7

Nuclear Cytology ¹

Allen B. Griffen

Throughout the history of animal nuclear cytology the consensus among investigators has been that the nuclei in the cells of the "germ line" must be the most dependable sources of chromosome counts which will reveal the true diploid chromosome number for the species. This was, and still is, based upon the logical assumption that the progenitors of the germ cells contain, in as nearly inviolate a state as is possible under the exigencies of early development and differentiation, a reserved portion of the individual's unique inheritance from the past, to be used only as the source of his contributions to the future. These cells are the "gonial" cells: oogonia in the female, and spermatogonia in the male.

Quite in keeping with their being on reserve, the chromosomes of gonial cells at metaphase are invariable highly condensed and individually discrete, providing the ideal condition for absolute chromosome counts. In the mouse, both oogonia and spermatogonia provide an absolute count of 40. Figures 7-1 and 7-2 illustrate oogonial and spermatogonial metaphases, respectively. These photographs, taken from Sudan Black squash preparations, clearly show the sharp definition of gonial metaphase chromosomes; they also reveal the prevailing lack of individuality of gonial metaphase chromosomes of the mouse, all of which appear as blunt rod-like elements differing only in length. When gonial metaphase pictures are dismembered and the chromosomes paired in serial order according to length, the resulting idiograms show the presence of two "smallest" elements in the female ( Figure 7-3) and three smallest elements in the male. For one of these elements in the male there is no mate of the same size ( Figure 7-4). Instead there is a long chromosome, left over after all pairings have been made, which is the only available partner for the small element. This pair of unlike bodies in the male is concluded to be the XY pair, the shorter being the Y since it is not present in the female. The first diploid counts from gonial cells were made by Cox ( 1926) and verified by Painter ( 1927) and by Cutright ( 1932). Aside from revealing the basic chromosome number and, in the male, the sex chromosome pair, gonial nuclei are of little diagnostic use in the mouse. All metaphase elements appear to be telocentric, showing neither constrictions nor accompanying small arms or satellites which might indicate that the centromeres have subterminal positions. The prophase chromosomes are thick and heavy, without marked chromomeric appearance. Anaphase and telophase stages always show compact chromosome clusters in which individual elements are not readily discernable.

In the female, oogonia complete their mitoses before birth. The ovary of the newborn individual shows nearly all gonial cells to be in the final interphase during which occurs the growth in volume immediately preceding meiosis. Borum ( 1961) and Ohno ( 1963) stated that oocytes appear on the 14th day of fetal life. Borum added that all are in pachytene by the 18th day; by the fourth or fifth day after birth all oocytes are in the arrested dictyotene or dictyate stage.

In the male, spermatogonia are visible from the last third of fetal life through old age. In the fully formed spermatogenic tubule the spermatogonia appear as a thin layer of cells with compact deeply staining nuclei forming a poorly defined row immediately internal to the wall of the tubule ( Figure 7-5). Most of these cells are type B spermatogonia, the last stage before the transformation into primary spermatocytes. Although occasional metaphases and anaphases are seen in this layer, most of the nuclei are in the resting or interphase stage, characterized by the presence of irregular blocks and "clots" of chromatin. Type A and intermediate spermatogonia are less prominent in the gonial layer, being far outnumbered by the type B spermatogonia. They are occasionally discerned in squash preparations. In the gonial layer, type B spermatogonia become transformed into primary spermatocytes. The inconspicuous spermatocytic interphase appears finely granular and may show a single pale vesicular nucleolus. Being of very short duration, spermatocytic interphases are usually outnumbered by spermatocytic leptotene, zygotene, and earliest pachytene nuclei ( Figure 7-6). By the time of the transformation of all preceding stages into the early pachytene stage, the row of primary spermatocytes is found to have migrated inward, allowing for the formation of a new gonial layer from the multiplication of type A spermatogonia.

Interphase nuclei

Interphase nuclei in type A spermatogonia have been described by Regaud ( 1901) as "dust-like," with reference to the appearance of their fine and evenly dispersed chromatin particles. A single prominent chromatin nucleolus, whose nature is not known, usually appears in an eccentric position. This body does not seem to represent a cluster of centromeres, as is the case for the chromatin nucleolus in Drosophila ( Warters and Griffen, 1958). Interphase nuclei in intermediate and type B spermatogonia are characterized by deeply staining peripheral spots and patches of chromatin which impart a "crust-like" appearance to the nuclei. A prominent nucleolus somewhat resembling a plasmosome, but surrounded by irregular crustlike chromatin bodies, usually appears in a central rather than a peripheral position in each nucleus. Oakberg (1956 a, b) provided descriptions of the several types of spermatogonia.

Prophase

Prophase in type B spermatogonia is characterized by the formation of thick, smooth threads which seem to fill the nucleus completely. As the nuclear membrane disintegrates, the final condensation and shortening of the chromosomes into their metaphase form occurs in a steady progression showing no clearly demarcated steps. Prophase in type A and intermediate cells apparently has not been described.

Metaphase

Metaphase in spermatogonia has the expected equatorial arrangement of the chromosomes, with no morphological indications of spindle attachment points. In side views the spindle is readily visible as a double cone with a broad common base (the equator) and short altitudes; often the spindle may appear rather barrel-shaped. At the poles, centrosomes of very small size often may be observed. Type A and type B spermatogonial metaphases are easily distinguished. In A cells the chromosomes are long and slender, whereas in B cells they are usually short and are rounded into bean-like shapes.

Anaphase

Anaphase is not often observed in sectioned material, but is frequently observed in spermatogonia in squash preparations. Chromatid separations appear to be both clean-cut and quick, with no lagging elements and no precocious passages to the poles. As at metaphase, A cells show long, slender chromosomes, and B cells show rounded, short chromosomes.

Telophase

Telophase in all types of spermatogonia is a fleeting stage seen occasionally in the extreme periphery of the spermatogenic tubules. It has no unusual characteristics other than location and scarcity. As the gonial layer becomes crowded through cell divisions, the daughter nuclei of B spermatogonia tend to remain associated in pairs. This association frequently appears to be carried over into the darkly staining sharp layer of early primary spermatocytes. Figures 7-7 and 7-8 illustrate spermatogonial mitoses.

In vivo

Aside from routine observations made in the regular course of histological and pathological studies, somatic mitosis in the mouse has received little attention. There have been no indications from sectioned material that somatic chromosome studies might be of any cytogenetic use, aside from the demonstrations of polyploidy, aneuploidy, anaphase bridging, multipolar mitoses, endomitosis, and other phenomena always associated with malignancy and tumorigenesis. Through the use of mechanical means of cell separation, combined with improved techniques for squashing and spreading, Bunker ( 1961, 1965) has produced diagnostically usable prometaphase and metaphase preparations from adult bone marrow, spleen, and kidney and from several fetal tissues. The chromosomes are regularly separated into component chromatids, held together at the subterminal centromere positions. Minute chromosome arms or satellites are regularly visible and make possible the construction of somatic idiograms in which most of the members of the chromosome complement can be characterized and "paired up" with accuracy. Figure 7-9 illustrates a typical metaphase from the adult spleen. Figure 7-10 is an idiogram of the same cell, showing the small arms and centromere positions in several of the component pairs. A late prophase nucleus from the spleen of an adult female is shown in Figure 7-11; Figure 7-12 is an idiogram of the same cell.

In vitro

The advent of somatic mitosis into great prominence as a source of cytogenetic information came with Hughes's ( 1952) suggestion that the hypotonic pretreatment of cells grown in vitro improved the opportunities for studying the metaphase chromosome complement of these cells. The independent discovery and immediate successful use of hypotonic pretreatment by Hsu and Pomerat ( 1953) upon cells grown in short-term tissue cultures opened new horizons for the cytology of all mammals, and the technique has been in many respects contributory to the clarification of sex determination in these animals. In the mouse, hypotonic pretreatment of actively dividing cells from cultures of any soft tissue, such as fetal gonads, kidney, and bone marrow, provides material which can be handled directly with squash techniques.

Somatic metaphases, whether derived from direct squashes or from cultures, reveal chromosome individualities not seen in gonial metaphases prepared by the same methods, presumably indicating that the "reserved" state of the gonia causes the occlusion of many fine points of chromosome structure. For example, the metaphase and idiograms of Figures 7-9, 7-10, 7-11 and 7-12 may be compared with those of Figure 7-1 and 7-2. All received hypotonic pretreatment, yet the somatic chromosomes show constrictions, satellites, and short arms never visible in the gonia. The X and Y seem to retain their gonial type of condensation, appearing more dense, more deeply-staining, and less frequently separated into component chromatids.

Allocycly of the X and Y

Although the X and Y under such conditions may be called "heteropycnotic," a better term is "allocyclic," indicating that these elements are not strictly in phase with other members of the chromosome complement. True allocycly, however, is best seen in XX cells such as those in which Ohno and Hauschka ( 1960) first pointed out the density of one X, whereas the other was hardly distinguishable from the autosomes in certain tumor samples. That the condensed X in such cases is not in phase with other elements is demonstrable through its late labeling with tritiated thymidine. The demonstration of allocycly was a direct forerunner of the inactive-X hypothesis of Lyon ( 1961, 1962). Lyon demonstrated genetically that an autosomal segment bearing the dominant allele of a coat color factor, when inserted into an X, is inactivated along with that of the X to permit a mosaic phenotypic expression of the recessive allele for color borne in the homologous intact autosome. Ohno and Cattanach ( 1962) provided cytological confirmation of the hypothesis through the study of the chromosomal constitution of the mosaic areas. Figure 7-13 illustrates the active and inactivated autosomal segment.

Somatic chromosome identification

Chromosome identification and chromosome mapping in somatic cells of mice have not been accomplished. However, with the development of techniques for spreading the chromosomes, somatic prophases may prove to be useful in these respects. Griffen ( 1960) was able to make tentative chromosome identifications in tumor cell prophases in which chromomeric structure was prominent. Figure 7-14 illustrates such a cell and indicates the identified chromosome parts.

Utilizing all information and available techniques, Levan et al. ( 1962) prepared somatic cell idiograms of the mouse, based upon tissue cultures. These idiograms represent a comprehensive approach to the problem of somatic chromosome identification. They also reveal the inadequacies of mouse metaphase chromosomes in this usage ( Figure 7-15).

Spermatogenesis

Spermatogonia. The location and appearance of the type B spermatogonia, which transform directly into primary spermatocytes, has been presented in the discussion of gonial mitosis. The thin peripheral layer of the spermatogenic tubule was shown to consist of these cells. This layer and several stages of the primary spermatocytes are shown in Figure 7-16.

Primary spermatocytes. The earliest stages of the primary spermatocytic prophase form a sharp, darkly staining row which consists of leptotene-zygotene or of early pachytene stages. The preceding interphase and similarly inconspicuous early leptotene stages seldom appear in this row. Internal to the dark row the later stages of the meiotic prophase are characterized by their large size, all nuclei from midpachytene through diakinesis being more than twice the diameter of the early pachytene nuclei. The growth period in primary spermatocytes is thus indicated to be the pachytene stage.

Interphase. The only well known interphase in meiosis in the male mouse is that of the primary spermatocyte, found in the gonial layer of tubules and distinguished from type B cells principally by the finely granular nucleus with its pale nucleolus. Griffen and McMahon (unpublished) have estimated the time of duration for this stage to be approximately 3 per cent of the total time of the spermatogenic cycle as determined by Oakberg ( 1956b). Since Oakberg found the length of the cycle to be 212 hours, the interphase exists for about 6 hours. The interphase and other early stages of meiosis as they appear in a sectioned tubule are illustrated in Figure 7-17.

Leptotene. The first stage of the meiotic prophase, the leptotene, appears in portions of the dark row of early spermatocytes. Leptotene nuclei are finely granular, with indications of fine strands connecting the chromatin particles (ultimate chromosomes?) for short distances. Eventually the nuclei appear as masses of lightly stainable beaded threads, among which the nucleolus appears as a clear space bounded by a distinct membrane.

Zygotene. In its early stages the zygotene is not distinguishable from the leptotene, except in the fact that thickened regions of the chromosome strands indicate the beginning of pairing. The late zygotene is readily identifiable through its many regions in which the double thickness of the chromosome strands indicates advanced pairing. Figure 7-18 illustrates the early stages of the meiotic prophase in a squash preparation.

Pachytene. The pachytene stage is characterized by complete pairing. With all homologous strands in synapsis, the chromosomes resemble minute railroad tracks winding about within the nuclear membrane with gently spiralled twists. The plasmosome is now sharply distinct and, according to Slizynski ( 1949) and Griffen ( 1955), is regularly attached through several heavy chromatic bodies to a particular member of the synapsed chromosome complement. Sachs ( 1955) and Geyer-Duszynska ( 1963) challenged this interpretation and stated that the plasmosome is not a part of a stranded XY pair, but is the sex vesicle, containing the X and Y in precondensed form. The nature of the vesicle or plasmosome and its true relation to the sex chromosomes is a matter of fundamental importance in chromosome mapping and in sex-chromosome inactivation studies. An early solution to the problem is therefore desirable.

Through the use of hypotonic solutions and squash methods in preparing slides, pachytene nuclei can be ruptured and the chromosomes separated with some success. Figure 7-19 illustrates a ruptured pachytene nucleus similar to those used by Slizynski and by Griffen for map construction. The chromomeric nature of the paired strands is clearly visible. Figure 7-20 shows 19 chromomeric chromosomes and the presumed X and Y in the sex vesicle, such as described by Geyer-Duszynska.

Diplotene. The passage from pachytene to diplotene is characterized by the relaxation of pairing, resulting in the disclosure of a four-stranded composition for each former pachytene pair. So gradual is the change that sharp demarcation of the diplotene is not possible. Early diplotene is almost identical with pachytene in superficial appearance, yet four strands can be discerned at numerous, if not all, levels along each chromosome pair. Chiasmata first appear at diplotene, occasionally visible as interchanges between members of the otherwise parallel pairs of chromatids. However, these exchanges are readily detectable in ordinary practice only at the onset of diakinesis. Figure 7-21 illustrates the diplotene nucleus. Diplotene and all preceding stages of the primary spermatocyte are notoriously difficult subjects for photography.

Diakinesis. At its most easily recognized stage diakinesis shows the tetrads of the diplotene in the process of condensation by means of coiling. Each tetrad has the appearance of a fuzzy, loose loop, ring, or cross, the configuration depending upon the position and the degree of terminalization of one or more chiasmata. Figure 7-22 shows several appearances of diakinesis, progressing from loose coiling to the compact coiling which approaches the condensation seen in metaphase I.

Metaphase I. The primary spermatocytic metaphase is the most easily recognized stage in meiosis. In the male mouse 20 discrete tetrads are visible, each usually contorted by the terminalization of chiasmata. Occasionally tetrads may be found which appear to have undergone complete terminalization, for no evidence of a chiasma is visible. Such tetrads usually appear as closely paired rod-like bodies, usually representing the smaller chromosome pairs. In contrast to those of diakinesis, metaphase I tetrads are highly condensed, especially deep-staining, and sharply smooth in outline. The XY pair is readily identified, the X appearing as a long rod and the Y as an elliptical short rod, in end-to-end synapsis. The X and Y seldom appear as dyads, the chromatids remaining closely appressed. In side views the meiotic spindle is clearly visible, having the same short form as that of the mitotic metaphase.

Anaphase I. The first meiotic anaphase is only occasionally seen in sections and squash preparations, being of very short duration. The spindle is usually quite distinct, and polar movement of the dyads is uneventful unless induced aberrations lend distortions in the form of dicentric bridging. The X and Y normally reduce (separate) at this stage, although reports and photographs indicate equational division of these bodies so that one monad of each body passes to each pole. Figure 7-23 illustrates reductional and equational separation as observed by Slizynski ( 1955b). Sachs ( 1955) and Ohno et al. ( 1959) have reviewed the significance of the terminal association of the X and Y at meiosis.

Telophase I. After anaphase I the two groups of dyads form tightly clumped aggregates at the poles, representing the first part of telophase I. As the cytoplasm constricts to form the two new cells, nuclear membranes become distinct and the dyads uncoil progressively to form the second meiotic interphase.

Interphase II. In studying the regeneration of the germinal epithelium after depopulation by irradiation, Griffen and Bunker (unpublished) found and described the interphase of the second meiotic division. Interphase II is always intimately associated with metaphase I, metaphase II, or both. In cursory examinations of sectioned testes, interphases II grossly resemble spermatids, with which they regularly have been confused; however, they are fully twice the diameter of spermatids and half as numerous. In the mouse, as in the rat ( Tjio and Levan, 1956), X-bearing interphase II nuclei show a large allocyclic element representing the X-chromosome; Y-bearing nuclei have a similar but smaller element.

Prophase II. The second meiotic prophase is of short duration and therefore is never prominent in either sections or squashed preparations. Having the same diameter as interphase II, prophase II is easily distinguished from late pachytene which it otherwise resembles. Second prophases probably have been classified usually as partial sections of pachytene nuclei.

Prometaphase II. The first recognizable stage in the second spermatocytic division is best described as a prometaphase. The lightly stainable dyads of fuzzy outline, often showing clear indications of the major condensation spiral, appear as ragged pairs held together only at the centromeres. Short arms or satellites are regularly found in many of the dyads. Figure 7-24 illustrates interphase II, prophase II, and metaphase II in a section. Prometaphase II is seen in Figure 7-29.

Metaphase II. The second spermatocytic metaphase is frequently observed and is readily identified through the presence of 20 small dyads not easily confused with the 20 large tetrads of metaphase I. In squash preparations sister cells in metaphase II often lie side by side, one showing the X and the other the small Y. Figure 7-25 illustrates metaphase II in two sister cells.

Anaphase II. The second spermatocytic anaphase is of short duration and therefore scarce in all preparations. In squash preparations the second spindle is distinct and more elongated than the first spindle. The chromosomes in passage to the poles are very small and seem to be approximately the same size.

Telophase II. The second telophase has not been recognized in the mouse, the transition from second spermatocyte to spermatid thus seeming to be inconspicuous.

Spermatids. The spermatids resulting from division II lie next inward beyond the spermatocyte layer in the spermatogenic tubules. Aside from their small size, approaching that of spermatogonia, and the presence of several prominent chromatin bodies within the otherwise finely granular nucleus, the spermatids are of little cytological interest.

Spermatozoa. Oakberg ( 1956) has comprehensively described the development of spermatozoa from spermatids. Mature spermatozoa appear in sections as deeply stained short rods which form the innermost layer of the spermatogenic tubule, the flagella extending into the lumen. In squash preparations sperm heads usually appear as arrowhead-like bodies with curved points. The flagella are seldom found attached to the heads.

Slizynski ( 1957a), Borum ( 1961), Ohno ( 1963), and Kuhlmann (1965, personal communication) have analyzed the nuclear phenomena of oogenesis. According to Borum the mouse gonad is first identified as such in the 10-day fetus. In the 13-day fetus the gonad can be identified as testis or ovary. In the ovary some gonial divisions are see ( Figure 7-1), but most cells appear to be in the interphase growth stage of the primary oocyte ( Figure 7-26A). On the 14th day most of the cells have entered meiosis and are in leptotene and zygotene; some oogonial divisions are still found. On the 15th day all cells are in meiosis, with zygotene and pachytene predominating. The 16th day shows practically all oocytes in pachytene, with many beginning the rather massive degeneration which will eventually limit the final number of potential ova. Degenerating cells have heavily staining clumped chromosomes which appear to be highly condensed pachytene elements. On the 17th day cells in pachytene and degenerating pachytene continue to be predominant. By the 18th day many of the innermost cortical cells become large and clear, entering the diplotene stage; they become surrounded by flattened stromal cells. The more peripheral cells remain in pachytene. On the 19th day diplotene nuclei are increased in number and vast numbers of degenerating pachytene stages are seen. A few hours after birth many cells are in late diplotene, representing the resting stage, known as dictyate or dictyotene. At 19 hours after birth, early and late diplotene and degenerating cells predominate. At 40 hours after birth pachytene nuclei become scare and diplotene nuclei are principally found. By 60 hours after birth many oocytes have enlarged and received their follicle cells. Further nuclear stages of meiosis await the onset of estrus.

Leptotene. Kuhlmann (1965, personal communication) has shown that leptotene nuclei contain 40 paired heterochromatic blocks from which the chromosome threads extend as very thin coiled strands ( Figure 7-26B). The blocks are considered to be the centromeric regions of the chromosomes, and the paired condition is interpreted as the beginning of synapsis.

Zygotene. At zygotene the chromosomal threads become progressively paired and thickened. Kuhlmann demonstrated the formation of a distinct "bouquet" stage (synizesis) at zygotene. The bouquet is doubly polarized, showing a cluster of the early staining centromeric ends of the chromosomes and beside it a cluster of non-centromeric ends. The pairing chromosomes extend from one cluster to the other in long loops ( Figure 7-26C).

Pachytene. Full synapsis at pachytene is generally similar to that in the male except for the pronounced polarization ( Figure 7-26D) and the absence of a large plasmosome (or sex vesicle). According to Slizynski ( 1957a) no plasmosome is found in early oocytes.

Diplotene. At diplotene, pairing relaxes slightly and four strands are detectable in each pair of synapsed elements; otherwise this stage closely resembles pachytene.

Dictyotene. Instead of entering diakinesis, which is a clearly defined stage in the primary spermatocyte, the primary oocyte passes from diplotene into the dictyotene or dictyate stage. This stage is peculiar to oogenesis and constitutes the long stage of arrest in which the oocyte nuclei remain from early infancy until the time of fertilization in the adult. At this stage in the mouse the tetrads become progressively denuded of nucleic acids, the major coiling of late diplotene disappears, and the nuclei assume an "empty" appearance. Chromosome organization becomes indistinct. A large nucleolus appears and persists as the outstanding feature of the nucleus. It is notable that dictyotene persists throughout the growth of the oocyte from the small primordial to the large mature state. Although invisible, the chromosomes remain in tetrad association and the chiasmata persist, as indicated by the presence of terminalizing chiasmata at the first meiotic metaphase and anaphase. Figure 7-26E illustrates the dictyotene nucleus in a primordial follicle of an adult mouse. Figure 7-26F shows the late dictyotene nucleus of an oocyte in a graafian follicle. The conspicuous large nucleolus is accompanied by several micronuclei and connecting eosinophilic strands.

Prometaphase I. In connection with studies on the radiosensitivities of oocytes at the time of estrus, Kuhlmann has defined and described several stages which augment the classic first and second metaphase, anaphase, and telophase stages. After dictyotene the first stage in the continuation of the meiotic cycle is prometaphase I, characterized by the disappearance of the large and small nucleoli, the dissolution of the nuclear membrane. the formation of chromatin mass I, and the formation of the first meiotic spindle. Figure 7-27A illustrates the formation of "chromatin mass I" and the weakening of the nuclear membrane. Figure 7-27B shows Kuhlmann's "transitional phase I" in which discrete chromosome bodies (tetrads) emerge from the irregular mass. Figure 7-27C is Kuhlmann's "forming spindle I," with the appearance of the spindle fibers and visible stretching of the bivalents.

Metaphase I. In Figure 7-27D the bivalents are becoming oriented in the equatorial plane of metaphase I. The bulging centers of the bivalents indicate the presence of chiasmata in the process of terminalization.

Anaphase I. At early anaphase I ( Figure 7-27E) terminalization is completed and the released homologous dyads separate toward the poles. At midanaphase I ( Figure 7-27F) the dyads approach the poles preparatory to the formation of "chromatin mass II" and the first polar body. At the equator the spindle fibers are thickened and present the first indication of the midbody which is a prominent feature of the first meiotic telophase. In Figure 7-27G late anaphase I shows "chromatin mass II" the continued thickening of the spindle elements at the equator, the beginning of polar body I, and the rotation of the spindle from the tangential toward a vertical position with reference to the surface of the oocyte.

Telophase I. The first oocytic telophase is shown in Figure 7-27H. "Chromatin mass II" is highly condensed, the midbody is condensed and is compressed by the constricting cell membranes, and polar body I is becoming well defined. The midbody is destined to be excluded from each cell; this structure constitutes the material which Ohno ( 1963) described as "non-DNA materials" accumulated during the long dictyotene growth phase and extruded by the condensed chromosomes at the equatorial plate.

Prometaphase II. Kuhlmann's prometaphase II begins with "chromatin mass II," a cup-shaped chromosome aggregation which formed at the inner pole of the first meiotic spindle ( Figure 7-27I). No spindle fibers are visible. In "transitional phase II" the dyads of the mass become discrete and somewhat rounded ( Figure 7-28A). At "forming spindle II" spindle fibers appear and the dyads become oriented toward the equatorial region ( Figure 7-28B).

Metaphase II. The second oocytic metaphase ( 7-28C) shows uniform equatorial arrangement of the dyads on the second spindle, in contrast to the irregular or staggered position of the tetrads on the first spindle. Metaphase II persists until sperm entry after ovulation.

Anaphase II. At sperm entry anaphase II begins and proceeds rapidly. Figure 7-28D shows late anaphase II and, at the bottom of the photograph, the head of the fertilizing spermatozoon.

Telophase II. At the second meiotic telophase a midbody is again formed at the equator of the spindle and is extruded. At the inner pole the definitive monads of the ovum form a dense aggregate in which individual chromosomes are not distinguishable. The second polar body contains a similar chromatin mass ( Figure 7-28E).

Coiling

The condensation of chromosomes through coiling occurs in the prophase of both mitosis and meiosis. In the mouse, as in other animals, three types of coiling are postulated: molecular, primary (or minor), and secondary (or major). All are best exemplified in the meiotic prophase because of the prolongation of this stage and the large size of the nuclei.

Molecular coiling. The initial step in the coiling sequences is classically specified as coiling at the molecular level, providing for the first condensation of individual interphase chromosomes which thereby become visible for the first time. The spermatocytic interphase ordinarily shows prominent chromomeres whose serial alignment can be inferred from the short stretches of such alignment which are visible at many points as the observer focuses upon successive levels through the interphase nucleus. As prophase begins, the chromomeres com closer together in serial fashion and are seen to be constituents of a stainable thread. At full leptotene the individuality of the chromomeric chromosomes is easily observed, yet no visible indication of coiling is detected. Accordingly, molecular coiling is presumed to have been in operation.

At zygotene the chromomeric homologous chromosomes begin the process of pairing, with further condensation and thickening of the involved homologues. Since there is still no visible coiling, this condensation represents a continuation of the initial type of condensation. Figure 7-18 illustrates the spermatocytic interphase, leptotene, and zygotene in squash preparations.

Primary coiling. At diplotene the first indications of primary or minor coiling are seen in the linear contraction of each of the two homologous dyads. Each dyad appears to be thickened and to have a finely serrated outline which, with proper optical resolution, is seen to result from the presence of a fine coil. The coiled state is best observed adjacent to the chiasmata which hold the homologous dyads together; at these points of cross-connection the tension causes stretching of the coils so that their gyres are quite distinct for short distances.

Secondary coiling. At diakinesis the secondary or major coil appears, consisting of gross gyres superimposed upon the primary coil. The major coil drastically shortens the dyads and brings them eventually to their highly condensed metaphase condition. Major coiling is especially clear in certain somatic prometaphases such as those of Figures 7-9 and 7-11. It is also visible at diakinesis ( Figure 7-22 and in the prometaphase of secondary spermatocytes ( Figure 7-29).

Relational coiling. The loose twining of one chromatid about another, the two being held together at the centromere positions, constitutes relational coiling. This type of spiralization is not readily demonstrable in the mouse, although it is suggested in well-spread squash preparations of the spermatocytic diplotene.

Inasmuch as the phenomenon of coiling is the subject of a large portion of nuclear cytology, discussions such as that of Swanson ( 1957) should be consulted for details and interpretations not obtainable from studies on the mouse.

Synapsis

In the mouse the synapsis of homologous chromosomes at zygotene through pachytene reveals no unusual phenomena. Crew and Koller ( 1932) have emphasized the nonrandom nature of synapsis, indicating that it regularly begins at the ends of leptotene threads and proceeds along the chromosomes until the pairing is complete. The starting points are presumed to be the centromere regions of many of the threads.

Somatic pairing

The phenomenon of somatic pairing or somatic association of homologous chromosomes is a classic feature of somatic nuclear cytology in organisms such as the Drosophila species. In the mouse there are strong, but not compelling, indications of extensive associations of homologues. The marked chromosome pairs of Figure 7-9 illustrate this presumed association, found too frequently to be accredited to chance. In squash preparations of testicular teratomas of the mouse Stevens and Bunker ( 1964) frequently observed pairs of chromosomes similar in size and with correspondingly curved chromatids. Gropp and Odunjo ( 1963) reported the same phenomenon in metaphases of cultured human leukocytes. Evidence clearly exists, therefore, that somatic association in mammalian cells may be a chromosomal feature with hitherto unexpected prevalence. An especially interesting aspect of the associations is the concordance of chromatid configurations, curvatures, and positions. There can be little doubt that somatic pairing in the mouse is far more extensive than is revealed in squash preparations.

Chiasma formation

Nature of chiasmata. At the diplotene stage in meiosis, chiasmata are the points of exchange between homologous (nonsister) chromatids and represent the physical evidence that genetical crossovers have occurred ( Figure 7-29). In the mouse the classic study of chiasmata was made by Crew and Koller ( 1932) who showed that, although diakinesis and metaphase configurations in the mouse have fewer chiasmata than can be counted at diplotene, the decrease in number and alteration of position are brought about by terminalization of chiasmata. Therefore, at diakinesis and metaphase, chiasma counts which include terminalizations provide a close approximation of genetically determined crossover frequencies in both males and females. Figure 7-30 illustrates Crew and Koller's drawings of the complete diplotene chromosome complement of a male. Below each bivalent are numerals indicating the total number of chiasmata and the number of terminal chiasmata.

Frequency in males. Figure 7-3 is a drawing of the complete male metaphase I complement as presented by Crew and Koller. Numerals indicate total/terminalized chiasmata as in Figure 7-30. The XY bivalent is the last bivalent in the second row. Comparison of the metaphase and diplotene drawings exemplifies the reduction in chiasma count at metaphase as compared with diplotene. Table 7-1 indicates the chiasma frequency as determined from five such metaphase cells in a fully mature male. Table 7-2 shows the higher chiasma frequency as determined at metaphase in six cells from a male 6 weeks old. These tables exemplify the decrease in chiasma frequency which occurs as age increases, a finding which is comparable with Dunn's ( 1920) demonstration of a decrease in crossing over as age increases. Fisher ( 1949) found a similar decrease in recombination in the agouti-undulated interval of the fifth linkage group in both sexes.

Crew and Koller presented the graph shown in Figure 7-32 as a comparison of their determinations of the number of chiasmata per bivalent in males at metaphase with information obtained in studies by Cox ( 1926) and Painter ( 1927). They attributed Painter's lower chiasmata frequency to the possible influence of a major chromosomal deficiency which was present in his material, the v/O of Gates.

Table 7-3 compares Crew and Koller's metaphase chiasma frequency for the male with the diakinesis chiasma frequencies found by Huskins and Hearne ( 1936) and by Slizynski ( 1955a). Since diakinesis and metaphase I are closely related in form and time, the comparisons are quite valid and compatible. Crew and Koller's value for a male 6 months old corresponds to Huskins and Hearne's and to Slizynski's values for CBA males. Crew and Koller's "old" male value corresponds to that found in males other than CBA by Huskins and Hearne and to that found in unspecified hybrids by Slizynski.

Frequency in females. Figure 7-33 shows Crew and Koller's drawing of a complete female metaphase I complement along with numerical indicators of total/terminalized chiasmata. The XX bivalent is not identifiable. Table 7-4 indicates the chiasma frequency in five metaphases from a female which was a littermate of the male of Table 7-1.

With these studies Crew and Koller proved a difference in the sex incidence of metaphase chiasma frequency corresponding with the established sex difference in crossover frequency ( Chapter 8).

Chiasmata and chromosome map length

Slizynski ( 1955a) counted the chiasmata in 50 diplotene and 56 diakinesis nuclei from male mice of the CBA strain and from hybrid males of unspecified origin. In comparing his diplotene data with those which Crew and Koller obtained from a single cell. Slizynski found that his counts indicated a total map length of 1,916 centi-Morgans, while Crew and Koller's single-cell count indicated 2,450 cM. By combining his diplotene data for CBA and hybrid males, Slizynski found the total map length of the male to be 1,954 cM. Average lengths of short, medium, and long chromosomes were 76, 106, and 127 cM, respectively. Cytologically based calculations of the female have not yet been made.

Chiasmata positions

Most of the mouse metaphase configurations are readily interpreted on the basis of the partial chiasmatype theory and terminalizations. Yet there is a class of configurations requiring clarification: namely, the small ring or donut-shaped metaphase bivalents such as those in the second row of Figure 7-31. Crew and Koller ( 1932) considered these to be produced by the terminalization of two chiasmata. Since the centromere is nearly terminal in all mouse chromosomes, this interpretation requires the assumption that chiasmata form in the extremely minute arms of the chromosomes with remarkably high frequency. An alternative explanation requires that one of the end associations be based upon failure of the homologous centromeres to separate at diakinesis with an even more remarkable high frequency. The first explanation appears to be preferable but must await confirmation.

According to the genic-balance principle of sex determination, long accepted for Drosophila (Bridges, 1922, 1932), autosomal factors for maleness are balanced against the female-determining capabilities of the X chromosome. This system of sex determination came to be accepted for higher animals, including mammals, in which cytogenetic experimentation had not become feasible. The first indication that mammalian sex determination was not of the Drosophila type came with the studies by Welshons and Russell ( 1959) and by Russell et al. ( 1959). In studying the aberrant features of the inheritance of the sex-linked gene scurfy, these investigators discovered that mice of the XO constitution were female and that the Y chromosome bears factors which determine maleness. Cattanach ( 1961a) found that XXY mice are males and thus firmly established the Y chromosome as the male determiner.

A similar system having maleness determined by the Y has long been known for a relatively obscure organism, the dioecious plant Melandrium dioicum. Warmke and Blakeslee ( 1940) showed that Y-bearing plants were male in all cases except those in which a great preponderance of X's existed; even in the XXXXY constitution the maleness factors are expressed, this combination being hermaphrodite. Comparisons of the Drosophila, Melandrium, and mouse situations are given in Table 7-5, where each letter A represents a complete haploid complement of autosomes. The eventual finding and cytological verification of sex modifications in polyploid mice can provide further information about the strength of the mouse Y, as well as any possible role of the autosomes, in determining maleness.

Radiation cytogenetics in the mouse began with Painter's ( 1928) cytological study of Little and Bagg's ( 1924) abnormal-eyed mice, discovered in the progeny of animals exposed to X-rays. No abnormal chromosomal conditions were revealed, the diploid number of 40 being present in cells of each of four males studied. No aberrant features were found in the study of separating dyads on the first meiotic spindle, employing the exceedingly difficult procedures previously successful in disclosing for the first time the phenomenon of deletion in Gates' "nondisjunction" v/O mice.

After Muller's discovery of the production of mutation in Drosophila by X-rays, several investigators accumulated data indicative of chromosome abnormalities in the offspring of irradiated mice. Russell ( 1962) summarizes most of the studies and provides extensive references. Snell ( 1932, 1933a, b, c) first reported the occurrence of semisterility in the offspring of irradiated males and attributed this effect to the presence of chromosome translocations. Snell et al. ( 1934), Snell ( 1935), and Snell and Picken ( 1935) related the production of abnormal embryos by the semisterile offspring of irradiated mice to the chromosomal imbalance resulting from the several possibilities of segregation in translocation heterozygotes. Figure 7-34 illustrates the production of aneuploid gametes by a generalized translocation heterozygote. This diagram postulates a slightly subterminal position for the centromeres and includes the classes of gametes producible through nondisjunction of homologous centromeres.

Koller and Auerbach ( 1941) conclusively demonstrated that semisterility in the offspring of irradiated mice was caused by reciprocal translocation readily detected in primary spermatocytes of semisterile males. Koller ( 1944) further explained the mechanical basis for semisterility, indicating that for any particular translocation the degree of fertility is correlated with the frequency of nondisjunctional arrangement of members of the translocation double tetrad or ring-of-ring. Slizynski ( 1949) prepared the first pachytene chromosome map for the mouse and employed it with success ( 1952) in making a cytological analysis of Snell's T(5;8)a translocation at pachytene. He extended the pachytene studies ( 1957b) in the analysis of several translocation produced by Carter et al. ( 1955). Jaffe ( 1952) reported segmental interchanges in primary spermatocytes of semisterile males derived from certain of Dunn's tailless series but he did not find evidence for inversions in any of these lines. Griffen ( 1958, 1964) found translocations, supernumerary fragments, trisomy and asynapsis to be common aberrations in the offspring of irradiated males. Deletions and inversions were not detected, since pachytene nuclei were not studied.

7-6 lists the gene-tagged translocations and other heritable chromosome abnormalities thought to be available in existing stocks in 1965.

Two-break aberrations induced in haploid cells

Translocations. The cytological investigations thus far mentioned have dealt primarily with segmental interchanges, which are often readily visible as rings-of-four or chains-of-four in the first meiotic division of spermatogenesis and are readily detected because of the reduced number of tetrads at diakinesis and at metaphase I. Whereas the normal number of tetrads at metaphase I is 20, the presence of one translocation involving two chromosomes reduces the count to 19 through the formation of a ring or chain "double tetrad." Two independent translocations, each resulting from two breaks and each involving two different chromosomes, reduces the count to 18, as in cases such as that of Falconer et al. ( 1952) and in those of Griffen ( 1964). As a general rule it may be stated that each translocation involving two chromosomes reduces by one the count of metaphase I bodies.

Inversions. Inversions have not been reported in the mouse, although they are among the most common of all spontaneous chromosome aberrations and also are of very frequent occurrence in experimental organisms exposed to ionizing radiations. We may presume that they have been produced in mice in the course of numerous radiation experiments. Inversions are readily detected in only two ways: (1) through the use of breeding tests which may reveal drastic modifications of the recombination frequencies of linked markers and (2) through the discovery of loops between inverted and noninverted homologous chromosomes at meiotic synapsis, particularly at pachytene. Since pachytene is extremely difficult to study in mouse cells, it has not been feasible to search the pachytene stage of individuals sired by irradiated males for the presence of inversions. Partial sterility may indicate the possible presence of inversions, particularly those of greater length, since crossing over within the limits of an inversion may produce dicentric and acentric fragments whose loss results in zygotic death through hypoploidy.

Deficiencies. Deficiencies or deletions have been reported as probable in the offspring of irradiated mice by Russell and Russell ( 1960) and by Kidwell et al. ( 1961). Cytological verifications have not been made for these deficiencies, although the genetic evidence for their occurrence is unimpeachable. Eventual pachytene demonstrations of irradiation-induced deletions may be expected to resemble the deficiencies of spontaneous origin studied by Geyer-Duszynska ( 1964) in a cytological analysis of the t series of alleles in chromosome IX. Three t "alleles" ( t⁰, t^w6, and t^w18) were shown to be represented by small loop formation at pachytene. Figure 7-35 provides a generalized example of synapsed deficient and normal chromosomes at pachytene.

External duplications. External duplication are those portions of the genome carried as supernumerary free fragments, the remnants of more or less grossly deleted two-break chromosomes. Such free bodies are partial trisomics and will be discussed in a later section.

Three-break aberrations induced in haploid cells

Translocations. Translocations involving three chromosomes, with one break in each, form especially prominent rings-of-six at metaphase I of meiosis ( Griffen, 1964).

Such aberrations are especially rare and may constitute the class of "progressive" translocations in which the attachments result in a 1-2, 2-3, 3-1 type of union ( Figure 7-36). Each aberration of this type reduces the count of metaphase I bodies by two. It should be noted that a ring-of-six and reduction of the count by two will also appear in the heterozygote formed by crossing two individuals, each carrying a single translocation and the two translocations having one chromosome in common. Thus the heterozygote for T(1;2) and T(2;3) shows a 1-2-3 ring of six and a count of 18 at metaphase I. Slizinski ( 1957b) illustrated such configurations in mice heterozygous for Carter, Lyon, and Philips' translocations 2/281, 5/7, 2/5, and others.

Transpositions. Transpositions result when the interval between two breaks in one chromosome becomes inserted between the ends of a single break in a second chromosome ( Figure 7-37). Ohno and Cattanach ( 1962) illustrated such an aberration in the "flecked" mouse, showing that a segment of chromosome I bearing the wild-type alleles of genes p and c is inserted into the X chromosome. Even though this transposition was chemically induced ( Cattanach, 1961b), it may nevertheless exemplify the similar instances of radiation-induced variegated aberrations reported by Russell and Bangham ( 1959, 1961) and by Russell et al. ( 1962). Cytological analyses of these cases have not been reported.

Segregational internal duplications. Internal duplications are those contained within some member of the regular chromosome complement and are derived from transpositions such as Cattanach's "flecked" transposition. Segregants having two entire chromosomes I plus an X carrying the inserted segment of I are viable and fertile in both sexes. (Duplications in Russell and Bangham's cases are lethal; legitimate inclusion of these cases must await cytological analyses.) Other types of internal duplication depend upon origination in diploid cells and are discussed in a following section.

Segregational deficiencies. Segregational deficiencies are the complements of segregational internal duplications. The "flecked" transposition, for example, readily segregates so as to produce a class of lethal zygotes presumed to include both males and females and to have one normal chromosome I, one deficient chromosome I, and two normal X's or a normal X and a normal Y.

Autosomal aneuploidy

Radiation-induced deviations from the normal haploid and diploid chromosome numbers usually may be defined as the gain or loss of a centromere, along with all or part of the portion of the genome normally transported by that centromere in cell division. Nondisjunction, the failure of centromeres to separate at the first meiotic division, is usually invoked in accounting for the production of aneuploidy.

Trisomy. Through nondisjunction, gametes may be formed which contain a single whole chromosome as a supernumerary element. Union with a normal haploid gamete results in a 2N + 1 zygote in which one linkage group is present in a triplicate condition in an otherwise diploid genome. The zygote is said to be trisomic for the triply represented linkage group. Four cases of autosomal trisomy have been reported for the mouse, one by Cattanach ( 1964) and three by Griffen and Bunker ( 1964). Cattanach's trisomic was a phenotypically normal, but sterile, male which resulted from chemically-induced nondisjunction of one of the smallest autosomal pairs at spermatogenesis in the male parent. Griffen and Bunker's three trisomics occurred among the offspring of irradiated males, presumably as the result of nondisjunction at spermatogenesis. All were phenotypically normal, but two were sterile and one semisterile. In each case the trisomy involved members of the smaller chromosome classes. A trisomic spermatogonial metaphase and its idiogram are shown in Figure 7-38.

Monosomy. Gametes lacking one complete member of the haploid chromosome set are produced by nondisjunction as the complements of gametes containing a supernumerary element. Union of a deficient with a normal gamete results in a 2N - 1 zygote in which one linkage group is present in the haploid state in an otherwise diploid genome. The zygote is said to be monosomic for the singly represented linkage group. No case of viable autosomal monosomy has been reported for the mouse.

Partial trisomy. Supernumerary free fragments, also classifiable as external duplications, result from the gross deletion of single chromosomes whose small remains, carried by centromeres, may become added to an otherwise normal diploid genotype through nondisjunction. Partial trisomics are of especially common occurrence in the offspring of irradiated mice ( Griffen, 1964). Partial trisomy may result also from reciprocal translocations whose reunion products consist of a very large plus a very small element. If the small element becomes a supernumerary body through nondisjunction, partial trisomy exists for a minute portion of each of two linkage groups rather than for a single linkage group. Partial trisomics cannot be classified as to origin unless, in the translocation type, the original intact segmental interchange has been recognized in precursors of the trisomic individuals. A spermatogonial metaphase showing a partial trisomic is illustrated in Figure 7-39.

Aneuploidy of the sex chromosomes

The X and Y chromosomes are the only elements of the mouse genome which, in the monosomic condition, are known to permit survival. The normal XY male is, by definition, monosomic for both the X and the Y. It is known that normal XX females, through the X-inactivation phenomenon, are functionally monosomic even though cytologically disomic. True cytological X-monosomy has been found by Russell et al. ( 1959), Welshons and Russell ( 1959), McLaren ( 1960), Cattanach ( 1961a), and Kindred ( 1961) who have described viable XO females, some of which were derived from irradiated sperm whose X or Y had been destroyed.

Hyperploidy for the sex chromosomes in the mouse is represented by sterile XXY males such as found by Russell and Chu ( 1961) and by Kindred ( 1961). XXY individuals cannot be considered as trisomics, since the X and Y cannot be equated cytologically or physiologically. True XXX female mice have not been reported; such individuals, if comparable to multiple-X human beings, would be functionally monosomic through inactivation of two of the three X's, although cytologically trisomic. Diagnostically useful sex chromatin bodies, such as those of humans and cats, have not been found in somatic cells of the mouse.

Chromosome breakage sensitivities in germinal cells

A comprehensive review of the radiosensitivity of germ cells by Mandl ( 1964) and a review of chromosome aberrations in experimental animals by Russell ( 1962) indicate the gross lack of precise information on cytologically demonstrable chromosome aberrations which are produced in germinal cells of precise morphological types and at definite mitotic and meiotic phases. Although the investigations of Oakberg and his colleagues (summarized by Oakberg and Clark, 1964) have made possible the accurate timing and identification of spermatogenic stages in the mouse, only sensitivity determinations have been made, as determined by cell survival. Extensive information on the production of aberrations in irradiated spermatogonia has been obtained by Griffen ( 1964), who found translocations, fragments, trisomics, and asynaptics among partially fertile individuals derived from treated gonia.

SUMMARY

The nuclear cytology of the mouse generally shows chromosomal features and activities conforming to the classic patterns for both animal and plant cytology. Deviations are seen in the prophase of oogenesis, presumably associated with the long stage of nuclear arrest and cytoplasmic growth.

Chromosome identifications are not feasible in gonial cells, nor in meiocytes except for the XY bivalent in primary spermatocytes. Identifications are possible in prometaphases of somatic cells grown in vivo and in vitro and in prophases of certain large tumor cells. Prophase somatic chromosomes can be identified to some extent with elements of the pachytene chromosome maps.

Allocycly of the X and Y chromosomes permits the location of these elements in somatic and tumor prophases. However, in female cells no sex chromatin body is diagnostically visible as it is in certain other mammals.

Evidence of somatic pairing in somatic cells is strongly but not conclusively indicated, along with an unexplained concordance of position, curvature, and other attributes of the chromatids of homologous chromosomes at prometaphase and metaphase.

Chiasma frequencies at diplotene, diakinesis, and metaphase can be correlated with crossover frequencies which are genetically detected. Terminalizations of chiasmata cause understandable discrepancies between the two phenomena. Like crossover frequencies, chiasma frequencies are lower in males than in females; no cytological explanation for this difference is available.

Sex determination in the mouse resembles the system in Melandrium, the Y chromosome being male-determining. There appears to be no male-determining role of the autosomes such as exists in the genic balance system of Drosophila and certain other organisms.

Chromosome aberrations in the mouse, principally induced by X-rays, include translocations, transpositions, deficiencies, and segregational internal duplications. Autosomal monosomy has not been found. Trisomy and partial trisomy have been demonstrated for several of the smaller autosomes.

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