Choi Y H, Landim-Alvarenga F C, Seidel G E, Squires E L
Department of Biomedical Sciences, Animal Reproduction and Biotechnology Laboratory, Colorado State University, Fort Collins 80523, USA.
J Anim Sci. 2003 Aug;81(8):2080-7. doi: 10.2527/2003.8182080x.
Experiments were conducted to study effects of macromolecules on stallion sperm capacitation and fertilization as determined by penetration of bovine zona-free and equine partially zona-removed oocytes. Stallion sperm were capacitated in TYH medium (modified Krebs-Ringer bicarbonate) supplemented with either 1 mg/mL of polyvinylalcohol (PVA) or 4 mg/mL of BSA. Capacitation was induced with 8 bromoadenosine cyclic monophosphate (8BrcAMP; 0.5 mM) alone or in combination with 0.1 microM of ionomycin. Intraspecies gametes were co-incubated in TYH/PVA or TYH/BSA for 18 to 20 h. For zona-free bovine oocytes, penetration rate (35%) with the combination of 8BrcAMP and ionomycin in PVA-containing medium was higher (P < 0.05) than any treatment in BSA-containing medium (5 to 6%). A similar study was conducted using equine oocytes with partially removed zonae. Sperm capacitated and used for in vitro fertilization (IVF) in PVA-containing medium had higher penetration rates (P < 0.01) than sperm in BSA-containing medium (54 vs. 11%). The effect of equine preovulatory follicular fluid on bovine oocyte penetration was assessed. Bovine oocytes were matured in tissue culture medium-199 with 0, 20, 50, or 100% equine preovulatory follicular fluid, and 1 IU/mL of equine chorionic gonadotropin. Stallion sperm were treated with 8BrcAMP + ionomycin in PVA- or BSA-containing media. The penetration rates of bovine zona-free oocytes by stallion sperm were again higher with PVA (47%) than BSA (18%; P < 0.01). Penetration rates of oocytes matured in 100% follicular fluid were higher (P < 0.05) than for oocytes matured with 0% follicular fluid. The effects of equine follicular fluid and PVA/BSA during sperm capacitation on standard bovine IVF were examined. Culture of bovine oocytes with equine follicular fluid did not affect oocyte maturation or penetration rates after IVF. Bovine sperm capacitated with heparin in PVA-containing medium yielded lower (P < 0.05) fertilization rates than those capacitated in BSA-containing medium when incubated with both zona-intact and zona-free bovine oocytes. In summary, PVA was superior to BSA for ionophore-induced capacitation of equine sperm for penetration of zona-free bovine oocytes or partially zona-removed equine oocytes, but not for standard bovine IVF with bovine sperm. Zona-free bovine oocytes may be useful for assaying in vitro capacitation and fertilization of stallion sperm.
进行了实验，以研究大分子对种马精子获能和受精的影响，这是通过牛无透明带卵母细胞和马部分去除透明带的卵母细胞的穿透情况来确定的。种马精子在添加了1 mg/mL聚乙烯醇（PVA）或4 mg/mL牛血清白蛋白（BSA）的TYH培养基（改良的克雷布斯-林格碳酸氢盐培养基）中获能。单独使用8-溴腺苷环磷酸（8BrcAMP；0.5 mM）或与0.1 microM离子霉素联合诱导获能。种内配子在TYH/PVA或TYH/BSA中共同孵育18至20小时。对于牛无透明带卵母细胞，在含PVA的培养基中，8BrcAMP和离子霉素联合使用时的穿透率（35%）高于含BSA培养基中的任何处理组（5%至6%，P < 0.05）。使用部分去除透明带的马卵母细胞进行了类似研究。在含PVA的培养基中获能并用于体外受精（IVF）的精子穿透率高于含BSA培养基中的精子（54%对11%，P < 0.01）。评估了马排卵前卵泡液对牛卵母细胞穿透的影响。牛卵母细胞在含有0、20、50或100%马排卵前卵泡液以及1 IU/mL马绒毛膜促性腺激素的组织培养基-199中成熟。种马精子在含PVA或含BSA的培养基中用8BrcAMP +离子霉素处理。种马精子对牛无透明带卵母细胞的穿透率再次显示，PVA组（47%）高于BSA组（18%；P < 0.01）。在100%卵泡液中成熟的卵母细胞的穿透率高于在0%卵泡液中成熟的卵母细胞（P < 0.05）。研究了马卵泡液和PVA/BSA在精子获能过程中对标准牛IVF的影响。用马卵泡液培养牛卵母细胞不影响IVF后的卵母细胞成熟或穿透率。当与完整透明带和无透明带的牛卵母细胞一起孵育时，在含PVA的培养基中用肝素使牛精子获能产生的受精率低于在含BSA的培养基中获能的精子（P < 0.05）。总之，对于离子霉素诱导的种马精子获能以穿透牛无透明带卵母细胞或部分去除透明带的马卵母细胞，PVA优于BSA，但对于用牛精子进行的标准牛IVF则不然。牛无透明带卵母细胞可能有助于检测种马精子的体外获能和受精情况。
