The effect of growth media and physical treatments on the adhesion properties of canine probiotics.

AIMS: The manufacturing processes have been reported to influence the properties of probiotics with potential impact on health properties. The aim was to investigate the effect of different growth media and inactivation methods on the properties of canine-originated probiotic bacteria alone and in combination mixture. METHODS AND RESULTS: Three established dog probiotics, Lactobacillus fermentum VET9A, Lactobacillus plantarum VET14A and Lactobacillus rhamnosus VET16A, and their combination mixture were evaluated for their adhesion to dog mucus. The effect of different growth media, one reflecting laboratory and the other manufacturing conditions, and inactivation methods (95°C, 80°C and UV irradiation) on the mucus adhesion of the probiotic strains was characterized. Evaluation of dog probiotics was supported by cell visualization using transmission electron microscopy (TEM). Higher adhesion percentage was reported for probiotic strains growing in laboratory rather than in manufacturing conditions (P < 0.05). Inactivation by heat (95°C, 80°C) decreased the adhesion properties when strains were cultivated in soy-based growth media compared with those grown in MRS broth (P < 0.05). TEM observations uncovered differences in cell-surface components in nonviable forms of probiotic strains as compared with their viable forms. CONCLUSIONS: Manufacturing process conditions such as growth media and pretreatment methods may significantly affect the adhesive ability of the tested strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Growth conditions, growth media, pretreatment methods and different probiotic combinations should be carefully considered for quality control of existing probiotics and for identification of new probiotics for dogs. These may also have an impact on health benefits for the host.

Morphometric and ultrastructural analysis of stage-specific effects of Sertoli and spermatogenic cells seen after short-term testosterone treatment in young adult rat testes.

The effects of testosterone treatment on spermatogenesis in the rat have been investigated by morphometric and structural analysis at the ultrastructural level in stages VII-IX. The aim has been to characterize the changes in Sertoli and spermatogenic cells to elucidate the mechanism of testosterone effects on spermatogenesis and to test the possibilities of developing male contraceptives. In stage VII, the morphometric parameters of volume and surface area in Sertoli cells (see abbreviations below): and the morphometric parameter of volume in the spermatogenic cells such as V(VPG,T), V(VPC,T), V(VrPT,T) and V(VelPT,T) decreased. In stage VIII, the respective values of Sertoli cells, VSN, and VSN/VSC decreased while SSJ increased, and the respective morphometric parameters in the spermatogenic cells, V(VPG,T), V(VPC,T), and V(VrPT,T) increased. In stage IX, in Sertoli cells VSC, VSN, VSN/VSC, and SSJ remained unchanged. In spermatogenic cells V(VPG,T), V(VPC,T), and V(VrPT,T) increased. Further, in all stages, a close apposition of mitochondria and rough endoplasmic reticulum in basolateral cytoplasm of Sertoli cells suggested active protein synthesis. In elongated spermatids in stage IX the microtubular manchette became disorganized. This disorganization and the unexpected shift after testosterone treatment from decrease in several morphometric parameters in stage VIII to increases in stage IX cannot be explained by alterations in testosterone (T), LH, FSH, and their respective receptors. Therefore, still unknown regulatory factors in spermatogenesis are apparently involved in the developmental interactions between Sertoli and spermatogenic cells.

Reactions of Leydig cells and blood vessels to lymphoblastic leukemia in the rat testis.

The testis is the third common site of relapse in childhood acute lymphoblastic leukemia (ALL). Despite the apparent clinical importance of testicular relapse, its pathogenesis is still unknown. The studies with an animal model of ALL have shown that many testicular factors are able to control the intratesticular infiltration and proliferation of leukemic lymphoblasts during the untreated course of ALL. In the present study, the ultrastructure of rat testicular interstitium infiltrated by leukemic lymphoblasts was studied in two groups of rats transplanted with rat T cell leukemia in early and late puberty. In both groups most of the leukemic cells infiltrating testicular interstitium were totally or partly enveloped by one or more Leydig cells, and the endothelial cells of capillaries, arterioles and venules were hypertrophic. The Leydig cells of the younger experimental group were by nuclear and cytoplasmic ultrastructure similar to the undifferentiated Leydig cells normally seen on the third postnatal week. The results suggest that Leydig cells bind leukemic lymphoblasts on their surface in vivo as also previously observed in vitro, and that ALL may disturb the pubertal maturation of Leydig cells. The occlusion of arterial and capillary lumina by folds of hypertrophic endothelial cells together with adhered leukemic lymphoblasts may impair the circulation of leukemic testes.

Structural proteins in sexual differentiation of embryonic gonads.

Sexual differentiation of embryonic gonads was studied by immunocytochemical analysis of cytoskeleton, basement membrane and extracellular matrix. The epithelial cells of the prospective gonadal region in both sexes contained vimentin and desmin intermediate filament proteins but not cytokeratin. Basement membrane components laminin, collagen types IV and V, heparan sulfate proteoglycan, and fibronectin were seen in an unorganized form in the extracellular space. The development of the gonads started by proliferation of the pregonadal epithelial cells, which formed separate clusters and loose mesenchyme. In the male gonad the clusters joined together into elongated cords, outlined by basement membrane components. The cord cells became polarized epithelial cells, and cytokeratin appeared with the disappearance of desmin in their cytoplasm. Desmin and vimentin remained in the interstitial cells. In the female gonad the clusters were smaller, and the cords were irregular in shape and size. Desmin disappeared from the cord cells and cytokeratin appeared, but more slowly and less well polarized than in the testis. The present results show that after common early development, the sexual differences in gonads emerge as different organization of the internal epithelial tissue with different timing of changes in intra- and extracellular components.

Differential distribution of laminin chains in the development and sex differentiation of mouse internal genitalia.

The distribution of laminin chains and basement membranes (BMs) in the ontogenesis and sex differentiation of male and female mouse gonads and mesonephros was studied by conventional and immunocytochemical light and electron microscopy. The alpha 1 (synonymous to A) chain was recognized with MAbs against fragment E3, and three chains of laminin with PAbs raised against EHS-laminin. BMs, which formed around the mesonephric duct, the mesonephric tubules, and the paramesonephric duct, contained the laminin alpha 1 chain. The alpha 1 chain appeared with epithelial differentiation in the developing gonads in both sexes. The alpha 1 chain was first evident around the embryonic gonadal cords and remained, after development, in the BMs of the testicular cords and ovarian follicles. The laminin alpha 1 chain was also detected in BMs of the myoid cells around the epithelial rete cords, and transiently in the surface epithelium and in the corpus luteum. Laminin beta-gamma chains were found in many locations where the alpha 1 chain was not detected. These included the mesenchyme of the early mesonephros, the BMs of blood vessels and surface epithelium in the differentiated testis and ovary, between the theca cells in the ovary, and in some corpora lutea. The morphological differentiation of the BMs of the embryonic testicular cords proceeded rapidly. In contrast, the BM of the ovarian cords remained relatively poorly differentiated during the prenatal phases, and developed concomitantly with the differentiation of the follicles. The results show that BMs in the differentiating internal genitalia are heterogeneous with respect to their laminin chains, and suggest that all known laminin chains must be analyzed in the differentiation of gonadal epithelia for a complete role of the BMs in gonadal sex differentiation.

Immunocytochemical localization of androgen-binding protein in the male rat reproductive tract.

The localization of androgen-binding protein (ABP) in the reproductive tract of young adult male rats was studied with the peroxidase-antiperoxidase technique using frozen sections and light microscopy. Within the seminiferous tubules, a positive reaction was noted in the apical portion of the epithelium, apparently in spermatids and/or Sertoli cells. ABP was localized in granules in the apical cytoplasm of the principal epithelial cells of the proximal part of the caput epididymis and in the epithelial cells of the ductuli efferentes. The cells in the distal part of the caput as well as the corpus and cauda of the epididymis did not contain ABP. Numerous coated vesicles and multivesicular bodies were present in the supranuclear cytoplasm of the epididymal epithelium where ABP was taken up. The results indicate that ABP is taken up from the lumen by epithelial cells of the ductuli efferentes and proximal part of the caput epididymis.

Lanthanide chelates as new fluorochrome labels for cytochemistry.

Anti-rabbit IgG labeled with a new fluorescent europium chelate was used to localize rabbit IgG to human smooth muscle myosin in a histological section. The antibody labeled with the europium chelate could be viewed with a conventional fluorescence microscope with a steady-state light source. This result encourages the development of a time-resolved fluorescence microscope, because a significant improvement in the signal-to-noise ratio can be anticipated.

Induced ablation of gonadotropins in transgenic mice expressing herpes simplex virus thymidine kinase under the FSH beta-subunit promoter.

Suppression of gonadotropins was induced by gancyclovir or acyclovir treatment in transgenic mice carrying 2.3 kb of bovine follicle-stimulating hormone beta (FSH beta) promoter fused to Herpes simplex virus thymidine kinase (tk) coding sequence. Transgenic tk and endogenous FSH beta were immunohistochemically co-localized in the same pituitary cells. In adult castrated transgenic males, gancyclovir treatment reduced plasma FSH (30%, P < 0.001). In intact juvenile gancyclovir treated mice, the reduction of pituitary FSH, and in males also of plasma FSH, was greater (50-70%, P < 0.05-0.01). A concomitant suppression of luteinizing hormone (LH) (50%, P < 0.01) was observed in female pups. The most pronounced reduction of gonadotropins was observed in newborn transgenic pups treated in utero with acyclovir. Both males and females had significantly lower pituitary levels of FSH (75-55%), LH (80-90%) or both (P < 0.05-0.01). Less pronounced decreases (30-40%, P < 0.01) were observed in plasma FSH. No apparent defects were seen in the testes of the transgenic, acyclovir treated, newborn pups. This mouse model is applied to study the dynamics of the gonadotropes and the role of gonadotropins in the maturation of the reproductive functions.

Experimental testicular teratoma promotes formation of humoral immune responses in the host testis.

The testis is an immunologically privileged site. Very little is known about the factors regulating formation of immune responses elicited by a neoplasm in the testis. We have studied the immune response of the host testis against experimental testicular teratoma in mouse by localizing adhesion molecules (CD106, CD54, CD49d/CD29, CD44, CD18, CD8 and CD4), cytokines (IL-2, IL-4, IL-6, IL-10 and IL-12), T-cell costimulators (CD80, CD86) and the lipid antigen presenting molecule CD1d in the testis of 129/SvJ mice with and without experimental testicular teratoma. The testicular teratomas were induced by grafting male gonadal ridges from 12-day-old 129/SvJ mouse fetuses into testes of adult mice from the same strain. The tumors cultured intratesticularly for 2, 3, 4 and 8 weeks (three animals per time point) were used for immunocytochemistry. CD1d was detected in Sertoli cells and in some degenerated tubules of the host testis surrounding the graft. In the tumor, CD1d was detected in glandular epithelia, smooth muscle and in thin fibers of neural origin. IL-2 was observed in some blood vessels of the host testis and of the tumor and in occasional cell infiltrates around these vessels. Some tubular structures of the tumor were also positive for IL-2. IL-6 was detected in Sertoli cells of the normal testis and in Sertoli cells and in solitaryinterstitial cells as well as in the walls of some blood vessels of the host testis. The reaction for IL-6 was more prominent in the tubules apparently damaged by the growing tumor. In the tumor IL-6 was detected in epithelial structures, muscle cells, in thin fibers of neural origin and in some blood vessels. IL-10 was detected in individual cells in the interstitium and in degenerating tubules of the host testis. In the tumor the epithelial structures were positive for IL-10. The interstitium of the host testis was positive for CD106 and the embryonic testicular cords in the graft were also positive, but the tumor was negative. CD44 and CD18 were observed in some blood vessels and in degenerated tubules of the host testis. In the tumor CD44 and CD18 were occasionally observed in cartilage and in epithelial structures. The results of the present study suggest that cytokine microenvironment in the testis containing neoplastic tissue promotes activation of humoral immune responses. In addition, as the damaged seminiferous tubules expressed increased amounts of two cytokines promoting humoral immune responses, IL-6 and IL-10, it is possible that also in other conditions with damage to the tubules, humoral immune responses predominate.

Mechanisms of kainate-induced region-specific neuronal death in immature organotypic hippocampal slice cultures.

Excessive activation of excitatory amino acid receptors has been implicated in neuronal death in a number of central nervous system insults. We have here investigated, the time course and mechanisms of kainate (KA)- induced neuronal death in immature organotypic hippocampal slice cultures (OHCs) using Fluoro-Jade B (FJB) staining as a marker of cell death, and immunoblotting, immunocytochemistry, and electron microscopy as methods to clarify the mechanisms. After 6 KA treatment (5 microM), no significant neuronal death was detected in any hippocampal subregion, whereas the treatment of 12, 24, and 48 h resulted in neuronal death in the CA3 regions, but not in CA1. The 48 h resting period in normal medium after KA-treatment did not rescue the cells but further increased the number of dead neurons in CA3 as compared to the corresponding acute phase. In Western blotting, the expression levels of the active, 17 kDa form of caspase-3, and the 84-85 kDa cleaved fragment of poly(ADP ribose)polymerase (PARP) were not altered from the control levels. Moreover, no active caspase-3 labelled cells were detected in immunocytochemical study 24 h after KA treatment either in the acute or resting groups. Electron microscopy showed non-apoptotic injury in the CA3a/b pyramidal neurons in KA-treated slices. Our results suggest that KA-induced neuronal death in immature OHCs is a strictly region-specific, irreversible, necrotic process.

Effect of etoposide on experimental testicular teratoma in 129/SvJ mice.

To study the effects of etoposide on experimental testicular teratoma in 129/SvJ mouse we analysed the tumour growth, differentiation, apoptosis and the localisation of mdr1 P-glycoprotein (mdr1-Pgp). In this model the implanted gonadal ridges developed into testicular teratomas in 17 out of 56 implanted testes (30%) and in 14 out of 28 mice (50%). The tumour-bearing mice were treated with etoposide on 4 successive days either 4 weeks or 6 weeks after implantation, and killed 7 days after the last dose. The mice in the control groups did not receive etoposide. The teratomas consisted mainly of neural tissue. The etoposide-treated 4-week teratomas, but not the 6-week teratomas, were significantly smaller than those in the corresponding control groups. The density of apoptotic cells and the distribution of the mdr1-Pgp were not altered by etoposide. The decreased proportion of immature neuroectodermal tissue components was observed in all treated teratomas, converting the histology towards that of a mature teratoma. In addition, a low proportion of immature tissue components was frequently combined with a low density of apoptotic cells. In conclusion, etoposide decreased the immature tissue components of teratomas, while mature tissues remained unaffected. These results may have clinical relevance in man, since they confirm that postchemotherapy mature teratomas cannot be treated with chemotherapy. Despite benign histology, the human residual tumours have a significant malignant potential and require complete surgical excision and close surveillance.

Sex ratio of human conceptuses.

The sex ratio in each developmental week was analyzed in 551 conceptuses from induced abortions for medical or social reasons in Southwest Finland. The ovulation age of the conceptuses varied from five to 24 weeks. There was a clear decrease in the sex ratios with increasing duration of pregnancy. The embryonic sex ratio was 164, the fetal ratio was 111, and the total ratio was 117. According to the regression analysis, the sex ratios at the ages of eight and 14 weeks were 119 and 105, respectively. The results suggest that higher mortality of male versus female conceptuses is restricted to the period of organogenesis. Seasonal or monthly variation in the sex ratio was not found in the present study.

Survival and degradation of Salmonella enterica serotype Enteritidis in intestinal epithelial cells in vitro.

The survival and fate of Salmonella enterica serotype Enteritidis in Henle-407 human intestinal epithelial cells was investigated during prolonged incubation to evaluate the persistence of causative microbes and the relationship to patients developing reactive arthritis. Most of the bacteria were killed and degraded quite soon after infection of the cells, but there were still live bacteria inside the cells for up to 14 days. These results suggest that in patients developing reactive arthritis the salmonellae could persist in the epithelial cells and spread within the host to the joint and be present there at the time of the inflammatory response. Production of marked amounts of nitric oxide was observed as a novel response to salmonella infection in human intestinal epithelial cells. The present experimental procedure appears to be a suitable model to further investigate host-bacteria interaction in HLA-B27 positive cells from patients developing reactive arthritis.

Ultrastructural study of separated cell and acrosomal membranes from bull spermatozoa.

Ejaculated bull spermatozoa were washed and incubated with a cationic detergent, Hyamine 2389, and the material removed was centrifuged on a three-step sucrose gradient. Five different bands were obtained, three of which contained cell and acrosomal membranes and also acrosomal material. The first of these, band III, was composed of two parts. The lower part contained U-shaped membrane profiles originating from the outer acrosomal membrane, acrosomal material attached to them, and vesicles of different sizes. The upper part, in addition to various vesicles (probably formed from cell membrane), contained acrosomal vesicles arranged in the form of aggregates or caps. The latter vesicles were formed by fusion and vesiculation of the cell and outer acrosomal membranes with tightly bound acrosomal material. Band IV was uniform and comprised various round vesicles with a smooth membrane, which had a typical unit-membrane structure. There were obviously formed from the cell membrane. The fifth band, in the saline portion with material unsedimented in the sucrose, included small vesicles and membrane fragments, the origin of which was obscure. The solubilized material was also left in this fraction.

Transplantation of newborn rat testis under the kidney capsule of adult host as a model to study the structure and function of Leydig cells.

Newborn rat testis was transplanted under the kidney capsule of adult castrated and uncastrated male rats to develop and characterize a model system for studies on Leydig cell development. Two weeks after transplantation, the number of Leydig cells and the size of their nuclei in the transplants had increased. Secretion of testosterone was indicated by increased seminal vesicle weights and decreased pituitary LH in the castrated host animals. Pituitary FSH content increased significantly in the uncastrated animals with transplants, which suggested production of an FSH-stimulating factor. Cells with the morphologic features characteristic of fetal- and adult-type Leydig cells were observed in the transplants. The seminiferous tubules with spermatocytes, incipient lumina, and significantly larger average diameters showed more advanced development than those in the normal 2-week-old testis. By the present morphologic and functional criteria, the kidney subcapsular transplantation technique provides a suitable model for studies of fetal and adult Leydig cell development.

Electron microscopy and immunolabeling of Yersinia antigens in human synovial fluid cells.

Recent findings have emphasized the involvement of bacterial antigens in synovitis in reactive arthritis. It is still unclear, however, in what form the microbial material exists in the joint. Both antigen-containing cells and intact bacteria have been proposed as candidates on the basis of immunohistochemical studies of the synovial membrane. This study addresses that question by electron microscopy and peroxidase immunolabeling of synovial fluid cells from three patients with reactive arthritis triggered by Yersinia enterocolitica O:3. For all three patients a diffuse reaction in the cytoplasm of phagocytes was interpreted as bacterial material in a degraded form. These results are consistent with our proposal that intact bacteria rarely, if ever, enter the joints of Yersinia-triggered reactive arthritis patients.

Ultrastructure of gonadal ridge in male and female pig embryos.

Pig embryos aged 21-22 days (d) obtained from artificially inseminated sows for an ultrastructural study of the development and sex differentiation of the gonadal ridge. The chromosomal sex of the embryos was identified by a chromosome analysis. At the age of 21 d the gonadal ridge consisted of three different tissues: the surface epithelium, the primitive cords, and the mesenchyme. The surface epithelial cells contained elongate mitochondria with lamellar cristae, granular endoplasmic reticulum (GER), the Golgi complex, free polysomes, coated vesicles and fine filaments. The epithelium was continuous with the primitive cords, which were composed of ultrastructurally similar cells. The epithelial basal lamina followed the cord surface, but covered it incompletely. Primordial germ cells (PGC) were located under the surface epithelium in the mesenchyme and in the cords. The nucleus with a prominent nucleolus, and the large mitochondria were round. The cytoplasm contained a pair of centrioles, the Golgi complex, solitary lamellae of the GER, free polysomes, lipid droplets and some coated vesicles. The PGC entered the gonadal cords before these were surrounded by the continuous basal lamina. A day later (22 d) the number and length of the cords were increased. The PGC were more frequent and seen also in deeper layers. All the structures studied at the age of 21-22 d showed that the gonadal ridge also at the ultrastructural level is at the sexually indifferent stage.

Glycogen accumulations in differentiating mesonephric ducts and tubuli in male human embryos.

Human mesonephric duct epithelial cells contained empty appearing regions in the infranuclear cytoplasm when prepared for transmission electron microscopy using glutaraldehyde and osmium fixation. The same regions stained positively with PAS in Epon sections for light microscopy suggesting that glycogen was present. Incubation with saliva abolished the reaction. For electron microscopy the glycogen stained very intensely if a mixture of osmium tetroxide and potassium ferrocyanide was used instead of osmium alone. Glycogen accumulations were present between the ages of 5 to 10 weeks and absent at the age of 15 weeks. Reports by others indicate that glycogen may be present in different reactive forms in relation to its staining behaviour after various fixatives. The present results, and similar studies in other tissues, indicate that osmium tetroxide-potassium ferrocyanide fixative should be used routinely for preservation of embryos and fetuses and where indicated, for ultrastructural identification of glycogen and cytoplasmic filaments in clinical specimens.

Immunohistochemical characterization of the basement membranes of the human oral mucosa.

Type IV and V collagens, laminin and heparan sulphate proteoglycan were localized in vascular and subepithelial basement membranes. Fibronectin was distributed in a reticular pattern throughout the lamina propria under the oral epithelium. The uniform distribution of basement membrane components and type V collagen in different regions suggests a similar molecular composition for the basement membranes under functionally-different oral epithelia. The more intense reaction in the vascular than in the subepithelial basement membranes, with diluted antibodies to type IV collagen and laminin apparently reflects chemical differences in these basement membranes. Occasional discontinuities in the subepithelial basement membranes were seen in inflamed gingival sulci and in tonsillar crypts. The destruction responsible affected all basement membrane components, except fibronectin, which maintained a reticular distribution even in the deep tonsillar tissue. The immunohistochemical method is useful in demonstrating different degrees of destruction in basement membranes associated with inflammation.

Hormonal regulation of differentiation of human fetal prostate and Leydig cells in vitro.

Trowell type of organ culture was used for correlative study of the human fetal prostate and Leydig cell differentiation at the ultrastructural level. Androgens accelerated the differentiation of human urethral epithelial cells into secretory prostatic cells and the ultrastructure resembled that in vivo. Also Leydig cells retained in organ culture the same ultrastructural features as in vivo and human chorionic gonadotropin (hCG) accelerated their differentiation. It is concluded that this type of culture technic can be used in the study of early differentiation of human genital organ and androgenes and hCG take part of human prostatic and Leydig cell differentiation.