Uroplakin 1a Interacts with Regucalcin and Proteasome Subunit Beta 1.

Uroplakins (UPKs) are specialized proteins that plan an important role in protecting the epithelium of the bladder from toxic waste. We recently demonstrated the expression pattern of UPKs in the male reproductive tract and their importance in sperm function in murine models. However, the exact mechanisms through which UPKs affect spermatogenesis are not reported. In this study, using yeast two-hybrid screening was conducted to determine the interaction partners of Uroplakin 1a (UPK1A). Y2H Gold yeast strain overexpressing UPK1A was mated with Y187 yeast strain overexpressing human testis cDNA library and the mutants were plated on SD agar plates containing selection media. Colonies that grew on SD/-Trp, SD/-Leu, SD/-His, and SD/-Ade plates were isolated and evaluated to identify the interacting partners of UPK1A. Regucalcin (RGN) and proteasome subunit beta 1 (PSMB1) were identified as potential interaction partners. Using HEK cells that overexpress UPK1A and RGN or PMSB1, the co-localization and interaction were estimated with high-resolution microscopy and Pearson's coefficient. In light of the fact that UPK1A knockout caused subfertility and that the role of RGN and PSMB1 in spermatogenesis is documented, an interaction between UPK1A and RGN or PSMB1 could be required for spermatogenesis.

Characterization of SPINK2, SPACA7 and PDCL2: Effect of immunization on fecundity, sperm function and testicular transcriptome.

Testicular factors play a vital role in spermatogenesis. We characterized the functional role of rat Spink2, Spaca7 and Pdcl2 genes. Their primary, secondary and tertiary structure were deduced in silico. The genes of rat Spink2, Spaca7 and Pdcl2 mRNA were predominantly expressed in the testis. SPINK2, SPACA7 and PDCL2 protein expression was evident in all the cell types of testis and on spermatozoa. Ablation of each of these proteins by active immunization resulted in reduced fecundity and sperm count. Damage to the anatomical architecture of testis and epididymis was evident. In SPINK2 immunized rats, 283 genes were differentially regulated while it was 434 and 872 genes for SPACA7 and PDCL2 respectively. Genes that were differentially regulated in the testis of SPINK2 immunized rats primarily belonged to extracellular exosome formation, extracellular space and response to drugs. SPACA7 ablation affected genes related to extracellular space, oxidation-reduction processes, endoplasmic reticulum membrane and response to drugs. Differential gene expression was observed for nuclear function, protein binding and positive regulation of transcription from RNA polymerase II promoter in testis of PDCL2 immunized rats. Results of our study demonstrate the role of SPINK2, SPACA7 and PDCL2 in spermatogenesis and in important molecular processes that may dictate testicular function and other physiological responses as well.

Uroplakin 1a Knockout Mice Display Marginal Reduction in Fecundity, Decreased Bacterial Clearance Capacity, and Drastic Changes in the Testicular Transcriptome.

Uroplakins (UPKs) form physical and chemical barriers in the bladder and other urinary tract tissues. We previously reported the identification and localization of UPKs in the male reproductive tract of rat. In this study, we characterized Upk1a knockout mice and report a marginal reduction in fecundity associated with significant decrease in sperm count. Upk1a mice had lower bacterial clearance capacity when challenged with uropathogenic Escherichia coli for 1 to 5 days. High-throughput analyses of testicular transcriptome indicated that 1128 genes that are expressed in testis of wild-type mice were completely absent in the knockout, while 2330 genes were found to be expressed only in the testis of knockout mice. Furthermore, differential regulation of 148 (67 upregulated and 81 downregulated) was observed. Gene ontology analyses indicated that processes related to integral components of membrane (plasma membrane), G-protein receptor activity and signaling, olfactory receptor activity and perception of smell, organization of extracellular space/region, immune and inflammatory responses to pathogens, spermatid development, meiotic cell cycle, and formation of synaptonemal complex were affected. Results of this study provide evidence on the possible multi-functional role of Upk1a in male reproductive tract and in other tissues as well.

A mixture of pyrethroids induces reduced fecundity and increased testicular genotoxicity in rats.

To achieve crop protection and higher agricultural yield, pesticides are used; and among them pyrethroid based ones are the most preferred choice because of their specificity on the pests. Uncontrolled use of pesticides resulted in contamination of food products. Presence of residual amounts of pyrethroids was reported in agricultural products sold in Indian market, indicating that humans are constantly exposed to these chemicals through food on a routine basis. Studies that determine the toxic effects of pyrethroids at doses equivalent to human exposure are rare. We orally administered a mixture of pyrethroids (detected in the rice and vegetables of Indian market) to male rats for 15 months to mimic the long-term exposure in humans. We observed reduced fecundity, sperm count and 13ß-hydroxysteroid dehydrogenase enzyme activity. The serum concentrations of hormones involved in male reproductive function were altered. Further, testicular genotoxicity as reflected by perturbations in the expression pattern of genes involved in the molecular processes of gametogenesis was evident. Such toxic effects may also be occurring in humans who consume agricultural products that contain residual amounts of pyrethroids on a regular basis throughout their lifetime.

Effect of a mixture of pyrethroids at doses similar to human exposure through food in the Indian context.

Residual amounts of pyrethroids were detected in rice and vegetables of the Indian market. Thus, consumers are exposed to a mixture of pyrethroids on a daily basis through food. Though a large number of studies reported the toxic effects of pyrethroids, there are no reports that used doses equivalent to human consumption. In this study, male Wistar rats were exposed daily to a mixture of pyrethroids for 1-15 months which is equivalent to the amount present in rice and vegetables consumed by an average Indian each day. The oxidant-antioxidant status (lipid peroxidation, nitric oxide; activities of catalase, glutathione peroxidase, glutathione S transferase, and superoxide dismutase) and anatomical changes in the general organs (liver, lung, and kidney) and male reproductive tract tissues (caput, cauda, testis, and prostate) were evaluated. Further, liver and kidney function tests, lipid profile, and complete blood picture were analyzed. Increased oxidative stress, perturbations in the antioxidant enzyme activities, and damage to the anatomical architecture were observed. Disturbances in the liver function and lipid profile were significant. Results of our study demonstrate that exposure to a mixture of pyrethroids at a dose that is equivalent to human consumption can cause systemic and reproductive toxicity, which may ultimately result in the development of lifestyle diseases. This first line of evidence will fuel further studies to determine the impact of food-based pyrethroid exposure on the long-term health of humans and to envisage policies to reduce pesticide content in food products.

Ablation of the sperm-associated antigen 11A (SPAG11A) protein by active immunization promotes epididymal oncogenesis in the rat.

Incidence of cancer in the epididymis is very rare. It is proposed that proteins specific to this organ may contribute to this unique property. We previously demonstrated that siRNA-mediated knockdown of SPAG11A mRNA resulted in increased proliferation of epididymal epithelial cells, whereas overexpression of this gene caused reduced proliferation in immortalized cell lines. In this study, we evaluated the oncogenesis-related anatomical and transcriptome changes in the epididymis of SPAG11A-immunized rats challenged with a low dose of diethyl nitrosamine (DEN). DEN treatment or SPAG11A immunization alone did not cause any histopathological changes in the epididymis. Interestingly, indications of oncogenesis were observed in SPAG11A-immunized + DEN-treated rats. Using high throughput sequencing, we observed that 3549 transcripts that were differentially expressed in the caput epididymis of DEN only-treated rats displayed similar differential expression in the caput epididymis of SPAG11A-immunized rats, indicating that the microenvironment that contributes to oncogenesis sets in when SPAG11A protein is ablated. Differential expression of genes that are involved in 10 major cancer related pathways was also analyzed. Majority of the genes related to these pathways that were differentially expressed in the caput epididymis of DEN only-treated rats also showed similar pattern in the caput epididymis of SPAG11A-immunized rats. For the first time, results of our study demonstrate that ablation of SPAG11A by active immunization renders the epididymis susceptible to oncogenesis and that this protein may be one of the factors that contributes to the rarity of epididymal cancer.

Transgenerational effects on the fecundity and sperm proteome in rats exposed to a mixture of pyrethroids at doses similar to human consumption.

Pyrethroid based pesticide usage for crop protection resulted in percolation of these compounds into the food chain. Toxicological studies that reflect exposure to pyrethroids through food in the human settings are rare. We conducted animal experimentations using a mixture of pyrethroids that is equivalent to the amount consumed by average individual through rice and vegetables in the Indian context. Male rats treated with a mixture of pyrethroids for 1-12 months displayed decreased transgenerational fecundity, sperm count, activities of 3ß- and 17ß-HSD and perturbed hormonal profile. At the transcriptome level, the expression of genes involved in spermatogenesis, steroidogenesis, germ cell epigenetic modulators and germ cell apoptosis were altered in the testis. In the sperm lysates of control rats, 506 proteins identified by mass spectrometry. The differential expression of these proteins (treated/control ratio) in the pyrethroid exposed rats was analyzed. Among the 506 proteins, 153 had a ratio of 0; 41 had a ratio ranging from >0 to <0.5; and 10 had a ratio >2.0. Interestingly, the differential expression was transgenerational. 26 proteins that were differentially expressed in the sperm of F0 treated rats continued to remain the same in the F1, F2 and F3 generations, while the differential expression was maintained up to F2 and F1 generations for 46 and 2 proteins respectively. Some of the proteins that continued to be differentially expressed in the later generations are reported to have critical roles in male reproduction. These results indicate that the reduced fecundity observed in the later generations could be due to the continued differential expression that was initiated by pyrethroid treatment in the F0 rats. Results of our study, for the first time, provide evidence that long-term exposure to pyrethroids affects transgenerational fecundity manifested by changes in sperm proteome.

Coiled-Coil Domain-Containing (CCDC) Proteins: Functional Roles in General and Male Reproductive Physiology.

The coiled-coil domain-containing (CCDC) proteins have been implicated in a variety of physiological and pathological processes. Their functional roles vary from their interaction with molecular components of signaling pathways to determining the physiological functions at the cellular and organ level. Thus, they govern important functions like gametogenesis, embryonic development, hematopoiesis, angiogenesis, and ciliary development. Further, they are implicated in the pathogenesis of a large number of cancers. Polymorphisms in CCDC genes are associated with the risk of lifetime diseases. Because of their role in many biological processes, they have been extensively studied. This review concisely presents the functional role of CCDC proteins that have been studied in the last decade. Studies on CCDC proteins continue to be an active area of investigation because of their indispensable functions. However, there is ample opportunity to further understand the involvement of CCDC proteins in many more functions. It is anticipated that basing on the available literature, the functional role of CCDC proteins will be explored much further.

Pyrethroid based pesticides - chemical and biological aspects.

Human and animal welfare primarily depends on the availability of food and surrounding environment. Over a century and half, the quest to identify agents that can enhance food production and protection from vector borne diseases resulted in the identification and use of a variety of pesticides, of which the pyrethroid based ones emerged as the best choice. Pesticides while improved the quality of life, on the other hand caused enormous health risks. Because of their percolation into drinking water and food chain and usage in domestic settings, humans unintentionally get exposed to the pesticides on a daily basis. The health hazards of almost all known pesticides at a variety of doses and exposure times are reported. This review provides a comprehensive summation on the historical, epidemiological, chemical and biological (physiological, biochemical and molecular) aspects of pyrethroid based insecticides. An overview of the available knowledge suggests that the synthetic pyrethroids vary in their chemical and toxic nature and pose health hazards that range from simple nausea to cancers. Despite large number of reports, studies that focused on identifying the health hazards using doses that are equivalent or relevant to human exposure are lacking. It is high time such studies are conducted to provide concrete evidence on the hazards of consuming pesticide contaminated food. Policy decisions to decrease the residual levels of pesticides in agricultural products and also to encourage organic farming is suggested.

Effect of oral administration of a mixture of pyrethroids at doses relevant to human exposure on the general and male reproductive physiology in the rat.

Studies on the effects of unintentional intake of pyrethroid pesticides that are akin to actual human exposure settings are very rare. Such an exposure is primarily by consuming the food products as routine diet that contain residual levels of pyrethroids. In this study, rats were orally administered for 15 months with a mixture of pyrethroids at a dose that is one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the residual levels commonly present in the average amount of rice and vegetables consumed by Indian population. Lipid profile, kidney and liver function were assessed. Lipid peroxidation, nitric oxide, antioxidant enzyme activities and histopathological changes were analyzed in the liver, lung, kidney, pancreas, testes, caput, cauda and prostate. The effect on the male reproductive system as a function of sperm count, enzyme activity of 3ß-HSD and 17ß-HSD and the expression profile of genes involved in spermatogenesis, steroidogenesis, genetic reprogramming and apoptosis of male gametes were evaluated. Significant increase in the relative organ weight, perturbations in the activities of antioxidant enzymes, lipid profile and liver function were observed in both LD and HD groups. Damage to the anatomical architecture was evident in all the tissues due to pyrethroid toxicity. Exposure to LD and HD of pyrethroid mixture resulted in decreased sperm count, activities of 3ß-HSD and 17ß-HSD, impaired capacitation and acrosome reaction and perturbations in the expression of genes that govern male gamete production. Results of our study indicate that exposure to pyrethroids for longer durations even at doses that are far below the residual levels present in the food consumed will result in severe damage to general physiological processes as well as reproductive function.

Lytic Bacteriophages Against Bacterial Biofilms Formed by Multidrug-Resistant Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus Isolated from Burn Wounds.

Background: Use of bacteriophages as antibiofilm agents to tackle multidrug-resistant bacteria has gained importance in recent years. Materials and Methods: In this study, biofilm formation by Staphylococcus aureus, Pseudomona aeruginosa, Klebsiella pneumoniae, and Escherichia coli under different growth conditions was studied. Furthermore, the ability of bacteriophages to inhibit biofilm formation was analyzed. Results: Under dynamic growth condition, wherein the medium is renewed for every 12 h, the amount of biomass produced and log10 colony-forming unit counts of all bacterial species studied was highest when compared with other growth conditions tested. Biomass of biofilms produced was drastically reduced when incubated for 2 or 4 h with bacteriophages vB_SAnS_SADP1, vB_PAnP_PADP4, vB_KPnM_KPDP1, and vB_ECnM_ECDP3. Scanning electron microscopy and confocal laser scanning microscopy analyses indicated that the reduction in biomass was due to the lytic action of the bacteriophages. Conclusions: Results of our study reinforce the concept of developing bacteriophages as alternatives to antibiotics to treat bacterial infections.

Effect of long-term treatment with a mixture of pyrethroids on the expression of genes that govern male germ cell production in rats.

Humans are exposed to pyrethroid-based pesticides through agricultural produce. In this study, male Wistar rats were orally treated for 9 to 12 months with a mixture of pyrethroids that is equivalent to one-fifth (high dose; HD) or one-twenty fifth (low dose; LD) of the amount of pyrethroids present in the cereals and rice consumed by an average Indian. In rats treated for 9 months, the spermatogenesis-associated genes Abp, Ar, Cd9, Dax1, Dazap1, Ddx3y, Gdnf, Gfra1, Grth, Inhb, Ovol1, P1, Plzf, Pygo2, Scf, Tgfb1, Tp1, Tp2, and Vim1 were downregulated in both LD and HD groups. In rats treated for 12 months Gdnf, Hsf2, Inhb, Tgfb1, Thy1, and Ybx2 expression was downregulated in both LD and HD groups. Steroidogenesis-associated genes 17-ß-Hsd, Gata4, Hmgcr, Hmgcs1, Pde4b, and Tspo gene expression were reduced in both LD- and HD-treated groups treated for 9 months. In 12-month-treated rats, Creb1 expression decreased in both LD and HD groups. The epigenetic reprogramming-associated genes, Dnmt1, Dnmt3a, Dnmt3b, Hdac10, Hp1bp3, Kat3a Kat3b, Mch2ta, Ncoa7, and Sirt1 were downregulated in both HD and LD groups of 9-months-treated rats. In rats treated for 12 months, Hdac10, Mch2ta, Ncoa7, and Sirt1 messenger RNA levels decreased in both the HD and LD groups. Thus, we demonstrate that long-term exposure to a mixture of pyrethroids caused aberrations in the transcriptome of factors involved in sperm production and development.

Transgenesis and active immunization mediated reduction of sperm associated antigen 11A mRNA and protein levels affect fecundity in the rat.

Spermatozoa acquire motility and fertilizing ability during their transit through the epididymis. A wide variety of proteins secreted into the epididymal lumen are added on to the sperm surface to allow morphological and molecular changes involved in sperm maturation. Proteins of the Sperm Associated Antigen 11 (SPAG11) family are known to be localized on the sperm surface. The rat SPAG11A protein was implicated in sperm maturation during epididymal transit in vitro. However, systematic analyses on the significance of SPAG11A in fertility and sperm function is not yet reported in vivo. In this study, using testicular electroporation, we generated transgenic rats that express shRNA to ablate endogenous Spag11a mRNA. Genotyping revealed the integration of the plasmid that expresses shRNA against Spag11a mRNA. Significant decrease in the mRNA levels of Spag11a and its encoded protein was observed in the caput epididymis of transgenic rats. We also generated an active immunization rat model to ablate endogenous SPAG11A protein by administering recombinant SPAG11A protein. Immunized rats had a high antibody titer in the serum and the tissue fluids of caput, cauda and testis. In both these model systems, the litter size and sperm count was significantly reduced. However, spermatozoa obtained from the transgenic or immunized rats underwent capacitation and acrosome reaction and the associated calcium release. Results of this study indicate the role of SPAG11A in fecundity and sperm production and not in sperm function, especially capacitation and acrosome reaction.

siRNA-mediated knockdown of sperm-associated antigen 11a (Spag11a) mRNA in epididymal primary epithelial cells affects proliferation: a transcriptome analyses.

Differential expression of a variety of proteins in the four major regions of the epididymis contributes to maturation of spermatozoa and region-specific cellular functions as well. Proliferation of epithelial cells of the epididymis is highly controlled and thus is one of the major reasons for the nonoccurrence of cancers in this organ system. The molecular mechanisms and the contribution of region-specific genes in epithelial cell proliferation are not yet fully understood. In this study, for the first time, we analyzed the role of sperm-associated antigen 11a (Spag11a), a caput-specific beta-defensin-like antimicrobial gene in governing epididymal cell proliferation and global gene expression. siRNA-mediated knockdown of Spag11a mRNA in epididymal primary epithelial cells resulted in increased cell proliferation. Out of the 68,842 genes analyzed, 4182 genes were differentially expressed (2154 upregulated and 2028 downregulated). A variety of genes that participate in different cellular processes and pathways were differentially regulated. Genes that are important for epithelial cell proliferation were found to be differentially regulated and these changes were confirmed by real-time PCR. Overexpression of Spag11a in immortalized rat caput epididymal cells resulted in decreased proliferation capacity. Results of this study indicate that Spag11a plays a crucial role in governing epididymal epithelial cell proliferation.

Tlr1-13, Nod1/2 and antimicrobial gene expression in the epididymis and testis of rats with alloxan-induced diabetes.

Pattern recognition receptors (PRRs) such as toll-like receptors (TLRs) and nuclear oligomerization domain (NOD) receptors along with antimicrobial proteins and peptides (AMPs) are crucial for innate immunity. The pathology of insulin-dependent diabetes mellitus is associated with the disrupted expression of TLRs, NODs and AMPs in the kidney, lungs and other organs. However, such a relation in the male reproductive tract is not yet investigated. In this study, we analysed the expression pattern of Tlr1-13, Nod1/2 receptors and AMPs (ß-defensins and defensin-like proteins of the Sperm-Associated Antigen 11 (Spag11) family) in the male reproductive tissues (caput, cauda and testis) obtained from diabetic or insulin-treated diabetic or untreated control rats. Alterations in the expression pattern of Tlr1-13, Nod 1/ 2, Defb1, 2, 21, 24, 27, 30 and Spag11a/ c/ t were observed under diabetic conditions. Administration of insulin to diabetic rats could modulate the expression pattern of only some these genes. Results of our study indicate perturbed gene expression profile of Tlrs, Nod1/2, Defbs and Spag11 isoforms in the epididymis and testis of diabetic rats, and this could be one of the important reasons for the increased risk of infections in the male genital tract.

Male reproductive tract antimicrobial expression in the extremes of ages of rats.

Alterations in the global gene expression profile are considered to contribute to the various physiological and pathological changes during the course of ageing. Genes that code for the molecular components of the innate system are alter markedly as ageing occurs; and this may define the susceptibility of very young and very old individuals to reproductive tract infections. The expression pattern of genes that code for beta-defensins (effectors of innate immune response) in male reproductive tract tissues of different stages of ageing is not yet reported. Further, the induction of beta-defensins during endotoxin challenge and whether epigenetic modulators can influence the expression of these genes in different stages of ageing are not reported. We analyzed the basal mRNA levels of beta-defensins and defensin-like proteins (Sperm Associated Antigen 11 (SPAG11) family members), their induction during endotoxin challenge and modulation by epigenetic modifiers (Trichostatin A and Azacytidine) in the caput, cauda, testis, prostate and seminal vesicle of rats that represent early stage to late stages of life (20 day to 730 day old). We observed differential basal gene expression pattern in the male reproductive tract tissues and the induction by LPS was not consistent neither among the age groups not the tissues analyzed. Trichostatin A and Azacytidine also influenced antimicrobial gene expression and the pattern was not consistent in different tissues obtained from different age groups. Results of this study demonstrate that antimicrobial gene expression varies to a great extent during ageing and is strongly influenced by endotoxins and epigenetic modulators.

Long term oral administration of a mixture of pyrethroids affects reproductive function in rats.

Pyrethroid toxicity using dosages that are relevant to the human settings are not reported. Male Wistar rats were treated for 9 or 12 months daily with a mixture of pyrethroids equivalent to a fifth or a twenty-fifth of that is present in cereals and vegetables consumed by an average Indian adult. Altered oxidant and antioxidant status, severe anatomical damage in the testis, caput, cauda, prostate, liver, lung and kidney and increased serum SGPT, SGOT and ALP activity was evident in all the treatment groups. Decreased levels of 3ß- and 17ß-HSD activity, litter size and impaired acrosome reaction was observed in all the treatment groups. To the best of our knowledge, for the first time, we demonstrate that exposure to even very low levels of pyrethroids (relevant to human consumption) for longer durations could cause damage to tissues that are important in general and male reproductive physiology.

Uroplakin expression in the male reproductive tract of rat.

Uroplakins (UPKs) play an important role in the normal and pathophysiology of the urothelium. They protect the urothelium and play a crucial role during urothelial infections by Uropathogenic E. coli. However, their functions beyond this organ system remain unexplored. A wide variety of proteins secreted in the male reproductive tract tissues contribute to spermatogenesis, sperm maturation, fertilization and innate immunity. However, the presence of UPKs and their possible contribution to the male reproductive tract physiology is not yet reported. Hence, in this study, we characterized UPKs in the male reproductive tract of rats. To the best of our knowledge, for the first time, we report the expression of UPKs in the male reproductive system. Upk1a, Upk1b, Upk2 and Upk3b mRNA and their corresponding proteins were abundantly expressed in the caput, cauda, testis, seminal vesicles and the prostate. Their expression was not developmentally regulated. UPK protein expression was also localized on the spermatozoa, suggesting a role for these proteins in sperm function. To study the role of UPKs in innate immunity, Upk mRNA expression in response to endotoxin challenge was evaluated in vitro and in vivo. In the rat testicular and epididymal cell lines, Upk mRNA levels increased in response to lipopolysaccharide challenge. However, in the caput, cauda, testes, seminal vesicle and prostate obtained from LPS treated rats, Upk mRNA expression was significantly reduced. Results of this study indicate a role for UPKs in male reproductive physiology and innate immune responses.

Characterization of isolated rat caput epididymal primary epithelial cells: A molecular biology approach.

Sperm maturation in the epididymis is a tightly regulated process which involves secretion and addition of a variety of proteins onto the sperm surface. The molecular mechanisms governing these processes has gained interest in the last decade. In vitro model systems to study the role of epididymal proteins in sperm maturation and other physiological process are very important. Isolation of epididymal cells, culture of epididymal explants and generation of immortalized cells were standardized to be used as in vitro models to study epididymal function. However, isolation and maintenance of primary cultures of epididymal epithelial cells seems to be the best option because of its closeness to the in vivo conditions. Though structural and morphological characterization of primary cultures of epididymal epithelial cells were carried out, the same were not conducted at the molecular level. In this study, we isolated adult rat epididymal primary epithelial cells (EPECs) and characterized them for their purity and cell specific expression of molecular markers. Isolated EPECs exhibited normal cell morphology and were sub cultured and maintained up to 3 weeks. EPECs expressed the epithelial marker, E-cadherin and their purity was estimated to be 73% using flow cytometry. EPECs abundantly expressed CRISP1, Urp1a, Pate-F, Crisp1, Ar and Spag11e, markers of epididymal cells and were negative for Urp1b and Pate, markers negative for epididymis. Results of our study provide a systematic characterization of EPECs at the molecular level and thus a refinement to the previously reported characterization methods.