M.Sousa et al.
Table I. Overview of different types of Ca 2+ response observed after
injection of different types of cells into oocytes and subsequent challenge
with Ca 2+ ionophore
Type of cell injected
into oocytes
Leukocyte
Primary spermatocyte
Secondary spermatocyte
Round spermatid
Spermatozoon
No. of oocytes
injected
10
4
16
17
10
\
No. (%) of oocytes showing
different types of reaction3
Non-response
Oscillatory
response
10(100)
4(100)
16 (100)
4(24)
2(20)
0(0)
0(0)
0(0)
13(76)
8(80)
"Only changes occurring during a 3 h time period following the exposure to
Ca 2+ ionophore were taken into account.
'
0
C
10 20 30 40 50 60 70 80 90 130140150
Time (min)
V
0
10 20 30 40 50 60 70 80 90 130 140 150
Time (min)
Figure 2. Evidence of a normal responsiveness to SSF of oocytes
failing to produce Ca 2+ oscillations after the injection of human
polymorphonuclear leukocytes and secondary spermatocytes. (a)
Lack of the typical Ca 2+ oscillatory response in an oocyte injected
with a polymorphonuclear leukocyte and subsequently challenged
with Ca 2+ ionophore (addition and removal marked with arrows)
followed by typical Ca 2+ oscillations after the later injection of
SSF (arrow), (b) Lack of the typical Ca 2+ oscillatory response in
an oocyte injected with a secondary spermatocyte and subsequently
challenged with Ca 2+ ionophore (addition and removal marked
with arrows) followed by typical Ca 2+ oscillations after the later
injection of SSF (arrow).
spermatozoa (Tesarik, 1994; Swann, 1996), these results suggest that human round spermatids contain sufficient SSF
activity to sensitize the Ca 2 + oscillation mechanism of
human oocytes.
It is important to note that these Ca 2 + responses were
856
Figure 3. Representative electron micrographs of oocytes injected
with different types of cells, (a) Oocyte injected with a
polymorphonuclear leukocyte. This picture shows the tabulated
nucleus (N) and the cytoplasm of a polymorphonuclear leukocyte
surrounded by the oocyte cytoplasm. The plasma membrane of the
injected leukocyte (large arrows) is still apparent. Small arrows
indicate an area in which the leukocyte plasma membrane is broken
allowing a direct contact between cytoplasmic components of both
cells, (b) Oocyte injected with a secondary spermatocyte (the
corresponding Ca 2+ record is shown in Figure lb). This picture •
shows the spermatocyte nucleus (N) surrounded by an incomplete
nuclear envelope (arrows) and the adjacent oocyte cytoplasm. No
structural components of the spermatocyte cytoplasm are visible,
(c) Oocyte injected with a round spermatid (the corresponding Ca 2+
record is shown in Figure lc). This picture shows the spermatid
chromatin (C) with occasionally attached remnants of the nuclear
envelope (large arrows) and with intermingled fragments of the
axonemal structures (small arrows). The adjacent oocyte cytoplasm
does not contain any identifiable structural components of the
spermatid cytoplasm. Scale bar = 2 um.
Oocyte calcium responses
obtained after ionophore addition. In comparison with the
experiments with mouse spermatid injection reported by
Kimura and Yanagimachi (1995 a) who were able to obtain
activation of spermatid-injected oocytes only after application
of an extra electrical stimulus, the present data suggest the
possibility that mouse spermatids may contain sufficient SSF
only to sensitize Ca 2+ oscillations but not to induce them. The
Ca 2+ oscillations in the sensitized oocytes would thus be
triggered by the electroactivation stimulus. If this hypothesis is
correct, electroactivation of spermatid-injected mouse oocytes
might lead to the development of Ca 2+ oscillations although
electroactivation of non-sensitized oocytes does not. This
situation may be analogous to the action of Ca 2+ ionophore
upon human oocytes; in fact the ionophore treatment triggers
Ca 2+ oscillations in oocytes that have been previously injected
with spermatozoa but never in control oocytes (Tesarik and
Testart, 1994). Unlike the mouse, the SSF content in human
round spermatids may not be limiting because identical Ca 2+
responses were observed in this study in sperm-injected and
spermatid-injected oocytes.
In contrast to sperm-injected and spermatid-injected oocytes,
Ca 2+ oscillations did not develop in oocytes injected with
primary or secondary spermatocytes, and the Ca 2+ records
from these oocytes were similar to those observed in oocytes
injected with non-germ cells. Because the same oocytes
developed a typical oscillatory response after subsequent
injection of SSF, and disintegration of the plasma membrane
of injected cells was confirmed by electron microscopy, this
lack of Ca2+-oscillatory response was due to a deficiency of
SSF activity in the injected cells and not to the lack of the
oocyte's ability to respond to SSF or to the failure of SSF to
escape from the injected cells and reach the oocyte cytoplasm.
Together, these data show that SSF activity develops between
the secondary spermatocyte and round spermatid stages of
human spermatogenesis. This explains why the boosting procedure (ooplasmic aspiration producing a Ca 2+ influx) used
for oocyte activation after sperm injection (Tesarik and Sousa,
1995) is also sufficient to activate human oocytes injected
with round or elongated spermatids (Tesarik et al., 1995;
Tesarik and Mendoza, 1996). On the other hand, SSF activity
is deficient in human secondary spermatocytes. Looking
towards the future of assisted conception therapy for patients
whose spermatogenesis cannot progress beyond the secondary
spermatocyte stage, this raises the question of whether SSF
deficiency may influence embryo quality after fertilization with
secondary spermatocytes. Further study is needed to understand
the relationship between the action of SSF, the form of the
fertilization-associated Ca 2+ signals and the control of early
embryonic development.
References
Bermudez, D., Escalier, D., Gallo, J.M. el al. (1994). Proacrosin as a
marker of meiotic and post-meiotic germ cell differentiation: quantitative
assessment of human spermatogenesis with a monoclonal antibody.
J. Reprod. Fertil., 100, 567-575.
Berridge, M.J. (1996) Regulation of calcium spiking in mammalian oocytes
through a combination of inositol trisphosphate-dependent entry and release.
Mol. Hum. Reprod., 2, 386-388.
Ciapa, B. and Epel, D. (1991) A rapid change in phosphorylation on tyrosine
accompanies fertilization of sea urchin eggs. FEBS Lett., 293, 110-115.
Dale, B., DeFelice, L.J. and Ehrenstein, G. (1985) Injection of a soluble sperm
extract into sea urchin eggs triggers the cortical reaction. Experientia, 41,
1068-1070.
Dozortsev, D., Rybouchkin, A., De Sutter, P. et al. (1995) Human oocyte
activation following intracytoplasmic sperm injection: the role of the sperm
cell. Hum. Reprod., 10, 403-407.
Escalier, D., Gallo, J.-M., Albert, M. etal. (1991). Human acrosome biogenesis:
immunodetection of proacrosin in primary spermatocytes and of its
partitioning pattern during meiosis. Development 113, 779-788.
Fishel, S., Green, S., Bishop, M. et al. (1995) Pregnancy after intracytoplasmic
injection of spermatid. Lancet, 345, 1641-1642.
Fishel, S., Aslam, I. and Tesarik, J. (1996) Spermatid conception: a stage too
early, or a time too soon? Hum. Reprod., 11, 1371-1375.
Foltz, K.R., Partin, J.S. and Lennarz, W.J. (1993) Sea urchin egg receptor for
sperm: sequence similarity of binding domain and hsp70. Science, 259,
1421-1425.
Homa, S.T. and Swann, K. (1994) A cytosolic sperm factor triggers calcium
oscillations and membrane hyperpolarizations in human oocytes. Hum.
Reprod., 9, 2356-2361.
Kimura, Y. and Yanagimachi, R. (1995a) Mouse oocytes injected with testicular
spermatozoa and round spermatids can develop into normal offspring.
Development, 121, 2397-2405.
Kimura, Y. and Yanagimachi, R. (1995b) Development of normal mice
from oocytes injected with secondary spermatocyte nuclei. Biology of
Reproduction, 53, 855-862.
Moore, G.D., Kopf, G.S. and Schultz, R.M. (1993) Complete mouse egg
activation in the absence of sperm by stimulation of an exogenous G
protein-coupled receptor. Dev. Bioi, 159, 669-678.
Ogura, A., Matsuda, J. and Yanagimachi, R. (1994) Birth of normal young
after electrofusion of mouse oocytes with round spermatids. Proc. Natl.
Acad. Sci. USA, 91, 7460-7462.
Parrington, J., Swann, K., Shevchenko, V.I. et al. (1996) Calcium oscillations
in mammalian eggs triggered by a soluble protein. Nature, 379, 364—368.
Shilling, F.M., Carroll, D.J., Muslin, A.J. et al. (1994) Evidence for both
tyrosine kinase and G-protein-coupled pathways leading to starfish egg
activation. Dev. Bioi, 162, 590-599.
Sousa, M. and Tesarik, J. (1994) Ultrastructural analysis of fertilization failure
after intracytoplasmic sperm injection. Hum. Reprod., 9, 2374—2380.
Swann, K. (1994) Ca 2+ oscillations and sensitization of Ca 2+ release in
unfertilized mouse oocytes injected with a sperm factor. Cell Calcium, 15,
331-339.
Swann, K. (1996) Soluble sperm factors and Ca 2+ release in eggs at
fertilization. Rev. Reprod., 1, 33-39.
Swann, K. and Lawrence, Y. (1996) How and why spermatozoa cause calcium
oscillations in mammalian oocytes. Mol. Hum. Reprod., 2, 388—390.
Taylor, C.T., Lawrence, Y.M., Kingsland, C.R. el al. (1993) Oscillations in
intracellular free calcium induced by spermatozoa in human oocytes at
fertilization. Hum. Reprod., 8, 2174-2179.
Tesarik, J. (1994) How the spermatozoon awakens the oocyte: lessons from
intracytoplasmic sperm injection. Hum. Reprod., 9, 977-978.
Tesarik, J. and Sousa, M. (1994) Comparison of Ca 2+ responses in human
oocytes fertilized by subzonal insemination and by intracytoplasmic sperm
injection. Fertil. Steril, 62, 1197-1204.
Tesarik, J. and Testart, J. (1994) Treatment of sperm-injected human oocytes
with Ca 2+ ionophore supports the development of Ca 2+ oscillations. Bioi.
Reprod., 51, 385-391.
Tesarik, J. and Sousa M. (1995) Key elements of a highly efficient
intracytoplasmic sperm injection technique: Ca 2+ fluxes and oocyte
cytoplasmic dislocation. Fertil. Steril., 64, 770-776.
Tesarik, J. and Mendoza, C. (1996) Spermatid injection into human oocytes.
I. Laboratory techniques and special features of zygote development. Hum.
Reprod., 11, 772-779.
Tesarik, J. and Sousa, M. (1996) Mechanism of calcium oscillations in human
oocytes: a two-store model. Mol. Hum. Reprod., 2, 383-390.
Tesarik, J., Sousa, M. and Testart, J. (1994) Human oocyte activation after
intracytoplasmic sperm injection. Hum. Reprod., 9, 511-518.
Tesarik, J., Mendoza, C. and Testart J. (1995) Viable embryos from injection
of round spermatids into oocytes. N. Engl. J. Med., 333, 525.
Tesarik, J. Rolet, F., Brami, C. el al. (1996) Spermatid injection into human
oocytes. II. Clinical application in the treatment of infertility due to
nonobstructive azoospermia. Hum. Reprod., 11, 780-783.
Received on July 27, 1996; accepted on October 10, 1996
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