Differential endocrine regulation of genes enriched in initial segment and distal caput of the mouse epididymis as revealed by genome-wide expression profiling.

We have performed genome-wide expression profiling of endocrine regulation of genes expressed in the mouse initial segment (IS) and distal caput of the epididymis by using Affymetrix microarrays. The data revealed that of the 15 020 genes expressed in the epididymis, 35% were enriched in one of the two regions studied, indicating that differential functions can be attributed to the IS and the more distal caput regions. The data, furthermore, showed that 27% of the genes expressed in the IS and/or distal caput epididymidis are under the regulation of testicular factors present in the duct fluid, while bloodborne androgens can regulate for 14% of them. This is in line with the high testis dependency of epididymal physiology. We then focused on genes with moderate or strong expression, showing strict segment enrichment and strong dependency on testicular factors. Analyses of the 59 genes, including upregulated and downregulated genes, fulfilling the criteria indicated that the expression of 18 (17 downregulated genes; 1 upregulated gene) of 19 gonadectomy-responsive genes enriched in the IS was not maintained by the androgen treatment, whereas the expression of all six downregulated genes enriched in the distal caput and the majority of those with no strict segment enrichment of expression (28 of 34; consisting of 23 downregulated and 5 upregulated genes) were maintained by androgens. Hence, it is evident that testicular factors other than androgens are important for the expression of IS-enriched genes, whereas the expression of distal caput-enriched genes is typically regulated by androgens. Identical data were obtained by independent clustering analyses performed for the expression data of 3626 epididymal genes. Several novel genes with putative involvement in epididymal sperm maturation, such as a disintegrin and metallopeptidase domain 28 (Adam28) and a solute carrier organic anion transporter family, member 4C1 (Slco4c1), were identified, indicating that this approach is successful for identifying novel epididymal genes.

Identification of novel epididymal genes by expression profiling and in silico gene discovery.

We have used both the UniGene RIKEN epididymal EST library and the Affymetrix microarray profiling for identifying novel epididymal genes in mouse. The use of ESTs is a complementary approach to Affymetrix arrays for identifying novel epididymal genes, while only 32% and 28% of ESTs of unknown genes were present in the U74v.2Set and MG 430 2.0 version Affymetrix arrays, respectively. As expected, the probe set for a notably larger proportion of known genes was present in the Affymetrix arrays, and the coverage was greatly improved by the newer array version. Furthermore, many genes with more than five ESTs in the UniGene library showed variable levels of expression in both versions of the Affymetrix arrays. However, both the Affymetrix and EST data correlated well with that obtained by quantitative RT-PCR, and thus, we conclude that the findings of high EST number but only limited expression in the arrays could be considered as false negatives in the Affymetrix arrays.

Fetal but not adult Leydig cells are susceptible to adenoma formation in response to persistently high hCG level: a study on hCG overexpressing transgenic mice.

We have previously demonstrated that male transgenic (TG) mice overexpressing human chorionic gonadotropin (hCG+) develop reproductive organ defects, but no tumors, in adult age. In this study, the effects of persistently elevated hCG were followed in TG males between day 5 postpartum and adulthood. Leydig cell (LC) adenomas were found in prepubertal mice, most prominently at the age of 10 days, but not in adult age. Serum testosterone concentrations were significantly increased in TG males at all ages studied. The phenotype of the prepubertal hCG+ males resembled that found in boys upon expression of constitutively activating luteinizing hormone (LH) receptor mutations. The temporal expression patterns of the fetal LC marker gene, thrombospondin 2, and those of adult LCs, hydroxysteroid dehydrogenase-6, delta5-3-beta and prostaglandin D synthase, were similar in wild-type and hCG+ males. Hence, the postnatal adenomas resemble functionally fetal LCs, and only these cells are susceptible to hCG-induced tumorigenesis. Our findings demonstrate a novel intriguing difference between the fetal and adult LC populations and provide further insight into the potential tumorigenic effects of gonadotropins.

Ca2+-dependent potentiation of muscarinic receptor-mediated Ca2+ elevation.

Muscarinic receptor-mediated increases in Ca(2+) in SH-SY5Y neuroblastoma cells consist of an initial fast and transient phase followed by a sustained phase. Activation of voltage-gated Ca(2+) channels prior to muscarinic stimulation resulted in a several-fold potentiation of the fast phase. Unlike the muscarinic response under control conditions, this potentiated elevation of intracellular Ca(2+) was to a large extent dependent on extracellular Ca(2+). In potentiated cells, muscarinic stimulation also activated a rapid Mn(2+) entry. By using known organic and inorganic blockers of cation channels, this influx pathway was easily separated from the known Ca(2+) influx pathways, the store-operated pathway and the voltage-gated Ca(2+) channels. In addition to the Ca(2+) influx, both IP(3) production and Ca(2+) release were also enhanced during the potentiated response. The results suggest that a small increase in intracellular Ca(2+) amplifies the muscarinic Ca(2+) response at several stages, most notably by unravelling an apparently novel receptor-activated influx pathway.

Ceramide 1-phosphate enhances calcium entry through voltage-operated calcium channels by a protein kinase C-dependent mechanism in GH4C1 rat pituitary cells.

Sphingomyelin derivatives modulate a multitude of cellular processes, including the regulation of [Ca2+]i (the intracellular free calcium concentration). Previous studies have shown that these metabolites often inhibit calcium entry through VOCCs (voltage-operated calcium channels). In the present study, we show that, in pituitary GH4C1 cells, C1P (C2-ceramide 1-phosphate) enhances calcium entry in a dose-dependent manner. The phospholipase C inhibitor U73122 attenuated the response. C1P invoked a small, but significant, increase in the formation of inositol phosphates. Pre-treatment of the cells with pertussis toxin was without an effect on the C1P-evoked increase in [Ca2+]i. The effect of C1P was critically dependent on extracellular calcium, since no increase in [Ca2+]i was observed when cells in a calcium-free buffer were stimulated with C1P. Furthermore, if the cells were retreated with 300 nM of the VOCC inhibitor nimodipine, the effect of C1P was almost totally abolished. In addition, ceramide C8-1-phosphate evoked an increase in [Ca2+]i, but the onset of the response was slow compared with that of C1P. In cells treated with 1 mM thapsigargin for 15 min, C1P still evoked an increase in [Ca2+]i. In patch-clamp experiments in the whole-cell mode, C1P enhanced calcium entry through the VOCCs compared with vehicle-treated cells. Dialysis of the cells with C1P did not enhance the calcium current. On-cell patch-clamp experiments showed an enhanced probability of the VOCCs being open (P(open)) in the presence of C1P. Inhibition of PKC (protein kinase C) with GF109203X and down-regulation of PKC with PMA attenuated the C1P-evoked increase in [Ca2+]i. Furthermore, down-regulation of PKC abolished the effect of C1P on P(open). This is the first report showing that a sphingomyelin derivative enhances calcium entry through VOCCs.

Immortalization of epididymal epithelium in transgenic mice expressing simian virus 40 T antigen: characterization of cell lines and regulation of the polyoma enhancer activator 3.

In the present study epididymal epithelium was immortalized in transgenic mice by expressing simian virus 40 T antigen under a 5.0-kb mouse glutathione peroxidase 5 promoter (GPX5-Tag1). Epididymal tumorigenesis was associated with an increase in c-Myc expression, and a marked decrease in B-Myc expression, with a 500-fold lower level in the GPX5-Tag1 caput epididymis compared with wild-type caput. Furthermore, B-Myc was undetectable in the immortalized corpus and cauda epididymis. Hence, it is possible that the normally high B-Myc expression in the epididymis is one of the factors contributing to the highly resistant nature of epididymis toward immortalization. Morphologically different epithelial cell lines were generated from the immortalized epididymides, and the cells expressed several genes typical for epididymal epithelium, such as mouse epididymal 1, mouse epididymal protein 9, androgen and estrogen receptors, anion exchangers 2 and 4, retinoic acid receptor alpha, and polyoma enhancer activator 3 (PEA3). This indicated the differentiated status of the cells and their usefulness for analyzing epididymal gene expression in vitro. As PEA3 is considered to be one of the transcription factors responsible for epididymal gene expression, we further studied its regulation in epididymal cells in vitro. The data showed that PEA3 mRNA expression is regulated in the epididymis via protein kinase A and ERK signaling cascades. Inhibiting protein kinase A resulted in up-regulation and inhibiting ERK resulted in down-regulation of PEA3 mRNA, whereas no significant effect on PEA3 expression was found by modulating the protein kinase C, stress-activated p38, phosphoinositol 3-kinase and p70 S6 kinase cascades.

Distinct recognition of OX1 and OX2 receptors by orexin peptides.

In this study, we have compared the abilities of orexin-A and orexin-B and variants of orexin-A to activate different Ca(2+) responses (influx and release) in human OX(1) and OX(2) receptor- expressing Chinese hamster ovary cells. Responses mediated by activation of both receptor subtypes with either orexin-A or -B were primarily dependent on extracellular Ca(2+), suggesting similar activation of Ca(2+) influx as we have previously shown for orexin-A and OX(1) receptors. Amino acid-wise truncation of orexin-A reduced its ability to activate OX(1) and OX(2) receptors, but the response mediated by the OX(2) receptor was more resistant to truncation than the response mediated by the OX(1) receptor. We also performed a sequential replacement of amino acids 14 to 26 with alanine in the truncated orexin-A variant orexin-A(14-33). Replacement of the same amino acids produced a fall in the potency for each receptor subtype, but the reduction was less prominent for the OX(2) receptor. The most marked reduction was produced by the replacement of Leu20, Asp25, and His26 with alanine. Interestingly, extracellular Ca(2+) dependence of responses to some of the mutated peptides was different from those of orexin-A and -B. The mutagenesis also suggests that although the determinants required from orexin-A for binding to and activation of the receptor are highly conserved between the orexin receptor subtypes, the OX(2) receptor requires fewer determinants. This might in part explain why orexin-B has the affinity and potency equal to orexin-A for this subtype, although it has 10- to 100-fold lower affinity and potency for the OX(1) receptor.

Adenosine triphosphate induces Ca2+ signal in epithelial cells of the mouse caput epididymis through activation of P2X and P2Y purinergic receptors.

In the present study, we developed a novel method to analyze the calcium (Ca2+) signal in living slices of mouse caput epididymides by applying calcium imaging on Fura-2-loaded vibratome slices. The data revealed that in epithelial cells of mouse caput epididymides, ATP induces a rapid Ca2+ signal that is sustained after 60 sec. Preincubating the sections in Ca2+-free medium in the presence of EGTA did not affect the amplitude of the ATP-induced Ca2+ signal, indicating the presence of P2Y type purinergic receptors and phospholipase C activity. Furthermore, ATP induced a similar Ca2+ signal in the different subregions of caput epididymides. The P2X type ion-gated purinergic receptors could also be responsible for the ATP-induced Ca2+ signal because immunohistochemical and reverse transcriptase-polymerase chain reaction analyses showed that P2X1, P2X2, P2X4, P2X7, P2Y1, and P2Y2 receptors were expressed in the epididymis. We propose that P2X and P2Y receptor expression is vital for the normal function of epididymal epithelium and sperm maturation. Furthermore, the method we developed allows us to analyze the activity of various G protein-coupled receptors in intact epithelial cells of mouse epididymides, and other reproductive tissues as well.