Food-grade titanium dioxide (E171) by solid or liquid matrix administration induces inflammation, germ cells sloughing in seminiferous tubules and blood-testis barrier disruption in mice.

Food-grade titanium dioxide labeled as E171 has been approved for human consumption by the Food and Drug Administration (USA) and by the European Union for five decades. However, titanium dioxide has been classified as a possible carcinogen for humans by the International Agency of Research in Cancer raising concerns of its oral intake and the translocation to bloodstream, which could disturb barriers such as the blood-testis barrier. There is evidence that titanium dioxide by intragastric/intraperitoneal/intravenous administration induced alterations on testosterone levels, testicular function and architecture, but studies of the E171 effects on the testicle structure and blood-testis barrier are limited. E171 is contained not only in foods in liquid matrix but also in solid ones, which can exert different biological effects. We aimed to compare the effects of E171 consumption in a solid matrix (0.1%, 0.5% and 1% in pellets) and liquid suspension (5 mg/kg body weight) on testis structure, inflammation infiltrate and blood-testis barrier disruption of male BALB/c mice. Results showed that none of the administration routes had influence on body weight but an increase in germ cell sloughing and the infiltrate of inflammatory cells in seminiferous tubules, together with disruption of the blood-testis barrier were similar in testis of both groups even if the dose received in mice in liquid matrix was 136 or 260 times lower than the dose reached by oral intake in solid E171 pellets in 0.5% E171 and 1% E171, respectively. This study highlights the attention on matrix food containing E171 and possible adverse effects on testis when E171 is consumed in a liquid matrix.

Mice spermatogonial stem cells transplantation induces macrophage migration into the seminiferous epithelium and lipid body formation: high-resolution light microscopy and ultrastructural studies.

Transplantation of spermatogonial stem cells (SSCs), the male germline stem cells, in experimental animal models has been successfully used to study mechanisms involved in SSC self-renewal and to restore fertility. However, there are still many challenges associated with understanding the recipient immune response for SSCs use in clinical therapies. Here, we have undertaken a detailed structural study of macrophages elicited by SSCs transplantation in mice using both high-resolution light microscopy (HRLM) and transmission electron microscopy (TEM). We demonstrate that SSCs transplantation elicits a rapid and potent recruitment of macrophages into the seminiferous epithelium (SE). Infiltrating macrophages were derived from differentiation of peritubular monocyte-like cells into typical activated macrophages, which actively migrate through the SE, accumulate in the tubule lumen, and direct phagocytosis of differentiating germ cells and spermatozoa. Quantitative TEM analyses revealed increased formation of lipid bodies (LBs), organelles recognized as intracellular platforms for synthesis of inflammatory mediators and key markers of macrophage activation, within both infiltrating macrophages and Sertoli cells. LBs significantly increased in number and size in parallel to the augmented macrophage migration during different times post-transplantation. Our findings suggest that LBs may be involved with immunomodulatory mechanisms regulating the seminiferous tubule niche after SSC transplantation.

Immune response to bacteria in seminiferous epithelium.

The luminal part of the seminiferous epithelium, a tissue compartment protected by the blood-testis barrier, has been considered a site of immune privilege. However, there are reports describing the production of anti-microbial peptides and the expression of Toll-like receptors in cells present in the seminiferous epithelium, evoking the possibility that this tissue compartment is immunologically active at least with regard to the innate immune response. To test this, we injected Escherichia coli into seminiferous tubules of live mice and examined the fate of bacteria, the production of chemokines and inflammatory cytokines, and the infiltration of neutrophils. The bacteria actively propagated and reached a maximal level in a day, but started to decrease after 5 days and completely disappeared in 2 months. The expression of macrophage inflammatory protein-2 and tumor necrosis factor-alpha became evident in macrophages present in the interstitial compartment of testes as early as 1-3 h after the inoculation of bacteria. Neutrophils first accumulated in the interstitial space at 9-12 h and entered the tubules after a day. On the other hand, impairment of spermatogenesis was observed a day after bacteria injection and seemed unrecoverable even after the bacteria were eliminated. By contrast, bacteria injected into the interstitial compartment were more rapidly cleared with no damage in the seminiferous epithelium. These results suggest the existence of immunity against invading microbes in the seminiferous epithelium although its effectiveness in maintaining tissue homeostasis remains equivocal.

Immunomorphological aspects of the tubuli recti and the surrounding interstitium in normal mice.

The tubuli recti (TR) are immunologically special, because the lymphocytes preferably accumulate around them during the course of T-cell dependent testicular autoimmunity in mice. This finding implies that the testicular interstitium around the TR is where autoreactive lymphocytes can gain access to autoimmunogenic germ cell antigens. In the present study, the histoarchitecture of the TR was minutely examined in normal mice. Three-dimensional analysis showed that 14-16 TR appeared to be connected to the rete testis (RT). Electron microscopical analysis revealed that the epithelial cells in the TR formed protruding cytoplasmic strings, with active endocytosis of degenerated spermatozoa, and exhibited three different morphological characteristics. Furthermore, a few macrophages were found to have penetrated into the TR. Immunohistochemical image analysis revealed that more macrophages specifically accumulated around the TR than the RT or seminiferous tubules. These findings indicate that macrophages preferentially accumulate around the TR, where contact between germ cell antigens and macrophages may occur under normal and pathological conditions.

Fas and Fas-ligand expression in seminomatous testes.

Sixteen seminomas with surrounding tissue containing normal and precancerous (cis) seminiferous tubules were examined for the expression of Fas (CD95, APO-1) and Fas ligand (FasL) (CD95L). This was done by analyzing frozen specimens using immunohistochemistry with antibodies directed against Fas and FasL. The study showed that varying numbers (mean approx. 20%) of Fas-positive lymphocytes were present among tumor-infiltrating lymphocytes, but very few FasL-positive lymphocytes. Fas was not expressed by normal seminiferous tubules and only occasional Fas-positive epithelial cells were seen in cis tubules. FasL was expressed in 9 out of 10 cases in virtually all normal seminiferous tubules, mainly as a thin layer at the base of the seminiferous epithelium. In precancerous tubules, this layer was discontinuous and less pronounced. Rete testis expressed FasL in 2 out of 2 cases with rete present and Fas in 1 out of 1 case. Invasive tumor cells did not express Fas or FasL. The data are discussed in relation to immune reactions to seminomas and to the concept of the testis being an immunologically privileged area.

Expression of the proliferation-associated Ki-67 antigen in bovine testicular tubular cells during the seminiferous epithelial cycle, demonstrated with the MIB-1 antibody.

Ki-67 expression in the seminiferous tubule of the bovine testis was studied by immunohistochemistry during the seminiferous epithelial cycle using the monoclonal antibody MIB-1. Spermatogonial proliferation is most obvious in stages 5-7, and 8, when B-spermatogonia divide. A lower rate of spermatogonial propagation is observed preceding or during meiosis in stages 1-4. The MIB-1 antibody also gives positive results with some post-spermatogonial tubular cells. Preleptotenes passing through S-phase in stage 1 reveal positive nuclei. During prophase of meiosis I pachytenes react strongly, diplotenes react in an attenuated manner, while leptotenes and zygotenes stay negative. Secondary spermatocytes seen in stage 4 are positive as are the chromosomes during meta- and anaphase of the meiotic divisions. Post-meiotic spermatids are also decorated but stop Ki-67 expression abruptly at the end of stage 4. Sertoli cells are negative.

The molecular weight of aspermatogenic antigen as determined by equilibrium ultracentrifugation.

The extraction of antigen from testis and sperm is described. This antigen, when administered in microgram amounts in a single injection, induces aspermatogenesis in 100% of guinea pigs so treated. The deletion of the seminiferous cell line is specific (no cross-reactions) and provides a tool for investigating inhibition of cell replication as well as reproductive control. The antigen is a polypeptide-polysaccharide complex and has been determined to have a molecular weight of 10,520, using the equilibrium ultracentrifugation method.