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In order to elucidate the ultrastructural dynamics of endometrium differentiation, uterine samples of fetuses aged 14 to 22 weeks of gestation (WG) were analyzed. Samples were processed for light (LM), transmission (TEM) and field-emission scanning electron microscopy (FE-SEM). Initial stratification of the uterine wall occurred at 14 WG: endometrial, myometrial, and perimetrial primordia were identified. At this age, the endometrial epithelium was simple columnar to pseudostratified and consisted of microvillous cells. Blood capillaries developed mainly in the stroma and between the myometrium and perimetrium primordia. At 18-20 WG the endometrial epithelium became clearly pseudostratified, with active ciliogenesis and a predominance of microvillous cells. Primordia of tubular glands were present at 20 WG. Microvillous cells still predominated in the endometrial epithelium at 21-22 WG and showed morphological features of apoptosis. The endometrial stroma at this stage was organizing into a thick lamina propria provided with subepithelial capillary plexuses. However, the stroma was formed by still undifferentiated mesenchymal cells during the whole period of study. Our data showed that the epithelial differentiation and distribution in the uterus occur in the human fetus in a similar way as in the adult. The above events are likely the expression of an early developmental patterning and related to future reproductive processes, such as the regulation of gamete passage and blastocyst implantation. Because the structure of the adult uterus is determined by the degree of paramesonephric duct fusion, septum absorption, and differentiation of the uterine primordial layers, our study may contribute toward clarifying to normal urogenital development.

The aim of this article is to summarize and update, through an integrated analysis by transmission electron microscopy (TEM) and high resolution scanning electron microscopy (SEM) after osmium-dimethyl sulfoxide-osmium (ODO) maceration, the studies of our research group on the morphodynamics of oocyte-follicle cell associations during follicle development in humans. In resting oocytes, follicular cells project few and short cytoplasmic processes in the perioocytic space. They often form bulbous terminals very close to the oolemma where zonulae adherentes, maculae adherentes, and gap junctions are present. The oolemma mostly appears smooth with short and scanty microvilli. In early growing follicles, follicular cell projections appear as (a) long and tortuous microvilli or (b) large and short extensions. The oolemma shows numerous short microvilli. By TEM, long and thin follicular "intraooplasmic processes" have been seen to penetrate deeply into some oolemma invaginations. In macerated samples, they are observed by SEM to come very close to the nucleus and contact different oocyte organelles. These processes are more likely involved in early oocyte growth. In late growing follicles, oocyte-somatic cell interactions-now established through the interposition of the zona pellucida (ZP)-preserve the general features of early growth stage, with the exceptions of "intraooplasmic processes," which are no more present. In mature follicles subjected to a long ODO maceration, corona cells appear to contact the oocyte through an apical plume of numerous very long "curly hair-like microvilli." Corona cell microvilli, quite likely provide a sort of cytoplasmic skeleton for the ZP and they are possibly involved in (a) release of nutrients or removal catabolites to/from oocyte and vice versa and (b) transfer of substances to build up ZP. In conclusion, among oocyte and somatic cells a structural and functional association is revealed. This association, certainly highly dynamic in vivo, plays a key role in regulating the healthy folliculogenesis to assure a correct and timed oocyte maturation and ovulation.

Human zona pellucida (ZP) is maintained up to the blastocyst stage prior to hatching. In in vitro fertilized (IVF) embryos, it eventually acts as a morphodynamic interface between the cultured embryo and its microenvironment. Ultrastructural data on the ZP of IVF blastocysts are scarce in humans. We employed correlated phase contrast microscopy (PCM) and scanning electron microscopy (SEM) to study retrospectively the ultrastructural morphology of the ZP outer surface of 20 IVF human blastocysts from 16 Japanese patients (28-44 years of age, average 36.7+/-4.2) with a history of infertility. Blastocysts were derived from conventional in vitro fertilization (cIVF) (n = 10) and from intracytoplasmic sperm injection (ICSI) (n = 10). Both cIVF and ICSI groups included "clear blastocysts" (n = 5) and "dark blastocysts" (n = 5). By PCM, the clear blastocysts exhibited a regular, round-shaped contour and consisted of clear and voluminous cells. By SEM, they displayed a spongy ZP with numerous fenestrations formed by networked filaments. By PCM, dark blastocysts appeared irregularly shaped and often collapsed, and comprised dark cells and debris. By SEM, their ZP were smooth with remnants of compact fenestrations. In conclusion, viable blastocysts presented a normal ZP outer surface ultrastructure, whereas unhealthy blastocysts showed an altered ZP outer surface, comparable to that of immature/atretic oocytes. Such alterations could reflect sub-optimal culture conditions and/or could be related to blastocyst degenerative processes. The blastocyst ZP surface ultrastructure was unaffected by the fertilization technique (cIVF or ICSI). These data suggest that blastocyst survival in vitro is related to ZP ultrastructure maintenance.

This review updates the three-dimensional (3-D) microanatomic evolution of the ageing human ovary in a novel dynamic fashion, taking advantage of integrated scanning, transmission, corrosion casts and correlated techniques developed by ourselves. Tissues were collected from ovaries of women (aged between 28 and 70 years) during open surgery or endoscopic procedures. Our findings are as follows: the volume of the postmenopausal ovary was about one half of that present during reproductive years; postmenopausally the ovary became progressively atrophic and fibrotic. Particularly it showed a decrease of papillae and crypts which made its surface smoother than that of premenopausal tissue. In perimenopausal women (aged 45–55 years) the superficial cells covering the ovary were irregularly polyhedral and flattened. Their cytoplasm showed few organelles and in some instances apoptotic and/or necrotic cells. These features increased in most aged ovaries. Consequently the zones of the ovarian cortex (tunica albugina) could occasionally appear as consisting of dense fibrous collagen structures. Here only a few altered and swollen small follicles could be found. In the medullary regions variously sized cysts, some of which were blood filled (corpora atretica and hemorragica), were present along with retracted scars (corpora albicantia). In older women (aged 55–70 years) follicles were never observed. Instead, areas of thick density of collagen fibres and numerous sized scars (corpora albicantia) were present. A drastic reduction of the ovarian vasculature along with thickened vessel walls was commonly found. These typical features were already noted in perimenopausal women, but to a lesser extent. The detailed fine cell alterations of the ovary would appear to reflect the progressive decline in steroid hormone production as the ovary ages.

The detailed three-dimensional ultrastructural features of the reproductive organs of menopausal and postmenopausal women were studied by means of integrated transmission and scanning electron microscopy (SEM) and reported in a new colored microtopographical fashion. These methods revealed significant alterations in the microanatomy of the various reproductive organs specifically related to the decline of plasma estrogen levels. In particular, the ovary progressively showed characteristic wide areas of loss of epithelium with consequent exposure of the underlying connective tissue. Both endometrial and tubal mucosa demonstrated a gradual but often dramatic decrease in the number of ciliated cells which was more evident in the tube. In addition, the non-ciliated (microvillous secretory) cells of the uterus, including both endocervix and tubal mucosa, became flattened and, in some instances, their apical poles developed unusual wrinkles (microridges or microplicae). The ectocervix and vaginal squamous cells presented a reduction in the number of their microridges and changes in the typical structural organization. These microtopographical results showed that the decline of estrogen during the menopause and postmenopause induces important and complex structural changes of the woman's reproductive system, much more detailed than those revealed to date by the use of only conventional optical and transmission electron microscopy (TEM). The three-dimensional findings offer the opportunity to re-evaluate the classic histopathology of the above aging organs using more refined microtopographical and morphophysiopathological parameters.

This presentation, on both printed copy and CD-ROM, summarizes a series of original data on the ultrastructure of human reproduction produced by our research group. In particular, female germ cell behaviour at the time of migration and colonization of the gonad and germ-somatic cell interactions inside the developing ovary are reviewed from a morphodynamic point of view. The results mostly consist of black-and-white transmission and scanning electron microscopy (TEM and SEM) images. Artificially coloured SEM pictures, light microscopy images and drawings have also been selected for iconography to render complex microanatomical details and their morphofunctional relationships more comprehensible. In all, 35 images are presented in this article, each related to a concise text section and accompanied by a self-explaining caption. A list of pertinent references is also provided.

The ultrastructure of the follicle-oocyte complex in rodents and humans was revealed by high resolution scanning electron microscopy (SEM) following the Osmium-DMSO-Osmium maceration method (TANAKA and NAGURO, 1981). In primary follicles, the majority of oocyte organelles such as mitochondria and Golgi complex components are concentrated in a juxtanuclear area. In particular, many spherical mitochondria are oriented all around the nucleus. After maceration of the ooplasm matrix, most of these mitochondria appear intermingled with numerous microtubules (MT) and associated with many Golgi vesicles. Such a nuclear polarization of organelles, essential to the oocyte metabolism, might depend upon a MT activity. MT might guide mitochondria to gather in the perinuclear region and further maintain their close associated to the nuclear envelope. A similar relationship among microtubules, vesicular Golgi complex and mitochondria has been also observed when, in maturing oocytes, these organelles migrate and gather in other areas of the ooplasm. A pattern common only to human developing follicles appears in the occurrence of long microvilli projected from follicle cells deep into the oocyte. These unusual microvilli running within the ooplasm are surrounded by several vesicles of the Golgi complex and endoplasmic reticulum, and often end close to the nucleus. In the antral follicle, the microvilli of corona cells, directed toward the oocyte (after their full exposure through the chemical dissolution of the zona pellucida matrix) are extremely numerous (up to 70/cell), long (up to 7/10 microns) and tortuous. They resemble epididymal stereocilia, may be ramified and possess bulbous tips. In contrast, oocyte microvilli are thin and short.(ABSTRACT TRUNCATED AT 250 WORDS)

The development and differentiation of the coelomic epithelium lining the paramesonephric ducts in human fetus, that gives rise to the female genital organs, have been ultrastructurally examined. The epithelium appeared pseudostratified, consisting of basal, microvillous and ciliated cells. In younger fetuses (12th gestational week) ciliogenic elements could be detected mainly on the developing tubal fimbriae, but most of the cells showed microvilli and often single cilia. In the subsequent phases of development, morphodynamics of cell renewal were documented by aspects of apoptosis. Fully ciliated cells were numerous on the fimbriae and at the utero-tubal junction, but not in the uterus; however, these were less abundant than those showing microvillous. In older fetuses (31st gestational week) microapocrine secretion by microvillous cells, in the form of droplets contacting cilia, could be observed. In the same fetuses the ectocervix was covered by a mature squamous epithelium, made up of polygonal flat desquamating cells, showing labyrinthine surface microplicae. Our observations demonstrated that ciliation in the human female genital organs, like that of other systems, is neither simultaneous nor uniform, and ciliated cells are gathered preferentially in strategic sites, to mediate germ cell migration and blastocyst implantation in adult life. These ultrastructural data seem to indicate that the female genital tract epithelium, at least in its general features, is sketched since fetal life, and cell morphodynamics, including microvillous and ciliated cell differentiation, as well as the secretory activity, are the morphological expression of the complex molecular mechanisms, involved in developmental biology and reproductive physiology.

Female reproductive activity depends upon cyclic morphofunctional changes of the ovarian tissue during the female's fertile period, but the primum movens of an active gonadal rearrangement can be found from early phases of embryo development. To offer a basic account of the main steps of ovarian dynamics, we review the morphofunctional behavior of the follicular-luteal complex in an integrated study using light microscopy and transmission and scanning electron microscopy as well as through the use of numerous drawings. Particular emphasis is given to some reproductive aspects including (1) germ-somatic cell relationships and onset of folliculogenesis during early gonadal development; (2) follicular development and oocyte-follicle cell associations through adult folliculogenesis, finally leading to ovulation; (3) morphodynamics of corpus luteum formation, development, and regression, and (4) degenerative processes involving germ and somatic cells. The results reported, many of which originated in our laboratory, arise from some experiments on laboratory mammals but mostly from a large selection of human specimens. The data obtained are integrated and correlated with classic reports as well as with current views. Crucial biochemical, histophysiological, and clinical aspects are also emphasized.

Objective: To evaluate the microanatomical dynamics of the ovary during postreproductive life. Design: Retrospective, observational research study. Setting: Institutional (university). Patient(s): Eleven (43- to 72-year-old) women in perimenopause, menopause, or postmenopause. Intervention(s): Biopsies of ovaries obtained from patients undergoing gynecologic surgery or diagnostic procedures. Main Outcome Measure(s): Analysis of the 3-dimensional microanatomy of the ovary by transmission and high-resolution scanning electron microscopy. Result(s): The surface epithelium gets gradually flatter and is always present, even in advanced age. The surface appears smoother because of a smaller number of papillae and crypts as well as a decreased number and shortening of microvilli on surface epithelial cells. Signs of atrophy and fibrosis are evident. Primordial follicles are usually absent in postmenopause, whereas corpora atretica, hemorrhagica, and albicantia, scar tissue, and simple follicular cysts are common after menopause. Apoptotic and necrotic cells appear frequently within the surface epithelium. Major common features are a marked reduction in number and caliber of blood vessels with thickening of the vascular walls and changes in endothelial cells. Conclusion(s): Scanning electron microscopy studies are a useful complement to ordinary gynecologic diagnostic methods. Variations among patients of the same age range or functional status should be considered.

Our study reviews and ultrastructurally characterises human pre-Sertoli cells between the 6th and the 20th week of gestation by means of integrated light microscopy, transmission electron microscopy and high resolution scanning electron microscopy (standard or following ODO maceration). The morphofunctional differentiation of Sertoli cells defines testicular differentiation. These somatic cells are mostly of mesonephric origin and can be first morphologically recognised in 7 week-old embryos altogether with the formation of testicular cords. The latter organise as primordial germ cells surrounded by pre-Sertoli cells. Due to the great synthetic activity of pre-Sertoli cells the rough endoplasmic reticulum develops. The basal lamina of the cords becomes distinguishable at 7 to 8 weeks of gestation. Both prespermatogonia and pre-Sertoli cells actively proliferate but the latter greatly outnumber prespermatogonia. Many interdigitations and cytoplasmic processes are observed between neighbouring pre-Sertoli cells. Due to cell proliferation a sort of compartmentalisation is established inside the cords in which pre-Sertoli cells tend to localise closer to the basal membrane embracing prespermatogonia with long and thin cytoplasmic processes. One of the main typical features of differentiating pre-Sertoli cells is the irregular nucleus and the prominent nucleolus. When the embryo is 14 to 20 weeks-old pre-Sertoli cells maintain their general morphology whereas the most significant change is the maximum development of Leydig cells. Testicular cords do not show any lumen at all so they cannot be termed "tubules".

The present study on in vitro fertilization in humans demonstrates that three-dimensional (3-D) fine morphology by high resolution scanning electron microscopy (SEM) combined with parallel light and transmission electron microscopy (TEM) can reveal a number of new cellular detailed findings which cannot be detected with other methods. In this study the following aspects have been investigated in early human embryos. 1. Micro-topographical features of the zona pellucida (ZP), surface blastomeres and polar body. 2. Intracytoplasmic features of mature and healthy oocyte, in vitro fertilized (IVF) oocyte and early embryo development. 3. Comparison of general views of intracytoplasmic sperm injection (ICSI) and conventional IVF (C-IVF) of early embryos. 4. Presence of unusual large tubular smooth endoplasmic reticulum (SER) aggregates in 4, 5, and 6-cell embryos after ICSI. 5. Inside views of 3-D blastocysts such as inner cell mass and trophoblast. To our knowledge, this is the first time that such images are reported by using these techniques.

When the embryo is 6-week-old the gonad is composed mostly of migrating primordial germ cells, surface coelomic mesothelium and mesenchymal cells. At 7 weeks of gestation (wg) testicular cords consist of prespermatogonia, larger, with a more regular outline and higher mitotic activity than primordial germ cells, embraced by somatic pre-Sertoli cells. The morphofunctional development of Sertoli cells defines testicular differentiation. Towards 7.5 wg the gonad is finally in its differentiated stage; the basal lamina of the cords becomes distinguishable, testicular cords radially branch and elongate, and Leydig cells are clearly recognized. Primordial germ cells are large and spherical, with rounded and eosinophilic nuclei and large nucleoli. Pre-Sertoli cells, in turn, show round or columnar nuclei and rough endopasmic reticulum. Prespermatogonia and mostly pre-Sertoli cells actively proliferate. Many interdigitations and cytoplasmic processes are observed between neighboring pre-Sertoli cells and between pre-Sertoli cells and prespermatogonia. A sort of com partmentalization is established inside the cords in which pre-Sertoli cells tend to localize closer to the basal membrane embracing the prespermatogonia with long and thin cytoplasmic processes. Between 14 and 16 wg the most significant changes besides maximum development of Leydig cells are differentiation of mesenchymal cells around the cords into future peritubular cells and maturational changes of pre-Sertoli cells. These likely reveal a peak coinciding in time with reported increases in either testosterone production and Müllerian Inhibiting Substance secretion. During the period herein considered testicular cords show no lumen neither any sign of canalization so they cannot be termed "tubules".

The aim of this study has been to observe, by electron microscopy, the morphological changes affecting mitochondria and associated organelles in the human female germ cell during oogenesis, maturation and fertilization. In the primordial germ cell (PGC), rounded mitochondria with a pale matrix and small vesicular cristae are disposed near the nucleus and significantly increase in number during PGC migration and settlement in the gonadal ridge, where they differentiate into oogonia. In these early stages of mammalian oogenesis, aggregates of mitochondria are typically clustered around or in close relationship with the nuage. In oocytes at early prophase stage, mitochondria proliferate while aligned along the outer surface of the nuclear membrane, contain a more dense matrix than before, and have lamellar cristae. Oocytes of primordial and primary follicles mostly contain round or irregular mitochondria whose matrix has become very light. These mitochondria show typical parallel, arched cristae, and are clustered near the nucleus with other organelles forming the Balbiani's vitelline body. When follicles grow, the mitochondria of the oocytes become even more numerous and are dispersed in the ooplasm. Both paranuclear accumulation and subsequent dispersion of mitochondria in the cytoplasm are likely to be regulated by microtubules. By ovulation, mitochondria are the most prominent organelles in the ooplasm. They form voluminous aggregates with smooth endoplasmic reticulum (SER) tubules and vesicles. These mitochondrial-SER aggregates (M-SER) and the mitochondrial-vesicle complexes (MV) could be involved in the production of a reservoir of substances or membranes anticipating subsequent fertilization and early embryogenesis. Just after fertilization, the mitochondria of the oocyte undergo a further substantial change in size, shape, and microtopography. In the pronuclear zygote, mitochondria concentrate around the pronuclei. During the first embryonic cleavage divisions, round or oval mitochondria with a dense matrix and few arched cristae are gradually replaced by elongated ones with a less dense matrix and numerous transverse cristae. A progressive reduction in size and number of M-SER aggregates and MV complexes also occurs. In summary, oocyte mitochondria show dynamic morphological changes as they increase in number and populate different cell domains within the oocyte. They form complex relationships with other cell organelles, according to the different energetic -metabolic needs of the cell during differentiation, maturation, and fertilization, and are ultimately inherited by the developing embryo, where they eventually assume a more typical somatic cell form.

In order to understand the fine structure and distribution of the interstitial glandular cells (IGCs) and associated elements in the human fetal ovary, we studied human fetal ovaries at 16 weeks post fertilization (p. f.) by transmission electron microscopy. Semithin sections revealed voluminous typical IGCs usually grouped in clusters, located in the interstitium among the ovigerous cords. Isolated primordial follicles were seen in the cords located close to the interstitium in which IGCs were present. Besides the main ultrastructural characteristics of steroid secreting cells, the IGCs showed lipofuscin granules and stacks of annulate lamellae in their cytoplasm. Fibrocytes, macrophages and mast cells were detected close to the IGCs. In particular, the fibrocytes were located around the IGCs, with which they occasionally formed focal cell contacts. Fibrocytes issued numerous long projections, which, together with collagen fibers, surrounded the clusters of IGCs and small vessels (mainly capillaries), often extending into the intercellular spaces among IGCs. These data indicated that, already at the initiation of folliculogenesis, the IGCs are present numerously in a close association with the ovigerous cords. The morphological aspects of IGCs were comparable to that of fetal testis interstitial (Leydig) cells and hilar cells in adult ovary, and suggest that fetal IGCs may be source of adult ovary hilar cells. In addition, we have here demonstrated for the first time that IGCs are associated with stromal cells whose distribution seems to support IGCs microtopography. Fetal ovarian fibrocytes revealed a structural arrangement similar to that of the “compartmentalizing cells” previously described in the adult testis. Macrophages and mast cells presumably have a role as local modulators of steroid synthesis. Mast cells may also affect fibrocyte organization and vascular permeability. We thus suggest that IGCs and associated cells may form a glandular unit in the human fetal ovary similar to that in the adult testis, and this structure is likely involved in early steroid secretion during gonadal differentiation.