Ultrastructural Observations on Human Oocytes Fertilized in Vitro*

Vol. 25, No.1, January 1974 Printed in U.S.A.

Fr::RTlLITY AND STERILITY

Copyright

©

19i4 The American Fertility Society

ULTRASTRUCTURAL OBSERVATIONS ON HUMAN OOCYTES FERTILIZED IN VITRO':' PIERRE SOUPART, M.D., PH.D.,

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PATRICIA ANN STRONG, M.A.

Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232

Since the first reported attempts at human fertilization in vitro by Rock and Menkin 1 in 1944, investigators have consistently been confronted with the problem of authenticating fertilization they claimed was obtained in culture. Cleavage alone, a widely used criterion, is not enough. Unfertilized mammalian ova often undergo degenerative fragmentation that may mimic cleavage. If chromatin stammg helps distinguish fragmented from cleaved ova, the presence of chromatin does not rule out cleavage due to artificial activation. Workers generally agree on three criteria as convincing evidence of fertilization of the mammalian ovum: (1) the presence of two or more polar bodies in the perivitelline space, (2) the presence of two pronuclei within the ooplasm, and (3) the presence of remnants of the fertilizing sperm flagellum within the ooplasm. A careful review of the evidence up to 1969 led Mastroianni and Noriega" to conclude that, if rigid criteria were applied, in vitro fertilization had not yet been accomplished in the human. Since then, research has progressed rapidly, however. Fertilization was unquestionably demonstrated for the first time when Bavister et aP identified by light microscopy the middlepiece and tail of a spermatozoon in a pronuclear ovum fixed and stained

between 11 and 14.5 hours after insemination. Numerous ultrastructural studies on mammalian fertilization, both in vivo and in vitro, have led to a detailed description of the morphologic criteria for fertilization" Although the ultrastructure of a tubal human pronuclear ovum was described earlier," it provided no information on how the spermatozoon penetrated the zona pellucida. The first requirement for mammalian fertilization is the availability of mature oocytes (those having completed their first meiotic division). The second requirement is the availability of capacitated sperm (sperm having acquired the ability to penetrate through the zona pellucida of the oocyte). Only when investigators recognized the need for sperm capacitation S - U was in vitro fertilization in a mammal (the rabbit) successfully achieved." o,l1 According to current concept,"2 capacitation is a conditioning of the sperm surface, at least around the anterior two-thirds of the acrosome, occurring in the female genital tract. It permits the acrosome reaction in the immediate vicinity of the zona pellucida."" This reaction is an orderly vesiculation process 14 exposing the inner acrosomal membrane, which appears to be the site 15 of the acrosomal proteinase 1G (an enzyme needed for sperm to pass through the zona pellucida). Because morphologic evidence of capacitation is unknown, capacitation must be demonstrated functionally. However,

Received April 30, 1973. 'Supported by U.S.P.H.S. Contrast NIH-70-2162 and Ford Foundation Grant 630-0141A.

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for a spermatozoon traversing the zona pellucida to have undergone the acrosome reaction implies that it had to be capacitated. The need for capacitation of human sperm was suggested by fertilization of mature human oocytes17 ,18 using a modification of a culture medium that had proven effective in capacitating hamster sperm.19 Previous studies from our laboratory20 provided further functional evidence of the concept of human sperm capacitation. Sperm-penetrating ability through the zona pellucida was enhanced by the presence of gonadotropins in the fertilization culture medium. The capacitation process was thought to be mediated by gonadotropin-stimulated follicle cells. However, authentication of fertilization was hampered by our failure to demonstrate by light microscopy the remnants of the fertilizing sperm flagellum within the ooplasm examined at a late pronuclear stage (28 hours after insemination). The present study, designed to remedy this, describes the fine morphology of the human sperm traversing the zona pellucida of the human ovum in culture, the ultrastructural criteria of human fertilization in vitro, and other ultrastructural features of the human zygotes obtained in culture. MATERIALS AND METHODS

Oocyte Recovery arullnitial Evaluation. Oocytes were recovered by direct aspiration of follicles on ovaries in situ during elective gynecologic surgery, or on excised ovaries, using the ovum recovery unit previously described. 21 Oocyte recovery was performed at random (i.e., without scheduling surgery to coincide with ovulation and without treatment to induce ovulation). Quality evaluation at oocyte recovery was performed by examination with the 16x objective of a microscope equipped with differential interference contrast optics (Nomarski's

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optics) . The oocytes were classified as preovulatory, nonovulatory, and degenerate, on the basis of morphologic criteria previously described. 20 Maturation and Fertilization Cultures. Unless otherwise specified, immature oocytes were cultured in Ham's FlO medium for 48 hours, using a two-stage maturation system. To compensate for the lability of pyruvate ions in solution, 0.6 mM sodium pyruvate was added to the medium before use. Immature oocytes were primed with 0.1 p.g/ml of 17f3estradiol in this medium for 4 hours, then cultured for the rest of the maturation period in the same medium containing 0.1 p.g/ ml 1 7a- h ydroxyprogesterone instead of estradiol. The details of this technique will be described elsewhere. 2.2 Preovulatory oocytes were kept in the second maturation medium for the minimum time (about 4 hours) necessary to obtain semen and prepare the fertilization medium. Sperm suspensions were prepared, and fertilization cultures were carried out as previously described,20 using a modification of the medium used by Edwards et al.17 Morphologic Analysis. Mter 24 hours in fertilization culture, ova were fixed for 10 minutes in 3% glutaraldehyde in 0.1 M cacodylate buffer, and rinsed in the same buffer. The specimens were postfixed in 1 % osmium tetroxide in Zetterqvist's salt solution,23 as formulated by Sjostrand. 24 The specimens were dehydrated in ethanol series and embedded either in Spurr's medium or in Araldite by Luft's method. 25 Survey sections (0.51.0 microns) were stained with 1 % toluidine blue on a glass slide and examined with a Zeiss photomicroscope equipped with differential interference contrast optics. Silver-colored thin sections, obtained with an LKB Ultratome III, were collected on a Formvar film on one-hole copper grids. They were stained with uranyl acetate and lead citrate, and ex-

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FIG. 1. High-resolution light micrograph of a survey section (0.5-1.0 ~m) through a pronuclear human ovum matured and fertilized in culture. The male pronucleus (mPN) and the second polar body (PB.) are visible on this section. The zona pellucida (ZP) is incompletely surrounded by corona cells (CC). Processes from the corona cells have retracted from the zona pellucid a, creating a space that separated the zona from the corona cell. The ooplasm (0) contains numerous discrete vacuoles, which could be a sign of early degeneration. The enlargement of the perivitelline space (pvs) at lower right is where the first polar body was found deeper in this specimen. The female pronucleus, not visible on this section, was also found deeper in the specimen and located subcortically to the right of the second polar body. Maturation culture: 24 hours; fertilization culture: 24 hours. (x650.)

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amined with a Zeiss 9S-2 electron microscope equipped with low magnification pole pieces. Each specimen was serially sectioned from pole to pole. OBSERVATIONS

Number of Specimens. Sixteen specimens were sectioned serially and analyzed. Fourteen were apparently normally fertilized. Another had undergone dispermic fertilization and the last was unfertilized but showed signs of abnormal activation. In none of the fertilized specimens were supplementary sperm found in the perivitelline space. Supplementary sperm were observed within the zona pellucida in 13 of the apparently normally fertilized ova, their number varying from one to three. All fertilized specimens, fixed 24 hours after insemination, were found to be at late pronuclear stages. Sperm with reacted or unreacted acrosomes, as well as the described relationship between sperm and corona cells, were observed in all fertilized specimens. General Organization of the Human Ovum Fertilized in Culture. The general organization of human oocytes matured and fertilized in culture is illustrated by Figure 1. The ovum may be surrounded by corona cells. The processes of the corona cells retracted from the zona pellucida, forming a separating space. Several sperm were observed, either near the zona, lying with the flat surface of the head on its outer surface, or touching it by the tip of the head (Fig. 1). Most of the sperm, however, were trapped between or sequestered by corona cells. The perivitelline space was somewhat irregular but generally crescent-shaped. The section plane passed through a polar body, which, owing to the virtual absence of cortical granules was identified as the second polar body. The vitellus had short microvilli at the surface of the vitelline membrane, almost no cortical

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granules, organelles tending to migrate toward the center of the ooplasm, and a pronucleus. The pronucleus was located subcortically and almost diametrically opposite the second polar body. Although no remnants of the fertilizing sperm flagellum could be identified by light microscopic examination of the survey section shown in Figure 1, a thin section taken at this level (Fig. 2) revealed a cross section through the coarse fibers of the sperm flagellum, close to the edge of the pronucleus. This proximity to the pronucleus strongly suggested that the structure was, indeed, the male pro-

FIG. 2. Cross section through the coarse outer fibers (cf) of the fertilizing sperm middlepiece, located very close to the edge of the pronuclear membrane (pnm). One of the nucleoli (n) of the pronucleus is also visible. Same specimen as Figure 1. (x29,900.)

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nucleus. Deeper survey sections revealed another polar body located in the second enlargement of the perivitelline space (Fig. 1) and containing numerous cortical granules. Thus, it was identified as the first polar body. Still deeper in the same specimen, another pronucleus was found (Fig. 3). It was located in a subcortical area close to the two polar bodies in the perivitelline space. This was identified as the female pronucleus, confirming the identification of the other one as the male pronucleus. The two pronuclei remained widely separated 24 hours after insemination, pos-

FIG. 3. Female pronucleus (fPN) with two of its nucleoli visible, located subcortically and very close to the second polar body (not visible) in the enlarged area of the perivitelline space. ZP = zone pellucida; CC = corona cells. Same specimen as Figures 1, 2. (x1,960.)

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sibly due to a short maturation culture period (see Discussion). All other serial sections through this ovum, up to its opposite pole, revealed no other nuclear structure or sperm

FIG. 4. Longitudinal section through the head and proximal part of the neck of one of the most normal-appearing spermatozoa observed in the space between zona pellucida and corona cells. The characteristic components are: the slightly undulated sperm plasma membrane (pm), the anterior acrosome (aa), the equatorial segment of the acrosome (eq), the postacrosomal cap (pac), the striated band (sb), the redundant nuclear membrane (rnm) , the basal or implantation plate (bp), the sperm proximal centriole (pc), a striated column (sc), and the mitochondrial spiral (ms). The principal piece of the flagellum of another spermatozoon, cut transversally, is also visible. (x22,900.)

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flagellum. No supplementary sperm were found, either in the perivitelline space or within the zona pellucida of this specimen. Very few supplementary sperm were observed in all other specimens, and then only within the zona pellucida. Apparently, then, the specimen illustrated by Figures 1-3 had undergone monospermic fertilization in culture. The Acrosome Reaction and Passage of Sperm Through the Zona Pellucida. Two sperm types were consistently observed in the space created by retraction of the corona cell processes from the zona pellucida (Fig. 1). The first type, illustrated by Figure 4, shows a longitudinal section through a spermatozoon head and neck. The chromatin, nearly homogenous in this specimen, looked different in other spermatozoa, varying from coarsely granulated to nearly homogenous. There were several vacuoles of various sizes in the nucleus, the larger ones being most often located in the anterior pointed part of the nucleus. These vacuoles were not bounded by a membrane. Their content was finely granular, but irregular membranous structures were also sometimes present. Some vacuoles opened directly in the subacrosomal space. The chromatin was bounded by a nuclear membrane containing nuclear pores 26 (of difficult resolution and not visible on Fig. 4) . The acrosome covered at least the anterior two-thirds of the nucleus, from which it was separated by a thin layer of structureless cytoplasm of constant thickness. The homogenous, electrondense content of the acrosome was bounded by inner and outer membranes paralleling each other. The distance separating these membranes was about 700 A in the anterior or reactive part of the acrosome,26 about 350 A in the posterior part sometimes called the "unchangeable" equatorial segment of the acrosome. 26 These two segments of the acrosome are clearly illustrated by Figure

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5, showing a para sagittal section of a sperm head lying on the outer surface of the zona pellucida. The sperm plasma membrane (appearing slightly undulated in Fig. 4) ran parallel to the outer acrosomal membrane and was separated from it by a thin layer of structureless cytoplasm. At the posterior margin of the acrosome, the nucleus was deeply indented. Posterior to the acrosome, the postacrosomal cap was parallel to the nuclear surface. It was separated from it by a thin layer of structureless cytoplasm, considered an extension of the subacrosomal cytoplasm. 26

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FIG. 5. Longitudinal section through the head of a spermatozoon with unreacted acrosome, illustrating the difference in width of the anterior acrosome (aa) and of the equatorial segment (eq). (x21,400.)

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The postacrosomal cap is formed by two distinct components (not distinguishable in Fig. 4): the plasma membrane and, underneath it, a layer of dense material with transversally oriented ridges,26 constituting the postacrosomal lamina. 27 ,28 The posterior boundary of the postacrosomal cap is marked by the posterior ring, characterized by the striated band.29 At this level, the sperm plasma membrane evaginates, allowing the neck cytoplasm to surround the basal part of the nucleus. From the level of the striated band, the redundant nuclear membrane detaches itself from the chromatin to form the posterior nuclear space. The redundant nuclear membrane is continuous with the basal or implantation plate. The implantation plate consists of an outer thick lamina and an mner thin lamina, both electron-dense. It is separated from the chromatin by a thin space of uniform width and low electron density, similar to that separating the two laminae of the implantation plate. Posterior to these structures, one of the segmented colunms, the proximal centriole (cut transversally), proximal mitochondria, and the initial mitochondria of the mitochondrial spiral can also be seen on Figure 4. Thus, sperm of this type, found close to the zona pellucida 24 hours after insemination in culture, are structurally identical to the most normal-appearing spermatozoa described in the human ejaculate. 26 -3o It should be noted that the human spermatozoon lacks the apical subacrosomal material described as perforatorium 31 in other mammalian species. The second type of sperm was found at the surface of the zona pellucida (Fig. 6). The rostral half of the sperm head was flanked by a shroud of vesicles, apparently formed by intermittent fusion of the plasma membrane with the outer acrosomal membrane.14 The equatorial segment of the acrosome appeared unaffected by vesiculation. Such sperm, which have

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undergone the acrosome reaction, were those seen starting (Fig. 7) or having almost completed their penetration through the zona pellucida (Figs. 8, 9). Figure 7 shows a sperm at the initial stage of zona penetration. Several mem-

FIG. 6. Longitudinal section through the head of a spermatozoon, lying on the outer surface of the zona pellucida (ZP). It has undergone the acrosome reaction, a process of intermittent fusion of sperm plasma membrane and outer acrosomal membrane, resulting in the formation of vesicles (ves) and exposing the inner acrosomal membrane (iam). A transversal section through the middlepiece of another spermatozoon is also visible, showing the sperm plasma membrane, the mitochondrial sheath, the nine outer dense fibers, and the axial filament complex. (x22,200.)

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branous vesicles, resulting from the acrosome reaction, were being discarded at the zona surface as the sperm head slid inward along a curved penetration pattern, starting almost tangential to the outer zonal surface. The inner acrosomal membrane at the tip of the sperm head was in intimate contact with the filamentous material of the zona. The outer wall of the acrosome equatorial segment, formed by the outer acrosomal membrane fused to the sperm plasma

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membrane, appeared everted. The postacrosomal cap, with its electron-dense postacrosomallamina, appeared intact. Figure 8 shows a sperm head, seen from profile, in the last segment of its curved penetration slit. The tip of the sperm head was emerging in· the perivitelline space. The angle of emergence makes it clear that the flat surface of the sperm head had to slide on the vitelline membrane during the final stage of sperm

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.. FIG. 7. Parasagittal section through the head of a spermatozoon with reacted acrosome, starting its penetration through the zona pellucida (ZP) at a very acute angle. The inner acrosomal membrane is in intimate contact with the fibrous material of the zona. Vesiculated acrosomal products (ves) are being discarded by the head and left at the zona surface. On either side of the nucleus, the outer wall of the equatorial segment (oweq) of the acrosome is everted. In the sperm nucleus, a large vacuole opens directly into the subacrosomal space and contains granular and membranous material. (x42,600.)

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penetration through the zona pellucida. The sperm shown in Figures 8 and 9 are two of the few supplementary sperm observed in this study. The ova, in which

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supplementary sperm were seen, had already been activated, as shown by the absence of cortical granules under the vitelline membrane and the short micro-

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FIG. 8. Longitudinal section through the head (seen from profile) and proximal part of the neck of a spermatozoon with reacted acrosome emerging into the perivitelline space. Only vestiges (v) of the equatorial segment remain trapped in a circular indentation of the nucleus. The inner acrosomal membrane (iam) is in intimate contact with the fibrous material of the zona (ZP). The sharpness of the penetration slit (PS) strongly suggests that the acrosomal proteinase is firmly bound to the inner acrosomal membrane. This is a supplementary sperm, and the absence of electron dense cortical granules underneath the membrane of the oocyte (0) indicates that the ovum is already activated. Maturation culture: 48 hours; fertilization culture: 24 hours. (x20,850.)

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villi at the membrane surface. However, the characteristics of the sperm head penetrating the zona pellucida are obvious. The inner acrosomal

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membrane closely contacted the zona material. The penetration slit from the sperm passage was extremely s,harp, as seen in the area immediately posterior to the sperm head. Also, vestiges of only the outer wall of the acrosome equatorial segment seemed to remain trapped in the circular indentation of the sperm head (Fig. 8). In other sperm observed within the zona pellucida, the outer wall of the acrosome equatorial segment had completely disappeared (Fig. 9). The postacrosomal cap was still intact. The posterior part of the nucleus was surrounded by a layer of neck cytoplasm. The redundant nuclear membrane was continuous with the implantation plate (Fig. 8). The longitudinal section through the neck region (Fig. 8) shows the segmented columns and their relationship to the coarse outer fibers of the flagellum, as well as the initial gyri of the mitochondrial spiral. The cross section through the mitochondria of the third and fourth gyri suggests that the mitochondrial structure was being altered, and that the sperm might have lost its motility at this stage of zona penetration. The Criteria of Fertilization and Other Ultrastructural Attributes of Fertilized Ova. The criteria of fertilization consist of the presence of two or more polar bodies in the perivitelline space, the absence of cortical granules in the vitellus, the presence of two pronuclei and of remnants of the fertilizing sperm flagelFIG. 9. Oblique section through the head and proximal part of the neck of a spermatozoon with reacted acrosome, deeply embedded in the zona pellucida (ZP), but not yet, reaching the perivitelline space (pvs). The curved penetration pattern, starting tangent to the outer surface of the zona and ending tangent to the inner surface of the zona, is obvious. The oocyte (0) has already been activated by the fertilizing sperm, as shown by the absence of cortical granules underneath the vitelline membrane. Coated vesicles or acanthosomes, in formation or already formed, are visible at the base of the microvilli. Maturation culture: 48 hours; fertilization culture: 24 hours. (x21,400.)

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lum within the ooplasm. In favorably oriented sections, such as that illustrated by Figure 10, the two polar bodies were generally found side by side in a lenticularly distended area of the perivitelline space. The first polar body was irregular in shape, to the point that it often appeared fragmented (as it probably often becomes with aging). However, because of its irregular profile, a thin section through the superficial aspects of the first polar body could give the impression of multiple fragments, as shown by the profile of an unfragmented first polar body in a mature but unfertilized oocyte (Fig. 11). The plasma membrane of the first polar body was slightly ruffled (Fig. 12), with few microvilli. The first polar body chromosomes appeared as filamentous chromatin devoid of a membrane. In Figure 12, they seemed to be scattered in several fragments of the polar body, and were associated with residual microtubules from the first meiotic spindle. Because the first polar body was released before activation of the oocyte by

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the fertilizing sperm, it contained numerous electron-dense cortical granules. The presence of filamentous chromatin devoid of a membrane and numerous cortical granules as well as the scarcity of other oocyte organelles (Figs. 11, 12) constitutes specific criteria for identification of the first polar body' or its fragments. The second polar body differed in appearance (Fig. 13). It was ovoid in shape. The filamentous chromatin was being surrounded by a double membrane, with nuclear pores in some areas (Fig. 13, inset). Since the second polar body had extruded after the cortical reaction 32 (which was triggered by the fertilizing sperm establishing contact with the vitelline membrane), it had few, if any, cortical granules. Some microtubules from the second meiotic spindle were observed. The plasma membrane of the second polar body was only slightly ruffled and almost devoid of microvilli. The absence of cortical granules and the reconstitution of a nucleus are criteria that distinguish the second polar body

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FIG. 10. Typical appearance of the polar bodies, located in a lenticular enlargement of the perivitelline space, between the zona pellucid a (ZP) and the oocyte (0). The first polar body (PB1) appears fragmented. It contains fibrillar chromatin, residual microtubules from the first meiotic spindle, electron dense cortical granules, and a few other oocyte organelles. The second polar body (PB.) is ovoid. It contains a reforming nucleus, residual microtubules of the second meiotic spindle, but no cortical granules. A tight bundle of microtubules (arrow) from the second meiotic spindle possibly containing the midbody, appears isolated from both oocyte and the second polar body. Maturation culture: 48 hours; fertilization culture: 24 hours. (x3,900.)

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from the first.4 Apparently, a tight bundle of second meiotic spindle microtubules, possibly containing the midbody, can be excluded from both the oocyte and the second polar body. Occasionally, the button-like terminations (Fig. 14) of corona cell processes were seen in the perivitelline space. They appeared as ovoid globules of cytoplasm, bounded by a plasma membrane and filled almost completely with microfibrils. Several dense bodies, bounded by a single membrane and resembling lysosomes, were lined up parallel to the plasma membrane area facing the vitelline membrane. In the area of close approximation of the corona cell process and the vitelline membrane, a junctional zone was

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observed, characterized by increased electron density of both corona cell plasma membrane and vitelline membrane, and by the presence of tight junctions (Fig. 14). The vitelline membrane displayed numerous short microvilli as well as some blunt projections protruding into the perivitelline space (Figs. 12-14). Microvilli were longer and thinner in the area of the membrane where polar bodies had been extruded (Figs. 10-13). Large numbers of cytoplasmic processes, vesicles, and membranous profiles also occupied this area of the perivitelline space. The vitelline membrane exhibited marked pinocytotic activity, and numerous coated vesicles (acanthosomes) were observed just underneath the vitelline membrane

FIG. 11. Section through the first polar body (PB,) of a mature but unfertilized oocyte, located in an enlargement of the perivitelline space between the zona pellucida (ZP) and the oocyte (0). The first polar body has an irregular profile, and a thin section through its superficial aspects could make it appear fragmented. It contains cortical granules (cg), filamentous chromatin (chr) , and residual microtubules (t) from the first meiotic spindle. The oocyte has not been activated as evidenced by the presence of cortical granules (cg) underneath the vitelline membrane. Maturation culture: 48 hours; fertilization culture: '24 hours. (x6,200.)

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(Figs. 8, 9). Numerous organelles were present throughout the ooplasm, with a marked tendency to concentrate near the pronuclei (Fig. 15). Mitochondria were of two different types: spheroidal (Fig. 16) and dumbbellshaped (Fig. 17). The spheroidal mitochondria was almost exclusively present in late pronuclear stages. These organelles contained but a few cristae arranged either parallel to the outer mitochondrial membrane or transversally in the matrix. Many of the spheroidal mitochondria had arch-like cristae located at one of their poles (Fig. 16). The spheroidal mitochondria were often seen close to variablesize vesicles of the endoplasmic reticulum,

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which were filled with a granular material of low electron density. Large and small endoplasmic reticulum vesicles were distributed throughout the ooplasm, with clusters of the smaller vesicles surrounding the larger ones. The dumbbellshaped mitochondria (Fig. 17), characterized by transversally oriented cristae and symmetrical constriction in the central region, were extremely few in late pronuclear stages. They were most conspicuous in early pronuclear stages, where they appeared to be involved in the synthesis of pronuclear membranes. 33 Golgi complexes, clusters of closely packed vesicles surrounding arrays of parallel tubules, were distributed through-

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FIG. 12. Details of the fragmented first polar body (PB i ) of a fertilized ovum (0). Filamentous chromatin (chr) is present in several fragments, all containing cortical granules (CG). Note the absence of cortical granules underneath the vitelline membrane. ZP = zona pellucida. Maturation culture: 48 hours; fertilization culture: 24 hours. (x8,400.)

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out the ooplasm, but were most abundant in the interpronuclear area (Fig. 18). Annulate lamellae were present in considerable numbers (Fig. 18), but were located in the immediate vicinity of the pronuclei. They were arranged in stacks consisting of 3 to 25 parallel units. In sections perpendicular to their surface (Fig. 19), the lamellae appeared to consist of two parallel smooth membranes delimiting flat cisternae. Both ends of the cisternae were dilated. The membrane limiting the lamellae joined each other at regular intervals, the junction being characterized by increased electron density. Material of medium electron

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density appeared to connect the pore areas of successive annulate lamellae in a given stack (Fig. 19) . In sections tangential to the pores of the lamellae, the ring-shaped configuration of the annuli and their hexagonal arrangement was clearly visible (Fig. 20). Crystalline inclusions were observed only rarely (Fig. 21). They were seen as parallel arrays of 10 to 15 rods, arranged in bundles with pointed extremities. The pronuclei were eccentrically (Figs. 1, 3) or centrally (Fig. 15) located. The distance between them varied according to pronuclear age. In specimens fixed at about 24 hours after insemination, the

FIG. 13. Details of the second polar body (PB.) of a fertilized ovum (0). A nucleus (N) is reforming, and the chromatin is being surrounded by a nuclear membrane (nm, inset). The second polar body contains mitochondria (m) but no cortical granules. ZP = zona pellucida. Maturation culture: 48 hours; fertilization culture: 24 hours. (x8,400; inset x19,700.)

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distance separating the pronuclei was often of 10 to 20 microns, whereas in an older specimen20 (28 hours after insemination) the spherical pronuclei were closely apposed to each other, with a flattened surface in the area of apposition. The two pronuclei were about the

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same size, with identical characteristics. They appeared elliptical in cross section when still widely separated from each other (Fig. 15). Their outline was regular and limited by a typical nuclear membrane but with fewer nuclear pores than somatic cell nuclear membrane.

FIG. 14. The button-like termination of a corona cell process, persIstmg in the perivitelline space of a fertilized ovum (0), after retraction of the corona cell processes. It consists of a tight bundle of microfibrils (mf) of variable orientation. A row of electron dense bodies, resembling lysosomes, is lined up parallel to the area of contact between corona cell process and vitelline membrane. The area of contact exhibits a junctional zone (jz) characterized by increased electron density of both membranes. A tight junction (ti) is indicated by the arrow. On both sides, microfibrils and cytoplasmic filaments terminate into the membrane areas of increased electron density. ZP = zona pellucida. Maturation culture: 48 hours; fertilization culture: 24 hours. (x21,400.)

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From place to place, outpocketings of the outer leaflet of the nuclear membrane toward the ooplasm were observed (Fig. 22). They contained rounded or elongated vesicles bounded by an independent membrane, containing granular material of medium electron density. The pronuclear

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chromatin was highly hydrated, but with localized condensations of finely fibrillar material of high electron density (Fig. 18). Up to eight nucleoli were present in each of the older pronuclei. 20 They were spheroidal in shape, and made of highly compacted granular or fibrillar

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FIG. 15. Concentration of organelles in the interpronuclear area of the ooplasm in a fertilized ovum. PN = pronucleus. Maturation culture: 'loB hours; fertilization culture: 24 hours. (x1,BOO.)

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FIG. 16. Spheroidal mitochondria, with archlike cristae at one pole (arrow), and closely associated with large endoplasmic reticulum vesicle (ERV). (x33,500.)

FIG. 17. A dumbbell-shaped mitochondrion, with transversal cristae (arrows), in close association with small endoplasmic reticulum vesicles and cisternae. (x23,900.)

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material of high electron density. A few short, single annulate lamellae were observed in the pronucleoplasm, often close and parallel to the pronuclear membrane

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(Fig. 22). They resembled the annulate lamellae in the ooplasm but were never arranged in stacks as in Figures 18 and 19. Remnants of the fertilizing sperm flagellum were observed in various locations. Except when a portion of the sperm flagellum was cut longitudinally (Fig. 23), identification of flagellum remnants usually required patient, meticulous scanning of thin sections in order to disclose cross sections through the axial filameJ;1t complex. Some of these cross sections were smaller than the smallest mitochondria (Figs 24, 25) . In specimens fixed 24 hours or more after insemination, only the axial filament complex had retained its original 9 + 2 organization. Sometimes one could observe the outer row

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FIG. 19. Section perpendicular to the surface of annulate lamellae showing the pores, distended end of each lamella, and the interlamellar material of medium electron density which connects the edges of the pores in successive lamellae. (x32,400.)

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FIG. 20. Section tangent to the surface of annulate lamellae, showing the ring-shaped structure of the pores and their hexagonal arrangement (arrow). (x19,700.)

FIG. 21. Crystalline inclusions (ci), a tight bundle of rods with pointed ends. Also shown here is a grazing section through a curvature of the principal piece (pp) of the fertilizing sperm flagellum. The ribs of the fibrous sheath are cut through longitudinally and transversally. A portion of the axial filament complex is cut through longitudinally. (x19,500.)

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IN VITRO FERTILIZED OOCYTES

of nine dense fibers that originally surrounded the axial filament complex (Fig. 2) , but the mitochondria of the mitochondrial spiral had disappeared and the coarse fibers of the middle and principal piece were pulling away from the axial filament complex (Fig. 25). In favorable sections, the apparently unchanged sperm proximal centriole could also be observed (Fig. 25). The proximity of these structures to the membrane of one of the pronuclei suggested that such

29

pronucleus could be the male one (Fig. 25). Also, the presence of a microtubule, extending from the centriole toward the membrane of one of the pronuclei, suggested that the sperm proximal centriole might be involved in the mechanism that brings the two pronuclei together in the center of the ovum. The Relationship Between Sperm and Corona Cells at the Beginning and End of Fertilization Culture. Direct observation of the events during in vitro insemination was hampered by the presence of corona cells surrounding the oocytes. They either blurred or completely masked the zona pellucida surface. In some of the cultured oocytes, however, the shell of corona cells was incomplete and some areas of the zona surface were directly exposed to the sperm suspension. This

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FIG. 22. Section through the edge of a pronucleus (PN) showing the pores of the pronuclear membrane (pnm), an intrapronuclear annulate lamella (ial), and a nucleolus (N). Note the striking morphologic similarity between pronuclear membrane and intrapronuclear annulate lamella. (x24,800.)

FIG. 23. Longitudinal section through a wavy segment of the fertilizing sperm flagellum axial filament complex (afc). Maturation culture: 48 hours; fertilization culture: 24 hours. (x22,250.)

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FIG. 24. Section through the interpronuclear area of a fertilized ovum, showing two pronuclei (PN), and a cross section through the axial filament complex (AFe) sperm flagellum. An outpocketing of the outer leaflet of the pronuclear membrane an elongated vesicle of granular material. Maturation culture: 48 hours; fertilization (xlW,850.)

the edges of the of the fertilizing (arrow) contains culture: 24 hours.

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IN VITRO FERTILIZED OOCYTES

facilitated direct observation, with an inverted phase-contrast microscope, of the early relationship between sperm and exposed areas of the zona surface, as well as between sperm and masses of corona cells. Vigorously swimming sperm, hitting exposed areas of the zona surface, remained attached by the tips of the heads to the zona for a few seconds and ap-

31

peared to effect drilling motions centered around the tip of their head. However, they soon detached themselves and swam away with what appeared to be utter disregard for their target. Vigorously swimming sperm hitting masses of corona cells appended to the oocytes behaved differently. Although some sperm appeared to hit corona cells and bounce back swimming outward, a great many sperm

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FIG. 25. The edge of a pronucleus, is tentatively identified as the male pronucleus (mPN) because of the close proximity of the sperm proximal centriole (SPC) and of the axial filament complex (AFC). Coarse outer fibers (CF) are pulling away from the axial filament complex. Vestiges of a microtubule (mt) seem to extend from the male pronuclear membrane toward the sperm proximal centriole, which is cut through longitudinally and appears as the blind end of a flat-bottomed tube . Maturation culture: 48 hours; fertilization culture: 24 hours. (x20,100.)

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FIG. 26. Section through the peripheral aspects of a cluster of corona cells showing the many sperm deeply embedded between thick corona cell processes (CCP). The sperm nucleus (SN) contains a vacuole opening in the subacrosomal space. This spermatozoon, like most sperm seen in association with corona cells, has undergone the acrosome reaction. The inner acrosomal membrane is beginning to break down. At lower right, a section through the tail of an abnormal spermatozoon exhibits in the same plane four sections at different levels of the flagellum, all enclosed in the same plasma membrane. The fine granular material is probably the decondensing chromatin of the sperm nucleus. (x14,OOO.)

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33

,~

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FIG. 27. Partial and complete sequestration of spermatozoa by corona cells. Like the vast majority of sperm associated with corona cells, these three sperm have a reacted acrosome, but their membranes are breaking down. Chromatin decondensation begins in the posterior area of the nucleus. (x9,200.)

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FIG. 28. A spermatozoon sequestered by a corona cell. It has retained all its membranes. The acrosome is swollen, and the acrosomal material has collected on one side, in a hanging drop manner toward the tip of the nucleus. (x9,700.)

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buried themselves deeper between corona cells, while retaining vigorous flagellum movements. Despite dir~ct observation during the first 5 hours of fertilization cultures, immediate or early penetration of sperm through the zona pellucida was never recorded. On specimens fixed at the end of fertilization cultures, the relationship between sperm and corona cells was easily observed. Almost all sperm had undergone the acrosome reaction and were either deeply embedded between corona cell processes (Fig. 26) or sequestered by corona cells (Fig. 27). The chromatin of sequestered sperm appeared to undergo decondensation. The process apparently started at the posterior end of the nucleus and extended forward. Occasionally, a spermatozoon was observed in a sequestration vacuole, having retained all its membranes in the anterior region of the acrosome (Fig. 28). The acrosome was swollen, and the acrosomal material appeared to have collected in one area of the inner acrosomal membrane, in a hanging-drop manner. In contrast with the dismantling of the structural elements of the flagellum observed in the ooplasm of fertilized ova, all components of the flagellum seen within sequestration vacuoles in corona cells had retained their original reciprocal organization (Fig. 28). Thin sections were meticulously scanned for evidence of fusion between corona cells and spermatozoa without success. Occasionally, an isolated centriole was observed in the cytoplasm of corona cells (Fig. 29). This probably represented either the basal corpuscle of the single cilium that these cells seem to possess 34 or one of the two cell centrioles, since it was observed equally close to the nucleus and to the cell membrane. The second possibility seems more likely, owing to the presence of microtubules that seem to converge toward the centriole (Fig. 29). Consistently, corona

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FIG. 29. Cross section through a corona cell centriole, showing the nine triplets and the electron dense connecting material. Microtubules (arrows) are converging toward the centriole. (x68,900.)

cells in clusters were observed to have retained several sharply defined electrondense junctional zones. Anomalies. Two abnormal specimens were observed. The first was a dispermic ovum, exhibiting three pronuclei (Fig. 30). Remnants of two differently oriented flagella were found. This ovum appeared normal in every other aspect: two polar bodies were present in the perivitelline space, cortical granules had disappeared, and the zona pellucida was intact. The second abnormal specimen was a mature unfertilized oocyte, in which the characteristic first polar body was present in the perivitelline space. The vitellus had a full complement of cortical granules but, instead of the expected second meiotic spindle, two pronuclear-like structures of dissimilar size were present. They were close to each other and in the center of the ooplasm (Fig. 31). Except for their different size, these pronuclear-like structures had all the attributes of normal pronuclei. They contained nucleoli, a few intranuclear annulate lamellae were present, and their chromatin was highly hydrated but with localized condensa-

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35

FIG. 30. The three pronuclei of a dispermic ovum. (x2,200.)

FIG. 31. Two pronuclear-like structures of unequal size m the central area of a mature but unfertilized oocyte. (x2,200.)

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tions of finely fibrillar material of high electron density. Many ooplasmic annulate lamellae and Golgi complexes were present, mainly in the area separating the two pronuclear-like structures. Systematic serial sectioning of this specimen from pole to pole failed to reveal the presence of a sperm flagellum. DISCUSSION

Pronuclear human ova were analyzed by high-resolution light microscopy and electron microscopy to evaluate the conditions for maturation and fertilization of oocytes in culture. Only the morphologic aspects of the problem of fertilizing human ova in culture will be discussed here. The quantitative aspects of the problem will be reported in a forthcoming publication. 22 Our primary objective was to authenticate fertilization by demonstrating remnants of sperm flagellum in human ova at late pronuclear stages. Study specimens were obtained by semination in culture of matured oocytes in vitro. Previous studies from our laboratory20 had convinced us of the unreliability of conventional methods for such experiments. The present observations explain our past failure to demonstrate by light microscopy remnants of the sperm flagellum at late pronuclear stages. The various structures of the sperm flagellum are progressively dismantled as pronuclear age increases. Obviously, the first elements to break away from the flagellum complex are the mitochondria. These were never observed in specimens fixed 24 hours after insemination, because they had dispersed throughout the ooplasm and disintegrated beyond recognition, even by means of electron microscopy. The next components to dissociate themselves from the flagellum complex were the nine outer dense fibers of the

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middlepiece and proximal principal piece. In only one instance (Figs. 1-3), did the dense fibers retain their spatial relationship to each other. However, this specimen had been cultured for only 24-hour maturation. Probably, maturation was incomplete at the time of insemination, it had continued during the beginning of fertilization culture, and the stage of fertilization at the time of fixation was early pronuclear, possibly due to late fertilization. This interpretation is suggested not only by the typical arrangement of the dense fibers (Fig. 2), but also by the wide distance separating the two pronuclei. Both were located subcortically but at sites diametrically opposed. In one instance only, a specimen cultured 48 hours for maturation and fixed 24 hours after insemination, could the outer dense fibers be identified. They were pulling away from the axial filament complex and were identifiable only because of their presence on several serial thin sections (Fig. 25). The fibrous sheath of the principal piece seemed to persist around the axial filament complex somewhat longer than the other outer components, because it was observed on several occasions, with the typical appearance illustrated by Figure 21. The only structural element to persist consistently in all specimens fixed 24 hours after insemination was the axial filament complex (Figs. 24, 25). How easily the axial filament complex was found depended upon its orientation with respect to the plane of section. When cut through longitudinally, it was seen immediately in favorable sections (Fig. 23). This was also true in high-resolution light-microscopic survey sections. When the axial filament complex was cut transversally, however, the thin sections required patient and meticulous scanning, because the cross-section area was smaller than that of most mitochondrial cross sections (Figs. 24, 25). Even when some

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of the most heavily staining elements, such as the dense outer fibers, were retained, cross sections through the middlepiece could not be identified by high-resolution light microscopy (Fig. 1). Our observations suggest that lightmicroscopic identification of the sperm flagellum described by Dickmann et al,35 Noyes et al,36 and Bavister et aP was obtained from ova at a much earlier pronuclear stage. In the first study,35 the tubal specimen was thought to be as young as 3 hours after ovulation, while in the last, 3 the specimens were fixed and analyzed from 11 to 14.5 hours after insemination in cultur.e. In both studies, however, the middle piece of the sperm flagellum was clearly visible on the published micrographs. Its typical short length and its large diameter (compared to that of the rest of the flagellum) suggest that the mitochondrial sheath had not yet dispersed when these specimens were fixed. In the tubal pronuclear ovum described by Zamboni et aV the sperm flagellum seemed to have disintegrated further, since the mitochondrial sheath was not observed, but the dense outer fibers had retained their characteristic spatial relationship with the axial filament complex . Our observations make it apparent that the fertilizing sperm flagellum remnants in human ova at a late pronuclear stage can be demonstrated reliably only by electron microscope . The penetration of the spermatozoon into the ovum starts a series of transformations involving the nuclear and cytoplasmic components of both ovum and sperm. 5 These transformations characterize the "stage of activation," which culminates in the pairing, on the equatorial plate of the first cleavage spindle, of male and female chromosomes that were duplicated during the pronuclear stage. In the specimens we analyzed, all changes leading to pronuclear transformation had already occurred, but none

37

of the specimens had reached the point at which the pronuclear membrane breaks down, the duplicated chromosomes from both pronuclei condense, and they rearrange themselves on the equatorial plate of the first cleavage spindle. For the discussion of cytoplasmic and nuclear components, the reader is referred to the analysis of a tubal pronuclear ovum by Zamboni et al, 5 since almost identical features were observed in our study. A few remarks, however, are worth adding here. In specimens fixed 24 hours after insemination, very few elongated or dumbbell-shaped mitochondria were found. It has been hypothesized that, in order to maintain a constant number of mitochondria in daughter cells, mitochondrial duplication has to precede the stage of cell cleavage. 5 It was also assumed that elongated mitochondria represented those about to divide. If this is correct, the fact that few elongated mitochondria were found in ova at late pronuclear stage might signify that mitochondrial duplication was completed in our specimens. By contrast, in a companion study,33 where early changes leading to the formation of both male and female pronuclei were observed, elongated mitochondria, although not in majority, were quite numerous, and were conspicuously involved in the energy-dependent process of pronuclear membrane synthesis. The early degeneration of sperm mitochondria had been considered extremely important, 5 because it implies that the mitochondrial endowment of the embryo would be exclusively maternal in derivation. We feel that this is not necessarily so. No evidence exists that sperm mitochondrial DNA is hydrolyzed and loses its identity. If it remained intact, despite the structural dislocation of sperm mitochondria, it could well be incorporated into ovum mitochondria, since a process of mitochondrial duplication seems to be actively proceeding in early pronuclear ova. If

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such were the case, cytoplasmic inheritance would not be uniquely of maternal derivation in the embryo. The same companion study 33 provided a likely interpretation for the presence of single annulate lamellae (Fig. 22) within the pronuclei. These could be no more than isolated stretches of pronuclear membrane, trapped within the pronuclei during the process of chromatin rehydration and expansion. The striking morphologic similarity between intrapronuclear annulate lamellae and pronuclear memhrane is evident in Figure 22. In the specimens analyzed in this study, there was no strongly suggestive evidence for a microtubular apparatus eventually responsible for the movement of pronuclei toward each other and toward the center of the ovum. In one thin section, however, what appeared to be vestiges of a microtubule were observed, apparently extending from the sperm proximal centriole toward the surface of one of the pronuclei (Fig. 25). The possibility of the transient existence of such a microtubular apparatus cannot therefore be excluded. The spheroidal aggregates of narrow tubules surrounded by a corona of mitochondria, described by Zamboni et aP7 as being numerous and prominent in human oocytes maturing in culture, were never observed in pronuclear ova. Probably, these aggregates serve a purpose necessary for the maturation process, which has ceased to exist in fertilized ova. Golgi complexes were few in peripheral areas of the ooplasm, but they were abundant in the interpronuclear area (Fig. 18). There were many cytoplasmic annulate lamellae but only in the interpronuclear area of the ooplasm (Fig. 18). These cytoplasmic annulate lamellae, the function of which is unknown (KesseP8), have been described as a feature of the tubal pronuclear human ovum by Zamboni et ai.5 More recently, Zamboni

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et aP7 suggested that the presence of annulate lamellae only in the degenerating or anomalous oocytes cultured for maturation, may be a sign of atretic degeneration. In the present study, however, cytoplasmic annulate lamellae were always observed with pronuclei, eithe;r in normally fertilized ova (Fig. 18); dispermic ovum (Fig. 30), or mature unfertilized ovum containing pronuclearlike structures (Fig. 31). None of these exhibited signs of advanced degeneration. We did not find cytoplasmic annulate lamellae in degenerating ova. We also did not find the other type of parallel lamellae, similar to annulate lamellae in morphology and spatial arrangement but lacking typical "annuli" or "pores" and associated with numerous ribosomes (described by Zamboni et aP7 as representing a form of transition or degradation of ergastoplasmic reticulum elements into annulate lamellae). Obviously, the significance of cytoplasmic annulate lamellae must be further investigated. Great care was exercised in the present study in order to detect early signs of degeneration. In none of the fertilized ova was gross vacuolization of the ooplasm observed. However, practically all specimens studied exhibited some degree of very discrete vacuolization (Figs. 1, 9, 10, 12, 15, 18, 24, 25, 30, 31), and occasionally these small vacuoles were seen to become confluent. Such a phenomenon may reflect the present inadequacy of culture media, especially that of the fertilization medium, which is a modified Tyrode solution. It is devoid of amino acids and other components present in maturation media. Further investigations will indicate whether vacuolization can be avoided by shortening the fertilization culture period to that necessary to obtain sperm penetration. The migration of entire corona cells into the perivitelline space and the sequestration of such cells by the oocytes,

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as described by Zamboni et al,34 were not observed. Detached button-like terminations of corona cell processes, however, were occasionally present in the perivitelline space (Fig. 14). The presence of junctional zones, with one or several tight junctions (Fig. 14), suggested the existence of a special arrangement, possibly destined to prevent diffusion into the perivitelline space of metabolites being transferred from corona cells to oocyte. Such junctional zones and tight junctions were also described by Zamboni et al. 34 Whether or not human spermatozoa must be capacitated before fertilization has been the subject of much speculation among researchers. Opinions vary from utter scepticism to strong belief in sperm capacitation as a feature of human fertilization. We have provided the first ultrastructural evidence that human sperm undergo capacitation just as any other mammalian sperm studied. Capacitation is a conditioning of the sperm surface 12 (at least in the area surrounding the anterior two-thirds of the acrosome) that sperm undergo in the female genital tract, permiting the acrosome reaction within the cumulus or corona cell mass or near the zona pellucida. 13 The acrosome reaction is an orderly vesiculation process resulting from intermittent fusion of the sperm plasma membrane and the outer acrosomal membrane. 14 In this process, the inner acrosomal membrane becomes exposed, and appears to be the site 15 of the acrosomal proteinase,16 an enzyme thought to be necessary for sperm to gain passage through the zona pellucida. In this study, two types of sperm were observed in the immediate vicinity of the zona pellucida. Sperm of the first type (Fig. 4) had retained all the ultrastructural features of the most normalappearing spermatozoa present in human ejaculates. 26 -3o Since all specimens were

39

fixed 24 hours after insemination, it appears that sperm do not necessarily undergo membrane breakdown from being washed, centrifuged, diluted to appropriate concentrations, and cultured for 24 hours. The second type of sperm (Fig. 6) had the flat surface of the head on the outer surface of the zona pellucida. The acrosome reaction had occurred or was occurring at the time of fixation, since the rostral half of the head was flanked by a shroud of acrosomal vesiculation products. The equatorial segment appeared unaffected by the vesiculation process. In sperm starting to penetrate through the zona pellucida (Fig. 7), the vesiculated products of the acrosome reaction were discarded by the sperm head at the zona surface. In sperm lying on the outer surface of the zona, the equatorial segment appeared to persist (Fig. 6). However, in sperm having begun to penetrate the zona, the outer wall of the segment was everted (Fig. 7). In sperm emerging into the perivitelline space, the segment was merely a vestige (Fig. 8). In sperm embedded in the zona, the segment had completely disappeared (Fig. 9). This is at variance with what has been observed in mammals other than huans: the equatorial segment remains unchanged in sperm in the zona, in the perivitelline space, or even in the early stage of gamete fusion (Bedford 39 ) . In this study, the inner acrosomal membrane was in direct contact with the zona material in all cases (Figs. 7-9) . The sharpness of the penetration slit (Fig. 8) strongly suggests that the acrosomal proteinase is firmly bound to the inner acrosomal membrane. The even electron density along the edges of the penetration slit clearly indicates the absence of diffusion of proteolytic activity from the inner acrosomal membrane. The enlargement of the penetration slit is due to the arching of the sperm, resulting from the flexion of the head around the

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articulation of the implantation plate. In a live specimen, one would expect the next flagellum beat to bring about a mirror image of Figure 8; the general curvature of the sperm would become tangent to the vitelline membrane, and the enlargement of the penetration slit would appear on the opposite side of the neck articulation and deeper into the zona pellucida. Thus, the human spermatozoon passing through the zona pellucida of the oocyte has undergone the acrosome reaction. In the light of the present concept of sperm capacitation, it follows that the human spermatozoon must have been capacitated to begin with. The spermatozoa seen passing through the zona pellucida, however, were all supplementary sperm (i.e., sperm other than the fertilizing one). These could not be observed because fertilization cultures were carried out for 24 hours. Supplementary sperm, however, were rarely seen. None of the analyzed specimens had supplementary sperm in the perivitelline space. Two fertilized specimens had only one supplementary sperm each, either within the zona (Fig. 8) or emerging in the perivitelline space (Fig. 9). In addition, one specimen was found to be dispermic, implying that two spermatozoa did pass through the zona (Fig. 30), but no other sperm was seen in the perivitelline space. Since oocytes were seminated individually in 50 p.l droplets of sperm suspension containing either 0.5 or 1.0 x 105 motile sperm, and since many sperm with reacted acrosomes were observed lying on the surface of the zona, it appears that the block to polyspermy was effected mainly at the zona. Such a zona reaction might develop from the diffusion through the zona of material released from cortical granules at oocyte activation by the fertilizing sperm, and the reaction of this material with the zona. This

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mechanism would make the zona resistant to the acrosomal proteinase. The zona reaction of hamster and mouse eggs can be produced in vitro by a trypsin-like protease from cortical granules. 40 Such a mechanism, however, does not exclude the possibility of other mechanisms acting on sperm that could reach the perivitelline space, or acting at the level of the vitelline membrane itself. No evidence that could support such a concept was obtained in this study. If the block to polyspermy relies only on the zona reaction, the dispermic ovum (Fig. 30) probably resulted from synchronous penetration of the zona by two spermatozoa, and synchronous fusion of these two spermatozoa to the oocyte, rather than from a failure of the block to polyspermy since, in the latter case, one would have expected a much higher degree of polyspermy. For the specimen in Figure 30, it was not possible to identify the female pronucleus and compare the two male pronuclei for size. Moreover, male and female pronuclei are of equal size at this stage of development, so a younger male pronucleus probably could not be identified on the basis of its size alone. The apparently smaller size of one of the pronuclei (Fig. 30) is simply due to the fact that the section plane coincided with the equatorial plane of only two of the pronuclei. Thus, it appears that the human zona pellucida is of extreme functional importance. It not only provides mechanical protection to one of the most voluminous cells of the mammalian organism (which otherwise would be very vulnerable after its release from the ovarian follicle), but it also seems to play a major role in the block to polyspermy. The latter concept Was strongly supported by another study made in our laboratory,33 in which semination of zona-free mature oocytes led to extreme polyspermy, some of the

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specimens exhibiting more than 100 developing male pronuclei. Edwards et aI's reported that sperm were not observed in the perivitelline space earlier than 7 to 7.5 hours after insemination. Our numerous direct observations during the first 4 to 5 hours of fertilization culture never disclosed early passage of sperm through the zona pellucida. It therefore appears that human sperm capacitation in the in vitro fertilization environment is not achieved before 5 to 7 hours after insemination. Edwards et al17 suggested that human sperm capacitation could be induced by suitable environmental conditions (Bavister's medium 19 ) and by allowing for the spontaneous occurrence of the process. Such a hypothesis does not seem likely. The preparation of sperm suspension for fertilization culture requires about an hour, during which time spermatozoa are suspended in Bavister's medium. Furthermore, definite evidence of sperm presence in the perivitelline space was not obtained (by Edwards et aP8) until 7 to 7.5 hours after insemination. This amounts to an 8-hour exposure of sperm to the fertilization medium before sperm passage through the zona pellucida (assuming that the passage requires very little time, as it does in other mammalian species). If capacitation were spontaneous, due to medium conditions alone, at least a few specimens would exhibit sperm passage through their zona sooner after insemination than has been observed. Our previous studies 20 found that human sperm capacitation in vitro is mediated by follicle cells surrounding the oocyte, and that the phenomenon is significantly enhanced by gonadotropin stimulation of the follicle cells in vitro. Consequently, particular attention was paid in this study to the relationship between sperm and corona cells at the end of fertilization cultures. Even after 24 hours in fertilization culture, corona

41

cells surrounding the ova did not disperse. Several clusters and layers of cells remained around the zona, separated from it by a space created by the retraction of the processes which formerly extended through the thickness of the zona. The vast majority of sperm outside the zona were found close to the corona cells. Most of these sperm had undergone the acrosome reaction (Fig. 26, 27). No vesiculated acrosomal products or strips of membranse were close to the sperm heads. Such products were probably left behind during sperm progression between corona cells. The inner acrosomal membrane, as well as the postacrosomal cap of these sperm, was beginning to disintegrate. These sperm were deeply embedded in thick corona cell processes, deeply buried in intercellular spaces, or sequestered by the corona cells. Their nuclei were undergoing chromatin decondensation, apparently starting at the posterior nuclear end. This has also been found to take place in the rabbit. 39 Some of the sequestered sperm had retained all their membranes. However, the acrosome was considerably swollen and the acrosomal contents had collected on one or both sides of the sperm nucleus (Fig. 28). Despite a meticulous search, no evidence of fusion of sperm and corona cell could be detected. This suggests that sperm with reacted acrosome are able to fuse with oocyte only. Although the relationship between sperm and corona cells was analyzed much too late to permit observation of early changes in sperm membranes, it appears that many sperm close to corona cells had undergone the acrosome reaction. This may indicate that the changes leading to the acrosome reaction occur while sperm are passing through the layers of corona cells. Sperm with intact membranes in the space between the corona cells and the zona (Fig. 4) probably gained access to that

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space without forcing their way through several layers of corona cells. Ovulated oocytes in the mouse, fixed by glutaraldehyde perfusion of the oviduct in situ prior to fertilization, exhibit a wide dispersion of their corona cells. 4 Mouse spermatozoa, however, appear to be easily capacitated in chemically defined media. 41 The degree of dispersion of corona cells around oocytes in the human oviduct is presently ill-defined. Of the two pronuclear tubal ova described thus far, one had to be freed from cumulus mass by means of hyaluronidase treatment 36 • 37 and its postovulation age was estimated to be 3 to 50 hours, while the other had only a few cells of the cumulus oophorus present outside the zona pellucida, 5 but its postovulation age was not estimated. In the mature but unfertilized specimen, which was observed to exhibit two pronuclear-like structures in its ooplasm (Fig. 31), it seems that abnormal activation of the second meiotic spindle did occur, without extrusion of a second polar body or of the contents of the cortical granules. Apparently, the second metaphase chromosomes did separate in two unequal numbers, and the process stopped during anaphase. Each group of chromosomes then appears to have undergone the process of female pronucleus formation, and two pronuclear-like structures of widely different size migrated toward the center of the ooplasm. If the cause of such abnormal behavior remains obscure, the abnormal character of this specimen was immediately detected by light microscopy, even prior to fixation, owing to the lack of one polar body and the size disparity of the "pronuclei." SUMMARY

Serial sectioning and high-resolution light microscopy combined with electron microscopy of human oocytes, matured

Vol. 25

and fertilized in culture and fixed 24 hours after insemination, has led to the following conclusions: 1. At late pronuclear stages, fertilization can be best authenticated by means of electron microscopy, conventional methods being unreliable. This is due to progressive dismantling of the ferilizing sperm flagellum. 2. Although the passage of the fertilizing sperm through the zona pellucida was not observed owing to late (24 hours) fixation, the supplementary sperm observed while negotiating their passage through the zona had undergone the acrosome reaction. Therefore, in the light of the current concept of capacitation, human sperm must be capacitated prior to fertilization. 3. The extreme scarcity of supplementary sperm within the thickness of the zona, and their total absence from the perivitelline space of fertilized specimens, implies that the block to polyspermy in human ova is effective mainly at the level of the zona, through the "zona reaction." 4. Although the early relationship between sperm and corona cells was not analyzed in the present study, the ultrastructural findings 24 hours after insemination strongly suggest that capacitation occurs when sperm are forcing their way between corona cells to reach zona pellucida. These findings, together with those of previous studies, strongly support the concept that human sperm capacitation is mediated through gonadotropin-stimulated corona cells in in vitro fertilization experiments. 5. Although all ultrastructural features of human ova fertilized in culture were found to be identical to those of a human tubal pronuclear ovum described in the literature, a discrete degree of ooplasmic vacuolization suggested that present culture conditions are still not entirely adequate and require further investigation.

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Acknowledgments. The authors wish to acknowledge the technical skills of John E. Repp and the secretarial skills of Anne J ett. REFERENCES

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r

t .,

Rock J, Menkin MF: In vitro fertilization and cleavage of human ovarian eggs. Science 100:105, 1944 2. Mastroianni L Jr, Noriega C: Observations on human ova and the fertilization process. Am J Obstet Gynecol 107:682, 1970 3. Bavister BD, Edwards RG, Steptoe PC: Identification of the mid-piece and tail of the spermatozoon during fertilization of human eggs in vitro. J Reprod Fertil 20: 159, 1969 4. Zamboni L: The Fine Morphology of Mammalian Fertilization. New York, Harper and Row, 1971 5. 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 6. Chang MC: Fertilization of rabbit ova in vitro. Nature (Lond) 184:466, 1959 7. Chang MC: Fertilizing capacity of spermatozoa deposited in the Fallopian tube. Nature ILond) 168:687, 1951 8. Austin CR: Observations on the penetration of sperm into the mammalian egg. Aust J Sci Res B4:581, 1951 9. Austin CR: The "capacitation" of the mammalian sperm. Nature (Lond) 170:326, 1952 10. Thibault C, Dauzier L, Wintenberger S: Etude cytologique de la fecondation in vitro de l'oeuf de la lapine. C R Soc BioI (Paris) 148:789, 1954 11. Chang MC: Fertilization of rabbit ova in vitro. Nature (Lond) 184:466, 1959 12. Austin CR: Capacitation of spermatozoa. Int .J Fertil 12:25, 1967. 13. Bedford JM: Ultrastructural changes in the sperm head during fertilization in the rabbit. Am .J Anat 123:329, 1968

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14.

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