Following the downregulation of testicular endocrine and germinative function by slow-release gonadotropin-releasing hormone (GnRH)-agonist implants, testicular functions are quickly restored after implant removal. As an intact blood-testis barrier (BTB) is crucial for normal spermatogenesis and its integrity is FSH- and androgen-dependent, alterations in the BTB gene and protein expressions during downregulation and subsequent restart seem inevitable. We investigated occludin (OCLN), claudin (CLDN) 3, 5, 11, and connexin (CX) 43 mRNA-, and CLDN11 and CX43 protein expressions during GnRH implant-induced downregulation (W0) and restart of spermatogenesis after implant removal (week, W, 3-12). Untreated juvenile (JG) and adult dogs (CG) served as controls. Sertoli cells were significantly affected by treatment (reduced nuclear area, OCLN, and CLDN5 expressions). All investigated genes (except CLDN3) differed significantly during restart (W0-12) compared with CG (p < 0.05). CLDN11 and CX43 immunopositive staining was absent or diffuse cytoplasmic at downregulation and relocated at W9, indicating disruption and subsequent restorage of BTB. As W0 and JG differed considerably, our results suggest that the model cannot mimic puberty. In conclusion, GnRH implant-induced long-term gonadotropin suppression disrupts testicular CX43 and CLDN11 distribution and changes gap and tight junction mRNA expression. Treatment effects are reversible suggesting re-establishment of the BTB.

Body size represents a complex phenotype driven by genetic variation and epigenetic regulation, with the molecular processes underlying this trait remaining a central challenge to disentangle. To elucidate these fundamental mechanisms, we apply a multi-omics approach that combines ROH-based selection mapping with growth plate epigenomics in pigs. Taking advantage of divergent selection that separates pigs into miniature and larger-sized groups, we target genomic regions under this intense selection for height, which harbour functional variants with pronounced effects. We assemble a multi-omics dataset, identifying homozygous alternative SNPs in Aachen Minipigs and Mini-LEWE predicted to affect cis-regulatory elements potentially interacting with differentially expressed genes that drive body size in breed-dependent ways. Our results point to an lncRNA (ENSSSCG00000048200) near SDR16C5 and PLAG1, HPX and NET-related pathways, as central players in the growth plates. In summary, our study offers a multi-layered characterisation of regulatory mechanisms in the growth plates in the pig model.

Genetically induced loss of the gap-junction protein Connexin 43 (Cx43) in murine Sertoli cells leads to an arrest of spermatogenesis at the level of spermatogonia, highly vacuolated tubules, and intratubular cell clusters. Transmission electron microscopy as well as 3D-reconstruction of Sertoli cells based on serial block-face scanning electron microscopy imaging revealed severe cell shape changes in Cx43 deficient Sertoli cells. Since the cytoskeleton is important for the transport of germ cells within the seminiferous epithelium and for keeping the cell shape, the study at hand aimed to reveal correlations of Cx43 loss and changes of cytoskeletal components and their spatial organization in the seminiferous epithelium. Immunohistochemistry, immunofluorescence, conventional transmission electron microcopy and immunogold labeling indicated alterations in microtubule and actin filament distribution patterns in Cx43 deficient Sertoli cells compared to wildtype mice. Firstly, microtubules seemed to be misoriented in mutant Sertoli cells. Secondly, the actin filament based basal ectoplasmic specializations were increased in spatial extension, but the apical ectoplasmic specialization was missing. Lastly, Sertoli cells of both genotypes immunostained positive for vimentin, the prevalent intermediate filament of Sertoli cells, but not for keratins, markers for Sertoli cell immaturity or dedifferentiation. In conclusion, Cx43 deficiency in Sertoli cells correlates not only with severe cell shape alterations but also with changes in microtubule and actin filament distribution patterns, while intermediate filament expression seems to be only negligibly influenced.

The giant anteater (superorder Xenarthra) is listed as Vulnerable by the International Union for Conservation of Nature (IUCN) and a low reproductive rate is considered one of the factors contributing to population decline of the species. Nevertheless, little is known on reproductive features in male giant anteaters and, in this regard, microscopic testis morphology and spermatogenesis were studied in roadkill specimens in Brazil. Characteristics of germ cell populations resembled descriptions in other eutherian mammals including other xenarthran species. Furthermore, eight stages of the seminiferous epithelium cycle could be defined. The proposed staging system offers baseline data for assessing impairments or seasonal changes in spermatogenesis and thus allows future studies on reproductive health and reproductive seasonality in male giant anteaters.

Adult male Sertoli cell-specific Connexin43 knockout mice (SCCx43KO) exhibit higher Sertoli cell (SC) numbers per seminiferous tubule compared to their wild type (WT) littermates. Thus, deletion of this testicular gap junction protein seems to affect the proliferative potential and differentiation of "younger" SC. Although SC have so far mostly been characterised as postmitotic cells that cease to divide and become an adult, terminally differentiated cell population at around puberty, there is rising evidence that there exist exceptions from this for a very long time accepted paradigm. Aim of this study was to investigate postnatal SC development and to figure out underlying causes for observed higher SC numbers in adult KO mice. Therefore, the amount of SC mitotic figures was compared, resulting in slightly more and prolonged detection of SC mitotic figures in KO mice compared to WT. SC counting per tubular cross section revealed significantly different time curves, and comparing proliferation rates using Bromodesoxyuridine and Sox9 showed higher proliferation rates in 8-day old KO mice. SC proliferation was further investigated by Ki67 immunohistochemistry. SC in KO mice displayed a delayed initiation of cell-cycle-inhibitor p27Kip1 synthesis and prolonged synthesis of the phosphorylated tumour suppressor pRb and proliferation marker Ki67. Thus, the higher SC numbers in adult male SCCx43KO mice may arise due to two different reasons: Firstly, in prepubertal KO mice, the proliferation rate of SC was higher. Secondly, there were differences in their ability to cease proliferation as shown by the delayed initiation of p27Kip1 synthesis and the prolonged production of phosphorylated pRb and Ki67. Immunohistochemical results indicating a prolonged period of SC proliferation in SCCx43KO were confirmed by detection of proliferating SC in 17-days-old KO mice. In conclusion, deletion of the testicular gap junction protein Cx43 might prevent normal SC maturation and might even alter also the proliferation potential of adult SC.

The southern tamandua (Tamandua tetradactyla) and the giant anteater (Myrmecophaga tridactyla) belong to the anteater family Myrmecophagidae and both species share basic morphological characteristics including the general features of reproductive organs. However, in female and male giant anteaters, persisting Wolffian and Müllerian ducts have been observed that have not been described in the southern tamandua, so far. Therefore, the present study evaluated whether those persisting genital ducts of the opposite sex can be observed in the southern tamandua as well. For this purpose, the reproductive organs of adult roadkill male and female specimens were studied in Brazil. In female southern tamanduas, persisting Wolffian ducts extended from the opening of the uterovaginal canal into the sinus urogenitalis in cranial direction through the ventral wall of the uterovaginal canal and the uterus and followed the course of the uterine tubes until the lateral pole of the ovaries. Those ducts showed the same characteristics as described in giant anteaters and revealed similarities to male epididymal and deferent ducts. Furthermore, glandular structures in the wall of the urethra and the sinus urogenitalis were observed that showed microscopic characteristics corresponding to male prostate and bulbourethral glands, similarly to observations in female giant anteaters. In male southern tamanduas, on the contrary, only rudimentary tubules were found in the prostatic urethral wall while well-differentiated Müllerian ducts have been previously described in the male giant anteater. In conclusion, well-developed Wolffian ducts are a shared characteristic in both female southern tamanduas and female giant anteaters whereas well-developed Müllerian ducts are unique to male giant anteaters and only rudimentary Müllerian vestiges were observed in the male southern tamandua. Data on those persisting genital ducts are of interest for studies on reproductive biology and physiology of southern tamanduas and sexual development of mammalian species in general.

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.

In a pig model, pancreatic duct ligation (PL) leads to a complete loss of exocrine function, causing an exocrine pancreatic insufficiency (EPI) without affecting endocrine function, allowing research of clinical effects and therapy options. This study aimed to investigate effects of experimentally induced EPI in juvenile pigs on digestion and intestinal morphology. Eight female juvenile cross-bred pigs (BW 54.8 kg at the start of the study) were included. Three animals were considered as a control (CON group), and in five animals the ductus pancreaticus accessorius was ligated (PL group). During the 10-week trial period, body weight and body measurements were recorded regularly. At the end of the trial, gastrointestinal tract (GIT) was investigated macroscopically and histologically and weight and digesta samples of individual segments were obtained. The pigs in the CON showed a significantly higher apparent total tract digestibility of crude protein and crude fat (87.8 and 79.9%, respectively) compared to PL (52.4 and 16.6%, respectively). Significant differences were noted in relative weights of duodenum, jejunum and colon (with and without digesta) and also in absolute weights of jejunum and colon. The mean number of nuclei in the transverse section in stratum circulare were significantly higher in all intestinal segments in CON compared to PL. Overall, EPI results in impaired nutrient digestibility with a greater filling of the GIT with digesta. The elongation of the small intestine does not represent “stretching” of the intestine, but rather increased synthesis of intestinal tissue.

Knowledge of reproductive health in wild southern tamanduas (Tamandua tetradactyla; Mammalia: Myrmecophagidae) is fragmentary. During necropsies of roadkill xenarthran species in Brazil, a case of ovarian filariasis in an adult female southern tamandua was observed. Macroscopically, both ovaries were irregularly enlarged and had numerous smooth protuberances. Histologically, the affected ovarian parenchyma presented adult nematodes (including females with microfilaria) surrounded by pleocellular inflammatory infiltrates. The morphological characteristics of the nematodes were consistent with the superfamily Filarioidea (order Spirurida). The adjacent ovarian parenchyma had developing and atretic follicles at different stages of maturation. Filarial nematodes were not observed in other tissues. The cause of death of this tamandua was fatal acute polytrauma as a consequence of the motor vehicle collision. This case adds to a prior report of ovarian filariasis in two southern tamanduas in Nicaragua and Guatemala, dating back almost 100 years, and suggests filarial infections could potentially have an impact on reproductive success in southern tamanduas and possibly other xenarthrans. Several xenarthran species are under different levels of threat and knowledge of their basic reproductive health is crucial for conservation programs.

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) was shown to be an effector of multiple histological changes in tubular morphology, resulting in germ cell loss through to a Sertoli-cell-only (SCO) phenotype and vacuolated seminiferous tubules containing SC-clusters. Our present study focused on the effects of Cx43 loss on SC ultrastructure. Using serial block-face scanning electron microscopy (SBF-SEM), we could confirm previous results. Ultrastructural analysis of Sertoli cell nuclei (SCN) revealed that these appear in clusters with a phenotype resembling immature/proliferating SCs in KO mice. Surprisingly, SCs of fertile wild type (WT) mice contained SCN with a predominantly smooth surface instead of deep indentations of the nuclear envelope, suggesting that these indentations do not correlate with germ cell support or spermatogenesis. SBF-SEM facilitated the precise examination of clustered SCs. Even if the exact maturation state of mutant SCs remained unclear, our study could detect indications of cellular senescence as well as immaturity, emphasising that Cx43 affects SC maturation. Moreover, Sudan III staining and transmission electron microscopy (TEM) demonstrated an altered lipid metabolism in SCs of Cx43 deficient mice.

The eukaryotic initiation factor 4E binding protein (4E-BP) family is involved in translational control of cell proliferation and pro-angiogenic factors. The zebrafish eukaryotic initiation factor 4E binding protein 3 like (eif4ebp3l) is a member of the 4E-BPs and responsible for activity-dependent myofibrillogenesis, but whether it affects cardiomyocyte (CM) proliferation or heart regeneration is unclear. We examined eif4ebp3l during zebrafish vascular development and heart regeneration post cryoinjury in adult zebrafish. Using morpholino injections we induced silencing of eif4ebp3l in zebrafish embryos, which led to increased angiogenesis at 94 h post fertilization (hpf). For investigation of eif4ebp3l in cardiac regeneration, zebrafish hearts were subjected to cryoinjury. Regenerating hearts were analyzed at different time points post-cryoinjury for expression of eif4ebp3l by in situ hybridization and showed strongly decreased eif4ebp3l expression in the injured area. We established a transgenic zebrafish strain, which overexpressed eif4ebp3l under the control of a heat-shock dependent promotor. Overexpression of eif4ebp3l during zebrafish heart regeneration caused only macroscopically a reduced amount of fibrin at the site of injury. Overall, these findings demonstrate that silencing of eif4ebp3l has pro-angiogenic properties in zebrafish vascular development and when eif4ebp3l is overexpressed, fibrin deposition tends to be altered in zebrafish cardiac regeneration after cryoinjury.

Testicular Connexin43 (Cx43) connects adjacent Sertoli cells (SC) and SC to germ cells (GC) in the seminiferous epithelium and plays a crucial role in spermatogenesis. However, the distinction whether this results from impaired inter-SC communication or between GC and SC is not possible, so far. Thus, the question arises, whether a GC-specific Cx43 KO has similar effects on spermatogenesis as it is general or SC-specific KO. Using the Cre/loxP recombinase system, two conditional KO mouse lines lacking Cx43 in premeiotic (pGCCx43KO) or meiotic GC (mGCCx43KO) were generated. It was demonstrated by qRT-PCR that Cx43 mRNA was significantly decreased in adult pGCCx43KO mice, while it was also reduced in mGCCx43KO mice, yet not statistically significant. Body and testis weights, testicular histology, tubular diameter, numbers of intratubular cells and Cx43 protein synthesis and localization did not show any significant differences in semi-quantitative Western blot analysis and immunohistochemistry comparing adult male KO and WT mice of both mouse lines. Male KO mice were fertile. These results indicate that Cx43 in spermatogonia/spermatids does not seem to be essential for successful termination of spermatogenesis and fertility as it is known for Cx43 in somatic SC, but SC-GC communication might rather occur via heterotypic GJ channels.

BACKGROUND:The giant anteater belongs to the supraorder Xenarthra which occupies a systematically isolated position among placental mammals. The species is categorized as Vulnerable by the International Union for Conservation of Nature, and understanding its reproductive characteristics is critical for future conservation efforts.METHODS:Gross and microscopic anatomy of the genital organs of 23 male and 21 female adult and young roadkill giant anteaters in Brazil were studied.RESULTS:Male giant anteaters presented a short conical penis, intraabdominal testes, and prostate, vesicular and bulbourethral glands. A tubular remnant of the partially fused Müllerian ducts extended from the seminal colliculus through the prostate gland, continued cranially in the genital fold, bifurcated, and attached with one elongation each to the left and right epididymal corpus. The structure presented a total length of up to 10 cm and contained a yellowish liquid in its lumen. Histologically, the caudal section of this structure resembled the female vagina, the middle portion corresponded to the uterus, and the extensions showed characteristics of uterine tubes. In adult female giant anteaters, ovoid ovaries with occasional seminiferous cord-like structures were observed. The animals possessed a simple uterus, which was directly continuous with the vaginal canal. The caudal portion of the vagina had two lumina, separated by a longitudinal septum and opening into two apertures into the vaginal vestibule, cranial to the urethral opening. In the urethral and the lateral vestibular wall, glandular structures with characteristics of male prostate and bulbourethral glands, respectively, were found. The vestibule opened through a vertical vulvar cleft to the exterior. A pair of well-differentiated Wolffian ducts with a central lumen originated ventrally at the vaginal opening into the vestibule and passed in a cranial direction through the ventral vaginal and uterine wall. Each duct extended highly coiled along the ipsilateral uterine tube until the lateral pole of the ovaries where it merged with the rete ovarii.DISCUSSION:The reproductive morphology of giant anteaters reveals characteristics shared with other Xenarthrans: intraabdominal testes, a simple uterus, and a double caudal vagina. The persistence of well-differentiated genital ducts of the opposite sex in both males and females, however, singles them out among other species. These structures are the results of an aberration during fetal sexual differentiation and possess secretory functions. The possibility of a pathological degeneration of these organs should be considered in reproductive medicine of the species.CONCLUSION:Knowledge of the unique reproductive characteristics of the giant anteater is essential for future reproductive management of the species. Additionally, further research on the peculiarities of the persisting genital duct structures might help to understand sexual differentiation in placental mammals in general.

BACKGROUND:The wild boar population in Europe is steadily growing, one of the reasons for this increase probably being the high reproductive potential of this large mammal. Population management is important to stabilise wild boar numbers and a great deal of attention is focusing on the reasons, which might contribute to the high reproductive rates. Understanding the timing of puberty attainment provides information required for proper management practices. Knowledge of the earliest expected time of sexual maturation in male wild boars is limited, research being mostly focused on females. Previous hunting references indicate that sexual maturity in males occurs in the second year after birth. In contrast, male domestic pigs become sexually mature from about seven months of age. Thus, aims of this study were to investigate (1) whether there is a physiological ability for reproduction also in male wild boars of a younger age and (2) whether the body weight of wild boar males has a more important role than age in driving the maturation of the testis.METHODS:Male wild boar individuals were sampled during hunting drives in the eastern part of Lower Saxony in Germany. Testes with epididymides from 74 males were collected and prepared for histological examination and immunohistochemistry. The reproductive status could be ascertained based on development/occurrence of different germ cell populations using histology and based on the immunohistochemical detection of the anti-Müllerian hormone and androgen receptor.RESULTS:In this study, male wild boars aged nine to ten months already passed puberty and were able to reproduce if they had reached the appropriate body condition of about 29 kg dressed weight. Immunopositivity to the anti-Müllerian hormone in Sertoli cells was evident only in prepubertal animals and decreased with the onset of puberty. No immunoreaction was evident at postpuberty. The androgen receptor was detected in Sertoli cells, peritubular cells and Leydig cells, surprisingly already in Sertoli cells of prepubertal wild boars as well depending on body weight. Moreover, two-thirds of young males aged about ten months were precociously reproductively mature, showing histologically the presence of spermatozoa in testes and epididymides.CONCLUSIONS:As piglets are mostly born in spring, also these young male individuals could target the heat of female wild boars in the winter months, resulting in the observed population increase. Therefore, a reduction in wild boar numbers should also focus on piglets of both sexes.

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.

Gastrointestinal infectious diseases remain an important issue for human and animal health. Investigations on gastrointestinal infectious diseases are classically performed in laboratory animals leading to the problem that species-specific models are scarcely available, especially when it comes to farm animals. The 3R principles of Russel and Burch were achieved using intestinal organoids of porcine jejunum. These organoids seem to be a promising tool to generate species-specific in vitro models of intestinal epithelium. 3D Organoids were grown in an extracellular matrix and characterized by qPCR. Organoids were also seeded on permeable filter supports in order to generate 2D epithelial monolayers. The organoid-based 2D monolayers were characterized morphologically and were investigated regarding their potential to study physiological transport properties and pathophysiological processes. They showed a monolayer structure containing different cell types. Moreover, their functional activity was demonstrated by their increasing transepithelial electrical resistance over 18 days and by an active glucose transport and chloride secretion. Furthermore, the organoid-based 2D monolayers were also confronted with cholera toxin derived from Vibrio cholerae as a proof of concept. Incubation with cholera toxin led to an increase of short-circuit current indicating an enhanced epithelial chloride secretion, which is a typical characteristic of cholera infections. Taken this together, our model allows the investigation of physiological and pathophysiological mechanisms focusing on the small intestine of pigs. This is in line with the 3R principle and allows the reduction of classical animal experiments.