Daniel B.C. Queiróz

Activation of Toll-Like Receptor 4 (TLR4) by In Vivo and In Vitro Exposure of Rat Epididymis to Lipopolysaccharide from Escherichia Coli

Abstract This study provides the first evidence that rat epididymis is fully capable of initiating an inflammatory response to lipopolysaccharide (LPS) from Escherichia coli through activation of Toll-like receptor 4 (TLR4). TLR4 functionality was demonstrated by in vivo LPS challenge, which induced a time- and dose-dependent activation of the transcription factor nuclear factor kappa B (NFKB) in caput and cauda epididymides. NFKB activation by LPS in caput epididymidis was abrogated when rats were pretreated with the NFKB inhibitor PDTC, confirming the specificity of this response. Within 2 h of LPS treatment (0.01 and 1 mg/kg, i.v.), NFKB activation in caput and cauda was accompanied by upregulation of Il1b, Nfkbia, and Cd14, but not Tlr4, mRNA. These effects, however, were not sustained after 24 h of LPS treatment. Lipopolysaccharide systemic effects were not restricted to epididymides, since Il1b, Nfkbia, and Cd14 mRNAs were also upregulated in other male reproductive tissues from LPS-treated rats (1 mg/kg, i.v., 2 h). Constitutive TLR4 was immunolocalized in some, but not all, epididymal epithelial cells and in interstitial cells, some of them identified as resident ED2-positive macrophages. No change in TLR4 immunostaining pattern was observed when epididymides from control and LPS-treated rats were compared (1 mg/kg, i.v., 2 h and 24 h). Significant NFKB activation was also achieved within 1 min of in vitro incubation of caput epididymidis with LPS (0.01–5 μg/ml), confirming that components for TLR4 signaling cascade activation are fully active in this tissue. This study contributes to a better understanding of the innate immune response in the epididymis and other tissues from the male reproductive tract.

Relaxin family peptide receptors Rxfp1 and Rxfp2: mapping of the mRNA and protein distribution in the reproductive tract of the male rat

BackgroundRelaxin is the endogenous ligand of the G-protein coupled receptor RXFP1, previously known as LGR7. In humans relaxin can also activate, but with lower affinity, the closely related receptor for the insulin-like peptide from Leydig cells, RXFP2, previously known as LGR8. The lack of relaxin impairs male fertility but the precise distribution and the function of relaxin receptors in the male reproductive tract is not known. We investigated the distribution of Rxfp1 and Rxfp2 in the reproductive tract of the male rat and the function of relaxin in the vas deferens, a tissue with high expression of both receptors.MethodsThe presence of mRNA for Rxfp1 and Rxfp2 was investigated in testes, cultured Sertoli cells, epididymis, vas deferens, seminal vesicle, prostate, and spermatozoa by RT-PCR and Southern blot. Protein expression in the testis, vas deferens, primary culture of Sertoli cells, and spermatozoa was assessed by immunohistochemistry and immunofluorescence. The role of relaxin in the vas deferens was evaluated by contractility studies and radioimmunoassay of cAMP production. The effect of relaxin on mRNA levels for metalloproteinase-7 was measured by Northern blot.ResultsTranscripts for Rxfp1 and Rxfp2 were present in almost all parts of the male reproductive tract, with high levels in testis and vas deferens. Both receptors were immunolocalized in late stage germ cells but not in mature spermatozoa, although mRNAs for both receptors were also present in mature spermatozoa. Rxfp1 but not Rxfp2 was detected in cultured Sertoli cells. Strong immunostaining for Rxfp1 and Rxfp2 was seen in muscular and epithelial layers of the vas deferens and in arteriolar walls. Relaxin did not affect contractility and cyclic AMP production of the vas deferens, but increased the levels of mRNA for metalloproteinase-7.ConclusionRxfp1 and Rxfp2 are widely and similarly distributed throughout the male reproductive tract. Our results suggest that Rxfp1 on spermatids and Sertoli cells may be important in spermatogenesis. Relaxin in the vas deferens does not affect contractility, but may affect vascular compliance and collagen and matrix remodeling.

Glucocorticoid receptor in the rat epididymis: Expression, cellular distribution and regulation by steroid hormones

Glucocorticoids regulate several physiological functions, including reproduction, in mammals. Curiously, little is known about glucocorticoid-induced effects on the epididymis, an androgen-dependent tissue with vital role on sperm maturation. Here, RT-PCR, Western blot and immunohistochemical studies were performed to evaluate expression, cellular distribution and hormonal regulation of glucocorticoid receptor (GR) along rat epididymis. The rat orthologue of human GRalpha (mRNA and protein) was detected in caput, corpus and cauda epididymis and immunolocalized in the nucleus and cytoplasm of different epididymal cells (epithelial, smooth muscle and interstitial cells) and nerve fibers. Changes in plasma glucocorticoid and androgen levels differentially regulated GR expression in caput and cauda epididymis by homologous and heterologous mechanisms. In vivo treatment with dexamethasone significantly changed the expression of glucocorticoid-responsive genes and induced ligand-dependent GR nuclear translocation in epithelial cells from epididymis, indicating that GR is fully active in this tissue. Heterologous regulation of androgen receptor expression by glucocorticoids was also demonstrated in cauda epididymis. Our results demonstrate that the epididymis is under glucocorticoid regulation, opening new insights into the roles of this hormone in male fertility.

α1-Adrenoceptor Subtypes in Rat Epididymis and the Effects of Sexual Maturation

Abstract We have characterized the expression of α1-adrenoceptor in epididymis from rats in different stages of sexual maturation: 40 (immature), 60 (young adult), and 120 (adult) days of age. Plasma testosterone levels were low in the immature animals but increased significantly in the 60- and 120-day-old animals. These changes were followed by a progressive increase in rat body weight and in caput and cauda epididymis relative weight. Reverse transcription polymerase chain reaction assay indicated that α1a-, α1b-, and α1d-adrenoceptor transcripts were present in both caput and cauda epididymis from adult rats. Ribonuclease protection assays further indicated that the expression of these α1-adrenoceptor transcripts differed with age and epididymal region analyzed. Prazosin (nonselective α1 antagonist), 5-methyl urapidil (α1A-selective), and BMY 7378 (α1D-selective) displaced [3H]prazosin binding curves in caput and cauda epididymis from 40- and 120-day-old rats. The potency order for these antagonists, as calculated from the negative logarithm of the inhibition constant (pKi) values for the high-affinity sites, indicated a predominant population of α1A-adrenoceptor subtype in caput and cauda epididymis from adult animals. Differences in pKi values in caput and cauda epididymis from immature and adult animals also suggested that the relative amount of α1-adrenoceptors, at the protein level, is modulated by sexual maturation. Taken together, the changes in α1-adrenoceptor expression during sexual maturation may suggest specific roles for these receptors in epididymal function.

Cells positive for microtubule-associated protein 1B (MAP 1B) are present along rat and human efferent ductules and epididymis.

Microtubule-associated protein 1B (MAP 1B) is a neuronal cytoskeleton marker with predominant expression in the developing nervous system. The present study provides evidence for the expression of this cytoskeleton protein in non-neuronal and neuronal cells along rat and human efferent ductules and epididymis (initial segment, caput, and cauda). Reverse transcription/polymerase chain reaction and Western blot analysis were used to confirm the presence of MAP 1B (mRNA and protein) in rat tissues. Immunohistochemical studies revealed MAP-1B–positive staining in columnar ciliated cells present in efferent ductules and in narrow cells located in the initial segment, in both rat and human. MAP-1B–positive basal cells, located underneath the columnar cells, were only identified in the initial segment and caput epididymidis of the rat. Qualitative analysis of tissues from 40–day-old and 120–day–old rats indicated that the number of MAP-1B–positive ciliated, narrow, and basal cells per tubule increased with sexual maturation. These immunoreactive cells did not stain for dopamine β–hydroxylase or acetylcholinesterase, indicating that they were not adrenergic or cholinergic in nature. Immunohistochemical studies also revealed the presence of MAP-1B–positive staining in interstitial nerve fibers in caput and cauda epididymidis from both rat and human. Thus, the expression of MAP 1B is not confined to a specific cell type in rat and human efferent ductules and epididymis. The functional significance of this cytoskeleton protein in tissues from the male reproductive tract requires further investigation.

Innate immunity and glucocorticoids: potential regulatory mechanisms in epididymal biology.

Inflammation is a primordial host response to invasion by pathogens or tissue injury. During infection, microbes can activate immune cells through pattern-recognition receptors, such as Toll-like receptors, an evolutionarily conserved family of receptors that mediate innate immunity in a wide range of organisms. Infection also triggers an increase in glucocorticoid levels as part of the stress response. The scenario indicates that these signals have to be well integrated to mount an effective host response to infection and injury. The mechanisms by which innate and adaptive immunity are regulated, as well as the intersection of these responses with glucocorticoids and the glucocorticoid receptor (GR) in the epididymis, an organ essential for the transport, maturation, storage, and protection of the spermatozoa, are not well understood. In this review we bring together recent data demonstrating the cellular and biochemical machinery involved in the response of the adult rat epididymis to a bacterial product challenge. We also illustrate the basic aspects of the expression, localization, function, and regulation of the GR by steroid hormones (androgens and glucocorticoids) within the epididymis. We conclude with considerations of controversial or still unanswered topics about GR, now emerging as a regulatory step in epididymal biology, its functional relationship with androgens and androgen receptor, and the innate immune response of the epididymis. How these topics may be of interest as part of future research in the area, and how they ultimately can help us to better understand the epididymal function under noninflammatory and inflammatory conditions, are also discussed.

Dynamic changes in the spatio-temporal expression of the β-defensin SPAG11C in the developing rat epididymis and its regulation by androgens

Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the β-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of β-defensins in the epididymis.