| Summary: | 碩士 === 國立臺灣大學 === 動物科學技術學研究所 === 105 === 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.
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