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A H Sathananthan

Publications and source records attributed to A H Sathananthan.

At least 19 recordsLinked to original sources
PubMed2000

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.

Centrioles↗
PubMed2000

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.

Blastocyst↗
PubMed1998

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.

Blastocyst↗
PubMed1998

Egg-sperm interactions in humans: ultrastructural aspects.

A large cumulus mass usually covers the human ovulated oocyte, and voluminous clusters of cumulus cells are still seen after fertilization around the egg. Cumulus cells surround oocytes and fertilized eggs also during in vitro fertilization (IVF) procedures. This study describes, by transmission and scanning electron microscopy, the morphology and the microtopography of the cells forming the human cumulus mass surrounding IVF samples (insemined but not fertilized oocytes and fertilized eggs). Particularly emphasized is their morphodynamic role in sperm-egg interactions. A comparison with the behavior in vivo of cumulus-enclosed oocyte/fertilized eggs has been also performed. All patients have given their informed consent to participate in this protocol. An inner layer (corona radiata cells) and an outer layer (proper cumulus cells) can be microtopographically recognized in the cumulus mass. Numerous cumulus-corona cells, particularly after fertilization, showed ultrastructural characteristics typical for steroid synthetic cells, thus undergoing a sort of "luteinization" parallel to that occurring in the sister granulosa cells of the postovulatory follicle. This steroid synthetic activity, particularly enhanced in vitro but present also in vivo, may be finalized to the release of small amount of steroids (estrogens and progesterone) in the oocyte/fertilized egg milieu. Various proteins, secreted by other cell subpopulations--as revealed in other studies by our research group--, may even enrich this milieu. Lymphocytes and macrophages were often found in the cumulus mass. They may modulate the steroid secretion of the neighboring cumulus cells by production of cytokines, mimicking what occurs in the ovarian follicle and, later, in the corpus luteum. Spermatozoa, both normal (acrosome-intact or--reacted) and abnormal, were frequently seen in the cumulus mass, free in the intercellular spaces or close to the cumulus cells, that can induce sperm capacitation and acrosome reaction. Leukocytes and cumuluscorona cells appeared both capable of actively phagocytizing supernumerary and/or abnormal sperms. Such spermiophagic response is present in a lesser extent around oocytes and eggs fertilized in vivo. In vitro, instead, cumulus spermiophagy leads to the elimination of a large part of the excess spermatozoa that have reached the oocyte, thus restoring in an extracorporeal medium the spermiophagic activity physiologically exerted by leukocytes and epithelial cells in the female and male genital tracts. In conclusion, the cumulus mass surrounding oocytes and fertilized eggs appears as a highly dynamic system, in which various subpopulations of cells cooperate in order to provide a suitable and healthy microenvironment for fertilization and early embryo development.

Blastomeres↗
PubMed1997

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.

Acrosome↗
PubMed1997

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.

Animals↗
PubMed1997

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.

Animals↗
PubMed1996

The sperm centriole: its inheritance, replication and perpetuation in early human embryos.

The inheritance, replication and perpetuation of the sperm centriole in the early human embryo are reported. Both normal monospermic and abnormal dispermic embryos (n = 127) were examined by transmission electron microscopy. Centrioles were traced from fertilization to the hatching blastocyst stage. The sperm proximal centriole is introduced into the oocyte at fertilization and remains attached to the expanding spermhead during sperm nuclear decondensation, as it forms the male pronucleus. A sperm aster is initially formed after the centriole duplicates at the pronuclear stage. At syngamy, centrioles occupy a pivotal position on opposite spindle poles, when the first mitotic figure is formed. Bipolar spindles were found in the majority of embryos, while tripolar spindles were seen in four dispermic embryos at syngamy. Two single centrioles were detected at two poles of two tripolar spindles, while two additional centrioles were located on the sides of a bipolar spindle of a dispermic embryo. Sperm tails were detected near spindle poles at syngamy and in later embryos. Typical centrioles showing the characteristic pin-wheel organization of nine triplets of microtubules were evident. During centriolar replication, the daughter centriole grows laterally from the parent and gradually acquires pericentriolar material (PCM). The two centrioles are surrounded by a halo of electron-dense PCM, which nucleates microtubules, thus making it a typical centrosome. The usual alignment of diplosomes at right angles to each other was maintained. Centrioles were detected at all stages of embryonic cleavage from the 1-cell through 8-cell stages, right up to the hatching blastocyst stage. They were closely associated with nuclei at interphase, when they were often replicating, and were prominently located at spindle poles during the first four cell cycles. In blastocysts, they were detected in trophoblast, embryoblast and endoderm cells respectively. It is evident that the sperm centrosome is the functional active centrosome in human, while the female is inactive but may contribute some centrosomal material to the zygote centrosome. It is very likely that the paternal centriole is the ancestor of the centrioles in fetal and adult somatic cells.

Blastocyst↗
PubMed1996

Centrifugation of bovine oocytes for nuclear micromanipulation and sperm microinjection.

The reproductive biotechnologies of intracytoplasmic sperm microinjection, nuclear transfer and DNA microinjection require the visualization of cytoplasmic components and nuclei of oocytes and early embryonic cells. Bovine oocytes were matured and fertilized in vitro, and then centrifuged at the germinal vesicle, metaphase II and pronuclear stages and at syngamy. These (n = 536) were examined using light and transmission electron microscopy. The organelles stratified in five distinct zones in a consistent pattern in both oocytes and zygotes, though relative fractions changed in organelle composition after fertilization. These comprised a centripetal lipid zone, below which was a vesicular zone, then a supra-equatorial band of smooth endoplasmic reticulum (SER), a clear zone and a centrifugal mitochondrial zone. Cortical granules were located peripherally, single or clumped together, in the clear and mitochondrial zones. The nuclei were usually found associated with the SER or Golgi membranes, and chromatin was clumped at one pole within the nucleus. The maturation spindles were often located beneath the oolemma in all zones, while the first mitotic spindle was usually located in the clear zone. Some of the oocytes were activated by centrifugation and completed maturation.

Animals↗
PubMed1994

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.

Animals↗
PubMed1994

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.

Centrioles↗
PubMed1994

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.

Acrosome↗
PubMed1992

The effects of cooling mouse oocytes.

The effects of cooling and warming on meiotic spindles of mouse oocytes have been assessed by transmission electron microscopy. Intact cumulus-oocyte complexes were immediately cooled from 37 to 15, 4, 0, and -7 degrees C (seeding temperature) for 15 min in a programmed biological freezer and fixed at these temperatures. Other complexes, cooled to these temperatures, were rapidly warmed to 37 degrees C and incubated for 2 hr before fixation at 37 degrees C. Of 334 oocytes assessed at various temperatures, at least 100 were examined for metaphase II spindles. Spindle microtubules completely disappear at 0 and -7 degrees C, while complete or partial depolymerization of microtubules was observed at 4 degrees C. Cooling to 15 degrees C did not cause major disruptions of spindle structure in most oocytes. Chromosomes tended to rotate or clump at lower temperatures but chromosome scatter outside the spindle zone was rarely observed. Centrosomal material was fragmented at 4 degrees C and occasionally at 15 degrees C and was not evident at the spindle poles at 0 and -7 degrees C. Kinetochores were seen at all temperatures. Spindle structure was evidently restored in the majority of oocytes on rewarming at 37 degrees C. Changes in the ooplasm induced by cooling were elongation and disruption of vesicular smooth endoplasmic reticulum, especially between lipid globules and disappearance of fibrillar inclusions. Cortical granule exocytosis was not observed on cooling, while microfilaments were intact. Swelling of membranous organelles was also observed in cumulus cells. Most of the cytoplasmic changes were also reversed on rewarming. The response of mouse oocytes to cooling is compared to that of human oocytes, reported previously.

Animals↗
PubMed1992

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.

Embryonic and Fetal Development↗
PubMed1991

Centrioles in the beginning of human development.

We demonstrate the presence of centrioles in fertilized human oocytes at syngamy. Single or double centrioles within centrosomes were detected by transmission electron microscopy at one pole of the first cleavage spindle in normal and dispermic embryos (25-26 hr after insemination). Sperm centrioles were also closely associated with the male pronucleus (16-20 hr after insemination) in pronuclear stage embryos. A tripolar spindle derived from a tripronuclear embryo is also demonstrated with two centrioles at one pole. The data provide evidence that human centrioles, as those in most other animals, and unlike the mouse, are paternally derived, thus supporting Boveri's classical theory. Furthermore, this study provides insights to the proposed mechanisms of aberrant cleavage patterns of dispermic human embryos.

Blastocyst↗
PubMed1990

Assessment of the human sperm acrosome reaction using concanavalin A lectin.

A method for assessment of the human sperm acrosome reaction is reported using fluorescein isothiocyanate (FITC)-conjugated Concanavalin A (ConA). The technique involved labelling prefixed spermatozoa, where only those spermatozoa that showed a complete loss of the acrosome bound FITC-ConA to the acrosomal region. Competitive sugar binding studies demonstrated that binding of ConA lectin to the acrosomal area of human spermatozoa was inhibited in the presence of 0.2 M D-mannose. Staining with the supravital stain Hoechst 33258 (H258) concomitantly with FITC-ConA allowed determination of only those spermatozoa that had undergone a true and not degenerative acrosomal loss. Incubation of human spermatozoa with 0, 1, 5, and 25 microM calcium ionophore, A23187, for 60 min demonstrated that changes in acrosomal status due to the different treatment protocols may be determined by the dual-staining method. Electron microscopy studies revealed that gold-conjugated ConA bound specifically to the surface of the inner acrosomal membrane of acrosome-reacted spermatozoa. A significant correlation (r = +.97) between transmission electron microscopy (TEM) and FITC-ConA labelling methods of acrosomal status assessment was achieved. The simple ConA labelling procedure reported here therefore provides a reliable method for quantitation of the physiological acrosome reaction of a population of human spermatozoa.

Acrosome↗
PubMed1990

Subzonal transfer of multiple sperm (MIST) into early human embryos.

Microinsemination sperm transfer (MIST) is a technique whereby sperm are transferred into the perivitelline space (PVS) with the aid of a micromanipulator. MIST is now used to investigate whether blastomere membranes of early human embryos are capable of fusing with the sperm as in the metaphase II oocyte. Between 10 and 30 sperm were transferred into 11 donated human embryos between pronuclear and 16 cell stage. After culture for 6-24 hr in vitro, the embryos were fixed for transmission electron microscopy (TEM). Both acrosome-intact and acrosome-reacted sperm were located in the PVS and between blastomeres. Sperm in the PVS were sometimes penetrating the inner regions of the zona. Sperm-blastomere membrane fusion was not observed, but sperm tail incorporation by phagocytosis was occasionally evident. Sperm heads incorporated into blastomeres were often located in membrane-bound vesicles. Both acrosome-intact and acrosome-reacted sperm heads were found in vacuoles. Acrosome-reacted sperm heads were lying passively in vacuoles or were undergoing degenerative changes at their surfaces. Sperm chromatin decondensation was not observed in any of the sperm heads that were detected in the blastomeres. The evidence presented clearly shows that sperm heads are incapable of expanding their chromatin to form typical male pronuclei following MIST into early human embryos.

Blastomeres↗