Factors affecting fertilization of porcine oocytes following intracytoplasmic injection of sperm

The objective of this study was to optimize intracytoplasmic sperm injection (ICSI), and to assess the effects of membrane-damaged sperm on development of porcine oocytes following ICSI. For optimization of development following the ICSI process, sperm injected oocytes were activated 0.5-1.0 hr after ICSI with 1 x 30 micros pulse of 1.2, 1.7, 2.2, and 2.7 kV/cm DC in experiment 1. After 7-days of culture ICSI oocytes activated with [x1]2.2 kV/cm produced more blastocyts ([x2]34.4%, P < 0.05) than other treatment groups. In experiment 2, oocytes were activated with 1 x 30 micros pulse of 2.2 kV/cm at either 0, 1.5, 3, or 6 hr after ICSI. Oocytes activated 1.5 hr after [x3]ICSI yielded more blastocysts (27.6%)[M4] than in other treatments. In experiment 3, sperm were briefly exposed to 0.1% Triton X-100 to induce membrane damage. Live-dead staining of Percoll-sorted untreated spermatozoa (frozen-thawed) used in this study showed that over 96% were "alive" whereas none were "alive" after Triton X-100 treatment. The rate of development to blastocyst of oocytes injected with Triton X-100 treated sperm combined with electrical activation (EA) at 1 x 30 micros pulse of 2.2 kV/cm (EA, 40.0%) was the best, when compared with those injected with untreated sperm plus EA (P < 0.05). In experiment 4, the development rate of oocytes to the blastocyst stage ([x5]32.1%) following injection of a sperm head only was not significantly different from that of oocytes injected with whole sperm (31.0%). In conclusion, we found that an intact membrane and tail structures of pig spermatozoa are not essential for embryo development by ICSI, and furthermore, dead porcine spermatozoa, at an early stage of necrosis caused by plasma membrane damage, support better embryo development than do live non-damaged sperm.     

Comparison of intracytoplasmic sperm injection for inbred and hybrid mice

We compared the results of intracytoplasmic sperm injection (ICSI) that leads to full term development of hybrid (B6C3F1 and B6D2F1) and inbred (C57BL/6) mouse embryos. Although fertilization and pre-implantation development of C57BL/6 eggs were similar to those of F1 hybrid eggs, post-implantation development of the embryos from C57BL/6 females was significantly poorer than those of the eggs from hybrid females. Reciprocal crosses of C57BL/6 and B6C3F1 gametes revealed that the low rate of post-implantation development of C57BL/6 embryos was due to oocyte factor(s), rather than the sperm factor.     

Fertilization of porcine oocytes following intracytoplasmic spermatozoon or isolated sperm head injection

We demonstrated normal fertilization processes (as determined by pronuclear formation, pronuclear apposition and syngamy) in porcine oocytes either following intracytoplasmic spermatozoon (ICSI) or isolated sperm head injection. Microtubule organization and chromatin configuration were investigated in these oocytes during the first cell cycle. Following ICSI, the microtubular aster was organized from the neck of the spermatozoon and filled the whole cytoplasm. These male-derived microtubules appear to move both pronuclei to the center of oocytes. These cytoskeletal changes are analogous to those seen following conventional fertilization. In contrast, following isolated sperm head injection, the sperm aster was not seen. Instead, the microtubule matrix was organized from the cortex and then filled the whole cytoplasm in all cases in normally fertilized oocytes following injection (n = 35). This organization is similar to what has been shown in the parthenogenetically activated oocytes. Chromosome analysis revealed that the oocytes injected with isolated sperm heads were fertilized normally. At 7 days following injection, the incidence of blastocoele formation following ICSI (38%) and isolated sperm head injection (22%) was higher than that following sham injection (2%). These results suggested that successful fertilization and preimplantation development occurred in porcine oocytes following either ICSI or isolated sperm head injection. Our results also indicated that fertilization processes can occur by self-assembled microtubules within cytoplasm in the absence of a sperm centrosome. Mol. Reprod. Dev. 51:436–444, 1998. © 1998 Wiley-Liss, Inc.     

Activation regimens to prepare bovine oocytes for intracytoplasmic sperm injection

Activation of bovine oocytes to produce a single haploid pronucleus in preparation for intracytoplasmic sperm injection (ICSI) has been investigated with various combinations of ionomycin and 6-dimethylaminopurine (DMAP). Effects were evaluated by immunocytochemical staining, chromosomal analysis and assessment of development in vitro. Oocytes matured in vitro were exposed to: ionomycin alone (single or repeated treatments, Groups 1 and 2 respectively), ionomycin followed by DMAP (immediately or after a 3-h delay, Groups 3 and 4), or no treatment (control, Group 5). They were then co-cultured in M199 with bovine oviductal epithelial cells. Activation rates were not significantly different among groups but significantly fewer oocytes in Group 3 extruded a second polar body than in Groups 1, 2, and 4. Most parthenotes (60% to 80%) in Groups 1, 2, and 4 were haploid, whereas 82% in Group 3 were mixoploid or polyploid. Most of the parthenotes (88%) in Group 4 formed a single pronucleus besides extruding the second polar body and were therefore more suitable for ICSI than those of Groups 1 and 2 in which condensed chromosomes predominated. The respective rates of oocyte cleavage in Groups 1 to 4 were 24%, 36%, 70%, and 75%; corresponding blastocyst rates were 1%, 5%, 17%, and 8%. There were significantly fewer cells in the parthenotes of Groups 1, 2, and 4 than of Group 3, or of embryos produced by in vitro fertilization. Thus, delaying the addition of DMAP after ionomycin decreases chromosomal abnormalities and produces a high proportion of activated oocytes suitable for ICSI. Mol. Reprod. Dev. 50:485–492, 1998. © 1998 Wiley-Liss, Inc.     

Sperm membrane incorporation into oolemma contributes to the oolemma block to sperm penetration: Evidence based on intracytoplasmic sperm injection experiments in the mouse

Mouse oocytes were fertilized by intracytoplasmic sperm injection (ICSI) and reinseminated after the removal of zonae pellucidae at pronuclear stage or at the 2-cell stage. Although these oocytes were activated normally by ICSI, as evidenced by resumption of meiosis and cortical granule exocytosis, they did not develop oolemma block to sperm penetration. They could be penetrated by spermatozoa at pronuclear stage and even at the 2-cell stage. This supports the notion that incorporation of sperm plasma membrane into oolemma contributes to the changes in oolemma that block sperm penetration. © 1996 Wiley-Liss, Inc.     

Inadequate function of sterile tw5/tw32 spermatozoa overcome by intracytoplasmic sperm injection

Mice carrying two t complementary haplotypes (t^w5/t^w32) are totally sterile. Their spermatozoa have poor motility and fertilize neither zone-intact nor zona-free oocytes, even though they are structurally indistinguishable from control (wild-type) spermatozoa. However, when injected directly into oocytes, these infertile spermatozoa are able to participate in normal development. This suggests that infertility of t^w5/t^w32 male (spermatozoa) is more likely to be due to poor sperm-oocyte interaction than to genetic incompetence of sperm nuclei. © 1996 Wiley-Liss, Inc.     

Investigation of assisted fertilization and biology of reproduction by sperm microinjection

Recent success in assisted fertilization mainly depended on the development of sperm microinjection methods: intracytoplasmic sperm injection and subzonal insemination. Some basic mechanisms that under-lie fertilization were revealed by using intracytoplasmic sperm injection. In respect to this, problems of fertility, oocyte activation, formation of pronuclei and practical aspects of intracytoplasmic sperm injection are discussed.     

Active demethylation of individual genes in intracytoplasmic sperm injection rabbit embryos

Intracytoplasmic sperm injection (ICSI), as an assisted reproduction technique, has been widely used in animal and human. However, its possible effect on epigenetic changes has not been well studied. To investigate whether ICSI can induce aberrant DNA methylation changes in rabbit preimplantation embryos, we examined the methylation status of the SP-A promoter region and the satellite sequence Rsat IIE by bisulfite-sequencing technology. The SP-A promoter region was extensively demethylated before the first round of DNA replication commences, and the unmethylated status was maintained until morula when dynamic remethylation occurred. A similar but more moderate demethylation process was observed in satellite sequence Rsat IIE. These results are in contrast with the previous reports of no active demethylation in normal rabbit embryos, suggesting that the active demethylation we observed may be induced by ICSI.     

Rhesus macaque blastocysts resulting from intracytoplasmic sperm injection of vacuum‐dried spermatozoa*

Background Sperm desiccation is an attractive approach for sperm preservation. In this study, we examined the feasibility and efficiency of intracytoplasmic sperm injection using vacuum‐dried rhesus macaque sperm in CZB medium supplemented with 10% fetal bovine serum. Methods A total of 109 MII oocytes were injected with 69 fresh ejaculated sperm and 40 vacuum‐dried sperm. Results Cleavage occurred in 97% of oocytes injected with fresh, motile sperm and in 88% of oocytes injected with vacuum‐dried sperm. Of the cleaved oocytes, 68% fresh sperm‐injected oocytes and 74% of dried sperm‐injected oocytes developed to the compact morula stage. Blastocyst development was comparable between fresh‐injected (16%) and vacuum‐dried‐injected (17%) oocytes. Differences between treatment groups were not significant. Transmission electron microscopic observation of the blastocysts indicated no detectable differences between fresh sperm and dried sperm‐derived embryos. Conclusions We conclude that vacuum‐dried rhesus macaque sperm are capable of inducing fertilization and development of pre‐implantation embryos when sperm were dried under vacuum and microinjected into normal viable oocytes.     

Understanding fertilization through intracytoplasmic sperm injection (ICSI)

Since the establishment of in vitro fertilization, it became evident that almost half of the couples failed to achieve fertilization and this phenomenon was attributed to a male gamete dysfunction. The adoption of assisted fertilization techniques particularly ICSI has been able to alleviate male factor infertility by granting the consistent ability of a viable spermatozoon to activate an oocyte. Single sperm injection, by pinpointing the beginning of fertilization, has been an invaluable tool in clarifying the different aspects of early fertilization and syngamy. However, even with ICSI some couples fail to fertilize due to ooplasmic dysmaturity in relation to the achieved nuclear maturation marked by the extrusion of the first polar body. More uncommon are cases where the spermatozoa partially or completely lack the specific oocyte activating factor. In this work, we review the most relevant aspects of fertilization and its failure through assisted reproductive technologies. Attempts at diagnosing and treating clinical fertilization failure are described.     

脱氢紫堇碱对正常和低氧豚鼠心肌细胞内钙的影响

目的 :探讨脱氢紫堇碱 (dehydeocorydaline,DHC)及维拉帕米 (verapamil,Ver)对豚鼠心肌细胞内游离钙浓度([Ca2 + ] i)变化的影响。方法 :采用离体豚鼠心脏Langendorff法灌注 ,用荧光指示剂方法 (Fure 2 /AM)标记心肌([Ca2 + ] i)变化。观察低氧后心肌 [Ca2 + ] i 的变化。结果 :①正常氧状态心肌 [Ca2 + ] i 均值为 (1 2 0 .5± 8.3)nmol/L(n =2 0 ) ;②正常氧条件下 ,DHC、Ver均使心肌 [Ca2 + ] i 明显下降。 (3)低氧状态下 ,心肌 [Ca2 + ] i 增加与缺氧时间(程度 )直线相关 (r=0 .98)。④DHC对低氧后心肌 [Ca2 + ] i 增加明显减缓。结论 :DHC在正常氧、低氧条件下阻止心肌细胞内钙超载 ,我们认为DHC可能提高心肌细胞的自我保护作用     

Influence of oocyte collection technique on initial chromatin configuration, meiotic competence, and male pronucleus formation after intracytoplasmic sperm injection (ICSI) of equine oocytes

There is a great variability in the success of horse oocyte maturation and fertilization among laboratories. This study was conducted to determine if the meiotic and developmental competence of horse oocytes could be dependent on the method of oocyte collection, i.e., aspiration of follicular fluid with a vacuum apparatus, or opening follicles and scraping the granulosa layer. Horse oocytes were recovered from abattoir ovaries by aspiration or scraping and classified as having compact (Cp), expanded (Ex), or partial (P) cumuli. In Experiment 1 (Part A in May and Part B in October), oocytes were fixed immediately after collection to assess whether the collection method influenced the initial chromatin configuration of oocytes. In Experiment 2, in vitro maturation rates of oocytes recovered by aspiration or scraping were compared. In Experiment 3, oocytes were matured in vitro and submitted to intracytoplasmic sperm injection (ICSI). Initial chromatin configuration differed according to collection method in that there was a significantly higher prevalence of diffuse chromatin within the germinal vesicle in oocytes recovered by scraping than in oocytes recovered by aspiration (29/87, 33% and 28/166, 17%, respectively; P < 0.01). Maturation of oocytes to metaphase II did not significantly differ between scraped and aspirated oocytes (56/101, 55.4 % vs. 65/106, 61.4%, respectively). The overall pronucleus formation rate after ICSI of oocytes recovered by scraping was not significantly different than that of oocytes recovered by aspiration (50/99, 52.6% vs. 50/85, 68.5 %, respectively); however, the rate of abnormal fertilization was significantly higher for oocytes collected by aspiration (14/73, 19% vs. 6/94, 6%, respectively; P <0.05). These results demonstrate that the collection method affects the population of recovered oocytes and may contribute to differences in results observed among laboratories working with horse oocytes.     

Sperm motility-initiating substance in newt egg-jelly induces differential initiation of sperm motility based on sperm intracellular calcium levels

Sperm motility-initiating substance (SMIS), a novel motility inducer from newt egg-jelly, is activated by the release from associated jelly substances at the beginning of internal fertilization and affects female-stored sperm. We examined motility initiation kinetics of newt sperm in response to SMIS by monitoring the changes of sperm intracellular calcium ([Ca2(+)](i)). In quiescent non-motile sperm loaded with the Ca2(+) indicator Fluo-4, intracellular free Ca2(+) was observed around mitochondria using confocal scanning laser microscopy. A slight increase in [Ca2(+)](i) occurred simultaneously and transiently at motility initiation in sperm treated with either heated jelly extract (hJE) containing activated SMIS, or a low osmotic solution, which naturally initiates motility in externally-fertilizing amphibians and can initiate motility in urodele sperm. When the increase of [Ca2(+)](i) at motility-initiation was monitored using spectrofluorometry, large increases in [Ca2(+)](i) occurred immediately in the low osmotic solution and within 1.5 min in the hJE. In the intact jelly extract (no heating), small increases of [Ca2(+)](i) irregularly occurred from around 1 min and for about 4 min, during which motility was differentially initiated among sperm. These results indicate that the SMIS induces differential initiation of sperm motility depending on the activational states of the SMIS and its overall activity. The motility initiation in the jelly extract was delayed in sperm whose intracellular Ca2(+) had been chelated with BAPTA-AM. The relative levels of [Ca2(+)](i) were variable with a mean of 414 ± 256 nmol/L among resting sperm, suggesting that the level of [Ca2(+)](i) in the resting sperm modulates the responsiveness to the SMIS.     

Full term development of rabbit oocytes fertilized by intracytoplasmic sperm injection

Intracytoplasmic sperm injection (ICSI) has been applied successfully in the treatment of male infertility in humans and in fertilization research in mice. However, the technique has had limited success in producing offspring in other species including the rabbit. The aim of this research was to test the in vitro and in vivo developmental of rabbit oocytes after ICSI. Sperm used for ICSI were collected from mature Dutch Belted buck and washed 2-3 times with PBS +0.1% polyvinyl alcohol (PVA) and then mixed with 10% polyvinyl pyrrolidone (PVP) prior to microinjection. Oocytes were collected from superovulated does 14-15 hr after hCG injection and were fertilized by microinjection of a single sperm into the ooplasm of each oocyte without additional activation treatment. After ICSI, the presumed zygotes were either cultured in KSOM +0.3% BSA for 4 days or transferred into oviducts of recipient does at the pronuclear or 2-cell stage. A high percentage of fertilization (78%, n = 114) and blastocyst development (39%) was obtained after ICSI. Control oocytes, receiving a sham injection, exhibited a lower activation rate (31%, n = 51) and were unable to develop to the blastocyst stage, suggesting that the blastocysts developed following ICSI were derived from successful fertilization rather than parthenogenetic development. A total of 113 embryos were transferred to six recipient does. Two recipients became pregnant and delivered seven live young. Our results demonstrated that rabbit oocytes can be successfully fertilized and activated by ICSI and can result in the birth of live offspring.     

整合素介导小鼠卵内钙离子增加

为了研究整合素是否作为跨膜信号传递受体介导小鼠卵[Ca^2 ]i的变化并探讨其机制。本实验采用甘-精-甘-天冬-丝-脯(GLY-ARG-GLY-ASP-SER-PRO,RGD肽)、纤连蛋A(fibronectin,Fn)及抗整合素α6、β1的单克隆抗体作用于负载了钙探针Fluo-3／AM的去透明带小鼠卵,用激光共聚焦显微镜检测小鼠卵的荧光强度以反映卵[Ca^2 ];用无钙液替代有钙液、或用酪氨酸激酶抑制剂或蛋白激酶C的抑制剂预先作用于卵,然后再观察RGD肽所致卵[Ca^2 ]i的变化。结果显示整合素配体RGD肽或Fn作用于去透明带小鼠卵可引起卵[Ca^2 ]i增加,增加的程度与精子作用相似;去除培养液中的Ca^2 后,再用RGD肽、Fn作用仍可引起卵[Ca^2 ]i增加：用功能性的抗小鼠整合素α6、β1的单克隆抗体也可引起不同程度的卵[Ca^2]i增加,尤其以抗小鼠整合素α6、β1单克隆抗体的作用明显;用酪氨酸激酶抑制剂预先作用于鼠卵,RGD肽或精子作用都不再引起卵[Ca^2 ]i增加;蛋白激酶C抑制剂预先作用鼠卵,RGD肽及Fn也不再引起卵[Ca^2 ]i增加。实验证明。小鼠卵膜整合素与其配体结合可使卵内贮存钙离子释放,引起卵[Ca^2 ]i增加这一卵激活的早期事件;整合素介导小鼠卵激活需要酪氨酸激酶信号转导途径的参与;蛋白激酶C也参与了整合素介导的卵激活。     

Starting a new life: sperm PLC-zeta mobilizes the Ca2+ signal that induces egg activation and embryo development: an essential phospholipase C with implications for male infertility

We have discovered that a single sperm protein, phospholipase C-zeta (PLCζ), can stimulate intracellular Ca(2+) signalling in the unfertilized oocyte ('egg') culminating in the initiation of embryonic development. Upon fertilization by a spermatozoon, the earliest observed signalling event in the dormant egg is a large, transient increase in free Ca(2+) concentration. The fertilized egg responds to the intracellular Ca(2+) rise by completing meiosis. In mammalian eggs, the Ca(2+) signal is delivered as a train of long-lasting cytoplasmic Ca(2+) oscillations that begin soon after gamete fusion and persist beyond the completion of meiosis. Sperm PLCζ effects Ca(2+) release from egg intracellular stores by hydrolyzing the membrane lipid PIP(2) and consequent stimulation of the inositol 1,4,5-trisphosphate (InsP(3) ) receptor Ca(2+) -signalling pathway, leading to egg activation and early embryogenesis. Recent advances have refined our understanding of how PLCζ induces Ca(2+) oscillations in the egg and also suggest its potential dysfunction as a cause of male infertility.     

In vivo survival of domestic cat oocytes after vitrification, intracytoplasmic sperm injection and embryo transfer

We evaluated: (1) cleavage rate after IVF or intracytoplasmic sperm injection (ICSI) of in vivo- and in vitro-matured oocytes after vitrification (experiment 1); and (2) fetal development after transfer of resultant ICSI-derived embryos into recipients (experiment 2). In vivo-matured cumulus-oocyte complexes (COCs) were recovered from gonadotropin-treated donors at 24 h after LH treatment. In vitro-matured oocytes were obtained by mincing ovaries (from local veterinary clinics) and placing COCs into maturation medium for 24 h. Mature oocytes were denuded and cryopreserved in a vitrification solution of 15% DMSO, 15% ethylene glycol, and 18% sucrose. In experiment 1, for both in vivo- and in vitro-matured oocytes, cleavage frequencies after IVF of control and vitrified oocytes and after ICSI of vitrified oocytes were not different (P > 0.05). After vitrification, blastocyst development occurred only in IVF-derived, in vitro-matured oocytes. In experiment 2, 18 presumptive zygotes and four two-cell embryos derived by ICSI of vitrified in vitro-matured oocytes and 19 presumptive zygotes produced from seven in vivo- and 12 in vitro-matured oocytes were transferred by laparoscopy into the oviducts of two recipients, respectively. On Day 21, there were three fetuses in one recipient and one fetus in the other. On Days 63 and 66 of gestation, four live kittens were born. In vivo viability of zygotes and/or embryos produced via ICSI of vitrified oocytes was established by birth of live kittens after transfer to recipients.     

Production of a transgenic piglet by a sperm injection technique in which no chemical or physical treatments were used for oocytes or sperm

As a method of producing transgenic animals, spermatozoa have been used to fertilize mammalian oocytes through natural copulation, artificial insemination (AI), and in vitro fertilization (IVF). Our objective was to produce live piglets expressing the enhanced green fluorescent protein (eGFP) by the modified ICSI procedure based on Yong et al. (2003) (Hum. Reprod. 18:2390) where this procedure resulted in an improvement in development in vitro as compared to conventional ICSI and IVF. After injecting frozen-thawed sperm, recovered from the descendant of a transgenic boar derived by oocyte transduction, into in vitro matured oocytes the injected oocytes were surgically transferred into the oviduct of six surrogate gilts. Two gilts (33%) became pregnant. One gave birth to a healthy male piglet. Expression of the eGFP was easily observed in the nose and hooves by direct epifluorescent examination in the newborn piglet. These results show the production of the first viable transgenic piglet by in vitro maturation and our new sperm injection method.     

Tyrosine phosphorylation signaling regulates Ca2+ entry by affecting intracellular pH during human sperm capacitation

Capacitation is a mandatory process for the acquisition of mammalian sperm fertilization competence and involves the activation of a complex and still not fully understood system of signaling pathways. Under in vitro conditions, there is an increase in both protein tyrosine phosphorylation (pTyr) and intracellular Ca²⁺ levels in several species. In human sperm, results from our group revealed that pTyr signaling can be blocked by inhibiting proline-rich tyrosine kinase 2 (PYK2). Based on the role of PYK2 in other cell types, we investigated whether the PYK2-dependent pTyr cascade serves as a sensor for Ca ²⁺ signaling during human sperm capacitation. Flow cytometry studies showed that exposure of sperm to the PYK2 inhibitor N-[2-[[[2-[(2,3-dihydro-2-oxo-1 H-indol-5-yl)amino]-5-(trifluoromethyl)-4-pyrimidinyl]amino]methyl]phenyl]- N-methyl-methanesulfonamide hydrate (PF431396) produced a significant and concentration-dependent reduction in intracellular Ca ²⁺ levels during capacitation. Further studies revealed that PF431396-treated sperm exhibited a decrease in the activity of CatSper, a key sperm Ca ²⁺ channel. In addition, time course studies during capacitation in the presence of PF431396 showed a significant and sustained decrease in both intracellular Ca ²⁺ and pH levels after 2 hr of incubation, temporarily coincident with the activation of PYK2 during capacitation. Interestingly, decreases in Ca ²⁺ levels and progressive motility caused by PF431396 were reverted by inducing intracellular alkalinization with NH ₄Cl, without affecting the pTyr blockage. Altogether, these observations support pTyr as an intracellular sensor for Ca ²⁺ entry in human sperm through regulation of cytoplasmic pH. These results contribute to a better understanding of the modulation of the polymodal CatSper and signaling pathways involved in human sperm capacitation.     

Staphylococcal serine proteinase increases intracellular free calcium concentration in human polymorphonuclear leukocytes

Staphylococcal serine proteinase (SSP) can influence various functions of human polymorphonuclear leukocytes (PMNL) including chemotaxis and phagocytosis. Since the rise in intracellular free calcium concentration is an important step in signal transduction leading to phagocyte activation, we tested the ability of SSP to increase the intracellular free calcium concentration in human PMNL using the fluorescent calcium indicator Fura-2AM. PMNL isolated from healthy donors responded to SSP in the concentration range of 10 to 100 µg/ml. The highest Ca²⁺ rise (104 ± 47 nM) was observed for 10 µg/ml SSP. It was mainly dependent (81 ± 11%) on extracellular calcium influx, however, SSP mobilized 68 ± 7% of Ca²⁺ from intracellular calcium stores. Boiling of SSP or preincubation with phenylmethylsulphonylfluoride (an serine proteinase inhibitor) did not change its ability to increase intracellular free calcium concentration in PMNL. It suggests that active center of SSP is not responsible for Ca²⁺ mobilization. Finally, PMNL responded to each of three consecutive stimulations with SSP independently of the presence of high or low extracellular Ca² concentration. This may be an additional mechanism responsible for activation of human PMNL and degradation of alveolar walls during the staphylococcal infection in the lower airways.