Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch.

Male factor infertility is a problem in today's society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GCspecific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., Dmrtb1, Sohlh1). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., Amh, Fshr). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation

Histamine 2 Receptor Agonism and Histamine 4 Receptor Antagonism Ameliorate Inflammation in a Model of Psoriasis

Psoriasis is a chronic inflammatory skin disorder characterized by hyperproliferative keratinocytes and immune cell infiltration into the skin, often accompanied by itch. Histamine, acting via histamine 1–4 receptors, is known to modulate immune responses in the skin and to induce itch. The aim of this study was to test the role of histamine 2 receptors and histamine 4 receptors in the imiquimod-induced psoriasis-like skin inflammation model. BALB/c mice were treated intraperitoneally with amthamine (histamine 2 receptor agonist), JNJ-39758979 (histamine 4 receptor antagonist), a combination of both, or vehicle twice daily in a preventive manner. Imiquimod was applied once daily onto the back skin for 10 consecutive days. Stimulation of histamine 2 receptors and blockade of histamine 4 receptors ameliorated imiquimod-induced skin inflammation. The combination of amthamine and JNJ-39758979 reduced skin inflammation even more pronounced, diminished epidermal hyperproliferation, and inhibited spontaneous scratching behaviour. A combination of histamine 2 receptor agonist and histamine 4 receptor antagonists could represent a new strategy for the treatment of psoriasis

Analysis of connexin 43, connexin 45 and N-cadherin in the human sertoli cell line FS1 and the human seminoma-like cell line TCam-2 in comparison with human testicular biopsies

Background: Germ cell tumors are relatively common in young men. They derive from a non-invasive precursor, called germ cell neoplasia in situ, but the exact pathogenesis is still unknown. Thus, further understanding provides the basis for diagnostics, prognostics and therapy and is therefore paramount. A recently developed cell culture model consisting of human FS1 Sertoli cells and human TCam-2 seminoma-like cells offers new opportunities for research on seminoma. Since junctional proteins within the seminiferous epithelium are involved in cell organization, differentiation and proliferation, they represent interesting candidates for investigations on intercellular adhesion and communication in context with neoplastic progression. Methods: FS1 and TCam-2 cells were characterized regarding gap-junction-related connexin 43 (Cx43) and connexin 45 (Cx45), and adherens-junction-related N-cadherin using microarray, PCR, Western blot, immunocytochemistry and immunofluorescence. Results were compared to human testicular biopsies at different stages of seminoma development via immunohistochemistry to confirm the cell lines’ representativeness. Furthermore, dye-transfer measurements were performed to investigate functional cell coupling. Results: Cx43, Cx45 and N-cadherin mRNA and protein were generally detectable in both cell lines via qualitative RT-PCR and Western blot. Immunocytochemistry and immunofluorescence revealed a mainly membrane-associated expression of N-cadherin in both cell lines, but gene expression values were higher in FS1 cells. Cx43 expression was also membrane-associated in FS1 cells but barely detectable in TCam-2 cells. Accordingly, a high gene expression value of Cx43 was measured for FS1 and a low value for TCam-2 cells. Cx45 was primary located in the cytoplasm of FS1 and TCam-2 cells and revealed similar low to medium gene expression values in both cell lines. Overall, results were comparable with corresponding biopsies. Additionally, both FS1 and TCam-2 cells showed dye diffusion into neighboring cells. Conclusion: The junctional proteins Cx43, Cx45 and N-cadherin are expressed in FS1 and TCam-2 cells at mRNA and/or protein level in different amounts and localizations, and cells of both lines are functionally coupled among each other. Concerning the expression of these junctional proteins, FS1 and TCam-2 cells are largely representative for Sertoli and seminoma cells, respectively. Thus, these results provide the basis for further coculture experiments evaluating the role of junctional proteins in context with seminoma progression

Influence of Reduced Protein Content in Complete Diets with a Consistent Arginine–Lysine Ratio on Performance and Nitrogen Excretion in Broilers

The current discussion concerning resource-efficient broiler production inevitably leads to diets with lowered crude protein (CP) levels. Therefore, the hypothesis was formed that crude protein reduction far below the recommended levels can significantly lower the nitrogen (N) content in litter, if essential amino acids are added and a constant lysine-arginine ratio is guaranteed. In a five-week feeding trial, 360 ROSS 308 broilers of both sexes were randomly assigned to four feeding groups with six replicates each with a standard three-phase feeding program (d 1⁻7, d 8⁻14, d 15⁻35). The control group was offered a complete diet with a common protein content found in practice (CP-% as fed; starter: 21.5, grower: 20.5, finisher: 20.0; lysine/arginine: 100/115). In the experimental diets the lysine/arginine ratio was constant, whereas the protein content was lowered in steps of 1.00 percent each with simultaneous supplementation of growth limiting amino acids. Feeding a diet with a 2.00 percent reduced protein content led to higher body weights after 34 days compared to the control (2329 g vs. 2192 g). The N content in the total litter decreased significantly with a 2.00 and 3.00 percent reduction in the CP content (51.2 vs. 46.2 or rather 36.2 g/kg dry matter (DM)). Meticulous balanced protein-reduced diets therefore allow a significant environmental relief

Feed Choice Led to Higher Protein Intake in Broiler Chickens Experimentally Infected With Campylobacter jejuni

In 2016, Campylobacter was the most commonly reported gastrointestinal bacterial pathogen in humans in the European Union with 246,307 reported cases. Of these cases, 83.6% were Campylobacter jejuni. The objective of the present study was to investigate to what extent an infection with C. jejuni alters the feed intake behavior of broiler chicks in terms of protein intake. This was done to see if, conversely, measures of control could be derived. In total, 300 commercial broilers of the Ross 308 line were allocated to four different groups, including five replications of 15 chickens each. In two groups, a conventional diet [216 g CP/kg dry matter (DM)] and in the two choice diet groups, diets with different levels of crude protein (286 and 109 g CP/kg DM, respectively) were fed between day 14 and day 42. An intake of both choice diets at a ratio of 3:2 resulted in a composition of consumed feed identical to that of the control concerning composition, energy and nutrient content. One group of each feeding concept was infected artificially with C. jejuni at day 21 by applying an oral C. jejuni-suspension containing 5.26 ± 0.08 log10 colony forming units of C. jejuni to three out of 15 chickens. No significant differences concerning C. jejuni prevalence and excretion could be seen. Broilers infected with C. jejuni chose a higher amount of the high protein choice diet in comparison to C. jejuni negative broilers. This resulted in a significantly (p < 0.0001) higher content of crude protein in the consumed diet (198 ± 3.09 g CP/kg DM and 208 ± 8.57 g CP/kg DM, respectively). Due to C. jejuni infection, a significant increase in crude mucin in excreta at day 42 was seen in experimentally infected groups (62.6 ± 4.62 g/kg DM vs. 59.6 ± 6.21 g/kg DM, respectively; p = 0.0396). There were significantly deeper crypts in infected birds (256 ± 71.6 vs. 234 ± 61.3 μm). In summary, C. jejuni infections significantly alter the feed intake behavior of broiler chickens regarding higher protein intake. Therefore, targeted manipulation of protein supply could be tested for limiting the spread of infection

Establishment and functional characterization of a murine primary Sertoli cell line deficient of connexin43

The Sertoli cell (SC) specific connexin43 (Cx43) knockout (SCCx43KO) mouse line is ideal to gain insight into the mechanistic gap junction formation in SC and the seminiferous epithelium. A method for developing primary SC cultures from these mice was established, validated and successfully characterized via polymerase chain reaction, immunohistochemistry, immunofluorescence (IF), and Western blots (WB). It was evident that both knockout (KO) and wild-type (WT) primary cell cultures were similar in morphology. These highly pure SC cultures were subjected to cell proliferation assays indicating no notable proliferation in cultures of both genotypes. Measurements of cell monolayer integrity indicated significant increases in transepithelial electrical resistance and consequently in tight junction expression of the KO cultures. Using semi-quantitative WB and IF, tight junction protein claudin-11 was analyzed. These results support a role for Cx43 in regulating blood-testis barrier (BTB) function, composition, and dynamics in vitro. Thus, the SC deficient Cx43 cell cultures may provide a valuable in vitro tool for a better understanding of the mechanistic role of Cx43 in spermatogenesis and BTB assembly

Influence of Different Methionine Sources on Performance and Slaughter Characteristics of Broilers

Sustainably produced poultry meat with consideration of animal health poses a challenge for broiler production. Low protein diets with high amounts of synthetic amino acids (AAs) like methionine (Met) are the consequence. In a five-week feeding trial, 360 broilers (Ross 308) assigned to four feeding groups were offered protein-reduced complete diets (starter: 20% crude protein (CP); grower: 18.5% CP; finisher: 17.5% CP), supplemented with essential AAs. The “MHA” group received DL-2-hydroxy-4-(methylthio) butanoic acid (DL-HMTBA; trade name: MHA®), groups “L” and “DL” the respective Met source in equivalent concentrations each exceeding the nutritional recommendations. “R-MHA” (“R” for “reduced”) received the minimum required level (using MHA as Met source). Performance exceeded performance goals without differences between the groups. The average feed conversion ratio (FCR) amounted to 1.35. The carcass/body weight ratio of R-MHA was significantly lower (0.782) compared to DL (0.808) and L (0.809). Breast meat of R-MHA contained significantly more fat (144 g/kg dry matter (DM)) compared to L (104 g/kg DM) and significantly lower CP content (R-MHA: 838 g/kg DM; L: 875 g/kg DM). The results indicated possible improvement in slaughter yield by protein-reduced diets supplemented with L-Met, thus recommending further research focusing on the Met influence

Evaluation of Methionine Sources in Protein Reduced Diets for Turkeys in the Late Finishing Period Regarding Performance, Footpad Health and Liver Health

Footpad dermatitis and hepatic lipidosis are health problems in fattening turkeys where a positive influence of higher methionine content in feed is discussed. The effects of the methionine supplements DL-methionine (DLM) and liquid methionine hydroxyl analogue free acid (MHA-FA) under the aspect of low protein diets were investigated in this study based on performance parameters, footpad health, liver health and oxidative stress. In this study, 80 female turkeys (B.U.T. Big 6) of 63 day-old, were randomly assigned to four groups characterising a 2 × 2 factorial design with five replicates each over five weeks. The groups were fed with diets differing in methionine source (DLM vs. MHA-FA, assuming a biological activity of MHA-FA of 65%) and crude protein content (15% vs. 18%) for 35 days. The results showed no significant interactions between the protein content and methionine source. Strong protein reduction significantly impaired water intake, feed intake, weight gain and feed conversation ratio, but improved footpad health. DLM and MHA-FA addition had no significant effect on weight gain, crude fat and protein contents in the liver, but DLM resulted in a significant increase in livers antioxidative capacity compared to MHA-FA. Although the protein reduction resulted in reduced performance, the study showed that MHA-FA can be replaced by DLM in a 100:65 weight ratio without compromising performance but with certain advantages in the antioxidative capacity of the liver

Loss of Cx43 in Murine Sertoli Cells Leads to Altered Prepubertal Sertoli Cell Maturation and Impairment of the Mitosis-Meiosis Switch.

Male factor infertility is a problem in today's society but many underlying causes are still unknown. The generation of a conditional Sertoli cell (SC)-specific connexin 43 (Cx43) knockout mouse line (SCCx43KO) has provided a translational model. Expression of the gap junction protein Cx43 between adjacent SCs as well as between SCs and germ cells (GCs) is known to be essential for the initiation and maintenance of spermatogenesis in different species and men. Adult SCCx43KO males show altered spermatogenesis and are infertile. Thus, the present study aims to identify molecular mechanisms leading to testicular alterations in prepubertal SCCx43KO mice. Transcriptome analysis of 8-, 10- and 12-day-old mice was performed by next-generation sequencing (NGS). Additionally, candidate genes were examined by qRT-PCR and immunohistochemistry. NGS revealed many significantly differentially expressed genes in the SCCx43KO mice. For example, GCspecific genes were mostly downregulated and found to be involved in meiosis and spermatogonial differentiation (e.g., Dmrtb1, Sohlh1). In contrast, SC-specific genes implicated in SC maturation and proliferation were mostly upregulated (e.g., Amh, Fshr). In conclusion, Cx43 in SCs appears to be required for normal progression of the first wave of spermatogenesis, especially for the mitosis-meiosis switch, and also for the regulation of prepubertal SC maturation

Expression of Tight Junction Proteins and Cadherin 17 in the Small Intestine of Young Goats Offered a Reduced N and/or Ca Diet

<div><p>Diets fed to ruminants should contain nitrogen (N) as low as possible to reduce feed costs and environmental pollution. Though possessing effective N-recycling mechanisms to maintain the N supply for rumen microbial protein synthesis and hence protein supply for the host, an N reduction caused substantial changes in calcium (Ca) and phosphate homeostasis in young goats including decreased intestinal transepithelial Ca absorption as reported for monogastric species. In contrast to the transcellular component of transepithelial Ca transport, the paracellular route has not been investigated in young goats. Therefore, the aim of the present study was to characterise the effects of dietary N and/or Ca reduction on paracellular transport mechanisms in young goats. Electrophysiological properties of intestinal epithelia were investigated by Ussing chamber experiments. The expression of tight junction (TJ) and adherens junction (AJ) proteins in intestinal epithelia were examined on mRNA level by <i>q</i>PCR and on protein level by western blot analysis. Dietary N reduction led to a segment specific increase in tissue conductances in the proximal jejunum which might be linked to concomitantly decreased expression of cadherin 17 mRNA. Expression of occludin (OCLN) and zonula occludens protein 1 was increased in mid jejunal epithelia of N reduced fed goats on mRNA and partly on protein level. Reduced dietary Ca supply resulted in a segment specific increase in claudin 2 and claudin 12 expression and decreased the expression of OCLN which might have been mediated at least in part by calcitriol. These data show that dietary N as well as Ca reduction affected expression of TJ and AJ proteins in a segment specific manner in young goats and may thus be involved in modulation of paracellular Ca permeability.</p></div