of total DNA D C B A Treatment DAZL : shRNA_DAZL = 1 : 1.5 DAZL : shRNA_DAZL = 1 : 0.5 DAZL : shRNA_DAZL = 1 : 0 0.8 + 1.2 0.6 + 0.9 0.4 + 0.6 0.2 + 0.3 3 0.8 + 0.4 0.6 + 0.3 0.4 + 0.2 0.2 + 0.1 2 0.8 + 0 0.6 + 0 0.4 + 0 0.2 + 0 1 2 μg 1.5 μg 1 μg 0.5 μg Max. amount of total DNA D C B A Treatment A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 M A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 DAZL GAPDH A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 M A1 A2 A3 B1 B2 B3 C1 C2 C3 D1 D2 D3 DAZL GAPDH 圖4 M. 100 bp DNA ladder 1. DA0: day 0 control 2. DB0: day 0 pcDNA3.1-DAZL
3. DC0: day 0 pcDNA3.1-DAZL/ shRNA-DAZL 4. DA1: day 1 control
5. DB1: day 1 pcDNA3.1-DAZL
6. DC1: day 1 pcDNA3.1-DAZL/ shRNA-DAZL
7. DA2: day 2 control 8. DB2: day 2 pcDNA3.1-DAZL
9. DC2: day 2 pcDNA3.1-DAZL/ shRNA-DAZL 10. DA3: day 3 control
11. DB3: day 3 pcDNA3.1-DAZL
12. DC3: day 3 pcDNA3.1-DAZL/ shRNA-DAZL M 1 2 3 4 5 6 7 8 9 10 11 12 DAZL M 1 2 3 4 5 6 7 8 9 10 11 12 DAZL 1 2 3 4 5 6 7 8 9 10 11 12 M GAPDH 1 2 3 4 5 6 7 8 9 10 11 12 M GAPDH 圖5
圖6
TUNEL analysis for cell apoptosis
Negative control Positive control Mixture
圖7 0 1 2 3 4 5 0 0.5 1 1.5 2 2.5 3 3.5 Control DAZL Knockdown
討論
DAZL 基因在許多物種的生殖功能上扮演重要的角色，若將老鼠的 dazl 基因除去， 會造成公鼠及母鼠都不孕。在果蠅若將 DAZL 之同源基因 boule 除去，雄性果蠅將完全 沒有精蟲。DAZ 基因是 DAZL 基因的同源基因，位於人類 Y 染色體上，兩者蛋白結構 有超過 80 %相同。實驗發現 DAZ 基因缺失，是造成男性不孕症重要的原因，但在人類 DAZL 究竟扮演何種角色，則尚不清楚。我們實驗室曾利用 QC-RT-PCR 測得黃體細胞有 DAZL RNA 之表現及證實 DAZL 蛋 白在人類黃體的表現。由先前的研究亦知道 DAZL 在黃體化顆粒細胞有表現。黃體化顆 粒細胞是形成黃體的主要細胞，而黃體分泌黃體素是維持早期懷孕的重要因素，由文 獻報告已知黃體素的產生和黃體細胞的凋亡有直接的相關性，於是推測 DAZL 應可藉由 調控黃體細胞分泌黃體素的功能，進而調控黃體細胞的凋亡。 本研究以人類黃體化顆粒細胞為模組，先將 DAZL 基因轉殖到此細胞內，使 DAZL 基因大量表現，接著再以 antisense oligomer 阻斷其 DAZL 蛋白的合成，觀察在這過程 中，DAZL 表現量與黃體素分泌量的關係、以及與細胞凋亡指數的相關性。由 RT-PCR 可以確認 DAZL 的表現量發生改變，但是與黃體素分泌有關係的 StAR 基因表現量，以 及與細胞淍亡有關的 Caspase 3 的表現量未見到差異。利用 TUNEL 偵測淍亡細胞再配 合細胞流速分析，結果顯示 DAZL 表現量與細胞凋亡的相關性並未達到顯著差異。 由於實驗的對照組其本身細胞凋亡的程度並不高，是否因此影響分析的顯著性， 仍待進一歩確認。而 DAZL 的 RNA 表現量發生改變是否與其蛋白質表現量同歩改變亦 待分析。未來我們將嚐試利用藥物誘導人類黃體化顆粒細胞的凋亡現象，配合利用 Western blot 偵測 DAZL 蛋白質表現量的改變，再次確認 DAZL 表現量與細胞凋亡的相 關性。
計畫成果自評
本計劃的執行已建立在細胞株及人類黃體化顆粒細胞進行基因轉殖的相關技術， 對於 RNAi 的操作：包括 oligomer 或 hair-loop 的設計及測試亦有初步的渉獵。並利用此 技術平台測試人類黃體細胞 DAZL 的表現和黃體素分泌的關係，及 DAZL 的表現和黃體細 胞的凋亡指數的相關性。
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in the human corpus luteum. Molecular Human Reproduction 8:540-545.
出席國際學術會議心得報告及發表之論文
歐洲生殖內分泌暨胚胎醫學會年會是每年一次歐洲地區最盛大的不孕症專家包括臨床、基 礎研究交換心得的聚會，除了增廣見聞也認識朋友，又可一窺目前最新的研究趨勢。綜觀 今年的重點是基因在胚胎分裂時期的調控及胚胎著床時的分子變化，大部分是一組研究團 隊的報告，鮮少有單打獨鬥的傑出報告，顯示財力、物力、人力的整合對研究的發展是重 要的影響因素。 Topic: Embryology, experimental Abstract title:Dazl expression in mouse preimplantation embryo
Abstract:
Introduction:
The misregulation of gene expression during gametogenesis contributes to infertility, a problem affecting 12-15%, of couples worldwide (de Kretser, 1997). The Daz (Deleted in Azoospermia) family consists of three genes: BOULE, Daz, and Dazl (Dazl-like). The genes of the Daz family encode proteins with a highly conserved RNA-binding motif and a unique Daz repeat. The proteins are believed to function in the post-transcriptional regulation of mRNA expression because they interact with RNA homopolymers through their
RNA-binding motif and form complexes with themselves, with each other or with proteins involved in mRNA transport, localization or in mRNA translation (Yen, 2004). Dazl gene is believed to function in the development of primordial germ cell and in germ cell differentiation and maturation. Recently, expression of DAZL has been reported in undifferentiated human embryonic stem (ES) cell lines and in embryonic bodies (EBs), suggesting a spontaneous differentiation of a subpopulation of ES cells into germ cells (Clark et al., 2004; Moore et al., 2004). The link between ES cells and germ cells is the preimplantation period during which a 2- cell embryo develops towards a blastocyst. In this study, we demonstrated the Dazl expression throughout this period.
Materials and Methods:
Female FVB mice (28-35-day-old) were induced to superovulate by intraperitoneal injection of gonadotropins pregnant mare’s serum gonadotropin (PMSG; Intervet) followed by the injection of human chorionic gonadotropin (HCG; Sigma). Female mice were sacrificed at 1.5, 2.5, 3.5, dpc (plug date, 0.5 dpc), to examine the Dazl expression by immunofluorescence staining and microscopy from 2-cell embryo to blastocyst.
Results:
When fluorescently labeled antibodies are used, the non-uniform cytoplasmic localization of the protein is evident. We show that the Dazl expressions were abundant in the cytoplasm from the 2-cell embryo to blastocyst. Strong signals of Dazl expression (green) were found in preimplantation embryos cytoplasm including 2-cell, 8-cell, morula, and blastocyst. The 4-cell stage embryo has a relative weak, but apparent, signal. The nuclear DNA is shown in blue. Merge of the two fluors is also shown.
Conclusions:
The mouse Dazl gene is essential for development and survival of germ cells in ovary. The preimplantation development comprise all stages from oocytes fertilization until blastocyst implantation. In this study, we demonstrated the expression of the germ cell marker Dazl throughout the preimplantation development. In contrast to the nuclear RBM protein, Dazl is cytoplasmic and so is unlikely to be a transcription or splicing factor, but may be involved in translational control by packaging or localizing mRNAs. The precise molecular role of these proteins in embryo development remains elusive, however, we had a further step to show their appearance in the cytoplasm of mouse preimplantation embryo from the 2-cell embryo to blastocyst.
Authors:
H. Pan1, R. Liao1, P. Kuo1.
1National Cheng-Kang University Hospital, Dept. of Ob/Gyn, Tainan, Taiwan R.O.C..
Keywords:
Dazl;embryo;RNA-binding protein;
Award:
Introduction:
The misregulation of gene expression during gametogenesis contributes to infertility, a problem affecting 12-15%, of couples worldwide (de Kretser, 1997). The Daz (Deleted in Azoospermia) family consists of three genes: BOULE, Daz, and Dazl (Dazl-like). The genes of the Daz family encode proteins with a highly conserved RNA-binding motif and a unique Daz repeat. The proteins are believed to function in the post-transcriptional regulation of mRNA expression because they interact with RNA homopolymers through their RNA-binding motif and form complexes with themselves, with each other or with proteins involved in mRNA transport, localization or in mRNA translation (Yen, 2004). Dazl gene is believed to function in the development of primordial germ cell and in germ cell differentiation and maturation. Recently, expression of DAZL has been reported in undifferentiated human embryonic stem (ES) cell lines and in embryonic bodies (EBs), suggesting a spontaneous differentiation of a subpopulation of ES cells into germ cells (Clark et al., 2004; Moore et al., 2004). The link between ES cells and germ cells is the preimplantation period during which a 2- cell embryo develops towards a blastocyst. In this study, we demonstrated the Dazl expression throughout this period. Materials and Methods:
Female FVB mice (28-35-day-old) were induced to superovulate by intraperitoneal injection of gonadotropins pregnant mare’s serum gonadotropin (PMSG; Intervet) followed by the injection of human chorionic gonadotropin (HCG; Sigma). Female mice were sacrificed at 1.5, 2.5, 3.5, dpc (plug date, 0.5 dpc), to examine the Dazl expression by immunofluorescence staining and microscopy from 2-cell embryo to blastocyst.
Results:
When fluorescently labeled antibodies are used, the non-uniform cytoplasmic localization of the protein is evident. We show that the Dazl expressions were abundant in the cytoplasm from the 2-cell embryo to blastocyst. Strong signals of Dazl expression (green) were found in
preimplantation embryos cytoplasm including 2-cell, 8-cell, morula, and blastocyst. The 4-cell stage embryo has a relative weak, but apparent, signal. The nuclear DNA is shown in blue. Merge of the two fluors is also shown.
Conclusions:
The mouse Dazl gene is essential for development and survival of germ cells in ovary. The preimplantation development comprise all stages from oocytes fertilization until blastocyst implantation. In this study, we demonstrated the expression of the germ cell marker Dazl throughout the preimplantation development. In contrast to the nuclear RBM protein, Dazl is cytoplasmic and so is unlikely to be a transcription or splicing factor, but may be involved in translational control by packaging or localizing mRNAs. The precise molecular role of these proteins in embryo development remains elusive, however, we had a further step to show their appearance in the cytoplasm of mouse preimplantation embryo from the 2-cell embryo to blastocyst.
