Effects of ICI 182780 on estrogen receptor expression, fluid absorption and sperm motility in the epididymis of the bonnet monkey

BACKGROUND: The importance of estrogen in regulation of fluid absorption and sperm maturation in the rodent epididymis has been established from studies on estrogen receptor-alpha knockout mice. However, functional studies on the role of estrogen in primate epididymis have been few. The main objective of this study was therefore to extend these observations and systematically analyze the presence and function of estrogen receptors in modulating the function of the primate epididymis, using the bonnet monkey (Macaca radiata) as a model system. METHODS: A steroidal estrogen receptor (ER) antagonist, ICI 182780 (ICI), was administered to adult male bonnet monkeys via mini-osmotic pumps for a duration of 30 to 180 days. The expression of key estrogen-regulated genes (ER-alpha, Na-K ATPase alpha-1 and Aquaporin-1) was examined at specific time points. Further, the effect of ICI in modulating fluid reabsorption in efferent ductules was monitored, and critical sperm-maturation parameters were also analyzed. RESULTS: Our studies in the bonnet monkey revealed that both ER-alpha and ER-beta were expressed in all the three regions of the epididymis. We observed an increase in ER-alpha mRNA and protein in the caput of ICI-treated monkeys. Steady state mRNA levels of the water-channel protein, Aquaporin-1, was significantly lower in the caput of ICI-treated monkeys compared to controls, whereas the mRNA levels of Na-K ATPase alpha-1 remained unchanged. In vitro incubation of efferent ductules with ICI resulted in two-fold increase in tubular diameter, indicating affected fluid reabsorption capacity. Furthermore, sperm from ICI-treated monkeys were immotile. CONCLUSION: Taken together, our results point to an integral role for estrogen in modulating the functions of the bonnet monkey epididymis. This study also demonstrates possible differences in the epididymal physiology of rodents and non-human primates, and thus underscores the significance of reports such as these, that examine the physiology of non-human primates (as opposed to rodents), in an attempt to understand similar events in the human

The functional epigenetic landscape of aberrant gene expression in molecular subgroups of newly diagnosed multiple myeloma

Background Multiple Myeloma (MM) is a hematological malignancy with genomic heterogeneity and poor survival outcome. Apart from the central role of genetic lesions, epigenetic anomalies have been identified as drivers in the development of the disease. Methods Alterations in the DNA methylome were mapped in 52 newly diagnosed MM (NDMM) patients of six molecular subgroups and matched with loci-specific chromatin marks to define their impact on gene expression. Differential DNA methylation analysis was performed using DMAP with a ≥10% increase (hypermethylation) or decrease (hypomethylation) in NDMM subgroups, compared to control samples, considered significant for all the subsequent analyses with p<0.05 after adjusting for a false discovery rate. Results We identified differentially methylated regions (DMRs) within the etiological cytogenetic subgroups of myeloma, compared to control plasma cells. Using gene expression data we identified genes that are dysregulated and correlate with DNA methylation levels, indicating a role for DNA methylation in their transcriptional control. We demonstrated that 70% of DMRs in the MM epigenome were hypomethylated and overlapped with repressive H3K27me3. In contrast, differentially expressed genes containing hypermethylated DMRs within the gene body or hypomethylated DMRs at the promoters overlapped with H3K4me1, H3K4me3, or H3K36me3 marks. Additionally, enrichment of BRD4 or MED1 at the H3K27ac enriched DMRs functioned as super-enhancers (SE), controlling the overexpression of genes or gene-cassettes. Conclusions Therefore, this study presents the underlying epigenetic regulatory networks of gene expression dysregulation in NDMM patients and identifies potential targets for future therapies

Microhomology-mediated end joining drives complex rearrangements and overexpression of MYC and PVT1 in multiple myeloma

MYC is a widely acting transcription factor and its deregulation is a crucial event in many human cancers. MYC is important biologically and clinically in multiple myeloma, but the mechanisms underlying its dysregulation are poorly understood. We show that MYC rearrangements are present in 36.0% of newly diagnosed myeloma patients, as detected in the largest set of next generation sequencing data to date (n=1,267). Rearrangements were complex and associated with increased expression of MYC and PVT1, but not other genes at 8q24. The highest effect on gene expression was detected in cases where the MYC locus is juxtaposed next to super-enhancers associated with genes such as IGH, IGK, IGL, TXNDC5/BMP6, FAM46C and FOXO3. We identified three hotspots of recombination at 8q24, one of which is enriched for IGH-MYC translocations. Breakpoint analysis indicates primary myeloma rearrangements involving the IGH locus occur through non-homologous end joining, whereas secondary MYC rearrangements occur through microhomology-mediated end joining. This mechanism is different to lymphomas, where non-homologous end joining generates MYC rearrangements. Rearrangements resulted in overexpression of key genes and chromatin immunoprecipitation-sequencing identified that HK2, a member of the glucose metabolism pathway, is directly over-expressed through binding of MYC at its promoter

Expression of functional aromatase in the epididymis: Role of androgens and LH in modulation of expression and activity

The primary source of 17beta-estradiol (E2) in the male is the testis, which expresses the enzyme complex aromatase that is involved in E2 biosynthesis. However, recent evidences suggest that the epididymis is also capable of E2 biosynthesis. Our results demonstrate the presence of cytochrome P450 aromatase (P450_A_R_O_M) and 17beta-hydroxysteroid dehydrogenase I messenger ribonucleic acid (mRNA) in the caput and cauda regions of rat epididymis. The androgenic substrates testosterone and androstenedione could be utilized by the rat epididymal aromatase for E2 biosynthesis as assessed by radioimmunoassay. (P450_A_R_O_M) expression is transcriptionally regulated in a tissue-specific manner by various factors including androgens and luteinizing hormone (LH). Androgens could positively modulate epididymal (P450_A_R_O_M) mRNA levels as assessed by castration studies, treatment with flutamide or in vitro incubation of tissue minces with 5alpha-dihydrotestosterone (DHT). Several extra-gonadal tissues including the epididymis are known to express LH receptors (LHR). Our study revealed a higher level of LHR mRNA expression in the cauda region compared to the caput. Caudal membrane extracts could bind human chorionic gonadotropin (hCG), which resulted in the production of cAMP. Interestingly, hCG could also regulate (P450_A_R_O_M) mRNA expression in vitro and enhance E2 biosynthesis. Together our results highlight the presence of a functional aromatase in the epididymis that is subject to regulation by LH and androgens

Differential expression and antibacterial activity of WFDC10A in the monkey epididymis

The ability of the epididymis to perform its diverse functions stems from its regionalized gene and protein expression patterns. The differences in the gene expression patterns of the caput and cauda regions of the bonnet monkey epididymis were compared using the technique of differential display reverse transcriptase polymerase chain reaction. A transcript showing homology to human whey acidic protein 10 (hWFDC10A) was highly expressed in the monkey caput region. A peptide P2 was designed spanning a region of the monkey WFDC10A (mWFDC10A), which could inhibit the growth of gram-negative bacterial strains of Escherichia coli. P2 could permeabilize the bacterial cell membrane but was unable to permeabilize mammalian cells as evidenced by the lack of hemolysis upon incubation with the peptide. Expression of genes such as mWFDC10A may be essential in providing the first line of defense against microbial infections to the epididymal tract and thus rendering protection to the male gametes sheltered within the epididymis

Differential expression and antibacterial activity of WFDC10A in the monkey epididymis

The ability of the epididymis to perform its diverse functions stems from its regionalized gene and protein expression patterns. The differences in the gene expression patterns of the caput and cauda regions of the bonnet monkey epididymis were compared using the technique of differential display reverse transcriptase polymerase chain reaction. A transcript showing homology to human whey acidic protein 10 (hWFDC10A) was highly expressed in the monkey caput region. A peptide P2 was designed spanning a region of the monkey WFDC10A (mWFDC10A), which could inhibit the growth of gram-negative bacterial strains of Escherichia coli. P2 could permeabilize the bacterial cell membrane but was unable to permeabilize mammalian cells as evidenced by the lack of hemolysis upon incubation with the peptide. Expression of genes such as mWFDC10A may be essential in providing the first line of defense against microbial infections to the epididymal tract and thus rendering protection to the male gametes sheltered within the epididymis

Delineating the role of estrogen in regulating epididymal gene expression

Maturation of sperm within the epididymis is a pre-requisite for fertilization in mammals. Epididymal function is controlled by a complex array of hormones and growth factors. While testosterone is the primary stimulus for epididymal development and sperm maturation, the importance of estrogen effects on efferent ductules has been increasingly recognized, and points to a need to clarify the role of estrogen receptor-mediated action in the epididymis. Estrogens modulate the expression of genes involved in fluid absorption in the efferent ductules and the epididymis. The present review highlights the role of estrogen in regulation of epididymal gene expression