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Molecular Human Reproduction vol.3 no.4 pp. 367–374, 1997
Human sperm cytosolic factor triggers Ca2F oscillations and
overcomes activation failure of mammalian oocytes
Gianpiero D.Palermo1,3, Ori M.Avrech1, Liliana T.Colombero1, Hua Wu2, Yvonne M.Wolny1,
Rafael A.Fissore2 and Zev Rosenwaks1
1The
Center for Reproductive Medicine and Infertility, The New York Hospital–Cornell Medical Center, New York, 505 East
70th Street, HT-336, New York, NY 10021, and 2Department of Veterinary and Animal Sciences University of
Massachusetts, Amherst, MA, USA
3To
whom correspondence should be addressed
Among the possible mechanisms of oocyte activation after sperm penetration, it appears most likely that a
protein released by the spermatozoon elicits a calcium elevation in the ooplasm. To further test this idea,
cytosolic factors obtained from human spermatozoa by two different methods, freezing–thawing and
sonication, were injected into mouse oocytes following which intracellular calcium release was measured.
Of a total of 42 mouse oocytes, a pattern of calcium oscillations was observed in nine out of 16 oocytes
injected with sonicated fraction, in all of eight oocytes with the frozen–thawed fraction and in none of 18
control oocytes. Injection of the frozen–thawed fraction also produced regular calcium oscillations in all of
five in-vitro matured human oocytes. To assess the putative factor’s ability to support fertilization, human
oocytes that were not activated by prior intracytoplasmic injection of spermatozoa (ICSI) and round spermatids
were reinjected with the frozen–thawed sperm fraction. Of 23 human oocytes which remained unfertilized
after ICSI, 19 became activated after injection with sperm cytosolic factor; eight showed two pronuclei, three
one pronucleus and eight showed three or more pronuclei. Of 11 oocytes unfertilized after prior round
spermatid injection, two developed two pronuclei, four developed one pronucleus and two had three or more
pronuclei. Cytogenetic analysis by fluorescence in-situ hybridization confirmed the existence of a male
pronucleus in eight out of nine such zygotes displaying two or more pronuclei. Thus, human sperm extracts
activated mouse and human oocytes after injection, as judged by calcium flux patterns in conjunction with
male pronucleus formation.
Key words: calcium oscillations/fertilization/fluorescence in-situ hybridization/oocyte activation/sperm cytosolic factor
Introduction
Activation of the mature oocyte can be defined as a series of
events involving at least cortical granule exocytosis, resumption
of meiosis with extrusion of a second polar body, and other
changes in the cytoskeleton of the oocyte. The initial step
of activation, which is initiated by entry of the fertilizing
spermatozoon, is triggered by calcium released in a specific
oscillatory pattern from intracellular stores (Whittingam, 1980;
Jaffe, 1983; Whitaker and Steinhardt, 1985). These changes
in intracellular calcium ([Ca21]i) that occur during fertilization
of mammalian eggs present as a series of repetitive and
transient rises in [Ca21]i that persist for several hours after
fusion of the gamete membranes (Cuthbertson and Cobbold,
1985; Miyazaki, 1988).
Activation can be independent of the presence of the
spermatozoon since certain chemical, mechanical or thermal
stimuli can induce an activation with single pronucleus formation; this is sometimes even followed by parthenogenetic
development (Ozil, 1990).
Fertilization achieved by intracytoplasmic sperm injection
(ICSI) bypasses the normal steps of sperm–zona pellucida
© European Society for Human Reproduction and Embryology
binding, penetration and, especially in the present context,
sperm–oolemma fusion. However, notwithstanding its current
widespread use, the mechanism of oocyte activation following
ICSI is still controversial. It has been suggested that one
possible trigger might be mechanical stimulation by the glass
pipette (Perreault and Zirkin, 1982; Lanzendorf et al., 1988),
or that higher concentrations of calcium ions are carried into
the cytoplasm during the injection procedure (Edwards and
Van Steirteghem, 1993). However, it was later demonstrated
that the calcium flux produced solely by the injection procedure
in a normal culture medium is not sufficient to activate the
human oocyte (Tesarik et al., 1994). Furthermore, since oocytes
injected with spermatozoa are activated at a significantly
higher rate than after sham injection, it seems likely that the
spermatozoon itself plays a key role in oocyte activation
occurring after ICSI (Dozortsev et al., 1994).
An oocyte activating factor was indeed shown to be present
in the cytosolic fraction of rabbit, hamster, boar, and human
spermatozoa (Stice and Robl, 1990; Swann, 1990; Parrington
et al., 1996). In addition, it has been reported recently that
injected spermatozoa contribute to activation of the oocyte by
releasing a heat-sensitive, intracellular active factor that is not
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G.D.Palermo et al.
species specific (Dozortzev et al., 1995). In accordance with
that, a cytosolic 33 kDa protein isolated from hamster spermatozoa induces Ca21 oscillations when injected into mouse eggs.
Intracytoplasmic injection of mouse eggs with this component,
which has been named oscillin and is claimed to exist in the
equatorial segment of the sperm head, evokes Ca21 oscillations
in a pattern similar to that seen following fertilization
(Parrington et al., 1996). The signal transduction pathway
between the sperm–oocyte fusion site and intracellular calcium
stores most likely involves the participation of oscillin, and a
similar protein in structure and function may be present in the
human spermatozoon (Parrington et al., 1996; Swann, 1996).
The aim of this study was to find a simple and reliable
method of isolating a factor from human spermatozoa that is
able to activate oocytes. Two different methods of sperm cell
permeabilization were used for this purpose, the activity of
isolated fractions being assessed by intracellular free Ca21
release following injection into mouse eggs and human oocytes.
Among the latter, some had failed fertilization after ICSI and
round spermatid injection (RSI). To analyse the chromosomal
status of the embryos generated by this salvage procedure,
fluorescence in-situ hybridization (FISH) was performed on
individual blastomeres.
Materials and methods
4°C. The clear sperm cytosolic supernatant was then subjected to
ultrafiltration (Centricon-50 and Microcon-50 membranes; Amicon,
Beverly, MA, USA) to reach a final concentration of 10–20 mg/ml
of protein with a molecular weight ù50 kDa. Aliquots of 20 µl each
were stored at –80°C until further use.
Oocyte sources
Mouse oocytes
Mouse mature oocytes were collected from the oviduct of CD-1
female mice primed with 5 IU of pregnant mare serum gonadotrophin
(Sigma Chemical Co.) i.p. followed by 5 IU of human chorionic
gonadotrophin (HCG) 48 h later to induce ovulation. Oocytes were
recovered 13–14 h post-HCG and the cumulus oophorus was removed
with 1 mg/ml bovine testis hyaluronidase in culture medium (TCM199; Gibco, Grand Island, NY, USA). Oocytes were kept in 50 µl
drops of medium covered with paraffin oil and maintained in a
humidified atmosphere of 5% CO2 in air until the injection procedure
(Fissore and Robl, 1993).
Human oocytes
The ovulation stimulation protocols and the collection and preparation
of the oocytes for microinjection were as previously described
(Palermo et al., 1995). Oocytes originally collected for ICSI that
proved to be at germinal vesicle stage (GV) or metaphase I (MI)
stage were utilized after patients’ consent. In addition, oocytes that
failed to fertilize after ICSI or round spermatid injection were donated
for this study.
Sperm membrane permeabilization
Two distinct techniques were used to permeabilize spermatozoa: a
single freeze–thaw cycle (Dozortsev et al., 1995), and ultrasonic cell
disruption. Both procedures were carried out in intracellular-like
medium consisting of human tubal fluid (HTF; Irvine Scientific, Santa
Ana, CA, USA) with 10% synthetic serum substitute (SSS; Irvine
Scientific), 200 µM phenylmethylsulphonyl fluoride (PMSF), 10 µg/
ml leupeptin and 10 µg/ml pepstatin, pH 5 7.0. All reagents were
from Sigma Chemical Co (St Louis, MO, USA) (Swann 1990; 1994).
Freezing was performed by plunging the spermatozoa resuspended
in intracellular-like medium directly into liquid nitrogen at –156°C
without a cryoprotectant. They were thawed by incubating the vials
at room temperature for 1 h.
Ultrasonic homogenization was performed by Microson XL-2007
(Heat Systems Inc, Farmingdale, NY, USA). Sperm pellets were
diluted in an intracellular-like medium. The sperm suspensions were
sonicated for 25 min at 4°C with an output of 23 kHz and 2 W of
power. To avoid contact of the sample with the probe, a cup horn
accessory with a high-intensity water bath was utilized.
Injection of sperm cytosolic factor for calcium release assay
The ability of the sperm cytosolic factor (SCF) to induce a [Ca21]i
response after injection into mouse and human oocytes matured
in vitro was monitored using the fura-2 D fluorescence probe. A
microdrop of the Ca21-sensitive fluorescent dye, 0.5 mM fura-2
dextran (Fura-2 D; dextran 10 kDa, Molecular Probes, Eugene, OR,
USA) was sucked into a glass micropipette in a calcium-free injection
buffer solution of 70 mM KCl and 10 mM HEPES (pH 5 7.0), then
injected by pneumatic pressure (PLI-100, Medical System Corp.,
Great Neck, NY, USA), in a volume of ~5 pl (Fissore et al., 1992;
Fissore and Robl, 1992). SCF was injected starting 30 min after the
injection of Fura-2 D. Illumination was provided by a 75 watt xenon
arc lamp on a Nikon Diaphot microscope equipped with 340 UV oil
immersion objective (Nikon Inc., Melville, NY, USA). Excitation
wavelengths were at 340–380 nm and the emitted light, attenuated
32-fold by neutral density filters, was quantified by a photomultiplier
tube (Nikon) which equalized the fluorescence signal in the whole
egg. A rotating filter wheel and shutter apparatus were used to
alternate excitation wavelengths. The field of illumination/detection
was set to the diameter of the egg. The fluorescent measurements
were accumulated and processed by a modified Phoscan 3.0 software
(Nikon). Free [Ca21]i concentrations were determined every 4 s from
the 340/380 ratio of fluorescence and Rmin and Rmax were calculated
as previously described (Grynkiewicz et al., 1985; Poenie, 1990;
Fissore and Robl, 1993). Oocyte fluorescence ratios were monitored
for 5 min prior to injection of SCF, to establish baseline, and then
for 30–45 min to determine the response to SCF. The monitoring was
carried out in 40 µl drops of culture medium placed on a glass
coverslip covered with paraffin oil and maintained at 37°C.
Sperm factor extraction, concentration and storage
The sperm suspension, either freeze–thawed or sonicated was centrifuged twice at 1800 g at 25°C in order to remove particulate material
and then once at 100 000 g (Beckman ultracentrifuge L5-75B;
Beckman Instruments Inc, Palo Alto, CA, USA) for 60 min at
Injection of sperm cytosolic factor into human oocytes
The SCF was injected into human oocytes that remained unfertilized
as judged by absence of pronuclei 16–18 h after ICSI or RSI. The
SCF injection was performed ~24 h after the failed ICSI or RSI.
For injection, a thawed 5 µl aliquot of SCF was placed under oil
Semen collection and selection
Human semen was obtained from 32 proven fertile donors. The
samples were processed by washing and centrifugation together with
a swim-up procedure to enhance selection of spermatozoa with high
progressive motility. The samples were pooled and concentrated by
centrifugation at 1800 g for 5 min. The resulting pellets were diluted
for membrane permeabilization at a final concentration of 100–
2003106/ml spermatozoa (Avrech et al., 1996a,b).
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Sperm cytosolic factor activates mammalian oocytes
and adjusted to a final concentration of 1–2 sperm equivalents per
pl, the latter calculated according to Stice and Robl (1990). The
oocytes were prepared for microinjection as described elsewhere
(Palermo et al., 1993, 1995). Up to 5 pl of SCF containing 5–10 sperm
equivalents was injected. Since the procedure was not performed with
a picoinjector, the 5 pl volume was calculated according to the pipette
radius (3.75 µm) and the length of the fluid column in it (100 µm).
As a proximal indicator of the fluid column, a meniscus of an oil
droplet was used. The injection of SCF was performed carefully to
avoid any aspiration of the cytoplasmic organelles as it has been
clearly demonstrated that an aggressive cytoplasmic ‘dislocation’ is
responsible for the Ca21 release involved in oocyte activation (Tesarik
and Sousa, 1995). A similar injection method was used for the
unfertilized control oocytes (n 5 4).
Assessment of activation was performed at 16–18 h following
injection of the SCF. The activated oocytes were observed for 2 more
days for cleavage and embryonic development.
Fluorescence in-situ hybridization analysis
To assess the genetic status of embryos generated by injection of
SCF, multiprobe FISH was performed on single blastomeres collected
on day three of development. For this, the zona pellucida was removed
with 0.2% pronase (Sigma) in HTF supplemented with 6% SSS
(Irvine Scientific) (HTF-SSS) for ~5 min. Blastomeres were then
rinsed four times in HTF-SSS and allowed to recover for 30 min at
37°C in an incubator. The blastomeres of a single embryo were
isolated by a hand pulled micropipette and exposed to a hypotonic
solution (0.60 % sodium citrate in water, 4 mg bovine serum albumin/
ml; Sigma) for 5 min. Under a stereomicroscope, 10 µl of fixative
(1:3 v/v acetic acid/methanol) were dropped on top of each individual
blastomere on a clean microslide (Tarkowsky, 1966). The fixative
was delivered from a 30 1/2 gauge needle (Precision Glide; Becton
Dickinson & Co., Franklin Lakes, NJ, USA) on a tuberculin syringe
(Becton Dickinson) from a distance of 1.5 cm. The fluid was allowed
to dry by continuous and gentle blowing. Coincidentally with the
evaporation of the fluid, the blastomere membrane ruptured with
spreading and disappearance of the cytoplasm. Immediately after this
procedure, slides were assessed under phase contrast microscopy to
observe presence and proper fixation of nuclei. Slides were dehydrated
by subsequent passages in ethanol (70, 85, 95%, 2 min each) and
either analysed immediately or stored at –20°C for further analysis.
Multiprobe FISH was performed using probes for chromosomes
13/21, 18, X, and Y. DNA probes were alpha-satellite repeat clusters
in the centromeric region of 13/21, 18 and X chromosomes, and the
satellite-III DNA on the long arm of the Y-chromosome (Vysis,
Downers Grove, IL, USA). The probe for chromosome 13/21 was
labelled with digoxigenin (Oncor, Gaithersburg, MD, USA) and
visualized using rhodamine-labelled anti-digoxigenin antibodies; the
18-chromosome probe was directly labelled with a green chromosome
enumeration probe (CEP Spectrum Green; Vysis), the X-chromosome
probe was a 1:1 mixture of probes labelled with red and green
fluorochromes; and the Y-chromosome-specific probe was labelled
with a blue fluorochrome (CEP Spectrum Aqua; Vysis). The hybridization solution, consisting of 7 µl of Spectrum CEP hybridization buffer
(Vysis), 0.9 µl of digoxigenin chromosomes 13/21 (Oncor), 0.7 µl
green chromosome 18, 0.3 µl of CEP red chromosome X, 0.4 µl of
CEP green chromosome X, 0.9 µl of CEP aqua chromosome Y, and
0.2 µl of concentrated COT-DNA (Gibco BRL, Gaithersburg, MD,
USA), was added to the blastomeres. The slides were covered with
a coverslip, denatured for 3 min in a slide warmer at 80°C, sealed
with rubber cement and allowed to hybridize at 37°C in the dark for
at least 4 h. Excess probe was then washed off and the digoxigeninlabelled probes were demonstrated by rhodamine-labelled antibodies
as previously described (Munné et al., 1993). The slides were covered
with 49,6-diamino-2-phenylindole (DAPI) in antifade solution (Vysis)
and observed with a fluorescence microscope (Olympus B Max 60;
New York/New Jersey Scientific, NJ, USA). A DAPI filter (Olympus
U-C83360) was used to locate the nuclei on the slide. With a
tripleband-pass filter (Olympus U-C83103), the nuclei appeared blue,
the 13/21 chromosomes red, the 18 green and the X yellow. In order
to distinguish the blue Y chromosome from the DAPI counterstain,
a tripleband-pass filter aqua/green/orange (Vysis 30–152517) was
utilized. The scoring and interpretation of the signals were those of
Hopman et al. (1988). The FISH failure criteria and definition of
mosaicism were as previously described (Munné et al., 1994; Harper
and Delhanty, 1996). Briefly, mosaic diploids were those embryos in
which the majority of nuclei were diploid, and a small number of
blastomeres were aneuploid, haploid or tetraploid. In addition, sibling
blastomeres with a missing signal compensating for extra ones in
other blastomeres were considered compensated mosaics. The ploidy
status of these mosaic embryos was derived from the overall number
of chromosomes present in all blastomeres. When there was a
completely irregular distribution of chromosomes among the blastomeres, the overall chromosome count was matched to the closest
ploidy status.
Statistical analysis
The free [Ca21]i patterns were plotted using a spreadsheet program
and further stored on an optical disk. The [Ca21]i responses induced by
the injection of different agonists (frequency, duration and periodicity)
were subjected to one-way analysis of variance and Student’s t-test
using the SAS statistical software package (SAS, Cary, NC, USA).
Results are given as mean 6 SEM.
Results
The sonication and single freeze–thaw isolation methods both
yielded a final SCF concentration of at least one sperm
equivalent/pl allowing the injection of 5 pl volume of SCF
into each oocyte. However, the frozen–thawed fraction enabled
pooling of a larger number of samples.
Calcium oscillations following SCF injection into
mouse oocytes
A total of 24 mouse oocytes were injected with SCF, 16
with the sonicated fraction and eight with the frozen–thawed
fraction; 18 controls were injected with the culture medium
alone. Only nine out of 16 oocytes receiving the sonicated
fraction displayed [Ca21]i oscillations (Figure 1 middle panel),
whereas the frozen–thawed fraction induced a [Ca21]i response
in all eight oocytes injected (Figure 1 lower panel). The first
response to the sonicated fraction was a mean [Ca21]i peak of
716 6 72 nM, subsequent oscillations having a mean [Ca21]i
of 364 6 42 nM. The mean baseline value was 149 6 19 nM.
The oscillations occurred at a frequency of 0.4 6 0.2 per min.
The frozen fraction generated a first rise of 752 6 48 nM and
subsequent spikes had a mean peak [Ca21]i of 335 6 25 nM
at a frequency of 1.4 6 0.2 per min. For both fractions, the
oscillations lasted for at least 30 min. No similar [Ca21]i
release was observed in any of the 18 control oocytes injected
with medium. Among the latter the only response was a single
spike of a lower amplitude and duration than that seen after
SCF injection, irrespective of isolation method (Figure 1 upper
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G.D.Palermo et al.
Figure 1. Injection of human sperm cytosolic factor (SCF) elicits
Ca21 oscillations in mouse oocytes. Injection with culture
medium (upper) did not induce long-lasting Ca21 rises. Injection
of SCF obtained by sonication (middle) or by freezing–thawing
(lower) elicited persistent oscillations. The first arrow in each
graph indicates time of injection.
panel). No specific Ca21 repetitive transients were observed,
and the only calcium alteration registered was a single spike
at the time of pipette removal.
Calcium response of human oocytes to SCF injection
After loading of in-vitro matured human oocytes with fluorescent Ca21 dye Fura-2-dextran, injection of SCF prepared by
370
Figure 2. Injection of human sperm cytosolic factor (SCF) obtained
by a freezing–thawing method triggers Ca21 oscillations in human
oocytes. Injection with culture medium (upper) did not induce longlasting Ca21 rises. After withdrawal of the pipette, the injection of
1 pl of SCF (middle) induced an isolated Ca21 spike, while the
injection of 5 pl of SCF (lower) elicited persistent Ca21 oscillations.
The first arrow in each graph indicates time of injection.
freezing/thawing yielded high frequency calcium oscillations
in the fluorescent imaging assay that lasted .30 min in all of
those injected (Figure 2 lower panel). The amplitude of the
first spike was 617 6 53 nM and subsequent rises had a mean
peak [Ca21]i of 297 6 10 nM at a frequency of 0.8 6 0.4
per min. In the five oocytes injected with 5 pl of SCF solution
at a concentration of 1 sperm equivalent/pl, the incidence of
[Ca21]i oscillations was 100% (Figure 2 lower panel). In
Sperm cytosolic factor activates mammalian oocytes
Table I. Human oocyte activation following injection of sperm cytosolic
factor (SCF) after failed intracytoplasmic sperm injection (ICSI) and round
sperm injection (RSI)
Oocytes
ICSI
RSI
Injected
Survived
One pronucleus (%)
Two pronuclei (%)
Three or more pronuclei (%)
23
21
3 (14.0)
8 (38.0)
8 (38.0)
11
9
4 (44.4)
2 (22.1)
2 (22.1)
In regard to the ICSI-generated embryos, the zygotes with
a single pronucleus were haploid with no Y chromosome. Of
the multipronucleate zygotes, one was a mosaic diploid and
one was a mosaic triploid. The three bipronucleate zygotes
were all mosaic diploids (Table II).
Among the RSI-derived embryos, those that earlier had one
pronucleus proved to be simply activated, that is haploid. Of
the two polypronucleate zygotes, one was aneuploid because
of an additional chromosome 13 or 21, and one was diploid
but mosaic. Of the two bipronucleate zygotes, one was tetraploid and one was pentaploid, both mosaic (Table III).
Table II. Chromosomal complement of seven embryos generated by injection of sperm cytosolic factor (SCF) in unfertilized human oocytes following
intracytoplasmic sperm injection (ICSI)
Pronuclear (PN) pattern
No. embryos
Cytogenetic result
Karyotype
2PN
3
mosaic diploid
XX 1818 4[13,21] (2 blastomeres);
XXXX 181818 5[13,21]; 18 3[13,21]
XY 1818 4[13,21] (3 blastomeres);
X 1818 2[13,21]; Y0 2[13,21]
XX 1818 2[13,21]; 2[13,21];
XX 18 4[13,21]; X0 180
X0 180 2[13,21] (3 blastomeres)
X0 180 2[13,21] (2 blastomeres);
X0 180 1[13,21]; 1[13,21]
XY 180 2[13,21]; XY 5[18] 5[13,21]; XY 5[13,21]
XXX 180 5[13,21]; XXX 180;
XXXXX 1818 3[13,21]; X0 180 2[13,21]
mosaic diploid
mosaic diploid
1PN
2
1 haploid
1 mosaic haploid
ù3PN
2
1 mosaic diploid
1 mosaic triploid
contrast, the oocytes injected solely with culture medium (n 5
5) showed no oscillation pattern but only a single spike due
to the penetration of the pipette (Figure 2 upper panel), while
those receiving only 1 pl of SCF (n 5 2) showed a single
additional spike (Figure 2 middle panel).
Activation and early embryonic development of
human oocytes that failed to fertilize after ICSI and
RSI
Of a total of 23 human oocytes previously unfertilizable by
ICSI, after treatment with SCF, 21 survived and 19 (90%)
showed signs of activation (Table I). Of these, eight oocytes
revealed two pronuclei and three had one pronucleus. The
remaining eight oocytes had three or more pronuclei. In a
second experimental group of 11 oocytes unfertilized after
RSI, nine survived and eight displayed calcium release from
intracellular stores. Later, two oocytes developed two pronuclei,
and four a single pronucleus. By the third day of culture,
the cleavage rate (five out of eight) for the ICSI-treated
bipronucleate zygotes was 62.5% and two out of two for the
RSI oocytes which had cleaved. None of the four oocytes
injected in the same manner with culture medium showed any
sign of activation.
Cytogenetic analysis
After isolating 91 blastomeres from 18 human embryos, 74.7%
(68/91) proved to be multinucleated when observed with
Hoffman Modulation Contrast optics (Modulation Optics,
Greenvale, NY, USA), and 15 embryos were successfully fixed
and analysed for FISH. The stage of maturity of the sperm
cell injected as well the pronuclear pattern is shown in Tables II
and III.
Discussion
A human sperm cytosolic moiety that induces Ca21 oscillations
in eggs has been extracted using either sonication or freeze–
thawing; of the two, the freezing/thawing method had the
advantage of enabling pooling of a larger number of semen
samples.
The sonicated fraction evoked calcium oscillations in 56%
of mouse oocytes, whereas that obtained after the freezing/
thawing procedure activated 100% of the eggs. This outcome
led us to use only the latter fraction for human oocytes, in
which it produced an activation rate of 100%, as measured by
the intracellular Ca21 release. By contrast, mouse and human
oocytes injected only with culture medium displayed no
signs of Ca21 release. These results suggested that the Ca21
oscillations were caused by SCF, not by the injection procedure
itself or through introduction of Ca21 ions from the medium.
Mouse and human oocytes injected with control medium
evoked only single Ca21 spikes, lower in intensity and of
shorter duration than in SCF-treated oocytes. These observations are in concordance with other reports, where the injection
of culture medium into human and mouse eggs yielded only
a single brief Ca21 spike (Homa and Swann, 1994). The
concentration of protein injected was 10–20 mg/ml suggesting
that a similar amount of protein produced from a cell lysate
obtained from another tissue might also be able to induce
Ca21 oscillations. We have demonstrated that this is not the
case. In fact, a cytosolic preparation of brain tissue failed to
induce Ca21 release (Wu et al., 1997).
It is not clear as to the exact mechanism that allows
intracellular Ca21 release following the stimulus of the SCF.
It is unclear from these data whether the Ca21 release was
from the peripheral stores (Tesarik and Sousa, 1996) or from
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G.D.Palermo et al.
Table III. Chromosomal complement of eight embryos generated by injection of sperm cytosolic factor (SCF) in unfertilized human oocytes following round
sperm injection (RSI)
Pronuclear pattern
No. embryos
Cytogenetic result
Karyotype
2PN
2
1 mosaic tetraploid
XXXXY 181818 9[13,21]; XXXY 6[18] 8[13,21]
XY 181818 8[13,21]; XXY 181818 3[13,21]
XX 180 5[13,21]; XXXXYY 6[18] 10[13,21]
XXYY 180 8[13,21]; XXXY 8[18] 10[13,21]
X0 180 2[13,21] (2 blastomeres)
X0 180 2[13,21] (5 blastomeres)
X0 180 2[13,21] (2 blastomeres); XX 181818 1[13,21]
X0 180 2[13,21]; X0 1818 5[13,21];
X0; X0 180 1[13,21]
X0 180 3[13,21] (2 blastomeres)
XXX 181818 3[13,21]; X0 1[13,21];
X0 2[13,21]; 180 3[13,21]; XXX 1[13,21]
1 mosaic pentaploid
1PN
4
2 haploid
2 mosaic haploid
ù3PN
2
1 aneuploid (chromosome 13/21)
1 mosaic diploid
a calcium gradient between the periphery and the centre of
the cytoplasm (Berridge, 1996). In natural fertilization, the
SCF is released after sperm–egg fusion (Swann and Lawrence,
1996). In the ICSI procedure the release of the SCF is
facilitated from the membrane permeabilization performed
prior to the injection.
Since the Ca21 oscillation patterns were similar in mouse
and in human oocytes it appears that the effect of injected
SCF is not species specific. These characteristic repetitive
Ca21 oscillations were similar in frequency and pattern to
those observed when sperm fractions from different species
were injected into mouse, hamster and human oocytes in other
studies (Swann, 1990; Homa and Swann, 1994; Parrington
et al., 1996). According to immunohistochemical analysis, the
protein in its hexamer form could be located at an intracellular
site in the equatorial segment of the sperm head in hamster,
boar and human spermatozoa (Parrington et al., 1996). The
logic of this is seen in the fact that the equatorial region is
the site of the initial step of sperm–oocyte fusion (Bedford
et al., 1979). Another argument for the equatorial localization
of SCF can be found in studies where an isolated sperm head
was injected which was able to activate and fertilize oocytes
at the same rate as an intact spermatozoon (Colombero et al.,
1996; Kuretake et al., 1996).
To determine whether fertilization failure after ICSI is due
to the absence of an oocyte activating factor in the sperm cell,
or to a failure of its release, the sperm extract was injected
into human oocytes that had failed fertilization with ICSI. A
90% response in terms of pronuclear formation in any egg that
survived the procedure (19/21) suggested that the fertilization
failure was due to lack of an oocyte activator.
The outcome of ICSI is not influenced by semen parameters
(Palermo et al., 1993; Nagy et al., 1995; Palermo et al., 1995),
and ICSI has been successful with immature epididymal and
testicular spermatozoa (Tournaye et al., 1994; Palermo et al.,
1995). Pregnancies have been obtained even after the injection
of round spermatids (Tesarik et al., 1995) following the demonstration of this procedure first in the mouse (Ogura et al.,
1994). In the latter study, however, the activation of the oocytes
had to be established by their exposure to an electrical stimulus.
A present limitation to the use of round spermatids is the
difficulty in their identification, since they are only recognizable
372
according to their size and shape under the light microscope.
Currently, fertilization rates with RSI range between 20 and
30% (Tesarik et al., 1995; Chen et al., 1996), generating only
a limited number of embryos that can be analysed. The low
fertilization rate following RSI could be due to the low
concentration or absence of SCF, or because of the difficulty
of permeabilizing these cells, in contrast to mature spermatozoa
(Palermo et al., 1996). At all events, it appears that the routine
availability of SCF would enhance fertilization with these
immature cells. Since signs of activation were observed in
eight out of nine unfertilized RSI injected oocytes, this allowed
a genetic analysis in all of them.
FISH analysis of the embryos derived from the reactivation
of unfertilized oocytes showed that the zygotes with one
pronucleus were haploid, in accord with previous studies
(Staessen et al., 1993; Sultan et al., 1995). Though two out of
four multipronucleate zygotes were diploid, all displayed
two distinct polar bodies since these probably were pseudomultipronuclei, possibly originating from scattered chromatin
within the ooplasm. This observation is in agreement with the
high incidence of multinucleated blastomeres derived from all
these embryos. Multinucleation is associated with a high
incidence of mosaicism (Munné and Cohen, 1993; Kligman
et al., 1996), and here the bipronucleate zygotes generated
after reactivation of ICSI injected oocytes were all mosaic
diploids. This indicates that although chromosomes were
unevenly distributed among the blastomeres, the injection of
the SCF nevertheless enabled the sperm chromosomes to
participate in syngamy. Such zygotes which were generated
after RSI were polyploid, a condition that might be explained
by inadvertent injection of a primary spermatocyte or possibly
a multinucleated spermatid.
It is not clear why there was a high incidence of mosaicism
in these embryos, but this could have been due to prolonged
culture; to the two consecutive intracytoplasmic injections; or
to excessive Ca21 release triggered by excessive SCF, with
consequent disruption of the mitotic spindle leading to abnormal distribution of the chromosomes among the blastomeres.
In conclusion, at the present time the only limit to the ICSI
procedure is represented by the use of spermatozoa that do
not release SCF, either because of an insufficient permeabilization or because of an absence of the SCF. Immotile spermato-
Sperm cytosolic factor activates mammalian oocytes
zoa may have lost their SCF as a consequence of a damaged
membrane. Thus, the availability of SCF may well allow
a significant improvement in fertilization rate where ICSI
necessarily involves the use of spermatozoa lacking SCF, or
of immotile spermatozoa or round spermatids. Since production
of SCF is laborious, the definition of its character and synthesis
of an active form will be an important step in this regard.
Acknowledgements
The authors are grateful to the clinical and scientific staff of The
Center for Reproductive Medicine and Infertility; J.Michael Bedford
for his critical review of the manuscript; Miriam Feliciano, June
Hariprashad and Maria Oquendo for technical assistance; Richard
Larocco for preparation of the illustrations.
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Received on October 25, 1996; accepted on February 7, 1997
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