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Ultrastructure of Rabbit Ova Recovered from Ovarian Follicles and Inseminated in Vitro*
Vol. 26, No. 7, July 1975 Printed in U.S.A.
FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society
ULTRASTRUCTURE OF RABBIT OVA RECOVERED FROM OVARIAN FOLLICLES AND INSEMINATED IN VITRO* YON K. OH, D.V.M. M.S.,
AND
BENJAMIN G. BRACKE'IT, D.V.M., PH.D.t
Section of Clinical Reproduction, Department of Clinical Studies, Sclwol of Veterinary Medicine, and Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania Sclwol of Medicine, Philadelphia, Pennsylvania 19174
The ultrastructural morphology of rabbit follicular oocytes fertilized in vitro has not previously been chronologically documented. Most of the ultrastructural studies of fertilization described in mammalian species have been done under in vivo conditions, e.g., in the rabbit, 1 - 5 rat, 6 - 8 mouse, 9 • 10 hamster, 11 and pig. 12 Little information has been available on sperm interactions with egg investments and pronuclear formation under in vitro conditions, especially in the follicular oocytes of the rabbit. 13 The following study describes electron microscopic observations of sperm penetration of the egg a11d pronuclear formation and compares the ultrastructural criteria of fertilization with those for control ova, which were examined conventionally with light microscopy after cleavage to the two- or four-cell stages. Control ova were incubated in culture for 25 hours after insemination. · MATERIALS AND METHODS
Ova were obtained from preovulatory ovarian follicles of gonadotropin-treated New Zealand White does. Ovum donors received intramuscular injections of 150 IU of pregnant mare's serum, followed Received September 11, 1974. *Supported by National Institutes of Health Grant HDO 6274, Career Development Award HD 15861, and by a grant from The Ford Foundation. tReprint requests: Dr. B. G. Brackett, Section of Clinical Reproduction, New Bolton Center, R.D. #1, Kennett Square, Pa. 19348.
83 hours later by intravenous injection of 75 IU of human chorionic gonadotropin. Ten hours after treatment with human chorionic gonadotropin, ova were recovered from unruptured ovarian follicles, pooled, and then transferred into a suspension of spermatozoa recovered from the uterine horns of does which had been mated 18 hours previously. The procedure for in vitro fertilization was the same as that reported previously.14·15 The medium in these exeriments was the simple defined medium16 supplemented with 10-5 M pyruvate.15 The incubation atmosphere was moiRt 5% C02, 8% 02. and balance N2. Ova were removed from culture 0, 1% 3, 6, and 9 hours after exposure to the capacitated sperm. The ova were fixed in 2.0% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1% Os04, dehydrated, individually embedded in Epon 812, and serially sectioned for light and electron microscopy. For light microscopy, sections were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi llB electron microscope. Ova from the same donors that had been inseminated by sperm from the same capacitators were examined by light microscopy approximately 25 hours after insemination in vitro. The proportion of ova having undergone normal cleavage, i.e., cleaved ova with symmetrical blastomeres resembling those recovered from oviducts after in vivo fertilization, was
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FIG. 1. Portion of a secondary oocyte and the first polar body IRB 1). The first polar body contains chromosomes (CHr), mitochondria (M), cortical granules (CG), and endoplasmic reticulum (ER). In the cortical ooplasm (Oop), the metaphase II chromosomes (Chr) can be seen arranged in the equatorial plane. Note the absence of organelles in the area of the chromosomes (x8,000).
•
FIG. 2. Portion of a follicular oocyte showing the zona pellucida (ZP) and the cortical granules. Relatively evenly spaced cortical granules (CG) can be seen immediately beneath the vitelline membrane. Note also the mitochondria (M) and the vesicular structures (V) (X 1,200).
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compared with the proportion of ova exhibiting signs of undergoing fertilization, as determined by electron microscopy. In this work, an ovum was considered to be undergoing fertilization if sperm penetration had occurred and if characteristic morphologic changes, already well recognized in the fertilization process in vivo for several species, could be observed. Fertilization rates, i.e., proportions of inseminated ova showing signs of fertilization according to criteria observable by light and electron microscopy, were compared; data were statistically treated by x2 analysis. RESULTS
The features of rabbit oocytes recovered just prior to the expected time of ovulation are shown in Figures 1 and 2. The presence of a first polar body (Fig. 1) was previously demonstrated in approximately 72.9% of the oocytes. 14 The first polar body found within the perivitelline space was usually iocated within a shallow depression in the surface of the oocyte (Fig. 1). Chromosomes were present in the first polar body and in the subcortical area of the ooplasm, as a result of the first meiotic division. The first polar body contained cytoplasmic organelles, mitochondria, endoplasmic reticulum, and cortical granules (Fig. 1). Many spherically shaped, uniformly sized cortical granules were arranged at more or less regular intervals in the area immediately beneath the vitelline membrane (Fig. 2). In some areas near the cortex, they were also found in clusters (Fig. 3). Spheroidal mitochondria with peripheral crystae, vesicles, and Golgi complexes, evenly distributed around the ooplasm, were characteristic features of unfertilized ova at this early stage. Observations Made 1¥2 Hours after Insemination. Five of eight eggs that were serially sectioned following removal from the culture 1¥2 hours after insemination
FIG. 3. Portion of a secondary oocyte (Ooc) showing cortical granules (CG) clustered in the subcortical ooplasm and lipid inclusions (LP). Elements of endoplasmic reticulum CER) can be seen in the first polar body (PB 1) (x20,000).
in vitro were observed to be undergoing fertilization. At this time, sperm had already penetrated into the ooplasm; in three of these ova, the sperm nucleus was seen to be undergoing decondensation (Figs. 4 and 5). In two of the eggs, the sperm nucleus was located close to the maternal chromosomes (Fig. 5). Decondensation of the sperm nucleus occurred following loss of the nuclear membrane and presented a diffused and faintly staining appearance. Microtubules sur-
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FIG. 4. Ovum 1'h hours after insemination. A swollen sperm nucleus (SN), delineated by arrowheads, and sperm midpiece (SM) are evident within the ooplasm. The egg cortex is completely devoid of cortical granules. Also seen are ovum mitochondria (M) and the zona pellucida fZP) (14,000).
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FIG. 5. Sperm nucleus (SN) located near the maternal chromosomes (mChr) llh hours after insemination. Arrowheads delineate the limits of the sperm nucleus. Microtubules (MT) are seen in cross-section surrounding the sperm nucleus and in oblique section adjacent to the maternal chromosomes (x 10,000).
rounded the regions of sperm nuclear decondensation and the maternal chromosomes (Fig. 5). The sperm midpiece, mitochondria, and coarse fibers remained unchanged (Fig. 4). Ooplasmic organelles, mitochondria, vesicles, and endoplasmic reticulum surrounded the area of sperm decondensation (Figs. 4 and 5). Cortical granules were completely absent from the peripheral area of the ooplasm, where sperm penetration presumably had occurred (Fig. 4). The characteristic changes in sperm cells in the vicinity of the egg which comprised the acrosome reaction were seen in this study. These changes involved swelling of the plasma membrane, vesicu-
lation of the plasma membrane with the outer acrosomal membrane, and progression of these changes to apparent loss of the plasma and outer acrosomal membrane, exposing the inner acrosomal membrane (Fig. 6). These changes occurred within the interstices of corona cells and were completed by the time the sperm cell arrived at the surface of the zona pellucida (Fig. 7). Most of the sperm cells observed to be penetrating the zona pellucida were undoubtedly supplementary sperm, since cortical granules were absent from the periphery of the egg cytoplasm, indicating that activation was already under way. A penetration slit in the zona pellucida was observed pos-
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FIG. 6. Sperm undergoing the acrosome reaction in the interstices between corona cells (CC) 1 ¥z hours after insemination. In sperm 1 (f3 1 ), the plasma membrane !PM) is swollen. In sperm 2 (S 2), the plasma membrane and outer acrosomal membrane are undergoing vesiculation (V). In sperm 3 (S 3), the acrosome reaction has been completed, exposing the inner acrosomal membrane (JAM) (x32,000).
terior to the penetrating sperm head which provided an impression that the sperm cell was crossing tangentially toward the ooplasmic membrane (Fig. 8). Supplementary sperm found lying within the perivitelline space presented morphologic features similar to those within the matrix of the zona pellucida and those at the surface of the zona that had already completed the acrosome reaction (Fig. 9). Observations Made 3 Hours after Insemination. Five of six eggs serially sectioned following 3 hours in culture after in vitro insemination provided evidence that fertilization was under way.
Two of these eggs each had a developing male pronucleus, and the other three eggs were at the decondensation stage of the sperm nucleus. The presence of a sperm flagellum in close proximity to a pronucleus strongly suggested that this was the male pronucleus (Fig. 10). The long remnant of the sperm flagellum seen in the cortex of the ovum in Figure 10 presumably indicated the site of penetration. The second polar body was released at the completion of the second meiotic division. The early pronucleus possessed a double nuclear membrane with widely dispersed nuclear pores and faint, elec-
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FIG. 7. Portion of oocyte (Ooc), zona pellucida (ZP), and corona cells (CC) llh hours after insemination. The sperm resting on the zona pellucida has completed the acrosome reaction (xlO,OOO).
tron-opaque material aggregated randomly within the pronuclear boundries (Fig. 11). The sperm flagellum within the ooplasm exhibited mitochondria ~hich appeared somewhat swollen and possessed peripheral cristae (Fig. 12). Observations Made 6 Hours after Insemination. Nine of fifteen eggs serially
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sectioned following 6 hours in culture after in vitro insemination were found to be undergoing fertilization. Five of the eggs were at the pronuclear stage, while two eggs possessed only a male pronucleus; the remaining two eggs were at the decondensation stage of the sperm nucleus. At the early pronuclear stage observed at this time, the cytoplasmic region adjacent to the pronuclei was morphologically distinct from the cortex in that it contained more mitochondria, annulate lamellae, and vesicles which were closely associated with the Golgi complex (Fig. 13). Both pronuclear membranes had surfaces that appeared relatively smooth and had randomly dispersed nuclear pores. The male pronucleus was generally larger than the female pronucleus and frequently had a remnant of sperm flagellum associated with it. Observations Made 9 Hours after Insemination. Seven of seventeen eggs serially sectioned and examined following 9 hours in culture after in vitro insemination were in the pronuclear stage. By this time, the pronuclei had developed and migrated toward the center of the ovum (Fig. 14). When in juxtaposition, the pronuclear membrane projections approached each other through the intervening cytoplasm. Invagination of pronuclei extended toward the center of each pronucleus (Fig. 15). Sometimes neither pronuclear membrane could be discerned at a point of apposition, strongly suggesting an exchange of nuclear material from paternal and maternal origin at this stage (Fig. 16). Frequently, nuclear blebbing occurred in the pronuclear envelope, and polyribosomes were scattered around the region (Fig. 1 7). Annulate lamellae also appeared to be more abundant in this region than was the case 6 hours after insemination (Fig. 19). When oocytes were not activated by sperm, the number of cortical granules lining the periphery of the ooplasm was increased over that of the
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FIG. 8. Penetration of zona pellucida
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FIG. 9. Supernumerary sperm in the perivitelline space fPVS) of an ovum llh hours after insemination. The inner acrosomal membrane (lAM) and the equatorial segment CES) of the sperm are clearly evident (x29,000).
eggs recovered from the ovarian follicles and examined immediately (Figs. 18 and 2, respectively). The distance between the sperm remnant and the pronuclear envelope seemed to depend on the plane of sectioning of the oocyte. When the region proximal to the sperm neck was sectioned, the sperm remnant was found closer to the pronucleus (Fig. 19); when the distal portion of the sperm tail was sectioned, the remnant was farther from the pronucleus (Fig. 20). In some oocytes, regressive changes were seen in sperm mitochondria and in the axial filament complex of sperm remnants 9 hours after insemination (Fig. 21). Number of Sperm within the Matrix of the Zona Pellucida and in the Perivitelline Space. There was no obvious correlation between time interval following insemination and the number of sperm cells that had penetrated into and through the zona pellucida but had not taken part in fertilization. Also, no correlation was obvious between sperm concentration
and numbers of penetrating and supplementary sperm cells. In this work, one to nine sperm were found within the matrix of the zona pellucida and in the perivitelline spaces of individual eggs examined at 1 'h to 9 hours after in vitro insemination. Occasionally, several sperm cells were found assembled in a relatively small region of the perivitelline space (Fig. 22). Comparison of Fertilization Rates According to Ultrastructural Observations of Ova Undergoing Fertilization with Those Based on Observation of Cleaved Ova. Table 1 summarizes data from the above ultrastructural observations along with the corresponding cleavage rates observed in control ova cultured 25 hours in each experiment. From these data, 56.5% of 46 ova examined ultrastructurally were found to be undergoing fertilization, compared with 54.8% of 93 ova that cleaved normally in the control group. These proportions are very close, and are not statistically significantly different. Of the 51 control ova that
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FIG. 10. Early male pronuclear (mPN) formation 3 hours after insemination. The sperm midpiece (SM) is located near the male pronucleus. Note also the sperm tail (ST) in the ooplasm (Oop) and the second polar body
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FIG. 11. The pronuclear membrane 3 hours after insemination. The double membrane is seen with numerous well developed nuclear pores WP) separating the pronucleus (}'N) and ooplasm (Oop) (X 50,000).
cleaved, 45 had reached the four-cell stage, and only 6 remained in the two-cell stage when observed 25 hours after insemination in vitro. These data indicate that ultrastructural evidence of the fertilization process and light microscopic observation of proportions of normally cleaved ova following this in vitro insemination procedure provide two means for assessing the ability of rabbit gametes to undergo fertilization. DISCUSSION
Sperm Penetration and Pronuclear Formation. According to Bedford, 1 in vivo sperm penetration into the vitellus occurs 111h hours postcoitus, which is comparable to the 11h hours after insemination in vitro in the present work. Fraser et al. 13 reported the absence of cortical granules
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FIG. 12. Sperm midpiece in an activated ovum 3 hours after insemination. The sperm mitochondria ISM) with peripheral cristae appear swollen. The sperm fibers (SF) remain well organized (x30,000).
in ovum cytoplasm of tubal ova 1 hour after in vitro insemination; however, no sperm remnants were demonstrated in the ovum cytoplasm. Our findings 1¥2 hours postinsemination demonstrate that sperm had already penetrated the oocyte. Sperm were found at the nuclear decondensation stage, and the cortical granule reaction had already taken place (Fig. 4). ThibauW7· 18 reported similar results in light microscopic studies in the rabbit. In earlier work from our laboratory using ovulated rabbit ova recovered from oviducts, motile sperm were seen within the perivitelline space as early as 2 to 4% hours after in vitro insemination. 19 Variability in conditions, methods of observation, and even the gametes themselves contribute to reported differences in the sequence of events in the fertilization process in vitro.
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FIG. 13. Pronuclei in apposition 6 hours after insemination. The nuclear membrane WM) has reformed about both pronuclei (}'N), and nuclear pores WP) are numerous. The cytoplasm surrounding the pronuclei is rich in organelles such as Golgi complex (GC), mitochondria (M), annulate lamellae (AL), and vesicular structures (V) (x 10,000).
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FIG. 14. Pronuclei 9 hours after insemination. The pronuclei (J'N) are now very close, separated only by a narrow intermembranous space (x7,000).
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FIG. 15. Pronuclei (J'N) in close apposition showing several infoldings of the pronuclear envelopes. Elements of the Golgi complex (GC) are seen between the pronuclei, and intranuclear annulate lamellae (JAL) are evident in the lower pronucleus (x 12,000).
Sperm were found to be undergoing the plasma membrane vesiculation process, i.e., the acrosome reaction (Figs. 6 and 7), before penetration of the zona pellucida. 1 • 20 • 21 When the sperm were found penetrating the zona pellucida, the plasma and outer ascrosomal membranes had already disappeared. A well defined zona penetration slit was left behind (Fig. 8). The present observations reconfirmed the morphologic changes in sperm, including the acrosome reaction and pene-
tration through the zona and into the vitellus, reported to occur in vivo in the rabbit, L 20 • 22 - 25 pig, 12 rat, 6 • 8 hamster, n and mouse. 9 Similar observations have also been reported following in vitro studies in the hamster, 26 mouse, 27 and human. 28 As early as 3 hours after in vitro insemination, a small male pronucleus could be seen in a very early stage of formation (Fig. 10). This followed second polar body extrusion. In careful serial
FIG. 16. Internuclear communication in the late stage of pronuclei. Intranuclear annulate lamellae are evident within both pronuclei (J'N) and in the area of pronuclear fusion (arrows) (x 8,000).
(JAL)
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FIG. 17. Nuclear blebbing in the pronuclear envelope. Two membrane-bound structures WB) appear
to be forming between the inner and outer leaflets of the pronuclear (}'N} envelope. Several polyribosomes (}'Rb) can be seen in the cytoplasm. NP, Nuclear pore (x42,000).
sectioning of these oocytes, another pronucleus could not be found at this time, which leads to the conclusion that the male pronucleus forms before the female pronucleus. The time required after
sperm penetration for pronucleus formation in the rabbit egg was similar to that found in studies of in vivo fertilizationt. 20 • 29 • 30 or in other studies after in vitro inseminationY· 18 Female pronucleus formation apparently took place between 1% and 4¥2 hours after the sperm cell had penetrated the ooplasm. Under light microscopic examination, pronuclei must be larger to be seen; therefore, some previous studies have reported that pronuclear formation occurs later. 31
FIG. 18. Unfertilized egg 9 hours after insemination. The number of cortical granules (CG) has increased and mitochondria (M} remain in the egg cortex. Microvilli are seen projecting into the zona pellucida ~P) (x 24,000).
When the pronuclei are fully developed, it is difficult to distinguish the male pronucleus from the female pronucleus. As a general rule, the male pronucleus reportedly is larger, 17 although it may be nearly the same size. 4 It is larger in the rat. 7 • 32 The male pronucleus has usually been identified by the presence of nearby sperm remnants (Figs. 10, 19, and 20). As they develop, the male and female pronuclei move to the central part
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FIG. 19. Fertilized ovum 9 hours after insemination. A portion of the sperm tail (ST) and several stacks of annulate lamellae (AL) are seen close to one of the pronuclei (J>N). M, Mitochondrion (x 38,000).
of the oocyte and become surrounded by cytoplasmic organelles, including mitochondria, Golgi components, and annulate lamellae (Fig. 13). Similar morphologic changes have been described in vivo. 4 • 33 Characteristic of rabbit ova are the multiple, irregular infoldings of the pronuclear envelopes that form especially at sites where pronuclei come into apposition (Fig. 15). Some communication occurs between pronuclei through areas of direct union (Fig. 16), as was previously reported
Fro. 20. Sperm flagellum (SF) present distant to the pronucleus. The egg was fixed 9 hours after insemination. PN, Pronucleus (X 10,000).
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FIG. 22. A 1-11-m section of ovum (0), zona pellucida (ZP), and corona cells (CC) 3 hours after insemination. Supernumerary sperm (SS) are evident in the perivitelline space (x640).
FIG. 21. Portion of a fertilized ovum 9 hours after insemination. The sperm flagellum located close to one of the pronuclei (}'N) exhibits regressive changes in both the sperm mitochondria (SM) and sperm fibers (SF) (x25,000).
during in vivo fertilization. 33 Subsequently, there is complete breakdown of the pronuclear envelopes. 4 An exchange of nuclear materials from male and female pronuclei takes place just before cleavage to the two-cell stage. 34 Cortical Granules. The cortical granule reaction has drawn attention for its role as a manifestation of ovum activation early in fertilization and also for its role in the block to polyspermy. 35-38 The cortical granules are round, moderately homogeneous, and electron-dense organelles (0.08 to 0.2 p.m in diameter). It has been postulated that they are synthesized in the Golgi complex2 • 3 • 3s. 40, 4t and later migrate into the cortex of the oocyte, localizing just beneath the vi-
telline membrane (Fig. 2). 42 In the present study, most cortical granules were absent when sperm remnants of male pronuclei were present (Figs. 4 and 10). The precise time of cortical granule breakdown after sperm penetration is not known in the rabbit, but SzollosP8 found in the rat and hamster that the cortical granules are activated by the initial attachment of sperm to the vitelline membrane. In the unfertilized rabbit egg, the number of cortical granules at the periphery of the ooplasm increases during aging in culture media (Fig. 18), as was previously described by Hadek. 39 This is TABLE 1. Comparison of Fertilization Rates Using Different Criteria Light microscopic observation
Ultrastructural evidence Hours postinsemination
Ova undergoing fertilization/ ova inseminated
5/8 1Yl 5/6 3 9/15 6 7/17 9 Summary 27/46 (56.5%)
25 hr postinsemination Ova cleaved/ ova inseminated
9/15 9/15 12/24 21/39 51193 (54.8%)
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in contrast to unfertilized mouse tubal ova9 and hamster ova, in which the number of cortical granules apparently decreases when they are aged, having a close relationship to the fertilizability of the ova. 43 Annulate Lamellae. Annulate lamellae appear within 4lh hours after sperm penetration (Fig. 13) and become abundant at the late stage of pronuclear formation (Fig. 19). The presence of annulate lamellae has been reported in ova undergoing fertilization in vivo in the rabbit, 2· 3· 5 the pig, 12 and human44 and in human in vitro-fertilized ova. 28 Annulate lamellae were also described in primordial and follicular oocytes of the human, 4L 45 bovine, 46 and chimpanzeeY Gulyas48 observed no annulate lamellae in unfertilized rabbit ova soon after ovulation; but 5 to 6 hours later, they appeared. This is in agreement with the present observations of formation of annulate lamellae within 6 hours after in vitro insemination of ova recovered from mature follicles that are undergoing fertilization (Fig. 13). It has been proposed that annulate lamellae develop from stacks of endoplasmic reticulum 41 or from the nuclear envelopeY· 49 -51 Annulate lamellae are formed in late stages in both fertilized and unfertilized rabbit ova and may, therefore, be related to the aging of the oocyte or to the developmental stage of the egg. However, their function is unknown. 52 Sperm Flagellum in the Ovum Cytoplasm. The presence of a sperm flagellum in the cytoplasm of an oocyte has been used as one criterion of fertilization. Thibault and Dauzier53 have shown the sperm by hematoxylin staining, but this was at an early stage of sperm penetration. Light microscopic detection of the sperm flagellum is probably not a reliable criterion of routine determination of whether ova are undergoing fertilization in vitro, since the absence of the flagellum can be interpreted as failure to detect it.
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In the present work, transverse sectioning of the flagellum resulted in a structure smaller in size than the mitochondria of maternal origin (Fig. 19); and, even after longitudinal sectioning, the flagellum might easily be passed over as a staining artifact (Fig. 10) in light microscopic efforts to find it. Nevertheless, successful light microscopic identification of the sperm flagellum has been reported by previous investigators54-56 after appropriate fixation followed by staining with lacmoid stain 54 and examination by phase-contrast microscopy. However, in this method, the exact location of sperm was difficult to determine. 28 · 54 As the sperm flagellum in the cytoplasm changed morphologically, it seems logical that the mitochondria disintegrated first, followed by the axial filament complex. The sperm mitochondria were swollen 1lh hours after sperm penetration (Fig. 12), and degeneration was apparent 7lh hours after sperm penetration in the pronuclear stage (Fig. 21). The precise time for disintegration of the sperm flagellum in the cytoplasm is unknown. There may be species differences regarding complete sperm mitochondria degeneration, since it reportedly occurs early in the mouse 9 and in the latter part of cleavage in the rabbit, 24 rat, 57 · 58 human, 44 · 59 and pig. 12 The function of sperm mitochondria in the oocyte is controversial. Longo and Anderson4 suggested that they remain functional throughout embryogenesis, but others maintain that they do not contribute embryonic mitochondria to the ovum. 12 Supplementary Sperm in the Activated Ovum. The number of supplementary sperm in the rabbit in vivo-fertilized egg was usually 6 to 10. 60 In this work, fewer supplementary sperm were found. Supplementary sperm are normally found in the rabbit egg (Figs. 9 and 22) and are not considered harmful to the development of the embryo in the rat61 or mouse. 62 The fate of supplementary sperm within
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the rabbit ovum is unknown; most likely, some of them undergo degenerative change in the later stage (Fig. 9) in the perivitelline space, and most would be phagocytosed by the embryo blastomere. 62 Bedford60 also suggested that the number of supplementary sperm may be approximately proportional to the number of available spermatozoa in the vicinity of the egg at the time of in vivo fertilization. Indeed, Braden and Austin63 found a positive relationship in association with the number of sperm within the oviduct and number of supplementary sperm in the rat. The number of sperm present with ova in culture dishes in this work did not affect the number of supplementary sperm within the ova. Thibault 17 found that the average number of supplementary sperm in vitro was less than one, whereas the average number was eight at the same stage of in vivo fertilization. The stage of maturation of the ovum and culture medium components might influence sperm entry into the perivitelline space. At any rate, the mechanism between in vivo and in vitro regulation of sperm entry seems to be different. In this work, ultrastructural evidence is provided for the documentation of fertilization in vitro of rabbit ova recovered from ovarian follicles just prior to the anticipated time of ovulation, and the observations made at the electron microscope level compare very well with previously reported observations involving in vivo-fertilized rabbit ova. In addition, it is apparent from this work that the ultrastructural observations of stages of fertilization agree closely with the light microscopic observation of ovum cleavage, with regard to the proportions of ova undergoing fertilization or having been fertilized in vitro. This is not surprising, since rabbit ova handled under in vitro conditions in these experiments have never been observed to undergo spontaneous cleavage in the absence of spermatozoa, and also since similar propor-
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tions of in vitro-fertilized and cultured rabbit embryos and in vivo-fertilized embryos that have been cultured in vitro develop through gestation following transfer to recipient does. These observations made on rabbit ova undergoing fertilization in vitro provide a base for further studies to examine morphologic alterations imposed on the process of gamete union by various experimental manipulations. SUMMARY
Rabbit ova were obtained from presumably preovulatory ovarian follicles of gonadotropin-treated does and inseminated with uterine sperm in vitro. Ova were serially sectioned for light and electron microscopy 0, llh, 3, 6, and 9 hours after in vitro insemination. Control ova were maintained in culture for 25 hours and examined by light microscopy for cleavage. Little difference in fertilization rate (i.e., ova fertilized/ova inseminated) was observed when criteria were based on ultrastructural evidence (56.5%) and on light microscopic evidence 25 hours postinsemination provided by cleavage of control ova (54.8% ). Therefore, observations of cleavage (as in controls here) can be an accurate criterion for fertilization in the rabbit, at least under the in vitro conditions used in this study. After insemination of ova recovered from preovulatory follicles, the developmental sequence was as follows: after llh hours, sperm penetration into the vitellus; after 3 hours, male pronuclear formation and second polar body extrusion; after 6 hours, male and female pronuclear enlargement; and after 9 hours, pronuclei in apposition. This sequence compares favorably with the normal temporal sequence of events already well known for fertilization of ovulated ova in the rabbit. Penetrating sperm cells within the matrix of the zona pellucida and the supplementary sperm cells lying in the perivitelline space had undergone the acrosome reaction. Changes associated with the aero-
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some reaction took place before the penetration of the zona pellucida. The presence of a number of supplementary sperm, usually five to six per ovum, within the perivitelline space of the already activated ovum indicated that the block to polyspermy in these experiments operated mainly at the level of the vitelline membrane, as is the case in the normal fertilization process. The vitelline membrane block to polyspermy occurred rapidly after penetration of the fertilizing sperm into the vitellus and was associated with cortical granule breakdown. Ultrastructural details of rabbit ova recovered from ovarian follicles and inseminated in vitro revealed no distinguishable characteristics when compared with reported observations of ovulated rabbit ova undergoing fertilization in vivo. Acknowledgments. The authors thank Dr. Richard
10.
11. 12.
13.
14. 15.
16. 17.
A. Stark for his critical reading of the manuscript
and acknowledge the expert technical assistance provided by Mrs. Joan Trammell and Mr. George G. Jeitles, Jr. REFERENCES 1. Bedford JM: An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213, 1972 2. Zamboni L, Mastroianni L Jr: Electron microscopic studies on rabbit ova. I. The follicular oocyte. J Ultrastruct Res 14:95, 1966 3. Zamboni L, Mastroianni L Jr: Electron microscopic studies on rabbit ova. II. The penetrated tubal ovum. J Ultrastruct Res 14:118, 1966 4. Longo FJ, Anderson E: Cytological events leading to the formation of the two-cell stage in the rabbit: aBBOciation of the maternally and paternally derived genomes. J Ultrastruct Res 29:86, 1969 5. Gulyas BJ: The rabbit zygote: formation of annulate lamellae. J Ultrastruct Res 35:112, 1971 6. Piko L, Tyler A: Fine structural studies of sperm penetration in the rat. In Procedings of the Fifth International Congress on Animal Reproduction, Trento, Italy, Vol 2, 1964, p 372 7. Szollosi DG: Extrusion of nucleoli from pronuclei of the rat. J Cell Bioi 25:545, 1965 8. Szollosi DG, Ris H: Observations on sperm penetration in the rat. J Biophys Biochem Cytol10:275, 1961 9. Stefanini M, Oura C, Zamboni L Jr: Ultrastructure of fertilization in the mouse. 2.
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23.
24.
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Penetration of sperm into the ovum. J Submicrosc Cytol 1:1 1969 Zamboni L: Fertilization in the mouse. In Biology of Mammalian Fertilization and Implantation, Edited by KS Moghissi and ESE Hafez. Springfield, Ill, Charles C Thomas, 1972, p 213 Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head during fertilization. J Ultrastruct Res 31:465, 1970 Szollosi DG, Hunter RHF: Ultrastructural aspects of fertilization in the domestic pig: sperm penetration and pronucleus formation. J Anat 116:181, 1973 Fraser LR, Dandekar PV, Gordon MK: Loss of cortical granules in rabbit eggs exposed to spermatozoa in vitro. J Reprod Fertil 29:295, 1972 Brackett BG, Mills JA, Jeitles GG Jr: In vitro fertilization of rabbit ova recovered from ovarian follicles. Fertil Steril 23:898, 1972 Mills JA, Jeitles GG Jr, Brackett BG: Embryo transfer following in vitro and in vivo fertilization of rabbit ova. Fertil Steril 24:602, 1973 Brackett BG: In vitro fertilization of mammalian ova. Adv Biosci 4:73, 1969 Thibault C: Analyse comparee de Ia iecondation et de ses anomalies chez la brebis, la vache et Ia lapine. Ann Bioi Anim Biochem Biophys 7:5, 1967 Thibault C: In vitro fertilization of the mammalian egg. In Fertilization: Comparative Morphology, Biochemistry and Immunology, Vol 2, Edited by CB Metz and A Monroy. New York, Academic Press, 1969, p 405 Brackett BG: In vitro fertilization of rabbit ova: time sequence of events. Fertil Steril 21:169, 1970 Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329, 1968 Barros C, Bedford JM, Franklin LE, Austin CR: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell Bioi 34:C1, 1967 Austin CR: Acrosome loss from the rabbit spermatozoa in relation to entry into the egg. J Reprod Fertil 6:313, 1963 Bedford JM: Experimental requirement for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J Reprod Fertil [Suppl] 2:35, 1967 Bedford JM: Sperm capacitation and fertilization in mammals. Bioi Reprod [Suppl] 2:128, 1970 Hadek R: Submicroscopic changes in penetrating spermatozoa of the rabbit. J Ultrastruct Res 8:161, 1963 Yanagimachi R, Noda YD: Electron microscope studies on sperm incorporation into the golden hamster egg. Am J Anat 128:429, 1970
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27. Thompson RS, Smith DM, Zamboni L: Fertilization of mouse ova in vitro: an electron microscopic study. Fertil Steril 25:222, 1974 28. Soupart P, Strong PA: Ultrastructural observations on human oocytes fertilized in vitro. Fertil Steril 25:11, 1974 29. Austin CR, Bishop MWH: Fertilization in mammals. Biol Rev 32:297, 1957 30. Harper MJK: Factors influencing sperm penetration of rabbit eggs in vivo. J Exp Zool 47:144, 1970 31. Bedford JM: Techniques and criteria used in the study of fertilization. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman, 1971, p 37 32. Odor LD, Blandau RJ: Observation on fertilization and the first segmentation division in rat ova. Am J Anat 89:29, 1951 33. Gondos B, Bhiraleus P, Conner A: Pronuclear membrane alterations during approximation of pronuclei and initiation of cleavage in the rabbit. J Cell Sci 10:61, 1972 34. Gondos B, Bhiraleus P: Pronuclear relationship and association of maternal and paternal chromosomes in flushed rabbit ova. Z Zellforsch Mikrosk Anat 3:149, 1970 35. Austin CR: Cortical granules in hamster eggs. Exp Cell Res 10:533, 1956 36. Austin CR: The Mammalian Egg. Oxford, Blackwell Scientific Publishers, 1961 37. Austin CR: Ultrastructure of Fertilization. New York, Holt, Rinehart and Winston, 1968, p 51 38. Szollosi DG: Development of cortical granules and the cortical reaction in the rat and hamster egg. Anat Rec 159:431, 1967 39. Hadek R: Submicroscopic study on the cortical granules in rabbit ova. J Ultrastruct Res 8:170, 1963 40. Weakly BS: Electron microscopy of the oocytes and granulosa cells in the developing ovarian follicles of the golden hamster (Mesocricetus auratus). J Anat 100:503, 1966 41. Baca M, Zamboni L: The fine structure of human follicular oocytes. J Ultrastruct Res 19:354, 1967 42. Zeigel RF, Dalton AJ: Speculation based on the morphology of the Golgi system in several types of protein-secreting cells. J Cell Bioi 15:45, 1962 43. Yanagimachi R, Chang MC: Fertilizable life of golden hamster ova and their morphologic changes at time of losing fertilizability. J Exp Zool 148:185, 1961 44. Zamboni L, Mishell DR, Bell TH, Baca M: Fine structure of the human ovum in the pronuclear stage. J Cell Bioi 30:579, 1966 45. Hertig AT, Adams EC: Studies on the human oocyte and its follicle. I. Ultrastructural and
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histochemical observations on the primordial follicle stage. J Cell Bioi 34:647, 1967 46. Fleming WN, Saacke PG: Fine structure of the bovine oocyte from the mature Graafian follicle. J Reprod Fertil 29:203, 1972 47. Barton BR, Hertig AT: Ultrastructure of annulate lamellae in primary oocytes of chimpanzees (/'an troglodytes). Bioi Reprod 6:98, 1972 48. Gulyas BJ: The formation of annulate lamellae in the rabbit eggs. Anat Rec 166:311, 1970 49. Bernhard W: Ultrastructural aspects of nucleocytoplasmic relationship. Exp Cell Res [Suppl] 6:17, 1958 50. Hertig AT: The primary human oocyte: some observations on fine structure of Balbiani's vitelline body and the origin of the annulate lamellae. Am J Anat 122:107, 1968 51. Kessel RG: Electron microscope studies on the origin of annulate lamellae in oocytes of necturus. J Cell Biol 19:391, 1963 52. Kessel RG: Annulate lamellae. J Ultrastruct Res 25 (SuppllO):l, 1968 53. Thibault C, Dauzier L: Analyse des conditions de la lecondation in vitro de l'oeuf de la lapine. Ann Bioi Anim Biochem Biophys 1:277, 1961 54. Dickmann Z, Clewe TH, Bonney WA Jr, Noyes RW: The human egg in the pronuclear stage. Anat Rec 152:293, 1965 55. Noyes RW, Dickman Z, Clewe TH, Bonney WA: Pronuclear ovum from a patient using an intrauterine contraceptive device. Science 147:744, 1965 56. Bavister BD, Edwards RG, Steptoe PC: ldentffication of the mid-piece and tail of spermatozoon during fertilization of human eggs in vitro. J Reprod Fertil 20:159, 1969 57. Szollosi DG: The fate of sperm middle piece mitochondria in the rat egg. J Exp Zool159:367, 1965 58. Tachi S, Kraicer PF: Studies on the mechanism of nidation. XXVIT. Sperm-derived inclusions in the rat blastocyst. J Reprod Fertil 14:401, 1967 59. Soupart P, Morgenstern LL: Human sperm capacitation and in vitro fertilization. Fertil Steril 24:462, 1973 60. Bedford JM: Morphological reaction of spermatozoa in the female reproductive tract of the rabbit. J Reprod Fertil 6:245, 1963 61. Odor DL, Blandau RJ: The frequency of occurrence of supernumerary sperm in rat ova. Anat Rec 104:1, 1949 62. Thompson RS, Zamboni L: Phagocytosis of supernumerary spermatozoa by two-cell mouse embryos. Anat Rec 178:3, 1974 63. Braden AWH, Austin CR: The number of sperm about the eggs in mammals and its significance for normal fertilization. Aust J Bioi Sci 7:543, 1954
FERTILITY AND STERILITY Copyright~ 1975 The American Fertility Society
ULTRASTRUCTURE OF RABBIT OVA RECOVERED FROM OVARIAN FOLLICLES AND INSEMINATED IN VITRO* YON K. OH, D.V.M. M.S.,
AND
BENJAMIN G. BRACKE'IT, D.V.M., PH.D.t
Section of Clinical Reproduction, Department of Clinical Studies, Sclwol of Veterinary Medicine, and Division of Reproductive Biology, Department of Obstetrics and Gynecology, University of Pennsylvania Sclwol of Medicine, Philadelphia, Pennsylvania 19174
The ultrastructural morphology of rabbit follicular oocytes fertilized in vitro has not previously been chronologically documented. Most of the ultrastructural studies of fertilization described in mammalian species have been done under in vivo conditions, e.g., in the rabbit, 1 - 5 rat, 6 - 8 mouse, 9 • 10 hamster, 11 and pig. 12 Little information has been available on sperm interactions with egg investments and pronuclear formation under in vitro conditions, especially in the follicular oocytes of the rabbit. 13 The following study describes electron microscopic observations of sperm penetration of the egg a11d pronuclear formation and compares the ultrastructural criteria of fertilization with those for control ova, which were examined conventionally with light microscopy after cleavage to the two- or four-cell stages. Control ova were incubated in culture for 25 hours after insemination. · MATERIALS AND METHODS
Ova were obtained from preovulatory ovarian follicles of gonadotropin-treated New Zealand White does. Ovum donors received intramuscular injections of 150 IU of pregnant mare's serum, followed Received September 11, 1974. *Supported by National Institutes of Health Grant HDO 6274, Career Development Award HD 15861, and by a grant from The Ford Foundation. tReprint requests: Dr. B. G. Brackett, Section of Clinical Reproduction, New Bolton Center, R.D. #1, Kennett Square, Pa. 19348.
83 hours later by intravenous injection of 75 IU of human chorionic gonadotropin. Ten hours after treatment with human chorionic gonadotropin, ova were recovered from unruptured ovarian follicles, pooled, and then transferred into a suspension of spermatozoa recovered from the uterine horns of does which had been mated 18 hours previously. The procedure for in vitro fertilization was the same as that reported previously.14·15 The medium in these exeriments was the simple defined medium16 supplemented with 10-5 M pyruvate.15 The incubation atmosphere was moiRt 5% C02, 8% 02. and balance N2. Ova were removed from culture 0, 1% 3, 6, and 9 hours after exposure to the capacitated sperm. The ova were fixed in 2.0% glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1% Os04, dehydrated, individually embedded in Epon 812, and serially sectioned for light and electron microscopy. For light microscopy, sections were stained with toluidine blue. Thin sections were stained with uranyl acetate and lead citrate and examined with a Hitachi llB electron microscope. Ova from the same donors that had been inseminated by sperm from the same capacitators were examined by light microscopy approximately 25 hours after insemination in vitro. The proportion of ova having undergone normal cleavage, i.e., cleaved ova with symmetrical blastomeres resembling those recovered from oviducts after in vivo fertilization, was
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FIG. 1. Portion of a secondary oocyte and the first polar body IRB 1). The first polar body contains chromosomes (CHr), mitochondria (M), cortical granules (CG), and endoplasmic reticulum (ER). In the cortical ooplasm (Oop), the metaphase II chromosomes (Chr) can be seen arranged in the equatorial plane. Note the absence of organelles in the area of the chromosomes (x8,000).
•
FIG. 2. Portion of a follicular oocyte showing the zona pellucida (ZP) and the cortical granules. Relatively evenly spaced cortical granules (CG) can be seen immediately beneath the vitelline membrane. Note also the mitochondria (M) and the vesicular structures (V) (X 1,200).
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compared with the proportion of ova exhibiting signs of undergoing fertilization, as determined by electron microscopy. In this work, an ovum was considered to be undergoing fertilization if sperm penetration had occurred and if characteristic morphologic changes, already well recognized in the fertilization process in vivo for several species, could be observed. Fertilization rates, i.e., proportions of inseminated ova showing signs of fertilization according to criteria observable by light and electron microscopy, were compared; data were statistically treated by x2 analysis. RESULTS
The features of rabbit oocytes recovered just prior to the expected time of ovulation are shown in Figures 1 and 2. The presence of a first polar body (Fig. 1) was previously demonstrated in approximately 72.9% of the oocytes. 14 The first polar body found within the perivitelline space was usually iocated within a shallow depression in the surface of the oocyte (Fig. 1). Chromosomes were present in the first polar body and in the subcortical area of the ooplasm, as a result of the first meiotic division. The first polar body contained cytoplasmic organelles, mitochondria, endoplasmic reticulum, and cortical granules (Fig. 1). Many spherically shaped, uniformly sized cortical granules were arranged at more or less regular intervals in the area immediately beneath the vitelline membrane (Fig. 2). In some areas near the cortex, they were also found in clusters (Fig. 3). Spheroidal mitochondria with peripheral crystae, vesicles, and Golgi complexes, evenly distributed around the ooplasm, were characteristic features of unfertilized ova at this early stage. Observations Made 1¥2 Hours after Insemination. Five of eight eggs that were serially sectioned following removal from the culture 1¥2 hours after insemination
FIG. 3. Portion of a secondary oocyte (Ooc) showing cortical granules (CG) clustered in the subcortical ooplasm and lipid inclusions (LP). Elements of endoplasmic reticulum CER) can be seen in the first polar body (PB 1) (x20,000).
in vitro were observed to be undergoing fertilization. At this time, sperm had already penetrated into the ooplasm; in three of these ova, the sperm nucleus was seen to be undergoing decondensation (Figs. 4 and 5). In two of the eggs, the sperm nucleus was located close to the maternal chromosomes (Fig. 5). Decondensation of the sperm nucleus occurred following loss of the nuclear membrane and presented a diffused and faintly staining appearance. Microtubules sur-
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FIG. 4. Ovum 1'h hours after insemination. A swollen sperm nucleus (SN), delineated by arrowheads, and sperm midpiece (SM) are evident within the ooplasm. The egg cortex is completely devoid of cortical granules. Also seen are ovum mitochondria (M) and the zona pellucida fZP) (14,000).
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FIG. 5. Sperm nucleus (SN) located near the maternal chromosomes (mChr) llh hours after insemination. Arrowheads delineate the limits of the sperm nucleus. Microtubules (MT) are seen in cross-section surrounding the sperm nucleus and in oblique section adjacent to the maternal chromosomes (x 10,000).
rounded the regions of sperm nuclear decondensation and the maternal chromosomes (Fig. 5). The sperm midpiece, mitochondria, and coarse fibers remained unchanged (Fig. 4). Ooplasmic organelles, mitochondria, vesicles, and endoplasmic reticulum surrounded the area of sperm decondensation (Figs. 4 and 5). Cortical granules were completely absent from the peripheral area of the ooplasm, where sperm penetration presumably had occurred (Fig. 4). The characteristic changes in sperm cells in the vicinity of the egg which comprised the acrosome reaction were seen in this study. These changes involved swelling of the plasma membrane, vesicu-
lation of the plasma membrane with the outer acrosomal membrane, and progression of these changes to apparent loss of the plasma and outer acrosomal membrane, exposing the inner acrosomal membrane (Fig. 6). These changes occurred within the interstices of corona cells and were completed by the time the sperm cell arrived at the surface of the zona pellucida (Fig. 7). Most of the sperm cells observed to be penetrating the zona pellucida were undoubtedly supplementary sperm, since cortical granules were absent from the periphery of the egg cytoplasm, indicating that activation was already under way. A penetration slit in the zona pellucida was observed pos-
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FIG. 6. Sperm undergoing the acrosome reaction in the interstices between corona cells (CC) 1 ¥z hours after insemination. In sperm 1 (f3 1 ), the plasma membrane !PM) is swollen. In sperm 2 (S 2), the plasma membrane and outer acrosomal membrane are undergoing vesiculation (V). In sperm 3 (S 3), the acrosome reaction has been completed, exposing the inner acrosomal membrane (JAM) (x32,000).
terior to the penetrating sperm head which provided an impression that the sperm cell was crossing tangentially toward the ooplasmic membrane (Fig. 8). Supplementary sperm found lying within the perivitelline space presented morphologic features similar to those within the matrix of the zona pellucida and those at the surface of the zona that had already completed the acrosome reaction (Fig. 9). Observations Made 3 Hours after Insemination. Five of six eggs serially sectioned following 3 hours in culture after in vitro insemination provided evidence that fertilization was under way.
Two of these eggs each had a developing male pronucleus, and the other three eggs were at the decondensation stage of the sperm nucleus. The presence of a sperm flagellum in close proximity to a pronucleus strongly suggested that this was the male pronucleus (Fig. 10). The long remnant of the sperm flagellum seen in the cortex of the ovum in Figure 10 presumably indicated the site of penetration. The second polar body was released at the completion of the second meiotic division. The early pronucleus possessed a double nuclear membrane with widely dispersed nuclear pores and faint, elec-
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FIG. 7. Portion of oocyte (Ooc), zona pellucida (ZP), and corona cells (CC) llh hours after insemination. The sperm resting on the zona pellucida has completed the acrosome reaction (xlO,OOO).
tron-opaque material aggregated randomly within the pronuclear boundries (Fig. 11). The sperm flagellum within the ooplasm exhibited mitochondria ~hich appeared somewhat swollen and possessed peripheral cristae (Fig. 12). Observations Made 6 Hours after Insemination. Nine of fifteen eggs serially
671
sectioned following 6 hours in culture after in vitro insemination were found to be undergoing fertilization. Five of the eggs were at the pronuclear stage, while two eggs possessed only a male pronucleus; the remaining two eggs were at the decondensation stage of the sperm nucleus. At the early pronuclear stage observed at this time, the cytoplasmic region adjacent to the pronuclei was morphologically distinct from the cortex in that it contained more mitochondria, annulate lamellae, and vesicles which were closely associated with the Golgi complex (Fig. 13). Both pronuclear membranes had surfaces that appeared relatively smooth and had randomly dispersed nuclear pores. The male pronucleus was generally larger than the female pronucleus and frequently had a remnant of sperm flagellum associated with it. Observations Made 9 Hours after Insemination. Seven of seventeen eggs serially sectioned and examined following 9 hours in culture after in vitro insemination were in the pronuclear stage. By this time, the pronuclei had developed and migrated toward the center of the ovum (Fig. 14). When in juxtaposition, the pronuclear membrane projections approached each other through the intervening cytoplasm. Invagination of pronuclei extended toward the center of each pronucleus (Fig. 15). Sometimes neither pronuclear membrane could be discerned at a point of apposition, strongly suggesting an exchange of nuclear material from paternal and maternal origin at this stage (Fig. 16). Frequently, nuclear blebbing occurred in the pronuclear envelope, and polyribosomes were scattered around the region (Fig. 1 7). Annulate lamellae also appeared to be more abundant in this region than was the case 6 hours after insemination (Fig. 19). When oocytes were not activated by sperm, the number of cortical granules lining the periphery of the ooplasm was increased over that of the
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FIG. 8. Penetration of zona pellucida
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FIG. 9. Supernumerary sperm in the perivitelline space fPVS) of an ovum llh hours after insemination. The inner acrosomal membrane (lAM) and the equatorial segment CES) of the sperm are clearly evident (x29,000).
eggs recovered from the ovarian follicles and examined immediately (Figs. 18 and 2, respectively). The distance between the sperm remnant and the pronuclear envelope seemed to depend on the plane of sectioning of the oocyte. When the region proximal to the sperm neck was sectioned, the sperm remnant was found closer to the pronucleus (Fig. 19); when the distal portion of the sperm tail was sectioned, the remnant was farther from the pronucleus (Fig. 20). In some oocytes, regressive changes were seen in sperm mitochondria and in the axial filament complex of sperm remnants 9 hours after insemination (Fig. 21). Number of Sperm within the Matrix of the Zona Pellucida and in the Perivitelline Space. There was no obvious correlation between time interval following insemination and the number of sperm cells that had penetrated into and through the zona pellucida but had not taken part in fertilization. Also, no correlation was obvious between sperm concentration
and numbers of penetrating and supplementary sperm cells. In this work, one to nine sperm were found within the matrix of the zona pellucida and in the perivitelline spaces of individual eggs examined at 1 'h to 9 hours after in vitro insemination. Occasionally, several sperm cells were found assembled in a relatively small region of the perivitelline space (Fig. 22). Comparison of Fertilization Rates According to Ultrastructural Observations of Ova Undergoing Fertilization with Those Based on Observation of Cleaved Ova. Table 1 summarizes data from the above ultrastructural observations along with the corresponding cleavage rates observed in control ova cultured 25 hours in each experiment. From these data, 56.5% of 46 ova examined ultrastructurally were found to be undergoing fertilization, compared with 54.8% of 93 ova that cleaved normally in the control group. These proportions are very close, and are not statistically significantly different. Of the 51 control ova that
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FIG. 10. Early male pronuclear (mPN) formation 3 hours after insemination. The sperm midpiece (SM) is located near the male pronucleus. Note also the sperm tail (ST) in the ooplasm (Oop) and the second polar body
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FIG. 11. The pronuclear membrane 3 hours after insemination. The double membrane is seen with numerous well developed nuclear pores WP) separating the pronucleus (}'N) and ooplasm (Oop) (X 50,000).
cleaved, 45 had reached the four-cell stage, and only 6 remained in the two-cell stage when observed 25 hours after insemination in vitro. These data indicate that ultrastructural evidence of the fertilization process and light microscopic observation of proportions of normally cleaved ova following this in vitro insemination procedure provide two means for assessing the ability of rabbit gametes to undergo fertilization. DISCUSSION
Sperm Penetration and Pronuclear Formation. According to Bedford, 1 in vivo sperm penetration into the vitellus occurs 111h hours postcoitus, which is comparable to the 11h hours after insemination in vitro in the present work. Fraser et al. 13 reported the absence of cortical granules
675
FIG. 12. Sperm midpiece in an activated ovum 3 hours after insemination. The sperm mitochondria ISM) with peripheral cristae appear swollen. The sperm fibers (SF) remain well organized (x30,000).
in ovum cytoplasm of tubal ova 1 hour after in vitro insemination; however, no sperm remnants were demonstrated in the ovum cytoplasm. Our findings 1¥2 hours postinsemination demonstrate that sperm had already penetrated the oocyte. Sperm were found at the nuclear decondensation stage, and the cortical granule reaction had already taken place (Fig. 4). ThibauW7· 18 reported similar results in light microscopic studies in the rabbit. In earlier work from our laboratory using ovulated rabbit ova recovered from oviducts, motile sperm were seen within the perivitelline space as early as 2 to 4% hours after in vitro insemination. 19 Variability in conditions, methods of observation, and even the gametes themselves contribute to reported differences in the sequence of events in the fertilization process in vitro.
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FIG. 13. Pronuclei in apposition 6 hours after insemination. The nuclear membrane WM) has reformed about both pronuclei (}'N), and nuclear pores WP) are numerous. The cytoplasm surrounding the pronuclei is rich in organelles such as Golgi complex (GC), mitochondria (M), annulate lamellae (AL), and vesicular structures (V) (x 10,000).
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FIG. 14. Pronuclei 9 hours after insemination. The pronuclei (J'N) are now very close, separated only by a narrow intermembranous space (x7,000).
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FIG. 15. Pronuclei (J'N) in close apposition showing several infoldings of the pronuclear envelopes. Elements of the Golgi complex (GC) are seen between the pronuclei, and intranuclear annulate lamellae (JAL) are evident in the lower pronucleus (x 12,000).
Sperm were found to be undergoing the plasma membrane vesiculation process, i.e., the acrosome reaction (Figs. 6 and 7), before penetration of the zona pellucida. 1 • 20 • 21 When the sperm were found penetrating the zona pellucida, the plasma and outer ascrosomal membranes had already disappeared. A well defined zona penetration slit was left behind (Fig. 8). The present observations reconfirmed the morphologic changes in sperm, including the acrosome reaction and pene-
tration through the zona and into the vitellus, reported to occur in vivo in the rabbit, L 20 • 22 - 25 pig, 12 rat, 6 • 8 hamster, n and mouse. 9 Similar observations have also been reported following in vitro studies in the hamster, 26 mouse, 27 and human. 28 As early as 3 hours after in vitro insemination, a small male pronucleus could be seen in a very early stage of formation (Fig. 10). This followed second polar body extrusion. In careful serial
FIG. 16. Internuclear communication in the late stage of pronuclei. Intranuclear annulate lamellae are evident within both pronuclei (J'N) and in the area of pronuclear fusion (arrows) (x 8,000).
(JAL)
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FIG. 17. Nuclear blebbing in the pronuclear envelope. Two membrane-bound structures WB) appear
to be forming between the inner and outer leaflets of the pronuclear (}'N} envelope. Several polyribosomes (}'Rb) can be seen in the cytoplasm. NP, Nuclear pore (x42,000).
sectioning of these oocytes, another pronucleus could not be found at this time, which leads to the conclusion that the male pronucleus forms before the female pronucleus. The time required after
sperm penetration for pronucleus formation in the rabbit egg was similar to that found in studies of in vivo fertilizationt. 20 • 29 • 30 or in other studies after in vitro inseminationY· 18 Female pronucleus formation apparently took place between 1% and 4¥2 hours after the sperm cell had penetrated the ooplasm. Under light microscopic examination, pronuclei must be larger to be seen; therefore, some previous studies have reported that pronuclear formation occurs later. 31
FIG. 18. Unfertilized egg 9 hours after insemination. The number of cortical granules (CG) has increased and mitochondria (M} remain in the egg cortex. Microvilli are seen projecting into the zona pellucida ~P) (x 24,000).
When the pronuclei are fully developed, it is difficult to distinguish the male pronucleus from the female pronucleus. As a general rule, the male pronucleus reportedly is larger, 17 although it may be nearly the same size. 4 It is larger in the rat. 7 • 32 The male pronucleus has usually been identified by the presence of nearby sperm remnants (Figs. 10, 19, and 20). As they develop, the male and female pronuclei move to the central part
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FIG. 19. Fertilized ovum 9 hours after insemination. A portion of the sperm tail (ST) and several stacks of annulate lamellae (AL) are seen close to one of the pronuclei (J>N). M, Mitochondrion (x 38,000).
of the oocyte and become surrounded by cytoplasmic organelles, including mitochondria, Golgi components, and annulate lamellae (Fig. 13). Similar morphologic changes have been described in vivo. 4 • 33 Characteristic of rabbit ova are the multiple, irregular infoldings of the pronuclear envelopes that form especially at sites where pronuclei come into apposition (Fig. 15). Some communication occurs between pronuclei through areas of direct union (Fig. 16), as was previously reported
Fro. 20. Sperm flagellum (SF) present distant to the pronucleus. The egg was fixed 9 hours after insemination. PN, Pronucleus (X 10,000).
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FIG. 22. A 1-11-m section of ovum (0), zona pellucida (ZP), and corona cells (CC) 3 hours after insemination. Supernumerary sperm (SS) are evident in the perivitelline space (x640).
FIG. 21. Portion of a fertilized ovum 9 hours after insemination. The sperm flagellum located close to one of the pronuclei (}'N) exhibits regressive changes in both the sperm mitochondria (SM) and sperm fibers (SF) (x25,000).
during in vivo fertilization. 33 Subsequently, there is complete breakdown of the pronuclear envelopes. 4 An exchange of nuclear materials from male and female pronuclei takes place just before cleavage to the two-cell stage. 34 Cortical Granules. The cortical granule reaction has drawn attention for its role as a manifestation of ovum activation early in fertilization and also for its role in the block to polyspermy. 35-38 The cortical granules are round, moderately homogeneous, and electron-dense organelles (0.08 to 0.2 p.m in diameter). It has been postulated that they are synthesized in the Golgi complex2 • 3 • 3s. 40, 4t and later migrate into the cortex of the oocyte, localizing just beneath the vi-
telline membrane (Fig. 2). 42 In the present study, most cortical granules were absent when sperm remnants of male pronuclei were present (Figs. 4 and 10). The precise time of cortical granule breakdown after sperm penetration is not known in the rabbit, but SzollosP8 found in the rat and hamster that the cortical granules are activated by the initial attachment of sperm to the vitelline membrane. In the unfertilized rabbit egg, the number of cortical granules at the periphery of the ooplasm increases during aging in culture media (Fig. 18), as was previously described by Hadek. 39 This is TABLE 1. Comparison of Fertilization Rates Using Different Criteria Light microscopic observation
Ultrastructural evidence Hours postinsemination
Ova undergoing fertilization/ ova inseminated
5/8 1Yl 5/6 3 9/15 6 7/17 9 Summary 27/46 (56.5%)
25 hr postinsemination Ova cleaved/ ova inseminated
9/15 9/15 12/24 21/39 51193 (54.8%)
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in contrast to unfertilized mouse tubal ova9 and hamster ova, in which the number of cortical granules apparently decreases when they are aged, having a close relationship to the fertilizability of the ova. 43 Annulate Lamellae. Annulate lamellae appear within 4lh hours after sperm penetration (Fig. 13) and become abundant at the late stage of pronuclear formation (Fig. 19). The presence of annulate lamellae has been reported in ova undergoing fertilization in vivo in the rabbit, 2· 3· 5 the pig, 12 and human44 and in human in vitro-fertilized ova. 28 Annulate lamellae were also described in primordial and follicular oocytes of the human, 4L 45 bovine, 46 and chimpanzeeY Gulyas48 observed no annulate lamellae in unfertilized rabbit ova soon after ovulation; but 5 to 6 hours later, they appeared. This is in agreement with the present observations of formation of annulate lamellae within 6 hours after in vitro insemination of ova recovered from mature follicles that are undergoing fertilization (Fig. 13). It has been proposed that annulate lamellae develop from stacks of endoplasmic reticulum 41 or from the nuclear envelopeY· 49 -51 Annulate lamellae are formed in late stages in both fertilized and unfertilized rabbit ova and may, therefore, be related to the aging of the oocyte or to the developmental stage of the egg. However, their function is unknown. 52 Sperm Flagellum in the Ovum Cytoplasm. The presence of a sperm flagellum in the cytoplasm of an oocyte has been used as one criterion of fertilization. Thibault and Dauzier53 have shown the sperm by hematoxylin staining, but this was at an early stage of sperm penetration. Light microscopic detection of the sperm flagellum is probably not a reliable criterion of routine determination of whether ova are undergoing fertilization in vitro, since the absence of the flagellum can be interpreted as failure to detect it.
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In the present work, transverse sectioning of the flagellum resulted in a structure smaller in size than the mitochondria of maternal origin (Fig. 19); and, even after longitudinal sectioning, the flagellum might easily be passed over as a staining artifact (Fig. 10) in light microscopic efforts to find it. Nevertheless, successful light microscopic identification of the sperm flagellum has been reported by previous investigators54-56 after appropriate fixation followed by staining with lacmoid stain 54 and examination by phase-contrast microscopy. However, in this method, the exact location of sperm was difficult to determine. 28 · 54 As the sperm flagellum in the cytoplasm changed morphologically, it seems logical that the mitochondria disintegrated first, followed by the axial filament complex. The sperm mitochondria were swollen 1lh hours after sperm penetration (Fig. 12), and degeneration was apparent 7lh hours after sperm penetration in the pronuclear stage (Fig. 21). The precise time for disintegration of the sperm flagellum in the cytoplasm is unknown. There may be species differences regarding complete sperm mitochondria degeneration, since it reportedly occurs early in the mouse 9 and in the latter part of cleavage in the rabbit, 24 rat, 57 · 58 human, 44 · 59 and pig. 12 The function of sperm mitochondria in the oocyte is controversial. Longo and Anderson4 suggested that they remain functional throughout embryogenesis, but others maintain that they do not contribute embryonic mitochondria to the ovum. 12 Supplementary Sperm in the Activated Ovum. The number of supplementary sperm in the rabbit in vivo-fertilized egg was usually 6 to 10. 60 In this work, fewer supplementary sperm were found. Supplementary sperm are normally found in the rabbit egg (Figs. 9 and 22) and are not considered harmful to the development of the embryo in the rat61 or mouse. 62 The fate of supplementary sperm within
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the rabbit ovum is unknown; most likely, some of them undergo degenerative change in the later stage (Fig. 9) in the perivitelline space, and most would be phagocytosed by the embryo blastomere. 62 Bedford60 also suggested that the number of supplementary sperm may be approximately proportional to the number of available spermatozoa in the vicinity of the egg at the time of in vivo fertilization. Indeed, Braden and Austin63 found a positive relationship in association with the number of sperm within the oviduct and number of supplementary sperm in the rat. The number of sperm present with ova in culture dishes in this work did not affect the number of supplementary sperm within the ova. Thibault 17 found that the average number of supplementary sperm in vitro was less than one, whereas the average number was eight at the same stage of in vivo fertilization. The stage of maturation of the ovum and culture medium components might influence sperm entry into the perivitelline space. At any rate, the mechanism between in vivo and in vitro regulation of sperm entry seems to be different. In this work, ultrastructural evidence is provided for the documentation of fertilization in vitro of rabbit ova recovered from ovarian follicles just prior to the anticipated time of ovulation, and the observations made at the electron microscope level compare very well with previously reported observations involving in vivo-fertilized rabbit ova. In addition, it is apparent from this work that the ultrastructural observations of stages of fertilization agree closely with the light microscopic observation of ovum cleavage, with regard to the proportions of ova undergoing fertilization or having been fertilized in vitro. This is not surprising, since rabbit ova handled under in vitro conditions in these experiments have never been observed to undergo spontaneous cleavage in the absence of spermatozoa, and also since similar propor-
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tions of in vitro-fertilized and cultured rabbit embryos and in vivo-fertilized embryos that have been cultured in vitro develop through gestation following transfer to recipient does. These observations made on rabbit ova undergoing fertilization in vitro provide a base for further studies to examine morphologic alterations imposed on the process of gamete union by various experimental manipulations. SUMMARY
Rabbit ova were obtained from presumably preovulatory ovarian follicles of gonadotropin-treated does and inseminated with uterine sperm in vitro. Ova were serially sectioned for light and electron microscopy 0, llh, 3, 6, and 9 hours after in vitro insemination. Control ova were maintained in culture for 25 hours and examined by light microscopy for cleavage. Little difference in fertilization rate (i.e., ova fertilized/ova inseminated) was observed when criteria were based on ultrastructural evidence (56.5%) and on light microscopic evidence 25 hours postinsemination provided by cleavage of control ova (54.8% ). Therefore, observations of cleavage (as in controls here) can be an accurate criterion for fertilization in the rabbit, at least under the in vitro conditions used in this study. After insemination of ova recovered from preovulatory follicles, the developmental sequence was as follows: after llh hours, sperm penetration into the vitellus; after 3 hours, male pronuclear formation and second polar body extrusion; after 6 hours, male and female pronuclear enlargement; and after 9 hours, pronuclei in apposition. This sequence compares favorably with the normal temporal sequence of events already well known for fertilization of ovulated ova in the rabbit. Penetrating sperm cells within the matrix of the zona pellucida and the supplementary sperm cells lying in the perivitelline space had undergone the acrosome reaction. Changes associated with the aero-
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OH AND BRACKE'IT
some reaction took place before the penetration of the zona pellucida. The presence of a number of supplementary sperm, usually five to six per ovum, within the perivitelline space of the already activated ovum indicated that the block to polyspermy in these experiments operated mainly at the level of the vitelline membrane, as is the case in the normal fertilization process. The vitelline membrane block to polyspermy occurred rapidly after penetration of the fertilizing sperm into the vitellus and was associated with cortical granule breakdown. Ultrastructural details of rabbit ova recovered from ovarian follicles and inseminated in vitro revealed no distinguishable characteristics when compared with reported observations of ovulated rabbit ova undergoing fertilization in vivo. Acknowledgments. The authors thank Dr. Richard
10.
11. 12.
13.
14. 15.
16. 17.
A. Stark for his critical reading of the manuscript
and acknowledge the expert technical assistance provided by Mrs. Joan Trammell and Mr. George G. Jeitles, Jr. REFERENCES 1. Bedford JM: An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213, 1972 2. Zamboni L, Mastroianni L Jr: Electron microscopic studies on rabbit ova. I. The follicular oocyte. J Ultrastruct Res 14:95, 1966 3. Zamboni L, Mastroianni L Jr: Electron microscopic studies on rabbit ova. II. The penetrated tubal ovum. J Ultrastruct Res 14:118, 1966 4. Longo FJ, Anderson E: Cytological events leading to the formation of the two-cell stage in the rabbit: aBBOciation of the maternally and paternally derived genomes. J Ultrastruct Res 29:86, 1969 5. Gulyas BJ: The rabbit zygote: formation of annulate lamellae. J Ultrastruct Res 35:112, 1971 6. Piko L, Tyler A: Fine structural studies of sperm penetration in the rat. In Procedings of the Fifth International Congress on Animal Reproduction, Trento, Italy, Vol 2, 1964, p 372 7. Szollosi DG: Extrusion of nucleoli from pronuclei of the rat. J Cell Bioi 25:545, 1965 8. Szollosi DG, Ris H: Observations on sperm penetration in the rat. J Biophys Biochem Cytol10:275, 1961 9. Stefanini M, Oura C, Zamboni L Jr: Ultrastructure of fertilization in the mouse. 2.
18.
19. 20.
21.
22.
23.
24.
25.
26.
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Penetration of sperm into the ovum. J Submicrosc Cytol 1:1 1969 Zamboni L: Fertilization in the mouse. In Biology of Mammalian Fertilization and Implantation, Edited by KS Moghissi and ESE Hafez. Springfield, Ill, Charles C Thomas, 1972, p 213 Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head during fertilization. J Ultrastruct Res 31:465, 1970 Szollosi DG, Hunter RHF: Ultrastructural aspects of fertilization in the domestic pig: sperm penetration and pronucleus formation. J Anat 116:181, 1973 Fraser LR, Dandekar PV, Gordon MK: Loss of cortical granules in rabbit eggs exposed to spermatozoa in vitro. J Reprod Fertil 29:295, 1972 Brackett BG, Mills JA, Jeitles GG Jr: In vitro fertilization of rabbit ova recovered from ovarian follicles. Fertil Steril 23:898, 1972 Mills JA, Jeitles GG Jr, Brackett BG: Embryo transfer following in vitro and in vivo fertilization of rabbit ova. Fertil Steril 24:602, 1973 Brackett BG: In vitro fertilization of mammalian ova. Adv Biosci 4:73, 1969 Thibault C: Analyse comparee de Ia iecondation et de ses anomalies chez la brebis, la vache et Ia lapine. Ann Bioi Anim Biochem Biophys 7:5, 1967 Thibault C: In vitro fertilization of the mammalian egg. In Fertilization: Comparative Morphology, Biochemistry and Immunology, Vol 2, Edited by CB Metz and A Monroy. New York, Academic Press, 1969, p 405 Brackett BG: In vitro fertilization of rabbit ova: time sequence of events. Fertil Steril 21:169, 1970 Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329, 1968 Barros C, Bedford JM, Franklin LE, Austin CR: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell Bioi 34:C1, 1967 Austin CR: Acrosome loss from the rabbit spermatozoa in relation to entry into the egg. J Reprod Fertil 6:313, 1963 Bedford JM: Experimental requirement for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J Reprod Fertil [Suppl] 2:35, 1967 Bedford JM: Sperm capacitation and fertilization in mammals. Bioi Reprod [Suppl] 2:128, 1970 Hadek R: Submicroscopic changes in penetrating spermatozoa of the rabbit. J Ultrastruct Res 8:161, 1963 Yanagimachi R, Noda YD: Electron microscope studies on sperm incorporation into the golden hamster egg. Am J Anat 128:429, 1970
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27. Thompson RS, Smith DM, Zamboni L: Fertilization of mouse ova in vitro: an electron microscopic study. Fertil Steril 25:222, 1974 28. Soupart P, Strong PA: Ultrastructural observations on human oocytes fertilized in vitro. Fertil Steril 25:11, 1974 29. Austin CR, Bishop MWH: Fertilization in mammals. Biol Rev 32:297, 1957 30. Harper MJK: Factors influencing sperm penetration of rabbit eggs in vivo. J Exp Zool 47:144, 1970 31. Bedford JM: Techniques and criteria used in the study of fertilization. In Methods in Mammalian Embryology, Edited by JC Daniel. San Francisco, Freeman, 1971, p 37 32. Odor LD, Blandau RJ: Observation on fertilization and the first segmentation division in rat ova. Am J Anat 89:29, 1951 33. Gondos B, Bhiraleus P, Conner A: Pronuclear membrane alterations during approximation of pronuclei and initiation of cleavage in the rabbit. J Cell Sci 10:61, 1972 34. Gondos B, Bhiraleus P: Pronuclear relationship and association of maternal and paternal chromosomes in flushed rabbit ova. Z Zellforsch Mikrosk Anat 3:149, 1970 35. Austin CR: Cortical granules in hamster eggs. Exp Cell Res 10:533, 1956 36. Austin CR: The Mammalian Egg. Oxford, Blackwell Scientific Publishers, 1961 37. Austin CR: Ultrastructure of Fertilization. New York, Holt, Rinehart and Winston, 1968, p 51 38. Szollosi DG: Development of cortical granules and the cortical reaction in the rat and hamster egg. Anat Rec 159:431, 1967 39. Hadek R: Submicroscopic study on the cortical granules in rabbit ova. J Ultrastruct Res 8:170, 1963 40. Weakly BS: Electron microscopy of the oocytes and granulosa cells in the developing ovarian follicles of the golden hamster (Mesocricetus auratus). J Anat 100:503, 1966 41. Baca M, Zamboni L: The fine structure of human follicular oocytes. J Ultrastruct Res 19:354, 1967 42. Zeigel RF, Dalton AJ: Speculation based on the morphology of the Golgi system in several types of protein-secreting cells. J Cell Bioi 15:45, 1962 43. Yanagimachi R, Chang MC: Fertilizable life of golden hamster ova and their morphologic changes at time of losing fertilizability. J Exp Zool 148:185, 1961 44. Zamboni L, Mishell DR, Bell TH, Baca M: Fine structure of the human ovum in the pronuclear stage. J Cell Bioi 30:579, 1966 45. Hertig AT, Adams EC: Studies on the human oocyte and its follicle. I. Ultrastructural and
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histochemical observations on the primordial follicle stage. J Cell Bioi 34:647, 1967 46. Fleming WN, Saacke PG: Fine structure of the bovine oocyte from the mature Graafian follicle. J Reprod Fertil 29:203, 1972 47. Barton BR, Hertig AT: Ultrastructure of annulate lamellae in primary oocytes of chimpanzees (/'an troglodytes). Bioi Reprod 6:98, 1972 48. Gulyas BJ: The formation of annulate lamellae in the rabbit eggs. Anat Rec 166:311, 1970 49. Bernhard W: Ultrastructural aspects of nucleocytoplasmic relationship. Exp Cell Res [Suppl] 6:17, 1958 50. Hertig AT: The primary human oocyte: some observations on fine structure of Balbiani's vitelline body and the origin of the annulate lamellae. Am J Anat 122:107, 1968 51. Kessel RG: Electron microscope studies on the origin of annulate lamellae in oocytes of necturus. J Cell Biol 19:391, 1963 52. Kessel RG: Annulate lamellae. J Ultrastruct Res 25 (SuppllO):l, 1968 53. Thibault C, Dauzier L: Analyse des conditions de la lecondation in vitro de l'oeuf de la lapine. Ann Bioi Anim Biochem Biophys 1:277, 1961 54. Dickmann Z, Clewe TH, Bonney WA Jr, Noyes RW: The human egg in the pronuclear stage. Anat Rec 152:293, 1965 55. Noyes RW, Dickman Z, Clewe TH, Bonney WA: Pronuclear ovum from a patient using an intrauterine contraceptive device. Science 147:744, 1965 56. Bavister BD, Edwards RG, Steptoe PC: ldentffication of the mid-piece and tail of spermatozoon during fertilization of human eggs in vitro. J Reprod Fertil 20:159, 1969 57. Szollosi DG: The fate of sperm middle piece mitochondria in the rat egg. J Exp Zool159:367, 1965 58. Tachi S, Kraicer PF: Studies on the mechanism of nidation. XXVIT. Sperm-derived inclusions in the rat blastocyst. J Reprod Fertil 14:401, 1967 59. Soupart P, Morgenstern LL: Human sperm capacitation and in vitro fertilization. Fertil Steril 24:462, 1973 60. Bedford JM: Morphological reaction of spermatozoa in the female reproductive tract of the rabbit. J Reprod Fertil 6:245, 1963 61. Odor DL, Blandau RJ: The frequency of occurrence of supernumerary sperm in rat ova. Anat Rec 104:1, 1949 62. Thompson RS, Zamboni L: Phagocytosis of supernumerary spermatozoa by two-cell mouse embryos. Anat Rec 178:3, 1974 63. Braden AWH, Austin CR: The number of sperm about the eggs in mammals and its significance for normal fertilization. Aust J Bioi Sci 7:543, 1954