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研究生:黃伯達
論文名稱:分析比較FertilizationPromotingPeptide、Adenosine與Pentoxifylline對冷凍解凍後人類精子作用的研究
論文名稱(外文):Comparisons of Effects of Fertilization Promoting Peptide, Adenosine, and Pentoxifylline on Frozen - thawed Human Sperm
指導教授:周松男周松男引用關係楊友仕楊友仕引用關係何弘能何弘能引用關係
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:臨床醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2002
畢業學年度:90
語文別:中文
論文頁數:39
中文關鍵詞:精子冷凍電腦輔助精子分析儀
外文關鍵詞:capacitatiopnhyperactivationacrosome reactionfertilization promoting peptideadenosinecryopreservationchlortetracyclinecomputer aided sperm analyzer
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摘要
緒論
哺乳類動物的精子自雄性體內釋出時尚不具備受精能力,但於雌性生殖道中或在某些合適的體外環境下,其受精能力就會啟動。這過程稱作”capacitation”。Capacitation牽涉到某些精子表面與細胞內的變化,且有某些維持capacitation可能所需的物質已陸續被發現:
Fertilization promoting peptide ( FPP; pGlu-Glu-ProNH2 )是由前列腺製造而分泌入精液中的tripeptide,目前已發現存在於包括人類等哺乳類動物中。它於人類精液中以49.5±10.3 nM的濃度存在,且於射精時始接觸精子。FPP被認為是活體內capacitation的重要調節者,可誘發capacitation-dependent responses。FPP對uncapacitated sperm有促進capacitation的作用,而對capacitated sperm則有防止spontaneous acrosome reaction的作用。如此,FPP有助於使存在於雌性生殖道中尚未碰觸卵子的精子保有受精能力。
Adenosine是另一個精液的組成成分。它可誘發同於FPP的反應,故也被認為是活體內 capacitation的重要調節者。與FPP一樣,adenosine對uncapacitated sperm有促進capacitation的作用,而對capacitated sperm則有防止spontaneous acrosome reaction的作用。若將adenosine與FPP兩者各以低於其可個別引發反應的濃度予以混合,仍可引發顯著促進capacitation的作用;若adenosine與FPP兩者各以高於其可個別引發反應的濃度予以混合,則所引發的反應會較個別使用時為大。由此推論可知adenosine與FPP兩者是透過相同的signal transduction pathway(即adenylate cyclase / cAMP pathway),但鍵結於不同的external receptors來作用的。
曾於臨床上應用於不孕男性以增強精子活動力的pentoxifylline,是一種phosphodiesterase inhibitor,其作用即可能是透過提高精子細胞內的cAMP濃度來促成的。Pentoxifylline可於活體外改善hamster sperm的capacitation與受精率,並誘發hyperactivation與acrosome reaction的發生。而pentoxifylline則似乎可能應用於增強解凍後活動精子的活動力,且於細胞質內精子顯微注射( ICSI )時選擇存活的精子也有幫助。
基於體外實驗發現FPP與adenosine有助於增強新鮮人類精子的受精能力,而pentoxifylline是目前應用於臨床以改善人類精子品質的藥劑,且目前認為FPP、adenosine與pentoxifylline三者對於精子的作用機轉有部分同樣是透過改變細胞內cAMP的濃度來達成。我們嘗試以FPP、 adenosine與pentoxifylline處理解凍後的人類冷凍精子,分析FPP、 adenosine與pentoxifylline對經過冷凍與解凍過程的人類精子可能造成的作用,同時並評估三者對其受精能力是否亦具提升的作用。
研究方法與原理
Semen Sample
本實驗用之精液的採集乃經由志願男性以自慰方式取得。採得後之精液經冷凍處理後儲存數日,於實驗當日再行解凍。解凍後之精液等分為二至四份,其中一份不加任何試劑當作control,另外一至三份則分別加入FPP (100nM)、adenosine (10μM)或pentoxyfilline (1mg/mL)。精液於解凍並以各試劑處理後,於一小時與四小時兩個時間點,來自同一人的檢體或以chlortetracyclline ( CTC )螢光染色評估各組精子的capacitation status,或以電腦輔助精子分析儀( CASA )來分析其各組精子的motility characteristics。
Chlortetracycline ( CTC ) Fluorescence Assessment
本實驗共收取十六個個案之檢體,每份檢體等分為四份,其中一份不加任何試劑當作control,另外三份則分別加入FPP、adenosine與pentoxyfilline。
一般精子之染色法通常只能將精子區分為acrosome — intact cells 與acrosome — reacted cells。CTC螢光染色是目前唯一可進一步將acrosome — intact sperm區分為uncapacitated cells與capacitated cells之染色法:
F, with uniform fluorescence over the entire head, characteristic of uncapicitated, acrosome-intact cells
B, with a fluorescence - free band in the post-acrosomal region, characteristic of capicitated, acrosome - intact cells
AR, with dull or absent fluorescence over the sperm head, characteristic of capicitated, acrosome - reacted cells
另外,我們利用Hoechst 33258來與CTC共染,用以評估個別精子的存活與否,以避免將顯現Hoechst 33258 positive之死亡精子誤當成acrosome - reacted cells。
將染色後之檢體置於瑩光顯微鏡下,計數Hoechst 33258 negative cells約200隻,再計算各種CTC染色形態之精子所佔百分比。
Computer Aided Sperm Analyzer ( CASA )
本實驗所收集的檢體,視各個案解凍後的精液品質而定,將解凍後之精液等分為二至四份,其中一份不加任何試劑當作control,另外一至三份則分別加入FPP、adenosine或pentoxyfilline。經以FPP、adenosine或pentoxyfilline處理部份檢體的個案數分別為27、16、23。
利用CASA我們可以分析比較各組檢體中活動精子所佔的百分比與下列各motion parameters的平均值:
Straight — line velocity ( VSL ):the straight — line distance between the beginning and the end of the track divided by the time elapsed
Curvilinear velocity ( VCL ):the total distance between each position of the cell center of brightness ( CB ) for a given cell during the acquisition, divided by the time elapsed
Average path velocity ( VAP ):the smoothed average position of the CB and gives an average cell path velocity
Amplitude of lateral head displacement ( ALH ):the mean width of the head oscillation as the cell swims
Beat cross frequency ( BCF ):the frequency with which the cell track crosses the cell path in either direction
Straightness ( STR ):the departure of the cell path from a straight line
Linearity ( LIN ):the departure of the cell track from a straight line
Statistic Analyses
我們以paired t — test 分別對FPP — treated、adenosine — treated、pentoxifylline — treated各組相較於控制組的實驗結果作配對的統計分析比較。分析的變項包括在各檢體中經由:
1. CTC染色後在螢光顯微鏡下所計數的F、B、AR三種不同螢光呈色形態的精子所佔的百分比值,將其比值經arc — sine transformation處理後所得的數值。2. CASA所計算得的活動精子的各項平均參數值,包括VAP、VSL、VCL、ALH、BCF、STR、LIN等參數,與精子活動率(即motility rate)。
結果
第一部份、受精能力的評估
CTC螢光染色實驗在FPP — treated、adenosine — treated、pentoxifylline — treated三組相較於control group的結果並未達到統計顯著差異;我們發現人類冷凍精子在解凍後,其capacitation 與acrosome status並不因加入FPP、adenosine、或pentoxifylline而明顯地受影響,顯然其使卵子受精的能力並不因此而獲得改善。
第二部份、活動型態的評估
Adenosine與pentoxifylline都有加速解凍後活動精子VCL遊動速率的效果,adenosine — treated與pentoxifylline — treated兩組於1 H時,其VCL都顯著地較control group為大。另外, pentoxifylline並有減小LIN以促使解凍後活動精子遊動行徑偏離線性的作用,且此作用可以維持至4 H時。
FPP雖無加速解凍後活動精子遊動速率的效果,但確實修飾了部分的運動型態。FPP — treated group於1 H時,其BCF顯著地較control group為小;而於4 H時,其STR與LIN則明顯小於control group,此時該兩項運動參數的P — value甚至小於0.005。因此,FPP有促使解凍後活動精子的前進路線與遊動行徑皆偏離線性的作用。
至於解凍後人類精子的活動率,並不因為pentoxifylline的加入而受影響。但解凍後的人類精子於加入FPP或adenosine後於1 H時其活動率都明顯地較control group為差,而至4 H時該兩實驗組的活動率與control group相較,則已無顯著差別。
討論
由CTC螢光染色實驗中我們發現人類冷凍精子在解凍後,其capacitation 與acrosome status並不因加入FPP、adenosine或pentoxifylline而明顯地受影響。我們的研究顯示啟動並維持人類精子受精能力的生理作用會因冷凍與解凍過程的處理而遭受相當程度的損害。但FPP、adenosine與pentoxifylline三者對於解凍後的人類冷凍精子確實部分地保留了修飾其運動行為的作用。本實驗結果提供我們關於精子受精能力的調控機制的某些暗示:
Adenosine — treated與pentoxifylline — treated兩組於1 H時,其VCL都顯著地較control group為大,這表示精子的游動速率決定於精子細胞內cAMP的濃度。在某有效濃度範圍內,細胞內cAMP的濃度提高則精子的游動速率加快。而FPP因對解凍後活動的人類精子並無顯著加速VCL的作用,因此FPP作為一個first messenger,其對AC / cAMP signalling pathway應是間接運作的。然而,由FPP — treated group於4 H時其活動精子的前進路線與遊動行徑皆顯著地偏離線性看來,FPP於促進新鮮精子發生capacitation的同時,應該也與hyperactivation相關的運動途徑的表現有較adenosine更直接的關係,只是無法於已受過冷凍與解凍傷害的精子完全表現出來。另外,因為精子的活動率反映了精子整體的能量儲存,adenosine因活化了adenylate cyclase,將ATP消耗轉變為cAMP,因而可以解釋adenosine — treated group於1 H時的活動率之所以明顯下降。然而相較於1 H時VCL游動速率明顯較快的pentoxifylline — treated group,同樣於1 H時活動率顯著下降的FPP — treated group,則顯然有除了游動之外額外的能量耗費,而這同時也應該是於1 H時FPP — treated group的活動精子的運動型態不若control group活潑有力的原因。而不論是adenosine — treated或FPP — treated group,因其活動率於4 H時,與control group已無顯著差別,這表示有相當部份的精子因為adenosine與FPP的作用導致能量耗盡而提前死亡。
精子的細胞膜對於capacitation的發生與hyperactivation的表現無疑是相當重要的,然而精子細胞膜的功能與性質會因冷凍處理而改變。精子於冷凍時,離子與水對細胞膜的通透性都發生了變化,這會導致解凍後精子細胞內的離子濃度偏離了正常情況下適於受精的生理濃度。而其中精子細胞內的Ca++濃度不但與精子受精能力的表現息息相關,細胞中的Ca++ / calmodulin — dependent protein kinases因為調節了cytoskeleton的功能,也會影響細胞的運動。過去FPP於動物實驗的研究結果皆指出FPP具有啟動並保有哺乳動物精子的受精能力與引導發生hyperactivation的作用,而本實驗結果又顯示FPP對於解凍後的人類活動精子具有修飾其運動行為的作用,且其作用伴隨有能量的耗失。考慮冷凍技術對於精子細胞膜與細胞內離子濃度的影響,我們可以合理地推論,FPP對於精子的作用應該是透過調節其細胞膜上的Na+ - K+ATPase與/或Ca++ATPase,以維持精子細胞內Ca++濃度的恆定來達成的。而Ca++則是細胞內除了cAMP以外,調節capacitation與acrosome reaction發生的另一個重要的second messenger。
雖然經FPP處理過的解凍後活動精子因為能量分流之故,以致於細胞內的cAMP濃度無法明顯提昇,而於1 H時其運動的表現不如control group的活潑有力。然而,如同FPP,pentoxifylline亦有促使解凍後活動精子的遊動行徑顯著地偏離線性的效果。因此,於Ca++ / calmodulin與AC / cAMP 兩個訊息傳導機制間應有交互影響的作用存在。
我們認為FPP在與精子細胞膜上的特定接受器結合後,活化了細胞質內的某些G protein,這些G protein引發了後續一連串的細胞反應。其中有些G protein幫助調控細胞內的Ca++濃度,活化了Ca++ / calmodulin — dependent protein kinases,並因此促成了cytoskeleton的重組而使精子發生hyperactivation;另外有些G protein則可能活化了adenylate cyclase,啟動了AC / cAMP signalling pathway。雖然過去的文獻指出FPP對於促進人類精子發生capacitation的作用於FPP加入後一個小時內即有顯著的顯現,然而對於解凍後存活的人類精子,其中應有相當部分其細胞內的離子濃度偏離正常的生理濃度,以致於FPP在調控細胞內的離子濃度的同時不但耗費較多的能量,也花費了較長的時間才使得細胞內的離子濃度回復或接近適於受精的濃度,因而在我們的實驗結果中,經FPP處理的解凍後活動精子,其運動的表現不但於1 H時因能量分流之故,顯得不及control group的活潑有力,而且FPP對於精子運動行為的修飾作用也遲至一個小時後才表現出來。而解凍後存活的精子也因細胞膜與cytoskeleton受到冷凍處理的破壞而難以表現或僅有少數表現出正常新鮮精子的capacitation與hyperactivation。
另外,精子細胞內Ca++與cAMP都於精子受精能力的調控上扮演了second messenger的角色,它們於精子細胞內藉由一連串交互作用的訊息傳遞機制擴大了精子對於FPP與adenosine等等外在調節因子的特定細胞反應,同時也耗損了相當的能量,這應該是capacitation的生理意義所在。
展望
我們的實驗結果架構了FPP對於哺乳類動物精子的可能運作機制,提供了另一個關於精子capacitation與acrosome reaction發生機轉的研究方向。為了證實我們的假設,我們初步可以分別利用digitalis與vanadate來驗證FPP與精子細胞膜上的Na+ - K+ATPase與Ca++ATPase的關係。因為digitalis是Na+ - K+ATPase的抑制劑,digitalis的使用會導致細胞內Ca++濃度的提高;而vanadate則具有穩定Ca++ATPase以減少Ca++經其通透的作用。我們可以試著個別對於控制組精子與經過FPP、digitalis / vanadate、FPP + digitalis / vanadate處理後的精子,如同本實驗,利用CTC fluorescence assessment與CASA來分別測量各組精子的受精能力與各項運動參數的變化,藉由分析比較來證實並釐清FPP對於調節精子細胞內Ca++濃度的可能運作機轉。
另外,最近證實ceramide對於Ca++ATPase的活性有刺激作用,而sphingosine則有抑制Ca++ATPase活性的作用。這些sphingolipids應也可以考慮用於評估FPP與精子細胞膜上Ca++ATPase的作用關係。
除此之外,利用nifedipine、verapamil、diltiazem等鈣離子通道抑制劑( calcium channel blockers ),應該也有助於我們釐清鈣離子通道的活性在capacitation與acrosome reaction的發生機制中所扮演的角色。
由於精子所載有的DNA與RNA等遺傳物質也可能因為冷凍處理而受損,並進而損害精子的受精能力。為了了解參與FPP與adenosine訊息傳導途徑與運作機轉中的特定蛋白質及其功能,我們可以分別以新鮮與冷凍精蟲為對象,嘗試利用microarray來偵測加入FPP與adenosine後,特定mRNA的表現。
若FPP確實具有調節細胞膜上Na+ - K+ATPase與/或Ca++ATPase的作用,則無疑地於生物醫學上將有廣大的應用空間。
Summary
Introduction
Upon release from the male, mammalian sperm are nonfertilizing, but become functionally competent cells with the capacity to fertilize physiologically both in the female reproductive tract and / or under appropriate conditions in vitro, a process so-called “capacitation”. Capacitation involves both sperm surface and intracellular changes. Certain requirements for the modulation of capacitation have been identified :
Fertilization promoting peptide ( FPP; pGlu-Glu-ProNH2 ), a tripeptide produced by the prostate gland and secreted into seminal plasma in several mammals, is a potential modulator of capacitation in vivo. At ejaculation, nonfertilizing mammalian sperm come into contact with FPP, which elicits capacitation — dependant responses, i.e. stimulating capacitation in uncapacitated sperm and then inhibiting spontaneous acrosome reaction in capacitated cells. FPP thus initiates and maintains fertilizing potential of sperm until they contact an oocyte in the female reproductive tract.
Adenosine, another compound found in mammalian seminal plasma, elicits similar capacitation — dependant responses in sperm cells to FPP. The combination of FPP plus adenosine, whether mixed at low, non-stimulatory concentrations or high, maximally-stimulatory concentrations, was more effective in promoting capacitation than either compound used individually. This suggests that the two molecules act via separate external receptors and modulate a common signal transduction pathway.
Adequate sperm motility is known to be an essential prerequisite for successful fertilization. Pentoxifylline, clinically used to enhance motility of sperm from infertile human subjects, is a phosphodiesterase inhibitor leading to an increase in intracellular cAMP level. In hamsters, pentoxifylline has been shown to induce capacitation, hyperactivation and acrosome reaction, and also to improve fertilization rates in vitro. The application of pentoxifylline could contributes to enhancement of post — thawed sperm motility and selection of viable sperm in intracytoplasmic sperm injection.
Both FPP and adenosine may act as a first messenger, via adenylate cyclase ( AC ) / cAMP signaling pathway, to modulate the fertilizing ability of mammalian sperm. Cyclic AMP, therefore, plays an essential role in the mechanisms of capacitation and acrosome reaction, which are modulated by either external signal molecules such as FPP and adenosine, or by artificial sperm stimulants such as pentoxifylline. Herein, we try to evaluate the effects of FPP, adenosine and pentoxifylline on the fertilizing ability and motility characteristics of frozen — thawed human sperm.
Materials and Methods
Semen Sample
Semen obtained by masturbation was provided by volunteer donors. After liquefaction and basic seminalysis, the donated semen sample was stored by cryopreservation. On the day of examination, the frozen semen sample was thawed, and the post — thawed sperm suspension was then divided into 2 to 4 aliquotes, one of which was used as the control. Just after thawing, the other aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. Thereafter, we assessed the effects of these three reagents on frozen — thawed human sperm at 1 hour and 4 hours of incubation.
Part I. Chlortetracycline ( CTC ) Fluorescence Assessment
We collected 16 semen samples, all which were divided into 4 aliquotes of sperm suspension after thawing. One of the 4 aliquotes was used as the control, and the other aliquots were added with reagents to make 100nM FPP - treated, 10μM adenosine - treated, and 1 mg/mL pentoxyfilline - treated sperm suspensions respectively.
Most cytological techniques uased to determine the effects of treatment on sperm capacitation only identify acrosome — intact cells and acrosome — reacted cells。With CTC, the acrosome — intact category can be subdivided into two on the basis of apparent differences in their functional state, i.e. uncapacitated and capacitated:
F, with uniform fluorescence over the entire head, characteristic of uncapicitated, acrosome-intact cells
B, with a fluorescence - free band in the post-acrosomal region, characteristic of capicitated, acrosome - intact cells
AR, with dull or absent fluorescence over the sperm head, characteristic of capicitated, acrosome - reacted cells
In addition to CTC , we use Hoechst 33258 to assess the live / dead status of the sperm.
In each sample, 200 Hoechst 33258 negative cells were assessed for CTC staining patterns under Nikon’s eclipse E600 microscope equipped with phase — contrast and epifluorescent optics.
Part II. Computer Aided Sperm Analyzer ( CASA )
Depending on the post — thawed semen qualities, we divided the sperm suspensions into 2 to 4 aliquotes. One aliquote was used as the control, and the other 1 to 3 aliquots were treated to make 100nM FPP, 10μM adenosine, or 1 mg/mL pentoxyfilline sperm suspensions. There were 27 cases with FPP — treated aliquotes, 16 cases with adenosine — treated aliquotes, and 23 cases with pentoxyfilline — treated aliquots in this experiment.
Using CASA, we assessed the following motion parameters in each sample:
Straight — line velocity ( VSL ):the straight — line distance between the beginning and the end of the track divided by the time elapsed
Curvilinear velocity ( VCL ):the total distance between each position of the cell center of brightness ( CB ) for a given cell during the acquisition, divided by the time elapsed
Average path velocity ( VAP ):the smoothed average position of the CB and gives an average cell path velocity
Amplitude of lateral head displacement ( ALH ):the mean width of the head oscillation as the cell swims
Beat cross frequency ( BCF ):the frequency with which the cell track crosses the cell path in either direction
Straightness ( STR ):the departure of the cell path from a straight line
Linearity ( LIN ):the departure of the cell track from a straight line
Statistic Analyses
All statistic analyses were carried out by paired t — test. Percentage data of the CTC results were subjected to arc — sine transformation before statistic analyses. Data were expressed as mean ± SEM and P≦0.05 was considered to be statistically significant.
Results
Part I. Chlortetracycline ( CTC ) Fluorescence Assessment
CTC fluorescence assessment showed no significant differences between control group and FPP — treated, adenosine — treated, pentoxifylline — treated groups. All FPP, adenosine, and pentoxifylline failed to improve the fertilizing ability of frozen — thawed human sperm.
Part II. Computer Aided Sperm Analyzer ( CASA )
At 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated groups were significantly greater than that of control group. In addition, pentoxifylline significantly lessened LIN up to 4 hours of incubation.
Although FPP did not speed the frozen — thawed human sperm, it did modify the motility characteristics. At 1 hour of incubation, the BCF of FPP — treated group was significantly smaller than that of control group. At 4 hours of incubation, FPP made the STR and LIN much less than that of the control group.
Pentoxifylline did not affect the motility rate of the frozen — thawed human sperm. However, the motility rates of both the FPP — treated and adenosine — treated groups were unexpectedly lower than that of the control group at 1 hour of incubation.
Discussion
CTC fluorescence assessment showed that none of FPP, adenosine and pentoxifylline have significant effects on the capacitation and acrosome status of frozen — thawed human sperm. While comparing the motility characteristics of reagent - treated cells and non - treated cells with CASA, however, we found that all these three reagents did modify the swimming behaviors of frozen — thawed human sperm. The results of our studies gave us some hints of mechanisms about the modulation of sperm fertilizing ability:
After 1 hour of incubation, the VCL of adenosine — treated and pentoxifylline — treated cells are significantly greater than that of non — treated cells. This indicated that the intracellular cAMP level determines swimming velocity of sperm. Within the physiologically effective range, the higher the intracellular cAMP level, the larger the VCL. Because the swimming velocities of frozen — thawed cells did not be affected by the addition of FPP, we concluded that FPP, as a first messenger, may act on AC / cAMP signaling pathway indirectly. Despite of no effects on the velocities of the post — thawed sperm cells, FPP made the departure of cell tracks and paths significantly farther away from a straight line after 4 hours of incubation. Such a finding suggested that FPP is more closely than adenosine related to the alterations of swimming tracks associated with hyperactivation, which most of post- thawed sperm failed to express because of the damages resulting from freezing and thawing procedure. In addition, it is reasonable that the motility rates reflected the overall energy reserve of sperm population in our study. By activating adenylate cyclase, adenosine converted ATP to cAMP and thus contributed to the significant fall in the motility rate of post — thawed cells after 1 hour of incubation.. In contrast to pentoxifylline, which accelerated the movement of frozen — thawed cells without affecting motility rate, FPP caused a significant decrease in motility rate without speeding the sperm cells. There must be additional energy expenses other than those required for swimming in the FPP — treated cells. These additional energy expenses, which resulted in intracellular “ energy shift “, also accounted for the less active sports ( less BCF without greater velocities ) in the FPP —treated cells after 1 hour of incubation. After 4 hours of incubation, either of the adenosine — treated and FPP — treated groups had a smaller, but not significantly different motility rate from that of control group. This indicated that a substantial proportions of cryopreservation — damaged cells, because of energy exhaustion resulting from the addition of FPP or adenosine, did not survive as long as if not exposed to any reagent post — thawed.
Sperm cell membrane plays an essential role in capacitation and acrosome reaction. Cryopreservation causes damage to the integrity and function of the sperm cell membrane and thus alters the permeabilities of ion and water, leading to sperm intracellular ion concentrations out of the physiologic ranges optimal for fertilization. It is well known that the intracellular Ca++ concentration is closely correlated with the fertilizing ability of spermatozoa, and rearrangements of cytoskeletal elements modulated by intracellular Ca++ / calmodulin — dependent protein kinases lead to modifications of cell movement. Because the previous literature revealed that FPP is a potential modulator of capacitation and hyperactivation, and our studies showed that FPP could modify swimming behaviors with additional energy expenses in frozen — thawed sperm cells with non - optimal intracellular ion concentrations, we propose that FPP potentiates fertilizing ability and prevents spontaneous acrosome loss, via regulating membrane — bound Na+ - K+ATPase and / or Ca++ATPase, by keeping the sperm intracellular Ca++ concentration within the “ capacitation “ range. In addition to cAMP, the intracellular Ca++ is another second messenger in modulation of capacitation and acrosome reaction of mammalian spermatozoa.
FPP failed to speed frozen — thawed cells because the intracellular “ energy shift “ resulted in no significant increase in the cAMP level. However, pentoxifylline also made the departure of cell tracks significantly farther away from a straight line as FPP did. Therefore, there may be “ cross — talk “ between Ca++ / calmodulin and AC / cAMP signaling pathway.
While acting on the specific receptor on the sperm cell membrane, FPP may make conformational changes of the cytoplasmic domain of its receptor which then activate G proteins. Some of these G proteins help modulate the intracellular Ca++ level, activate the Ca++ / calmodulin — dependent protein kinases, and thus lead to rearrangements of cytoskeletal elements associated with hyperactivation. Other G proteins modulate AC / cAMP and the consequent protein phosphorylation. Both Ca++ / calmodulin and AC / cAMP signaling pathway interact with each other and thus modulate the expression of capacitation and hyperactivation.
Although Green et al. reported that FPP induces capacitation in fresh human sperm within one hour of incubation, our study showed that the physiologic effects on the motion characteristics of frozen — thawed cells caused by FPP appeared beyond one hour of incubation. Such a delay resulted from a longer time it took for FPP to modulate the intracellular ion concentrations, which might be far away from the physiologic ranges, in the viable frozen — thawed sperm cells to the optimal levels for fertilization. Because cryopreservation disrupted sperm cell membrane and cytoskeleton, only a few “ healthier “ post — thawed sperm cells could express capacitation and hyperactivation. This is why none of the three reagents used in our studies made any difference in the fertilizing ability between reagent —treated and control groups.
We suggest that FPP resets the intracellular ion concentrations of sperm cells at levels optimal for fertilization at ejaculation. Both the intracellular Ca++ and cAMP act as second messengers in the modulation of sperm fertilizing ability. There may be branching and cross — talk between pathways involved in capacitation to regulate and coordinate a sperm cell’s response to specific signal molecules such as FPP and adenosine. It consumes energy to regulate the intracellular ion concentrations and to amplify the cell’s specific response to the incoming information, so is the physiologic significance of the phenomenon of “ capacitation “.
Perspectives
To verify the mechanism of FPP in modulating the fertilizing ability of mammalian sperm, we could try to use digitalis and vanadate to evaluate the possible effects of FPP on Na+ - K+ATPase and Ca++ATPase on the sperm cell membrane. Digitalis is an inhibitor of Na+ - K+ATPase, which leads to an elevation of intracellular Ca++ level. By stablizing the structure of Ca++ATPase, vanadate slows down the passage of Ca++. Comparisons of the effects of FPP, digitalis / vanadate, and FPP + digitalis / vanadate on spermatozoa, we will clarify the possible mechanisms of FPP we proposed on the sperm intracellular ion concentrations.
In addition, it was recently demonstrated that ceramide stimulates the Ca++ATPase in a dose — dependent manner and sphingosine has conversely an inhibitory effect on Ca++ATPase activity. Such sphingolipids may also be considered in assessing whether or not FPP has any effects on membrane — bound Ca++ATPase of sperm.
Calcium channel blockers such as nifedipine, verapamil, and diltiazem may also help us understand the role of calcium channel in capacitation and acrosome reaction.
By using microarray, we may try to specify the proteins and their functions in the signal pathways responded to FPP and adenosine.
Once proved to contribute to regulation of membrane — bound Na+ - K+ATPase and Ca++ATPase, FPP could be widely applied in biomedical science.
目錄
中文摘要 1
緒論 7
研究方法與材料 12
結果 18
討論 19
展望 25
英文簡述 27
參考文獻 33
圖表 38
參考文獻
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