Talk:Paper - the primary human oocyte - some observations on the fine structure of Balbiani's vitelline body and the origin of the annulate lamellae

From Embryology


Department of Pathology, Harvard Medical School, Boston, Massachusetts

1 The present communication forms the historical and scientific basis of the addressgiven by the author after the banquet of the American Association of Anatomists in Kansas City, Mo., April 6, 1967 entitled: “Human Oocytes, Past Present and Future; a Resurrection of Balbianfs Body.”

2 This work was supported U.S.P.H.S. grant I-ID-00137.


The ultrastructural details of human oocytes from four primordial

follicles and one early primary follicle are presented. A fifth primordial follicle is represented by a paraflin section stained by hematoxylin and eosin. The paranuclear Balbiani vitelline body, contsisting of a centrosome surrounded by endoplasmic reticulum, Golgi complexes, compound aggregates, annulate lamellae, and mitochondria is described. The annulate larnellae arise as an evagination from the outer leaflet of the nuclear envelope and interdigitate with folds of the endoplasmic reticulum which also is continuous with the outer leaflet of the nuclear envelope. Structural aspects of annulate lamellae are discussed in relationship to current ideas of nuclear membrane ultrastructure and to their possible role in nucleo-cytoplasmic transfer.

A biographical note on the life of Edouard Gérard Balbiani is presented.

The conspicuous paranuclear mitochondrial mass surrounding the centrosome of vertebrate and invertebrate oocytes has been studied by light microscopists for many years. According to I-Ienneguy (1887) it was described in the spider oocyte by von Wittich in 1845 and was named the “Dotterkern” by Came in 1848. Balbiani described it in detafl in oocytes of spiders and myriapods (1864a, b). He summarized his extensive observations in 1893. This structure was designated by Henneguy —— Balbianifs student -- as “Balbiani’s vesicle” in 1887 and as the “yolk body of Balbiani” in 1893. Van der Stricht in his classic monograph of 1923 on the developmental stages of oocytes in mammals distinguishes clearly between the central vitelline or Balbiani body and the surrounding vitellogenic or mitochondrial bed. Aykroyd (’38), following the example of Brambell, Whose 1925 paper she quotes, designated these areas as the “pallial layer” and “couche vitellogéne” respectively. These authors believed that in the past Balbiani’s name has been applied indiscriminately to either or both the components of this paranuclear complex. Beams and Sheehan (’41) designate it as the “yolk nucleus complex” whereas Raven (’61) believes that it should be called Balbiani’s vitelline body. We agree with this latter designation and apply that term to the entire paranuclear complex of apparently interrelated organelles in human oocytes within primordial and early transitional primary follicles. Four of the oocytes from primordial follicles illustrated here were included in a larger group previously reported in detafl (Hertig and Adams, 67). One previously unpublished primordial oocyte is illustrated and, in addition, one oocyte from an early transitional primary follicle (H35-2) will be described (table 1).

During the preparation of this paper and the address based upon it, the author became interested in the life and Works of Edouard Gérard Balbiani, the cytologist, comparative embryologist and supplier of medical eponyms. A biographical summary of this remarkable man is included here.

Edouard Gérard Balbiani (fig. 17) came from an Italian family of ancient lineage. During the reign of Francis the first his ancestors left Italy to reside in various European countries. Edouard descended from the German branch; his banker father having emigrated to Haiti. Balbiani was born, apparently" in Port-auPrince, about 1823. His classical schooling took place in Frankfurt-am-Main and in Paris. There he went on to study law, natural science and medicine; obtaining his M.D. in 1854 (Henneguy, ’00; Boyer, ’48; Bibliographic Anatomique, 1899; Nature, 1899).

Balbiani immediately embarked upon a scientific career. He was a patient, skillful dissector and manipulator of living cells and unicellular organisms which he observed for hours; recording the effect upon them of various reagents. He developed techniques of microdissection and coined the term “merotomy.” During his first study of the ciliated infusoria he observed their fission and conjugation. Believing that such animals Were complete and that the macronucleus was an ovary and the micronucleus a “testicule,” he totally misunderstood the process of mitosis. According to Hughes, (plate X, fig. G., ’59) the original plates of Balbiani’s 1861 paper, however, show several phases of mitotic division; the spindle fibers and chromosomes unfortunately being interpreted as a bundle of spermatozoa! Thus, although Balbiani was the first to observe and record the vital process of mitosis, it remained for Biitschli in 1876 to interpret correctly the role of the micronucleus in the sexual reproduction of ciliates. Ba.lbiani’s skills brought him to the attention of Claude Bernard who appointed him in 1867 as Director of Histological Studies of the Laboratory of Physiology at the Museum of Natural Sciences. He held this post until 1873 when he succeeded Coste in the Professorship of Comparative Embryology at the College de France.

The complex, subsequently to be called the “yolk body of Balbiani” by Henneguy in 1893, was first studied by Balbiani in spiders and myriapods ( 1864a, b). His interpretation of these structures was obscured by his belief in the hermaphroditism which he thought he had established for the ciliates. In applying such concepts to the germ cells of metazoa he attributed a role in the formation of the “germ” to the yolk nucleus. Later (1893) he was to regard the “yolk nucleus” (Dotterkern) as homologous to the Nebenkern (centrosome of Platner) of germinal cells and of the centrosome of somatic cells. He believed that the yolk nucleus originated from the nucleus of the immature oocyte and that during its formation, a more or less thick layer of yolk condensed around it. (It is now clear that what he was observing was the centrosome with its surrounding mass of organelles, undoubtedly Golgi bodies and mitochondria. Please refer to figure 1, an intact oocyte from the myriapod, Geophilus longicornis drawn by Balbiani himself and figure 18, the microscope with which he presumably studied such oocytes.)

As an applied scientist he identified the causative sporozoan of the silk-worm disease, pebrme, whose cure added to Pasteur’s growing fame. Balbiani also Worked out the life cycle of the root louse, Phylloxem vastatrix, with its parthenogenetic generations, which was infesting the French vineyards (Henneguy, ’O0; Boyer, ’-48). Although Balbiani recommended decortication (“décorticage des ceps”) of the vine stocks, and application of insecticides to the roots to control the overwintering louse eggs, the more practical solution to this pressing problem lay elsewhere. Resistant root stocks of Vitis rupestris were imported from American vineyards and upon these Were grafted the native French Vitis vinifera (Amerine, ’66).

Perhaps Balbianfs greatest cytological achievement lay in his discovery and description of the giant chromosome with its surrounding puffs or rings in the larval salivary glands of the midge, Chironomus plumosus (fig. 19). He named this structure a “cordon” (Balbiani, 1881a, b) and it is later referred to as a “filament nucléin— ien” (Bibliographic Anatomique, 1899). It is not clear whether it was accepted as a chromosome or not, even though Balbiani himself (1876) explicitly identified it as being homologous with the filaments which he had earlier observed in the nuclei of dividing cells in the ovary of the grasshopper, Stenobrothus pmtorum. He also related the “cordon” to the nuclear filaments of cells in karyokinesis as described in the salamander by Pfitzner ( 1881). [The term “mitosis” was introduced in 1882 by Flemming and the term “chromosome” by Waldeyer in 1888. It was of historic interest that at this time Miescher (1871) and Altmann (1889) were doing their pioneer work on the nucleic acids.

Balbiani described at magnifications of 100-150, the irregularly convoluted or folded cord of 15 u in diameter with its dark cross striations and discoid pale expansions. The relationship of the “cordon” to the nucleoli is seen in all three of his drawings (fig. 19). The cross striations were stained by methyl green although the intervening areas and rings were unstained. Carmine and hematoxylin, however, left the bands only slightly stained but stained the rings and the nucleoli intensely. It is ironic that Balbiani refused to speculate (1881b) on the function of the “cordon” and never returned to it in any of his subsequent publications. Nevertheless, he was the first to describe the morphologic counterpart of the gene concept as formulated by the geneticists.

  • It is of interest that both Mendel’s and Balbiani’s observations lay unnoticed for many years. By 1915 the location of 50 genes for the four single chromosomes of Drosophila had been mapped (Morgan et al., ’15, as cited by Hughes, ’59). It was not until 1933, however, that Heitz and Bauer realized that the giant chromosomes of dipterous larvae, described by Balbiani in 188-1 under the light microscope, were equivalent to the interphase chromosomes of ordinary cells. This led Painter and others to compare the band patterns of giant chromosomes to established genetic maps in larval Drosophila. There also emerged the chemical nature of Balbiani’s rings or puffs (Beermann, "52; Beermann and Bahr, ’54; Clever and Karlson, ’60).

The bands contain DNA whereas the puifs or rings contain RNA (Beermann and Clever, ’64).

Henneguy (’00) summarized Balbiani’s failure to understand the significance of his classic observations in these words,

... "if he did not fully profit in terms of scientific renown from his discoveries it is because he made them too soon.”


The ovarian material was obtained at elective operation from four fertile women whose essential clinical data are shown in table 1. Since this is, in essence, the summary of a speech, the interested reader is referred to our previous paper for the detafls of preparation of our material (Hertig and Adams, ’6'7).


Light microscopy

If the plane of section happens to pass through the center of the nucleus and through the center of the cytocentrum, Balbiani’s vitelline body is clearly seen in the conventional histologic sections (fig. 2) as well as in electron micrographs (fig. 3). stained by hematoxylin and eosin it is a pale, crescent shaped eosinopihlic complex applied closely to the nuclear membrane as shown in figure 2. The cytocentrum appears as a dark sphere surrounded by a pale halo and flanked by dark granules.

With Sudan black stain, sudanophfla is present throughout Balbianifs vitelline

TABLE 1 Clinical data from patients whose oocytes up-pear in this study

Indication for

No. Age Para Grav

Endornetrial No.


operation phase oocytes

H-35 28 V VII Carcinoma in Mid-proliferative 6, 7, 8 2

situ of 9, 10, 11

Cervix uteri 12 and 14 H-43 37 VIII XII Caesarean Term (35 3/ 7 Weeks)

section pregnancy 13 1 H48 34 V VI Carcinoma in

situ of

Cervix uteri 19—20th day 2, 3, 4 2 H-57 34 I III Pelvic pain,


2 ectopic

pregnancies 25th day 15 1 110

body. This apparently represents the phospholipid of the complex, since there was no detectable difference between Sudan black preparations of frozen and of paraffin sections. The PAS stain reveals a pale center and a darker periphery, possibly associated with compound aggregates.

Etectron microscopy

These oocytes have a spherical nucleus of 22 to 24 u in diameter with a typical envelope whose double leaflet is interrupted by pores containing electron dense material (figs. 4, 8, 9, 12, 13, 14, 15, 16).

Closely packed, parallel spiral fibrillae are occasionally seen attached to but apparently not arising from, the outer leaflet of the nuclear membrane (fig. 8). There is a suggestive evidence that the nuclear pores may be more closely spaced in conjunction With these fibrils than elsewhere. These clusters of closely packed fibrillae are also seen occasionally in the cytoplasm. Arising from the outer nuclear leaflet are profiles of endoplasmic reticulum (figs. 9, 12, 14, 16) and stacks of annulate lamellae (figs. 12, 13, 14, 16). These will be described more in detail later.

The cytocentrum (centrosome) at the center of Balbiani’s body (figs. 3, 4, 7, 8) is conspicuous as a cluster of organelles with a spherical dense center and lighter periphery. Mitochondria are conspicuous by their absence in the center and scarcity at the periphery. The prominent center, measuring up to 4.5 u in diameter (probably the counterpart of the dense structure seen by H and E stain in fig. 2) is composed of electron opaque deposits embedded within a matrix of fine fibrils. These dense granules may become periodically aligned to form radiating fibrils which apparently merge with peripheral coarse fibers measuring up to 0.13 u in diameter. Their maximum length is not possible to estimate. In oocytes of primordial follicles, (fig. 4) the peripheral coarse fibers appear in favorable serial sections to be straight and to be interwoven as a basket-like network about the periphery of the cytocentrum. In the early primary follicle (fig. 8) they appear more curved so that they conform to the contour of the cytocentrum. Vesicles either in discrete aggregates or dispersed among the amor ARTHUR T. HERTIG

phous deposits are also characteristic of the cytocentrum (fig. 4). At the periphery of the ctoycentrum there is a network of vesicular or tubular endoplasmic reticulum which becomes continuous with that in the more peripheral areas of Balbiani’s body (figs. 4, 8). No centrioles were seen Within the cytocentrum in spite of careful search. Microtubules can be found throughout Balbianfs vitelline body and also less frequently in the peripheral cytoplasm.

Surrounding the cytocentrum are multiple Golgi complexes, prominent compound aggregates, a mass of mitochondria and a single stack or coil of annulate lamellae. Endoplasmic reticulum that is sparsely granular is found throughout this area. The ovoid or spherical mitochondria measure 0.5 to 1.8 u and contain prominent arched cristae. They are frequently clustered in rosette fashion, about a dense somewhat reticulated mass (Hertig and Adams, ’67). In some oocytes in primordial follicles, the mitochondria appear to be entirely localized within Balbiani’s vitelline body (fig. 3) whereas in other oocytes a variable number together with Golgi complexes and elements of endoplasmic reticulum are sparsely distributed around the entire circumference of the nucleus (Hertig and Adams, ’67).

In oocytes of both primordial and early transitional primary follicles the endoplasmic reticulum is intimately associated with the mitochondria in Balbiani’s vitelline body (figs. 4, 8). In the stage of early transitional primary follicle illustrated, the endoplasmic reticulum is also prominent as a basket-like shell at the periphery of Balbiani’s body (figs. 7, 8,). The membranes of this shell appear to be formed by multiple evaginations from the outer leaflet of the nuclear membrane (fig. 9). In this primary stage, the concentration of Golgi complexes appears to have shifted from the periphery of the cytocentrum (fig. 4) to the periphery of Balbiani’s body where their membranes are in continuity with those of the endoplasmic reticulum (figs. 8, 10).

The large, electron-opaque compound aggregates at the periphery of the cytocentrum or within the mitochondrial mass are composed of vacuoles surrounded by finely divided, electron-opaque membranous or granular material (figs. 4, 8). The vacuoles contain a homogeneous material. These compound - aggregates vary in size and appearance (fig. 4): some are membrane-bound while others are not; some are ballooned and appear to have imbided a clear fluid in which the elements of the aggregates are dispersed. Similar structures are frequently seen in the peripheral cytoplasm of the oocyte and in the cytoplasm of the follicular (figs. 3, 11) and cortical cells of the surrounding ovarian tissue. Evidence for the possible transport, either from oocyte to follicle or vice versa, of the material in the compound aggregates has been presented previously (Hertig and Adams, ’67).

Annulate lamellae may be attached or immediately adjacent to the nucleus (figs. 12-16) or be free within Balbiani’s vitelline body. In the latter situation it may be adjacent to the cytocentrum (figs. 7, 8) or be Within the surrounding mass of mitochondria (fig. 4). These structures may be stacked (figs. 12, 14, 16) or concentric (figs. 13, 15). They consist of smooth surfaced paired membranes which at their periphery are connected to the more granular endoplasmic reticulum, the latter in turn closely associated with mitochondria. These paired membranes mimic or duplicate the two leaflets of the nuclear membrane. The pores of the latter are often in register with some of those of the attached lamellae although the lamellae have more pores per unit of membrane than does the nuclear envelope. Within the pores of the nuclear envelope and connected lamellae are cores of moderately dense material, presumably the extended or expanded material of the “pore-complex” (Watson, ’59). Close inspection of some of the annuli seen in a fortunate plane of section suggests that this material within the pore is a hollow cylinder rather than a solid band or rod (figs. 14, 15). Moreover, its external diameter seems to be somewhat larger than that of the pore and its internal diameter slightly less than that of the pore. The appearance of such annulate lamellae, whether attached, detached, stacked or concentric is one of a multilaminated structure duplicating the nuclear envelope and apparently precisely aligned so that the pores are in register. That the “pore complex” of these annulate lamellae duplicates that of the nuclear membrane is shown by their similarity when they are sectioned in a tangential plane (figs. 4, 8).

When annulate lamellae are in the coiled form and are immediately adjacent to the nuclei, the ctyoplasm within the coil will occasionally (twice in our experience) show multiple collections of moderately fine, dense, almost amorphous material surrounded by finer, more widely dispersed, halos (fig. 15). These appear to be morphologically simflar, and maybe analagous to the material within the pore complex of the nuclear membrane and annulate lamellae. In another oocyte from a primordial follicle similar deposits were found within the cytopalsmic center of comparable concentric annulate lamellae. These were illustrated in a previous paper (fig. 32 in Hertig and Adams, ’67) although another section through this same structure, illustrated here in figure 13, does not contain them.

By a fortunate plane of section through one primordial oocyte the continuity of the annulate lamellae with the outer leaflet of the nuclear envelope could be clearly demonstrated (figs. 12, 14, 16). The outer leaflet evaginates into an expanded perinuclear space forming a series of folds which are at first single and then double. The latter feature owes its origin to the invagination of the primary fold by secondary folds derived from the endoplasmic reticulum. The latter also arises from the external nuclear leaflet. Thus there is formed a very striking replication of the external nuclear leaflet which, in its early stage is connected to both the nucleus and the endoplasmic reticulum. A simplified diagrammatic reconstruction of annulate lamellae, in the act of formation, is to be seen in figure 16.

The granulosa cells of the primordial follicle are of variable thickness and density. A crescent-shaped cap of thicker cells appears at one side (fig. 3). The early or transitional primary follicle, however, has cuboidal epithelium (figs. 5-7). Neither of these closely related stages shows evidence of mitotic activity in the follicular epithelium. The latter contains dense vesicular compound aggregates (fig. 112

11) which are indistinguishable from those seen within Balbiani’s vitelline body (figs. 3, 4, 7, 8, 10). Such deposits or structures are also to be seen in cortical and even serosal cells of the ovary. The granulosa cells are characterized by villous projections which only slightly indent the ooplasm of the enclosed oocyte (fig. 111). Such microvilli are most prominent be tween adjacent granulosa cells at the granulosa-oocyte junction.


Balbiani’s vitelline body has been observed for well over a hundred years and extensively studied by light microscopists in both invertebrate and vertebrate oocytes. Raven (’61) summarizing the voluminous comparative observations on oocytes has this to say about primordial stage, “the earliest oocyte often contains a distinct cytocentrum, situated on one side against the nuclear membrane, sometimes in an indentation of the latter. It consists of one or two small granules, the centrioles, surrounded by a sphere of dense cytoplasm, the archoplasm or idiosome. Sometimes there is an indication of astral radiations in the latter. The archoplasm is often encircled by a ring of Golgi bodies, which in turn is surrounded by a dense cloud of mitochondria. The whole structure thus outlined is known as Balbiani’s vitelline body, often wrongly called yolk nucleus.”

Rebhun (’65a), in reporting on the ultrastructure and basophilia of the periodic lamellae and nuclear envelope, cites literature from 1919 to 1949 and says, “The mitochondria and Golgi apparatus in the early germ cell are located in a perinuclear ring or crescent so closely applied to the nuclear membrane as to appear attached to it.” He emphasizes that as the oocyte increases in size these structures migrate throughout the ooplasm. This dispersal of organelles has also been no-ted by Akyroyd (’38) and Beams and Sheehan (’41).

As a pathologist, examining routine hematoxylin and eosin sections of ovaries, the author has seen an occasional Balbiani vitelline body in an ovo—testes of an 8-months stillborn; in newborns and in a child of 18 months. Judging from such routine observations over the years, oocytes at this stage of human development are apparently uudergoing atresia or “degeneration” (pathologist’s terminology) in great numbers-. That this actually is so is shown by Baker’s (’63) precise quantitative and cytological studies of germ cells in human ovaries from the early fetal stage through seven years of age. The difficulty of finding a Balbiani vitelline body in routine sections of any ovary prepared in a pathology laboratory is compounded of many factors; the generally poor fixation of the large watery oocyte, the random plane of section and the question: -do oocytes in primordial follicles of the adult continue to undergo atresia?

By electron microscopy the paranuclear complex of organelles forming Balbiani’s vitelline body is particularly striking in the oocytes of adult human primordial follicles. Comparable though less striking paranuclear aggregates are also found in other mammalian species. They are regarded as large Golgi complexes in the rat (Sotelo, ’59); guinea pig (Anderson and Beams, ’60; Adams and Hertig, ’64); the rabbit (Blanchette, ’61; Zamboni and Mastroianni, "66); the hamster (Odor, ’65; Weakley, ’66). Fetal human ovaries containing oocytes in early meiotic prophase show a paranuclear concentration of mitochondria and Golgi complexes (Lanzavecchia and Mangioni, ’64; Stegner and Wartenberg, "63). A

All of the oocytes in primordial follicles studied by Hertig and Adams (’67) contained similar arrangements of the organelles within Balbiani’s vitelline body. In some oocytes, however, the annulate lamellate were attached to the nuclear membrane and in others they were free in the cytoplasm. It was noted that those oocytes with annulate lamellae attached to the nuclear membrane also had some mitochondria, Golgi complexes and elements of the endoplasmic reticulum dispersed around the entire circumference of the nuclear membrane, whereas in those with detached annulate lamellae these organelles appeared to be mo-re concentrated in or near Balbiani’s vitelline body. Furthermore the prevalence of attached annulate lamellae together with the dispersal of some organelles around the nucleus in ovaries removed during the estrogen dominant or follicular phase of the menstrual cycle suggested the need for further study of some cyclical hormonal effect upon the oocyte nucleus and its adjacent organelles.

Wilson’s ("66) radioautographic studies on the localization of estradiol-17B-1,2-H3 on the lampbrush chromosomes of the ova from the newt Tritums viridlescens seem germane in this regard. She showed that “radioactivity is concentrated in the areas of the loops which have been demonstrated to correspond to the areas of RNA transcription in these cells.” She concluded that this result (and others) “are compatible with the concept that these steroid hormones may be bound selectively to the sites of gene transcription in the nuclei of target tissues.”

Ancla and DeBrux (’65) in reporting the ultrastructure of the endometrium from a series of 40 patients, most of them infertility problems, said, “annulate lamellae may app-ear before ovulation but are more frequently seen during the secretory phase if there is hyperestrogenism.” This observation suggests some relationship of annulate lamellae to the endocrine events in the human menstrual cycle.

It is not surprising that the oocytes in primordial human follicles and in one early primary follicle, (Adams and Hertig, ’65), should have annulate lamellae since later stages of human oocyte development contain annulate lamellae (Wartenberg and Stegner, ’60) and the early pronucleus stage of the human fertilized ovum contains such structures both in the cytoplasm and pronuclei (Zamboni et al., ’66).

The interested reader is referred to the original articles of the various authors who have made significant observations on the origin, form and possible function of this amazing structure which simulates the nuclear membrane so precisely. Swift ("56) originally named these structures annulate lamellae but pointed out that others had seen them before. According to Swift they were probably fiISlZ seen by McCulloch in 1952 in Arbacia. Afzelius (’55) considered them fragments of nuclear membrane of the sea urchin oocyte. Palade (’52) observed them in the rat spermatid; Dalton and Felix (’54) in the mouse epididymis; and Gay (’55) in Drosophila. It was Swift (’56) who pointed out their basophilia and presumed high RNA con 113

tent. He observed and described annulate lamellae in the oocytes of the surf clam, Spisula solidissimtz; the pulmonate helicid snail, Otala lactea; the spermatocytes of the Sprague-Dawley rat of four months; and the larval pancreas of Amblystoma. His succinct summary is worth recording here. “These structures are alike in possessing numerous rings or annuli; resembling those in the nuclear membrane. Thus the name ‘annulate lamellae’ has been proposed for them. It is suggested that they may function in the transfer of specificities from nucleus to cytoplasm.”

Rebhun (’56a, b, ’61) and Ruthman (’58) have stressed the basophilia of these structures. Ruthman has . studied them cytochemically in the spermatocyte of the crayfish. Although annulate lamellae are strongly basophilic, they are relatively free of ribosome-like particles. Ruthman observed that annulate lamellae connect with endoplasmic reticulum. This is in agreement with our findings in human oocytes and suggests a potential mechanism for the transfer of material from nuclear-derived membranes to the cytoplasm via the endoplasmic reticulum.

The favorable chance finding of masses within the cytoplasm surrounded by a coil of annulate lamellae (fig. 15) further suggests that this structure, replicating nuclear membrane, is transporting or elaborating material that simulates that found within the pore complex of the nuclear membrane.

Annulate lamellae have been described in oocytes of the sea urchin Psammechinus miliaris by Afzelius (’55); of the sand dollar Dendmster excemtricus by Merriam (’59); of the surf clam Spisula solidz'ssima and the pulmonate snail Otczla lactea by Rebhun (’56a, b, ’61); of the newt Triturus viridescens by Wischnitzer (’58) and of the tunicate Ciomt éeztestémzlés by Mancusco (’64).

The latter author cites literature which shows that annulate lamellae also occur in germ cells of other forms as well as in other types of cells but are to be seen in cells having the “common characteristic of a particularly active metabolism.”

Kessel, in a classic series of papers, has reported his investigations on the origin of annulate lamellae in oocytes of such diverse forms as Necturus (’63), the echino— derm Thyone birareus (’64) and various tunicates (’65). He has shown that annulate lamellae probably arise by blebbing of the external lamella of the nuclear envelope; the blebs subsequently aligning themselves to simulate a segment of nuclear membrane. Such contiguous flattened vesicles then arrange themselves parallel to their fellows, thus forming stacks of annulate lamellae; the annulae of which are in register. Kessel (’65) indicates there is some morphologic evidence that the annuli are patent. The outer nuclear leaflet of the tunicate oocyte also contributes to the formation of the vesicular and granular endoplasmic reticulum.

Annulate lamellae have also been seen in a Variety of somatic cells. For a recent review see the report by Frasca et al. ("67) who found these structures in normal bronchial cells from 17 of 90 human males.

The mechanism of origin of annulate lamellae of oocytes in various specimens is to be sure somewhat different but the morphologic, and presumably functional features are comparable; the replication of nuclear envelope material which ultimately lies within the cytoplasm. Whether, as Swift suggested in 1956, annulate lamellae are involved in nucleo-cytoplasmic transfer is still unknown (Gall, ’64). Nevertheless there is no doubt that in the human primordial oocyte the annulate lamellae are continuous with and appear to arise from the nuclear envelope, interdigitate with the endoplasmic reticulum (fig. 16) and appear to be cast off periodically into the ooplasm. It would seem that this transfer to the cytoplasm of enormous amounts of stacked replicated nuclear membrane with its pore complexes or annulae in perfect register might be an ideal mechanism for transfer of genetic information by some form of RNA.

The transfer of material from nucleus to cytoplasm and vice versa has been investigated by many techniques. For a recent complete symposium on the nucleus, the reader is referred to the collection of articles collectively entitled “The Nuclear Membrane and Nucleoplasmic Interchange” by Feldherr and Harding, Gall, Goldstein, Loewenstein and Mirsky (’64) respectively. In discussing the fine structure of the nuclear envelope, Gall concludes that “there is both circumstantial and direct evidence of an association between the nuclear envelope and membrane systems of the cytoplasm, and it is possible that transport of materials may involve the membranes.”

Loewenstein (’64) summarizes his and his colleagues’ elegant microelectrical measurements of nuclei from the giant salivary glands of Drosophila and Chimnemus and from oocytes of amphibia (Xenopus and Triturus); the starfish (Astericzs); the seaworm (Nereis); the clam (Spisula); and the coelenterate (Hydractinia). The gland cell nuclei are much less permeable than those of oocytes although the ultrastructural morphology of these nuclei give no clue as to reason for such electrophysical discrepancy. That the pores per se in nuclear membranes do not offer free communication between the nucleoplasm and ooplasm of gland nuclei is shown by the fact that a simple “ ‘porous’ membrane would have a resistance of 10"3 Q cm”, three orders of magnitude smaller than the observed membrane resistance in gland cell nuclei,” (Loewenstein and Kano, "63). Nevertheless the oocyte nuclei tested by Loewenstein behave “merely like a small droplet of nucleoplasm without the additional surface resistance‘ of a membrane.”

Kessel (’66) has reported morphologic evidence of nucleocytoplasmic exchange in the oocytes of the tunicate, Céomz intestinalis. He demonstrates the transfer of Feulgen negative material from nucleoplasm to ooplasm directly through the nuclear pores. His superb pictures are of interest in that the material as it lies within the pores (and presumably is going through the pores) is similar in appearance to this non-granular pore material -- cylinders or stacked annuli — of the human oocyte. Nevertheless the adjacent material in the nucleoplasm and in the cytoplasm (Kessel, fig. 3) is obviously coarsely granular and suggests “a subsequent transformation of this material into particulate ribosomes.” Anderson and Beams ("56) first observed direct transfer of nuclear material to the cytoplasm in the nuclei of oocyte nurse cells of the true Reduviid bug Rhodmius prolixus (their fig. 2). The nuclear pores are 400 A and contain hour-glass shaped aggregates of granular material which appears identical to the Feulgen negative material of the nucleolus.

Fawcett (’66a, b) emphasizes very succintly that the nuclear pore is a complicated structure and subject to variation in different animal forms. It is usually regarded as being round although Gall (’67) gives evidence that it may be octagonal in such diverse forms as the Starfish (Henrécia), frog (Rama) and newt (Triturus). These observations may well be related to those of Wischnitzer (’58) who demonstrated 8 “microcylinders” within the tubes or annuli associated with each nuclear pore. Callan and Tomlin (’50) first described annuli associated with nuclear pores in the newt, Triturus cristatus, and the toad Xenopus laevis using the technique of spreading the nuclear envelopes onto grids followed by shadow casting and examination under the electron microscope. Students of the nuclear pore are generally agreed that there is a cylinder or annulus associated with each pore. They are moreover in general agreement that it is amorphous, moderately electron-opaque and without discrete particles of 150 A sizeconsistent with RNP granules although dense bodies in the center of the pore are often noted. Such cylinders are about 1000-1500 A in diameter but may be smaller. The pore is variable but is about 800-1000 A in diameter and formed by the fusion of the two nuclear leaflets. The latter are about 75 A in thickness and separated by a perinuclear cisterna or space about 150 A in Width. VVhat these observers are not agreed upon, however, is the precise relationship of the cylinders or annuli to the pores. Gall (’64) has summarized in a diagram the conclusions of the various observers. Thus Afzelius (’55) believes that the cylinder or annulus is entirely beyond the pore; Watson (’55, ’59) believes that the less dense center of the hollow cylinder coincides with the pore diameter resulting in an “intranuclear channel;” Wischnitzer (’58) believes that the cylinder is entirely within the pore, whereas Gall C64) believes that the cylinder extends beyond the pore but its lumen is smaller than that of the pore. Watson (’59) designates the cylindrical formation penetrating the nuclear envelope as a “pore complex.”

It is of interest to us that the distribution of the pore material (equivalent to the cylinders, superimposed annuli or pore complexes) in our human oocyte nucleus appears to agree with the views of Gall (’64). The external diameter of the annulus is slightly larger than, but the internal diameter slightly smaller than, the nuclear pore. Although we have not studied the annuli, appearing as slightly electron-opaque cores or suggestive hollow cylinders in the nuclear envelope (figs. 13, 15) or in the annulate lamellae (figs. 12-15), at higher magnifications than 42,000, it would appear that this pore material extends into the nucleoplasm and out into the cytoplasm (figs. 13, 15). This is so whether the pore material is associated with a bare nuclear envelope or the registered stacks of attached or detached annulate lamellae. Such pore material appears to be equivalent to the cylinders described in oocytes of lower forms. The question is now a very simple one. Is this annulus or cylinder or pore material - Watson’s “pore complex” - indeed a potential or actual mechanism for the transfer of genetic information from nucleoplasm to cytoplasm? The answer is obviously unknown but the circumstantial evidence is in favor of an affirmative answer.

That nature has many potentially if not actually different mechanisms for transference of genetic information to the cytoplasm must be obvious to all (even to the newcomer in ultrastructure as is the author). It is of more than passing interest, however, that annulate lamellae —- a potential mechanism for the transfer of a genetic message ———- should appear in the human oocyte, be prominent in the oocytes of lower forms but seemingly absent in oocytes of all of the laboratory mammals thus far studied. The student of evolution could certainly make something of this strange and interesting phenomenon!


The author is grateful to many persons for their various contributions to this after dinner speech and to its definitive published form. The after dinner speech drew upon the skills of Stuart Little, a mouse described by E. B. White, drawn by Garth Williams and published by Harper and Row. Stuart helped the author to explore the perinuclear space and the nuclear porecomplex. Dr. A. James French made availabel a series of lantern slides depicting the famous Mary Toft, “The Rabbet Lady of Godalming,” a hoax perpetrated during the early eighteen century and immortalized by Hogarth in one of his prints. This material, appropriate and fun to assemble for the speech, does not, however, seem suitable for a scientific article. The preparation of the speech was fun but the preparation of this manuscript has been hard work. To Donald Duncan, medical classmate and long time friend, I am grateful for his invitation to give the banquet address.

Without the superb interpretive and technical Work of Miss Elearnor C. Adams, my long time colleague and collaborator, this material would not have been available for ultrastructural study. Her technical associates, Miss Harriet Jopson and Mrs. Barbara Barton, have also contributed significantly. Our research photographer, Mrs. Audrey Hadfield, Printed the micrographs as well as the other illustrations. Miss Adams’ group also did much searching of the pertinent literature on Balbiani’s vitelline body and also on the annulate lamellae.

Dr. Stephen M. Shea of this department diligently searched the scientific and biographic literature concerning Balbiani and his several significant contributions. The resume of Balbiani’s life is a much condensed version prepared by Dr. Shea.

I am grateful to the editorial staff on the Journal of Cell Biology (The Rockefeller University Press) for permission to use a small portion of the material published by Hertig and Adams in the August 1967 issue of that journal (Vol. 34; pp. 647-675). None of the pictures here used is identical to any of those in our other paper. figure 3 is a survey micrograph of a primordial oocyte, a portion of which appears as figure 10 in the J CB article. Similarly figure 12 is from the same stack of attached annulate lamellae as appears in figure 27 of the J CB article, although the cropping is somewhat different in the two illustrations. figure 13 is from the same stack of coiled annulate lamellae as in figures 29-32 of the JCB article but is from a different serial section.

Dr. Morris J. Karnovsky has made many helpful criticisms during the interpretive and preparatory phases of this paper. And finally my thanks are due to Mrs. Nancy Cote for her patience and typing skills during the preparation of this manuscript.

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figures 1-3 represent three epochs in the investigation of Balbiani’s vitelline body.

1 A drawing made by Balbiani himself of the oocyte of the miriapod, Geophilus

longicornis. His original legend says, “figure 18. Ovule de G. longicornis, observé en mars. Outre la vésicule germinative vg, il renferme un volunineux noyau vitellin D, compose’ d’un corps central sphérique, etouré d’une épaisse couche de protoplasma vitellin homogéne (archoplasma); tg, tache germinative; p, prolongement stoloniforme de la vésicule gerrninative qui a donné naissance au noyau vitellin (voir mon Memoire in Zool. A722,, 1883, nos. 155 et 156).” Translated it becomes “Oocyte of G. longicornis, observed in the month of March. Besides the germinal vesicle '09, it contains a voluminous yolk nucleus ’D, composed of a central spherical body, surrounded by a thick layer of homogeneous vitelline protoplasm (archoplasm); tg germinal spot; p beak—like prolongation of the germinal vesicle which has given birth to the yolk nucleus (see my memoir in Zool. Anzeiger, 1883, numbers 155 and 156).” From plate III, Balbiani (1893).

A paraffin section of a primordial human oocyte from the left ovary of a patient whose, endometrial histology was that of the nineteenth to twentieth day. Note the characteristic crescent-shaped paranuclear complex known as Balbiani’s vitelline body, closely applied to the spherical nucleus. The cytocentrum (centrosome) is the dark spot surrounded by a halo which in turn is flanked by massed mitochondria. Case H-48, hematoxylin and eosin X 1200.

Another primordial human oocyte from the right ovary (corpus luteum side) of the same patient as figure 2, oriented in much the same position to show the ultrastructural components of Balbiani’s vitelline body. A higher power detail of a section, two grids beyond this one is to be seen in figure 4. The granulosa cells are of variable thickness and density. No mitoses are present. Note cytocentrum surrounded by dense fibers, the surrouding halo of endoplasmic reticulum, the dense, vacuolated, compound aggregates, the massed mitochondria and the dispersed vesicular endoplasmic reticulum. Case H-48, Oocyte no. 1 X 2400. ULTRASTRUCTURE OF THE PRIMORDIAL HUMAN OOCYTE Arthur T. I-Iertig PLATE 2


4 A nearby section (2 grids removed) to that seen in figure 3. Note nuclear envelope to right; the prominent dense, vacuolated compound

aggregates With and without surrounding membranes; the cytocentrum composed of dense granules and closely packed vesicles with peripheral dense ‘fibers; the surrounding halo of smooth endoplasmic reticulum; the scattered Golgi complexes; the annulate larnellae (upper left) cut tangentially and therefore resembling the nuclear pores and annulae; and the clusters of mitochondria intimately connected with endoplasmic reticulum. Case H-48, Oocyte no. 1 X 10,000.

122 PLATE 2



figures 5-7 show the evolution of techniques used to study the organelle complex of human oocytes designated as Balbiani’s vitelline body.

5 A primary human follicle from van der Stricht’s classic monograph

of 1923, figure 6, plate VII. (The definitions are translated from his description.) This represents a section of a small follicle from the ovary of an adult woman, fixed in concentrated aqueous sublimate solution and stained by iron hematoxylin. Note the several parts of the Balbiani vitelline body as seen in light microscopic preparation. (Courtesy of Archives de Biologie, Brussels, Belgium, published by Masson and Cie, Paris and Liege.)

Photograph by phase microscopy of thick epon section from a comparable human early primary follicle removed from a patient on the tenth day of her menstrual cycle. Note similarity of Balbiani’s vitelline body to that drawn in figure 5. The ultrastructure of this same oocyte is to be seen in figures 7-11. Case H-85, Oocyte no. 2, phase microscopy X 950.

A survey electron micrograph of the same early transitional primary Oocyte seen in figure 6. Note cuboidal granulosa cells which, however, do not yet show mitotic activity. The paranuclear complex of Balbiani’s vitelline body is well seen below the nucleus. Case H~35, Oocyte no. 2 X 2700. ULTRASTBUCTURE OF THE PRIMORDIAL HUMAN OOCYTE PLATE 3 Arthur T. I-Iertig



8 A more detailed micrograph of the section adjacent to that seen in

figure 7. The nuclear membrane is at upper right. Note cluster of closely packed spiral fibrils attached to the nuclear envelope. The cytocentrum, at center, is composed of dense granules, some arranged periodically on fine fibers, and small vesicles, with a peripheral zone of endoplasmic reticulum and dense fibers. Surrounding the cytocentrum are massed mitochondria and compound aggregates. A stack of a-nnulate lamellae is out somewhat tangentially. Note the prominent endoplasmic reticulum is close association with multiple Golgi complexes at periphery of Balbiani’s vitelline body. See figures 9-11 for higher powered details of this primary oocyte. Case H—35, Oocyte no. 2 X 10,500.



The edge of the paranuclear cluster of mitochondria surrounded by a network of endoplasmic reticulum continuous with the outer leaflet of the nuclear envelope. The nuclear envelope is at the right, the cytoplasm at upper part of micrograph. Note close affiliation of endoplasmic reticulum, to both margin of mitochondrial mass and to individual mitochondria. Note concentration of microtubules, cut in cross section, along the network of endoplasmic reticulum that encloses Balbiani’s Vitelline body at this stage of development of the oo-cyte.

The endoplasmic reticulum has relatively few RNP particles. Case H-35, Oocyte no. 2 X 13,500.

A detail of the meshwork of sparsely granular endoplasmic reticulum forming a meshwork surrounding Balbiani’s vitelline body and in continuity with the numerous prominent Golgi complexes. Note prominent, longitudinally oriented microtubules-. Note compound aggregate at top and the membrane bound mass of granules at bottom. Case H-35, Oocyte no. 2 X 13,500.

Two granulosa cells below and oocyte above. Note dilated, sparsely granular endoplasmic reticulum of oocyte; multiple villi of granulosa cells, thepinocytic Vesicle attached to oolemma at far right of micrograph; and the dark vesicular compound aggregate within the cytoplasm of the granulosa cell to the right. Case H-35, Oocyte no. 2 X 1,500.



figures 12-14 show details of the origin from and/ or attachment of annulate lamel lae to the nucleus of two primordial oocytes.




The nucleus is at the left. The external leaflet of the nuclear envelope has evaginated by multiple folds into the expanded perinuclear cisterna seen best at the lower portion of the micrograph and in somewhat more detail in figure 14. Note that the fold nearest the nucleus is single whereas the "next four folds are double. The reason for this is the secondary invagination by folds of the endoplasmic reticulum, also continuous with the external leaflet "of the nuclear envelope. This feature is best seen at the top of the micrograph. Note several connections of this large stack of annulate lamellae with the endoplasmic reticulum in the adjacent cytoplasm. Note that many annuli of the lamellae are in register and that occasional annuli are in continuity with those of nuclear membrane. The three-dimensional drawing of those several interrelationships is shown in figure 16. Case H-35, Oocyte no. 4 X 22,000.

A concentric stack of annulate lamellae apparently in the process of peeling off from the nucleus seen in the upper part of the picture. Note pore material or

annuli Within the nuclear pores as well as Within the annulate lamellae. Case H-43 Oocyte no. 1 X 21,000.

A high power detail of the edge of a stack of annulate lamellae continuous with the external leaflet of the nuclear envelope. The entire stack is seen in figure 12.

Note expanded perinuclear cisternae; one single folded lamina next to nucleus; four double folded laminae; and communication of annulate lamellae with endoplasmic reticulum. Note further that the pores and electron-opaque annuli of the annulate lamellae are in register within the stack and occasionally connect with

the nuclear pores. Case H-35, Oocyte no. 4 X 33,000. ULTRASTRUCTURE OF THE PRIMORDIAL HUMAN OOCYTE PLATE 6 Arthur T. Hertig

A ‘ 9-,‘ I _ .' '?e.«~; "5? 3" PLATE '7


15 A coiled stack of annulate lamellae from a primordial oocyte. The nuclear membrane is at the top of the picture and the ooplasm below. Note nuclear pores, at least four of which show core material extending into both the nucleoplasm and ooplasm. Within the coiled annulate lamellae are numerous pores, often in register and containing electron-opaque material in the form of a cylinder. Note the numerous masses of amorphous material, possibly representing concentrations of core material, lying within the ooplasm enclosed by the coiled annulate lamellae. The sparsely granular endoplasmic reticulum, apparently connected with the annulate lamellae, is. dispersed throughout the ooplasm. Case H-57, Oocyte no. 1, X 31,250.



16 A three—dimensional drawing by Mr. Joshua Clark, made from a sketch

by the author. The drawing is oriented in the same position as figures 12 and 14 from which the drawing was made. Illumination is represented as being from the left, exposing the inner surface of the nuclear envelope (N). The pore material (PM) or annulus is indicated by a dotted line. The pore (P) contains a diaphragm formed by coalescence of the two nuclear leaflets. Only two evaginated folds of outer nuclear leaflet in the expanded perinuclear cisterna or space (PS) are shown; the one nearest the nucleus is single whereas the more distal one, containing an invagination of the endoplasmic reticulum (ER) is double. The cytoplasm (C) is stippled. Note multiple connections of annulate lamellae with endoplasmic reticulum and,-” or the outer nuclear leaflet. Case I-I-35, Oocyte no. 4, magnified about 50 0-00 diameters. PLATE 8



figures 17-21 represent some aspects of the life and work of Professor Edouard Gérard Balbiani.

17 A photograph of Balbiani. (Courtesy of Mme. Matte, librarian of the College de France.)

18 Microscope used by Balbiani, now in the museum of The Faculty of Medicine of;__ the University of Paris. (Courtesy of Dr. Guido Majno and photographed by Gabriel of Paris.)

19 Line drawings, made by Balbiani, of giant chromosomes with puffs from salivary glands of the larval midge, Chironomus plumosus (Balbiani, 1881).

20 A giant chromosome IV from the larval salivary glands of the midge Ch-ironomus tentmzs. This structure, originally photographed in color after staining for DNA and counterstained green for protein. The bands (or genes) are positive for DNA, the puffs contain protein and RNA as seen-- in figure 21. (Courtesy of Dr. Ulrich Clever. From Beermann and Clever, Scientific American, 210: 50-58, ’64.)

21 A giant larva]. salivary chromosome IV of the midge Chironomus tentcms, stained for RNA by toluidine blue. The pufi‘ is positive for RNA whereas the bands contain DNA. (Courtesy of Dr. Claus Pelling. From Beermann and Clever, Scientific American, 210: 50—58, ’64.)