Cleavage Spindle Research Articles

The role of unequal cleavage and the polar lobe in the segregation of developmental potential during first cleavage in the embryo of Chætopterus variopedatus.

The inequality of the first cleavage division of the Chætopterus embryo is caused by the production of a small polar lobe and the internal shifting of the first cleavage spindle. This division produces a two-celled embryo containing a small AB and a large CD blastomere. These blastomeres have different morphogenetic potentials. Only the larvae resulting from isolated CD blastomeres are able to form bioluminescent photocytes, eyes and lateral hooked bristles. The removal of the polar lobe during first cleavage does not have a great effect on development. These lobeless embryos display a normal pattern of cleavages through the time of mesentoblast formation. The resulting larvae are essentially normal, however they do not form functional photocytes. If the CD cell is isolated after the removal of the first polar lobe, the resulting larva is virtually identical to those formed by the intact CD cell except it lacks the photocyte cells. These results indicate that two separate pathways are involved in the segregation of developmental or morphogenetic potential which takes place during first cleavage. One set of factors, which are necessary for photocyte formation, are associated with the first polar lobe. Other factors that are necessary for the formation of the eyes and lateral hooked bristles are segregated by the unequal cleavage which results from an internal shifting of the cleavage spindle. The removal of a large portion of the vegetal region of the embryo during first cleavage leads to the production of larvae which display a decreased ability to form eyes and lateral hooked bristles. These embryos frequently display an abnormal pattern of cleavages. They do not form the primary somatoblast or the mesentoblast. These results indicate that the vegetal region of the CD cell of Chætopterus is analogous to polar lobes which have been studied in other species, and is therefore important in the specification of the D quadrant. These features of the first cleavage of Chætopterus are a combination of those displayed by forms with direct unequal cleavage and other forms which cleave unequally through the production of large polar lobes. The significance of these findings is discussed relative to the origins of these different types of unequal cleavage.

Formation of the first cleavage spindle in nematode embryos

The distribution of microtubules and microtubule organizing centers in the events leading up to the establishment of the first asymmetric cleavage furrow in nematode embryos was followed using indirect immunofluorescence of antibodies to tubulin. Oocytes arrest in meiotic prophase then undergo two meiotic reduction divisions after fertilization. At both of these divisions barrel-shaped spindles were observed. Initially a single microtubule organizing center was seen adjacent to the sperm pronucleus following fertilization in Caenorhabditis elegans, but later two sperm asters were distinguished. These increased in size as the egg pronucleus migrated toward the sperm pronucleus and reached maximum size, with fascicles of microtubules extending to the cortex, once the pronuclei had become juxtaposed. The first cleavage spindle formed following rotation and migration of the juxtaposed pronuclei back toward the center of the embryo. The distribution of microtubules in a temperature-sensitive mutant that fails in both pronuclear migration and rotation was also examined. Asters in the mutant embryos at the nonpermissive temperature contained only short microtubules suggesting that the morphology of the asters is important for directing the movement of the pronuclei. In Panagrellus redivivus sperm asters were not detected by anti-tubulin staining until the female pronucleus had migrated to the centrally placed sperm pronucleus. Asters then increased in size and formed the first cleavage spindle.

The sperm centriole persists during early egg cleavage in the insect Chrysopa carnea (Neuroptera, Chrysopidae).

The giant centriole of the sperm of Chrsyopa carnea persists in the fertilized egg. The giant centriole appears together with a regular small centriole at one pole of the first cleavage spindle; two small centrioles are found at the other pole. During the subsequent divisions, the giant centriole appears either at one pole of one of the spindles, as in the first cleavage, or close to one inter-kinetic nucleus. All the other centrioles are regular in size. Therefore, the assumption that the mature spermatozoa of insects contain no true centrioles is not valid, at least for Chrysopa.

‘Cleavage fields’: Hypothesis on early embryonic development

The hypothesis is put forward that events of the early embryonic development can be interpreted on the basis of a radial distribution of cytoplasmic components, i.e. of a ‘cleavage field’, progressively established during the growth of the oocytes. The orientations of the cleavage spindles and the corresponding furrows' positions are assumed to be correlated to the field's temporal evolution which, in turn, is determined by flows of cytoplasmic components originated by the changes in the membrane shape. From this viewpoint, a simple explanation of egg regulation is proposed, and the particular case of the sea urchin embryo is briefly discussed.

Cytological events leading to the cleavage of golden hamster zygotes.

Fertilized golden hamster eggs were examined between 6 and 20 hours post-ovulation to determine the events leading to the two-cell stage. Following their migration the pronuclei remain in the central region of the zygote for approximately ten hours. The morphologically, indistinguishable male and female pronuclei remain relatively unchanged during this period, i.e., they do not interdigitate or fuse with one another as described for the zygotes of other organisms. Following this period and at the time of pronuclear breakdown elongate vesicles appear along the nucleoplasmic surface of the pronuclear envelopes. Later the pronuclear envelopes fragment into elongate cisternae; these and the vesicles formed along the inner lamina of the pronuclear envelopes remain closely associated and constitute quadrilaminar structures. The chromosomes which condense prior to and during pronuclear envelope breakdown, migrate to the equatorial plate of the forming cleavage spindle. After cytokinesis the chromosomes in the blastomere nuclei disperse. Increase in the nuclear envelope to accommodate this dispersion may involve the addition of membrane from the quandrilaminar structures.

Timing of the first cleavage division of haploid mouse eggs, and the duration of its component stages.

ABSTRACT Mouse eggs were activated by treatment with hyaluronidase which removed the follicle cells, followed by culture in vitro, and examined at the first cleavage mitosis. Second polar body extrusion usually occurred and haploid parthenogenesis was initiated. Air-dried chromosome preparations were made between 11 and 15·5 h after activation. Out of the 308 eggs examined 74 had already progressed to the 2-cell stage; the remaining 234 at the 1-cell stage were examined in detail. All chromosome preparations of the first cleavage mitosis were classified into groups corresponding with the stages of prometaphase, metaphase (early or ‘pre-chromatid’, ‘chromatid’ and ‘late chromatid’) and anaphase. An indirect estimate was made of the duration of the first cleavage mitosis and of its com-ponent stages from the incidence of stages observed at different time intervals after activation. Similar eggs were also observed at 37 °C by time-lapse cine-photography and the interval between the disappearance of the pronucleus to the beginning of telophase of the first cleavage division was determined. The results of timing studies on the haploid eggs were compared with results obtained from similar observations on the first cleavage division of fertilized eggs which would of course normally be diploid. Artificially activated eggs with2 pronuclei, resulting from second polar body suppression, were also examined, and serial chromosome preparations during mitosis showed that the 2 pronuclear chromosome groups unite on the first cleavage spindle and divide to give a hetero-zygous diploid 2-cell embryo.

An ultrastructural analysis of fertilization in the surf clam, Spisula solidissima. II. Development of the male pronucleus and the association of the maternally and paternally derived chromosomes

The events of fertilization involving the incorporation of the spermatozoa, the development of the male pronucleus, and the association of the maternally and paternally derived pronuclei have been investigated in the egg of Spisula solidissima. At the site of gamete plasma membrane fusion, a small fertilization cone is produced through which the contents of the spermatozoon passes. Entrance of the spermatozoon is accompanied by the vesiculation of sperm nuclear envelope. Subsequently the sperm chromatin is dispersed. A male pronuclear envelope and sperm aster are not formed until the maternal chromatin completes meiosis. Following these events, migration occurs and the male and female pronuclei come into proximity with one another. Later the pronuclear envelopes vesiculate and, concomitantly, the chromosomes condense into large reticular aggregates. The two groups of chromosomes become associated on what will become the metaphase plate of the first cleavage spindle. Thus, as in the rabbit and the mussel, no zygote nucleus is produced.

Cytokinesis in the gonocysts of the drone honey bee (Apis mellifera L.)

Gonocyst growth in the drone honey bee (Apis mellifera, L.) is characterized by a series of mitoses in which cytokinesis is incomplete. After karyokinesis, the division products are reoriented in such a way that the cleavage furrow and spindle remnants point towards the cyst center. Throughout the larval period the cyst center contains depositions of spindle plaques with distinct microtubules and other cytoplasmic components which are associated with the furrowing membranes.Cleavage in the lining cells of the gonocysts is complete. The membranes are closely associated with fibrils and microtubules appear to abut directly on these membranes even in late stages of cytokinesis. In the gonial cells, the innermost regions of the membranes in the furrows are subtended by, but not closely associated with, accumulations of fibrillar material. It is suggested that incomplete cytokinesis in the gonial cells may be mediated by restricted movement of the furrows and that the loose association of fibrillar materials with the furrowing membranes may be indicative of aspects of this process.

Cytological events leading to the formation of the two-cell stage in the rabbit: Association of the maternally and paternally derived genomes

The cytological transformations leading to the establishment of the two-cell stage in the rabbit have been studied with techniques of light and electron microscopy. Subsequent to their migration the male and female pronuclei become centrally located and juxtaposed. Each pronucleas appears to be nearly identical in size and in structure and contains a fibrous nucleoplasm in which fine-textured and granular aggregations are embedded. Later the pronuclei become closely apposed and their proximal surfaces flatten and become highly convoluted. Along the proximal margin of the pronuclear envelopes small vesicular structures are produced. They are filled with a flocculent material and appear to be budded-off into the cytoplasm. During further morphogenesis, the pronuclei produce an extensive array of projections which interdigitate with one another to form complex configurations. Concomitantly, the blebbing activity of the pronuclear envelopes ceases and the chromatin condenses into large reticular aggregations, primarily in the region where the pronuclei are intimately associated. Association of the maternal and paternal genomes is effected when the two pronuclear envelopes break down at widely spaced intervals leaving regions of intact double-membrane structures (pronuclear envelope). Microtubules (spindle) are associated with the condensed chromatin and course through the disrupted regions of the pronuclear envelope. The two groups of chromosomes move together and assume positions on the first cleavage spindle. Thus, in the rabbit, there is an absence of a zygote nucleus.

SOME CYTOLOGICAL ASPECTS OF FERTILIZATION IN THE CROSS BETWEEN THE FUNA (CARASSIUS CARASSIUS, CYPRINIDAE) AND THE LOACH (MISGURNUS ANGUILLICAUDATUS, COBITIDAE)

1. Insemination and early cleavages were cytologically investigated in the cross between the funa (Carassius carassius) amid the loach (Misgurnus anguillicaudatus), belonging to different families. Eggs of the fumia were artificially insemimiated with bach sperm at water temperatures from 26° C. to 28° C.2. Monospermic insemination was cytologically proved; a simigle spermatozoon of the bach entered the funa egg through the micropyle. Following insemination, the second maturation division of the egg proceeded. About 15 minutes after insemination, the extrusion of the second polocyte occurred. Then the formation of female and male pronuclei followed. About 20 to 25 minutes after insemination, the meeting of the male and female pronuclei took place. In conjugation. both pronuclei came into close contact, side by side, with the nuclear membrane intact, without evidence of actual fusion.3. It was difficult to distinguish clearly groups of chromosomes of paternal and maternal origin or to count the chromosome number in the first cleavage metaphase. During the period from anaphase to telophase, separation of the chromosomes took place symichronously, without evidence of lag or elimination of chromosomes. The first cleavage was completed 35 to 40 minutes after insemination.4. Early cleavages took place at intervals of 15 to 20 minutes after insemination. No abnormality was detected in the cleavage spindle. The second polar body was found lying in its original position at the first cleavage.5. Evidence presented indicates that both insemination ard early cleavage followed normal cytological patterns, and consequently that there is no cytological evidence in this cross to show that the sperm acted as a parthenogenetic agent, in spite of time maternal characters noted in larvae resulting from this cross.

ON THE ORIENTATION OF CENTRIOLES IN DIVIDING CELLS, AND ITS SIGNIFICANCE: A NEW CONTRIBUTION TO SPINDLE MECHANICS

1. The centrioles of the egg of Polychoerus carmelensis, at first meiotic metaphase, second meiotic metaphase, and resting first cleavage metaphase, are slightly curved rods which are usually oriented at right angles to each other and to the main axis of the spindle.2. Centriole orientation and behavior in the spermatocyte divisions of Gryllidae and Hemiptera, as described by Johnson (1931) and Payne (1927), in relation to the arrangements of daughter cells, are compared with centriole orientation and predicted behavior in the egg of Polychoerus.3. These considerations (on centriole orientation and behavior) constitute the basis for a new hypothesis, as follows:a. The orientation of the centrioles at any given division determines the position in which the daughter centrioles will separate from each other.b. The path of separation of daughter centrioles determines the position of the main axis of the spindle for the next division.c. The axis of the spindle determines the relative positions of the daughter cells with respect to each other.d. This arrangement of the daughter cells is maintained, for a time at least, by the primary cell connective, of which the spindle remnant is the significant portion.e. These relations obtain in the absence of secondary intervening factors.4. The inherent right-angle orientation of the centrioles at the two poles of the first cleavage spindle of Polychoerus is thus interpreted in causal relation to the alternating dexiotropic and leiotropic divisions in spiral cleavage.

Activation of Eggs by Hypothermia in Rats and Hamsters

ABSTRACT Hamster eggs appear unique among known mammalian eggs in that nearly 80% were found to undergo spontaneous activation, extruding the second polar body, within 24 hr. of ovulation. Often the eggs showed spontaneous early parthenogenesis the development of one or occasionally two nuclei and the formation of a haploid first cleavage spindle took place in a manner that closely resembled the corresponding stages in fertilization. Time relations, too, were much the same. Rarely, however, does the cleavage spindle pass into mitosis, but instead it breaks up and the chromosomes become scattered. Only one normal-looking 2-cell egg was found. Rat and hamster eggs were very resistant to activation by hypothermia while still in the ovary. After ovulation, however, 100% of rat eggs were activated by body temperatures between 0 and 1° C. and about 60-70% of hamster eggs by temperatures between 0 and 16° C. In view of the high incidence of spontaneous activation in hamster eggs, the effect of chilling in this animal can be regarded as merely that of hastening the change. Hypoxia alone activated 36% of rat eggs. Relative tissue anoxia is therefore probably the immediate effective agent in activation by anaesthetics and is partly responsible for activation by low temperatures. Cold-shock itself, however, is evidently a more potent activator. Hamster eggs were not activated by hypoxia, probably because a less severe tissue anoxia was induced. Rat eggs activated by hypothermia showed less tendency to early parthenogenesis than those activated by local chilling of the Fallopian tube. Nucleus and spindle formation was rather better in artificially than in spontaneously activated hamster eggs, but the prospects of extensive parthenogenetic development did not seem in any way improved. Only two 2-cell eggs resembling fertilized eggs were seen. Nuclei in activated hamster eggs achieved a volume similar to that of male or female pronuclei at full development. Total nucleolar volume was equal to the sum of the nucleolar volumes of male and female pronuclei.

THE CHEMICAL AND EXPERIMENTAL EMBRYOLOGY OF LIMNAEA

Summary1. A method is described for obtaining egg masses of Limnaea stagnalis at the moment desired, and the nature of the egg‐laying stimulus is analysed.2. The oogenesis of Limnaea has been studied in detail, with special reference to the formation of various egg substances.3. The insemination of the eggs and the formation of the egg mass are described.4. The development of the fertilized egg from oviposition until the trochophore stage has been studied; special attention has been given to the cytochemical changes in the egg and embryo. A distinction is made between the ‘chemo‐differentiation’, consisting of a redistribution of preformed substances during early development, and the ‘chemical differentiation’, accompanying the histological differentiation. The chemical differentiation leads in all three germ layers to a separation into two types of cells, which specialize in different directions and are destined to form larval and adult organs respectively.5. The osmotic properties of the egg have been studied. The recently laid egg is, on an average, isotonic with a 0.093 M solution of non‐electrolytes.6. With the aid of centrifuge experiments, the swelling of protein granules, the viscosity and tension at the surface of the eggs, their changes during the uncleaved stage and the first cleavages, and the influence of lithium and calcium chloride solutions on these physical properties have been studied. Eggs centrifuged during the uncleaved stage develop into normal young snails in a large percentage of cases. The redistribution of the egg substances after centrifuging has been studied. It is concluded from the experiments that this redistribution is caused by factors residing in the relatively unmovable egg cortex. Apparently the proteid yolk does not play an important part in the determination of the cells.7. The normal cleavage of the egg is dependent on the presence of Ca‥‐ions, but these may be replaced by Lï‐ions in optimal concentration. The Ca‥‐ions influence the properties both of the vitelline membrane and of the egg cortex proper.8. By treatment with lithium chloride solutions both exogastrulation and cyclopean malformations may be induced. For both types of malformation specific periods of susceptibility exist. In the embryos of the cyclopean series the differentiation of the most animal cells of the embryo is suppressed. It is concluded that lithium acts in Limnaea on a polar gradient field. Cyclopean malformations can still be induced at the 24‐cell stage; evidently the pattern of determination has not yet been laid down irrevocably at this stage.9. Recent experiments suggest that two groups of factors are involved in the conformation of the polar gradient field: (a) factors governing the evolution of the mitotic apparatus, which may be influenced by hypertonic solutions; (b) factors directing the cytoplasmic movements, which are susceptible to the influence of lithium.10. Sodium sulphocyanate does not influence the pattern of determination in Limnaea. It causes a wine‐red coloration, the nature of which is still obscure.11. Treatment of the eggs with thiourea causes cleavage abnormalities. Cases of irregularities in cleavage, with deviations in the position of the cleavage spindles, suggest that the factors governing the succession of spindle positions in normal development change relatively independent of the other developmental processes.

Cytological observations on fertilization in the carp, <i>Cyprinus</i> <i>carpio</i> L.

Employing the carp, Cyprinus carpio L. as the meterial, the processes of maturation of the egg and the phenomena of fertilization were microscopically observed in this study. Observations were carried on in the sections of the eggs, fixed at different intervals ranging from five minutes to 2 hours after fertilization. The majority of eggs were fixed in Carothers' solution and some were preserved in Gilson's fluid. For sectioning the Gilson material proved to be better than the Carothers material. Sections were made following the usual paraffin method. They were stained with Ehrlich's haematoxylin. The results of observations are briefly outlined as follows:1) The first maturation division of the egg is completed prior to ovulation within the ovary. After the extrusion of the first polar body, the chromosomes remaining in the egg are immdiately drawn into the equatorial plate forming the metaphase spindle of the second maturation division (Fig. 4).2) The spawning and fertilization take place in the water at the same time.3) At the time of insemination the second maturation division of the egg persists in the stage of metaphase, and then it gradually advances in further course of division (Fig. 8). About 25 to 30 minutes after insemination the second division is completed, resulting in the formation of the second polar body (Fig. 9).4) During the ovulation process the course of the second division does not proceed being arrested in metaphase. Stages subsequent to the metaphase of the second division take place only after the entry of the spermatozoon into the egg. This fact emphasizes that insemination is a necessary antecedent to the second maturation division to advance further from metaphase.5) Only a single spermatozoon enters an egg (Fig. 7). Thus the carp egg is monospermic in fertilization under the normal condition.6) About 40 to 50 minutes after insemination there is found at the animal pole of the egg the already metamorphosed female pronucleus. It assumes a spherical shape lying in naked state in the ooplasm (Fig. 13).7) As early as 50-60 minutes after entrance, the head of the spermatozoon is converted into the male pronucleus attaining nearly spherical form (Figs. 10-12). At this stage there is no visible demarkation between the male and female pronuclei, so far as their structure and size are concerned. But there is present a ramarkable character peculiar to the male pronucleus. That is the pronounced appearance of the sperm-aster.8) After completion both the pronuclei begin to move towards the deeper part of the animal pole of the egg to conjugate with each other (Fig. 14). The conjugation of the pronuclei generally takes place about 1 hour after insemination occurs (Figs. 15-17).9) After the two pronuclei conjugate together, they do not actually fuse, but lie side by side in closs contact with the nuclear membrance intact (Figs. 15-16). Thus the maternal and paternal chromosome materials are separated in distinct groups during the stages prior to the first cleavage division. The chromosomes are formd independently in each vesicle (Fig. 17).10) In the egg about 11/2 hours after fertilization, the first cleavage spindle is generally formed (Fig. 18).

THE INFLUENCE OF CENTRIFUGAL FORCE ON THE BILATERAL DETERMINATION OF THE SPIRALLY-CLEAVING EGG OF URECHIS

1. The eggs of Urechis caupo were ultracentrifuged for short periods with forces ranging from 6,000 to 75,000 gravities before fertilization and following fertilization at various times before cleavage.2. In the trochophores 75 per cent of the total recorded embryos had the centripetal oil in the ventral median or oblique position, in slightly more than 20 per cent it was found laterally, and in less than 5 per cent dorsally.3. The data are essentially the same for unfertilized eggs and all eggs centrifuged at various times following fertilization and before cleavage, and irrespective of the force applied.4. From these data it is concluded that the bilateral axis of the embryo is induced or influenced by the stratification of material substances in the egg. In centrifuged eggs it is unrelated to either the sperm entrance point or the first cleavage plane.5. There is no polar orientation in the centrifuge tubes before fertilization or following fertilization until the germinal vesicle breakdown. But following the germinal vesicle breakdown the eggs tend to orient in the centrifuge tubes so that the pole comes to lie most commonly near the centrifugal end.6. Normal trochophores are produced from eggs centrifuged before fertilization and at certain times following fertilization, but during some miotic and mitotic stages centrifuging suppresses polar body formation and disturbs the cleavage spindles so that few normal embryos are produced.

Cytological observations on the fertilization of the egg of <i>Hynobius retardatus</i> DUNN

The fertilization phenomena in Hynobius retardatus are observed in sections of the eggs, fixed at different intervals after insemination of the spermatozoa. The results are briefly outlined as below:1) About 1 to 1 1/2 hours after insemination of the spermatozoon the the second polar division is completed, and formation of the second polocyte follows about 1 1/2 to 2 hours after insemination.2) About 2 1/2 to 3 hours after insemination there is found at the periphery of the egg the already metamorphosed female pronucleus, which moves away from the surface towards the interior of the egg.3) The spermatozoon enters the egg as early as 15 minutes after insemination and then, within 1 hour, it metamorphoses into the male pronucleus.4) The conjugation of the male and female pronuclei generally takes place between the 5th and 6th hour after fertilization. They meet at a distance of about 1/4 to 1/3 the egg diameter from the animal pole, along an egg-axis joining the second polocyte with the center of the egg.5) At the time of the conjugation, both pronuclei are quite similar in their structure, size and staining condition; they are nearly spherical with a smooth nuclear membrane and are filled with colourless nuclear sap. Generally they measure 0.038-0.042mm in diameter.6) After the two pronuclei meet together, they do not actually fuse, but lie side by side in close contact with the nuclear membrane intact. The maternal and paternal nuclear elements are separated in distinct groups during the stages preparatory to the first cleavage division and the chromosomes are formed independently in each respective nuclear vesicle.7) In the egg about 7 hours after insemination, the first cleavage spindle is usually found in the process of division.

On the unsegmented ovum of Echidna (Tachyglossus)

ABSTRACT In this communication is described for the first time the unsegmented germinal disc of the monotreme Echidna (Tachyglossus), and measurements are also given of living intra-uterine eggs at early stages Segmentation is not initiated in Echidna until the egg has arrived in the uterus The polar bodies are much smaller than those described for Ornithorhynchus. The first is much larger than the second but, contrary to what has been reported to occur in Ornithorhynchus, remains undivided The germ nuclei, immediately before the formation of the first cleavage spindle, undergo conjugation and fusion. Contrary to what is described for Ornithorhynchus, polyspermy does not normally occur in the fertilization of the ovum of Echidna.

On the Development of Cucumaria echinata v. Marenzeller.

On the Development of Cucumaria echinata v. Marenzeller.

ON THE SUPERPOSITION OF FERTILIZATION ON PARTHENOGENESIS

1. Normal Arbacia eggs exposed to butyric acid (50 c.c. sea water + 2.8 c.c. N/10 butyric acid) and subsequently fertilized, reveal a curve of fertilization falling from the normal reaction to the optimum exposure to butyric acid for membrane production: rising from the optimum exposure (20 sec.) to an over-exposure of 2½-3 minutes; and again falling off until no further fertilization is possible—about 10 minutes.2. Eggs having been exposed for the optimum time to butyric acid, have acquired an unfertilizable condition. If the membranes are completely removed, sperm enter the eggs but do not lead to development. The eggs, though sperm have entered them, are not fertilized.3. Sperm having entered these eggs appear only as foreign bodies. They do not cause the production of asters, nor are cleavage spindles produced.4. The lysin theory of fertilization is found inadequate to explain the results.5. Eggs over-exposed to butyric acid are still capable of a certain amount of fertilization, though the process is usually very abnormal. No membranes are produced. Polyspermy is extremely frequent.6. Exposure to butyric acid for ten minutes leads to a non-fertilizable condition of the egg. Sperm enter but are non-effective and non-effected.7. Eggs subjected to a temperature of 35° C. for 10 minutes are rendered non-fertilizable. Sperm enter the eggs, part disintegrating in fragments, part forming large clear vesicles and part apparently remain unaffected.8. Where fertilization is possible fertilizin has always been found present. Where fertilization is not possible fertilizin has never been found.9. The quantitative aspect of fertilization is very definitely indicated.10. Superposition of fertilization upon parthenogenesis, where activation has been complete (indicated by full membrane production and absence of fertilizin) is impossible.Where activation by parthenogenetic agents has been only partially completed, partial fertilization is yet possible, but development is usually abnormal.

A STUDY OF THE EFFECTS OF INJURY UPON THE FERTILIZING POWER OF SPERM

1. Nereis eggs, inseminated with sperm cells which have been injured by one of several methods, may fail to develop in a normal manner.2. In some cases the egg does not form a fertilization cone, attachment granules are lacking and the sperm head is not drawn into the egg but remains outside attached to the vitelline membrane. There are two classes of such eggs, neither of which segment, viz.: (a) Those which slowly undergo maturation without forming jelly. (b) Those which form both jelly and polar bodies.3. Those eggs in which the sperm head enters, form the first cleavage spindle in an apparently normal fashion but may fail to complete the division of the cytoplasm. Those which complete this division may develop abnormalities in later stages.4. The eggs of Arbacia exhibit a similar series of abnormalities when fertilized by weakened sperm cells.5. There is no indication of specificity in the action of the agents used in injuring the sperm cells.6. These experiments demonstrate that eggs fertilized with sperm cells injured by alcohol and by other means may produce abnormal forms. Taken in connection with the demonstration by others that the germ cells of mammals may be exposed to injurious conditions this has an important bearing upon the relation of alcoholism to the production of defectives.7. There seem to be at least two factors involved in the process of fertilization. The one has to do with membrane formation and certain other changes, the other with the internal stimulus.8. In Nereis the presence of the two germ nuclei within the egg is not necessarily sufficient as an internal stimulus for normal development.