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Fertilization of Mouse Ova in Vitro: An Electron Microscopic Study*
Copyright
Vol. 25, No.3, March, 1974 Printed in U.S.A.
© 1974 The American Fertility Society
FERTILITY AND STERILITY
FERTILIZATION OF MOUSE OVA IN VITRO: AN ELECTRON MICROSCOPIC STUDY" ROBERT STANLEY THOMPSON, PH.D., DIANNE MOORE SMITH, PH.D·,t AND LUCIANO· ZAMBONI, M.D.
Department of Pathology, Harbor .General Hospital, Torrance, California 90509 and University of California School of Medicine, Los Angeles, California
Numerous studies have been concerned with the ultrastructural phenomena occurring in mammalian spermatozoa and ova shortly before and during fertilization, and in zygotes during activation.1-38 These studies have been of importance in understanding the basic mechanisms of mammalian fertilization; this is because this process involves mostly organelles and components which are beyond the resolution attainable by conventional methods of morphologic investigation. However, little is known of the fine structural changes associated with gamete interaction and early embryonic development in vitro; the only information is that of Yanagimachi and Noda on early stages of in vitro penetration of hamster eggs,39,40 and that of Fraser et al on loss of cortical granules from rabbit eggs inseminated in vitro.41 The purpose of this article is to illustrate and discuss the most salient events associated with in vitro fertilization of mouse eggs from the stage of early sperm penetration to development of a two-cell embryo. Mouse ova were selected for this study because we know that they undergo fertilization in vitro,42-45 even when taken from follicles during early stages of maturation,46 ,47 and that, following transfer Received July 3, 1973. *Supported by U.S.P.H.S. research grant R01HD05725 and research contract 69-2220. tPresent address: Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts.
to foster mothers, mouse ova fertilized in vitro can develop into viable fetuses. 42 ,44 Moreover, we expected that our previous studies on the ultrastructural aspects of fertilization of mouse ova in vivo would considerably facilitate a comparative analysis of in vivo and in vitro fertilization and early embryonic development in this species. MATERIALS AND METHODS
Female Swiss white mice were superovulated with 5 or 10 international units (IV) each of pregnant mare serum (PMS) and human chorionic gonadotropin (RCG) . Vnmated mice to be used as egg donors were sacrificed 13 to 15 hours after RCG injection (about the time of ovulation) .48 Ova were collected from the ampullae under sterile conditions and placed in groups of 20 to 40 in culture medium under oil. The medium used was a modification of that used by Whittingham,45 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. Gonadotropin - stimulated mice mated with proven breeder males were utilized as sperm donors. Spermatozoa were obtained from one or both uterine horns 2 to 5 hours after vaginal plug formation. The sperm were microscopically examined for motility and then diluted with 0.5 ml of culture medium. Approximately
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FIG. 1. Nonpenetrated oocyte cultured for 3 hours displaying a prominent peripheral collar of cortical granules and uniform distribution of other organelles. The first polar body is in an advanced stage of degeneration. G = Golgi complex; C = polar body chromosomes. (x6,100)
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FIG. Z. Chromosomes aligned on the metaphase 2 spindle of a nonpenetrated oocyte cultured for 9 hours. The area encompassed by the spindle is mostly devoid of organelles. The ooplasm just below the oolemma is occupied exclusively by a thin layer of finely filamentous material. The oolemma in this area lacks microvilli. (x15,600)
50 p.l of this sperm suspension was added to each group of ova which were then cultured at 37°C in an atmosphere of 5% carbon dioxide in air. Insemination followed recovery of eggs by periods not exceeding 1 hour. Ova were cultured from 1 to 45Y2 hours after insemination. Studies were made on 270 ova and 77 twocell embryos. One group consisted of all ova incubated from 1 to 19Y2 hours; the other consisted exclusively of the two-cell forms found in dishes incubated from 21 Y2 to 45 Y2 hours. Ova and embryos were fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1 % OS04, rapidly dehydrated, individually embedded in Epon 812, and serially sectioned for electron microscopy. Sections were stained with lead citrate and examined with Hitachi HU11C and HU11E electron microscopes. RESULTS AND DISCUSSION
Fertilization. Of the 270 ova studied from 1 to 19 Y2 hours after insemination, 161 (60%) were nonactivated and 90 (33 % ) were activated. This percentage
of fertilization is about 20% higher than that of Iwamatsu and Chang"3 for in vitro fertilization of mouse ova in the presence of follicular fluid. In our study, 19 ova (7 % ) were eliminated from the study for technical reasons. The nonactivated ova had morphologic characteristics indistinguishable from those of virgin ova in the fallopian tube,2s,29,31 except for the condition of the first polar body (vide infra). The organization of the cytoplasm appeared normal; prominent Golgi complexes were present in various locations, and the ooplasm just below the plasma membrane contained numerous cortical granules (Fig. 1). The chromosomes were arranged on the equatorial plate of the spindle of the second meiotic metaphase (Fig. 2); the spindle was subcortical in location and oriented para tangentially to the egg surface. The oolemma over and around this region was smooth and without microvilli; the cortical ooplasm lacked organelles and was occupied by a uniform layer of finely fibrillar material of increased electron opacity.
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FIG. 3. A nonpenetrated oocyte showing a discontinuous collar of cumulus cells. One of the cumulus cells is degenerating. A spermatozoon lies outside of the follicle cells. This oocyte had been in culture for 3 hours. (x3,900)
The penetrated ova were found in stages of activation ranging from early conjugation with the fertilizing spermatozoon to first cleavage. Under the experimental conditions used in our study, sperm penetration mostly occurred within the first few hours after insemination. No fertilized ova were found from dishes incubated up to 2Y2 hours. Of 52 pene-
trated ova incubated from 3 to 5Y2 hours after insemination, 5 were in the pronuclear stage, 40 in earlier stages of activation, and 7 were still in the process of conjugation with the fertilizing spermatozoon. Between 8 and 19Y2 hours, the majority of the activated ova were in the pronuclear stage. Earliest prophases of first cleavage occurred at 19Y2 hours
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FIG. 4. Phagocytosis of sperm by follicle cells associated with a nonpenetrated oocyte. Note that the spermatozoa in this micrograph have all undergone the acrosome reaction. This oocyte was cultured for 3 hours. (x8,OOO)
after insemination. Two-cell embryos were first seen at 21 Yz hours (vide infra). The timing of sperm penetration into the eggs agrees with the results of Iwamatsu and Chang 43 ; the timing of all other fertilization events appears to compare well with that of Donahue 49 (on a large series
of tubal ova undergoing fertilization in vivo). Degeneration of the first polar body was noted in about 80% of the ova studied, nonactivated as well as activated. The degree of polar body degeneration ranged from simple regression of organ-
FIG. 5. Spermatozoon with reacted acrosome passing through cumulus of a nonpenetrated oocyte. One of the follicle cells has a cilium (arrow). The oocyte had been in culture for 4 hours. (x12,OOO)
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FIG. 6. Spermatozoa in various stages of penetration of the zona pellucida. Two sperm, one fully within the zona and the other partially in the perivitelline space, have undergone the acrosome reaction. The other sperm, two of which are beginning to penetrate the zona, have intact acrosomes. Nonpenetrated oocyte cultured for 4 hours. Arrows point to cortical granules. (x15,300)
elles, mitochondria, and cortical granules especially, to generalized involvement. In the latter case, the polar body appeared as an extremely vacuolated mass of cytoplasm containing only a few degenerating organelles and regressing chromosomes (Fig. 1). First polar body degeneration had not been observed in our previous studies on tubal ova obtained from mice
stimulated with 2 IU of gonadotropins,21,37 but was observed by Donahue 49 in most of 2,643 tubal ova obtained from mice superovulated with 15 IU of PMS and BeG. Since the mice used in the present study were also superovulated, we must conclude that first polar body degeneration is related to accelerated maturation of an exaggerated number of follicular
FIG. 7. Stage of incipient sperm penetration into an oocyte cultured for 4 hours. The sperm is oriented paratangentially to the egg surface and initial membrane fusion between the two gametes is underway. Accessory spermatozoa are evident both in and around the zona pellucida. Resumption of the second meiotic division has not y et commenced. (x5,400)
oocytes not yet physiologically prepared. In the study of Donahue 49 and under the experimental conditions used in our investigation, accelerated maturation was brought about by stimulation with large doses of gonadotropins. There are reasons to believe, however, that any other condition that would induce accelerated maturation of nonphysiologically prepared oocytes could result in first polar body degeneration. One such situation is the maturation in vitro of oocytes obtained from young follicles; these would not have matured in such a short time had they remained in the follicular cavity; in fact, Moore-Smith (unpublished data) observed disintegration of first polar bodies within 2 to 4 hours of their formation from in vitro matured oocytes obtained from nonstimulated mice. That degeneration of first polar bodies may occur under certain experimental conditions should be kept in mind when trying to establish by means other than electron microscopy whether ova have or have not been fertilized. The presence of only one polar body does not necessarily prove that fertilization has not occurred. Upon recovery from the oviducts, ova were surrounded by a prominent cumulus oophorus which became looser and partially dispersed shortly after insemina-
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tion. At the time they were harvested from culture, all ova were associated with only a few residual cumulus cells which no longer encircled the eggs in a continuous fashion; the cells being separated from one another by wide, irregular lacunae (Fig. 3). In no instances were cytoplasmic processes of these cells observed extending through the zona pellucida. A few cumulus cells were degenerating (Fig. 3) but mitoses were not infrequent. Phagocytosis of spermatozoa by cells of the cumulus oophorus was frequently observed, irrespective of the length of time the ova remained in culture (Fig. 4). All ova, activated and nonactivated, were associated with varying numbers of spermatozoa outside of and within the cumulus oophorus (Figs. 3 to 5), in the zona pellucida, and the perivitelline space (Figs. 6 and 7). This situation drastically contrasts with that prevailing in the lumen of the tubal ampulla where, at the time of penetration and during early stages of activation, the number of ova is higher than or equal)o that of sperm, and each egg being fertillzed is associated with only one (the fertilizing) spermatozoon.·Z1 ,31 Sperm found in association with ova could be subdivided into two groups: those which lacked the acrosome and exhibited the classic signs of the acrosome reaction 2 (ie, presence of vesiculated remnants of plasma and outer acrosomal membranes) , and those which retained an intact acrosome surrounded by a continuous or only focally interrupted plasma membrane. It was impossible to establish any definitive correlation between sperm localization and the condition of the acrosome. All sperm whose heads were entirely in the perivitelline space lacked the acrosome; acrosome loss also was evident in many sperm in the thickness of the zona pellucida (Fig. 6). Sperm with "reacted"
FIG. 8. Sperm penetration into an oocyte cultured for 5 hours. This sperm is oriented perpendicular to the egg surface. The membrane resulting from the fusion of egg and sperm plasma membranes is plainly visible in the postnuclear cap region (large arrows). The anterior two thirds of the sperm nucleus, devoid of a plasma membrane, is contained within an ooplasmic vacuole limited by the oolemma. Small arrows point to vesicles considered to represent vestigia of the acrosome reaction. Note two persisting cortical granules. (x31 ,300)
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acrosomes were also numerous far away from the zona, that is, outside or between the cumulus cells (Figs. 4 and 5). Sperm with intact acrosomes were usually found outside the zona where they were just as numerous as those with reacted acrosomes; however, they also were present in the zona (Fig. 6) as well as near, or partially in the perivitelline space. In these locations, the acrosome-retaining sperm were considerably fewer than those with reacted acrosomes, but they were by no means exceptional. It is difficult to account for such a variability in the morphology of these sperm; in any case, the nonexceptional presence of sperm with nonreacted acrosomes close to the surface of eggs without detectable discontinuities of their envelopes could be an indication that at least some spermatzoa may penetrate through the egg investments without undergoing acrosomal changes. Incorporation of the spermatozoon and early transformation of its chromatin in the ooplasm were observed in seven ova; with only minor exceptions, the various phases of sperm penetration evolved just as they do in vivo, thus testifying to the rigorous uniformity of this process. The sperm entered into the egg with the sagittal axis of the head oriented either tangentially (Fig. 7) or, less frequently, almost perpendicular to the egg surface (Fig. 8). Sperm head incorporation occurred by two mechanisms: 1) fusion of gamete plasma membranes which involved that region of the sperm extending from the posterior margin of the acrosome to the caudal tip of the flagellum, and 2) engulfment of the structures of the anterior two thirds of the sperm head within an ooplasmic vacuole. All spermatozoa found to be engaged in the ova penetration process had undergone acrosomalloss (Fig. 8); the organelle appeared to be lacking entirely, including its thin equatorial segment (acrosome
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collar in the terminology of Yanagimachi and Noda 39 ). In this regard, the present observations, as well as those of a previous study (demonstrating complete loss of the acrosome in sperm penetrating mouse ova in the fallopian tube 21 ) do not agree with those of Pik6 16 and Franklin et al. 9 This suggests that an intact equatorial segment surrounded by the plasma membrane still persists in rat and hamster spermatozoa in the zona pellucida and perivitelline space. Loss of the main bulk of the acrosome, but retention of an intact or nearly intact equatorial segment, was reported by Bedford 7 in rabbit sperm found in close association with ova. We also entertained the possibility that in sperm of this species the thin acrosome segment may enter into the egg. However, this hypothesis was not confirmed; we instead found total loss of acrosome in mouse sperm that were in association with the egg plasma membrane. All spermatozoa engaged in various phases of penetration into ova had small vesicles around the original acrosome region, especially at the anterior third of the head (Figs. 8 and 12). In our opinion, these vesicles represent the remnants of the plasma and outer acrosomal membranes, an interpretation previously presented also by Stefanini et a1,21 However, Bedford 7 demonstrated absence of vesiculated membrane remnants in rabbit sperm at all stages of zona pellucida penetration. The sperm established first contact with the egg surface in the postacrosome region. The membrane coverings of the sperm in this region (ie, the plasma membrane and the thick underlying lamina which is a consistent component of the postacrosomal cap region of the mammalian sperm head 5o ,51) first became closely apposed against the oolemma and then fused with it. Gamete membrane fusion in the postacrosomal region resulted in the formation of a single thick lamina which extended from the posterior
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FIG. 9. Nucleus of the fertilizing spenn fully incor. porated in the ooplasm. The nucleus still retains its elongated configuration and is surrounded by remnants of the dense material of the subacrosomal space (large arrows). The nucleus is undergoing denudation and only fragments of the nuclear membrane are still evident (encased area). The oocyte had been in culture for 5 hours. (x43,700)
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margin of the acrosome to the base of the head (Fig. 8). Shortly after its formation, this lamina underwent fragmentation and dispersion into a granular form, causing the posterior sperm nucleus to be directly exposed to the ooplasm. The anterior two thirds of the sperm head, which was limited only by the inner acrosomal membrane surrounded by remnants of the plasma and outer acrosomal membranes (see above), was incorporated into the ooplasm by a different mechanism. It became surrounded by tongues or flaps of egg cytoplasm (Fig. 12), or contained within deep invaginations of the egg surface (Fig. 8). The latter situation occurred in cases of almost perpendicular orientation of the' sperm to the egg surface. The portions of ooplasm surrounding the anterior two
thirds of the sperm head subsequently fused; this resulted in a vacuole in which these structures remained enclosed (Fig. 8). This process was identical to that previously described for the rat/ 6 ,H rabbit,6,7 hamster 39 ,4o and mouse. "",3<; The pattern of formation and the characteristics of these vacuoles appeared very similar also to those of phagocytic vacuoles. In our opinion, incorporation of the anterior two thirds of the sperm head by "phagocytosis" could be explained by the absence of the plasma membrane in this region which is limited only by the inner acrosomal membrane. This hypothesis would indicate that the oolemma may conjugate only with the plasma mem~ brane of the sperm and that no other membranes of the male gamete may be· come part of the mosaic membrane of
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FIG. 10. This micrograph magnifies the encased area in Fig. 9. A fragment of condensed nuclear membrane is attached to a portion of the sperm nucleus (SN). Nuclear pores are clearly visible (arrows) . 0 = oocyte cytoplasm. (x87,400)
the zygote. Moreover, the ability of the oolemma to fuse with extraneous plasma membranes would appear to be restricted solely to that of the spermatozoon. When somatic cells, such as follicle cells, come into contact with the oolemma, they may be incorporated into the ooplasm but exclusively by means of phagocytosis. 52 Recently incorporated sperm heads were located a few microns from the cell surface (Figs. 12 and 16). The anterior region of the sperm nucleus was usually surrounded by a layer of the dense material of the subacrosomal space and vestigia of the inner acrosomal membrane (Figs. 9, 11, and 12). The posterior third was instead surrounded by the thick lamina (Fig. 8), or fragments of it (Figs. 9 and 11), resulting from the fusion of the two gamete membranes. At this stage, the sperm nucleus still retained its elongated shape and showed a relatively compact chromatin organization (Fig. 9); this phase coincided also with the denudation of the sperm nucleus, a process which has never been previously documented. The two leaflets of the
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nuclear membrane became closely apposed against one another and formed a single, rather thick lamina of increased electron opacity in which "pores" could still be identified (Fig. 10); they also fragmented and separated from the chromatin (Fig. 9) undergoing degeneration in the adjacent ooplasm in granular form. This was followed by dispersion of the sperm chromatin which assumed a filamentous organization (Fig. 11) with strands diffusing freely in the ooplasm (Fig. 12). The process, which initiated in the posterior region and spread anteriorly, transformed the originally compact sperm nucleus into an irregular mass of filamentous material of considerably decreased electron density (Figs. 13, 14, and 16). This chromatin mass, which was usually associated with residual material of the subacrosomal space and vestigia of the inner acrosomal membrane (Figs. 12 and 16) and segments of the flagellum (Figs. 13, 14, and 16), was at the periphery of the egg, a few microns from the oolemma. The cortical ooplasm above and around this region was devoid of organelles, occupied exclusively by a layer of finely filamentous material and limited by a smooth portion of oolemma without microvilli (Figs. 13, 14, and 16). Since the organization of this area appeared identical to that of the region above and around the second meiotic spindle (see above and Fig. 2), presence of two such areas is a pathognomonic ultrastructural sign of activation that can be determined even in the absence of sperm remnants in the plane of section. At the time of penetration of the head, the flagellum of the fertilizing spermatozoon was mostly outside the zona pellucida. This differs from the in vivo situation where, at a comparable phase of fertilization, the flagellum is entirely in the perivitelline space and wrapped closely around the egg surface. 21 ,31 The flagella displayed regressive changes af-
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233 FIG. 11. Nucleus of the fertilizing sperm undergoing chromatin dispersion in the ooplasm. This oocyte was in culture for 8Yz hours. (x46smO)
FIG. 12. Nucleus of the fertilizing sperm in advanced stage of chromatin dispersion. A part of the anterior portion of the sperm head is still partially outside the egg but is almost completely surrounded by ooplasmic projections. The almost fully dispersed chromatin appears as a finely filamentous mass. This oocyte had been cultured for 5 hours and was in anaphase of the second meiotic division. (x28,600)
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FIG. 13. Fertilizing sperm nucleus fully dispersed into a mass of finely filamentous chromatin. Arrow points to a segment of the sperm middlepiece with degenerating mitochondria. The sperm chromatin lies very close to the oocyte surface. The area immediately above (see also Figs. 14 and 16) is morphologically identical to that overlying the second metaphase spindle. This oocyte was cultured for 5 hours and was in telophase of second meiosis. (x15,700)
fecting some of the mitochondria of the middlepiece. These changes, which were evident in segments yet to be incorporated, consisted of swelling, fragmentation, and rarefaction of the cristae with markedly decreased electron opacity of the matrix (Fig. 15) . The matrix often contained large dense granules presumed to be toxic. 53 Stefanini et aPl noted identical changes in vivo and postulated that such a precocious mitochondrial degeneration could be related, causally or consequentially, to the considerably diminished motility of the flagellum 54 throughout the relatively long period of time that the fertilizing sperm spends in the perivitelline space prior to incorporation into the vitellus. 55 Possibly comparable conditions prevail in vitro also and as soon as the sperm head becomes engaged in penetration of the ooplasm, tail motility either decreases considerably or ceases simultaneously with regression of middlepiece mitochondria.
The incorporation of the flagellum into the vitellus appeared to be associated with pronounced surface activity of the egg. This was seen mostly in the form of elevation of the cortical ooplasm around segments to be incorporated (Fig. 15) with progressive fusion, in zipper-like fashion, of the oolemma with the sperm plasma membrane. The pattern and direction of the spreading surface activity progressed to reach the various segments of the flagellum and was identical to the "incorporation wave" described in ova undergoing fertilization in vivo. 21 It involved a very wide region of the egg surface and occasionally included the second polar body (Figs. 16 to 18). The sperm chromatin and a long segment of the middlepiece are inside the egg (Fig. 16) but the distal portion of the tail is in the bridge connecting the egg to the second polar body (Figs. 16 and 17) as well as in the polar body cytoplasm (Figs. 17 and 18) with a segment of the prin-
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FIG. 14. Fully dispersed sperm nucleus in intimate contact with the basal plate and connecting piece of the flagellum. This oocyte was cultured for 3 hours and was in telophase of the second meiotic division. (x19,500)
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cipal piece bisecting the polar body chromosomes (Fig. 18). Penetration of the sperm into the egg triggered the resumption of the second meiotic division (Figs. 19 and 20) which appeared to evolve in a manner identical to that described previously for mouse ova fertilized in the tubal lumen. 22, 31Meiosis, which was completed by the time the sperm chromatin had undergone total dispersion (Fig. 16), culminated in the formation of a second polar body. This did not undergo degeneration within the time periods included in this study and had normal morphologic characteristics. Cortical granules were obviously absent, and the nuclear complement was organized in the form of a nucleus, complete with a double membrane envelope (Fig. 20) and nucleoli. Reconstitution of a
typical nucleus appeared to occur rapidly; naked chromosomes were seen exclusively in very early stages of formation (Figs. 17 to 19), and fully reconstituted nuclei were already present in polar bodies still connected to the eggs by a cytoplasmic bridge (Fig. 20). Formation of a pronuclear envelope around the sperm and egg chromatill, appeared to be mediated by the ergastoplasmic reticulum whose vesicular elements congregated closely around the parental chromatin masses and fused with one another. This formed a continuous double membrane envelope whose outer leaflet was frequently studded with ribosomes. The morphogenesis and ultrastructural characteristics of parental pronuclei in the activated eggs (Figs. 21 to 23) ap-
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FIG. 15. Fertilizing spenn flagellum during incorporation into the egg. This segment of the spenn tail is contained within an elongated ooplasmic pseudopod, has lost its plasma membrane, and is directly exposed to the egg cytoplasm. Numerous mitochondria of the middlepiece show signs of degeneration such as swelling and rarefaction of the matrix (arrows). The oocyte was in telophase of second meiosis and had been in culture for 4 hours. (x17,500)
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peared identical to those of pronuclei descrihed in mammalian ova fertilized in vivo. 1o ,14,32,37 In advanced stages of activation, parental pronuclei did not show any salient differences and distinction hetween male and female pronuclei was usually impossihle (Fig. 23). In early stages of formation, the male pronucleus could he identified hy its close topographic relationship with sperm flagellum remnants (Fig. 21), and the female hy its association with residual microtuhules of the meiotic spindle (Fig. 22). In late stages of activation and shortly hefore the onset of first cleavage, the parental pronuclei occupied central regions of the ooplasm, where they were in close apposition to one another (Fig. 23). Contact hetween parental pronuclei was never ohserved. First cleavage division hegan and evolved in a manner identical to that descrihed recently for mouse ova cleaving in the tuhal lumen. 37 Chromatin condensation into chromosomes occurred while the pronuclear memhranes were still intact or had just hegun to dismantle hy fragmentation; this demonstrated once more that mammalian pronuclei enter prophase independently and that the first cleavage division in mammalian zygotes is not preceded hy syngamy14,37 as is the case for ova of some invertehrates. 56 Simultaneously with fragmentation of the memhranes, pronuclei were invaded by microtuhules of the developing spindle. Serial sectioning through the region from where the microtubules appeared to originate again failed to find centrioles, thus confirming the presently accepted hypothesis that mammalian oocytes and zygotes "lack typical centriolar structures, and that meiotic and mitotic divisions in these cells occur in their ahsence. 28 ,37,57 The only fertilization anomalies noted in our study were one polyspermic egg and one case of sperm penetration into an intact first polar hody.
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FIG. 16
FIG. 17
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FIG. 18 FIGs. 16, 17, 18. These three micrographs illustrate a spermatozoon which has penetrated an egg (Fig. 16) but whose flagellum is passing through the bridge connecting the forming second polar body with the oocyte (Figs. 16 and 17) and is bisecting the polar body chromosomes (Fig. 18). The sperm chromatin is fully dispersed and intimately associated with the connecting piece of the flagellum as well as residual material of the subacrosomal space (arrow, Fig. 16). Note the early degeneration of some of the sperm mitochondria (Figs. 16 and 17) . This oocyte had been cultured for4 hours. (Fig. 16, x10,OOO; Fig. 17, x6,700; Fig. 18, x23,500)
FIG. 19. Telophase of the second meiotic division in a penetrated oocyte which had been in culture for 4 hours. Extrusion of the second polar body is in progress. Remnants of the first polar body are visible in the lower right of the micrograph. (x4,600)
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FIG. ZOo Cytoplasmic bridge between second polar body and oocyte. A prominent midbody is evident in the bridge. The recently formed polar body already has a fully reconstituted nucleus (N). The principal piece of the fertilizing sperm is evident in the ooplasm adjacent to the cytoplasmic bridge (arrow). This oocyte was in culture for 10 hours and was in the pronuclear stage of activation. (x8,400)
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The polyspermic egg had three pronuclei (Fig. 24) and flagella remnants of two spermatozoa in different locations in the ooplasm. The extremely low incidence of polyspermy is comparable to that (0.3%) found by Donahue49 in a very large number of eggs undergoing fertilization in vivo. This indicates that mouse ova have a very efficient block to polyspermy, even when the sperm to egg ratio at fertilization is considerably higher than the one to one ratio prevailing under in vivo conditions. 21 ,sl Polar body "fertilization" involved a first polar body associated with a penetrated egg (Fig. 25). It is likely that
spermatozoa penetration into both egg and polar body occurred simultaneously because they were at the same stage of chromatin dispersion. Sperm penetration into polar bodies is a rare anomaly which has been observed also in a study of a large series of mouse ova undergoing fertilization in vivo (Zamboni, unpublished). Mosaicisms resulting from sperm penetration into polar bodies have been described in mice and humans. 58 Two-Cell Embryos. Two-cell embryos were first seen 21 Y2 hours after insemination. Because nonactivated ova can fragment or cleave parthenogenetically into cytoplasmic masses which are often
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FIG. 21. Young male pronucleus in the same oocyte as Fig. 20. This pronucleus is identified as male because of its close association with the sperm flagellum and remnants of the anterior structures of the sperm head (arrows). (x7,OOO)
indistinguishable from blastomeres, we thought it important to serially section all the two-cell embryos. This enabled us not only to study their ultrastructural characteristics, but also to screen them for remnants of the fertilizing sperm. It is, in fact, known from previous studies on in vivo fertilization of rar!4 and mouse ova 59 that sperm remnants can still be
discerned in two-cell embryo blastomeres. Of 77 embryos studied, 67 showed sperm remnants in various locations in the cytoplasm of one or both blastomeres (Fig. 28) and/or within the bridge connecting the two cells. In most cases, only fragments of flagellar fibers were still evident, the other components having undergone complete degeneration. In a
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few instances, flagellar fibers were associated with still recognizable mitochondria of the middlepiece (Fig. 28). Absence of sperm components in the nine remaining embryos was probably due to our failure to detect them, even though the possibility that at least some eggs
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FIG. 22. Young female pronucleus in an oocyte cultured for 5 hours . This pronucleus is identified as female due to its close association with the residual microtubules of the meiotic spindle. Recent formation of this pronucleus is evidenced also by its small size and condensed appearance. The stump of the bridge connecting the egg to the second polar body and the cytoplasmic debris resulting from previous cytokinesis are evident (arrow) . (x5,800)
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cleaved parthenogenetically cannot be ruled out. The blastomeres were enclosed within a continuous zona pellucida; the perivitelline space was evident and contained a second polar body and varying numbers of supplementary spermatozoa in various stages of degeneration. Some of these spermatozoa were seen to undergo phagocytosis by the blastomeres, a phenomenon which has been described in detail in a separate report. GO Blastomeres were all of a comparable size; their spheroidal profiles appeared slightly flattened only in the area where the surface of one blastomere was apposed against that of the other (Fig. 26). The plasma membrane was slightly undulated and provided with a few microvilli which were more prominent in the. areas of mutual apposition. The space between the two blastomeres was occupied by a cytoplasmic bridge (Fig. 29), or remnants thereof, and cytoplasmic debris resulting from ooplasmic fragmentation at the time of cytokinesis (Fig. 29). The cytoplasmic organization of the blastomeres was not different from that of zygotes (Figs. 26 and 29) and need not be described in detail. The blastomere nuclei were spheroidal, regular in outline and provided with numerous and prominent nucleoli (Fig. 26). Each nucleus contained several lamellar structures (Figs. 26 and 27) which were preferentially localized at the nuclear periphery where one of their extremities appeared to be in contact with the nuclear envelope (Fig. 26). The structures consisted of pairs of parallel membranes, each element of the pair separated from the other by a narrow cisterna comparable in width to the nuclear space (Fig. 27). The paired membranes showed frequent areas of mutual apposition which, in tangential planes, appeared as "pores" indistinguishable from those of the nuclear envelope. Structures morphologically identical to those described above have
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THOMPSON ET AL
FIG. 23. Close association between parental pronuclei in an egg in advanced stage of activation. Identification of either pronucleus as male or female is impossible at this stage. This egg had been cultured for 19~ hours. (x10,200)
been observed in the blastomere nuclei of Z- and 4-cell mouse embryos developed
boni et aP4 ovum.
III
the human pronuclear
in vivo,s,13,37 in anomalous "blastomerelike" nuclei of human oocytes in culture,as SUMMARY and in pronuclei of rabbitl l ,12,14,32 and Serial sectioning for electron microhuman zygotes. 34 In most cases, these scopy was used to investigate the fine structures were identified as annulate structural changes associated with mouse lamellae. In our opinion, and in that of gamete interaction and early embryonic Calarco and Brown,s the lamellar strucdevelopment in vitro and to compare tures in the nuclei of early embryo these processes with those occurring in blastomeres more probably represent segvivo. Two hundred and seventy mouse ova ments of nuclear envelope which had recultured from 1 to 19Y2 hours after inmained trapped inside the newly ~econ semination and 77 two-cell mouse emstituted nuclei at the time of their forbryos developed in culture were studied. mation. Fertilization in vitro occurred and In a few blastomeres, the perinuclear evolved following patterns similar to ooplasm contained from one to a few those observed in vivo. Minor differences crystalline inclusions of fibrillar material which depended upon the were noted in a perfectly regular spatial arrangespecial conditions inherent in in vitro ment and geometric organization. These were similar to the crystalloid inclusions systems. These differences, however, did destribed by Enders and Schlafke61 in not prevent fertilization from evolving in mammalian blastocysts and to those ob- a normal fashion in most cases. The essential normality of fertilization served by Hillman and Tasca1.3 in early and early embryonic development in vitro mouse embryo blastomeres and by Zam-
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FIG. 24. Polyspermic egg with three pronuclei (PN) and flagellar remnants of two sperm in the ooplasm (not included in the plane of section). This egg had been in culture for 19~ hours. Note the intact second polar body in lower right. (x3,900)
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FIG. 25. Sperm penetration into a first polar body (PB). The chromatin of the sperm is dispersing in a filamentous manner in the polar body cytoplasm and segments of the flagellum are also evident. The oocyte (0) associated with this polar body had been in culture for 5 hours, had been penetrated, and was in telophase of the second meiosis. (x13,700)
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FIG. 26. Two-cell embryo developed in flagellum of the fertilizing sperm in the Fig. 28. Blastomere nuclei contain dense mal ultrastructural characteristics and the
245
vitro within 28 hour~ after insemination. Remnants of the cyptoplasm of a blastomere of this embryo are shown in nucleoli and intranuclear lamellae (arrows). Note the noroverall healthy appearance of the embryo. (x3,700)
FIG. 27. Structural organization of the intranuclear lamellae typical of blastomere nuclei. The configuration of these lamellae and the multiple areas of mutual apposition between leaflets, which appear as "pores" in tangential planes, are similar to those of the nuclear envelope. This two-cell embryo developed within 45Yz hours after insemination. (x30,500)
FIG. 28. Same embryo as Fig. 26. The flagellum of the fertilizing sperm is in an advanced stage of dissolution. The sperm mitochondria (arrows) are swollen and highly rarefied, clearly distinguishable from those of the blastomere. The organization of the flagellar fibers is disrupted. (x40,600)
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3.
4.
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12. FIG. 29. Two-cell embryo developed in vitro within 28 hours after insemination. The blastomeres are still connected by an intact cytoplasmic bridge. The space between the blastomeres contains cytoplasmic debris resulting from cytokinesis. (x4,900)
13.
14.
noted in this study accounts for the fact that in vitro fertilization of mouse ova often results in normal pre- and postimplantation development. 42 ,44,45 Anomalies of fertilization noted in this study consisted only of one polyspermic egg and one instance of sperm penetration into a first polar body.
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REFERENCES 1. Austin CR: Acrosome loss from the rabbit spermatozoon in relation to entry into the egg. J Reprod Fertil 6:313, 1963 2. Barros C, Bedford JM, Franklin LE, et al: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell BioI 34:C1, 1967
17.
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Barros C, Franklin LE: Behavior of the gamete membranes during sperm entry into the mammalian egg. J Cell BioI 37:C13, 1968 Bedford JM: Experimental requirements for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J Reprod Fertil (Suppl) 2:35, 1967 Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329, 1968 Bedford JM: Sperm capacitation and fertilization in mammals. BioI Reprod (Suppl) 2: 128, 1970 Bedford JM: An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213, 1972 Calarco PG, Brown EH: An ultrastructural and cytological study of preimplantation development of the mouse. J Exp Zool 171: 253, 1968 Franklin LE, Barros C, Fussell EN: The acrosomal region and the acrosome reaction in sperm of the golden hamster. BioI Reprod 3: 180, 1970 Gondos B, Bhiraleus P: Pronuclear relationship and association of maternal and paternal chromosomes in flushed rabbit ova. Z Zellforsch Mikrosk Anat 111:149, 1970 Gulyas BJ: The rabbit zygote: formation of annulate lamellae. J Ultrastruct Res 35: 112, 1971 Gulyas BJ: The rabbit zygote. II. The fate of annulate lamellae during first cleavage. Z Zellforsch Mikrosk Anat 133:187, 1972 Hillman N, Tasca RJ: Ultrastructural and autoradiographic studies of mouse cleavage stages. Am J Anat 126: 151, 1969 Longo FJ, Anderson E: Cytological events leading to the formation of the two-cell stage in the rabbit: association of the maternally and paternally derived genomes. J Ultrastruct Res 29:86, 1969 Dura C, Chakraborty J, Ste£anini M, et al: The fine structure of the post-coital oviduct of the mouse. In Proc Seventh Intern Congr Electr Microsc. Edited by P Favard. Grenoble, 1970, Vol 3, p 625 Pika L: Mechanism of sperm penetration in the rat and the Chinese hamster based on fine structural studies. In Proc Fifth Intern Cong Anim Reprod. Trento, 1964, Vol 7, p 301 Pika L: Gamete Structure and Sperm Entry in Mammals, 1st edition. Edited by CB Metz and A Monroy. r-few York, 1969, Vol 2, p 325
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Pika L, Tyler A: Fine structural studies of sperm penetration in the rat. In Proc Fifth Intern Cong Anim Reprod. Trento, 1964, Vol 2, p 372 Sotelo JR, Porter KR: An electron microscope study of the rat ovum. J Biophys Biochem Cytol 5:327, 1959 Stefanini M, Hando T, Zamboni L: Elec~n microscope studies on mammalian fertilization. Uterine and tubal spermatozoa of the mouse. In Proc Fourth Europ Conf Electr Microsc. Edited by DS Bocciarelli. Rome, 1968, Vol 2, p 317 Stefanini M, Dura C, Zamboni L: Ultrastructure of fertilization in the mouse. II. Penetration of sperm into the ovum. J Submicr Cytol 1: 1, 1969 Stefanini M, Oura C, Zamboni L: Meiotic cleavage of the mammalian ovum. In Proc Ninth Ann Meet Am Soc Cell BioI, 1969, p 138 Stefanini M, Oura C, Zamboni L: The fme morphology of activated mouse ova. In Proc Seventh Intern Congr Electr Microsc. Edited by P Favard. Grenoble, 1970, Vol 3, p 665 Szollosi DG: The fate of sperm middle piece mitochondria in the rat egg. J Exp Zool 159:367, 1965 Szollosi DG: Development of cortical granules and the cortical reaction in rat and hamster eggs. Anat Rec 159:431, 1967 Szollosi DG, Ris H: Observations on sperm penetration in the rat. J Biophys Biochem Cytol 10:275, 1961 Zamboni L: Electron microscope studies on mammalian fertilization. A new approach to the problem. In Proc Fourth Europ Conf Electr Microsc. Edited by DS Bocciarelli. Rome, 1968, Vol 2, P 313 Zamboni L: Ultrastructure of mammalian oocytes and ova. BioI Reprod (Suppl) 2:#, 1970 Zamboni L: Fine Morphology of Mammalian Fertilization, 1st edition. New York, Harper & Row, 1971 Zamboni L: Acrosome reaction in the mammalian spermatozoon. A clarification. J Ultrastruct Res 34:401, 1971 Zamboni L: Fertilization in the mouse. In Biology of Mammalian Fertilization and Implantation. Edited by KS Moghissi and ESE Hafez. Springfield, Illinois, Charles C Thomas, Publisher, 1972, p 213 Zamboni L, Mastroianni L Jr: Electron microscope studies on rabbit ova. II. The penetrated tubal ovum. J Ultrastruct Res 14: 118, 1966
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Zamboni L, Stefanini M: On the configuration of the plasma membrane of the mature spermatozoon. Fertil Steril 19:570, 1968 Zamboni L, Mishell DR Jr, Bell JH, et al: Fine structure of the human ovum in the pronuclear stage. J Cell BioI 30:579, 1966 Zamboni L, Stefanini M, Hando T: Electron microscope studies on fertilization in the mouse. In Proc Sixth Intern Meet Anim Reprod Artif Insem. Edited by C Thibault. Paris, 1968, Vol 1, p 639 Zamboni L, Stefanini M, Oura C, et al: The pattern of sperm penetration into the mouse egg. In Proc Seventh Intern Cong Electr Microsc. Edited by P Favard. Grenoble, 1970, Vol 3, p 663 Zamboni L, Chakraborty J, Smith D: First cleavage division of the mouse zygote. An ultrastructural study. BioI Reprod 7: 170, 1972 Zamboni L, Thompson RS, Moore-Smith D: Fine morphology of human oocyte maturation in vitro. BioI Reprod 7:425, 1972 Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head during fertilization. J Ultrastruct Res 31:465, 1970 Yanagimachi R, Noda YD: Electron microscope studies of sperm incorporation into the golden hamster egg. Am J Anat 128:429, 1970 Fraser LR, Dandekar PV, Gordon MK: Loss of cortical granules in rabbit eggs exposed to spermatozoa -in vitro. J Reprod Fertil 29:295, 1972 Cross PC, Brinster RL: In vitro development of mouse oocytes. BioI Reprod 3:298, 1970 Iwamatsu T, Chang MC: In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature (Lond) 224:919,1969 Mukherjee AB, Cohen MM: Development of normal mice by in vitro fertilization. N ature (Lond) 228:472, 1970
I
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Whittingham DG: Fertilization of mouse eggs in vitro. Nature (Lond) 220:592, 1968 46. Iwamatsu T, Chang MC: Factors involved in the fertilization of mouse eggs in vitro. J Reprod Fertil 26: 197, 1971 47. Iwamatsu T, Chang MC: Sperm penetration in vitro of mouse oocytes at various times during maturation. J Reprod Fertil 31:237, 1972 48. Edwards RG, Gates AH: Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophin. J Endocrinol 18:292, 1959
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Donahue RP: Fertilization of the mouse oocyte: sequence and timing of nuclear progression to the two-cell stage. J Exp Zool 180:305, 1972 Fawcett DW, Ito S: The fine structure of bat spermatozoa. Am J Anat 116:567, 1965 Zamboni L, Zemjanis R, Stefanini M: The fine structure of monkey ami human spermatozoa. Anat Rec 169:129, 1971 Zamboni L, Moore-Smith D, Thompson RS: Migration of follicle cells through the zona pellucida and their sequestration by human oocytes in vitro. J Exp Zool 181:319, 1972 Trump BF, Goldblatt PJ, Howell RE: Studies on necrosis of mouse liver in vitro. Ultrastructural alterations in the mitochondria of hepatic parenchymal cells. Lab Invest 14: 343, 1965 Blandau RJ, Odor LD: Observations on sperm penetration into the ooplasm and changes in the cytoplasmic components of the fertilizing spermatozoon in rat ova. Fertil Steril 3: 13, 1952 Austin CR, Braden A WH: Time relation and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust J BioI Sci 7:179, 1954
249
Longo FJ, Anderson E: The fine structure of pronuclear development and fusion in the sea urchin, Arbacia punctulata. J Cell BioI 39:339, 1968 57. Szollosi DG, Calarco P, Donahue RP: Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci 11: 521, 1971 58. Austin CR: Variations and anomalies in fertilization. In Fertilization. Edited by CB Metz and A Monroy. New York, Academic Press, 1969, Vol 2, p 437 59. Smith DM, Oura C, Zamboni L: Fertilizing ability of structurally abnormal spermatozoa of the mouse. Nature (Land) 227:79, 1970 60. Thompson RS, Zamboni L: Phagocytosis of supernumerary spermatozoa by two-cell mouse embryos. Anat Rec 178:3, 1974 61. Enders AC, Schlafke SJ: The fine structure of the blastocyst: some comparative studies. In Preimplantation Stages of Pregnancy. Edited by GEW Wolstenholme and M O'Connor. Boston, Little, Brown & Co, 1965, p 29 56.
Vol. 25, No.3, March, 1974 Printed in U.S.A.
© 1974 The American Fertility Society
FERTILITY AND STERILITY
FERTILIZATION OF MOUSE OVA IN VITRO: AN ELECTRON MICROSCOPIC STUDY" ROBERT STANLEY THOMPSON, PH.D., DIANNE MOORE SMITH, PH.D·,t AND LUCIANO· ZAMBONI, M.D.
Department of Pathology, Harbor .General Hospital, Torrance, California 90509 and University of California School of Medicine, Los Angeles, California
Numerous studies have been concerned with the ultrastructural phenomena occurring in mammalian spermatozoa and ova shortly before and during fertilization, and in zygotes during activation.1-38 These studies have been of importance in understanding the basic mechanisms of mammalian fertilization; this is because this process involves mostly organelles and components which are beyond the resolution attainable by conventional methods of morphologic investigation. However, little is known of the fine structural changes associated with gamete interaction and early embryonic development in vitro; the only information is that of Yanagimachi and Noda on early stages of in vitro penetration of hamster eggs,39,40 and that of Fraser et al on loss of cortical granules from rabbit eggs inseminated in vitro.41 The purpose of this article is to illustrate and discuss the most salient events associated with in vitro fertilization of mouse eggs from the stage of early sperm penetration to development of a two-cell embryo. Mouse ova were selected for this study because we know that they undergo fertilization in vitro,42-45 even when taken from follicles during early stages of maturation,46 ,47 and that, following transfer Received July 3, 1973. *Supported by U.S.P.H.S. research grant R01HD05725 and research contract 69-2220. tPresent address: Laboratory of Human Reproduction and Reproductive Biology, Harvard Medical School, Boston, Massachusetts.
to foster mothers, mouse ova fertilized in vitro can develop into viable fetuses. 42 ,44 Moreover, we expected that our previous studies on the ultrastructural aspects of fertilization of mouse ova in vivo would considerably facilitate a comparative analysis of in vivo and in vitro fertilization and early embryonic development in this species. MATERIALS AND METHODS
Female Swiss white mice were superovulated with 5 or 10 international units (IV) each of pregnant mare serum (PMS) and human chorionic gonadotropin (RCG) . Vnmated mice to be used as egg donors were sacrificed 13 to 15 hours after RCG injection (about the time of ovulation) .48 Ova were collected from the ampullae under sterile conditions and placed in groups of 20 to 40 in culture medium under oil. The medium used was a modification of that used by Whittingham,45 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. Gonadotropin - stimulated mice mated with proven breeder males were utilized as sperm donors. Spermatozoa were obtained from one or both uterine horns 2 to 5 hours after vaginal plug formation. The sperm were microscopically examined for motility and then diluted with 0.5 ml of culture medium. Approximately
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FIG. 1. Nonpenetrated oocyte cultured for 3 hours displaying a prominent peripheral collar of cortical granules and uniform distribution of other organelles. The first polar body is in an advanced stage of degeneration. G = Golgi complex; C = polar body chromosomes. (x6,100)
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FIG. Z. Chromosomes aligned on the metaphase 2 spindle of a nonpenetrated oocyte cultured for 9 hours. The area encompassed by the spindle is mostly devoid of organelles. The ooplasm just below the oolemma is occupied exclusively by a thin layer of finely filamentous material. The oolemma in this area lacks microvilli. (x15,600)
50 p.l of this sperm suspension was added to each group of ova which were then cultured at 37°C in an atmosphere of 5% carbon dioxide in air. Insemination followed recovery of eggs by periods not exceeding 1 hour. Ova were cultured from 1 to 45Y2 hours after insemination. Studies were made on 270 ova and 77 twocell embryos. One group consisted of all ova incubated from 1 to 19Y2 hours; the other consisted exclusively of the two-cell forms found in dishes incubated from 21 Y2 to 45 Y2 hours. Ova and embryos were fixed in 2.5 % glutaraldehyde in 0.1 M cacodylate buffer, postfixed in 1 % OS04, rapidly dehydrated, individually embedded in Epon 812, and serially sectioned for electron microscopy. Sections were stained with lead citrate and examined with Hitachi HU11C and HU11E electron microscopes. RESULTS AND DISCUSSION
Fertilization. Of the 270 ova studied from 1 to 19 Y2 hours after insemination, 161 (60%) were nonactivated and 90 (33 % ) were activated. This percentage
of fertilization is about 20% higher than that of Iwamatsu and Chang"3 for in vitro fertilization of mouse ova in the presence of follicular fluid. In our study, 19 ova (7 % ) were eliminated from the study for technical reasons. The nonactivated ova had morphologic characteristics indistinguishable from those of virgin ova in the fallopian tube,2s,29,31 except for the condition of the first polar body (vide infra). The organization of the cytoplasm appeared normal; prominent Golgi complexes were present in various locations, and the ooplasm just below the plasma membrane contained numerous cortical granules (Fig. 1). The chromosomes were arranged on the equatorial plate of the spindle of the second meiotic metaphase (Fig. 2); the spindle was subcortical in location and oriented para tangentially to the egg surface. The oolemma over and around this region was smooth and without microvilli; the cortical ooplasm lacked organelles and was occupied by a uniform layer of finely fibrillar material of increased electron opacity.
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FIG. 3. A nonpenetrated oocyte showing a discontinuous collar of cumulus cells. One of the cumulus cells is degenerating. A spermatozoon lies outside of the follicle cells. This oocyte had been in culture for 3 hours. (x3,900)
The penetrated ova were found in stages of activation ranging from early conjugation with the fertilizing spermatozoon to first cleavage. Under the experimental conditions used in our study, sperm penetration mostly occurred within the first few hours after insemination. No fertilized ova were found from dishes incubated up to 2Y2 hours. Of 52 pene-
trated ova incubated from 3 to 5Y2 hours after insemination, 5 were in the pronuclear stage, 40 in earlier stages of activation, and 7 were still in the process of conjugation with the fertilizing spermatozoon. Between 8 and 19Y2 hours, the majority of the activated ova were in the pronuclear stage. Earliest prophases of first cleavage occurred at 19Y2 hours
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FIG. 4. Phagocytosis of sperm by follicle cells associated with a nonpenetrated oocyte. Note that the spermatozoa in this micrograph have all undergone the acrosome reaction. This oocyte was cultured for 3 hours. (x8,OOO)
after insemination. Two-cell embryos were first seen at 21 Yz hours (vide infra). The timing of sperm penetration into the eggs agrees with the results of Iwamatsu and Chang 43 ; the timing of all other fertilization events appears to compare well with that of Donahue 49 (on a large series
of tubal ova undergoing fertilization in vivo). Degeneration of the first polar body was noted in about 80% of the ova studied, nonactivated as well as activated. The degree of polar body degeneration ranged from simple regression of organ-
FIG. 5. Spermatozoon with reacted acrosome passing through cumulus of a nonpenetrated oocyte. One of the follicle cells has a cilium (arrow). The oocyte had been in culture for 4 hours. (x12,OOO)
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FIG. 6. Spermatozoa in various stages of penetration of the zona pellucida. Two sperm, one fully within the zona and the other partially in the perivitelline space, have undergone the acrosome reaction. The other sperm, two of which are beginning to penetrate the zona, have intact acrosomes. Nonpenetrated oocyte cultured for 4 hours. Arrows point to cortical granules. (x15,300)
elles, mitochondria, and cortical granules especially, to generalized involvement. In the latter case, the polar body appeared as an extremely vacuolated mass of cytoplasm containing only a few degenerating organelles and regressing chromosomes (Fig. 1). First polar body degeneration had not been observed in our previous studies on tubal ova obtained from mice
stimulated with 2 IU of gonadotropins,21,37 but was observed by Donahue 49 in most of 2,643 tubal ova obtained from mice superovulated with 15 IU of PMS and BeG. Since the mice used in the present study were also superovulated, we must conclude that first polar body degeneration is related to accelerated maturation of an exaggerated number of follicular
FIG. 7. Stage of incipient sperm penetration into an oocyte cultured for 4 hours. The sperm is oriented paratangentially to the egg surface and initial membrane fusion between the two gametes is underway. Accessory spermatozoa are evident both in and around the zona pellucida. Resumption of the second meiotic division has not y et commenced. (x5,400)
oocytes not yet physiologically prepared. In the study of Donahue 49 and under the experimental conditions used in our investigation, accelerated maturation was brought about by stimulation with large doses of gonadotropins. There are reasons to believe, however, that any other condition that would induce accelerated maturation of nonphysiologically prepared oocytes could result in first polar body degeneration. One such situation is the maturation in vitro of oocytes obtained from young follicles; these would not have matured in such a short time had they remained in the follicular cavity; in fact, Moore-Smith (unpublished data) observed disintegration of first polar bodies within 2 to 4 hours of their formation from in vitro matured oocytes obtained from nonstimulated mice. That degeneration of first polar bodies may occur under certain experimental conditions should be kept in mind when trying to establish by means other than electron microscopy whether ova have or have not been fertilized. The presence of only one polar body does not necessarily prove that fertilization has not occurred. Upon recovery from the oviducts, ova were surrounded by a prominent cumulus oophorus which became looser and partially dispersed shortly after insemina-
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tion. At the time they were harvested from culture, all ova were associated with only a few residual cumulus cells which no longer encircled the eggs in a continuous fashion; the cells being separated from one another by wide, irregular lacunae (Fig. 3). In no instances were cytoplasmic processes of these cells observed extending through the zona pellucida. A few cumulus cells were degenerating (Fig. 3) but mitoses were not infrequent. Phagocytosis of spermatozoa by cells of the cumulus oophorus was frequently observed, irrespective of the length of time the ova remained in culture (Fig. 4). All ova, activated and nonactivated, were associated with varying numbers of spermatozoa outside of and within the cumulus oophorus (Figs. 3 to 5), in the zona pellucida, and the perivitelline space (Figs. 6 and 7). This situation drastically contrasts with that prevailing in the lumen of the tubal ampulla where, at the time of penetration and during early stages of activation, the number of ova is higher than or equal)o that of sperm, and each egg being fertillzed is associated with only one (the fertilizing) spermatozoon.·Z1 ,31 Sperm found in association with ova could be subdivided into two groups: those which lacked the acrosome and exhibited the classic signs of the acrosome reaction 2 (ie, presence of vesiculated remnants of plasma and outer acrosomal membranes) , and those which retained an intact acrosome surrounded by a continuous or only focally interrupted plasma membrane. It was impossible to establish any definitive correlation between sperm localization and the condition of the acrosome. All sperm whose heads were entirely in the perivitelline space lacked the acrosome; acrosome loss also was evident in many sperm in the thickness of the zona pellucida (Fig. 6). Sperm with "reacted"
FIG. 8. Sperm penetration into an oocyte cultured for 5 hours. This sperm is oriented perpendicular to the egg surface. The membrane resulting from the fusion of egg and sperm plasma membranes is plainly visible in the postnuclear cap region (large arrows). The anterior two thirds of the sperm nucleus, devoid of a plasma membrane, is contained within an ooplasmic vacuole limited by the oolemma. Small arrows point to vesicles considered to represent vestigia of the acrosome reaction. Note two persisting cortical granules. (x31 ,300)
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acrosomes were also numerous far away from the zona, that is, outside or between the cumulus cells (Figs. 4 and 5). Sperm with intact acrosomes were usually found outside the zona where they were just as numerous as those with reacted acrosomes; however, they also were present in the zona (Fig. 6) as well as near, or partially in the perivitelline space. In these locations, the acrosome-retaining sperm were considerably fewer than those with reacted acrosomes, but they were by no means exceptional. It is difficult to account for such a variability in the morphology of these sperm; in any case, the nonexceptional presence of sperm with nonreacted acrosomes close to the surface of eggs without detectable discontinuities of their envelopes could be an indication that at least some spermatzoa may penetrate through the egg investments without undergoing acrosomal changes. Incorporation of the spermatozoon and early transformation of its chromatin in the ooplasm were observed in seven ova; with only minor exceptions, the various phases of sperm penetration evolved just as they do in vivo, thus testifying to the rigorous uniformity of this process. The sperm entered into the egg with the sagittal axis of the head oriented either tangentially (Fig. 7) or, less frequently, almost perpendicular to the egg surface (Fig. 8). Sperm head incorporation occurred by two mechanisms: 1) fusion of gamete plasma membranes which involved that region of the sperm extending from the posterior margin of the acrosome to the caudal tip of the flagellum, and 2) engulfment of the structures of the anterior two thirds of the sperm head within an ooplasmic vacuole. All spermatozoa found to be engaged in the ova penetration process had undergone acrosomalloss (Fig. 8); the organelle appeared to be lacking entirely, including its thin equatorial segment (acrosome
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collar in the terminology of Yanagimachi and Noda 39 ). In this regard, the present observations, as well as those of a previous study (demonstrating complete loss of the acrosome in sperm penetrating mouse ova in the fallopian tube 21 ) do not agree with those of Pik6 16 and Franklin et al. 9 This suggests that an intact equatorial segment surrounded by the plasma membrane still persists in rat and hamster spermatozoa in the zona pellucida and perivitelline space. Loss of the main bulk of the acrosome, but retention of an intact or nearly intact equatorial segment, was reported by Bedford 7 in rabbit sperm found in close association with ova. We also entertained the possibility that in sperm of this species the thin acrosome segment may enter into the egg. However, this hypothesis was not confirmed; we instead found total loss of acrosome in mouse sperm that were in association with the egg plasma membrane. All spermatozoa engaged in various phases of penetration into ova had small vesicles around the original acrosome region, especially at the anterior third of the head (Figs. 8 and 12). In our opinion, these vesicles represent the remnants of the plasma and outer acrosomal membranes, an interpretation previously presented also by Stefanini et a1,21 However, Bedford 7 demonstrated absence of vesiculated membrane remnants in rabbit sperm at all stages of zona pellucida penetration. The sperm established first contact with the egg surface in the postacrosome region. The membrane coverings of the sperm in this region (ie, the plasma membrane and the thick underlying lamina which is a consistent component of the postacrosomal cap region of the mammalian sperm head 5o ,51) first became closely apposed against the oolemma and then fused with it. Gamete membrane fusion in the postacrosomal region resulted in the formation of a single thick lamina which extended from the posterior
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FIG. 9. Nucleus of the fertilizing spenn fully incor. porated in the ooplasm. The nucleus still retains its elongated configuration and is surrounded by remnants of the dense material of the subacrosomal space (large arrows). The nucleus is undergoing denudation and only fragments of the nuclear membrane are still evident (encased area). The oocyte had been in culture for 5 hours. (x43,700)
•
margin of the acrosome to the base of the head (Fig. 8). Shortly after its formation, this lamina underwent fragmentation and dispersion into a granular form, causing the posterior sperm nucleus to be directly exposed to the ooplasm. The anterior two thirds of the sperm head, which was limited only by the inner acrosomal membrane surrounded by remnants of the plasma and outer acrosomal membranes (see above), was incorporated into the ooplasm by a different mechanism. It became surrounded by tongues or flaps of egg cytoplasm (Fig. 12), or contained within deep invaginations of the egg surface (Fig. 8). The latter situation occurred in cases of almost perpendicular orientation of the' sperm to the egg surface. The portions of ooplasm surrounding the anterior two
thirds of the sperm head subsequently fused; this resulted in a vacuole in which these structures remained enclosed (Fig. 8). This process was identical to that previously described for the rat/ 6 ,H rabbit,6,7 hamster 39 ,4o and mouse. "",3<; The pattern of formation and the characteristics of these vacuoles appeared very similar also to those of phagocytic vacuoles. In our opinion, incorporation of the anterior two thirds of the sperm head by "phagocytosis" could be explained by the absence of the plasma membrane in this region which is limited only by the inner acrosomal membrane. This hypothesis would indicate that the oolemma may conjugate only with the plasma mem~ brane of the sperm and that no other membranes of the male gamete may be· come part of the mosaic membrane of
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FIG. 10. This micrograph magnifies the encased area in Fig. 9. A fragment of condensed nuclear membrane is attached to a portion of the sperm nucleus (SN). Nuclear pores are clearly visible (arrows) . 0 = oocyte cytoplasm. (x87,400)
the zygote. Moreover, the ability of the oolemma to fuse with extraneous plasma membranes would appear to be restricted solely to that of the spermatozoon. When somatic cells, such as follicle cells, come into contact with the oolemma, they may be incorporated into the ooplasm but exclusively by means of phagocytosis. 52 Recently incorporated sperm heads were located a few microns from the cell surface (Figs. 12 and 16). The anterior region of the sperm nucleus was usually surrounded by a layer of the dense material of the subacrosomal space and vestigia of the inner acrosomal membrane (Figs. 9, 11, and 12). The posterior third was instead surrounded by the thick lamina (Fig. 8), or fragments of it (Figs. 9 and 11), resulting from the fusion of the two gamete membranes. At this stage, the sperm nucleus still retained its elongated shape and showed a relatively compact chromatin organization (Fig. 9); this phase coincided also with the denudation of the sperm nucleus, a process which has never been previously documented. The two leaflets of the
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nuclear membrane became closely apposed against one another and formed a single, rather thick lamina of increased electron opacity in which "pores" could still be identified (Fig. 10); they also fragmented and separated from the chromatin (Fig. 9) undergoing degeneration in the adjacent ooplasm in granular form. This was followed by dispersion of the sperm chromatin which assumed a filamentous organization (Fig. 11) with strands diffusing freely in the ooplasm (Fig. 12). The process, which initiated in the posterior region and spread anteriorly, transformed the originally compact sperm nucleus into an irregular mass of filamentous material of considerably decreased electron density (Figs. 13, 14, and 16). This chromatin mass, which was usually associated with residual material of the subacrosomal space and vestigia of the inner acrosomal membrane (Figs. 12 and 16) and segments of the flagellum (Figs. 13, 14, and 16), was at the periphery of the egg, a few microns from the oolemma. The cortical ooplasm above and around this region was devoid of organelles, occupied exclusively by a layer of finely filamentous material and limited by a smooth portion of oolemma without microvilli (Figs. 13, 14, and 16). Since the organization of this area appeared identical to that of the region above and around the second meiotic spindle (see above and Fig. 2), presence of two such areas is a pathognomonic ultrastructural sign of activation that can be determined even in the absence of sperm remnants in the plane of section. At the time of penetration of the head, the flagellum of the fertilizing spermatozoon was mostly outside the zona pellucida. This differs from the in vivo situation where, at a comparable phase of fertilization, the flagellum is entirely in the perivitelline space and wrapped closely around the egg surface. 21 ,31 The flagella displayed regressive changes af-
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233 FIG. 11. Nucleus of the fertilizing sperm undergoing chromatin dispersion in the ooplasm. This oocyte was in culture for 8Yz hours. (x46smO)
FIG. 12. Nucleus of the fertilizing sperm in advanced stage of chromatin dispersion. A part of the anterior portion of the sperm head is still partially outside the egg but is almost completely surrounded by ooplasmic projections. The almost fully dispersed chromatin appears as a finely filamentous mass. This oocyte had been cultured for 5 hours and was in anaphase of the second meiotic division. (x28,600)
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FIG. 13. Fertilizing sperm nucleus fully dispersed into a mass of finely filamentous chromatin. Arrow points to a segment of the sperm middlepiece with degenerating mitochondria. The sperm chromatin lies very close to the oocyte surface. The area immediately above (see also Figs. 14 and 16) is morphologically identical to that overlying the second metaphase spindle. This oocyte was cultured for 5 hours and was in telophase of second meiosis. (x15,700)
fecting some of the mitochondria of the middlepiece. These changes, which were evident in segments yet to be incorporated, consisted of swelling, fragmentation, and rarefaction of the cristae with markedly decreased electron opacity of the matrix (Fig. 15) . The matrix often contained large dense granules presumed to be toxic. 53 Stefanini et aPl noted identical changes in vivo and postulated that such a precocious mitochondrial degeneration could be related, causally or consequentially, to the considerably diminished motility of the flagellum 54 throughout the relatively long period of time that the fertilizing sperm spends in the perivitelline space prior to incorporation into the vitellus. 55 Possibly comparable conditions prevail in vitro also and as soon as the sperm head becomes engaged in penetration of the ooplasm, tail motility either decreases considerably or ceases simultaneously with regression of middlepiece mitochondria.
The incorporation of the flagellum into the vitellus appeared to be associated with pronounced surface activity of the egg. This was seen mostly in the form of elevation of the cortical ooplasm around segments to be incorporated (Fig. 15) with progressive fusion, in zipper-like fashion, of the oolemma with the sperm plasma membrane. The pattern and direction of the spreading surface activity progressed to reach the various segments of the flagellum and was identical to the "incorporation wave" described in ova undergoing fertilization in vivo. 21 It involved a very wide region of the egg surface and occasionally included the second polar body (Figs. 16 to 18). The sperm chromatin and a long segment of the middlepiece are inside the egg (Fig. 16) but the distal portion of the tail is in the bridge connecting the egg to the second polar body (Figs. 16 and 17) as well as in the polar body cytoplasm (Figs. 17 and 18) with a segment of the prin-
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235
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FIG. 14. Fully dispersed sperm nucleus in intimate contact with the basal plate and connecting piece of the flagellum. This oocyte was cultured for 3 hours and was in telophase of the second meiotic division. (x19,500)
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cipal piece bisecting the polar body chromosomes (Fig. 18). Penetration of the sperm into the egg triggered the resumption of the second meiotic division (Figs. 19 and 20) which appeared to evolve in a manner identical to that described previously for mouse ova fertilized in the tubal lumen. 22, 31Meiosis, which was completed by the time the sperm chromatin had undergone total dispersion (Fig. 16), culminated in the formation of a second polar body. This did not undergo degeneration within the time periods included in this study and had normal morphologic characteristics. Cortical granules were obviously absent, and the nuclear complement was organized in the form of a nucleus, complete with a double membrane envelope (Fig. 20) and nucleoli. Reconstitution of a
typical nucleus appeared to occur rapidly; naked chromosomes were seen exclusively in very early stages of formation (Figs. 17 to 19), and fully reconstituted nuclei were already present in polar bodies still connected to the eggs by a cytoplasmic bridge (Fig. 20). Formation of a pronuclear envelope around the sperm and egg chromatill, appeared to be mediated by the ergastoplasmic reticulum whose vesicular elements congregated closely around the parental chromatin masses and fused with one another. This formed a continuous double membrane envelope whose outer leaflet was frequently studded with ribosomes. The morphogenesis and ultrastructural characteristics of parental pronuclei in the activated eggs (Figs. 21 to 23) ap-
1 236
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FIG. 15. Fertilizing spenn flagellum during incorporation into the egg. This segment of the spenn tail is contained within an elongated ooplasmic pseudopod, has lost its plasma membrane, and is directly exposed to the egg cytoplasm. Numerous mitochondria of the middlepiece show signs of degeneration such as swelling and rarefaction of the matrix (arrows). The oocyte was in telophase of second meiosis and had been in culture for 4 hours. (x17,500)
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peared identical to those of pronuclei descrihed in mammalian ova fertilized in vivo. 1o ,14,32,37 In advanced stages of activation, parental pronuclei did not show any salient differences and distinction hetween male and female pronuclei was usually impossihle (Fig. 23). In early stages of formation, the male pronucleus could he identified hy its close topographic relationship with sperm flagellum remnants (Fig. 21), and the female hy its association with residual microtuhules of the meiotic spindle (Fig. 22). In late stages of activation and shortly hefore the onset of first cleavage, the parental pronuclei occupied central regions of the ooplasm, where they were in close apposition to one another (Fig. 23). Contact hetween parental pronuclei was never ohserved. First cleavage division hegan and evolved in a manner identical to that descrihed recently for mouse ova cleaving in the tuhal lumen. 37 Chromatin condensation into chromosomes occurred while the pronuclear memhranes were still intact or had just hegun to dismantle hy fragmentation; this demonstrated once more that mammalian pronuclei enter prophase independently and that the first cleavage division in mammalian zygotes is not preceded hy syngamy14,37 as is the case for ova of some invertehrates. 56 Simultaneously with fragmentation of the memhranes, pronuclei were invaded by microtuhules of the developing spindle. Serial sectioning through the region from where the microtubules appeared to originate again failed to find centrioles, thus confirming the presently accepted hypothesis that mammalian oocytes and zygotes "lack typical centriolar structures, and that meiotic and mitotic divisions in these cells occur in their ahsence. 28 ,37,57 The only fertilization anomalies noted in our study were one polyspermic egg and one case of sperm penetration into an intact first polar hody.
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FIG. 16
FIG. 17
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FIG. 18 FIGs. 16, 17, 18. These three micrographs illustrate a spermatozoon which has penetrated an egg (Fig. 16) but whose flagellum is passing through the bridge connecting the forming second polar body with the oocyte (Figs. 16 and 17) and is bisecting the polar body chromosomes (Fig. 18). The sperm chromatin is fully dispersed and intimately associated with the connecting piece of the flagellum as well as residual material of the subacrosomal space (arrow, Fig. 16). Note the early degeneration of some of the sperm mitochondria (Figs. 16 and 17) . This oocyte had been cultured for4 hours. (Fig. 16, x10,OOO; Fig. 17, x6,700; Fig. 18, x23,500)
FIG. 19. Telophase of the second meiotic division in a penetrated oocyte which had been in culture for 4 hours. Extrusion of the second polar body is in progress. Remnants of the first polar body are visible in the lower right of the micrograph. (x4,600)
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FIG. ZOo Cytoplasmic bridge between second polar body and oocyte. A prominent midbody is evident in the bridge. The recently formed polar body already has a fully reconstituted nucleus (N). The principal piece of the fertilizing sperm is evident in the ooplasm adjacent to the cytoplasmic bridge (arrow). This oocyte was in culture for 10 hours and was in the pronuclear stage of activation. (x8,400)
•
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The polyspermic egg had three pronuclei (Fig. 24) and flagella remnants of two spermatozoa in different locations in the ooplasm. The extremely low incidence of polyspermy is comparable to that (0.3%) found by Donahue49 in a very large number of eggs undergoing fertilization in vivo. This indicates that mouse ova have a very efficient block to polyspermy, even when the sperm to egg ratio at fertilization is considerably higher than the one to one ratio prevailing under in vivo conditions. 21 ,sl Polar body "fertilization" involved a first polar body associated with a penetrated egg (Fig. 25). It is likely that
spermatozoa penetration into both egg and polar body occurred simultaneously because they were at the same stage of chromatin dispersion. Sperm penetration into polar bodies is a rare anomaly which has been observed also in a study of a large series of mouse ova undergoing fertilization in vivo (Zamboni, unpublished). Mosaicisms resulting from sperm penetration into polar bodies have been described in mice and humans. 58 Two-Cell Embryos. Two-cell embryos were first seen 21 Y2 hours after insemination. Because nonactivated ova can fragment or cleave parthenogenetically into cytoplasmic masses which are often
240
THOMPSON ET AL
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FIG. 21. Young male pronucleus in the same oocyte as Fig. 20. This pronucleus is identified as male because of its close association with the sperm flagellum and remnants of the anterior structures of the sperm head (arrows). (x7,OOO)
indistinguishable from blastomeres, we thought it important to serially section all the two-cell embryos. This enabled us not only to study their ultrastructural characteristics, but also to screen them for remnants of the fertilizing sperm. It is, in fact, known from previous studies on in vivo fertilization of rar!4 and mouse ova 59 that sperm remnants can still be
discerned in two-cell embryo blastomeres. Of 77 embryos studied, 67 showed sperm remnants in various locations in the cytoplasm of one or both blastomeres (Fig. 28) and/or within the bridge connecting the two cells. In most cases, only fragments of flagellar fibers were still evident, the other components having undergone complete degeneration. In a
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few instances, flagellar fibers were associated with still recognizable mitochondria of the middlepiece (Fig. 28). Absence of sperm components in the nine remaining embryos was probably due to our failure to detect them, even though the possibility that at least some eggs
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FIG. 22. Young female pronucleus in an oocyte cultured for 5 hours . This pronucleus is identified as female due to its close association with the residual microtubules of the meiotic spindle. Recent formation of this pronucleus is evidenced also by its small size and condensed appearance. The stump of the bridge connecting the egg to the second polar body and the cytoplasmic debris resulting from previous cytokinesis are evident (arrow) . (x5,800)
241
cleaved parthenogenetically cannot be ruled out. The blastomeres were enclosed within a continuous zona pellucida; the perivitelline space was evident and contained a second polar body and varying numbers of supplementary spermatozoa in various stages of degeneration. Some of these spermatozoa were seen to undergo phagocytosis by the blastomeres, a phenomenon which has been described in detail in a separate report. GO Blastomeres were all of a comparable size; their spheroidal profiles appeared slightly flattened only in the area where the surface of one blastomere was apposed against that of the other (Fig. 26). The plasma membrane was slightly undulated and provided with a few microvilli which were more prominent in the. areas of mutual apposition. The space between the two blastomeres was occupied by a cytoplasmic bridge (Fig. 29), or remnants thereof, and cytoplasmic debris resulting from ooplasmic fragmentation at the time of cytokinesis (Fig. 29). The cytoplasmic organization of the blastomeres was not different from that of zygotes (Figs. 26 and 29) and need not be described in detail. The blastomere nuclei were spheroidal, regular in outline and provided with numerous and prominent nucleoli (Fig. 26). Each nucleus contained several lamellar structures (Figs. 26 and 27) which were preferentially localized at the nuclear periphery where one of their extremities appeared to be in contact with the nuclear envelope (Fig. 26). The structures consisted of pairs of parallel membranes, each element of the pair separated from the other by a narrow cisterna comparable in width to the nuclear space (Fig. 27). The paired membranes showed frequent areas of mutual apposition which, in tangential planes, appeared as "pores" indistinguishable from those of the nuclear envelope. Structures morphologically identical to those described above have
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March 1974
THOMPSON ET AL
FIG. 23. Close association between parental pronuclei in an egg in advanced stage of activation. Identification of either pronucleus as male or female is impossible at this stage. This egg had been cultured for 19~ hours. (x10,200)
been observed in the blastomere nuclei of Z- and 4-cell mouse embryos developed
boni et aP4 ovum.
III
the human pronuclear
in vivo,s,13,37 in anomalous "blastomerelike" nuclei of human oocytes in culture,as SUMMARY and in pronuclei of rabbitl l ,12,14,32 and Serial sectioning for electron microhuman zygotes. 34 In most cases, these scopy was used to investigate the fine structures were identified as annulate structural changes associated with mouse lamellae. In our opinion, and in that of gamete interaction and early embryonic Calarco and Brown,s the lamellar strucdevelopment in vitro and to compare tures in the nuclei of early embryo these processes with those occurring in blastomeres more probably represent segvivo. Two hundred and seventy mouse ova ments of nuclear envelope which had recultured from 1 to 19Y2 hours after inmained trapped inside the newly ~econ semination and 77 two-cell mouse emstituted nuclei at the time of their forbryos developed in culture were studied. mation. Fertilization in vitro occurred and In a few blastomeres, the perinuclear evolved following patterns similar to ooplasm contained from one to a few those observed in vivo. Minor differences crystalline inclusions of fibrillar material which depended upon the were noted in a perfectly regular spatial arrangespecial conditions inherent in in vitro ment and geometric organization. These were similar to the crystalloid inclusions systems. These differences, however, did destribed by Enders and Schlafke61 in not prevent fertilization from evolving in mammalian blastocysts and to those ob- a normal fashion in most cases. The essential normality of fertilization served by Hillman and Tasca1.3 in early and early embryonic development in vitro mouse embryo blastomeres and by Zam-
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FIG. 24. Polyspermic egg with three pronuclei (PN) and flagellar remnants of two sperm in the ooplasm (not included in the plane of section). This egg had been in culture for 19~ hours. Note the intact second polar body in lower right. (x3,900)
244
THOMPSON ET AL
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FIG. 25. Sperm penetration into a first polar body (PB). The chromatin of the sperm is dispersing in a filamentous manner in the polar body cytoplasm and segments of the flagellum are also evident. The oocyte (0) associated with this polar body had been in culture for 5 hours, had been penetrated, and was in telophase of the second meiosis. (x13,700)
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FIG. 26. Two-cell embryo developed in flagellum of the fertilizing sperm in the Fig. 28. Blastomere nuclei contain dense mal ultrastructural characteristics and the
245
vitro within 28 hour~ after insemination. Remnants of the cyptoplasm of a blastomere of this embryo are shown in nucleoli and intranuclear lamellae (arrows). Note the noroverall healthy appearance of the embryo. (x3,700)
FIG. 27. Structural organization of the intranuclear lamellae typical of blastomere nuclei. The configuration of these lamellae and the multiple areas of mutual apposition between leaflets, which appear as "pores" in tangential planes, are similar to those of the nuclear envelope. This two-cell embryo developed within 45Yz hours after insemination. (x30,500)
FIG. 28. Same embryo as Fig. 26. The flagellum of the fertilizing sperm is in an advanced stage of dissolution. The sperm mitochondria (arrows) are swollen and highly rarefied, clearly distinguishable from those of the blastomere. The organization of the flagellar fibers is disrupted. (x40,600)
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6.
7.
8.
9.
10.
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12. FIG. 29. Two-cell embryo developed in vitro within 28 hours after insemination. The blastomeres are still connected by an intact cytoplasmic bridge. The space between the blastomeres contains cytoplasmic debris resulting from cytokinesis. (x4,900)
13.
14.
noted in this study accounts for the fact that in vitro fertilization of mouse ova often results in normal pre- and postimplantation development. 42 ,44,45 Anomalies of fertilization noted in this study consisted only of one polyspermic egg and one instance of sperm penetration into a first polar body.
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REFERENCES 1. Austin CR: Acrosome loss from the rabbit spermatozoon in relation to entry into the egg. J Reprod Fertil 6:313, 1963 2. Barros C, Bedford JM, Franklin LE, et al: Membrane vesiculation as a feature of the mammalian acrosome reaction. J Cell BioI 34:C1, 1967
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Barros C, Franklin LE: Behavior of the gamete membranes during sperm entry into the mammalian egg. J Cell BioI 37:C13, 1968 Bedford JM: Experimental requirements for capacitation and observations on ultrastructural changes in rabbit spermatozoa during fertilization. J Reprod Fertil (Suppl) 2:35, 1967 Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am J Anat 123:329, 1968 Bedford JM: Sperm capacitation and fertilization in mammals. BioI Reprod (Suppl) 2: 128, 1970 Bedford JM: An electron microscopic study of sperm penetration into the rabbit egg after natural mating. Am J Anat 133:213, 1972 Calarco PG, Brown EH: An ultrastructural and cytological study of preimplantation development of the mouse. J Exp Zool 171: 253, 1968 Franklin LE, Barros C, Fussell EN: The acrosomal region and the acrosome reaction in sperm of the golden hamster. BioI Reprod 3: 180, 1970 Gondos B, Bhiraleus P: Pronuclear relationship and association of maternal and paternal chromosomes in flushed rabbit ova. Z Zellforsch Mikrosk Anat 111:149, 1970 Gulyas BJ: The rabbit zygote: formation of annulate lamellae. J Ultrastruct Res 35: 112, 1971 Gulyas BJ: The rabbit zygote. II. The fate of annulate lamellae during first cleavage. Z Zellforsch Mikrosk Anat 133:187, 1972 Hillman N, Tasca RJ: Ultrastructural and autoradiographic studies of mouse cleavage stages. Am J Anat 126: 151, 1969 Longo FJ, Anderson E: Cytological events leading to the formation of the two-cell stage in the rabbit: association of the maternally and paternally derived genomes. J Ultrastruct Res 29:86, 1969 Dura C, Chakraborty J, Ste£anini M, et al: The fine structure of the post-coital oviduct of the mouse. In Proc Seventh Intern Congr Electr Microsc. Edited by P Favard. Grenoble, 1970, Vol 3, p 625 Pika L: Mechanism of sperm penetration in the rat and the Chinese hamster based on fine structural studies. In Proc Fifth Intern Cong Anim Reprod. Trento, 1964, Vol 7, p 301 Pika L: Gamete Structure and Sperm Entry in Mammals, 1st edition. Edited by CB Metz and A Monroy. r-few York, 1969, Vol 2, p 325
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Zamboni L, Stefanini M: On the configuration of the plasma membrane of the mature spermatozoon. Fertil Steril 19:570, 1968 Zamboni L, Mishell DR Jr, Bell JH, et al: Fine structure of the human ovum in the pronuclear stage. J Cell BioI 30:579, 1966 Zamboni L, Stefanini M, Hando T: Electron microscope studies on fertilization in the mouse. In Proc Sixth Intern Meet Anim Reprod Artif Insem. Edited by C Thibault. Paris, 1968, Vol 1, p 639 Zamboni L, Stefanini M, Oura C, et al: The pattern of sperm penetration into the mouse egg. In Proc Seventh Intern Cong Electr Microsc. Edited by P Favard. Grenoble, 1970, Vol 3, p 663 Zamboni L, Chakraborty J, Smith D: First cleavage division of the mouse zygote. An ultrastructural study. BioI Reprod 7: 170, 1972 Zamboni L, Thompson RS, Moore-Smith D: Fine morphology of human oocyte maturation in vitro. BioI Reprod 7:425, 1972 Yanagimachi R, Noda YD: Ultrastructural changes in the hamster sperm head during fertilization. J Ultrastruct Res 31:465, 1970 Yanagimachi R, Noda YD: Electron microscope studies of sperm incorporation into the golden hamster egg. Am J Anat 128:429, 1970 Fraser LR, Dandekar PV, Gordon MK: Loss of cortical granules in rabbit eggs exposed to spermatozoa -in vitro. J Reprod Fertil 29:295, 1972 Cross PC, Brinster RL: In vitro development of mouse oocytes. BioI Reprod 3:298, 1970 Iwamatsu T, Chang MC: In vitro fertilization of mouse eggs in the presence of bovine follicular fluid. Nature (Lond) 224:919,1969 Mukherjee AB, Cohen MM: Development of normal mice by in vitro fertilization. N ature (Lond) 228:472, 1970
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Whittingham DG: Fertilization of mouse eggs in vitro. Nature (Lond) 220:592, 1968 46. Iwamatsu T, Chang MC: Factors involved in the fertilization of mouse eggs in vitro. J Reprod Fertil 26: 197, 1971 47. Iwamatsu T, Chang MC: Sperm penetration in vitro of mouse oocytes at various times during maturation. J Reprod Fertil 31:237, 1972 48. Edwards RG, Gates AH: Timing of the stages of the maturation divisions, ovulation, fertilization and the first cleavage of eggs of adult mice treated with gonadotrophin. J Endocrinol 18:292, 1959
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Donahue RP: Fertilization of the mouse oocyte: sequence and timing of nuclear progression to the two-cell stage. J Exp Zool 180:305, 1972 Fawcett DW, Ito S: The fine structure of bat spermatozoa. Am J Anat 116:567, 1965 Zamboni L, Zemjanis R, Stefanini M: The fine structure of monkey ami human spermatozoa. Anat Rec 169:129, 1971 Zamboni L, Moore-Smith D, Thompson RS: Migration of follicle cells through the zona pellucida and their sequestration by human oocytes in vitro. J Exp Zool 181:319, 1972 Trump BF, Goldblatt PJ, Howell RE: Studies on necrosis of mouse liver in vitro. Ultrastructural alterations in the mitochondria of hepatic parenchymal cells. Lab Invest 14: 343, 1965 Blandau RJ, Odor LD: Observations on sperm penetration into the ooplasm and changes in the cytoplasmic components of the fertilizing spermatozoon in rat ova. Fertil Steril 3: 13, 1952 Austin CR, Braden A WH: Time relation and their significance in the ovulation and penetration of eggs in rats and rabbits. Aust J BioI Sci 7:179, 1954
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Longo FJ, Anderson E: The fine structure of pronuclear development and fusion in the sea urchin, Arbacia punctulata. J Cell BioI 39:339, 1968 57. Szollosi DG, Calarco P, Donahue RP: Absence of centrioles in the first and second meiotic spindles of mouse oocytes. J Cell Sci 11: 521, 1971 58. Austin CR: Variations and anomalies in fertilization. In Fertilization. Edited by CB Metz and A Monroy. New York, Academic Press, 1969, Vol 2, p 437 59. Smith DM, Oura C, Zamboni L: Fertilizing ability of structurally abnormal spermatozoa of the mouse. Nature (Land) 227:79, 1970 60. Thompson RS, Zamboni L: Phagocytosis of supernumerary spermatozoa by two-cell mouse embryos. Anat Rec 178:3, 1974 61. Enders AC, Schlafke SJ: The fine structure of the blastocyst: some comparative studies. In Preimplantation Stages of Pregnancy. Edited by GEW Wolstenholme and M O'Connor. Boston, Little, Brown & Co, 1965, p 29 56.