Epididymal lithiasis in roosters: in the middle of the way there was a stone

The epididymal region plays an important role in the reproduction of roosters, as it is the site of functions important in the maintenance of fertility, including fluid and calcium reabsorption and sperm surface modifications. About 10 years ago, a reproductive dysfunction characterized by the formation of luminal calcium stones in the epididymal region of roosters was described. This anomaly, known as epididymal lithiasis, is associated with a significant decrease in the fertility of affected roosters. This reproductive anomaly has been observed in multiple countries and is thought to negatively impact the poultry industry; however, the cause of epididymal lithiasis has not been fully determined. Several hypotheses have been proposed to explain the origin of epididymal lithiasis, including the presence of an infectious agent within the epididymal region, an autoimmune response, increased dietary calcium and vitamin D3 intake and the presence of genetic susceptibility factors; however, none of these has been proven to be the primary cause of the calcium stone formation. Nonetheless, considerable evidence suggests that regardless of the primary cause of epididymal lithiasis, this anomaly could result from a hormonal imbalance or a local impairment of calcium homeostasis in the epididymal region. The objectives of this mini-review are to 1) summarize the reproductive alterations observed in animals affected by epididymal lithiasis, 2) discuss the hypotheses proposed to explain the cause of luminal stone formation and 3) provide perspectives for future studies of this reproductive disorder.     

Immunolocalization of androgen and vitamin D receptors in the epididymis of mature ram (Ovis aries)

This study illustrated the immunohistochemical distribution of androgen and vitamin D receptors of epididymis in 20 sexually mature ram (Rahmani breed) with average age ranged from (2^_4) years and average weight ranged from (50^_65kg). Androgen receptor was localized in the cytoplasm of both ciliated and non ciliated cells of efferent ductules, besides the principal cells via the entire epididymal duct. The principal cells of both corpus and proximal cauda epididymis showed the highest immunoreactivity to androgen receptors. Furthermore, vitamin D receptor was localized in the cytoplasm of all epithelium of the efferent ductules besides principal cells of all epididymal regions, however the immunoreaction was significantly higher in the efferent ductules, distal caput and distal cauda epididymis. In conclusion, these results suggest that the function of ram epididymis is regulated by both androgen and Vitamin D.     

Ion transporters for fluid reabsorption in the rooster (Gallus domesticus) epididymal region

Testicular fluid is highly condensed during its passage through the epididymal region in the avian species. In the present study, major ion transporters that are responsible for condensation mainly by water resorption in the reproductive tract as identified in the mammalian epididymis were localized within the rooster (Gallus domesticus) epididymis by immunohistochemistry. The results show that the efferent ductule epithelium expressed sodium-potassium ATPase (Na(+),K(+)-ATPase), carbonic anhydrase II (CAII) and sodium hydrogen exchanger isoform 3 (NHE3) and that the connecting ductule and epididymal duct epithelia expressed Na(+),K(+)-ATPase and CAII. These data suggest that a model proposed for reabsorption in mammalian efferent ductules can be applied to avian efferent ductules.     

Targeted deletion of the epididymal receptor HE6 results in fluid dysregulation and male infertility

Human epididymal protein 6 (HE6; also known as GPR64) is an orphan member of the LNB-7TM (B(2)) subfamily of G-protein-coupled receptors. Family members are characterized by the dual presence of a secretin-like (type II) seven-transmembrane (7TM) domain and a long cell adhesion-like extracellular domain. HE6 is specifically expressed within the efferent ductules and the initial segment of the epididymis, ductal systems involved in spermatozoon maturation. Here, we report that targeted deletion of the 7TM domain of the murine HE6 gene results in male infertility. Mutant mice reveal a dysregulation of fluid reabsorbtion within the efferent ductules, leading to a backup of fluid accumulation in the testis and a subsequent stasis of spermatozoa within the efferent ducts. The fertility phenotype of HE6 knockout mice identifies this receptor as a potential nonsteroidal, nontesticular target for future male contraceptives and identifies an in vivo function for a member of this unusual gene family.     

Aquaporin-1 and -9 are differentially regulated by oestrogen in the efferent ductule epithelium and initial segment of the epididymis

BACKGROUND INFORMATION: Efferent ductules reabsorb more than 90% of the rete testis fluid, a process that involves ion transporters and AQP (aquaporin) water channels. Oestrogen has been shown to modulate the expression of the ion transporters involved in this activity, but reports of AQP regulation in the male tract have been confounding. To understand better the regulation of AQP1 and AQP9, we investigated their expression in rat efferent ductules and initial segment of the epididymis after treatment with the pure antioestrogen ICI 182,780 or bilateral efferent duct ligation, or castration, followed by hormone replacement. RESULTS: Results show that AQP9 is modulated by oestrogen in the efferent ductule epithelium, but not in the initial segment of the epididymis. DHT (5alpha-dihydrotestosterone) also modulated AQP9 in efferent ductules. AQP9 was down-regulated by the antioestrogen in efferent ductules on day 45 post-treatment, which occurred before the non-ciliated cells had shown significant loss of microvilli. DHT, but not oestradiol, modulated AQP9 expression in the initial segment of the epididymis. In contrast, testosterone, DHT, oestrogen or the antioestrogen did not alter AQP1 staining, indicating constitutive expression of AQP1 in the efferent ductule epithelium. AQP1 expression was induced in peritubular cells of efferent ductules and in the initial segment of the epididymis after castration and long-term treatment with the antioestrogen. Although peritubular AQP1 staining in efferent ductules was partially reversed by the androgens, it was not reversed after any treatment in the initial segment of the epididymis. CONCLUSIONS: These results demonstrate that efferent ductules are unique in requiring both oestrogen and androgen to regulate an important mediator of fluid reabsorption, whereas the initial segment is dependent only on androgen stimulation.     

Lipopolysaccharide negatively modulates vitamin D action by down-regulating expression of vitamin D-induced VDR in human monocytic THP-1 cells

Vitamin D3, an important seco-steroid hormone for the regulation of body calcium homeostasis, promotes immature myeloid precursor cells to differentiate into monocytes/macrophages. Vitamin D receptor (VDR) belongs to a nuclear receptor super-family that mediates the genomic actions of vitamin D3 and regulates gene expression by binding with vitamin D response elements in the promoter region of the cognate gene. Thus by regulating gene expression, VDR plays an important role in modulating cellular events such as differentiation, apoptosis, and growth. Here we report lipopolysaccharide (LPS), a bacterial toxin; decreases VDR protein levels and thus inhibits VDR functions in the human blood monocytic cell line, THP-1. The biologically active form of vitamin D3, 1alpha,25-dihydroxy vitamin D3 [1,25(OH)2D3], induced VDR in THP-1 cells after 24 h treatment, and LPS inhibited 1,25(OH)2D3-mediated VDR induction. However, LPS and 1,25(OH)2D3 both increased VDR mRNA levels in THP-1 cells 20 h after treatment, as observed by real time RT-PCR. Moreover, LPS plus 1,25(OH)2D3 action on VDR mRNA level was additive and synergistic. A time course experiment up to 60 h showed an increase in VDR mRNA that was not preceded with an increase in VDR protein levels. Although the proteasome pathway plays an important role in VDR degradation, the proteasome inhibitor lactacystin had no effect on the LPS-mediated down-regulation of 1,25(OH)2D3 induced VDR levels. Reduced VDR levels by LPS were accompanied by decreased 1,25(OH)2D3/VDR function determined by VDR responsive 24-hydroxylase (CYP24) gene expression. The above results suggest that LPS impairs 1,25(OH)2D3/VDR functions, which may negatively affect the ability of 1,25(OH)2D3 to induce myeloid differentiation into monocytes/macrophages.     

Nuclear receptor antagonists designed based on the helix-folding inhibition hypothesis

Here we review our studies on the molecular design of nuclear receptor antagonists, including retinoic acid receptor (RAR) antagonists, retinoid X receptor (RXR) antagonists, androgen receptor (AR) antagonists, and vitamin D receptor (VDR) antagonists, based on inhibition of folding of helix 12, which contains a co-activator binding site. Recent progress in structural development studies of peroxisome proliferator-activated receptor (PPAR) ligands is also reviewed.     

Morphologic study of the efferent ductules of the pigeon (Columba livia)

The efferent ductules of the pigeon are localized in the epididymal region and are topographically divided into proximal and distal, both portions being lined with stereociliated pseudostratified epithelium. Transmission electron microscopy shows five distinct cell types: light, dark, and angular non-ciliated cells with possible apocrine secretory role cells and halo cells, possibly intraepithelial leucocytes. The proximal efferent ductules have the widest diameter among all ductules in the epididymal region.     

Where is the vitamin D receptor?

The vitamin D receptor (VDR) is a member of the nuclear receptor superfamily and plays a central role in the biological actions of vitamin D. VDR regulates the expression of numerous genes involved in calcium/phosphate homeostasis, cellular proliferation and differentiation, and immune response, largely in a ligand-dependent manner. To understand the global function of the vitamin D system in physiopathological processes, great effort has been devoted to the detection of VDR in various tissues and cells, many of which have been identified as vitamin D targets. This review focuses on the tissue- and cell type-specific distribution of VDR throughout the body.     

Ligands with a 3,3-diphenylpentane skeleton for nuclear vitamin D and androgen receptors: Dual activities and metabolic activation

Ligands possessing dual vitamin D(3) (VD(3))-agonistic and androgen-antagonistic activities with various activity spectra were prepared based on a substituted 3,3-diphenylpentane (DPP) skeleton. Among the compounds, (R,S)-DPP-1023 [(R,S)-7b] and (S,S)-DPP-0123 [(S,S)-7c] showed the most potent vitamin D(3)-agonistic activity [with potency comparable to that of 1alpha,25-dihydroxyvitamin D(3) (1,25-VD(3))] and nuclear androgen receptor (AR)-binding activity (with higher affinity than that of hydroxyflutamide), respectively. Metabolic activation (reduction of the carbonyl group) of pivaloyl analogs [DPP-1113 (3a), DPP-1013 (3b), DPP-0113 (3c), and DPP-0013 (3d)] in HL-60 cells was found to be necessary for binding to nuclear vitamin D(3) receptor (VDR).     

Deletion of deoxyribonucleic acid binding domain of the vitamin D receptor abrogates genomic and nongenomic functions of vitamin D

The vitamin D hormone 1,25-dihydroxyvitamin D(3) [1,25-(OH)(2)D(3)], the biologically active form of vitamin D, is essential for an intact mineral metabolism. Using gene targeting, we sought to generate vitamin D receptor (VDR) null mutant mice carrying the reporter gene lacZ driven by the endogenous VDR promoter. Here we show that our gene-targeted mutant mice express a VDR with an intact hormone binding domain, but lacking the first zinc finger necessary for DNA binding. Expression of the lacZ reporter gene was widely distributed during embryogenesis and postnatally. Strong lacZ expression was found in bones, cartilage, intestine, kidney, skin, brain, heart, and parathyroid glands. Homozygous mice are a phenocopy of mice totally lacking the VDR protein and showed growth retardation, rickets, secondary hyperparathyroidism, and alopecia. Feeding of a diet high in calcium, phosphorus, and lactose normalized blood calcium and serum PTH levels, but revealed a profound renal calcium leak in normocalcemic homozygous mutants. When mice were treated with pharmacological doses of vitamin D metabolites, responses in skin, bone, intestine, parathyroid glands, and kidney were absent in homozygous mice, indicating that the mutant receptor is nonfunctioning and that vitamin D signaling pathways other than those mediated through the classical nuclear receptor are of minor physiological importance. Furthermore, rapid, nongenomic responses to 1,25-(OH)(2)D(3) in osteoblasts were abrogated in homozygous mice, supporting the conclusion that the classical VDR mediates the nongenomic actions of 1,25-(OH)(2)D(3).     

Inactivation of the 25-hydroxyvitamin D 1alpha-hydroxylase and vitamin D receptor demonstrates independent and interdependent effects of calcium and vitamin D on skeletal and mineral homeostasis

We employed a genetic approach to determine whether deficiency of 1,25-dihydroxyvitamin D (1,25(OH)2D) and deficiency of the vitamin D receptor (VDR) produce the same alterations in skeletal and calcium homeostasis and whether calcium can subserve the skeletal functions of 1,25(OH)2D and the VDR. Mice with targeted deletion of the 25-hydroxyvitamin D 1alpha-hydroxylase (1alpha(OH)ase-/-) gene, the VDR gene, and both genes were exposed to 1) a high calcium intake, which maintained fertility but left mice hypocalcemic; 2) this intake plus three times weekly injections of 1,25(OH)2D3, which normalized calcium in the 1alpha(OH)ase-/- mice only; or 3) a "rescue" diet, which normalized calcium in all mutants. These regimens induced different phenotypic changes, thereby disclosing selective modulation by calcium and the vitamin D system. Parathyroid gland size and the development of the cartilaginous growth plate were each regulated by calcium and by 1,25(OH)2D3 but independent of the VDR. Parathyroid hormone secretion and mineralization of bone reflected ambient calcium levels rather than the 1,25(OH)2D/VDR system. In contrast, increased calcium absorption and optimal osteoblastogenesis and osteoclastogenesis were modulated by the 1,25(OH)2D/VDR system. These studies indicate that the calcium ion and the 1,25(OH)2D/VDR system exert discrete effects on skeletal and calcium homeostasis, which may occur coordinately or independently.     

Effect of hormones and development on the expression of the rat 1,25-dihydroxyvitamin D3 receptor gene. Comparison with calbindin gene expression

We have used specific cDNAs to the rat vitamin D receptor (VDR) and to the mammalian vitamin D-dependent calcium-binding proteins (calbindin-D9k in intestine and calbindin-D28k in kidney) in order to obtain a better understanding of the regulation of the VDR gene and its relationship to calbindin gene expression. Hormonal regulation and development expression of the rat VDR gene were characterized by both Northern and slot blot analyses. Administration of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3; 25 ng/day for 7 days) to vitamin D-deficient rats resulted in an increase in calbindin mRNA in intestine and kidney but no change in VDR mRNA in these tissues. Vitamin D-deficient rats responded to dexamethasone treatment (100 micrograms/100 g of body weight/day for 4 days) with a 2.5-fold increase in intestinal VDR mRNA which was accompanied by a 4-fold decrease in intestinal calbindin-D9k mRNA. Developmental studies indicated a pronounced increase in renal VDR mRNA and calbindin-D28k mRNA between birth and 1 week of age. In the intestine, an induction of VDR and calbindin-D9k gene expression was observed at a later time, during the 3rd postnatal week (the period of increased duodenal active transport of calcium). Taken collectively, our data indicate that in the adult rat, target tissue response to hormone is not modified by a corresponding alteration in new receptor synthesis. However, developmental studies indicate that the induction of 1,25(OH)2D3 receptor mRNA is correlated with the induction of calbindin gene expression. Our results also demonstrate that glucocorticoid administration can result in an alteration in intestinal calbindin and VDR gene expression.     

Alpha-mannosidase activity in stallion epididymal fluid and spermatozoa

The expression of α-D-mannosidase activity was fluorometrically and electrophoretically assessed in spermatozoa, epididymal fluid and homogenates of stallion epididymal tissue. Enzyme activity had regional differences; it was higher (P < 0.05) in samples from the cauda epididymal region than in samples from the proximal caput region (largely composed of efferent ducts). Based on enzyme activity, as a function of pH of the assay substrate, electrophoretic analysis in native and native/SDS-PAGE conditions, and the effect of inhibitors or activators, we inferred the presence of at least two catalytically active forms of α-D-mannosidase. The neutral form of the enzyme (α-mannosidase II) was activated by Co²⁺, whereas the acid form (optimum pH 3.5 to 4.0) was sensitive to swainsonine (an inhibitor of α-mannosidase I), stabilized or stimulated by Zn²⁺, and not activated by Co²⁺ (activator of the neutral form). The activity of the acid form of the enzyme was highest in the epididymal fluid, where it seemed to be mainly in a secretory form. This form of the enzyme may have a role in plasma membrane remodeling associated with sperm maturation. In contrast, the activity of α-mannosidase II was higher in mature spermatozoa. It has been postulated that α-mannosidase II may act as a receptor in the recognition and binding of the complementary carbohydrate moieties present on the zona pellucida. With non-denaturing electrophoresis, α-D-mannosidase had an electrophoretic mobility of 0.35 and 0.24. When resolved by 1D and 2D SDS-PAGE (under denaturing conditions) the enzyme had a major protein band of molecular weight 154 kDa in spermatozoa and epididymal samples. Based on its properties under native conditions, we inferred that this enzyme might interact with other proteins and form transitory aggregates.     

Vitamin D Receptor Is Not Essential for Extracellular Signal-Related Kinase Phosphorylation by Vitamin D(3) in Human Caco-2/TC7 Cells

Vitamin D(3) initiated rapid extracellular signal-related kinase (ERK) phosphorylation, but the contribution of vitamin D receptor (VDR) to this event is unclear. We investigated the use of RNA interference (RNAi) to knockdown VDR. RNAi downregulated VDR as well as its targeted gene expression, but vitamin D(3) dependent ERK phosphorylation remained. Thus VDR might not be involved in ERK phosphorylation by vitamin D(3).     

Effects of androgen on androgen receptor expression in rat testicular and epididymal cells: a quantitative immunohistochemical study

Androgen is essential for maintenance of spermatogenesis in the testis and for maturation of spermatozoa in the epididymis. The effects of androgen are mediated through its receptor (AR), the levels of which are, in turn, regulated by androgen. Previous studies have shown that AR concentrations in Leydig and Sertoli cells are differentially regulated during development. The aim of the present study was to determine if cell-type-specific regulation of AR by androgen occurs in testicular and epididymal cells during adulthood. Adult male rats were treated with the LHRH-antagonist Azaline B (100 g/day) by osmotic pump for 1, 2, 3, 4, or 8 wk to suppress endogenous androgen, with identical numbers of intact control animals at each time period. An androgen replacement group was simultaneously treated with the antagonist and a synthetic androgen, 7 alpha-methyl-19-nortestosterone (MENT), during the final 4 wk of the experiment. Levels of nuclear AR protein in specific cell types were quantified by immunohistochemistry in conjunction with computer-assisted image analysis. Levels of AR in testicular cells declined sharply after treatment with the LHRH antagonist. In Sertoli cells, nuclear AR levels decreased to 8% of control (P < 0. 01) after 4 wk treatment; and to 12% and 17% of control (P < 0.01) in Leydig and myoid cells, respectively. Androgen replacement resulted in complete recovery of nuclear AR levels in Sertoli cells (93%, P > 0.05) but in only partial recovery in myoid (69%, P < 0. 01) and Leydig cells (56%, P < 0.01). In the epididymis, tubular epithelial cells and stromal cells differed in their responses to the LHRH antagonist. After 1 wk, nuclear AR levels in caput stromal cells decreased dramatically to 34% of control (P < 0.01) and in cauda stromal cells to 43% (P < 0.01). In contrast, the decline of AR levels in epididymal epithelial cells was not as dramatic as that in stromal cells. After 1 wk, the decline in the caput and cauda was to 87% and 76% of control, respectively. After 8 wk, nuclear AR levels in stromal cells further declined to 1.1% in caput and 1.4% in cauda, whereas in the epithelial cells, a smaller decline in nuclear AR was noted (to 30% in the caput and 45% in the cauda). After androgen replacement with MENT, nuclear AR levels recovered to more than 90% of control in both epididymal cell types. These results indicate that AR levels in the nuclei of adult Sertoli cells depend mainly on the level of androgen, whereas in the adult Leydig and myoid cells, the androgen dependency is more limited. The results also indicate that in the epididymis, stromal cells are more sensitive than epithelial cells to the regulation of AR levels by androgen.