Three-dimensional imaging of human stem cells using soft X-ray tomography.

Three-dimensional imaging of human stem cells using transmission soft X-ray tomography (SXT) is presented for the first time. Major organelle types--nuclei, nucleoli, mitochondria, lysosomes and vesicles--were discriminated at approximately 50 nm spatial resolution without the use of contrast agents, on the basis of measured linear X-ray absorption coefficients and comparison of the size and shape of structures to transmission electron microscopy (TEM) images. In addition, SXT was used to visualize the distribution of a cell surface protein using gold-labelled antibody staining. We present the strengths of SXT, which include excellent spatial resolution (intermediate between that of TEM and light microscopy), the lack of the requirement for fixative or contrast agent that might perturb cellular morphology or produce imaging artefacts, and the ability to produce three-dimensional images of cells without microtome sectioning. Possible applications to studying the differentiation of human stem cells are discussed.

Fine structure of human oogonia in the foetal ovary.

Foetal ovarian tissue is now being cultured or frozen, to generate oocytes for assisted reproduction, an emerging technology. This study examines the ultrastructure of oogonia at 13-15 weeks of gestation, which could be used as a control for culture and freezing of foetal ovaries. Oogonia are largely located in the ovarian cortex, whilst primordial germ cells (PGC) and somatic follicle cells compose the surface epithelium. Oogonia and PGC have large vesicular nuclei with clear cytoplasm, compared to dense follicle cells, which have polymorphic nuclei. Follicle cells intermingle with oogonia and establish close contacts - beginning of folliculogenesis. Nuclei of oogonia contain one to three highly reticulated nucleoli, reflecting high levels of RNA synthesis at the onset of growth. Rough endoplasmic reticulum (RER) form stacks of cisternae associated with numerous ribosomes. Prominent organelles in the ooplasm are elongated mitochondria with dense matrices and tubular cristate presenting a multilocular appearance. Typical Golgi complexes, dense bodies and clear vacuoles are present and microfilaments are located beneath the plasma membrane. The most remarkable feature of oogonia is that they have typical juxtanuclear centrioles (diplosomes) with dense pericentriolar material, which nucleate microtubules, characteristic of functional centrosomes organizing the cytoskeleton. The mature oocyte has no centrioles, since the maternal centrosome is inactivated or reduced, while the paternal is dominant. Centrioles are most likely involved in mitosis of oogonia.

Mitosis in the human embryo: the vital role of the sperm centrosome (centriole).

The pattern of sperm centrosomal (centriolar) inheritance, centrosomal replication and perpetuation during mitosis of the human embryo is reviewed with a series of electron micrographs. Embryonic cleavage involves repeated mitoses, a convenient sequence to study centriolar behaviour during cell division. After the paternal inheritance of centrioles in the human was reported (Sathananthan et al., 1991a), there has been an upsurge of centrosomal research in mammals, which largely follow the human pattern. The human egg has an inactive non-functional centrosome. The paternal centrosome contains a prominent centriole (proximal) associated with pericentriolar material which is transmitted to the embryo at fertilization and persists during sperm incorporation. Centriolar duplication occurs at the pronuclear stage (interphase) and the centrosome initially organizes a sperm aster when male and female pronuclei breakdown (prometaphase). The astral centrosome containing diplosomes (two typical centrioles) splits and relocates at opposite poles of a bipolar spindle to establish bipolarization, a prerequisite to normal cell division. Single or double centrioles occupy pivotal positions on spindle poles and paternal and maternal chromosomes organize on the equator of a metaphase spindle, at syngamy. Bipolarization occurs in all monospermic and in most dispermic ova. Dispermic embryos occasionally form two sperm asters initially and produce tripolar spindles (tripolarization). Anaphase and telophase follows producing two or three cells respectively, completing the first cell cycle. Descendants of the sperm centriole were found at every stage of perimplantation embryo development and were traced from fertilization through cleavage (first four cell cycles) to the morula and hatching blastocyst stage. Centrioles were associated with nuclei at interphase, when they were often replicating and occupied pivotal positions on spindle poles during mitosis. Sperm remnants were associated with centrioles and were found at most stages of cleavage. Centrioles were found in trophoblast, embryoblast and endoderm cells in hatching blastocysts. Pericentriolar, centrosomal material nucleated astral and spindle microtubules. Abnormal nuclear configurations observed in embryos reflect mitotic aberrations. The bovine embryo closely resembles the human embryo in centriolar behaviour during mitosis. It is concluded that the sperm centrosome is the functional active centrosome in humans and is likely the ancestor of centrioles within centrosomes in foetal and adult somatic cells. The role of the sperm centrosome in embryogenesis and male infertility is discussed, since it is of clinical importance in assisted reproduction.

The origin and distribution of cortical granules in human oocytes with reference to Golgi, nucleolar, and microfilament activity.

The origin and distribution of cortical granules were investigated in human preovulatory oocytes at various phases of maturation. Twenty-five oocytes obtained from unstimulated small antral follicles and from stimulated large antral and mature follicles were examined by transmission electron microscopy. Ovarian stimulation in women was accomplished by administering Clomid followed by hMG or hCG or both. Small antral follicle oocytes were dissected from ovarian biopsies, while the other oocytes were recovered by laparoscopy. Some oocytes were allowed to mature in Ham's F-10 or Whittingham's T-6 media before routine fixation in glutaraldehyde/osmium. Cortical granules originate from typical, hypertrophic Golgi complexes during early maturation and continue till its completion. Evidently there are two waves of cortical granule synthesis, the first more prolific than the second. The first occurred in small antral follicle oocytes, when there was a peak in Golgi activity, and the second was observed at the germinal vesicle stage, particularly at the onset of resumption of meiosis. Golgi complexes became progressively scarce as oocytes completed first maturation. Golgi membranes were also involved in the formation of lysosomes. A well-defined band of microfilaments was detected in small antral follicle oocytes which seemed to prevent the cortical granules, organized in a single layer, from migrating to the periphery. This band gradually became disorganized at the germinal vesicle stage as oocytes resumed meiosis, when cortical granules were apparently migrating to the surface. Metaphase I and mature oocytes had one to three discontinuous layers of cortical granules beneath the oolemma. The general organization of oocytes was also investigated and the roles of the nucleolus and endoplasmic reticulum in relation to Golgi activity and cell secretion were discussed.

Ultrastructural changes during meiotic maturation in mammalian oocytes: unique aspects of the human oocyte.

This paper reviews the process of peri-ovulatory oocyte maturation and the ultrastructural organization of the human egg and compares it with that of the mouse. The main thrust of the paper is on the human, since there are several reviews on the mouse. Both preovulatory and postovulatory events at fertilization, as well as some of the aberrant features of maturation are covered. Some changes induced by oocyte culture and cooling in the human are also included. The report attempts to focus on unique features of the human oocyte and shows a variety of ultrastructural differences between human and murine oocytes, which may well reflect differences in their physiology and biochemistry. Based on these differences and further observations on the process of fertilization of both species, particularly with respect to the inheritance of paternal centrioles, it is concluded that the mouse may not be a suitable model for the development and refinement of current procedures in human assisted reproductive technology.

The ultrastructure of the preovulatory human egg fertilized in vitro.

Preovulatory eggs in cumulus were inseminated in vitro with washed spermatozoa which had been preincubated for 1.5 hours. After 3 hours, three eggs were processed for electron microscopy and each was sectioned serially from pole to pole. In the two eggs which had been fertilized, the expanded chromatin of the fertilizing sperm head and the chromatin of the ovum were almost completely surrounded by a developing pronuclear envelope. In one of the penetrated eggs the developing male pronucleus and associated midpiece and sperm tail were located within an incorporation cone. The surface of the cone was free of microvilli but contained a zone of microfilaments immediately beneath the plasma membrane. A similar zone of microfilaments was present beneath the advancing surface of the extruding second polar body (PB2) which was connected to the ovum by an interbody and microtubules of the meiotic spindle. Cortical granules were completely absent from the fertilized eggs but were present in the unfertilized egg. PB2 contained a nucleus at a stage of development similar to that of the early pronuclei.

Early sperm-egg interaction after sperm microinjection.

The early events of sperm-egg interaction occurring 1-3 h after multiple sperm injection into the perivitelline space (PVS) and after direct injection into the ooplasm of human oocytes are reported. The sperm acrosome reaction occurred in the PVS but was not detected within the ooplasm. Sperm in the PVS were incorporated into the ooplasm in the usual manner described in vitro, after completion of the acrosome reaction, and a block to polyspermy was evident at the oolemma. However, sperm incorporation into the ooplasm was not clearly defined and requires further investigation. Sperm were also incorporated into oolemma-bound vacuoles within the ooplasm and breaches in the ooplasm were seen after intracytoplasmic injection. Both normal and abnormal sperm were found in the PVS and ooplasm, even though sperm from donors with normal semen parameters were used. The effect of freezing in liquid nitrogen on demembranation of sperm for microinjection is also reported.

Is the acrosome reaction a prerequisite for sperm incorporation after intra-cytoplasmic sperm injection (ICSI)?

There is debate as to whether the acrosome reaction is necessary for sperm incorporation after intra-cytoplasmic sperm injection (ICSI). Ultrastructural evidence is presented to show that the acrosome reaction could occur in the ooplasm before sperm incorporation in mature human oocytes or the acrosome could be discarded intact before sperm incorporation in immature oocytes, matured in vitro. Both germinal vesicle and growing follicular oocytes showed sperm chromatin decondensation, with discarded acrosomes close to the sites of incorporation, and were able to form male pronuclei. This is probably the first report of microfertilization of a growing oocyte with a reticulate nucleolus by ICSI. The acrosome reaction, when it occurs, is preceded by acrosome swelling and is followed by vesiculation of surface membranes exposing the inner acrosome membrane, as observed on the surface of the zona during IVF or in the perivitelline space after subzonal sperm injection. These sperm were probably capacitated at the time of ICSI. There was subtle evidence of leaching of the acrosomal matrix from intact discarded acrosomes and from partially depleted acrosomes attached to decondensing spermheads. These sperm were probably not fully capacitated at the time of ICSI. It is concluded that both the acrosome reaction and acrosome deletion are possible prerequisites to sperm incorporation after ICSI.

Ultrastructure of the human egg.

This report on the fine structure of human oocyte is based on 20 years research where over 2000 eggs were examined by TEM in conjunction with our research on various methods of assisted reproduction. The eggs were routinely fixed in glutaraldehyde/osmium tetroxide, flat embedded in araldite, serially sectioned and examined by TEM. The oocytes were usually recovered after gonadotrophin stimulation. The general organisation of the mature human oocyte conforms to that of other mammals but has some unique features. The oocyte has the basic cell organelles such as mitochondria, lysosomes, two types of smooth endoplasmic reticulum (SER)-vesicular and tubular aggregates, multivesicular residual bodies, lipofuschin, microfilaments and microtubules. Golgi, RER and ribosomes are very rare and the egg has no yolk. It's surface has few microvilli, pinocytolic caveolae and 1-3 layers of cortical granules. The zona pellucida is composed of fine fibrils and granules embedded in an amorphous matrix and encloses a perivitelline space containing polar bodies. Remnants of corona cell junctions may be found at the oolemma. The metaphase II spindle is often oriented perpendicular to the surface and is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs since the maternal centrosome is inactive. The sperm centrosome organizes mitotic spindles of the embryo after fertilization, whereas in mice the maternal centrosome is active and dominant during cleavage. The stages of peri-ovulatory maturation and differences in oocyte structure during maturation are also presented. Oocytes ageing in culture show progressive swelling of vesicular SER culminating in vacuolation, denser mitochondria (clouding together or associated with vacuoles) peripheral conglomerations or centripetal migration of cortical granules and increased lysosomal activity. Prolonged culture also causes displacement and disorganisation of metaphase II spindles, loss of microtubules and consequent displacement of chromosomes. Evidently the cytoskeleton becomes disorganized. Some observations on oocyte maturation in vitro and spontaneous activation of oocytes are included.

Understanding the fundamentals of embryology in assisted reproductive technology.

This paper emphasises the need to understand the fundamentals of embryology in relation to in vitro fertilisation (IVF) and associated assisted reproductive technologies (ART). It introduces the reproductive technologist and others involved in such programmes to the events that occur during early embryogenesis, which have led to recent developments in IVF/ART. It also covers some of the contributions made by IVF/ART in understanding early events of development, particularly during the first week of human life. The reader is referred to some of the widely used embryology texts and audio-visual aids and also to selected reviews and papers published recently that would help toward a better understanding of early development and ART.

Rediscovering Boveri's centrosome in Ascaris (1888): its impact on human fertility and development.

We rediscover and review the brilliant work of Theodore Boveri, over a 100 years ago, on the centrosome of the round worm Ascaris and show how it impacts on our understanding of human fertilization and embryogenesis. Boveri was able to make fundamental predictions on the mechanics of fertilization and the dominant role of the sperm centrosome (Boveri's rule), which is now applicable to most animals. Using advanced digital imaging by light and electron microscopy, we explore centrosomal dynamics during Ascaris fertilization and the first cell cycle during cleavage. Twenty figures are presented in this visual publication. Humans follow Boveri's rule, as do most mammals excluding some rodents, and there is a remarkable similarity of the events of fertilization and cleavage in Ascaris and humans, the latter of which has been documented since 1991. The role of the sperm centrosome (centriole) in egg activation, polarity, embryogenesis, infertility and cancer is discussed. An attempt is made to portray the images Boveri may have visualized, in his painstaking drawings presented in his thesis in 1888. We now know the origins of the centrosome in human somatic cells--predominantly from the sperm cell. The impact of Boveri's work on human development is highlighted in this age of assisted reproductive technology.

Human embryonic stem cells and their spontaneous differentiation.

Human embryonic stem cells (hESCs) usually grow in saucer-shaped colonies with thickened rims and can form spherical human embryoid bodies (hEBs) under non-adherent conditions. A problem associated with ES cell culture is the spontaneous differentiation of cells into a variety of cell types representing all three germ layers, which is evident in both hESC colonies and hEBs. This presentation deals with the precise origins of hESCs and their spontaneous differentiation in vitro. We have used advanced digital microscopy, including transmission electron microscopy (TEM) to define the fine structure of these cells. We present images of undifferentiated hESCs and their spontaneous differentiation into basic embryonic cell types such as nerve, muscle, connective tissue, epithelium, and digestive tract progenitors, representing all three primary germ layers: embryonic ectoderm, mesoderm and endoderm. It appears that hESCs work in concert and interact with one another, as in tissue formation of the embryo. Our fine structural observations agree mostly with those of the Thomson group. Digital microscopy of plastic sections and TEM are invaluable tools in the precise characterization of cells forming these tissues and a combined study with immunofluorescent markers is most desirable.

Functional competence of abnormal spermatozoa.

The processes of abnormal sperm penetration and incorporation into human oocytes during IVF and after sperm microinjection, assessed by TEM, are reviewed. A spectrum of morphologically abnormal sperm with head, neck and midpiece defects penetrate the egg vestments of oocytes (1-3 h after insemination with sperm from normal donors) in both unfertilized and normally fertilized oocytes. Sperm with aberrant head shapes, acrosomal and nuclear defects penetrate the outer zona pellucida but are rarely encountered in the inner zona and perivitelline space, showing that the zona prevents abnormal sperm penetration. Grossly abnormal sperm are, however, incorporated into the ooplasm of zona-denuded oocytes. Microinjection of poor-quality sperm from male-factor patients into the perivitelline space or directly into the ooplasm of oocytes also reveals a variety of structural defects conforming to those observed in washed sperm pellets, highlighting the difficulties of selection of 'normal' sperm for microinjection. Abnormal sperm have been seen to interact and fuse with the oocyte in the perivitelline space, and to be incorporated into the ooplasm. Those with nuclear and neck (centriolar) defects are of particular significance, as they might contribute to aberrant development. The functional competence of immotile, round-headed and epididymal sperm is also briefly discussed.

Degeneration of possible serotonergic nerves in the myenteric plexus of Mytilus induced by 5,7-dihydroxytryptamine (5,7-DHT).

Initial observations on the effects of 5,7-DHT on the myenteric plexus of Mytilus reveal extensive lesioning of monoamine nerves containing dense-cored vesicles. At least, two types of monoaminergic nerves could be recognized among the degenerating axons. The most affected were profiles with a predominance of large granular vesicles (56-200 nm) which resemble possible indoleamine nerves in the lamprey gut. The other type had small granular vesicles (40-56 nm) resembling catecholamine nerves of vertebrates. These two types of nerves possibly represent serotonergic and dopaminergic nerves, in accordance with pharmacological, biochemical and physiological evidence available in molluscs. There were, however, nerves with a population of medium-sized granular vesicles (56-130 nm) which were also damaged. Two other types of nerve profiles were unaffected by the drug. One conforms to cholinergic nerves while the other resembles "purinergic" nerves in the vertebrate gut. These results confirm previous studies with dihydroxytryptamines on molluscan smooth muscle.

Degeneration of monoamine nerves in anterior byssus retractor muscle of Mytilus induced by 5,6-dihydroxytryptamine.

Preliminary ultrastructural studies on the effects of 5,6-Dihydroxytryptamine (5,6-DHT) on the anterior byssus retractor muscle (ABRM) of Mytilus show degeneration of 2 types of monoaminergic nerves after 10 days of drug treatment. One type contained large granular vesicles (560-1,680 A) possibly represent serotonergic and dopaminergic nerves, thought to innervate this muscle. Two other types of profiles seemed to be unaffected by the drug. One conforms to cholinergic nerves while the other has a predominance of large opaque vesicles (1,200-2,500 A). The significance of these findings is discussed in the light of recent observations on the neurotoxic effects of 5,6-DHT on vertebrate and molluscan nerves.

Paternal centrosomal dynamics in early human development and infertility.

PURPOSE: Our purpose was to demonstrate the dynamics of the human sperm centrosome during fertilization and cleavage. METHODS: Human gametes, fertilized oocytes, and preimplantation embryos were examined by transmission electron microscopy. RESULTS: The functional sperm centrosome containing a typical centriole (proximal) is inherited at fertilization and forms a sperm monoaster. It then replicates and is perpetuated during cleavage. It organizes the mitotic apparatus at each stage of cleavage up to the hatching blastocyst stage. Bipolar spindles are formed in all monospermic and most dispermic embryos. Occasionally, two sperm asters and tripolar spindles are formed in dispermic embryos. Centrioles are associated with pronuclei and nuclei at interphases when they duplicate and occupy pivotal positions at spindle poles during mitoses. The maternal centrosome is not functional. CONCLUSIONS: The human embryo shows paternal centrosome inheritance and perpetuation like most other animals. Inheritance of defective centrosomes may lead to abnormal cleavage and contribute to infertility.

Early penetration of human sperm through the vestments of human eggs in vitro.

Penetration of human sperm through the vestments of human oocytes during the first 3 h after insemination was investigated to determine the time taken for sperm capacitation, which precedes the acrosome reaction and fertilization. Twelve oocytes from women who became pregnant by IVF, recovered at laparoscopy after appropriate stimulation, were examined by electron microscopy. Follicular development was controlled by administration of clomiphene citrate, hMG, and hCG. Oocytes were cultured in Whittingham's T6 medium for 7-14 h before insemination and were fixed 1-3 h after insemination with preincubated sperm obtained from fertile men. All oocytes had matured and eight were normally fertilized 2-3 h after insemination. The acrosome reaction had already begun 1 h after insemination when 10-30% of sperm had reacted. Sperm-oocyte membrane fusion occurred 2 h after insemination, and sperm decondensation and pronuclear formation were in progress 3 h after insemination. Variable numbers of sperm (30-60%) had reacted acrosomes after 2-3 h of insemination. Many sperm penetrating the cumulus were intact or had partially reacted acrosomes. Intact, partially and fully reacted sperm were found at the surface of the zona. Intact sperm were bound to the zona by their plasma membranes. Sperm penetrating the zona had reacting or reacted acrosomes exposing their inner acrosome membranes. Those approaching the perivitelline space had a persistent equatorial vestige of the acrosome with intact plasma membrane. The acrosome reaction involved multiple fusions of the sperm plasma and outer acrosome membranes, resulting in vesiculation. This study shows that human sperm could complete capacitation and initiate the acrosome reaction within 1 h of insemination in vitro.

Ultrastructure of early cleavage and yolk extrusion in the marsupial Antechinus stuartii.

The fertilized egg and the two-cell stage and four-cell stage of the marsupial Antechinus stuartii were studied by transmission electron microscopy. The features that make the fertilized egg of Antechinus stuartii different from those of any eutherian mammal are (1) the presence of a shell and (2) the relatively large quantity and polarized distribution of cytoplasmic inclusions, including lipid, protein yolk bodies, and protein fibers. Mitochondria and vesicles of smooth endoplasmic reticulum are also polarized in distribution. Early cleavage differs from that of eutherians in several ways: (1) it occurs in the uterus; (2) there is extrusion of a large, single, membrane-bound yolk mass at first cleavage; and (3) blastomeres become separated after the second cleavage division and thus do not adhere by cell-to-cell contacts. Prior to the second division, blastomeres are connected to each other by remnants of the midbody and to the yolk mass by remnants of a cytoplasmic bridge. The yolk mass after extrusion is surrounded by plasma membrane and contains inclusions of lipid, protein yolk bodies, and fibers, as well as mitochondria and smooth endoplasmic reticulum. The blastomeres of the two-cell and four-cell stages also show intracellular polarization in the distribution of retained inclusions and organelles. Vesicles developing at the periphery of blastomeres and discharging their contents extracellularly increase in size and number from the fertilized egg to the four-cell stage. The discharged contents may be implicated in early development of the blastula cavity.

Mitochondrial morphology during preimplantational human embryogenesis.

The structure, distribution, and function of mitochondria during human oogenesis and early development is reported. Oogonia show a sparse and even distribution of mitochondria, which are oval or elongated. Except around nuclei, growing oocytes from small antral follicles have more dense rounded or oval mitochondria, associated with the rough endoplastic reticulum. Mitochondria in fully grown, germinal vesicle (GV) oocytes present an inert appearance, with a dense matrix and a few arch-like or transverse cristae. At this stage mitochondria are usually absent from the cortical part of the cytoplasm. Mitochondria in metaphase I and II oocytes, including fertilized oocytes, present a similar structure, but they are numerous and evenly spread in the ooplasm, associating closely with vesicles or aggregates of tubular smooth endoplasmic reticulum. The most substantial change in distribution occurs at the pronuclear stage, when there is a central conglomeration of mitochondria around the pronuclei in both monospermic and dispermic embryos, which persists up to syngamy. In structure and distribution, mitochondria in blastomeres of 2-16-cell embryos remain virtually unchanged and resemble those of mature oocytes, though perinuclear aggregation can be evident. Mitochondria are usually excluded from meiotic and mitotic spindles but locate peripherally, apparently providing energy for centrosomal, cytoskeletal, and chromosomal activity during cell division. Morphogenetic changes in mitochondrial structure occur in the 8-cell cleaving embryo, the morula and the blastocyst (apparently accompanying the onset of nuclear and mitochondrial transcription), when they become progressively less electron dense and often develop clear areas in their matrices. Elongating mitochondria with inner mitochondrial membranes arranged into transverse cristae appear in expanding blastocysts, in the trophoblast, embryoblast, and endodermal cells. These mitochondria seem to play a role in blastocyst differentiation, expansion, and hatching, with their morphological changes reflecting increased cellular activity.

The application of electron microscopy in the evaluation of two- to four-cell human embryos cultured in vitro for embryo transfer.

Eighteen two- to four-cell embryos, cultured in vitro for 32-65 hr after insemination, were examined by transmission electron microscopy to assess their normality and developmental potential. These stages are now being widely used for embryo transfer in in vitro fertilization clinics. They were obtained by inseminating preovulatory oocytes aspirated at laparoscopy, with or without ovarian stimulation, by methods which have yielded normal pregnancies. The organization of seven embryos was apparently normal and their blastomeres had cellular organelles usually present in fertilized ova. Details of their ultrastructure including subtle changes observed on prolonged culture are described. Other embryos showed some normal and obvious abnormal features, such as partial fragmentation and multinucleated blastomeres, or evidence of degeneration.