Morphological, Glycohistochemical, and Immunohistochemical Studies on the Embryonic and Adult Bovine Testis

In the present study, the testes of 32 bovine embryos with different crown-rump length (2.5- 90 cm CRL) and of 15 sexually mature bulls (Deutsches Fleckvieh) were investigated using light- and electron microscope as well as glycohistochemical and immunohistochemical methods. The gestation period was divided into 3 stages; early, mid, and late gestation. Developmental changes in the testicular morphogenesis were therefore analyzed in details during these phases. Generally, embryonic development of bovine testis involves the same mechanism described in other mammals. At the first stage of this study (2.5 cm CRL/43 dpc), the anlage of the testes protruded to the coelomic cavity as paired bean-shaped structures on either side of the dorsal mesentery medial to the mesonephros. It consists of primitive testicular cords, interstitium, and rete testis blastema. Proceeding with fetal age, these basic testicular structures are further differentiated. The tunica albuginea is separated into two layers: an outer fibrous layer (tunica fibrosa) with some mesenchymal cells, numerous fibroblast, and much fibrous content and an inner cellular layer with several blood vessels (tunica vasculosa). The testicular cords are surrounded by a marked basal lamina and peritubular cells and lined by two types of cells: a large number of dark polygonal cells with irregular nuclei, pre-Sertoli cells and small number of large light round cells with relatively round nuclei, the prespermatogonia. The average number of the germ cells per cross section of cord increases, particularly form 3.5 to 14 cm CRL, resulting in a germ cell maximum at the end of this stage (14 cm CRL). Although most of the germ cells are located toward the periphery of the cord, some are also found in the center. Pre-Sertoli cells form a complete layer at the periphery of the cords. Generally, these cells are irregular in shape and numerous but considerably smaller than the germ cells. Unlike prespermatogonia, mitotic figures are seen in pre-Sertoli cells during the whole embryonic life. As a consequence of the expansion in the interstitium, the seminiferous cords are progressively separated from each other. The testicular interstitium is rapidly differentiated and is composed of several islets or clusters of polygonal Leydig cells, peritubular flattened cells surrounding the testicular cords, connective tissue cells, and numerous blood vessels. In the present study, fetal Leydig cells were first recognized at 3.5 cm CRL. Thereafter, the average number of these cells is rapidly increased to attain their maximum with the end of the first gestation period (14 cm CRL). This generation of Leydig cells however dedifferentiates progressively with developmental age. A continuous system of basal lamina joins the testicular cords with rete strands from 10 cm CRL and onwards. This system establishes the first connection between these two testicular components via ill-developed uncanalized straight tubules (tubuli recti). Rete testis channels are lined by simple layer of cuboidal epithelium with round nuclei occupying most of the cytoplasm and enclosed by well-defined basal lamina. The adult bovine testis is enclosed by a connective tissue capsule, tunica albuginea, composed predominantly of collagen fibers and few elastic fibers. Most of the testicular parenchyma is made up of the convoluted seminiferous tubules (tubuli seminiferi contorti), two-ended convoluted loops, with both ends opening into the rete testis via specialized terminal segments. The seminiferous tubules of sexually mature bulls are enclosed by a distinct lamina propria and are lined by two cell populations, non-proliferating Sertoli cells and highly proliferating spermatogenic cells. The bovine lamina propria consists of basal lamina, collagen and elastic fibers, and 3-5 layers of partially overlapping myofibroblasts. Additionally, fibrocytes, collagen fibrils, and fibroblasts-like cells form the outermost border of the tubulus. Sertoli cells are easily identifiable elements of the seminiferous epithelium. Adult Sertoli cells are large irregularly shaped cells with their broad bases resting on the basal lamina while the remaining cytoplasmic processes extend upward to the tubular lumen. They are characterized by round or oval euchromatin-rich nuclei situating in the basal portion near the basal lamina of the seminiferous tubules. Adult bovine germ cells are present in four morphologically different groups, i.e., spermatogonia, spermatocytes, spermatids, and spermatozoa. The seminiferous cycle stages are identified using changes in the germ cell nuclei as well as location and shape of spermatids. According to this method, eight stages are defined in the seminiferous epithelium of bovine. The interstitial or intertubular tissue of adult bovine testis consists of Leydig cells, macrophages, scattered lymphocytes and plasma cells, and contains numerous blood and lymph vessels. Not all Leydig cells have contact to blood or lymph capillaries. The excurrent duct system of the adult bovine testis consists of terminal segment of the convoluted seminiferous tubules, straight tubules, and rete testis. The terminal segment can be further subdivided into a proximal (transitional) region, middle portion, and distal part (terminal plug). The proximal region is lined by typical Sertoli cells while the last two parts are lined by modified Sertoli cells. The tubulus rectus of adult bovine testis is composed of three morphologically different regions: a proximal cup-shaped region, a middle narrow stalk, and a distal festooned portion. The rete testis is a complicated centrally positioned meshwork of intercommunicating channels that lies within the mediastinum testis parallel to the long axis of epididymis. The simple cuboidal epithelium of straight tubules and rete testis is shown to contain some lymphocytes and macrophages. The cellular distribution of glycoconjugates within the fetal and adult bovine testis was investigated using thirteen (ConA, PSA, LCA, PNA, GSA-I, ECA, DBA, SBA, HPA, VVA, WGA, UEA-I, LTA) different fluorescein isothiocyanate (FITC) conjugated lectins. In fetal testes, detection of sugar moieties by lectins was carried out on Bouin õ s-fixed paraffin-embedded sections while in adult it was performed on both Bouin õ s-fixed paraffin-embedded and acetone-fixed frozen sections. Only five lectins (PSA, PNA, GSA-I, DBA, WGA) showed a positive reaction in the embryonic testes. PNA, GSA-I, DBA, and WGA were detected in the germ cells whereas PSA, DBA and WGA labeled the fetal Leydig cells. None of the lectins used was observed in the pre-Sertoli cells. Further on, some lectins were seen in tunica albuginea (PSA, PNA, GSA-I, WGA), basal lamina of testicular cords (PSA, WGA), interstitial blood vessels (PSA, GSA-I, WGA), mediastinum testis (PSA, PNA, WGA) and rete testis epithelium (PNA). In adult animals, spermatogonia and spermatocytes were positively stained with PSA, LCA, DBA, SBA, and VVA. All the lectins investigated except that of the fucose-binding lectin (UEA-I and LTA) were definitely detected in the acrosome of round and elongated spermatids. These results indicate a role for carbohydrates in spermiogenesis. Apical Sertoli cells processes and Leydig cells were weakly stained with PSA and LCA as well. DBA binding sites were also seen in the Leydig cells. Immunohistochemical studies were performed using the Avidin-Biotin-Peroxidase Complex (ABC) method for localization of fibroblast growth factor-1 (FGF-1), fibroblast growth factor-2 (FGF-2), S-100, laminin, alpha-smooth muscle actin (á -SMA), vascular endothelial growth factor (VEGF), connexin 43 (Cx43), CD4, CD8, CD68, angiotensin-converting enzyme (ACE), and galactosyltransferase (GalTase) in the bovine testis. The expression of FGF-1 and FGF-2 was further investigated in the adult bovine testis using in situ hybridization and PCR. Immunohistochemically, FGF-1 was seen in the Sertoli cells, Leydig cells, endothelium of the blood vessels, and epithelium of straight tubules and rete testis of fetal and adult testis. It was additionally detected in spermatogonia and spermatids of sexual mature animals. FGF-2 exhibited a striking positive reaction in fetal (from 6 to 30 cm CRL) and adult Leydig cells. Moreover, it showed marked reaction in the endothelium of blood vessels and in the epithelium of tubulus rectus and rete testis. FGF-2 was also localized in some spermatogonia, and myofibroblasts. By means of in situ hybridization, FGF-1 and FGF-2 mRNA were found in Leydig and Sertoli cells as well as in the modified Sertoli cells of the terminal segment. FGF-1 transcripts were additionally recognized in the straight tubules and rete testis epithelium. Distinct S100 immunostaining was observed in the Sertoli cells, endothelium of blood vessels and in the rete testis epithelium of fetal and adult testis. Laminin was localized to the basal lamina of seminiferous tubules, blood vessels, myofibroblasts, and rete testis. Although á -SMA was detected in smooth muscle cells of the blood vessels, no immunoreactivity was seen in the peritubular cells during the whole gestation period. The myofibroblasts surrounding the seminiferous tubules and rete testis showed intense positive reaction for á -SMA in the adult testis. VEGF was detected in the acrosomes of the elongating spermatids. Connexin 43 was localized to gap junctions between Leydig cells in the fetal and adult life as well as to the seminiferous epithelium apical to spermatogonia and basal to spermatocytes, a position correlating with Sertoli-Sertoli cell junctions. The detection of cells positive for CD4, CD8, CD68 within the adult testis interstitium clearly indicate the presence of lymphocytes and macrophages within this testicular compartment. GalTase showed striking positive reaction in the Golgi complex of Sertoli cells, Leydig cells, and some spermatocytes as well as at the cell membrane of elongating spermatids and in the simple cuboidal epithelium of rete testis. ACE positive reaction was found in the prespermatogonia (only at 6-10 cm CRL) and in fetal and adult testicular blood vessels. The functional significance of these immunocytochemically-demonstrated proteins is discussed

Validation of a method to elute viruses from different types of face masks

Due to the SARS-CoV-2 pandemic, it is crucial to study the efficiency of face masks in retaining viruses for the upcoming years. The first objective of this study was to validate a method to elute viruses from polyester and cotton face masks. We observed that deionized water followed by 3% beef glycine (pH 9.5 or pH 7.2) was significantly more efficient (p < 0.05) in eluting the bacteriophage phiX174 virus from polyester (4.73% ± 0.25% to 28.67% ± 1.89%), polyester/cotton (3% ± 0.33%), and cotton (1.7% ± 0.21%) face masks than 3% beef glycine only (pH 9.5 or pH 7.2) as a single eluent (3.4% ± 0.16% to 21.33% ± 0.94% for polyester, 1.91% ± 0.08% for polyester/cotton, and 1.47% ± 0.12% for cotton face masks). Also, deionized water was significantly less efficient as a single eluent for eluting bacteriophage phiX174 from all the studied face mask types. The polyethylene glycol (PEG) precipitation method was substantially more efficient (p < 0.05) as a second step concentration method for the viruses in the eluates than the organic flocculation (OF) method. Higher viral loads were eluted from polyester face masks than cotton ones. We also found varying viral loads in the eluate solutions from different commercial polyester face masks, with the highest percentage seen for the N95 face mask. The second objective was to apply the validated method to study the effect of autoclaving on the different face mask materials. Results of the study did not show any significant differences in the viral loads eluted from the studied face masks before and after one and five autoclaving cycles. Moreover, a scanning electron microscope (SEM) analysis revealed no changes in the yarns, elongation, tensile strength, and contact angle measurements of the polyester or cotton materials after one or five autoclaving cycles

Amelioration effect of 18β-Glycyrrhetinic acid on methylation inhibitors in hepatocarcinogenesis -induced by diethylnitrosamine

Aimsuppression of methylation inhibitors (epigenetic genes) in hepatocarcinogenesis induced by diethylnitrosamine using glycyrrhetinic acid.MethodIn the current work, we investigated the effect of sole GA combined with different agents such as doxorubicin (DOX) or probiotic bacteria (Lactobacillus rhamanosus) against hepatocarcinogenesis induced by diethylnitrosamine to improve efficiency. The genomic DNA was isolated from rats’ liver tissues to evaluate either methylation-sensitive or methylation-dependent resection enzymes. The methylation activity of the targeting genes DLC-1, TET-1, NF-kB, and STAT-3 was examined using specific primers and cleaved DNA products. Furthermore, flow cytometry was used to determine the protein expression profiles of DLC-1 and TET-1 in treated rats’ liver tissue.ResultsOur results demonstrated the activity of GA to reduce the methylation activity in TET-1 and DLC-1 by 33.6% and 78%, respectively. As compared with the positive control. Furthermore, the association of GA with DOX avoided the methylation activity by 88% and 91% for TET-1 and DLC-1, respectively, as compared with the positive control. Similarly, the combined use of GA with probiotics suppressed the methylation activity in the TET-1 and DLC-1 genes by 75% and 81% for TET-1 and DLC-1, respectively. Also, GA and its combination with bacteria attenuated the adverse effect in hepatocarcinogenesis rats by altering potential methylomic genes such as NF-kb and STAT3 genes by 76% and 83%, respectively.ConclusionGA has an ameliorative effect against methylation inhibitors in hepatocellular carcinoma (HCC) by decreasing the methylation activity genes

Xeno-free trans-differentiation of adipose tissue-derived mesenchymal stem cells into glial and neuronal cells.

Mesenchymal stem cells (MSCs) are undifferentiated cells that have the ability of self-renewal and trans-differentiation into other cell types. They hold out hope for finding a cure for many diseases. Nevertheless, there are still some obstacles that limit their clinical transplantation. One of these obstacles are the xenogeneic substances added in either proliferation or differentiation media with subsequent immunogenic and infectious transmission problems. In this study, we aimed to replace fetal bovine serum (FBS), the main nutrient source for MSC proliferation with xeno-free blood derivatives. We tested the effect of human activated pure platelet-rich plasma (P-PRP) and advanced platelet-rich fibrin (A-PRF) on the proliferation of human adipose derived-MSCs (AD-MSCs) at different concentrations. For the induction of MSC neural differentiation, we used human cerebrospinal fluid (CSF) at different concentrations in combination with P-PRP to effect xeno-free/species-specific neuronal/glial differentiation and we found that media with 10% CSF and 10% PRP promoted glial differentiation, while media with only 10% PRP induced a neuron-like phenotype

Gene Expression Profiling of Embryonic Human Neural Stem Cells and Dopaminergic Neurons from Adult Human Substantia Nigra

Neural stem cells (NSC) with self-renewal and multipotent properties serve as an ideal cell source for transplantation to treat neurodegenerative insults such as Parkinson's disease. We used Agilent's and Illumina Whole Human Genome Oligonucleotide Microarray to compare the genomic profiles of human embryonic NSC at a single time point in culture, and a multicellular tissue from postmortem adult substantia nigra (SN) which are rich in dopaminergic (DA) neurons. We identified 13525 up-regulated genes in both cell types of which 3737 (27.6%) genes were up-regulated in the hENSC, 4116 (30.4%) genes were up-regulated in the human substantia nigra dopaminergic cells, and 5672 (41.93%) were significantly up-regulated in both cell population. Careful analysis of the data that emerged using DAVID has permitted us to distinguish several genes and pathways that are involved in dopaminergic (DA) differentiation, and to identify the crucial signaling pathways that direct the process of differentiation. The set of genes expressed more highly at hENSC is enriched in molecules known or predicted to be involved in the M phase of the mitotic cell cycle. On the other hand, the genes enriched in SN cells include a different set of functional categories, namely synaptic transmission, central nervous system development, structural constituents of the myelin sheath, the internode region of axons, myelination, cell projection, cell somata, ion transport, and the voltage-gated ion channel complex. Our results were also compared with data from various databases, and between different types of arrays, Agilent versus Illumina. This approach has allowed us to confirm the consistency of our obtained results for a large number of genes that delineate the phenotypical differences of embryonic NSCs, and SN cells

Transcription factors expressed in embryonic and adult olfactory bulb neural stem cells reveal distinct proliferation, differentiation and epigenetic control

AbstractTF genomic markers associated with neurogenesis, proliferation, differentiation, and epigenetic control in human embryonic neural stem cells (hENSC(, and adult human olfactory bulb neural stem cells (OBNSC) were studied by immunohistochemistry (IHC) and DNA microarray. The biological impact of TF gene changes in the examined cell types was estimated using DAVID to specify a different GO class and signaling pathway based on KEGG database. Eleven, and twenty eight TF genes were up-regulated (fold change≤2–39) in OBNSC, and hENSC respectively. KEGG pathway analysis for the up-regulated TF genes revealed significant enrichments for the basal transcription factor pathway, and Notch signaling pathway in OBNSCs, and hENSCs, respectively. Immunofluorescence analysis revealed a significantly greater number of β-tubulin III (TUBB3), MAP, glial fibrillary acidic protein (GFAP), and O4 in hENSC when compared to those in OBNSC. Furthermore, the expression of epigenetic-related TF-genes SMARCC1, TAF12, and UHRF1 increased significantly in OBNSC when compared with hENSC

Morphological Features of the Testis among Autoimmune Mouse Model and Healthy Strains

Autoimmune diseases play a critical role in the progression of infertility in both sexes and their severity has been reported to increase with age. However, few reports have discussed their effect on the morphological features of the testis. Therefore, we compared the morphological alterations in the testes of autoimmune model mice (MRL/MpJ-Fas(lpr)) and the control strain (MRL/MpJ) with those of their background strain (C57BL/6N) at 3 and 6 months. Furthermore, we analyzed the changes in spermatocytes, Sertoli cells, immune cells, and Zonula occludens-1 junctional protein by immunohistochemical staining. The MRL/MpJ-Fas(lpr) mice showed a significant increase in the serum Anti-double stranded DNA antibody level, relative spleen weight, and seminiferous luminal area when compared with other studied two strains. In contrast, a significant decrease in the relative testis weight, and numbers of both Sertoli, meiotic spermatocyte was observed in MRL/MpJ-Fas(lpr) and MRL/MpJ mice compared with C57BL/6N mice especially at 6 months. Similarly, Zonula occludens-1 junctional protein positive cells showed a significant decrease in the same strains at 6 months. However, no immune cell infiltration could be observed among the studied three strains. Our findings suggest that the increase in autoimmune severity especially with age could lead to infertility through loss of spermatogenic and Sertoli cells, rather than the disturbance of the blood-testis barrier

Melissa officinalis extract palliates redox imbalance and inflammation associated with hyperthyroidism-induced liver damage by regulating Nrf-2/ Keap-1 gene expression in γ-irradiated rats

Abstract Background Melissa officinalis (MO) is a well-known medicinal plant species used in the treatment of several diseases; it is widely used as a vegetable, adding flavour to dishes. This study was designed to evaluate the therapeutic effect of MO Extract against hyperthyroidism induced by Eltroxin and γ-radiation. Methods Hyperthyroidism was induced by injecting rats with Eltroxin (100 µg/kg/ day) for 14 days and exposure to γ-radiation (IR) (5 Gy single dose). The hyperthyroid rats were orally treated with MO extract (75 mg/kg/day) at the beginning of the second week of the Eltroxin injection and continued for another week. The levels of thyroid hormones, liver enzymes and proteins besides the impaired hepatic redox status and antioxidant parameters were measured using commercial kits. The hepatic gene expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and its inhibitor Kelch-like ECH-associated protein-1(Keap-1) in addition to hepatic inflammatory mediators including tumor necrosis factor-α (TNF- α), Monocyte chemoattractant protein-1 (MCP-1) and fibrogenic markers such as transforming growth factor-beta1 (TGF-β1) were determined. Results MO Extract reversed the effect of Eltroxin + IR on rats and attenuated the thyroid hormones. Moreover, it alleviated hyperthyroidism-induced hepatic damage by inhibiting the hepatic enzymes’ activities as well as enhancing the production of proteins concomitant with improving cellular redox homeostasis by attenuating the deranged redox balance and modulating the Nrf2/Keap-1 pathway. Additionally, MO Extract alleviated the inflammatory response by suppressing the TNF- α and MCP-1 and prevented hepatic fibrosis via Nrf2-mediated inhibition of the TGF-β1/Smad pathway. Conclusion Accordingly, these results might strengthen the hepatoprotective effect of MO Extract in a rat model of hyperthyroidism by regulating the Nrf-2/ Keap-1 pathway

Effects of estrogen on Survival and Neuronal Differentiation of adult human olfactory bulb neural stem Cells Transplanted into Spinal Cord Injured Rats

In the present study we developed an excitotoxic spinal cord injury (SCI) model using kainic acid (KA) to evaluate of the therapeutic potential of human olfactory bulb neural stem cells (h-OBNSCs) for spinal cord injury (SCI). In a previous study, we assessed the therapeutic potential of these cells for SCI; all transplanted animals showed successful engraftment. These cells differentiated predominantly as astrocytes, not motor neurons, so no improvement in motor functions was detected. In the current study we used estrogen as neuroprotective therapy before transplantation of OBNSCs to preserve some of endogenous neurons and enhance the differentiation of these cells towards neurons. The present work demonstrated that the h-GFP-OBNSCs were able to survive for more than eight weeks after sub-acute transplantation into injured spinal cord. Stereological quantification of OBNSCs showed approximately a 2.38-fold increase in the initial cell population transplanted. 40.91% of OBNSCs showed differentiation along the neuronal lineages, which was the predominant fate of these cells. 36.36% of the cells differentiated into mature astrocytes; meanwhile 22.73% of the cells differentiated into oligodendrocytes. Improvement in motor functions was also detected after cell transplantation