Cryoprotectant-free vitrification of human spermatozoa in new artificial seminal fluid.

Vitrification is a new method that has been recently introduced in Assisted Reproduction Technique programs. The aim of this study was to design a new medium similar to normal human seminal fluid (SF), formulation artificial seminal fluid (ASF), and to compare the cryoprotective potency of this medium with SF and human tubal fluid (HTF) medium. Thirty normal ejaculates were processed with the swim-up technique and sperm suspensions were divided into four aliquots: (i) fresh sample (control); (ii) vitrification in HTF medium supplemented with 5 mg/mL human serum albumin and 0.25 mol sucrose (Vit HTF); (iii) vitrification with patients' SF (Vit SF); and (iv) vitrification in ASF (Vit ASF). After warming, sperm parameters of motility, viability, and morphology were analyzed using WHO criteria. Also, sperm pellets were fixed in 2.5% glutaraldehyde and processed for scanning electron microscopy and transmission electron microscopy observations. The results showed that progressive motility (46.09 ± 10.33 vs. 36.80 ± 13.75), grade A motility (36.59 ± 11.40 vs. 16.41 ± 11.24), and normal morphology (18.74 ± 8.35 vs. 11.85 ± 5.84) and viability (68.22 ± 10.83 vs. 60.86 ± 11.72) of spermatozoa were significantly higher in Vit ASF than in Vit HTF. All parameters were better in Vit ASF than in Vit SF, but only viability was significantly different (p = 0.006). After cryopreservation, deep invagination in cytoplasm and mechanically weak point sites and folded tail were commonly observed. But, this phenomenon was more significant in Vit HTF and Vit SF than in ASF (p < 0.05). In transmission electron microscopy evaluation, acrosome damage, plasma membrane loss, chromatin vacuolation, and disruption of mitochondria arrangement and structures were observed in all vitrified groups. Adherence of several tail sections together was also seen in all cryo groups. But this was seen more in Vit HTF and Vit SF than in ASF (p < 0.05). In conclusion, vitrification of human spermatozoa with ASF can effectively preserve the quality of sperm motility in comparison with Vit HTF.

Ultrastructural markers of quality are impaired in human metaphase II aged oocytes: a comparison between reproductive and in vitro aging.

PURPOSE: Childbearing delay contributes to the increase of subfertile couples that require assisted reproductive technology (ART). Subfertility relates with reproductive aging (RA). In vitro aging (IvA) (due to extended culture) may also impair oocyte competence. Aims of this study were to evaluate and compare the oocyte ultrastructure after RA and IvA. METHODS: Cumulus-oocyte complexes (COCs) (n = 68), with metaphase II oocyte and expanded cumulus, from consenting patients (<35 years old and ≥35 years old, n = 36), were selected by phase contrast microscopy and fixed at pick up, or after 24 h culture. COCs (n = 44) were studied by light and qualitative/morphometric transmission electron microscopy. Two-way ANOVA, with age and culture as grouping factors, was applied for statistical analysis (p < 0.05). Metaphase II cumulus-free oocytes (n = 24) were selected for confocal microscopy observations. RESULTS: Significant decrease of mitochondria-smooth endoplasmic reticulum aggregates, increase of mitochondria-vesicle complexes size and amount, decrease of cortical granules and microvilli, and alterations of the spindle structure characterized both RA and IvA oocytes. These changes were significantly more evident in the RA oocytes submitted to IvA. RA oocytes also showed changes of the zona pellucida and occurrence of vacuoles after culture. Cumuli appeared re-compacted after culture, irrespective of the age of the patients. CONCLUSIONS: These data demonstrated that aging is related to decay of oocyte ultrastructural quality, and that oocytes from elder women are more sensitive to prolonged culture (IvA) than the oocytes from younger women. These morphological results should be considered when applying ART in aged patients, rescue ICSI, or artificial oocyte activation.

Ultrastructure of human mature oocytes after vitrification.

Since the introduction of human assisted reproduction, oocyte cryopreservation has been regarded as an attractive option to capitalize the reproductive potential of surplus oocytes and preserve female fertility. However, for two decades the endeavor to store oocytes has been limited by the not yet optimized methodologies, with the consequence of poor clinical outcome or of uncertain reproducibility. Vitrification has been developed as the promising technology of cryopreservation even if slow freezing remains a suitable choice. Nevertheless, the insufficiency of clinical and correlated multidisciplinary data is still stirring controversy on the impact of this technique on oocyte integrity. Morphological studies may actually provide a great insight in this debate. Phase contrast microscopy and other light microscopy techniques, including cytochemistry, provided substantial morphofunctional data on cryopreserved oocyte, but are unable to unraveling fine structural changes. The ultrastructural damage is one of the most adverse events associated with cryopreservation, as an effect of cryo-protectant toxicity, ice crystal formation and osmotic stress. Surprisingly, transmission electron microscopy has attracted only limited attention in the field of cryopreservation. In this review, the subcellular structure of human mature oocytes following vitrification is discussed at the light of most relevant ultrastructural studies.

Ultrastructural markers of quality in human mature oocytes vitrified using cryoleaf and cryoloop.

This study describes and compares the possible effects of vitrification on the ultrastructural morphology of 20 human mature oocytes vitrified using two different supports, cryoleaf (n = 10) and cryoloop (n = 10). Fresh human mature oocytes (n = 15) were used as controls. Fresh and vitrified-warmed oocytes appeared rounded, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Sparse microvacuolization was only occasionally detected in fresh and vitrified-warmed oocytes, to the same extent. About 50% of the vitrified oocytes contained atypical, small and slender mitochondria-smooth endoplasmic reticulum aggregates, whereas a non-homogeneous microvillar pattern was observable in only 30% of the oocytes subjected to vitrification, regardless of the support utilized. Cortical granule content appeared generally reduced after vitrification, but cryoleaf-supported oocytes contained more cortical granules than cryoloop-supported oocytes (P < 0.05). Thus good overall preservation and virtual absence of cytoplasmic vacuolization seem to be the most relevant markers of quality in vitrified-warmed oocytes, using either support. In addition, cryoleaf-supported oocytes retained a higher number of cortical granules than cryoloop-supported oocytes. The variety of ultrastructural alterations recorded emphasizes the need for further studies aimed at assessing the actual tolerance of human oocytes to vitrification.

Ultrastructure of human mature oocytes after slow cooling cryopreservation with ethylene glycol.

The morphological characteristics of frozen-thawed human mature oocytes (n = 12) were studied by light and transmission electron microscopy following cryopreservation using a slow cooling protocol including increasing concentrations of ethylene glycol (0.5-1.5 mol/l) and sucrose 0.2 mol/l in the freezing solution. Fresh human mature oocytes (n = 12) were used as controls. Fresh and frozen-thawed oocytes appeared rounded in section, with a homogeneous cytoplasm, an intact oolemma and a continuous zona pellucida. Disorganization of mitochondria-smooth endoplasmic reticulum aggregates and a decreased complement of microvilli and cortical granules were frequently observable in frozen-thawed oocytes. Increased density of the inner zona pellucida, possibly related to the occurrence of zona 'hardening', was sometimes found associated with a reduced amount of cortical granules. In addition, delamination of the zona pellucida was evident in some frozen-thawed samples. Finally, numerous vacuoles and secondary lysosomes were detected in the ooplasm of most frozen-thawed oocytes. In conclusion, frozen-thawed oocytes treated with ethylene glycol may show a variety of ultrastructural alterations, possibly related, at least in part, to the use of this cryoprotectant. Thus, the ethylene glycol-based protocol of slow cooling herein described does not seem to offer significant advantages in terms of oocyte structural preservation.

Ultrastructure of human mature oocytes after slow cooling cryopreservation using different sucrose concentrations.

BACKGROUND: We studied the ultrastructural characteristics of human mature oocytes frozen/thawed (F/T) using different concentrations of sucrose. Fresh human mature oocytes were used as controls. METHODS: The oocytes (n = 48) were fixed in 1.5% glutaraldehyde at sampling (n = 16) or after freeze/thawing performed using a slow cooling method with propane-1,2-diol 1.5 mol/l and sucrose at either 0.1 mol/l (n = 16) or 0.3 mol/l (n = 16) in the freezing solution. The oocytes were then processed for electron microscopy observations. RESULTS: Fresh and F/T oocytes belonging to both study groups were regularly rounded in sections, with a homogeneous cytoplasm and an intact zona pellucida (ZP). Organelles (mainly mitochondria-smooth endoplasmic reticulum aggregates and mitochondria-vesicle complexes) were abundant and uniformly dispersed in the ooplasm. The amount and density of cortical granules appeared to be abnormally reduced in some F/T samples, independently of the sucrose concentration in the freezing solution: this feature was frequently associated with an increased density of the inner ZP, possibly related to the occurrence of zona 'hardening'. Furthermore, slight to moderate microvacuolization was revealed in the ooplasm of some F/T oocytes, particularly in those treated with sucrose 0.3 mol/l. CONCLUSIONS: Freeze/thawing procedures are associated with ultrastructural alterations in specific oocyte microdomains, presumably linked to the reduced developmental potential of mature cryopreserved oocytes. Further work is needed to determine whether or not a high concentration of sucrose plays a role, at least in part, in producing the above alterations.

Sucrose concentration influences the rate of human oocytes with normal spindle and chromosome configurations after slow-cooling cryopreservation.

BACKGROUND: Recently described slow-cooling cryopreservation protocols involving elevated sucrose concentration have improved survival frequencies of human oocytes, potentially overcoming a major hurdle that has limited the adoption of oocyte storage. Because implantation rates of embryos from frozen oocytes remain generally low, it is still debated whether, irrespective of survival rates, this form of cryopreservation leads inevitably to the disruption or complete loss of the metaphase II (MII) spindle. METHODS: Human oocytes with an extruded polar body I (PBI) were cryopreserved using a slow-cooling method including 1.5 mol/l propane-1,2-diol (PrOH) and alternative sucrose concentrations (either 0.1 or 0.3 mol/l) in the freezing solution. Fresh control and frozen-thawed survived oocytes were analysed by confocal microscopy to evaluate MII spindle and chromosome organizations. RESULTS: Of the 104 oocytes included in the unfrozen group, 76 (73.1%) displayed normal bipolar spindles with equatorially aligned chromosomes. Spindle and chromatin organizations were significantly affected (50.8%) after cryopreservation involving lower sucrose concentration (61 oocytes), whereas these parameters were unchanged (69.7%) using the 0.3 mol/l sucrose protocol (152 oocytes). CONCLUSIONS: Partial disruption of the MII spindle and associated chromosomes accompanies inadequate cryopreservation during slow cooling. However, protocols adopting higher sucrose concentration in the freezing solution promote the retention of an intact chromosome segregation apparatus comparable in incidence to freshly collected oocytes.

In vitro culture of mammalian early ovarian follicles. Morphofunctional correlates and future perspectives.

In vitro growth culture systems of mammalian oocytes have been developed with the aims of studying regulative processes occurring during oogenesis and folliculogenesis, and of preserving fertility. Although in large mammals IVG technology does not still assure the co-ordinate development of both somatic and germinal cells and the production of high number of viable offspring, their improvement may represent an important therapeutic tool for restoring fertility in women undergoing premature menopause or cancer treatments. Morphological studies of in vitro grown follicles were not performed extensively, especially by means of scanning electron microscopy. In the present paper preliminary ultrastructural observations of in vitro cultured follicles are presented.

A micro-anatomical model of the distribution of myocardial endomysial collagen.

Myocardial connective tissue probably provides passive support for regulating heart tensile strength and stiffness and ultimately for controlling heart mechanics through its endomysial part. However, endomysial collagen micro-arrangement is still a matter of debate. In order to define the fine distribution of left ventricle endomysial collagen, we applied the NaOH-scanning electron microscopy (SEM) maceration method (one of the techniques of choice for studying collagen micro-arrangement) to rabbit heart. Gomori-reticulum staining was used for correlated light microscopy (LM) observations. The SEM-NaOH method allowed isolation of collagen by removing other extracellular matrix components and cells and preserved collagen structure and position. Endomysial collagen appeared arranged in laminae that delimited the lacunae that were left empty by macerated myocytes and small vessels (mostly capillaries). These laminae were formed by reticular fibers, as confirmed by LM observations of Gomorireticulum-stained samples, and were organized in irregularly meshed networks made of thin (single) and thick (composed) filaments. In longitudinal views, collagen laminae extended the entire length of lacunae. In transversal views, the cut surface of the laminae appeared to be made of collagen bundles. These observations provide an updated microanatomical view of endomysial collagen distribution, which integrates previous studies. This model is based on the evidence that collagen laminae enveloped the surface of small vessels and myocytes. Thus, a type of myocyte-myocyte or capillary-myocyte "laminar connection" anchored to the entire cell length here is emphasized, rather than a type of "strut connection" anchored to defined loci, as usually described. This structure explains better how endomysium may provide the necessary support for heart compliance and protection against overstretch.

Ultrastructural dynamic features of in vitro fertilization in humans.

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.

The human zona pellucida and scanning electron microscopy. Reality or artifacts?

The surface micro-morphology of the zona pellucida (ZP) was investigated in 158 inseminated but unfertilized mature human oocytes derived from assisted reproduction trials (ART) by means of traditional scanning electron microscopy (SEM) techniques (gold coating and conductive staining methods) and saponin-ruthenium red-osmium tetroxide-thiocarbohydrazide method (Sap-RR-Os-TC). The main aspect of the ZP by traditional SEM (122 oocytes) consisted in a porous, net-like structure (97 oocytes), whereas a nearly smooth or compact structure of ZP was detected in 25 oocytes (79.5% vs 20.5%). Using Sap RR-Os-TC method on 36 oocytes, 31 oocytes showed ZP with alternating tight and large meshed networks, whereas 5 oocytes displayed only tight meshed network (86.1% vs 13.9%). Due to our well standardized procedures, to the stabilizing action of the conductive staining on the zona material and similar results obtained with the use of Sap RR-Os-TC method, we confidentially regard the ZP changes, occurring in oocytes of various groups, as genuine features, likely related to their actual maturation status, rather than as artifacts. In addition, we emphasize the concept that a modern view of the ZP surface implies the best evidence of crossing filaments' network. We think that the ZP "spongy" or "compact" appearance is only the result of microfilaments network collapse, not the true three-dimensional (3-D) representation of ZP structure.

In vitro fertilized human eggs. An electron microscopic study.

This paper describes by scanning and transmission electron microscopy the ultrastructure of the human fertilized egg and its vestments (cumulus oophorus and zona pellucida). Data are reported on the ultrastructure of a. conventional in vitro fertilized eggs (pronuclear eggs and cleaving eggs at two-to-four cell stage); b. eggs at the same developmental stage deriving fro intracytoplasmic sperm injection. The present results showed that: 1. The cumulus-enclosed fertilized egg is a highly dynamic structure in which egg vestments play a crucial role, positively affecting fertilization and healthy embryo development; 2. Intracytoplasmic sperm injection technique does not seem to significantly alter fertilized egg morphology.

Ultrastructure and distribution of interstitial glandular cells and associated elements in human fetal ovaries.

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.

Mitochondrial morphology in human fetal and adult female germ cells.

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.

Human embryo development and pregnancies in an homologous granulosa cell coculture system.

PURPOSE: Our purpose was to determine the effects of the coculture of embryos on human granulosa cells (GCs) in patients in the first cycle of IVF-ET treatment and in patients with repeated implantation failures and to investigate the presence of specific proteins in a 48-hr GC conditioned medium and the GC ultrastructural characteristics. METHODS: Eighteen patients with tubal or idiopathic infertility were enrolled in this study: 7 patients (Trial 1) were in the first cycle of IVF-ET treatment and 11 patients (Trial 2) had repeated implantation failures (one to five). Embryos from each patient were cocultured randomly either on homologous granulosa cells or on a conventional culture medium. RESULTS: At the end of the coculture period (day 5 or 6), 50% of the embryos (Trial 1) reached the blastocyst stage, with respect to 35% in Trial 2. The pregnancy rate per retrieval was 14.2 and 9%, respectively, in Trial 1 and in Trial 2. Many conditioned media showed proteins of 24-29 kDa. and some of them showed additional proteins of 90 kDa. The ultrastructural analysis of GCs showed healthy, metabolically active, protein-synthesizing, and mostly steroidogenic cells. CONCLUSIONS: GC cultures improve embryo development but not pregnancy rates both in Trial 1 and in Trial 2.

Bovine cumulus-corona cells in maturing oocytes. A scanning electron microscopic study.

The ultrastructural features of cumulus-corona cells surrounding maturing oocytes in bovine were studied by means of scanning electron microscope in order to provide a detailed description of their surface changes during oocyte maturation. Cumulus corona cell complexes of immature oocytes showed a compact aspect with narrow intercellular spaces. The spaces around mature oocytes enlarged because they were progressively filled with abundant microfibrillar extracellular matrix. In cumulus corona cells complexes of immature oocytes very numerous long and filiform microvilli were observed, whereas the cumulus corona cell surface surrounding mature oocytes showed occasional large cytoplasmic protrusions along with scanty microvilli and numerous blebs.

The microvasculature of the corpus luteum in pregnant rabbit. A scanning electron microscopy study of corrosion casts.

The vascular network of pregnant rabbit ovaries was studied by means of scanning electron microscopy (SEM) of corrosion casts, in order to evaluate the morphofunctional changes of the microcirculation of corpus luteum (CL). Pregnant rabbit ovary showed an overwhelming vascularization. Ovarian hilus displayed an increase in the arterial spirallisation. The arterial spiral pattern was present along the entire vessel course, up to CL tissues. The CL of pregnancy was supplied by wide vascular plexuses (2-5 plexuses were found in each pregnant ovary) whose major axis was about 2 mm. Luteal capillaries showed a tortuous course and were arranged in a three-dimensional, wide and rounded-meshed network. Postcapillary venoconstrictions were present. The venous drainage appeared more developed then the arterial supply. Tight artero-venous contacts in hilar, juxtamedullar and medullar regions of the ovary were observed. These results clearly show that the morphofunctional expression of CL of pregnancy is greatly dependent on its hemodynamic control. In particular, the increase of spirallisation exhibited by the arteries during pregnancy is likely to be considered a significant functional change. The spirallisation likely is a device for reducing the blood pressure through the CL. The artero-venous contacts, also previously described in hCG stimulated (pseudopregnant) ovaries, may support a counter-current like system that may allow a veno-arterial exchange of small molecules through the wall of the facing vessels. In addition, in 10-day pregnant rabbit CL the consolidation of a well-developed capillary network was revealed, which is a sign that the CL of pregnancy reached the full morphofunctional maturation. Furthermore, the CL of 10-day pregnant rabbit did not present significant capillary permeabilization and dilation or angiogenic processes, aspects that were previously found in stimulated periovulatory ovaries. Indeed, changes of the arterial supply and venous drainage of the CL of pregnancy were demonstrated. This suggests that the control of the blood flow through the CL of pregnancy may be transferred from the local capillary microcirculation to the regional artero/venous circulation. This may be probably related to the significant increase of the ovarian blood flow necessary for the maintenance of CL endocrine functions during pregnancy.

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.

Ultrastructural features of bovine cumulus-corona cells surrounding oocytes, zygotes and early embryos.

Integrated transmission and scanning electron microscopic (TEM and SEM) techniques have provided the first detailed description of the ultrastructural features of the bovine cumulus-corona (CC) cells surrounding oocytes at the time of final maturation, zygotes and early cleaving embryos (2/4 to 6/8 blastomeres). TEM revealed the presence of rough endoplasmic reticulum and Golgi complexes in the cytoplasm of CC cells surrounding immature, mature and fertilized eggs, and also revealed an increasing amount of smooth endoplasmic reticulation membranes, lipid droplets and mitochondria with villiform and/or tubular cristae in the cytoplasm of CC cells during maturation and fertilization of the oocyte. In addition, a loss of cell-to-cell junctions between CC cells was evident. TEM also demonstrated that a few residual CC cells were still associated with early embryos and that these cells showed rather degenerative or apoptotic patterns, the latter pattern also observed on cells associated with fertilized eggs. SEM revealed that the complex of CC cells of immature oocytes was compact with narrow intercellular spaces, which progressively enlarged in size around mature oocytes. This phenomenon is mostly due to the production of abundant extracellular matrix. Immature CC cell complexes possessed characteristic long and filiform microvilli whereas the surface of CC cells surrounding mature oocytes showed numerous blebs and occasional large cytoplasmic protrusions as well as microvilli. Zygotes and early embryos were covered with a few polyhedral CC cells possessing scarce and short microvilli and a large amount of pleomorphic blebs. This study demonstrated a precocious luteinization occurring in bovine CC cells at ovulation until zygote segmentation, and this process was associated with a progressive apoptotic mechanism that ended in the complete denudation of the zona pellucida covering the early embryo. The presence of CC cells around the maturing oocyte and fertilized egg could have important functions related to the microenvironmental requirements of ovum maturation as well as facilitating activities related to fertilization.

Histology of the exocrine pancreas.

The morphology of the exocrine secretory unit of the pancreas, i.e. the pancreatic acinus, is reviewed. The histological features of the acini and their relation with the duct system are described. The acinar three-dimensional architecture was studied by means of different ultrastructural techniques, some of which are complementary. The fine structure and morphodynamics of the acinar cells are also described. In addition, the location of the organelles in specific cytoplasmic domains and their close morphofunctional relationship with the sequential stages of secretion of the digestive enzymes are specially emphasized. Finally, morphological approaches are suggested to achieve a better comprehension of the physiological and pathological pancreatic activities whose morphodynamics need to be further elucidated or are almost totally unknown.