Age related Histology and Immunohistochemistry of some intermediate filaments in the Testis of the African Catfish (Clarias gariepinus).

The African Catfish (Clarias gariepinus) are important source of protein for local consumers in developing countries in Africa and have also been reported to have enormous commercial potential. Several works have been done on plethora of general histological, biochemical and hormone changes which accompany puberty in African Catfish. Other studies have touched the effects of ecotoxins on the histological and reproductive parameters of the mature African Catfish. This study is an attempt to use immunohistochemical and basic histology to elucidate the baseline information on the general structural differences between the testes of immature and post-pubertal catfish with respect to some intermediate filaments arrangement within the testicular tissue. Ten (10) each of mature male catfish (4-5 months old) and immature male catfish (3 months old) were used in the study. The fish were subjected to cold shock and decapitated before the testes were harvested from both groups. These tissues were fixed in Bouins fluid for 24 hours and subsequently transferred into 70% Ethanol. Testicular tissues from both groups were processed for paraffin embedding for routine staining. Another set of tissues were fixed in Neutral Buffered Formalin for testicular immunostaining techniques for expression of Vimentin, Desmin, Cytokeratin and Smooth Muscle Actin. There is an increase in seminiferous luminal area in the mature catfish testis with the presence of mature spermatozoa in the lumen when compared with immature catfish testis which has small size of lumen with absence of mature spermatozoa. Testicular interstitium thickness remain relatively unchanged. SMA was markedly expressed in the cytoplasm of interstitial Leydig cells in the immature catfish testis whereas it was weak in its expression in the mature catfish. However, SMA was not expressed in the connective tissue proper in the testicular interstitium. Cytokeratin expression was also marked in the testicular capsule of immature catfish but was weak to absent in the mature catfish, however, both mature and immature catfish had moderate cytokeratin expression in their seminiferous tubule basement membrane. Desmin was strongly expressed in cytoplasm of immature germinal cells in the immature catfish testis but was moderate in its expression in the mature catfish testis. Vimentin expression was marked in the cytoplasm of immature germinal cells in both immature and mature catfish testis but weak in its expression in the Sertoli cell cytoplasm of both groups. This study infers that ultra-structural and protein changes can be related to age changes alone apart from the contribution of seasonality and external interference by ecotoxins. The age-related changes seen in this study could set ``baseline information. The extent of contribution of season and other external factors will be better understood. Though the age-related difference might be peculiar to the species of current interest, the differences elucidated are a sound background for relational studies, especially on the effect of ecologic toxins on immature testis, as separate from the mature testis.

In vivo activity and in vitro specificity of CD4+ Th1 and Th2 cells derived from the spleens of diabetic NOD mice.

CD4+ T cell lines were generated from the spleens of diabetic NOD mice against crude membrane preparations derived from a rat insulinoma. Adoptive transfer of these lines into neonatal mice confirms that overt diabetes is induced by gamma-IFN-secreting Th1 cells, whereas transfer of IL-4-secreting Th2 cells resulted in a nondestructive peri-islet insulitis. Analysis of the antigens recognized by individual T cell clones from the Th1 line included reactivity against an insulinoma membrane fraction enriched in proteins of approximately 38 kD. Immune responses to the same antigen preparation have been associated with T cell clones derived from human insulin-dependent diabetes mellitus. The specificity of Th2 cells includes reactivity to a fraction enriched in proteins of 30 kD. The data suggest that in insulin-dependent diabetes mellitus the balance between beta cell destruction, associated with intra-islet infiltration, and nondestructive (potential protective) peri-islet insulitis may depend on both the antigens recognized, and the prevailing cytokine environment.

The morphology of the efferent ducts of the testis of the ostrich, a primitive bird.

The efferent duct of the ostrich consists of two segments, the proximal efferent duct (PED) and the distal efferent duct (DED) that are continuous, as in some other birds. Both segments of the duct possess an epithelium comprising non-ciliated and ciliated cells in varying proportions between the two segments. The non-ciliated cell (type I) of the PED contains a well-developed, subapical endocytic apparatus of apical tubules and endocytic vacuoles, a solitary, large, heterogeneous lipid droplet, and numerous, oval, dense bodies in the supranuclear region of the cell. Mitochondria tend to concentrate in the basal part of the cell. Intercellular spaces between the non-ciliated cells are enlarged, especially in the basal half of the epithelium. Together, these morphological features confer on the PED an efficient fluid absorption capability. The DED epithelium displays the type II non-ciliated cell whose poorly developed subapical endocytic apparatus as well as the absence of dilated basal intercellular spaces indicate its limited fluid absorptive capacity.

Development of a procedure for the direct cloning of T-cell epitopes using bacterial expression systems.

Although single bacterial recombinant antigens have been used successfully to stimulate individual T-cell clones and elicit recall responses in peripheral lymphocytes, the broader use of molecular cloning systems for the identification of autoantigens recognised by the cellular arm of the immune system has met with only limited success. In a systematic approach to address this problem, a series of bacterial expression vectors were examined for their potential use as cloning vectors to elicit a proliferative response in vitro from a non-obese diabetic (NOD) mouse T-cell clone which recognises the immunodominant ovalbumin epitope (aa 323-339). The use of the vector pRSET, which produces a hexa-histidine tagged fusion protein, was confounded by non-specific responses to bacterial protein contaminants. pGEX, which generates a glutathione-S-transferase hybrid, avoided this problem but suffered from the disadvantage that a universally applicable purification procedure for the hybrid antigen could not be easily developed. A practical screening protocol was developed using the pUEX expression system (beta-galactosidase hybrid) and purification based upon electroelution of the hybrid protein from purified inclusion bodies subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). This system can be used to screen expression libraries for the detection of T-cell epitopes provided that the T-cell clones give low background responses to irrelevant pUEX recombinant proteins. Low abundance antigens may be obtained using this system in combination with subtractive hybridisation to construct cDNA libraries enriched in the target antigen.

Wound healing activities of methanolic extracts Ocimum gratissimum leaf in Wistar rats - a preliminary study.

The wound healing effect of leaf extracts of Ocimum gratissimum was investigated in adult male Wistar rats. Two groups of adult male Wistar rats, average body weight 170g, had a 2cm by 2cm square wound inflicted on the dorsolateral aspect of their trunk with Paniculus Carnosus removed. Experimental group had their wound dressed with methanolic leaf extracts of Ocimum gratissimum while control group had their wounds dressed with normal saline dressing. All animals had wound dressing done every five days; wound dimension measured and, wound morphometry assessed. Wound biopsy was done by random selection in each group on day 10 and the day of complete re-epithelisation. Routine paraffin wax processing was done, slides stained with haematoxylene and eosin for histological assessment of fibroblast count, neovascularisation and granulation tissue profile. The result revealed significant wound contraction (P<0.05) on day 10 in the experimental group (mean 73.40 +/- 3.30)cm2 compared with the control group (mean 53.50 +/- 4.32)cm2. Histology of the healed scar showed non-significant (P>0.05) decrease in the mean fibroblast count forthe experimental group (83.80 +/- 5.70) relative to fibroblast count of 90.20 +/- 17.90 in the control group. The mean blood vessel count was also non-significantly lowered (P>0.05) in the experimental group (9.20 +/- 1.20) relative to the control group (13.40 +/- 2.40). Granulation tissue histology on day 10 showed denser inflammatory infiltrate as reflected by increased cellularity in the control group relative to that of the experimental group which though appeared adequate was not as dense as the control group. Thus we suggest that the methanolic extracts of O. gratissimum could be a potential wound healing agent due to its ability to enhance wound contraction.

Comparative evaluation of the sperm characteristics and morphology of adult Wistar rats fed either low or normal protein-energy diets and orally dosed with aqueous Cuscuta australis extracts.

Cuscuta australis (C. australis) seed and stem are commonly used as dietary supplements in a maize-meal, "Ogi", by the local population for the management of male and female reproductive dysfunctions. This study, as a part of on-going efforts, therefore, evaluated and compared the effects of Low Protein-energy (LP) and Normal Protein-energy (NP) diets on the sperm morphology and characteristics of adult Wistar rats orally dosed aqueous extracts of C. australis seed (LPSE and NPSE) and stem (LPST and NPST), 300 mg of extract/kg body weight of rat/day, for seven days. The control groups (LPWA and NPWA) received vehicle, water. Live-dead ratio and percentage of sperms with curved tail were significantly decreased (p<0.01) in the NPST relative to the NPWA, LPWA, LPST, NPSE and LPSE. Total abnormal sperm counts, acephalic sperms and tailless head sperms were significantly decreased (p<0.001, p<0.05 and p<0.001, respectively) in the LPST and NPST relative to LPSE, NPSE, LPWA and NPWA. The LPSE, LPST and NPST showed significantly decreased (p<0.05) percentages of sperms with either bent mid-piece or curved mid-piece relative to the LPWA. Significantly decreased (p<0.05) percentage of sperms with curved mid-piece was also observed in the NPSE relative to LPWA. Protein-energy diet significantly influenced (at least p<0.05) the effect of each extract on sperm motility and percentage of sperms with curved tail. Stem extract significantly decreased (p<0.01) the percentages of acephalic sperms and tailless head sperms. Diet-stem extract interaction significantly influenced (p<0.05) live-dead ratio. Our data suggest that orally administered aqueous extracts of C. australis generally enhanced the sperm morphology and characteristics of the male Wistar rat and that the stem extract maintained sperm morphology better than the seed extract. It also showed that the stem extract decreased live-dead ratio and that the efficacy of orally administered aqueous C. australis stem extract may be affected by variations in dietary protein-energy levels.

Comparative morphophysiological evaluation of the testis of adult Wistar rats fed low protein-energy diet and dosed with aqueous extracts of Cuscuta australis.

Cuscuta australis (C. australis) seed and stem are historically used by the local population as dietary supplement for the management of infertility. This study, therefore, evaluated the effect of orally administered aqueous extracts of C. australis seed and stem, 300 mg/kg body weight/day for seven days, on the testis of the adult Wistar rat fed either low or normal protein-energy diets. The control group received water. The relative weight of the testis was non-significantly increased (p>0.05) in the Low Protein-energy diet-Water-treated (LPWA), Low Protein-energy diet-Seed-treated (LPSE) and Normal Protein-energy diet-Seed-treated (NPSE) groups relative to the Normal Protein-energy diet-Water-treated (NPWA). The weight of the testis was also non-significantly increased (p˃0.05) in the Low Protein-energy diet-Stem-treated (LPST), but decreased in the Normal Protein-energy diet-Stem-treated (NPST), relative to LPWA and NPWA. Heights of germinal epithelium were significantly decreased (p<0.05) in the LPWA, LPSE and LPST relative to the NPWA, NPSE and NPST. Diet significantly influenced (p<0.001) the effect of stem extract on the height of germinal epithelium. The NPSE, LPSE, NPST, LPST and LPWA showed significantly decreased (p<0.001) plasma levels of luteinizing hormone (LH) and follicle stimulating hormone (FSH) relative to NPWA. The LPWA, LPSE and NPST also showed significantly decreased (p<0.001) levels of testosterone relative to NPWA and LPST. Diet significantly influenced (p<0.001) the effect of seed on the level of LH. Seed-diet interactions significantly affected the levels of FSH (p<0.001) and LH (p<0.05), but not testosterone. Diet significantly influenced (p<0.001) the effects of stem extract on the levels of FSH, LH and testosterone. Stem-diet interactions significantly affected (p<0.001) the levels of FSH, LH and testosterone. Our data suggest that the aqueous extract of C. australis stem is more potent than the seed extract and that dietary protein-energy intake may influence the efficacy of orally administered aqueous extracts of C. australis.

Components and development of the centriolar complex during and beyond spermiogenesis in a passeridan bird, the Masked weaver (Ploceus velatus).

The fate of the proximal centriole in passeridan birds is an area of controversy and relative lack of knowledge in avian spermatogenesis and spermatology. This study examines, for the first time, spatiotemporal changes in the centriolar complex in various phases of spermiogenesis in a passerine bird, the Masked weaver (Ploceus velatus). It also describes the configuration of the centriolar complex and the relationship between it and the granular body in both intra- and extra-testicular spermatozoa. It is shown that the proximal centriole is retained and attaches, at its free end, to the granular body of spermatids in every step of spermiogenesis, as well as in mature intra-testicular and post-testicular spermatozoa, including those in the lumen of the seminal glomus. As the centriolar complex, along with its attached granular body, approaches the nucleus in the early spermatid, the proximal centriole articulates with the distal centriole at an acute angle of about 45°, and thereafter, both centrioles, still maintaining this conformation, implant, by means of their articulating proximal ends, at the implantation fossa of the nucleus. In the mature spermatid and spermatozoon, the granular body winds itself helically around the centriolar complex in the neck/midpiece region of the cell, and, thus, becomes the granular helix. The significance of this observation must await future studies, including possible phylogenetic re-evaluation and classification of birds.

The cytoskeletal proteins in the contractile tissues of the testis and its excurrent ducts of the passerine bird, Masked Weaver (Ploceus velatus).

The cellular composition of the testicular capsule, seminiferous peritubular tissue, the epithelia as well as periductal muscle cell layers of the excurrent ducts was studied, in sexually mature and active Masked Weaver (Ploceus velatus) birds of the passerine family, Ploceidae. Ultrastructure of the contractile cells in the testicular capsule, peritubular and periductal tissues showed that these cells were smooth muscles of typical morphological characteristics. Variability in the immunohistochemical co-expression of microfilaments and intermediate filaments in the different tissues was evident. Actin and desmin proteins were co-expressed immunohistochemically in the testicular capsule and seminiferous peritubular smooth muscle layer. Actin was singly and very weakly expressed in the rete testis epithelium while cytokeratins and desmin were co-expressed in the epithelium of the excurrent ducts. The periductal muscle layer of all ducts of the epididymis, the ductus deferens as well as the seminal glomus, strongly co-expressed actin and desmin. Vimentin was absent in all cells and tissue types studied. There is clear evidence that the tissues of the male gonad and its excurrent ducts in the Masked Weaver, as has been reported for members of the Galloanserae and Ratitae, contain well-formed contractile tissues whose function would include the transportation of luminal through-flow from the testis into, and through, its excurrent ducts. The microtubule helix in the head and of the mid-piece, of elongating spermatids, as well as of the mature spermatozoa in the various excurrent ducts, including some spermatozoa in the seminal glomus, also co-expressed these three proteins.

The excurrent ducts of the testis of the emu (Dromaius novaehollandiae) and ostrich (Struthio camelus): Microstereology of the epididymis and immunohistochemistry of its cytoskeletal systems.

The volumetric proportion of the various ducts of the epididymis of the emu and ostrich and the immunohistochemistry of actin microfilaments, as well as cytokeratin, desmin and vimentin intermediate filaments, were studied in the various ducts of the epididymis of the emu and ostrich. The volumetric proportions of various ducts, which are remarkably different from those of members of the Galloanserae monophyly, are as follows: the rete testis, 5.2 +/- 1.4% for the emu and 2.4 +/- 1.8% for the ostrich; efferent ducts, 14.2 +/- 2.3% (emu) and 11.8 +/- 1.8% (ostrich); epididymal duct unit, 25.8 +/- 5.8% (emu) and 26.1 +/- 4.1% (ostrich) and connective tissue and its content, 54.7 +/- 5.8% (emu) and 60.0 +/- 4.9% (ostrich). Unlike in mammals and members of the Galloanserae monophyly, only vimentin was immunohistochemically demonstrated in the rete testis epithelium of the emu, and none of the cytoskeletal protein elements in the ostrich rete testis. The epithelium of the efferent ducts of the emu co-expressed actin, cytokeratin and desmin in the non-ciliated type I cells, and vimentin in the ciliated cell component. The ostrich demonstrated only cytokeratin in this epithelium. The ratite epididymal duct unit is different from that of mammals in lacking actin (only weaky expression in the ostrich), desmin and cytokeratin, and a moderate/strong immunoexpression of vimentin in the basal cells and basal parts of the NC type III cell in the epididymal duct unit. Immunoexpression of the microfilaments and intermediate filaments varied between the two ratite birds, as has been demonstrated previously in birds of the Galloanserae monophyly, and in mammals.

Immunohistochemistry of the cytoskeleton in the excurrent ducts of the testis in birds of the Galloanserae monophyly.

The presence, location and degree of immunoexpression of various microfilament (MF) and intermediate filament (IF) systems (actin, cytokeratins, desmin, vimentin) were studied in the excurrent ducts of the testis in sexually mature and active galliform (Japanese quail, domestic fowl, turkey) and anseriform (duck) birds. These proteins were variably expressed between the epithelia and periductal tissue (periductal smooth muscle cell layer and interductal connective tissue) types and between species. Variable heterogeneous co-expression of filament systems was also found in the various duct epithelia and periductal tissue types: co-expression of filament systems was the rule rather than the exception. In the duck, neither vimentin nor cytokeratin was present in any of the tissues, whereas actin and desmin (absent in the rete testis) were co-expressed in the efferent ducts and epididymal duct unit (comprising the ductus conjugens, ductus epididymidis and ductus deferens). Actin, desmin and vimentin were generally co-expressed in the rete testis, efferent ducts and epididymal duct unit of the quail, domestic fowl and turkey, with vimentin being more strongly immunoreactive than actin and desmin in the epididymal duct unit, but more weakly immunoexpressed in the efferent ducts. Cytokeratin was present and co-expressed with actin, desmin and vimentin in the rete testis, efferent ducts and epididymal duct unit of the domestic fowl and turkey, but not in the quail and duck. The periductal smooth muscle cell layer and interductal tissue co-expressed actin, desmin and vimentin variably in all birds. Luminal spermatozoa of both the turkey and duck were immunonegative for all protein systems, whereas those of the quail and domestic fowl co-expressed actin, desmin and vimentin moderately or strongly. The tissues of the reproductive tract of male birds thus contain cytoskeletal protein systems that are variably but mostly co-expressed and whose contractile ability appears necessary and sufficient for transportation through the various excurrent ducts of the voluminous testicular fluid and its high sperm content, characteristic features of male avian reproduction.

Morphological and immunohistochemical study of testicular capsule and peritubular tissue of emu (Dromaius novaehollandiae) and ostrich (Struthio camelus).

The testicular capsule and peritubular boundary tissue of the emu and ostrich, as typical representatives of ratite birds, were studied in sexually mature and active birds. The testicular capsule was much thicker (578.1+/-73.4 microm for the free surface of the ostrich testis, and 176.2+/-57.5 microm for the emu) than those of members of the Galloanserae. The cellular composition of both testicular capsule and peritubular tissue was similar generally to that of members of the previously studied Galloanserae and of mammals. The tunica albuginea of the testicular capsule mainly comprised smooth-muscle-like or myoid cells mostly running in one direction and occurring in one main mass. Unlike the Galloanserae, the tunica albuginea contained more collagen fibres than smooth muscle cells, especially in the ostrich. Peritubular tissue was similarly composed of smooth-muscle-like cells distributed in several layers. Actin microfilaments and desmin and vimentin intermediate filaments were variably immunoexpressed in these two tissue types in both birds, with a clear dichotomy in the peritubular tissue. Thus, taken together with studies of some members of the Galloanserae, avian testes clearly contain a morphological mechanism that is represented partly by the smooth muscle cells of the testicular capsule and peritubular tissue for transporting the testicular fluid, which is usually copious in birds, and its cellular content from the testis into the excurrent duct system; this mechanism is similar to that found in mammals.

Structural and immunohistochemical features of the epididymal duct unit of the ostrich (Struthio camelus).

The epididymal duct unit, comprising the ductus conjugens, ductus epididymidis and ductus deferens, was studied histologically, ultrastructurally and immunohistochemically in five sexually mature and active birds. The main morphological features of the pre-dominant non-ciliated (type III) cell of the epithelial lining of this duct unit include, but are not limited to, a moderately abundant smooth or sparsely granulated endoplasmic reticulum, electron-dense secretory granules and numerous mitochondria in the supranuclear zone of the cytoplasm. A single, large heterogeneous lipid droplet, of unknown function, was characteristically situated immediately proximal to the nucleus. The epithelium is obviously secretory and specifically, of the merocrine, and not apocrine, type of secretion. The epithelium of the epididymal duct unit was only focally and weakly to moderately immunopositive to both actin MF and desmin IF, while the duct unit was immunonegative to cytokeratin and vimentin intermediate filaments. The peritubular muscular layer was moderately to strongly positive to both actin and desmin, and negative to cytokeratins and vimentin.

The testicular capsule and peritubular tissue of birds: morphometry, histology, ultrastructure and immunohistochemistry.

The testicular capsule was studied histologically, morphometrically, ultrastructurally and immunohistochemically in the Japanese quail, domestic fowl, turkey and duck (all members of the Galloanserae). The testicular capsule was, relative to mammals, thin, being 81.5 +/- 13.7 microm in the quail, 91.7 +/- 6.2 microm in the domestic fowl, 104.5 +/- 29.8 microm in the turkey and 91.8 +/- 18.9 microm in the duck. The orchido-epididymal border (hilus) of the capsule was much thicker than elsewhere in all birds (from 233.7 +/- 50.7 microm in the duck to 550.0 +/- 147.3 microm thick in the turkey). The testicular capsule, other than the tunica serosa and tunica vasculosa, comprised, in the main, smooth muscle-like or myoid cells running mainly in one direction, and disposed in one main mass. Peritubular tissue was similarly composed of smooth muscle-like cells disposed in several layers. Actin and desmin intermediate filaments were immunolocalized in the inner cellular layers of the capsule in the quail, domestic fowl and duck, but uniformly in the turkey. Vimentin intermediate filament immunoreaction in the capsule was moderately and uniformly positive in the testicular capsule of only the quail. Actin and desmin, but not vimentin (except very faintly in the turkey) or cytokeratin, were immunolocalized in the peritubular tissue of all birds. The results therefore establish, or complement, some previous observations that these birds have contractile cells in their testicular capsule and peritubular tissue, whose function probably includes the transport of testicular fluid into the excurrent duct system.

Mycobacteria precipitate an SLE-like syndrome in diabetes-prone NOD mice.

Non-obese diabetic (NOD) mice spontaneously develop organ-specific autoimmunity and are widely used as a model for diabetes. Aged NOD mice also exhibit some features of non-organ-specific autoimmune rheumatic disease such as anti-nuclear antibodies and late-onset haemolytic anaemia. Here, we report that a single dose of 2.6 x 10(7) heat-killed bacillus Calmette-Guérin (BCG) i.v. in 8-week-old NOD mice prevented diabetes but precipitated a syndrome similar to systemic lupus erythematosus (SLE). Treated mice developed haemolytic anaemia, anti-DNA and anti-Sm anti-nuclear autoantibodies and an increased severity of sialadenitis. Perivascular lymphocytic infiltration in the kidneys and glomerular immune complex deposition were also found. The action of BCG appeared to be mediated by an adjuvant-like activity as treated mice showed a substantial increase in reticuloendothelial cell function and enhanced antigen presentation capacity.

Tolerance induction as a therapeutic strategy for the control of autoimmune endocrine disease in mouse models.

This chapter aims to describe ways in which autoimmunity can be prevented or reversed and 'self-tolerance' re-established. To this end we have largely restricted our overview to the two main autoimmune disease models with which we are involved, i.e. IDDM in NOD mice and EAT in H-2k mice although, where appropriate and to demonstrate a particular point, other models are mentioned. The chapter has been divided into sections covering protection afforded by 1) transgenes, 2) autoantigen and 3) by reagents targetting T-cell surface molecules. Where established, the mechanism by which protection or tolerance is achieved is described but where, as in most cases, it is unknown the possibilities are discussed. Investigations using T-cell lines and clones and on islet regeneration which are currently being followed as part of a comprehensive approach to the study of autoimmunity are included as separate sections and their relevance discussed.

Aspartate at position 57 of nonobese diabetic I-Ag7 beta-chain diminishes the spontaneous incidence of insulin-dependent diabetes mellitus.

MHC class II genes have been shown to influence the development of the autoimmune disease insulin-dependent diabetes mellitus (IDDM) in the nonobese diabetic (NOD) mouse. In human IDDM it has been suggested that the presence of an aspartate at position 57 of the DQ beta-chain might be important in determining resistance to development of IDDM. The involvement of MHC class II genes in IDDM was investigated through the introduction of MHC encoding transgenes. We show that introduction of a mutated I-Ag7 Ab gene which encodes an aspartate at position 57 reduces the incidence of IDDM but does not prevent insulitis, sialadenitis, or the development of insulin and nuclear autoantibodies.