Simultaneous removal of sperm plasma membrane and acrosome before intracytoplasmic sperm injection improves oocyte activation/embryonic development

Abstract

Direct injection of a single spermatozoon into an oocyte (ICSI) can produce apparently normal offspring. Although the production of normal offspring by ICSI has been successful in mice and humans, it has been less successful in many other species. The reason for this is not clear, but could be, in part, due to inconsistent activation of oocytes because of delayed disintegration of sperm plasma membrane within oocytes and incorporation of the acrosome containing a spectrum of hydrolyzing enzymes. In the mouse, the removal of sperm plasma membrane and acrosome was not a prerequisite to produce offspring by ICSI, but it resulted in earlier onset of oocyte activation and better embryonic development. The best result was obtained when spermatozoa were demembranated individually immediately before ICSI by using lysolecithin, a hydrolysis product of membrane phospholipids.mouse ͉ human ͉ Ca 2ϩ oscillations ͉ fertilization ͉ lysolecithin D irect injection of a single spermatozoon into an oocyte, commonly called intracytoplasmic sperm injection (ICSI), can produce apparently normal offspring even though it bypasses a number of biological processes necessary for normal fertilization. As long as the sperm nucleus has intact genetic integrity, ICSI can produce healthy offspring regardless of concentrations, morphology, and motility of spermatozoa (1, 2). Only one genomically normal spermatozoon is needed to fertilize one oocyte. A salient difference between natural and ICSI fertilization is that, in the latter, the sperm plasma membrane as well as acrosome (which contains a spectrum of powerful hydrolyzing enzymes) are introduced into an oocyte. For species with small acrosomes, injection of the acrosome into an oocyte apparently does not produce serious problems, but for species like the hamster, with very large acrosomes, injection inevitably results in death of the oocyte (3). We had demonstrated that the contents of the acrosome are potentially harmful to oocytes (4). A notable difference between normal and ICSI fertilization is that repetitive transient increases in intracellular Ca 2ϩ concentration of the oocyte (Ca 2ϩ oscillations), the pivotal signal for oocyte activation (5-8), begins much more slowly in ICSI oocytes than in normally fertilized oocytes. In the mouse, for instance, Ca 2ϩ oscillations begin 1-3 min after plasma membrane fusion between a fertilizing spermatozoon and an oocyte (9), whereas oscillation begins 15-30 min after ICSI (10, 11). In human oocytes, Ca 2ϩ oscillations start 4-12 h after ICSI, when a plasma membrane-intact spermatozoon is injected after immobilization by touching the terminal part of the tail (12). Ca 2ϩ oscillations begin faster (14 Ϯ 6 min) when a spermatozoon is immobilized by applying several piezo pulses to the proximal one-third of the sperm tail before injection (13). Kasai et al. (14) reported that oocyte activation, assessed by the completion of meiosis, occurred earlier when spermatozoa were freed from the plasma membrane before ICSI. Increased fertilization rates aft...

Chromosome analysis of mouse zygotes produced by intracytoplasmic injection of spermatozoa exposed to acrosome reaction inducing agents methyl-β-cyclodextrin and calcium ionophore A23187

“…These spermatozoal structures have been suggested to affect sperm chromatin remodeling in mice [2], pigs [3] and rhesus monkeys [4][5][6]. While injection of uncapacitated spermatozoa with intact acrosomes affects development of mouse ICSI embryos [7], simultaneous removal of the plasma membrane and acrosome before ICSI improves embryonic development [8]. Supportive evidence has been presented for the rat [9].…”

Plasma membrane and acrosome loss before ICSI is required for sheep embryonic development

“…However, despite the fact that the damaging of the plasma membrane positively influences the success of ICSI, its complete extraction by Triton X-100 incubation turned out to be deleterious for embryonic development, in contrast to what has been described in mouse [18]. The dissolution of the plasma membrane by detergent could induce the loss of the spermactivating factor, abolishing its activation ability, or perhaps it might denature the centriole (an issue not explored here).…”

“…Several studies in mice have stressed the importance of the membrane and acrosome's removal before ICSI [18,25]. Furthermore, the retention of spermatozoa membranes is known to delay oocyte activation in mouse and human model [19,38], and the acrosomal enzymes are harmful to hamster oocytes [25,37].…”

The ability of freeze-dried bull spermatozoa to induce calcium oscillations and resumption of meiosis

“…Since the stability of sperm function after freeze-drying and long-term storage has not been fully investigated, the first experiment of the present study was designed to examine the ability of FD bull spermatozoa to induce calcium oscillations using interspecies ICSI assay. The interspecies ICSI assay using hybrid mouse oocytes, that was originally developed by Rybouchkin et al [34], has been used to investigate the SOAF activity of different spermatogenic cells in hamster, rat, rabbit, human [35] whale [29] and cynomolgus monkey [36] and chemically membrane-damaged spermatozoa in cattle, pig and human [37]. The advantages of using B6D2F1 mouse oocytes are their abilities to be loaded easily with calcium-sensitive fluorescent dye and their resistance to sham-injection treatment in term of oocyte activation.…”

“…1a) may reflect a qualitatively or quantitatively higher SOAF activity in each spermatozoon, because such a short-cycled oscillation pattern has been reported when a non-physiologically larger volume of porcine [38,39] and equine [40] sperm extracts, or isolated sperm heads from bulls and whales but not from mice [29] were microinseminated into mouse oocytes. Moreover, human spermatozoa treated with lysolecithin induced calcium oscillations with higher frequent spikes [37], so the frequency of calcium oscillation may be related to factors controlling release of SOAF into the ooplasm. The process of SOAF release into ooplasm requires destruction of plasma membrane and exposure of perinuclear theca [41].…”