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Phagocytosis of supernumerary spermatozoa by two-cell mouse embryos.

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Phagocytosis of Supernumerary Spermatozoa by
Two-cell Mouse Embryos '
ROBERT S. THOMPSON AND LUCIAN0 ZAMBONI
Department o f Pathology, Harbor General Hospital, Torrance, California
90509 and University of California, School of Medicine, Los Angeles,
California 90024
ABSTRACT
Phagocytosis of supernumerary spermatozoa contained within
the perivitelline space was observed in 7 of 28 two-cell mouse embryos cultured
for 45 hours post-insemination (approximately 20-24 hours after cleavage).
The spermatozoa were incarcerated as a result of elevations of the blastomere
cytoplasm which gradually surrounded the sperm, overlapped and fused, thus
forming a typical phagocytic vacuole. Phagocytosis was not observed in two-cell
embryos cultured for less than 20-24 hours after cleavage; this indicates that
the blastomeres of two-cell mouse embryos in vitro require approximately 24
hours to develop one of the characteristics of somatic cells, i.e., the ability to
recognize and phagocytize foreign material.
Spermatozoa not directly involved in
fertilization are disposed of and eliminated
from the female reproductive tract by several mechanisms. One of the most frequent
means is phagocytosis by leucocytes in the
uterine cavity (Austin, '60; Bedford, '63;
Mahajan and Menge, '66, '67; Moyer et al.,
'65, '67; Stefanini et al., '68; Yanagimachi
and Chang, '63; Zamboni, '68, '71) and
cervix (Moyer et al., '70), and by epithelial
cells of the endometrium (Moyer et al.,
'67; Zamboni, '68, '71), endometrial gland
(Austin, '60) and oviductal mucosa (Austin, '60; Zamboni, '71). Accessory spermatozoa in the tubal ampulla can be phagocytized by the cells of the corona radiata
(Bedford, '68; Zamboni, '71). It has been
reported recently that spermatozoa may
be found also within phagoeytic vacuoles
of the trophoblast cells of mouse and rat
blastocysts (Tachi and Kraicer, '67; Tachi
et al., '70; McReynolds and Hadek, '71).
The purpose of this report is to present
evidence, for the first time, that blastomeres of two-cell mouse embryos developed
in vitro can phagocytize supernumerary
spermatozoa present in the perivitelline
space.
MATERIALS AND METHODS
Adult female, Swiss white mice were
superovulated by injection of 5 IU of PMS
followed 48 hours later by 5 IU of HCG.
ANAT. REC., 178: 3-14.
Ova were collected from the oviduct under
sterile conditions approximately 13 hours
after HCG injection and placed in groups
of 20-40 in microdrops of medium under
oil (Whittingham, '68). The medium used
was a Krebs Ringer bicarbonate supplemented with lactic acid (25 mM), sodium
pyruvate (0.5 mM), glucose (5.56 mM),
albumin (30 mg/ml), penicillin and streptomycin.
Semen was obtained from the uteri of
gonadotrophin-stimulated mice mated approximately three hours previously with
proven breeder males. Small aliquots of
semen were mixed with equal volumes of
culture medium and 50 pl of the suspension added to each group of oocytes. The
inseminated ova were cultured at 37" C
in an atmosphere of 5% CO, in air for
periods ranging from 19 to 45 hours. At
the end of culture, they were fixed in 2.5%
glutaraldehyde in 0.1 M cacodylate buffer,
post-fixed in 1% OsO,, dehydrated, embedded in Epon 812 and serially sectioned for
el$ctron microscopy. The sections were
stained with lead citrate and examined
with Hitachi HUllC and HUllE electron
microscopes.
Received April 30, '73. Accepted July 17, '73.
1 This study was supported by a U.S.P.H.S. Research
Grant (ROlHD05725) and Research Contract (69-
2220 ).
3
4
ROBERT S. THOMPSON AND LUCIAN0 ZAMBONI
tions of the sperm being incorporated;
whereas
fairly large protrusions were seen
Phagocytosis of supernumerary spermatozoa was observed in 7 of a group of around the sperm head (fig. 4), the tail
28 two-cell embryos which had been in cul- was usually surrounded by numerous, thin
ture for 45 hours after insemination (ap- cytoplasmic projections comparable in size
proximately 20-24 hours after cleavage); to microvilli (figs. 6-8). Following incarceno evidence of phagocytosis was obtained ration, the vacuole containing the phagoin embryos cultured for shorter periods. cytized sperm showed a tendency to move
The embryos involved in phagocytosis all deeper into the blastomere cytoplasm
appeared to result from a "'normal" fertili- (figs. 1, 5, 10).
Fusion of the blastomere plasma memzation process (Zamboni et al., '72a); the
blastomeres were of equal size and mor- brane with that of any incarcerated sperm
phologically normal (fig. 1). Their nuclei segment was never seen. Also, no indicadid not show any abnormal features and tion was obtained that any given sperm
the cytoplasm appeared healthy; it pos- segment was incorporated with greater fresessed a uniformly distributed complement quency or in any specific sequence than
of organelles which included endoplasmic others. Degradation of the phagocytized
reticulum vesicles, Golgi complexes, mito- sperm components within the cytoplasmic
chondria and occasional lipid droplets vacuoles was not observed.
(fig. 1). Flagellar components of the ferDISCUSSION
tilizing sperm were found in all embryos
The observations made in this study
studied. The embryos were enclosed within
normal appearing, unbroken zonae pellu- demonstrate for the first time that supercidae (fig, 1) and were occasionally asso- numerary spermatozoa in the perivitelline
ciated with a few degenerating follicle space of cultured two-cell mouse embryos
cells. Varying numbers of supernumerary may be disposed of by phagocytosis and
spermatozoa were present within the peri- subsequent incorporation within vacuoles
vitelline space of these embryos. The in the blastomere cytoplasm; the phagospermatozoa all showed varying signs of cytic process noted in this study appeared
degeneration (fig. 2 ) ; the acrosome was identical to that occurring in somatic cells
absent and vesiculated remnants of the engaged in ingestion of foreign material.
plasma and outer acrosomal membranes The time period considered in this study
surrounded the structures of the anterior prevented us from determining the ultiregion of the head. The mitochondria dem- mate destiny of the phagocytized speronstrated varying degrees of swelling and matozoa; however, i t is probable that they
rarefaction of the matrix with a numerical eventually undergo enzymatic degradation
within the phagocytic vacuoles. A less
reduction of the cristae.
Sperm phagocytosis initiated with the likely alternative is that the phagocytized
formation of a depression of the blasto- sperm could persist, more or less intact,
mere surface in the vicinity of the sperm until relatively advanced stages of embryto be incorporated (figs. 3, 4, 6). The onic development; this could explain the
plasma membrane of the blastomere in this presence of phagocytized sperm remnants
area displayed a moderate degree of sur- observed in the trophoblast cells of mouse
face activity mostly in the form of micro- and rat blastocysts (Tachi and Kraicer,
villar projections which were more numer- '67; Tachi et al., '70; McReynolds and
ous when the sperm tail, rather than the Hadek, '71 ).
An example of the attitude of ova for
head, was being engulfed (figs. 3, 4, 6, 7).
As the depression gradually deepened, the phagocytosis occurs at fertilization and inedges of this area began to surround the volves the incorporation into the ooplasm
of the most anterior region of the nucleus
sperm with cytoplasmic folds (figs. 4,7-9).
Eventually, these folds overlapped and of the fertilizing spermatozoon. The structheir membranes fused, thus incarcerating tures of this region lack the plasma memthe sperm within a cytoplasmic vacuole brane as a consequence of the acrosome
(figs. 5, l o ) . The cytoplasmic folds varied reaction (Barros et al., '67); therefore
considerably in size around different por- they cannot be incorporated into the oocyte
RESULTS
SPERM PHAGOCYTOSIS BY TWO-CE:LL MOUSE EMBRYOS
5
ture of the mammalian acrosome reaction. J.
through the process of gamete membrane
Cell. Biol., 34: Cl-C5.
fusion and instead become enclosed within Bedford, J. M. 1963 Morphological reaction of
an ooplasmic vacuole (Bedford, '70, '72;
sptmnatozoa in the female reproductive tract of
the rabbit. J. Reprod. Fert., 6: 245-255.
Zamboni et al., '70) which develops simi1968 Ultrastructural changes i n the
larly to the phagocytic vacuoles described -sperm head during fertilization in the rabbit.
in this report. A more definitive demonAm. J. Anat., 123: 329-358.
1970 Sperm capacitation and fertilizastration that ova are capable of phagocytic -tion in mammals, Biol. Reprod. (Suppl.), 2:
activity was recently provided by a study
125-158.
showing that human oocytes maintained ___ 1972 A n electron microscopic study of
sperm penetration into the rabbit egg after
in culture can phagocytize follicle cells
natural mating. Am. J. Anat., 133: 213-254.
which migrate through the zona pellucida Mahajan,
S. C., and A. C. Menge 1966 Factors
(Zamboni et al., '72b); the phagocytic
infuencing the disposal of sperm and the leukocyiic response in the rabbit uterus. Int. J. Fert.,
process evolved i n a manner not dissimilar
1 1 . 373-380.
from that described in the present study. __
-1967 Influence of gonadal hormones
Our present observations indicate that
and leukocytes on sperm removal from the rat
no phagocytosis seems to occur in blastauterus. Int. J. Fert., 11: 384-390.
meres before 20-24 hours after cleavage. McReynolds, H. D., and R. Hadek 1971 A study
on sperm tail elements i n mouse blastocysts.
It is possible that during this period the
J. Reprod. Fert., 24: 291-294.
spermatozoa undergo modifications of their Moyer, D. L., G. M. Kunitake and R. M. Nakamura 1965 Electron microscopic observations
surface components that would render
on phagocytosis of rabbit spermatozoa in the
them susceptible to phagocytosis. In our
female genital tract. Experientia, 21: 6-7.
opinion, however, a more likely possibility Moyer, D. L., G. Legorreta, H. Maruta and V. Henderson 1967 Elimination of homologous speris that fertilization is followed by a relamatozoa i n the female genital tract of the
tively long period of time during which
rabbit: a light and electron-microscope study.
the oolemma is unresponsive to the presJ. Path. Bact., 94: 345-350.
ence of foreign material. Supernumerary Moyer, D. L., S. Rimdusit and D. R. Mishell, Jr.
19'70 Sperm distribution and degradation in
spermatozoa would remain undisturbed in
the human female reproductive tract. Ob. Gyn.,
the perivitelline space of penetrated ova
35. 83 1-840.
through activation and cleavage, and only Stefanini, M., T. Hando and L. Zamboni 1968
hours after development of a two-cell emElectron microscope studies of mammalian
fertilization. Uterine and tuba1 spermatozoa of
bryo would the blastomeres reach a degree
the mouse. Proc. Fourth European Reg. Conf.
of differentiation sufficient to enable them
Electron Mlcroscopy. D. S. Bocciarelli, ed.
to recognize foreign material at or near
voi. 11: 317-318.
their plasma membrane and to dispose of Tachi, S., and P. F. Kraicer 1967 Studies on the
mechanism of nidation. XXVII. Sperm-derived
it by phagocytosis. That this ability seems
inclusions i n the rat blastocyst. J. Reprod. Fert.,
to develop approximately 20-24 hours after
14. 401405.
in vitro cleavage is further indicated by the Tachi, S . , C. Tachi and H. R. Lindner 1970 U1trastructural features of blastocyst attachment
fact that the embryos described in this reand trophoblastic invasion in the rat. J. Reprod.
port were engaged exclusively in stages of
Fert., 21: 37-56.
phagocytosis which did not go beyond early Whittingham, D. G. 1968 Fertilization of mouse
incarceration of the spermatozoa within
eggs in vitro. Nature, 220: 592-593.
Yanagimachi, R., and M. C. Chang 1963 Infilcytoplasmic vacuoles.
ACKNOWLEDGMENT
The authors wish to acknowledge the
collaboration of Dr. Dianne Moore Smith
during the in vitro culture phase of this
study.
LITERATURE CITED
Austin, C. R. 1960 Fate of spermatozoa i n the
female genital tract. J. Reprod. Fert., 3: 151156.
Barros, C., J. M. Bedford, L. E. Franklin and C. R.
Austin 1967 Membrane vesiculation as a fea-
tration of leucocytes into the uterine lumen of
the golden hamster during the oestrous cycle
and following mating, J . Reprod. Fert., 5:
389-396.
Zamhoni, L. 1968 Electron microscope studies
on mammalian fertilization. A new approach
to the problem. Proc. Fourth European Reg.
Conf. Electron Microscopy. D. S. Bocciarelli, ed.
Vol. 11: 313-316.
-1971 Fine morphology of Mammalian
Fertilization. Harper and Row, New York.
Z a m l ~ n i L.,
, J. Chakraborty and D. Moore Smith
1972a First cleavage division of the mouse
zygote. A n ultrastructural study. Biol. Reprod.,
7: 170-193.
6
ROBERT S. THOMPSON AND LUCIAN0 ZAMBONI
Zainboni, L., D. Moore Smith and R. S. Thompson
1972b Migration of follicle cells through the
zona pellucida and their sequestration by human oocytes i n vitro. J. Exp. Zool., 181:
3 19-340.
Zamboni, L., M. Stefanini, C. Oura and D. Smith
1970 The pattern of sperm penetration into
the mouse egg. Proc. Seventh Internat. Cong.
Electron Microscopy, P. Favard, ed. Vol. 3:
663-664.
PLATE 1
EXPLANATION OF FIGURE
1
A two-cell mouse embryo with a phagocytized sperm head i n the cytoplasm of one blastomere (arrow). Note the overall healthy appearance of the blastomeres and the uniform distribution of the organelles.
( N ) blastomere nucleus, (ZP) zona pellucida. x 4,900.
SPERM PHAGOCYTOSIS BY TWO-CELL MOUSE EMBRYOS
Robert S. Thompson and Luciano Zamboni
PLATE 1
7
PLATE 2
EXPLANATION O F FIGURE
2
a
Degenerating supernumerary sperm in the perivitelline space of a
two-cell mouse embryo. Note absence of the acrosome and presence
of vesiculated membrane remnants around the anterior portion of
the sperm head. The plasma membrane around the flagellum is absent
or fragmented. There is also a noticeable rarefaction of the mitochondrial matrix. X 13,700.
SPERM PHAGOCY TOSIS BY TWO-CELL MOUSE EMBRYOS
Robert S. Thompson and Luciano Zamboni
PLATE 2
9
PLATE 3
E X P L A N A T I O N O F FIGURES
Figures 3-5 depict sequential stages of incorporation of the heads of
supernumerary spermatozoa by two-cell mouse embryos.
3
Incipient stage of sperm phagocytosis. The posterior portion of the
nucleus of this sperm, which is contained in the space between the
blastomeres, is accommodated within a depression in the surface of
one of them. Cytoplasmic projections from the blastomere surface
are evident. y, 33,000.
4
In a more advanced stage of phagocytosis, the anterior region of this
sperm head is contained within a deeper depression in the blastomere
surface than that shown i n the previous micrograph. X 24,400.
5
At the end of the engulfment process, the sperm head is fully enclosed within a phagocytic vacuole deeper i n the blastomere cytoplasm. Arrow points to the blastomere surface over the incorporation
area which has now regained its original smooth conformation.
>: 47,500.
SPERM PHAGOCYTOSIS BY TWO-CELL MOUSE EMBRYOS
Robert S. Thompson and Lucian0 Zamboni
PLATE 3
11
PLATE 4
EXPLANATION OF FIGURES
Figures 6-10 illustrate sequential stages of phagocytosis of tails of
supernumerary sperm by two-cell mouse embryos. The events depicted
occur i n the same manner regardless of the portion of the sperm tail to
be phagocytized.
12
6
In a n early stage, the flagellum is partially contained in a slight
depression of the blastomere surface which shows only a moderate
degree of surface activity. y, 30,400.
7
I n a slightly more advanced stage of engulfment, there is deepening
of the depression and presence of a greater number of elongated cytoplasmic projections which partially surround the segment of flagellum t o be incorporated. Y, 37,400.
8
The segment of the flagellum is now completely encircled by elongated cytoplasmic protrusions from the blastomere surface. x 40,300.
9
Section of principal piece fully enclosed within a nearly complete
phagocytic vacuole whose projections have started to fuse. x 37,700.
10
Sperm midpiece incarcerated i n a completely closed phagocytic vacuole
deep in the blastomere cytoplasm. The wall of the vacuole appears
multilaminar due to the multiple wrapping over one another of the
cytoplasmic projections which initially surrounded the sperm tail.
;< 19,200.
SPERM FHAGOCYTOSIS BY TWO-CELL MOUSE EMBRYOS
Robert S. Thompson and Lucian0 Zamboni
PLATE 4
13
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