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Acrosome loss in fertilizing mammalian spermatozoa: A clarification
© 1971 by Academic Press, Inc.
J. ULTRASTRUCTURE RESEARCH 34,
401-405 (1971)
401
L E T T E R TO T H E E D I T O R
Acrosome Loss in Fertilizing Mammalian Spermatozoa: A Clarification LUCIANO ZAMBONI
Department of Pathology, School of Medicine, University of California Los Angeles, California 90024 Received July 29, 1970 Some points discussed by Yanagimachi and Noda in a recent article entitled "Ultrastructural changes in the hamster sperm head during fertilization" published in the Journal of Ultrastructure Research [31, 465-485, 1970] require clarification. Such clarification is necessary not only because, in my opinion, the conclusions reached and presented by these authors add confusion to an already nebulous subject, but also and mainly because, in the note added in the proof, the authors cite a recent study performed in my laboratory on the ultrastructure of fertilization of mouse eggs (1) and state that our observations are in good agreement with the results of their investigation. The omission of many of our conclusions may induce readers to believe that our ideas about the pattern of acrosome loss by the fertilizing spermatozoon indeed coincide with those of Yanagimachi and Noda. Nothing could be farther from the truth. The main difference between our study and that of Yanagimachi and Noda is that we made every possible effort (by perfusing with glutaraldehyde the whole reproductive tracts of female mice at various times after coitus and ovulation and by serially sectioning for electron microscopy the undisturbed contents of tubal ampullae) to study the morphological changes, if any, undergone by the fertilizing spermatozoon during its progression through the egg envelopes. In spite of the large numbers of ova studied in this fashion, we failed to observe any phase of the progression of the fertilizing spermatozoon through the cumulus oophorus and the zona pellucida. The earliest stage of fertilization that we were able to observe was that of initial fusion of the gamete membrane, at which time the fertilizing sperm appeared to be deprived of the acrosome. Thus, we concluded the following: 1. The progression of fertilizing spermatozoa through the envelopes of nonactivated ova must be very rapid. This assumption is in good agreement with the opinion of classic investigators who attempted to study this stage of the fertilization of mam-
402
ZAMBONI
malian ova by means of light and phase microscopy. In 1951, Austin (2) stated, "The penetration of the sperm into the egg is a very rapid process. It probably takes no more than a few minutes at most for the head of the sperm to pass through the zona, judging by the fact that among over 1,200 eggs examined none showed a sperm head in the thickness of the zona." In 1955, Austin and Braden (3) wrote, "Little can be said of the passage of the spermatozoon through the zona pellucida as it is very rare to find an egg with a spermatozoon head still in the thickness of the zona. The rarity of such a finding bears witness to the rapidity with which passage through the zona must occur." 2. At the time of earliest contact between gametes, the fertilizing spermatozoon lacks the acrosome. However, due to our failure to observe the progression of the fertilizing sperm through the cumulus oophorus and the zona pellucida, we added that we could not say exactly at what stage prior to contact with the egg membrane acrosome loss occurs. In my opinion, the study of the ultrastructural changes of the fertilizing spermatozoon during its progression through the egg envelopes is a formidable task. If one considers several points (a) that in natural conditions the egg to be fertilized is associated only with the fertilizing sperm (1, 4), (b) that we have no information as to the exact time this sperm arrives at the cumulus of the egg, (c) that we cannot predict from where around the cumulus the fertilizing spermatozoon begins the last leg of its journey toward the egg, and (d) that the fertilizing sperm has to move through a layer not exceeding 50 # in thickness one must conclude that it is highly unikely that this phase of fertilization can be observed in a few thin sections for electron microscopy. For these reasons we clearly publicized our deep conviction that most, if not all, of the copious acrosome-deprived spermatozoa found by other investigators in the envelopes of ova flushed from the fallopian tubes (5-8) are not fertilizing spermatozoa but represent supernumerary, dislocated, or degenerated sperm. I think that such is also the case for the spermatozoa in the study by Yanagimachi and Noda. The purpose of these authors was to investigate the fine morphological changes of hamster spermatozoa shortly before their arrival at the surface of the egg, and during the phase of early contact with the latter. Their study involved two experimental models: (a) ejaculated sperm in association with ova flushed from the oviducts of female hamsters mated at vaguely defined intervals in relationship to spontaneous ovulation and killed 5-7 hours after coitus, and (b) epididymal spermatozoa maintained in vitro for 3-4 hours in association with ova which had been obtained by superovulation and cultured for unspecified periods of time. It is evident that the spermatozoa in these two models attained maturity under very different circumstances (under natural conditions in the reproductive organs vs. in vitro) and that they were associated with 2 classes of ova (spontaneously ovulated monospermic eggs vs. polyspermic eggs
ACROSOME LOSS IN FERTILIZING MAMMALIAN SPERMATOZOA
403
ovulated in excessive numbers following gonadotropin stimulation). Yet, Yanagimachi and Noda indiscriminately blend the information derived from studying such different experimental models and, in the text as well as in the micrographs of their article, little mention is made about the origin of the spermatozoa whose morphological features are described and illustrated. In the discussion section, the differences between the two experimental models, and their possible impact on sperm behavior and morphology, have been completely ignored. In my opinion, any study directed at describing the morphological changes of spermatozoa in the process of interacting with ova should at least define the condition of the ova in association with the sperm whose particular ultrastructural features are being described. The micrographs should show morphologic features which permit the reader to establish whether the ova are activated or not and, if activated, what stage of activation has been attained. The features, which should be illustrated are the presence or the absence of cortical granules, the number of polar bodies extruded, and the configuration, arrangement, and localization of the egg chromosomes. In the article by Yanagimachi and Noda, none of these parameters is mentioned in the text. In the illustrations, with the exception of Fig. 16, the only elements of egg structure shown are a few microvilli and minute areas of cortical cytoplasm lacking dense granules. Most of their micrographs illustrate acrosome-deprived sperm heads in unrecognizable loci in relationship to the eggs. That Yanagimachi and Noda do not consider the stage of activation of the ova around which the sperm were found as an important parameter for the interpretation of their observations is demonstrated by the fact that for the examination of the ultrastructure of these spermatozoa cumuli isolated from flushed tubal ova were utilized (Materials and Methods, p. 465). In the observations section, Yanagimachi and Noda report having studied the acrosome changes in spermatozoa that were about to penetrate, or in the act of penetrating, the zona pellucida 3-4 hours after being added to cultured ova (p. 468). In consideration of the fact that the authors themselves state that spermatozoa came into direct contact with the zona almost immediately after insemination and that 3-4 hours after, they were in various stages of penetration into the eggs (Materials and Methods, p. 466), and in consideration of the fact that passage of the fertilizing sperm through the egg envelopes is very rapid (see above), it is evident that the sperm they found in the act of penetrating through the zona 3-4 hours after insemination likely were supernumerary spermatozoa around polyspermic ova in advanced stages of activation. Very probably, thus, Yanagimachi and Noda have investigated supernumerary spermatozoa. Nevertheless, the ultrastructural features of these sperm have been considered as if they were those of fertilizing spermatozoa, a mistake which may be very serious indeed, and against which Chang in 1967 (9) gave the following warning, "The electron microscopy of spermatozoa in the cumulus clot, in between the
404
ZAMBONI
corona cells, in the zona pellucida or in the vitellus is also of interest, but I must mention here that to get the fertilizing spermatozoon, not the supplementary or accessory spermatozoa, in the egg is not an easy task; and we have to be careful about our conclusion. Besides the possibility of mistaking one structure for another, any ultrastructure of a few spermatozoa may not represent the whole story." In describing the effect of fusion by vesiculation of the sperm plasma membrane the authors state, "Obviously the vesiculated outer acrosome and plasma membranes of the acrosome cap region, as well as the contents of the acrosome cap, had been completely discarded before the spermatozoa started to enter the zona pellucida" (p. 468). I take the liberty of disagreeing with the authours on the use of the adverb "obviously" since, as emphatically stated and photographically documented in our study, the vesiculated remnants of sperm plasma and outer acrosomal membranes, i.e., the structural elements of a reaction which is presumed to occur outside the zona, are consistently present around the heads of fertilizing spermatozoa in the perivitelline space at the time of their incipient conjugation with the ova. In conclusion, the results of the study by Stefanini et al. (1) concerning the loss of the acrosome in fertilizing spermatozoa do not agree with the views expressed by Yanagimachi and Noda. The present state of our knowledge on the subject allows us to say only that, at the time of initial contact of the gametes, the head of the fertilizing sperm lying on the egg membrane lacks an intact acrosome. However, since the passage of the fertilizing spermatozoon through the cumulus cells and the zona pellucida of a nonactivated mammalian ovum remains to be described and illustrated, we are in no position to say exactly where, when, and by what mechanisms the fertilizing spermatozoon loses its acrosome. It is evident from reports of our studies and from this letter that I am not at all convinced that acrosome loss in the fertilizing spermatozoon occurs during its progression through the egg envelopes for the purpose of liberating enzymes destined to depolymerize the matrix cementing the cells of the cumulus oophorus and the substance of the zona pellucida. M y incredulity has been vigorously boosted by the second article by Yanagimachi and Noda entitled "Physiological changes in the post-nuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells" appearing on p. 486 of the same issue of this Journal. In this article, the authors studied the differential ability of "capacitated" and "uncapacitated" spermatozoa to enter "naked" eggs, i.e., eggs freed of cumulus cells by treatment with hyaluronidase, and of zona pellucida by treatment with tripsin. Figures 2 and 3 of their article illustrate a capacitated spermatozoon conjugating with one of these eggs. The spermatozoon has lost its acrosome in spite of the fact that cumulus cells and zona pellucida were not present and, thus, did not need to be depolymerized.
ACROSOME LOSS IN FERTILIZING MAMMALIANSPERMATOZOA
405
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
STEFANINI,M., OURA, C. and ZAMBONI,L., J. Submicrosc. Cytol. 1, 1 (1969). AUSTIN, C. R., Aust. J. Sci. Res., Ser. B, 4, 581 (1951). AUSTIN,C. R. and BRADEN,A. W. H., J. Exp. Biol. 33, 358 (1956). STEI~ANINI,M., OURA, C. and ZAMBONI,L., Abstr., 2nd Meet. Soe. Study Reprod., 1969, p. 27. HADEK, R., J. Ultrastruet. Res. 8, 161-169 (1963). BARROS,C., BEDFORD,J. M., FRANKLIN,L. E. and AUSTIN,C. R., J. Cell Biol. 34, C1-C5 (1967). BEDFORD, J. M., J. Reprod. Fertil., Suppl. 2, 35-48 (1967). BEDFORD,J. M., Amer. J. Anat. 123, 329 (1968). CHANG, M. C., J. Reprod. Fertil., Suppl. 2, 143 (1967).
J. ULTRASTRUCTURE RESEARCH 34,
401-405 (1971)
401
L E T T E R TO T H E E D I T O R
Acrosome Loss in Fertilizing Mammalian Spermatozoa: A Clarification LUCIANO ZAMBONI
Department of Pathology, School of Medicine, University of California Los Angeles, California 90024 Received July 29, 1970 Some points discussed by Yanagimachi and Noda in a recent article entitled "Ultrastructural changes in the hamster sperm head during fertilization" published in the Journal of Ultrastructure Research [31, 465-485, 1970] require clarification. Such clarification is necessary not only because, in my opinion, the conclusions reached and presented by these authors add confusion to an already nebulous subject, but also and mainly because, in the note added in the proof, the authors cite a recent study performed in my laboratory on the ultrastructure of fertilization of mouse eggs (1) and state that our observations are in good agreement with the results of their investigation. The omission of many of our conclusions may induce readers to believe that our ideas about the pattern of acrosome loss by the fertilizing spermatozoon indeed coincide with those of Yanagimachi and Noda. Nothing could be farther from the truth. The main difference between our study and that of Yanagimachi and Noda is that we made every possible effort (by perfusing with glutaraldehyde the whole reproductive tracts of female mice at various times after coitus and ovulation and by serially sectioning for electron microscopy the undisturbed contents of tubal ampullae) to study the morphological changes, if any, undergone by the fertilizing spermatozoon during its progression through the egg envelopes. In spite of the large numbers of ova studied in this fashion, we failed to observe any phase of the progression of the fertilizing spermatozoon through the cumulus oophorus and the zona pellucida. The earliest stage of fertilization that we were able to observe was that of initial fusion of the gamete membrane, at which time the fertilizing sperm appeared to be deprived of the acrosome. Thus, we concluded the following: 1. The progression of fertilizing spermatozoa through the envelopes of nonactivated ova must be very rapid. This assumption is in good agreement with the opinion of classic investigators who attempted to study this stage of the fertilization of mam-
402
ZAMBONI
malian ova by means of light and phase microscopy. In 1951, Austin (2) stated, "The penetration of the sperm into the egg is a very rapid process. It probably takes no more than a few minutes at most for the head of the sperm to pass through the zona, judging by the fact that among over 1,200 eggs examined none showed a sperm head in the thickness of the zona." In 1955, Austin and Braden (3) wrote, "Little can be said of the passage of the spermatozoon through the zona pellucida as it is very rare to find an egg with a spermatozoon head still in the thickness of the zona. The rarity of such a finding bears witness to the rapidity with which passage through the zona must occur." 2. At the time of earliest contact between gametes, the fertilizing spermatozoon lacks the acrosome. However, due to our failure to observe the progression of the fertilizing sperm through the cumulus oophorus and the zona pellucida, we added that we could not say exactly at what stage prior to contact with the egg membrane acrosome loss occurs. In my opinion, the study of the ultrastructural changes of the fertilizing spermatozoon during its progression through the egg envelopes is a formidable task. If one considers several points (a) that in natural conditions the egg to be fertilized is associated only with the fertilizing sperm (1, 4), (b) that we have no information as to the exact time this sperm arrives at the cumulus of the egg, (c) that we cannot predict from where around the cumulus the fertilizing spermatozoon begins the last leg of its journey toward the egg, and (d) that the fertilizing sperm has to move through a layer not exceeding 50 # in thickness one must conclude that it is highly unikely that this phase of fertilization can be observed in a few thin sections for electron microscopy. For these reasons we clearly publicized our deep conviction that most, if not all, of the copious acrosome-deprived spermatozoa found by other investigators in the envelopes of ova flushed from the fallopian tubes (5-8) are not fertilizing spermatozoa but represent supernumerary, dislocated, or degenerated sperm. I think that such is also the case for the spermatozoa in the study by Yanagimachi and Noda. The purpose of these authors was to investigate the fine morphological changes of hamster spermatozoa shortly before their arrival at the surface of the egg, and during the phase of early contact with the latter. Their study involved two experimental models: (a) ejaculated sperm in association with ova flushed from the oviducts of female hamsters mated at vaguely defined intervals in relationship to spontaneous ovulation and killed 5-7 hours after coitus, and (b) epididymal spermatozoa maintained in vitro for 3-4 hours in association with ova which had been obtained by superovulation and cultured for unspecified periods of time. It is evident that the spermatozoa in these two models attained maturity under very different circumstances (under natural conditions in the reproductive organs vs. in vitro) and that they were associated with 2 classes of ova (spontaneously ovulated monospermic eggs vs. polyspermic eggs
ACROSOME LOSS IN FERTILIZING MAMMALIAN SPERMATOZOA
403
ovulated in excessive numbers following gonadotropin stimulation). Yet, Yanagimachi and Noda indiscriminately blend the information derived from studying such different experimental models and, in the text as well as in the micrographs of their article, little mention is made about the origin of the spermatozoa whose morphological features are described and illustrated. In the discussion section, the differences between the two experimental models, and their possible impact on sperm behavior and morphology, have been completely ignored. In my opinion, any study directed at describing the morphological changes of spermatozoa in the process of interacting with ova should at least define the condition of the ova in association with the sperm whose particular ultrastructural features are being described. The micrographs should show morphologic features which permit the reader to establish whether the ova are activated or not and, if activated, what stage of activation has been attained. The features, which should be illustrated are the presence or the absence of cortical granules, the number of polar bodies extruded, and the configuration, arrangement, and localization of the egg chromosomes. In the article by Yanagimachi and Noda, none of these parameters is mentioned in the text. In the illustrations, with the exception of Fig. 16, the only elements of egg structure shown are a few microvilli and minute areas of cortical cytoplasm lacking dense granules. Most of their micrographs illustrate acrosome-deprived sperm heads in unrecognizable loci in relationship to the eggs. That Yanagimachi and Noda do not consider the stage of activation of the ova around which the sperm were found as an important parameter for the interpretation of their observations is demonstrated by the fact that for the examination of the ultrastructure of these spermatozoa cumuli isolated from flushed tubal ova were utilized (Materials and Methods, p. 465). In the observations section, Yanagimachi and Noda report having studied the acrosome changes in spermatozoa that were about to penetrate, or in the act of penetrating, the zona pellucida 3-4 hours after being added to cultured ova (p. 468). In consideration of the fact that the authors themselves state that spermatozoa came into direct contact with the zona almost immediately after insemination and that 3-4 hours after, they were in various stages of penetration into the eggs (Materials and Methods, p. 466), and in consideration of the fact that passage of the fertilizing sperm through the egg envelopes is very rapid (see above), it is evident that the sperm they found in the act of penetrating through the zona 3-4 hours after insemination likely were supernumerary spermatozoa around polyspermic ova in advanced stages of activation. Very probably, thus, Yanagimachi and Noda have investigated supernumerary spermatozoa. Nevertheless, the ultrastructural features of these sperm have been considered as if they were those of fertilizing spermatozoa, a mistake which may be very serious indeed, and against which Chang in 1967 (9) gave the following warning, "The electron microscopy of spermatozoa in the cumulus clot, in between the
404
ZAMBONI
corona cells, in the zona pellucida or in the vitellus is also of interest, but I must mention here that to get the fertilizing spermatozoon, not the supplementary or accessory spermatozoa, in the egg is not an easy task; and we have to be careful about our conclusion. Besides the possibility of mistaking one structure for another, any ultrastructure of a few spermatozoa may not represent the whole story." In describing the effect of fusion by vesiculation of the sperm plasma membrane the authors state, "Obviously the vesiculated outer acrosome and plasma membranes of the acrosome cap region, as well as the contents of the acrosome cap, had been completely discarded before the spermatozoa started to enter the zona pellucida" (p. 468). I take the liberty of disagreeing with the authours on the use of the adverb "obviously" since, as emphatically stated and photographically documented in our study, the vesiculated remnants of sperm plasma and outer acrosomal membranes, i.e., the structural elements of a reaction which is presumed to occur outside the zona, are consistently present around the heads of fertilizing spermatozoa in the perivitelline space at the time of their incipient conjugation with the ova. In conclusion, the results of the study by Stefanini et al. (1) concerning the loss of the acrosome in fertilizing spermatozoa do not agree with the views expressed by Yanagimachi and Noda. The present state of our knowledge on the subject allows us to say only that, at the time of initial contact of the gametes, the head of the fertilizing sperm lying on the egg membrane lacks an intact acrosome. However, since the passage of the fertilizing spermatozoon through the cumulus cells and the zona pellucida of a nonactivated mammalian ovum remains to be described and illustrated, we are in no position to say exactly where, when, and by what mechanisms the fertilizing spermatozoon loses its acrosome. It is evident from reports of our studies and from this letter that I am not at all convinced that acrosome loss in the fertilizing spermatozoon occurs during its progression through the egg envelopes for the purpose of liberating enzymes destined to depolymerize the matrix cementing the cells of the cumulus oophorus and the substance of the zona pellucida. M y incredulity has been vigorously boosted by the second article by Yanagimachi and Noda entitled "Physiological changes in the post-nuclear cap region of mammalian spermatozoa: a necessary preliminary to the membrane fusion between sperm and egg cells" appearing on p. 486 of the same issue of this Journal. In this article, the authors studied the differential ability of "capacitated" and "uncapacitated" spermatozoa to enter "naked" eggs, i.e., eggs freed of cumulus cells by treatment with hyaluronidase, and of zona pellucida by treatment with tripsin. Figures 2 and 3 of their article illustrate a capacitated spermatozoon conjugating with one of these eggs. The spermatozoon has lost its acrosome in spite of the fact that cumulus cells and zona pellucida were not present and, thus, did not need to be depolymerized.
ACROSOME LOSS IN FERTILIZING MAMMALIANSPERMATOZOA
405
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
STEFANINI,M., OURA, C. and ZAMBONI,L., J. Submicrosc. Cytol. 1, 1 (1969). AUSTIN, C. R., Aust. J. Sci. Res., Ser. B, 4, 581 (1951). AUSTIN,C. R. and BRADEN,A. W. H., J. Exp. Biol. 33, 358 (1956). STEI~ANINI,M., OURA, C. and ZAMBONI,L., Abstr., 2nd Meet. Soe. Study Reprod., 1969, p. 27. HADEK, R., J. Ultrastruet. Res. 8, 161-169 (1963). BARROS,C., BEDFORD,J. M., FRANKLIN,L. E. and AUSTIN,C. R., J. Cell Biol. 34, C1-C5 (1967). BEDFORD, J. M., J. Reprod. Fertil., Suppl. 2, 35-48 (1967). BEDFORD,J. M., Amer. J. Anat. 123, 329 (1968). CHANG, M. C., J. Reprod. Fertil., Suppl. 2, 143 (1967).