Comparative transcriptomic analysis of ovary and testis reveals sex-related genes in roughskin sculpin Trachidermus fasciatus

The mechanisms underlying sex determination and differentiation have long intrigued researchers in the fields of development and evolutionary biology.The roughskin sculpin(Trachidermus fasciatus Heckel),displaying sexual dimorphism,provides an ideal model for studying the mechanisms.However,both genetic and genomic information concerning sex determination and differentiation,such as gonadal transcriptome data in roughskin sculpin,are lacking.Here,we present the first gonadal transcriptomes of roughskin sculpin and identify sex-related genes.We identified 8531 differentially expressed genes(DEGs),among them 4065 were upregulated in the ovary and 4466 upregulated in the testis.Several sex-related gene ontology(GO)terms were enriched in ovary-biased genes,including“binding of sperm to zona pellucida”,“egg coat formation”,“positive regulation of acrosome reaction”,“cell division”,and“cell cycle”,while the GO terms such as“spermatogenesis”,“sperm axoneme assembly”,“cilium assembly”,“cilium movement”,and“cilium movement involved in cell motility”were enriched in testis-biased genes.Moreover,six KEGG pathways were significantly enriched in the ovary,whereas only one was enriched in the testis.Of these DEGs,40 sex-related genes were identified,which including 26 testis-biased genes(such as Dmrtb 1,Gsdf,Sox 9 b,Wnt 4 b,Tcp 11 l 2,and Efhb),and 14 ovary-biased genes(such as Cyp 19 a 1 a,Foxh 1,Foxr 1,Gdf 3,Hsd 17 b 12,and Igf 2 bp 3).This gonadal transcript dataset would broaden our understanding of sex determination and differentiation mechanisms in roughskin sculpin,expand the genomic database,support future studies on sex-related gene functions,and facilitate molecular biology research into roughskin sculpin.

Testis Development and Ultrastructural Features During Spermatogenesis in Cultured Small Yellow Croaker,Larimichthys polyactis

The small yellow croaker Larimichthys polyactis is an economically important marine fish in Northeast Asia.Currently,its natural resources are threatened by overfishing and environmental pollution.Therefore,research on the reproductive system of the fish is crucial.Here,we studied the testis development and ultrastructural features of spermatogenesis in cultured L.polyactis using anatomical,histological,and ultrastructural techniques.A pair of testes,consisting of a central sperm duct and radial seminiferous lobules,were observed.The reproduction cycle of testes can be divided into stages I–VI.March to May was confirmed as the breeding season for male L.polyactis,while April is the ideal period for artificial breeding.The male L.polyactis can attain sexual maturity within 1 year.The spermatogenesis of L.polyactis comprised spermatogonium,spermatocyte,spermatid,and mature spermatozoon.The morphology of spermatogenic cells changed obviously during spermiogenesis,including nuclear shaping,midpiece and flagellum formation.The mature sperms consist of an ellipsoidal head,a short midpiece,and a long flagellum.The anterior of the head with a kidney-shaped nucleus can be distinguished.The midpiece is located posterior to the head and includes four to six spherical mitochondria.The flagellum has irregular lateral fins.The testis of L.polyactis is an unrestricted lobular type,with cystic spermatogenesis,type II spermiogenesis,and type II spermatozoa.These features are highly similar to those of other Sciaenid species.Our findings provide useful insights into the mechanism underlying testis development and spermatogenesis of L.polyactis,which can facilitate the artificial breeding of this species.

Expression of anti-Mullerian hormone receptor on the appendix testis in connection with urological disorders

The female internal sex organs develop from the paramesonephric （Mullerian） duct. In male embryos, the regression of the Mullerian duct is caused by the anti-Mullerian hormone （AMH）, which plays an important role in the process of testicular descent. The physiological remnant of the Mullerian duct in males is the appendix testis （AT）. In our previous study, we presented evidence for the decreased incidence of AT in cryptorchidism with intraoperative surgery. In this report, the expression of the anti-Mullerian hormone receptor type 2 （AMHR2）, the specific receptor of AMH, on the AT was investigated in connection with different urological disorders, such as hernia inguinalis, torsion of AT, cysta epididymis, varicocele, hydrocele testis and various forms of undescended testis. The correlation between the age of the patients and the expression of the AMHR2 was also examined. Reverse transcriptase-polymerase chain reaction （RT-PCR） and immunohistochemistry were used to detect the receptor＇s mRNA and protein levels, respectively. We demonstrate that AMHR2 is expressed in the ATs. Additionally, the presence of this receptor was proven at the mRNA and protein levels. The expression pattern of the receptor correlated with neither the examined urological disorders nor the age of the patients; therefore, the function of the AT remains obscure.

Testicular torsion in undescended testis:A persistent challenge

Objective:To evaluate the management and outcomes of patients who presented with torsion of an undescended testis and review the reported series in the literature.Methods:The case records of 13 patients operated for testicular torsion involving undescended testis were retrospectively reviewed.The medical records included age at presentation,medical history,physical examination,operative findings and the results of follow-up.The diagnosis of torsion of undescended testis was made clinically and confirmed by inguinal exploration.Results:In six cases the testis was preserved and orchiopexy was performed,while in seven cases orchidectomy was performed due to testicular gangrene in six patients and testicular tumor discovered peroperatively in one case.Mean duration of symptoms at time of surgery in the orchiopexy group was 6.5 h and in the orchidectomy group was 21.2 h.From six patients treated by orchiopexy,two patients suffered from testicular atrophy at a mean of 24 months.Conclusion:Testicular torsion in undescended testis is still diagnosed with delay which may affect testicular salvage.The importance of examination of external genital organs is highlighted which should be routinely included by emergency physicians in physical examination for abdominal or groin pain.

Mutations of t-complex testis expressed gene 5 transcripts in the testis of sterile t-haplotype mutant mouse

Aim： To determine the possible roles of the t-complex testis expressed gene 5 （Tctex5） on sperm functions, the fulllength sequence of mRNA was studied and compared in the testis between the normal wild-type and the sterile t-haplotype mutant mice. Methods： We applied rapid amplification of cDNA ends, Northern blot and reverse transcription polymerase chain reaction to analyze the full length of Tctex5 mRNAs isolated from testes of the wild-type and the t-haplotype mice. Reverse transcription polymerase chain reaction was used to semi-quantitatively compare expression of Tctex5 transcripts in the 16 tissues and 9.5 day stage embryos in the wild-type mice. E-translation was applied to estimate the amino acid sequences. Results： One long and one short transcript of Tctex5 mRNA were discovered in mouse testis of wild-type （Tctex5^long-＋ and Tctex5^short-＋） and t-haplotype （Tctex5^long-＋ and Tctex5^short-＋） mice, respectively. Being enhanced only in the testis, Tctex5^long-＋ had 17 point mutations and one 15-bp-deletion in the exon 1 region, comparing with the Tctex5^long-＋, whereas the Tctex5^short-＋ was similar to the Tctex5^short-＋. The short isoforms of Tctex5 mRNAs in the two models encoded exactly the same peptides, but the long isoforms did not. The estimated peptide encoded by Tctex5^long-＋ had significant mutations on putative sites of phosphorylation and PP1 binding. Conclusion： We established that mutations that occur in the Tctex5 long transcript of the t-haplotype mice are important for normal sperm function, whereas the short transcript of Tctex5 might have a conserved function among different tissues. （Asian J Androl 2008 Mar; 10： 219-226）

Expression of a novel pyridoxal kinase mRNA splice variant,PKH-T, in human testis

Aim: To identify the genes specifically expressed in human adult and fetal testes and spermatozoa. Methods: A human testis cDNA microarray was established. Then mRNAs of human adult and fetal testis and spermatozoa were purified and probes were prepared by a reverse transcription reaction with mRNA as the template. The microarray was hybridized with probes of adult and fetal testes and spermatozoa. The nucleic acid sequences of differentially expressed genes were determined and homologies were searched in the databases of GenBank. Results: A novel human testis-specific gene, PKH-T, was identified by hybridizing adult and fetal testis and spermatozoa probes with a human testis cDNA microarray. The cDNA of PKH-T was 1 069 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AY303972) and PKH-T was also determined as Interim GenSymbol (Unigene, HS.38041). PKH-T contained most PKH conserved motif. The 239 amino acid sequences deduced from the 719 bp open reading frame (ORF) had a homology with the gene PKH (U89606). PKH-T was specifically and strongly expressed in the testis. Comparison of the differential expressions of PKH and PKH-T in testes of different develop-mental stages indicated that PKH-T was expressed in the adult testis and spermatozoa, while PKH, in the adult, fetal and aged testes. PKH-T had no expression in the testis of Sertoli cell only and partially spermatogenic arrest patients. Conclusion: PKH-T is a gene highly expressed in adult human testis and spermatozoa. It may play an important role in spermatogenesis and could be related to male infertility. (Asian J Androl 2004 Jun; 6: 83-91)

Expression and role of P-element-induced wimpy testis-interacting RNA in diabetic-retinopathy in mice

BACKGROUND As one of the major microvascular complications of diabetes,diabetic retinopathy(DR)is the leading cause of blindness in the working age population.Because the extremely complex pathogenesis of DR has not been fully clarified,the occurrence and development of DR is closely related to tissue ischemia and hypoxia and neovascularization The formation of retinal neovascularization(RNV)has great harm to the visual acuity of patients.AIM To investigate the expression of P-element-induced wimpy testis-interacting RNA(piRNA)in proliferative DR mice and select piRNA related to RNV.METHODS One hundred healthy C57BL/6J mice were randomly divided into a normal group as control group(CG)and proliferative DR(PDR)group as experimental group(EG),with 50 mice in each group.Samples were collected from both groups at the same time,and the lesions of mice were evaluated by hematoxylin and eosin staining and retinal blood vessel staining.The retinal tissues were collected for second-generation high-throughput sequencing,and the differentially expressed piRNA between the CG and EG was detected,and polymerase chain reaction(PCR)was conducted for verification.The differentially obtained piRNA target genes and expression profiles were enrichment analysis based on gene annotation(Gene Ontology)and Kyoto Encyclopedia of Genes and Genomes.RESULTS In the CG there was no perfusion area,neovascularization and endothelial nucleus broke through the inner boundary membrane of retinap.In the EG,there were a lot of nonperfused areas,new blood vessels and endothelial nuclei breaking through the inner boundary membrane of the retina.There was a statistically significant difference in the number of vascular endothelial nuclei breaking through the inner retinal membrane between the two groups.High-throughput sequencing analysis showed that compared with the CG,a total of 79 piRNAs were differentially expressed in EG,among which 43 piRNAs were up-regulated and 36 piRNAs were down-regulated.Bioinformatics analysis showed that the differentially expressed piRNAs were mainly concentrated in the signaling pathways of angiogenesis and cell proliferation.Ten piRNAs were selected for PCR,and the results showed that the expression of piR-MMU-40373735,piRMMU-61121420,piR-MMU-55687822,piR-MMU-1373887 were high,and the expression of piR-MMU-7401535,piR-MMU-4773779,piR-MMU-1304999,and piR-MMU-5160126 were low,which were consistent with the sequencing results.CONCLUSION In the EG,the abnormal expression of piRNA is involved in the pathway of angiogenesis and cell proliferation,suggesting that piRNAs have some regulatory function in proliferative diabetic-retinopathy.

The expression and significance of CATSPER1 in human testis and ejaculated spermatozoa

Aim： To investigate the distribution of cation channel of sperm 1 （CATSPER1） protein and the presence of CATSPER1 mRNA in human testis and ejaculated spermatozoa. The influence of anti-human CATSPER1 antibody upon human sperm motility was used to evaluate the function of human CATSPER1 and to estimate its possible use as a target for immunocontraception. Methods： Human ejaculated sperm from normozoospermic donors （n = 12） and liquid nitrogen frozen human testis were used for the study of mRNA and protein expression of CATSPER1 by reverse transcription polymerase chain reaction （RT-PCR） and immunohistochemistry, respectively. Spermatozoa from normozoospermic donors （n = 12） were individually processed using a swim-up procedure and were then incubated with CATSPER1 antibody at final concentrations of 20, 4 and 0.8 μg/mL. After 1, 2 and 6 h incubation, progressive motility and fast progressive motility were measured by means of computer-assisted semen analysis. Results： CATSPER1 transcript was detected in both human testis and each human ejaculated semen sample. CATSPER1 protein expressed in the membrane of spermatid and was localized in the principal piece of the sperm tail. The application of CATSPER1 antibody at all concentrations significantly inhibited both progressive motility and fast progressive motility after 1, 2 and 6 h incubation, and significant dose-dependent changes were observed. Conclusion： CATSPER1 is meiotically and post-meiotically expressed in human testis tissue. CATSPER1 mRNA in human ejaculated spermatozoa could be a more feasible target for study and infertility screening than testis biopsy. In addition, our results suggest that human CATSPER1 could be a possible target for immunocontraception.

Effects of melatonin on lipid peroxidation and antioxidant enzymes in streptozotocin-induced diabetic rat testis

Aim： To examine the effects of melatonin treatment on lipid peroxidation （LPO） and the activities of antioxidant enzymes in the testicular tissue of streptozotocin （STZ）-induced diabetic rats. Methods： Twenty-six male rats were randomly divided into three groups as follows： group Ⅰ, control, non-diabetic rats （n = 9）; group Ⅱ, STZ-induced, untreated diabetic rats （n = 8）; group Ⅲ, STZ-induced, melatonin-treated （dose of 10 mg/kg·day） diabetic rats （n = 9）. Following 8-week melatonin treatment, all rats were anaesthetized and then were killed to remove testes from the scrotum. Results： As compared to group Ⅰ, in rat testicular tissues of grouap Ⅱ, increased levels of malondialdehyde （MDA） （P 〈 0.01） and superoxide dismutase （SOD） （P 〈 0.01） as well as, decreased levels of catalase （CAT） （P 〈 0.01） and glutathione peroxidase （GSH-Px） （P 〉 0.05） were found. In contrast, as compared to group Ⅱ, in rat testicular tissues of group Ⅲ, levels of MDA decreased （but this decrease was not significant, P 〉 0.05） and SOD （P 〈 0.01） as well as CAT （P 〈 0.05） increased. GSH-Px was not influenced by any of the treatment. Melatonin did not significantly affect the elevated glucose concentration of diabetic group. At the end of the study, there was no significant difference between the melatonin-treated group and the untreated group by means of body and testicular weight. Conclusion： Diabetes mellitus increases oxidative stress and melatonin inhibits lipid peroxidation and might regulate the activities of antioxidant enzymes of diabetic rat testes.

Histological changes of the testis and epididymis in adult rats as a result of Leydig cell destruction after ethane dimethane sulfonate treatment： a morphometric study

Aim： To quantitatively study the histological changes of the testis and epididymis as a result of a drastic reduction of testosterone secretion. Methods： Fourteen adult Sprague-Dawley rats were injected intraperitoneally with ethane dimethane sulfonate （EDS, 75 mg/kg） and the same number of animals were injected with normal saline as a control. At days 7 and 12 （after treatment）, respectively, half of the animals from each group were killed. The testes and epididymides were removed and tissue blocks embedded in methacrylate resin. The cell number per testis was estimated using the stereological optical disector and some other parameters were obtained using other morphometric methods. Results： The EDS treatment resulted in an almost complete elimination of Leydig cells but had no effect on the numbers of Sertoli cells per testis. At day 7 after EDS treatment, many elongated spermatids were retained in the seminiferous epithelium and many round spermatids could be seen in the epididymal ducts. At day 12, a looser arrangement of spermatids and spermatocytes became evident, with apparent narrow empty spaces being formed between germ cells in an approximately radial direction towards the tubule lumen; the numbers （per testis） of non-type B spermatogonia and spermatocytes were similar to controls, whereas that of type B spermatogonia increased by 59%, and that of early round, elongating and late elongated spermatids decreased by 37%, 72% and 52%, respectively. Conclusion： The primary spermatogenic lesions following EDS administration were （i） spermiation failure and （ii） detachment of spermatids and spermatocytes associated with impairment in spermiogenesis and meiosis.

Expression and localization of Smadl, Smad2 and Smad4 proteins in rat testis during postnatal development

<abstract>Aim: To study the expression and regulation of Smadl, Smad2 and Smad4 proteins (intracellular signaling molecules of transforming growth factor-β family) in rat testis during postnatal development. Methods: The whole testes were collected from SD rats aged 3, 7, 14, 28 and 90 (adult) days. The cellular localization and developmental changes were examined by immunohistochemistry ABC method with the glucose oxidase-DAB-nickel enhancement technique. Quantitative analysis of the immunostaining was made by the image analysis system. The Smads proteins coexistence in the adult rat testis was tested by the double immune staining for CD14-Smad4 and Smad2-Smad4. The protein expression of Smad during rat testicular development was examined by means of Western blots. Results: Smadl, Smad2 and Smad4 were present throughout testicular development. The immunostaining of Smadl and Smad2 were present in spermatogenic cells. A positive immunoreactivity was located at the cytoplasm, but the nucleus was negative. Smadl was immunolocalized at the d14, d28 and adult testes, while Smad2, at the d7, d14, d28 and adult testis. There was positive irnmunoreaction in the Sertoli cells and Leydig cells as well. The immunolocalization of Smad4 was exclusively at the cytoplasm of Leydig cells and the nuclei were negative throughout the testicular development. No expression was detected in the germ cells. The results of image and statistical analysis showed that generally the expression of Smadl, Smad2 and Smad4 in the testis tended to increase gradually with the growth of the rat. Conclusion: The present data provide direct evidences for the molecular mechanism of TGF-βaction in rat testes during postnatal development and spermatogenesis.

Apparent diffusion coefficient values of normal testis and variations with age

The usefulness of diffusion-weighted magnetic resonance imaging （DWI） in the evaluation of scrotal pathology has recently been reported. A standard reference of normal testicular apparent diffusion coefficient （ADC） values and their variations with age is necessary when interpreting normal testicular anatomy and pathology. We evaluated 147 normal testes using DWI, including 71 testes from 53 men aged 20-39years （group 1）, 67 testes from 42 men aged 40-69 years （group 2） and nine testes from six men older than 70years （group 3）. DWI was performed along the axial plane, using a single shot, multislice spin-echo planar diffusion pulse sequence and b-values of 0 and 900 s mm-2. The mean and standard deviation of the ADC values of normal testicular parenchyma were calculated for each age group separately. Analysis of variance （ANOVA） followed by post hoc analysis （Dunnett T3） was used for statistical purposes. The ADC values （x 10-3 mm2s-1） of normal testicular tissue were different among age groups （group 1：1.08 ± 0.13; group 2：1.15 ±0.15 and group 3：1.31± 0.22）. ANOVA revealed differences in mean ADC among age groups （F= 11.391, P〈 0.001）. Post hoc analysis showed differences between groups 1 and 2 （P= 0.008） and between groups 1 and 3 （P= 0.043）, but not between groups 2 and 3 （P= 0.197）. Our findings suggest that ADC values of normal testicular tissue increase with advancing age.

Effects of plants and plant products on the testis

For centuries, plants and plant-based products have been used as a valuable and safe natural source of medicines for treating various ailments. The therapeutic potential of most of these plants could be ascribed to their anticancer, antidiabetic, hepatoprotective, cardioprotective, antispasmodic, analgesic and various other pharmacological properties. However, several commonly used plants have been reported to adversely affect male reproductive functions in wildlife and humans. The effects observed with most of the plant and plant-based products have been attributed to the antispermatogenic and/or antisteroidogenic properties of one or more active ingredients. This review discusses the detrimental effects of some of the commonly used plants on various target cells in the testis. A deeper insight into the molecular mechanisms of action of these natural compounds could pave the way for developing therapeutic strategies against their toxicity.

Influence of selenium induced oxidative stress on spermatogenesis and lactate dehydrogenase-X in mice testis

Aim: To evaluate the effect of oxidative stress on the spermatogenesis and lactate dehydrogenase-X (LDH-X) activity in mouse testis. Methods: For creating different levels of oxidative stress in mice, three selenium (Se) level diets were fed in separate groups for 8 weeks. Group 1 animals were fed yeast-based Se-deficient (0.02 ppm) diet. Group 2 and Group 3 animals were fed with the same diet supplemented with 0.2 ppm and 1 ppm Se as sodium selenite, respectively. After 8 weeks, biochemical and histopathological observations of the testis were carried out. LDH-X levels in the testis were analyzed by western immunoblot and ELISA. Results: A significant decrease in testis Se level was observed in Group 1 animals, whereas it was enhanced in Group 3 as compared to Group 2. The glutathione peroxidase (GSH-Px) activity was significantly reduced in both the liver and testis in Group 1, but not in Group 2 and 3. A significant increase in the testis glutathione-S-transferase (GST) activity was observed in Group 1, whereas no significant change was seen in Groups 2 and 3. Histological analysis of testis revealed a normal structure in Group 2. A significant decrease in the germ cell population in Group 1 was observed as compared to Group 2 with the spermatids and mature sperm affected the most. Decrease in the lumen size was also observed. In the Se-excess group (Group 3), displacement of germ cell population was observed. Further, a decrease in the LDH-X level in testis was observed in Group 1. Conclusion: Excessive oxidative stress in the Se deficient group, as indicated by changes in the GSH-Px/GST activity, affects the spermatogenic process with a reduction in mature sperm and in turn the LDH-X level.

Cytokines in the BALB/c mouse testis in various conditions

Aim: To investigate whether testosterone, estrogens, vasectomy, experimental cryptorchidism, varicocele or agingwould induce changes in the cytokine environment of the mouse testis. Methods: In adult male BALB/c mice,testosterone implants, estradiol benzoate, vasectomy, unilateral cryptorchidism, unilateral varicocele were adminis-tered/performed. The mice were followed up for different periods of time and were then sacrificed with testes incisedfor examination. The control mice received the vehicle or sham-operation. Results: IL-10 was present in Leydigcells of nearly every testis and IL-10 + macrophages in 39% of testes. IL-6 was found in the testes of intact adultmice, mice treated with testosterone for 70 days, cryptorchid testes and sham-operated testes. Conclusion: Resultssuggest that IL-10 might be involved in the generation of the immunologically privileged microenvironment in the testis.(Asian J Androl 2001 Mar; 3: 9-19)

Protective effects of estrogens and caloric restriction during aging on various rat testis parameters

Aim： To investigate the effects of 17β-estradiol （E2）, Peganum harmala extract （PHE） and caloric restriction （CR） on various testis parameters during aging. Methods： Twelve-month-old male rats were treated for 6 months with either E2 or PHE, or submitted to CR （40%）. Results： Our results show that estrogens and CR are able to protect the male gonad by preventing the decrease of testosterone and E2 levels as well as the decrease of aromatase and estrogen receptor gene expressions. Indeed, E2, PHE and CR treatments induced an increase in the superoxide dismutase activities and decreased the activity of testicular enzymes： gamma-glutamyl transferase, alkaline phosphatase, lactate deshydrogenase as well as the aspartate and lactate transaminases in aged animals. In addition, the testicular catalase and gluthatione peroxidase activities were enhanced in E2, PHE and CR-treated rats compared to untreated animals at 18 months of age. Moreover, the positive effects of estradiol, PHE and CR were further supported by a lower level of lipid peroxidation. Recovery of spermatogenesis was recorded in treated rats. Conclusion： Besides a low caloric diet which is beneficial for spermatogenesis, a protective antioxydant role of estrogens is suggested. Estrogens delay testicular cell damage, which leads to functional senescence and, therefore, estrogens are helpful in protecting the reproductive functions from the adverse effects exerted by reactive oxygen species （ROS） produced in large quanti- ties in the aged testis.

CD99and CD 106(VCAM-1)in human testis

Aim: The expression of the cytokines IL-2, IL-6, IL-10, IFN-γ and TNF-α and the adhesion proteins CD99 and CD106 was studied in the human testis at the protein level. Methods: The expression of the cytokines and the adhesion proteins was assessed using immunohistochemistry and immunoblotting. Results: None of the cytokines studied was present in the human testis, but CD99 and CD106 (VCAM-1) strongly were expressed in all the testes investigated. CD99 was present in the interstitial tissue of the human testis as well as in the Sertoli cells. The identity of the CD99+ interstitial cells is unclear. CD106 (VCAM-1) was present in Leydig cells as well as the basal parts of the Sertoli cells in the seminiferous tubules. In immunoblotting, CD99 was demonstrated at molecular ratios of 46-57 (kD). This is a novel isoform of the molecule. Conclusion: The human testis produces both CD99 and CD106 and as CD106 mediates cell binding to lymphocytes, it is possible that the human Leydig cells adhere to lymphocytes like the rodent Leydig cells. (Asian J Androl 2002 Dec; 4: 243-248)

Expression of a novel bHLH-Zip gene in human testis

<abstract>Aim: To identify specifically expressed genes in the adult and fetal testes. Methods: A human testis cDNA microarray was established. Then the mRNA of adult and fetal testis was purified and probes were prepared by a reverse transcription reaction with the testis mRNA as template. The microarray was hybridized with probes of adult and fetal testes. The nucleic sequences of differentially expressed genes were determined and homologies were searched in the databases of the GenBank. Results: When hybridized with adult or fetal testis probes, the positive clones were 96.8 % and 95.4 %, respectively. Among these genes, one was a new testis-specific gene, which was named TSP1. TSP1 was highly expressed in human adult testis. The cDNA of TSP1 was 1,484 bp in length. The cDNA sequence of this clone was deposited in the Genbank (AF333098). TSP1 was also determined as Interim Gen Symbol (Unigene, No. Hs.98266). Protein analysis showed that TSP1 contained two functional domains: an N-terminal basic helix-loop-helix (bHLH) and a C-terminal leucine zipper (Zip). Homologous analysis showed that the 430 amino acid sequences deduced from the 1,293 bp open reading frame (ORF) had a homology with the human gene FLJ2509 (AK098575). TSP1 had also a sequence homology with Spz 1 protein of mouse. Expression profiles showed that TSP1 was specifically and strongly expressed in the testis. Conclusion: TSP1 is a gene highly expressed in adult testis. It may play an important role in spermatogenesis in the humans.

Identification of a novel testis-specific gene and its potential roles in testis development/spermatogenesis

Aim:To identify and characterize a novel gene with potential roles in testis development and spermatogenesis. Methods:A cDNA microarray was constructed from a human testis large insert cDNA library and hybridized with probes of human or mouse adult and fetal testes.Differentially expressed genes were isolated and sequenced.RT-PCR was used to test the tissue distribution of the genes of interest and in situ hybridization was performed to localize the gene expression in the mouse testis.A range of bioinformatical programs including Gene Runner,SMART,NCBI Blast and Emboss CpGPlot were used to characterize the new gene's feature.Results:A novel testis-specific gene, NYD-SP5,was differentially expressed in fetal and adult testes.The deduced protein structure of NYD-SP5 was found to contain an IQ motif (a short calmodulin-binding motif containing conserved lie and Gin residues),a Carbam- ate kinase-like domain,a Zn-dependent exopeptidase domain and a lactate dehydrogenase (LDH) C-terminal-like domain.RT-PCR analysis revealed that NYD-SP5 was predominantly expressed in the testis but not in other 15 tissues examined.In situ hybridization and RT-PCR examinations revealed that the expression of NYD-SP5 was confined in the male germ cell but not present in the somatic cell in the testes.Conclusion:NYD-SP5 is a newly found testis- specific gene with potential roles in testis development and spermatogenesis through a calmodulin-activated enzyme.

Targeting cancer testis antigens for biomarkers and immunotherapy in colorectal cancer: Current status and challenges

Colorectal cancer ranks third among the estimatedcancer cases and cancer related mortalities in United States in 2014. Early detection and efficient therapy remains a significant clinical challenge for this disease. Therefore, there is a need to identify novel tumor asso-ciated molecules to target for biomarker development and immunotherapy. In this regard, cancer testis antigens have emerged as a potential targets for developing novel clinical biomarkers and immunotherapy for various malignancies. These germ cell specific proteins exhibit aberrant expression in cancer cells and contribute in tumorigenesis. Owing to their unique expression profile and immunogenicity in cancer patients, cancer testis antigens are clinically referred as the most promising tumor associated antigens. Several cancer testis antigens have been studied in colorectal cancer but none of them could be used in clinical practice. This review is an attempt to address the promising cancer testis antigens in colorectal cancer and their possible clinical implications as biomarkers and immunotherapeutic targets with particular focus on challenges and future interventions.