Isolation, proliferation and characterization of endometrial canine stem cells.

In the last decade, progenitor cells isolated from dissociated endometrial tissue have been the subject of many studies in several animal species. Recently, endometrial cells showing characteristics of mesenchymal stem cells (MSC) have been demonstrated in human, pig and cow uterine tissue samples. The aim of this study was the isolation and characterization of stromal cells from the endometrium of healthy bitches, a tissue that after elective surgery is routinely discarded. Multipotent stromal cells could be isolated from all bitches enrolled in the study (n = 7). The multipotency of cells was demonstrated by their capacity to differentiate into adipocytic, osteocytic and chondrocytic lineages. Clonogenicity and cell proliferation ability were also tested. Furthermore, gene expression analysis by RT-PCR was used to compare the expression of a set of genes (CD44, CD29, CD34, CD45, CD90, CD13, CD133, CD73, CD31 CD105, Oct4) with adipose tissue-derived MSC. Stromal cells isolated from uterine endometrium showed similar morphology, ability of subculture and plasticity, and also expressed a panel of genes comparable with adipose tissue-derived MSC. These data suggest that endometrial stromal cells fulfil the basic criteria proposed by the "Mesenchymal and Tissue Stem Cell Committee of the International Society for Cellular Therapy" for the identification of mesenchymal stem cells. Although endometrial mesenchymal stem cells (EnMSC) showed a lower replicative ability in comparison with adipose tissue-derived MSC, they could be considered a cell therapeutic agent alternative to adipose tissue or bone marrow-derived MSC in dog.

Use of contrast-enhanced ultrasonography in chronic pathologic canine testes.

Contrast-enhanced ultrasound with sulphur hexafluoride microbubbles was performed in seven healthy dogs without a history of reproductive pathology and with histologically confirmed normal testes and in 42 dogs with chronic scrotal anomalies. All dogs underwent orchiectomy and histological examination. Enhancement patterns and perfusion parameters (peak intensity and regional blood flow) of testes of healthy dogs and testes with chronic lesions were compared. Fourteen non-pathologic and 60 pathologic testes were considered. Forty testes were neoplastic (24 interstitial cell tumours, 9 seminomas, 7 Sertoli cell tumours), 20 were non-neoplastic (16 testicular degenerations, 2 chronic orchitis, 1 testicular atrophy, 1 interstitial cell hyperplasia). In healthy dogs, the contrast medium flow had a rapid homogeneous wash-in and wash-out, with a short peak phase. With contrast ultrasound, testes that were inhomogeneous with a hyperenhancing pattern were associated with neoplasia (sensitivity: 87.5%, specificity: 100%). Lesions with persistent inner vessels and a hypo-to-isoechoic background were significantly associated with seminomas (sensitivity: 77.8%, specificity: 100%). Testes with non-neoplastic lesions were characterized by a scant/moderate homogeneous enhancement. Perfusion parameters were higher in neoplastic lesions. Contrast ultrasound was a feasible diagnostic tool in the assessment of testicular lesions, with hyperenhancement being an important feature in the diagnosis of malignancy.

Multiple symbiosis in the leafhopper Scaphoideus titanus (Hemiptera: Cicadellidae): details of transovarial transmission of Cardinium sp. and yeast-like endosymbionts.

Scaphoideus titanus is the insect vector of flavescence dorée (FD), a yellow disease of grapevines. Observations on adult females and nymphs of S. titanus showed that this insect is associated with a complex microbial community. Ultrastructural analysis showed that the fat body, salivary glands and ovary of the insect harbour microorganisms showing the brush-like structure typically observed in the genus Cardinium. In particular, it has been shown that these symbiotic bacteria are present both in the follicular cells and in the eggs. In addition, cells resembling bacteriocytes, harbouring numerous Cardinium symbionts in the cytoplasm, were observed in the apical portion of the ovary in adult females. These cells are likely responsible for bacterial transmission to the ovary. Optical microscopy showed that the fat body harbours an enormous population of yeast-like symbionts (YLSs). Ultrastructural observations showed that these symbionts are enclosed within specialized cells of the fat body and are also present in the ovary, where they are found in both the follicular cells and the eggs. There is thus evidence that both Cardinium and the YLSs are transovarially transmitted to the offspring. To our knowledge, S. titanus is the sole insect known to transmit two different kinds of symbionts to the eggs, a prokaryote and an eukaryote. Gene sequence analysis and in situ hybridization led to the identification of YLSs as members of the class Sordariomycetes (=Pyrenomycetes). Finally, ultrastructural observation of the midgut content revealed the presence, in both adult females and nymphs, of a complex microbial community, which include a phytoplasma-like microorganism, likely the agent of FD.

Ultrastructure of a novel Cardinium sp. symbiont in Scaphoideus titanus (Hemiptera: Cicadellidae).

An ultrastructural study of the novel symbiont Cardinium sp. was performed with particular attention to the description of the structure and organization of highly elaborated cytoplasmic complexes containing microtubule-like elements (MLC). Three major components were observed. The first was a system of microtubule-like elements (ML) arranged in parallel array extending from the plasma membrane into the cytosol of the bacterium. The second, an fibrous electrondense plaque (FEP), approximately 8 nm thick, located 7.5 nm away from the plasma membrane and parallel to it. The third component, not previously reported, was described for the first time in this paper. This consisted of a set of regularly distributed 8 nm electron-dense structures (ES), with a center-to-center spacing of about 12 nm, adhering to the outer leaflet of the plasma membrane. Often, the ES created a close connection between the plasma membrane and the outer membrane, so that in this area they became straight and stiff. The first and second component of these structures are compared to previously described microtubules and microfilaments.

Cell biology and invasion of the microsporidia.

Microsporidia are amitochondrial eukaryotic obligate intracellular parasites. They are reported to infect every animal group from protists to vertebrates, including humans. Microsporidia are of interest as opportunistic pathogens in humans and for certain characteristics which raise questions about their evolution and phylogenetic position. This review describes the basic biology and invasion mechanisms of microsporidian species infecting humans.

Evidence of H beta 58, a gene involved in mammalian placental development, in the three-toed skink, Chalcides chalcides (Squamata: Scincidae), a viviparous placentotrophic reptile.

The H beta 58 gene, whose disruption in mice causes reabsorption of the embryo at 9.5 days post-conception, is believed to be essential for development of the placenta. Although the H beta 58 gene is well conserved in some Amniota, nothing is known about its presence in reptiles, some species of which have developed a chorioallantoic placenta. In this work, we investigated the expression of H beta 58 mRNA and protein in the three-toed skink, Chalcides chalcides. H beta 58 protein expression was found in the uterine epithelium beginning from the peri-ovulatory stage. However, it increased strongly at the moment of placental formation, when a high level of expression of mRNA and protein was also observed in the extra-embryonic membranes. The expression of H beta 58 mRNA and protein was maintained, although to a lesser degree, in the placenta during late pregnancy. It was also present in the early embryo. Finally, cloning and sequencing of a gene fragment revealed strong homology of the reptile gene with that of mammals. The high degree of conservation of the gene in amniote vertebrates and its presence in a viviparous squamate reptile (as in mammals) indicates an important role of this gene in the chorioallantoic placenta formation and development.

Sperm morphology of salamandrids (Amphibia, Urodela): implications for phylogeny and fertilization biology.

Mature spermatozoa belonging to four salamander species, Salamandrina terdigitata, Triturus alpestris, Triturus carnifex and Triturus vulgaris, have been investigated by electron microscopy. The sperm ultrastructure of these species was compared with that of previously examined urodeles (36 species and 20 genera) and with that of anurans and caecilians. Many phylogenetic considerations may be inferred as a consequence of comparative spermatology. Urodela appears to be a monophyletic order characterized by three sperm synapomorphies: the acrosomal barb, nuclear ridge and marginal filament. Cryptobranchoidea are confirmed to form a monophyletic suborder having two synapomorphic characters: absence of mitochondria in the tail, and cylindrical shape of the tail axial rod. Within the family Salamandridae, sperm morphology confirms the phylogenetic distance between Salamandrina and Triturus, as already pointed out on the basis of molecular and morphological characters. The very complex ultrastructure of spermatozoa confirms a previous opinion that internal fertilization is the ancestral condition of the Amphibia.

Intercellular junctions in myriapods.

Tissue from the intestinal tract of myriapods, including millipedes, centipedes and pauropods were examined in tracer-impregnated sections and freeze-fracture replicas. The foregut and hindgut of all three classes exhibit pleated septate junctions; these display undulating intercellular ribbons in thin sections. In replicas they show discrete intramembranous particle (IMP) arrays aligned in rows in parallel; with one another. The tissues of the hindgut also possess scalariform junctions, characterized by cross-striated intercellular clefts in sections and IMP-enriched membranes in replicas. Gap junctions occur in all groups, but they are atypical in replicas in that their component IMPs do not always fracture onto the E face, as is characteristic of other arthropods; some IMPs cleave to the P face and others to the E face. The midgut of these organisms exhibits smooth septate junctions with conventional straight septal ribbons and occasional interseptal columns. However the intramembranous appearance in replicas is variable, particularly in centipedes, in that the rows of IMPs in chemically-unfixed propanecryofixed tissues, are prominent and adhere preferentially to the E face, with complementary P face grooves, while in fixed tissues the IMPs are much less distinct and fracture to either P face or E face. They tend not to protrude far beyond the mid-plane of the membrane bilayer and lie in rows which commonly take on the form of a network. Individual rows of the network sometimes curve to run beside a second row, over a short distance, before bending away into another part of the network. The aligned particle rows, which are much more prominent in millipedes, where they frequently lie in close parallel appositions, do not fuse into ridges as often occurs in insect tissues. The myriapod junctions, therefore, are of the same general kind as are found in the gut tract of other arthropod groups, but differ with respect to the subtleties of their intramembranous organization and disposition.

A symbiont of the tick Ixodes ricinus invades and consumes mitochondria in a mode similar to that of the parasitic bacterium Bdellovibrio bacteriovorus.

We have recently performed molecular characterisation of an intracellular alpha-proteobacterium, named IricES1, which resides in the ovarian tissue of female Ixodes ricinus ticks from Italy. A unique characteristic of this bacterium is its ability to invade the mitochondria of the cells in which it resides. Although some ultrastructural studies have been performed on close relatives of this bacterium from I. ricinus in England and Switzerland, a number of questions remain about its movement within ovarian tissues and mitochondria. We have performed the first detailed ultrastructural examination of IricES1 in engorged female adult I. ricinus. Among our findings was that the bacterium enters mitochondria in a similar way to that employed by the 'predatory' bacterium Bdellovibro bacteriovorus, that is, between the inner and outer membranes. It then appears to multiply, with the new 'colony' consuming the mitochondrial matrix. Despite having many of their mitochondria consumed, oocytes appear to develop normally, and the bacteria are likely to be vertically transferred to all eggs.

Some aspects of intracellular symbiosis during embryo development of Mastotermes darwiniensis (Isoptera: Mastotermitidae).

All examined species of cockroaches have been shown to harbour intracellular bacteria in specialized cells (bacteriocytes) of the fat body. In termites, bacteria in specialized cells have been observed only in Mastotermes darwiniensis (Isoptera: Mastotermitidae). All of these bacteria have been assigned to the same eubacterial lineage, with the bacteria of M. darwiniensis as the sister group to the cockroach bacteria. While the main steps of the life cycle of cockroach bacteria have been described, little is known about the bacteria of M. darwiniensis. More specifically, no data are available on their behaviour during the development of this termite. Using both optical and electron microscopy methods, we examined embryos of M. darwiniensis at different developmental stages. Our results show that the integration of bacteria during the development of M. darwiniensis is implemented in the same way as in cockroaches. In particular, we observed the aggregation of a large amount of bacteria in a single mass in the yolk sac, with vitellophage-associated bacterial lysis. In cockroaches, a similar process has been described in detail for Periplaneta americana (Blattaria: Blattidae), where the bacterial mass is referred to as the transitory mycetome. The formation of a transitory mycetome could thus be regarded as an ancestral condition for cockroaches and termites.

Early diagnosis of pregnancy in sows by ultrasound evaluation of embryo development and uterine echotexture.

Two operators attempted to detect pregnancy ultrasonographically in 196 sows daily from 15 to 25 days after insemination; 20 unbred sows were also investigated. The probe was applied transcutaneously on the right abdominal wall near the last three mammary glands. During each examination, the embryos were visualised and their transverse and longitudinal dimensions were measured. Pregnancy was confirmed by an ultrasonographic detection of embryos five days after the first ultrasound diagnosis and finally 30 to 32 days after insemination. The accuracy of diagnosis was less than 83 per cent on days 15, 16 and 17 but improved to more than 90 per cent from day 18 onwards. The uterine echotexture was studied in seven sows at oestrus and 15, 17, 19, 21, 23 and 25 days after insemination. The echotexture was more homogeneous from days 15 to 25 after insemination than at oestrus.

Complete uterine prolapse without uterine mucosal eversion in a queen.

A five-year-old female cat weighing 3 kg was presented by the owner after noticing a large pink, bilobed mass protruding through the vulva during labour. The cat was in good condition, with appropriate lactation, and the newborn kittens were nursing normally. The uterus was not reverted or invaginated at examination, and there was rupture of the mesovarium, mesometrium and uterine-vaginal connection around the cervix. Manual reduction of the prolapsed uterus was not possible because of torn ligaments. A coeliotomy was performed to remove the ovaries, and the apex of the uterine horns was passed by the vaginal route. The remaining part of the mesometrium was disconnected, and the prolapsed uterus was removed. The queen and kittens were discharged from the hospital on the second day after surgery. An unusual feature of this case is that the prolapse was complete, without eversion of any part of the uterus through a vaginal tear.

Fine structure of the female reproductive system in a viviparous insect, Glossina morsitans morsitans (Diptera, Glossinidae).

The female reproductive system of the tsetse fly Glossina morsitans morsitans is analysed by scanning electron microscopy (SEM). The study focuses in particular on the choriothete, a peculiar uterine structure involved in the viviparous mode of reproduction of Glossina morsitans morsitans. Under light microscopy, the choriothete appears formed by numerous tongue-like folds projecting towards the uterine lumen and lined by a thin cuticle. SEM analysis highlights for the first time a distinctive new feature that is not visible by traditional histological methods. That is a cuticular covering of the choriothete, which shows numerous thorns in the form of crest-like structures arranged in nearly parallel lines. The role of the choriothete in pregnancy and in larval nourishment is discussed.

Relationship between body fat and body condition score and their effects on estrous cycles of the Standardbred maiden mare.

Standardbred maiden mares are generally stressed and in poor physical condition because of their incorrect management at the end of their racing careers. The purpose of this study was to identify an objective assessment that, similar to body condition score (BCS) determination, is easy to measure and able to confirm or improve fattening status assessment, as well as to ascertain whether a relationship with reproduction efficiency exists in subjects destined for a first-time insemination program. The authors assessed 29 Standardbred maiden mares (7 +/- 2 years old) during the breeding season. On January 15 (day 0), the same operator performed the following on all subjects: the first gynecological and ultrasound examination, a BCS assessment (range 0 to 5), and an adiposity objective assessment (i.e., measurement of fat thickness by ultrasound scan). At day 0, all mares were in seasonal anestrous. On ovulation day, all subjects exited the study. Both techniques were shown to be significantly and reciprocally correlated (r = 0.976; P < 0.01) to the first seasonal ovulation in maiden mares (-0.772 and -0.805, respectively, for fat thickness and BCS; P < 0.01). Based on the results obtained, regression equations for the prediction of days to the first seasonal ovulation (y) were created. The best predictive equation was the following: y = 26.714x(3) - 202.44x(2) + 446.04x - 195.65 (R(2) = 0.783; SE = 17 d; P0.01), with an independent BCS variable. In conclusion, this study suggests that increasing the plane of nutrition (i.e., flushing), starting approximately 3 weeks before the breeding season, stimulates ovarian activity in stressed maiden mares.

Ultrastructural details of Sertoli cell junctional complexes in vivo and their modifications in tissue culture.

This study was carried out using thin sections, lanthanum tracing, and freeze-cleaving in order to investigate the Sertoli cell junctions of rat testis in vivo and their maintenance in culture. The presence of a new type of membrane specialization has been revealed. This consists of a close association between tight junctions and nexuses. The Sertoli cell contact specializations show a progressive disorganization in vitro correlated with the duration of the period in culture. The relationship between the morphological changes in Sertoli cell junctions and the lack of spermatogenesis in culture is discussed.

Electron microscopic study of interstitial cells in cryptorchid human testes. I: Interstitial cells in prepubertal age.

In order to establish the ultrastructural features of Leydig cells 50 biopsies or prepubertal human cryptorchid testes have been examined. Light and electron microscopic observations reveal the presence of two types of interstitial cells distinguishable on the basis of the peculiar ultrastructural characteristic of the nucleus and cytoplasm. In our opinion these two types of interstitial cells seem to represent different stages of cell differentiation. There are fibroblast-like cells and cells corresponding to Leydig cells. From our observations it may be inferred that the fibroblast-like cells are the precursor cells of the Leydig cells.

Action of gossypol on rat germinal cells. II. The acrosome.

The action of gossypol on the acrosomal complex in rats has been investigated by microscopical and submicroscopical methods. The drug displays its morphological action only on spermatozoa during the transit through the epididymis, causing malformations and vesiculations. It appears to exert a primary action on the S-S groups formation, disturbing morphological molding occurring in this period and inhibiting capacitation.

The Sertoli cell in lizards.

Sertoli cells of lizards are characterized by variable size, abundant smooth and rough endoplasmic reticulum, multivesicular bodies, lipid vacuoles probably related to the spermatogenic cycle, and mitochondria of normal size. The cytoskeleton contains actin, particularly abundant in the cell periphery, vimentin all around the nucleus and throughout the rest of the cytoplasm. Moreover, microtubules are distributed in the cell periphery. The junctional complexes demonstrate the presence of a very efficient blood-testis barrier, containing tight, gap, tight-gap, septate-like, desmosome-like, and "Sertoli-Sertoli" junctions. In the last, the actin layer interposed between the plasma membrane and the subsurface cistern is absent. The desmosome-like junctions are surrounded by 7-nm filaments and not by intermediate filaments.

In vitro efficacy of nikkomycin Z against the human isolate of the microsporidian species Encephalitozoon hellem.

Since 1985 microsporidia have been recognized as a cause of emerging infections in humans, mainly in immunocompromised human immunodeficiency virus-positive subjects. As chitin is a basic component of the microsporidian infective stage, the spore, we evaluated in vitro the susceptibility of a human-derived strain of Encephalitozoon hellem to nikkomycin Z, a peptide-nucleoside antibiotic known as a competitive inhibitor of chitin synthase enzymes. Transmission electron microscopy showed that this drug, at 25 microgram/ml, reduced the number of parasitic foci by about 35% +/- standard deviation after 7 days of culture (P < 0.0001) and induced cell damage of both mature and immature spores and also other sporogonic and merogonic stages. In particular, an irregular outline of the cell shape and an abnormally condensed cytoplasm in meronts and sporonts were documented. Also, the polar tubule and the polaroplast membranes appeared disarrayed in the sporoblast stage. The spore wall showed an enlarged endospore and delaminated exospore. Mature spores had a complete cytoplasmic disorganization and a swollen and delaminated cell wall. No ultrastructural cell damage was observed in uninfected control cultures treated with the drug.