Mortimer S T, Swan M A, Mortimer D
Department of Anatomy and Histology and Institute for Biomedical Research, University of Sydney, NSW, Australia.
Hum Reprod. 1998 Aug;13(8):2139-46. doi: 10.1093/humrep/13.8.2139.
While hyperactivated motility is known to be a concomitant of capacitation, and a prerequisite for fertilization, the specific interdependence of capacitation and hyperactivation in human spermatozoa has not been investigated. This study was designed to determine the effect of seminal plasma contamination on the expression of hyperactivated motility and the relationship between hyperactivation and capacitation, since seminal plasma contains decapacitation factor(s). Seminal plasma was obtained by centrifugation of aliquots of liquefied semen layered over 1.5 ml 40.5% Percoll and mixed with human tubal fluid (HTF) medium containing 30 mg/ml human serum albumin (HSA) (HTF) to a final concentration of 5% (v/v) seminal plasma (SP). Motile spermatozoa were isolated from the remainder of the semen by swim-up into either HTF or SP medium. Samples were taken from each treatment immediately post-harvest (0 h) and after 60 min at 37 degrees C (1 h) for hyperactivation and capacitation assessment. The treatments were then divided into two portions, centrifuged and resuspended in either HTF or SP, giving HTF control and SP control treatments and two crossover treatments, 1 h HTF then 1 h SP (H/SP) and 1 h SP then 1 h HTF (SP/H). All tubes were incubated for a further 60 min at 37 degrees C before aliquots were taken for hyperactivation and capacitation assessments. Hyperactivation was estimated using an IVOS v10.6t (Hamilton Thorne Research, Beverly, MA, USA) 60 Hz CASA instrument, and capacitation was estimated using the chlortetracycline (CTC) method. The presence of seminal plasma in the capacitation medium for 60-120 min post-swim-up inhibited the development of hyperactivated motility. This inhibition was reversible, and was not prevented by preincubation for 1 h in HTF medium. There was no difference in the CTC binding patterns between treatments at 2 h, indicating that the capacitation-associated membrane changes were not affected by the presence of a low concentration of seminal plasma. There was no correlation between percentage capacitated and percentage hyperactivated spermatozoa for any treatment. Since the proportions of hyperactivated spermatozoa and capacitated spermatozoa were not related, we conclude that the processes leading to hyperactivation and to the membrane changes associated with capacitation are not tightly interlinked and consider this finding to be due to hyperactivated motility being associated with flagellar movement, while the CTC assay assesses changes in the Ca2+ levels of the sperm head plasma membrane.
虽然已知超活化运动是获能的伴随现象，也是受精的先决条件，但人类精子获能与超活化之间的具体相互依存关系尚未得到研究。本研究旨在确定精浆污染对超活化运动表达的影响以及超活化与获能之间的关系，因为精浆中含有去能因子。通过将等分的液化精液离心，使其分层于1.5 ml 40.5%的Percoll上，获得精浆，并将其与人输卵管液（HTF）培养基混合，该培养基含有30 mg/ml人血清白蛋白（HSA）（HTF），使精浆最终浓度达到5%（v/v）（SP）。通过上游法将活动精子从其余精液中分离出来，置于HTF或SP培养基中。在收获后立即（0小时）以及在37℃孵育60分钟后（1小时）从每种处理中取样，用于评估超活化和获能情况。然后将处理分为两部分，离心并重新悬浮于HTF或SP中，得到HTF对照和SP对照处理以及两种交叉处理，即1小时HTF然后1小时SP（H/SP）和1小时SP然后1小时HTF（SP/H）。所有试管在37℃再孵育60分钟，然后取等分试样用于评估超活化和获能情况。使用IVOS v10.6t（美国马萨诸塞州贝弗利市汉密尔顿·桑恩研究公司）60 Hz的计算机辅助精子分析（CASA）仪器评估超活化情况，使用金霉素（CTC）方法评估获能情况。上游后在获能培养基中存在精浆60 - 120分钟会抑制超活化运动的发展。这种抑制是可逆的，并且在HTF培养基中预孵育1小时并不能阻止这种抑制。在2小时时各处理之间的CTC结合模式没有差异，这表明与获能相关的膜变化不受低浓度精浆存在的影响。对于任何处理，获能精子百分比与超活化精子百分比之间均无相关性。由于超活化精子比例和获能精子比例不相关，我们得出结论，导致超活化的过程与导致与获能相关的膜变化的过程并非紧密相连，并认为这一发现是由于超活化运动与鞭毛运动相关，而CTC检测评估的是精子头部质膜Ca2+水平的变化。
