臺灣博碩士論文加值系統

由下視丘、腦垂腺、性腺構成的HPG軸是控制動物的生殖系統的重要中樞，下視丘會藉由分泌促性腺激素釋放激素(GnRH)刺激腦垂腺分泌促黃體生成素(LH)與促濾泡成熟激素(FSH)，腦垂腺分泌的激素會進一步影響下游性腺的發育與成熟，調控包括雌性與雄性的性內泌素生合成與配子的形成等生理功能。而輔助生殖技術(ART)就是透過調控HPG軸操作動物的胚或是配子的一項技術。畜牧業經常使用外源性激性腺素使動物發情同期化或是誘導排卵是ART的範疇之一。業界經常使用的外源性激性腺素除了HPG軸上的內泌素GnRH、LH、FSH與它們的類似物之外，孕馬血清激性腺素(PMSG/eCG)與人類絨毛膜激性腺素(hCG)也是經常使用的激素。PMSG與hCG被廣泛用於取代FSH與LH的使用，除了前者具有後者類似的生物活性之外，也有更長的體內半衰期，使用上更加方便與經濟，而因此受到業界的喜愛。然而，多次接受PMSG或hCG處理的動物會因為體內受到外源性內泌的素刺激而產生抗體，繼而對處理不敏感或無反應。此現象在豬、牛、羊中都有紀錄，顯示利用PMSG與hCG調控動物生殖週期的方法雖然有效但是並不永續。因此，發展出一套有別於傳統內泌素刺激法又能夠延長動物使用年限的生殖週期調控技術是目前重要的研究方向。
另外一方面，負責調控動物HPG軸上游的一群胜肽kisspeptin近年來受到注意，其中最小的生物活性片段kisspeptin-10是由十個氨基酸組成，研究發現kisspeptin能夠影響GnRH與LH/FSH的分泌，對於動物生殖系統的功能與發育具有重要的角色。此外，kisspeptin在物種之間的相似度非常高，顯示kisspeptin在跨物種間的應用有不容易產生抗體的優點。
為了探究kisspeptin-10能否於動物輔助生殖技術中取代PMSG與hCG的使用，我們以小鼠的超級排卵為試驗模型。利用9至12週齡的ICR母鼠進行取代PMSG或是hCG的試驗。在取代PMSG的試驗中，以迷你幫浦作為緩釋媒介，給予動物三種不同濃度的kisspeptin-10 (1, 10, 100 μg/100 μl) ；而取代hCG的試驗中則是透過單劑腹腔注射給予不同濃度之kisspeptin-10。紀錄不同處理之間的動物排卵率與排卵數，卵巢組織取下後透過石蠟包埋切片觀察不同處理組之間的差異。在取代PMSG的試驗中，經過kisspeptin-10處理的小鼠排卵率達到83%；而控制組與正控制組的排卵率分別是50%與100%。傳統的超級排卵方法得到最多的排卵數，而kisspeptin-10與控制組得到的平均排卵數相似。在取代hCG的試驗中，控制組的排卵率為20%，而低劑量的kisspeptin-10 (1 μg/100 μl) 排卵率為25%，中高劑量的kisspeptin-10 (10, 100 μg/100 μl) 沒有任何一隻動物排卵。為了更加確認單劑kisspeptin對於排卵的影響，我們延長了收卵的時間，從本來打完hCG之後13~14小時收卵改成18~20小時收卵。結果顯示控制組與kisspeptin-10高劑量組的小鼠排卵率相同，而排卵個數雖然kisspeptn-10的組別較少，但是於統計上與控制組沒有顯著差異。
綜合以上，此研究證明了經由皮下長時間給予kisspeptin-10能夠刺激小鼠的卵巢濾泡發育，發情同期化，而誘發排卵的數量又與一般正常週期的小鼠相似。單劑腹腔注射的kisspeptin-10相較於hCG則有延長排卵的效果，此部分的詳細機制仍需更多試驗得知。而透過皮下給予kisspeptin-10長時間刺激動物能有效地影響濾泡發育，將來可應用於動物的生殖週期調控以及生產管理。

Animal reproductive system is controlled by the hypothalamic-pituitary-gonadal axis (HPG axis). The hypothalamus secretes gonadotropin-releasing hormone (GnRH) and regulates the anterior pituitary gland to produce luteinizing hormone (LH) and follicle-stimulating hormone (FSH). The gonads produce estrogen and testosterone in response to FSH/LH. Assisted Reproductive Technologies (ART), which refers to the various procedures and techniques involving the handling of animals’ sperm, oocytes and embryos. ART in livestock widely use exogenous gonadotropins to alter the activity of HPG axis, include GnRH, LH, FSH, pregnant mare serum gonadotropin (PMSG/eCG), human chorionic gonadotropin (hCG) and their artificial analogs. Among these hormones, PMSG and hCG are the preferred alternatives than FSH and LH with longer half-lives and lower cost. However, animals received multiple PMSG/hCG treatment would become insensitive in response. The development of antibodies against PMSG/hCG was reported in heifers, gilts, sheep and goats, which implies that the PMSG/hCG treatment in animal industry may be useful in time but not sustainable. Therefore, to develop alternatives to replace conventional protocol that could prolong the use of animal while sustaining their reproductive ability is relatively important.
On the other hand, kisspeptin-10, which is the smallest functional kisspeptin peptides made up of ten amino acids, is a potential candidate to replace the conventional hormones with its role in reproductive physiology - the gatekeeper of gonadotropins. Kisspeptin stimulates the secretion of LH/FSH by regulating the activity of GnRH neurons. Moreover, kisspeptin is highly conserved among species, which reduce the chance of antibody development.
To investigate the feasibility of using kisspeptin-10 in replacing PMSG/hCG, we modified the conventional superovulation protocol on ICR mice. Female ICR mice between 9~12 weeks of age were used, and given different doses (1, 10, 100 μg/100 μl) of kisspeptin-10 through time-released Alzet® micro-osmotic pump (1 μl/hr) to substitute for PMSG; and a single injection of kisspeptin-10 at different level (1, 10, 100 μg/100 μl) to displace hCG. The percentage of ovulated mice and the number of ovulated oocytes were counted, and the ovaries were collected for further tissue examination. In the trial of replacing PMSG, the ovulation rate of kisspeptin-10 administrated mice was 83% (n=6) while the untreated group and the conventional protocol group were 50% and 100% (n=6). The conventional protocol group had highest average retrieved oocyte numbers, while the others were similar to the untreated control group. The trial of replacing hCG, the ovulation rate was 20% (n=10) in saline control group, and the low dose of kisspeptin-10 (1 μg/100 μl) was 25% (n=4), while the medium dose and high dose (10, 100 μg/100 μl) groups were 0% (n=4) and 0% (n=8). The conventional protocol group was 100% (n=8). In another trial, to confirm the effect of single dose kisspeptin-10 to ovulation, the oocyte retrieval time was prolonged from 13-14 hour to 18-20 hour. The ovulation rate and ovulated oocyte was similar between saline control group versus kisspeptin-10 (100 μg/100 μl) group (33%, n=6).
In conclusion, our study demonstrated the effect of long-term peripheral kisspeptin-10 administration on mice reproduction, which has a follicular stimulated potential and synchronizes the estrous cycle with normally ovulated oocyte number, while a single dose of kisspeptin-10 at higher levels seems to prolong the ovulation. The results indicate that the long-term stimulation of kisspeptin-10 could affect the follicular development, which could be a new strategy in livestock reproduction management after some further investigation.

Table of Contents
中文摘要 iv
Abstract vi
Table of Contents ix
List of Figures and Tables xi
1. Introduction 1
2. Literature Review 3
2.1 Assisted Reproductive Technology 3
2.1.1 Exogenous gonadotropins 3
2.1.2 ART using PMSG/hCG 5
2.1.3 Drawback of the use of PMSG/hCG 7
2.2 Kisspeptin in reproduction 9
2.2.1 Kisspeptin regulates HPG-axis 11
2.2.2 Kisspeptin triggers puberty and maintain fertility 14
2.2.3 The feedback loop of steroid hormones 17
2.2.4 The local effects of Kisspeptin in gonads 19
2.2.5 Kisspeptin’s prospect in ART 22
2.3 Sustained release techniques 25
2.4 Histology of mouse ovary and follicle development 27
3. Materials and Methods 32
3.1 Animals 32
3.2 Chemicals 32
3.3 Time-Release of kisspeptin-10 33
3.4 Experimental Design 33
3.4.1 PMSG replacement trial 33
3.4.2 hCG replacement trial 34
3.5 Tissue collection and histology assessment 36
3.6 Statistical analyses 36
4. Results 37
4.1 The Effect of Long-term Subcutaneously Release of Kisspeptin-10 in Substituting for PMSG. 38
4.2 The Effect of Using Single Intraperitoneal Injection of Kisspeptin-10 to replace hCG. 40
5. Discussion 43
5.1 The Effect of Long-term Subcutaneously Release of Kisspeptin-10 in Substituting for PMSG. 43
5.2 The Effect of Using Single Intraperitoneal Injection of Kisspeptin-10 to replace hCG. 46
6. Conclusion 50
7. Figures and Tables 51
8. Reference 59

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