Comparison of three methods of DNA extraction from peripheral blood mononuclear cells and lung fragments of equines.

We compared three different protocols for DNA extraction from horse peripheral blood mononuclear cells (PBMC) and lung fragments, determining average final DNA concentration, purity, percentage of PCR amplification using beta-actin, and cost. Thirty-four samples from PBMC, and 33 samples from lung fragments were submitted to DNA extraction by three different protocols. Protocol A consisted of a phenol-chloroform and isoamylic alcohol extraction, Protocol B used alkaline extraction with NaOH, and Protocol C used the DNAzol((R)) reagent kit. Protocol A was the best option for DNA extraction from lung fragments, producing high DNA concentrations, with high sensitivity in PCR amplification (100%), followed by Protocols C and B. On the other hand, for PBMC samples, Protocol B gave the highest sensitivity in PCR amplification (100%), followed by Protocols C and A. We conclude that Protocol A should be used for PCR diagnosis from lung fragment samples, while Protocol B should be used for PBMC.

A new morpho-functional classification of the Fallopian tube based on its three-dimensional myoarchitecture.

The recent direct observations, under scanning electron microscopy (SEM), of the three-dimensional architecture of myosalpinx in different mammals allows us classify salpinxes according to the myoarchitecture of their tubo-uterine junction (TUJ) and isthmus segments. Based upon the myoarchitecture of the outer wall of the TUJ we could find barrier-like species (rat and sow), sphincter-like species type a (rabbit and ewe) and sphincter-like species type b (cow and woman). The different architecture of TUJ can be explained by the different nature of the mating process. Based upon the myoarchitecture of the isthmus we could distinguish type 1 (rat) and type 2 (rabbit, ewe, sow, cow and woman) salpinxes. In the latter the close fusion of musculature deriving from the meso (extrinsic musculature) with the musculature of salpinx (intrinsic musculature) suggests the existence of a unique mesosalpinx contractile system. The myosalpinx is mostly made up of a single network of muscular fibers. Such a plexiform structure, owing to the uneven distribution of fibers, rather than producing a series of regular contraction waves, is more likely to generate random contraction waves. The random propagation of muscular network contraction may deform the plexiform wall of the myosalpinx causing the stirring of tubal contents. By such a stirring movement the contact between hormones and nutrients and the eggs or embryos is intensified, thus favoring a correct fertilization and early embryo development. Taken all together, these systematic results probably suggest an additional and rather new function for the musculature of the tube, namely to increase fertility in a large number of species.

A three-dimensional study of human fetal endocervix with special reference to its epithelium.

The development of human fetal cervix has been systematically studied by SEM, obtaining a detailed map of its fine structure, particularly concerning the differentiation and maturation of the endocervical epithelium, including its "eversion" and "squamous metaplasia", normally occurring in postnatal life, but not yet observed in detail by electron microscopy in the fetus. Cervices from spontaneous abortion at 12, 15, 18, 20, 21 and 22 weeks and from intrauterine fetal death (hydrocephalus) at 31 weeks of development have been examined. At 12-15 weeks, as the canalization of the cervix proceeded, the endocervical epithelium consisted of high polyhedral cells, with regularly flattened or concave apices exhibiting scarce microvilli and often single primary cilia. Some narrow intercellular infoldings probably corresponded to primordial tubular glands. At the 18th week the epithelium was made up of a mosaic of flat or slightly raised polygonal cells, whose apical surface showed thin microplicae. At the 20th week a pseudostratified epithelium with many apically convex cells lined the cervical canal and the tubular glands. At 21 and 22 weeks "plicae palmatae" developed, covered by cells, often showing a smooth central area surrounded by microvilli, provided with a primary cilium and swollen by secretory material. This also formed rounded masses on the epithelium. In the lower part of the endocervix some very elongated cells showed short microplicae resulting from fusion of microvilli. At the 31st week secretion increased and its products spreading from the bottom of the glands contacted isolated ciliated cells at their openings and diffusely covered the surface epithelium. Most of the ectocervix exhibited squamous elements, with well-developed labyrinthine microplicae. These cells could overlap each other and also desquamate. The zone of the portio vaginalis around the os of the cervical canal appeared infolded and hypertrophic. Here, an indented squamo-columnar junction between the ectocervical and endocervical epithelium, caused by tongue-like prolongations of squamous epithelium directed toward the endocervix, was found. Their tips consisted of elongated cells, rich only in short microvilli. Our data indicate that the features of the microvillous cells are an expression of a hormone-dependent differentiative process. Thus, their secretion might be stimulated by progesterone. Similarly microplicae on the ectocervical epithelium (a sign of squamous maturation) might be promoted by estrogens. Furthermore, two aspects were significative: 1) the finding--in an early phase only (18th week)--of endocervically-located squamous cells, although devoid of microplicae; and 2) the occurrence--in the latest phase (31st week)--of an indented squamo-columnar junction on the surface of the portio. These features are in agreement with the caudal shift of the squamo-columnar junction near the uterine cavity to the ectocervix after cervico-vaginal demarcation; the squamous metaplasia of this everted endocervical epithelium has been reported by some authors. It is likely that these processes, occurring in fetal life as well as in pregnant women, are related to a common hormonal background, arising from the mother to her fetus.

The three-dimensional architecture of the myosalpinx in the rat (Rattus norvegicus) as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of myosalpinx in the rat has been investigated by means of scanning electron microscopy after microdissection and removing interstitial connective tissue with 6N NaOH digestion. In the extramural portion of tube-uterine junction the myosalpinx shows circularly arranged fibers originating from the uterus, together with oblique fibers typical for the salpinx, which occur more frequently in the deeper layers. As fibers approach the mucous folds they assume a plexiform arrangement, which is maintained through all tubal segments. In the isthmus surface fibers form wide muscle rings around the elbow of loops, peculiar to the rat tubal morphology. Surface fibers in the ampulla and pre-ampulla have an even circular course. Our 3-D results reveal that the muscular architecture of rat tube is mainly organized in concentric, monolayered shells with a plexiform arrangement tightly fastened together. Functionally, this muscular arrangement seems to be capable of stirring rather than pushing the embryo and gametes. Finally, such a plexiform network might work as a mechanism of "tube locking" in proximity of isthmic loops as well as at the level of the ampullary-isthmic junction.

Effects of captopril on the development of rat doxorubicin nephropathy.

The effects of a daily administration of an anti-converting enzyme inhibitor. Captopril (CPT) (100 mg/kg/orally), on the development of functional and morphological alterations induced in rats by a single injection (7.5 mg/kg/iv) of Doxorubicin (DXR) (Adriamycin*), were investigated. Twenty-four-hour protein excretion, urine output, food intake, water intake, and body weight gain were measured weekly for 30 days. Transmission and scanning electron microscopy observations were performed on kidney samples after 30 days. Four groups were studied. Group 1 were control rats. Group 2 were rats injected with DXR. Group 3 were rats injected with DXR and treated with CPT for 30 days. Group 4 were rats injected with DXR and treated with CPT for 15 days (CPT treatment started 15 days after DXR injection). Group 1 did not show significant functional or morphological changes. Group 2 showed severe proteinuria, significant increase in urinary volume within 2 weeks, significant body weight reduction and diffuse morphological changes. These changes mainly consisted of podocyte swelling, severe foot process fusion, and presence of casts within tubular lumen. Group 3, with respect to group 2, showed a significant reduction of the 24 h protein excretion and urine output. This group displayed morphological changes similar to those observed in group 2, but with a focal distribution. Group 4 showed functional and morphological changes comparable with those of group 2. It is concluded that CPT partially inhibits the development of the functional and morphological damage induced by DXR in the rat kidney. However, CPT did not influence the natural development of nephropathy when treatment started 15 days after DXR injection.

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.

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.

A technique for exposure of the glycoproteic matrix (zona pellucida and mucus) for scanning electron microscopy.

The fine structure of the zona pellucida (ZP) covering the oocyte and of the mucus covering the surface of the intestinal villi was investigated by using a new method employing ruthenium red (RR), saponin, and osmium-thiocarbohydrazide impregnation. The glycoproteic matrices both appeared constituted by thin filaments (ranging from 22 to 50 nm in thickness) anastomosed to form a very fine network. RR prevented the dissolution and/or alteration of glycoproteins and polyanionic carbohydrates induced by acqueous fixatives. Saponin was a detergent of the soluble proteins. Osmium-thiocarbohydrazide preserved the glycoproteic matrix filaments from the mechanical stress induced by dehydration and critical point drying and reduced filaments packing and shrinkage. The technical improvement was demonstrated by the following results: 1) a regular arrangement of the filaments network; 2) a thickness of mucus filaments smaller than that obtained with other methods of preparation; 3) a homogeneous thickness of ZP filaments. This method allowed a very detailed study of the fine structural organization of the ZP and intestinal mucus. Therefore, this technique can be useful for a better evaluation of the morphodynamic of these and other glycoproteic matrices.

The collagen skeleton of the human umbilical cord at term. A scanning electron microscopy study after 2N-NaOH maceration.

The organization of the collagen fibrils in the human umbilical cord at term is directly visualized by means of a scanning electron microscopy cell maceration method. This technique clearly reveals that there is a much more extensive collagen fibrillar architecture within the umbilical cord than that reported in the classical histological descriptions. The Wharton's jelly, in fact, appears as a spongy network of interlacing collagen fibres and small woven bundles apparently arranged at random and forming a continuous soft skeleton that encases the umbilical vessels. The collagen fibrillar network shows the presence of a wide system of interconnected cavities consisting of canalicular-like structures as well as cavernous and perivascular spaces. This system of cavities might play a mechanical role allowing the storing of the ground substance of the jelly and its diffusion during twisting or compression. Furthermore, it may have an important role facilitating the diffusion throughout the jelly of diffused materials (i.e. water and trophic metabolites) either from or to the umbilical vessels and the amniotic cavity, thus overcoming the lace of a proper vasculature with the jelly.

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.

Focal cell contacts detected by ruthenium red, triton X100 and saponin in the granulosa cells of mouse ovary.

Granulosa cells in growing follicles of mouse ovary, observed after treatment with ruthenium red (RR) as described by Luft (1971a, b), appeared to be covered by a continuous well-defined layer. On the contrary, treating granulosa cells with 1% Triton X100 (Vaccaro and Brody, 1981), followed by RR staining, resulted in the complete extraction of the plasma membrane coat (Triton does not affect the basement membrane and extracellular matrix proteoglycans). The use of 0.02% saponin together, with the RR stain, or 0.1% Triton X100 followed by RR staining, allows good visualization of follicular basement membrane and extracellular matrix proteoglycans without destroying cell morphology. Using this technique, we observed the extraction of the plasma membrane coat, but focal RR-stained condensations that were unaffected by saponin or 0.1% Triton X100 treatment were observed between plasma membranes of granulosa cells located around the periphery of large Graafian follicles. In some cases, RR condensations were located at the apex of plasmalemmal evaginations, in proximity to adjacent granulosa cells. Focal condensations of RR stain were never observed in secondary follicles. Present evidence suggests that focal cell contacts are mediated by transmembrane intercalated glycoproteins or proteoglycans and consequently play a role in cell adhesion. Their presence among granulosa cells of only very large Graafian follicles may be related to the maturation process of granulosa cells.

Elimination of germ cells during differentiation of the human ovary: an electron microscopic study.

Observations by scanning and transmission electron microscopy during ovarian development (from 7 wk until term of gestation) showed numerous germ cells within the superficial epithelial layer covering the ovary. In early stages of differentiation (between 7 and 11 wk), germ cells appeared to be actively migrating to the surface of the ovary by ameboid-like movements. Later, the transfer of the germ cells to the surface epithelium was also occurring passively, as a consequence of an intense proliferation of germ and somatic cells - arranged in clusters and cords - migrating towards the most superficial areas of the ovary. The present evidence suggests that a number of primordial germ cells - as well as oogonia and oocytes - which lie in the superficial epithelium can leave the ovary and ultimately rest freely upon its surface. Elimination of germ cells during differentiation of the ovary in humans was always paralleled by necrosis of oogonia and oocytes and atretic alteration of primitive follicles. The significance of these processes is discussed in relation to the reduction of the pool of oocytes at birth.

Natural history of the female germ cell from its origin to full maturation through prenatal ovarian development.

This paper contains a number of sketches concerning the main morphological ultrastructural features of the human female germ cell during the prenatal period. The morphodynamic outline of primordial germ cells has been traced, both in their extraembryonic site of origin and during their migration towards the developing ovary. After gonadal settlement, the intraovarian differentiation of the germ cells into primary oocytes through the stage of oogonia, as well as the dramatic fall in the number of germ cells before birth, is described. The presence of morphofunctionally relevant interactions between the differentiating female gamete and the surrounding somatic microenvironment has also been evaluated and discussed.

The mucosa of the rabbit vagina: a proposed experimental model for correlated morphofunctional studies in humans.

In order to suggest a valid experimental model for correlated studies regarding the complex phenomena of reproductive biology in humans, light (LM), transmission (TEM) and scanning electron microscopic (SEM) observations were carried out on vaginae of sexually mature New-Zealand albino rabbits, both in estrus and at 5 h after mating. To avoid artifacts depending upon the operative times for specimen removal and, in particular, to well visualize the inner surface of this organ, vaginal irrigation with fixative was performed prior to the surgical removal. Our data demonstrate that the upper 2/3 of the vaginal duct form a kind of 'cervico-vaginal canal' which shares many histological features with the human endocervix. In fact, this portion is lined within by a monostratified epithelium provided with tall microvillous and ciliated cells and often invaginated in crypts. After mating, the microvillous cells showed evidence of secretory activity, exhibiting dome-like luminal surfaces. Secretory droplets often contacted cilia and spermatozoa present in the mucosal furrows and crypts. Because of these singular features and the morphofunctional interactions between vaginal epithelium and male gametes, we believe the rabbit to be a helpful model for comparative biology in humans, concerning sperm capacitation and the general reactivity of the female genital tract during the reproductive cycle.

The architecture of the myosalpinx in the sow as revealed by scanning electron microscopy.

OBJECTIVE: To provide a definitive settlement of data on the architecture of myosalpinx in the sow in consideration of controversial data existing in literature. STUDY-DESIGN: To allow direct visualization of muscular architecture, segments of tube from fifteen sows were investigated by means of scanning electron microscopy after the removal of interstitial connective tissue with NAOH digestion. RESULTS: In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is mainly constituted by oblique bundles of variable length, which run around the tube and merge into the surrounding musculature, giving origin to a plexiform arrangement. In the ampulla the fibers join in short bundles variously oriented. CONCLUSION: The three-dimensional architecture of the sow myosalpinx consists of muscular bundles independent of one another which follow multiple spatial arrangements and form a complex network. Such a muscular structure is likely more suitable for stirring rather than pushing the embryo and gametes.

The extracellular matrix of healthy and atretic mouse ovarian follicles studied by lanthanum nitrate and polycation ruthenium red.

Changes occurring in the fine structure of Zona pellucida and follicular fluid in growing and atretic follicles in mice were examined by electron microscopy using lanthanum nitrate and ruthenium red. The results showed that follicular fluid has a dense globular aspect in growing follicles but becomes more dispersed and globular-fibrillar in atretic follicles. It is formed by rounded negatively charged particles (20 nm in diameter) that are closely packed in growing follicles, but spaced and united by filaments in atretic follicles. These changes may be related to a changed role of granulosa cells and also to an altered passage of nutritive substances from the plasma into the follicle. The Zona pellucida is intensely colorable only in the atretic follicles, where it is composed of a dense globular fibrous matrix, but not in growing follicles, where it reveals a weak reaction with ruthenium red. While in the Zona pellucida of growing follicles the globular negatively charged particles (20 nm in diameter) probably due to sialic acid moieties are rarely evident, these particles become highly numerous and connected by microfilamentous structures in atretic follicles. The altered electrical charge of polyanions may reflect a different chemical permeability of the Zona pellucida. Then the increase of negative charged particles in the zona may have a role in a process of sequestration of the oocyte during atreasia.

The three-dimensional architecture of the myosalpinx in the rabbit as revealed by scanning electron microscopy.

The three-dimensional (3-D) architecture of the myosalpinx in the rabbit has been investigated by means of scanning electron microscopy after microdissection and the removal of interstitial connective tissue with KOH digestion. In the extramural portion of the tubo-uterine junction, the myosalpinx presents outer longitudinal bundles which form a well-defined continuous muscular layer extending towards the ampulla. Underlying this layer there are single muscular bundles which follow an uneven circular arrangement. At a deeper level the same bundles present a plexiform arrangement. Several bundles reach the base of the mucous folds and follow a discontinuous, approximately longitudinal arrangement. In the isthmus, the myosalpinx shows superficial longitudinal strips that abruptly bend sideways as they approach the ampulla, thereby enveloping the underlying muscular structure. The latter is arranged in the same way as at the extramural portion of the tubo-uterine junction, except for the lack of the outer longitudinal layer originating from the uterus. Our 3-D results revealed that the architecture of the rabbit myosalpinx consists of muscular bundles independent of one another that follow multiple spatial arrangements and form a complex network. Such a type of structure seems to be capable of stirring rather than pushing the embryo and gametes.

The three-dimensional architecture of the myosalpinx in the cow as revealed by scanning electron microscopy.

The three-dimensional architecture of the myosalpinx in the cow has been investigated by means of scanning electron microscopy after removal of interstitial connective tissue with NaOH digestion. In the extramural portion of the tubo-uterine junction, in the isthmus and ampulla, the myosalpinx is made up of oblique bundles of variable orientation and length, which are loosely distributed in the tubo-uterine junction and densely packed in both isthmus and ampulla. These bundles intersect and merge into the surrounding musculature. Our observations demonstrate how myosalpinx consists mainly of bundles of muscular fibers independent one of another, which show a multiple spatial arrangement and form a complex network. Such a muscular architecture is likely more suitable for stirring rather than pushing the embryos and gametes through the tube.

Ovarian microvasculature in normal and hCG stimulated rabbits. A study of vascular corrosion casts with particular regard to the interstitium.

The microvasculature of rabbit ovaries, with special regard to the interstitial-stromal tissue, was studied by scanning electron microscopy (SEM) of vascular corrosion casts. The casting medium (Mercox) was injected in normal animals and in animals in which ovulation was induced by 100 I.U. of human chorionic gonadotropin (hCG) i.v. Vascular baskets of different size and architecture related to follicles in various developmental stages were observed in the ovarian cortex. Small (primary) follicles showed thin and thready capillaries. Larger (secondary and antral) follicles showed a progressive increase in number, size and tortuosity of round-meshed capillaries, related to adaptation of the growing follicle to the approaching ovulation. Capillary sprouts, due to the enhanced angiogenesis of growing follicles, were seen. These aspects were more evident in ovulatory follicles. In addition, numerous resin leakages, due to the increased permeability of the sinusoidal net, were seen in the cavities of ovulatory follicles. Interstitial-stromal tissue capillaries were diffusely distributed in the cortex among the follicular baskets. Their morphology remained unchanged after hCG stimulus. In the periphery of the cortex, the microvascular net showed large (70-90 microns) irregularly rounded meshes, with thin, thready capillaries often anastomosed with those of primary follicles. Inner cortex capillaries were thin, thready and arranged in polygonal meshes of 40-70 microns. The arrangement and the distribution of the interstitial-stromal capillaries may have some special role during the cyclic activity of the ovary.(ABSTRACT TRUNCATED AT 250 WORDS)