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.

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.

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 dispernic 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 dassical theory. Furthermore, this study provides insihts to the proposed mechanisms of aberrant cleavage patterns of dispermic human embryos.It is widely believed that mature mammalian oocytes and early cleavage stage embryos do not have centrioles (1-6). Most cells, however, do possess centrosomes, which are microtubule (MT) organizing centers at spindle poles (3). In his classical theory of fertilization, Boveri in 1900 (7) stated that unfertilized eggs derive their centrosomes from male gametes, and this has subsequently been shown to be the case in a number of animal species, including the sea urchin (2), where centrioles associated with centrosomes organize mitotic bipolar spindles (3). On the contrary, in mice, centrosomes are maternally derived (2) and this has been proposed to be true for other mammals.Meiotic spindles of mammalian oocytes are anastral, barrel shaped, and composed of numerous MTs (1, 2, 4). The structure of the human meiotic spindle has already been described to conform to the mammalian pattern (5,6,8,9). Mammalian meiotic spindles have centrosomes but no centrioles. Centrosomes and centrioles are both self-reproducing organelles and centrioles merely advertise the presence of centrosomes (3). After fertilization, the mitotic spindle of the sea urchin embryo is organized by paternally inherited centrioles and centrosomes (10, 11). In the sea urchin, each sperm carries two centrioles associated with centrosomes (12), which duplicate and separate to form a bipolar spindle during the first mitosis and are the ancestors of these organelles in all cells during subsequent development (2, 10, 11). In a fashion similar to sea urchin sperm, human sperm also have centrioles. A well-defined proximal centriole is present next to the basal plate of the sperm head (13-15), while the distal centriole (which is a remnant) gives rise to the sperm tail axoneme during spermiogenesis. The proximal centriole consists of nine triplets of MTs showing the typical 9 + 0 organization and is associated with osmiophilic centrosomal material. After gamete fusion, the sperm midpiece and tail are invariably incorporated into the ooplasm, and the centriolar region often remains attached to the decondensing sperm nucleus and persists after male pronuclear formation (16-18). This study demonstrates the presence of centrioles associated with centroso...

The fine structure of human oogonia and growing oocytes has been reviewed in fetal and adult ovaries. Preovulatory maturation and the ultrastructure of stimulated oocytes from the germinal vesicle (GV) stage to metaphase II (MII) stage are also documented. Oogonia have large nuclei, scanty cytoplasm with complex mitochondria. During folliculogenesis, follicle cell processes establish desmosomes and deep gap junctions at the surface of growing oocytes, which are retracted during the final stages of maturation. The zona pellucida is secreted in secondary follicles. Growing oocytes have mitochondria, Golgi, rough endoplasmic reticulum (RER), ribosomes, lysosomes, and lipofuscin bodies, often associated with Balbiani bodies and have nuclei with reticulated nucleoli. Oocytes from antral follicles show numerous surface microvilli and cortical granules (CGs) separated from the oolemma by a band of microfilaments. The CGs are evidently secreted by Golgi membranes. The GV oocytes have peripheral Golgi complexes associated with a single layer of CGs close to the oolemma. They have many lysosomes, and nuclei with dense compact nucleoli. GV breakdown occurs by disorganization of the nuclear envelope and the oocyte enters a transient metaphase I followed by MII, when it is arrested and ovulated. Maturation of oocytes in vitro follows the same pattern of meiosis seen in preovulatory oocytes. The general organization of the human oocyte conforms to that of most other mammals but has some unique features. The MII oocyte has the basic cellular organelles such as mitochondria, smooth endoplasmic reticulum, microfilaments, and microtubules, while Golgi, RER, lysosomes, multivesicular, residual and lipofuscin bodies are very rare. It neither has yolk nor lipid inclusions. Its surface has few microvilli, and 1-3 layers of CGs, aligned beneath the oolemma. Special reference has been made to the reduction and inactivation of the maternal centrosome during oogenesis. The MII spindle, often oriented perpendicular to the oocyte surface, is barrel-shaped, anastral and lacks centrioles. Osmiophilic centrosomes are not demonstrable in human eggs, since the maternal centrosome is nonfunctional. However, oogonia and growing oocytes have typical centrioles, similar to those of somatic cells. The sperm centrosome activates the egg and organizes the sperm aster and mitotic spindles of the embryo, after fertilization.

Preovulatory human oocytes were cooled to 0 degrees C at 1 degree C/min, with or without the cryoprotectant dimethyl sulphoxide (DMSO), to assess the effects of cooling on the meiotic spindles and on oocyte structure. Batches of oocytes, cultured for 3-9 h, were held at 0 degrees C for 20 or 60 min and then fixed for transmission electron microscopy (TEM) either at 0 or 8 degrees C. Control oocytes were not cooled and were fixed at 22 or 37 degrees C for comparison. TEM revealed that 80% of the oocytes were at metaphase II, while 20% were at metaphase I and most had resumed meiosis recently. Control oocytes had more or less barrel-shaped meiotic spindles composed of microtubules (MT), some associated with chromosomes at kinetochores. Both metaphase I and II spindles were disassembled when cooled and fixed at 0 degrees C, with or without DMSO, due to extensive depolymerization of MT. The few MT that survived were found at the poles or were bundled together or were associated with chromosomes. Kinetochores were not prominent. Some oocytes cooled with DMSO and fixed at 0 or 8 degrees C showed evidence of MT, but the spindles were still disorganized and were abnormal in structure. Chromosomes tended to clump together or were dislocated in the cortical ooplasm in cooled oocytes, but widespread scattering was not observed. This was particularly evident in the absence of DMSO. Elements of the endoplasmic reticulum, Golgi, mitochondria and the cytosol were also adversely affected in some of the cooled oocytes and their surrounding cumulus cells. The results show that meiotic spindles are very sensitive to simple cooling and that DMSO does not provide substantial stabilization of the meiotic spindle even at 0 degrees C. The findings are discussed with reference to recent work on frozen human and mouse oocytes.

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.

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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.