Deficiency of nucleosome-destabilizing factor GLYR1 dampens spermatogenesis in mice.

Aberrant sperm morphology hinders sperm motility and causes male subfertility. Spermatogenesis, a complex process in male germ cell development, necessitates precise regulation of numerous developmental genes. However, the regulatory pathways involved in this process remain partially understood. We have observed the widespread expression of Glyr1, the gene encoding a nucleosome-destabilizing factor, in mouse testicular cells. Our study demonstrates that mice experiencing Glyr1 depletion in spermatogenic cells exhibit subfertility characterized by a diminished count and motility of spermatozoa. Furthermore, the rate of sperm malformation significantly increases in the absence of Glyr1, with a predominant occurrence of head and neck malformation in spermatozoa within the cauda epididymis. Additionally, a reduction in spermatocyte numbers across different meiotic stages is observed, accompanied by diminished histone acetylation in spermatogenic cells upon Glyr1 depletion. Our findings underscore the crucial roles of Glyr1 in mouse spermiogenesis and unveil novel insights into the etiology of male reproductive diseases.

[Delivery of epididymis-specific mRNAs from mouse epididymosomes into N2a and TM4 cells].

OBJECTIVE: To investigate whether mouse epididymis-specific mRNAs Adam7 and Crisp1 can be delivered into N2a and TM4 cells, and to provide an experimental basis for exploring the function of epididymal mRNAs. METHODS: Using RT-PCR, we detected the presence of epididymis-specific genes (Adam7, Crisp1, Defb22, Wfdc2, and Wfdc9) in the testis, epididymis, epididymosome and sperm of adult male BALB/c mice as well as in the human testis, seminal vesicles and sperm. We isolated epididymosomes of BALB/c mice by low-speed centrifugation, filtration and ultracentrifugation, fluorescently labeled them by PKH26, co-incubated them for 1 hour with the N2a and TM4 cells after 24 hours of starvation culture, and observed whether they were fused with the N2a and TM4 cells and ingested using the epididymosomes without PKH26 labeling, PKH26 dye without epididymosomes, and non- epididymosome or -PKH26 dye as controls. Then we detected the epididymis-specific genes in the N2a and TM4 cells after 1-hour co-incubation by RT-PCR. RESULTS: Adam7 and Crisp1 were present in the mouse epididymis, epididymosomes and sperm, and in the human seminal vesicles and sperm as well, but not in the testes of either the mice or men. PKH26 and Hoechst33258 fluorescence double-labeling showed that the mouse epididymosomes were fused with the N2a and TM4 cells and ingested; RT-PCR revealed the mRNAs of Adam7 and Crisp1 in the N2a and TM4 cells after 1-hour co-incubation; and Western blot exhibited the CRISP1 protein in the N2a and TM4 cells incubated with epididymosomes. CONCLUSION: Epididymosomes can deliver epididymis-specific mRNAs Adam7 and Crisp1 into N2a and TM4 cells, where Crisp1 may be translated into proteins, though their function and significance need to be further studied.

Promising novel biomarkers and therapy targets: The application of cell-free seminal nucleotides in male reproduction research.

Liquid biopsy has the advantage of diagnosing diseases in a non-invasive manner. Seminal plasma contains secretions from the bilateral testes, epididymides, seminal vesicles, bulbourethral glands, and the prostate. These organs are relatively small and contain delicate tubes that are prone to damage by invasive diagnosis. Cell-free seminal nucleic acids test is a newly emerged item in liquid biopsy. Here, we present a comprehensive overview of all known cell-free DNA and cell-free RNAs (mRNA, miRNA, lncRNA, circRNA, piRNA, YRNA, tsRNA, etc.) and discuss their roles as biomarker candidates in liquid biopsy. With great advantages, including high stability, sensitivity, representability, and non-invasiveness, cell-free DNA/RNAs may be developed as promising biomarkers for the screening, diagnosis, prognosis, and follow-up of diseases in semen-secreting organs. Moreover, RNAs in semen may participate in important processes, including sperm maturation, early embryo development, and transgenerational disease inheritance, which may be developed as potential treatment targets for future clinical use.

[Molecular markers for predicting the success of micro-TESE in non-obstructive azoospermia].

Non-obstract azoospermia (NOA) is a serious male infertility disease. At present, testicular sperm extraction (micro-TESE) is performed in combination with intracytoplasmic sperm injection (ICSI) technology, NOA patients can have their own consanguine offspring. However, due to the invasiveness and uncertainty of micro-TESE surgery, it is difficult for patients to accept it. Therefore, finding an accurate method to predict the possibility of micro-TESE successful sperm retrival would be beneficial to azoospermia patients. Many genes are transcribed and expressed during spermatogenesis, and molecular assays have irreplaceable sensitivity and specificity in predicting the success sperm retrivel of micro-TESE. This article reviews the methods to predict the success sperm retrivel of micro-TESE including mRNA, non-coding RNA (piRNA, microRNA, cirRNA, tFRNAs) and some protein so far, to provide certain reference value for clinical and subsequent research.

Transcriptome and DNA methylome analysis of peripheral blood samples reveals incomplete restoration and transposable element activation after 3-months recovery of COVID-19.

Comprehensive analyses showed that SARS-CoV-2 infection caused COVID-19 and induced strong immune responses and sometimes severe illnesses. However, cellular features of recovered patients and long-term health consequences remain largely unexplored. In this study, we collected peripheral blood samples from nine recovered COVID-19 patients (median age of 36 years old) from Hubei province, China, 3 months after discharge as well as 5 age- and gender-matched healthy controls; and carried out RNA-seq and whole-genome bisulfite sequencing to identify hallmarks of recovered COVID-19 patients. Our analyses showed significant changes both in transcript abundance and DNA methylation of genes and transposable elements (TEs) in recovered COVID-19 patients. We identified 425 upregulated genes, 214 downregulated genes, and 18,516 differentially methylated regions (DMRs) in total. Aberrantly expressed genes and DMRs were found to be associated with immune responses and other related biological processes, implicating prolonged overreaction of the immune system in response to SARS-CoV-2 infection. Notably, a significant amount of TEs was aberrantly activated and their activation was positively correlated with COVID-19 severity. Moreover, differentially methylated TEs may regulate adjacent gene expression as regulatory elements. Those identified transcriptomic and epigenomic signatures define and drive the features of recovered COVID-19 patients, helping determine the risks of long COVID-19, and guiding clinical intervention.

Competitive CatSper Activators of Progesterone from Rhynchosia volubilis.

The root Rhynchosia volubilis was widely used for contraception in folk medicine, although its molecular mechanism on antifertility has not yet been revealed. In human sperm, it was reported that the cation channel of sperm, an indispensable cation channel for the fertilization process, could be regulated by various steroid-like compounds in plants. Interestingly, these nonphysiological ligands would also disturb the activation of the cation channel of sperm induced by progesterone. Therefore, this study aimed to explore whether the compounds in R. volubilis affect the physiological regulation of the cation channel of sperm. The bioguided isolation of the whole herb of R. volubilis has resulted in the novel discovery of five new prenylated isoflavonoids, rhynchones A - E (1:  - 5: ), a new natural product, 5'-O-methylphaseolinisoflavan (6: ) (1H and 13C NMR data, Supporting Information), together with twelve known compounds (7:  - 18: ). Their structures were established by extensive spectroscopic analyses and drawing a comparison with literature data, while their absolute configurations were determined by electronic circular dichroism calculations. The experiments of intracellular Ca2+ signals and patch clamping recordings showed that rhynchone A (1: ) significantly reduced cation channel of sperm activation by competing with progesterone. In conclusion, our findings indicat that rhynchone A might act as a contraceptive compound by impairing the activation of the cation channel of sperm and thus prevent fertilization.

Gut Microbiota May Not Be Fully Restored in Recovered COVID-19 Patients After 3-Month Recovery.

Coronavirus disease 2019 (COVID-19) has infected over 124 million people worldwide. In addition to the development of therapeutics and vaccines, the evaluation of the sequelae in recovered patients is also important. Recent studies have indicated that COVID-19 has the ability to infect intestinal tissues and to trigger alterations of the gut microbiota. However, whether these changes in gut microbiota persist into the recovery stage remains largely unknown. Here, we recruited seven healthy Chinese men and seven recovered COVID-19 male patients with an average of 3-months after discharge and analyzed their fecal samples by 16S rRNA sequencing analysis to identify the differences in gut microbiota. Our results suggested that the gut microbiota differed in male recovered patients compared with healthy controls, in which a significant difference in Chao index, Simpson index, and ß-diversity was observed. And the relative abundance of several bacterial species differed clearly between two groups, characterized by enrichment of opportunistic pathogens and insufficiency of some anti-inflammatory bacteria in producing short chain fatty acids. The above findings provide preliminary clues supporting that the imbalanced gut microbiota may not be fully restored in recovered patients, highlighting the importance of continuous monitoring of gut health in people who have recovered from COVID-19.

Transfer- or 'transmission'-RNA fragments? The roles of tsRNAs in the reproductive system.

Transfer-RNAs (tRNAs) help ribosomes decode mRNAs and synthesize proteins; however, tRNA fragments produced under certain conditions, known as tRNA-derived small RNAs (tsRNAs), have been found to play important roles in pathophysiological processes. In the reproductive system, tsRNAs are abundant in gametes and embryos and at the maternal-fetal interface, as well as in microvesicles like epididymosomes, seminal plasma exosomes, and syncytiotrophoblast-derived extracellular vesicles. tsRNAs can affect gamete cell maturation, zygote activation, and early embryonic development. tsRNAs can transmit epigenetic information to later generations. In particular, exposure to environmental factors such as nutrition, isoproterenol, and poly(I:C) may allow tsRNAs to transfer information to the gametes or placenta to alter offspring phenotype. The underlying mechanisms of tsRNAs action include transposon silencing, translation regulation, and target mRNA degradation. Herein, we review the currently reported tsRNAs in the reproductive system, their validated functions, and potential roles. A better understanding of this field may help to provide useful recommendations or develop strategies to increase fertility and conception of healthy babies.

[Functional polymorphism sites in non-coding regions of folate metabolism-related genes in the reproductive-aged population of Hubei Province].

Objective: To investigate the distribution of the functional polymorphisms in the non-coding regions of folate metabolism-related genes in the reproductive-aged population of Hubei Province. METHODS: Using Sanger sequencing, we examined the polymorphisms of the genes MTR (rs28372871 and rs1131450), MTRR (rs326119) and CBS (rs2850144) in 790 subjects before and during pregnancy from April 2020 to March 2021. We compared the distributions of the four loci between different populations. RESULTS: The distributions of the four genotypes of rs28372871, rs1131450, rs326119 and rs2850144 all conformed to the Hardy-Weinberg equilibrium (HWE). Statistically significant differences were observed in the polymorphism distribution of rs28372871 between Hubei and Jiangsu (P < 0.05), in that of rs1131450 between Hubei and Shanghai (P < 0.05), and in that of rs2850144 between Hubei and Yazd, Iran (P < 0.01). CONCLUSIONS: This was the first investigation on the distribution of MTR, MTRR and CBS gene polymorphisms in the reproductive-aged population of Hubei Province. The effects of the functional loci in both encoding and non-coding regions of folate metabolism-related genes have to be comprehensively considered so as to formulate an appropriate folate-supplementary protocol.

Comprehensive circRNA/miRNA/mRNA analysis reveals circRNAs protect against toxicity induced by BPA in GC-2 cells.

Aim: To identify the circRNAs expression pattern and roles in bisphenol A (BPA) induced germ cell apoptosis. Materials & methods: We performed circRNA/miRNA/mRNA-Seq in 120 µM BPA treated and nontreated GC-2 cells. Bioinformatic analysis, qPCR, apoptosis assays, luciferase report were done in the function analysis. Results: A large number of apoptosis related circRNAs/miRNAs/mRNAs were differentially expressed with competing endogenous RNA network constructed. Interestingly, most investigated upregulated circRNAs, including circDcbld2, circMapk1, circMpp6 and circTbc1d20 showed protective effects in antagonizing BPA toxicity, with the effects individually and synergistically observed. CircMapk1 may take its role by sponging miR-214-3p. Conclusion: circRNAs can play protective roles via sponging miRNAs in toxicity. Some circRNAs may serve as novel targets for BPA toxicity intervention or as biomarkers.

Intriguing circles: Conflicts and controversies in circular RNA research.

Circular RNAs (circRNAs) are covalently closed RNA circles without a 5' cap or 3' tail. Since the landmark discovery of ciRS-7/CDR1as functioning as a miR-7 sponge in 2013, circRNAs have become a hot topic in RNA research. CircRNAs have been found to play active roles in cancer, cardiovascular diseases, neurological disorders, and many other diseases. They can function as microRNA (miRNA) sponges, protein scaffolds, and even translation templates. However, as circRNA research expands, many divergent views have emerged. For example, are most circRNAs competent in serving as miRNA sponges? What kinds of circRNAs are most likely to sponge miRNAs? Apart from sponging miRNAs, what could the functions of most circRNAs be? What are the features of circRNAs that are translatable? Many researchers have claimed that circRNAs are abundant, stable, conserved, and specific molecules, which hold great potential in serving as biomarkers. However, circRNA abundance is variable and some circRNAs are abundant while others are not. In addition, their stability and conservation may vary under different circumstances. Furthermore, it is unclear whether circRNA biogenesis is more likely to be regulated by RNA binding proteins or transcription factors. All of these are open questions that remain to be answered by researchers in this field. Discussing and investigating these questions will advance the understanding of this class of novel molecules and may propel inspiring new ideas for future studies. This article is categorized under: Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs RNA in Disease and Development > RNA in Disease RNA Methods > RNA Analyses in Cells RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

[Correlation of sperm DNA fragmentation index with age and semen parameters in infertile men].

OBJECTIVE: To explore the correlation of the sperm DNA fragmentation index (DFI) with age, sperm concentration and sperm motility in infertile men. METHODS: We collected semen samples from 531 infertile males in our hospital from January 2016 to June 2017. We determined the semen parameters using the computer-assisted semen analysis system, measured the sperm DFI by sperm chromatin structure assay, and analyzed the correlation of the sperm DFI with the age, sperm concentration and sperm motility of the patients. RESULTS: With the increase of age, the infertile males showed a significantly decreased proportion of the sperm with a DFI ≤15% and elevated proportion of the sperm with a DFI ≥25%, with a positive correlation between age and sperm DFI (r = 0.653, P < 0.01). With the increase of sperm concentration and motility, however, the proportion of the sperm with a DFI ≤15% was remarkably increased while that of the sperm with 15%

Intestinal absorption of pallidifloside D are limited by P-glycoprotein in mice.

1. Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to be very effective in hyperuricemic control. But it is poorly bioavailable after oral administration. Here, we determined the role of P-glycoprotein (P-gp) in the intestinal absorption of Pallidifloside D. 2. We found that Pallidifloside D significantly stimulated P-gp ATPase activity in vitro ATPase assay with a small EC50 value of 0.46 µM. 3. In the single-pass perfused mouse intestine model, the absorption of Pallidifloside D was not favored in the small intestine (duodenum, jejunum and ileum) with a P*w value of 0.35-0.78. By contrast, this compound was well-absorbed in the colon with a P*w value of 1.23. The P-gp inhibitors cyclosporine significantly enhanced Pallidifloside D absorption in all four intestinal segments (duodenum, jejunum, ileum and colon) and the fold change ranged from 5.5 to 15.3. Pharmacokinetic study revealed that cyclosporine increased the systemic exposure of Pallidifloside D by a 2.5-fold after oral administration. 4. These results suggest that P-gp-mediated efflux is a limiting factor for intestinal absorption of Pallidifloside D in mice.

Identification and characterization of human testis derived circular RNAs and their existence in seminal plasma.

Circular RNAs (circRNAs) have emerged as novel molecules of interest in gene regulation as other noncoding RNAs. Though they have been explored in some species and tissues, the expression and functions of circRNAs in human reproductive systems remain unknown. Here we revealed the expression profiles of circRNAs in human testis tissue using high-throughput sequencing. The conformation of these testis-derived circRNAs in seminal plasma was also investigated, aiming to provide a non-invasive liquid biopsy surrogate for testicular biopsy. We predicted >15,000 circRNAs in human testis, with most of them (10,792; 67%) new. In all the 5,928 circRNA forming genes, 1,017 are first reported by us to generate circRNAs. Interestingly, these genes are mostly related to spermatogenesis, sperm motility, fertilization, etc. The sequence feature, chromosome location, alternative splicing and other characteristics of the circRNAs in human testis were also explored. Moreover, we found that these testis-derived circRNAs could be stably detected in seminal plasma. Most of them were probably bound with proteins in seminal plasma and were very stable at room temperature. Our work has laid the foundations to decipher regulation mechanisms of circRNAs in spermatogenesis and to develop circRNAs as novel noninvasive biomarkers for male infertile diseases.

Hypouricemic effect of allopurinol are improved by Pallidifloside D based on the uric acid metabolism enzymes PRPS, HGPRT and PRPPAT.

Allopurinol is a commonly used medication to treat hyperuricemia and its complications. Pallidifloside D, a saponin glycoside constituent from the total saponins of Smilax riparia, had been proved to enhanced hypouricemic effect of allopurinol based on uric acid metabolism enzyme XOD. In this study, we evaluated whether Pallidifloside D (5mg/kg) enhanced hypouricemic effect of allopurinol (5mg/kg) related to others uric acid metabolism enzymes such as PRPS, HGPRT and PRPPAT. We found that, compared with allopurinol alone, the combination of allopurinol and Pallidifloside D significantly up-regulated HGPRT mRNA expression and down-regulated the mRNA expression of PRPS and PRPPAT in PC12 cells (all P<0.01). These results strongly suggest that hypouricemic effect of allopurinol are improved by Pallidifloside D via numerous mechanisms and our data may have a potential value in clinical practice in the treatment of gout and other hyperuricemic conditions.

The plasma miR-125a, miR-361 and miR-133a are promising novel biomarkers for Late-Onset Hypogonadism.

Circulating miRNAs have been shown to serve as diagnostic/prognostic biomarkers in cancers and other diseases. However, the role of plasma miRNAs in Late-onset hypogonadism (LOH) diagnosis is still unknown. Using Illumina HiSeq2000 sequencing at discovery phase, and then two-step validated by reverse transcriptase polymerase chain reaction (RT-PCR) assays in verification phases. We verified that the expression levels of miR-125a-5p, miR-361-5p and miR-133a-3p were significantly altered in LOH group compared to the control group. The area under the receiver operating characteristic (ROC) curve (AUC) is 0.682, 0.698 and 0.765, respectively. The combination of three miRNAs showed a larger AUC (0.835) that was more efficient for the diagnosis of LOH. Among three miRNAs, miR-133a-3p had the best diagnostic value for LOH with 68.2% sensitivity and 77.3% specificity. Regression analyses show that miR-133a-3p level was negatively associated with the ageing males' symptoms (AMS) scale. However, miR-361-5p level was positively associated with serum testosterone concentrations. In summary, plasma miRNAs are differentially expressed between LOH and healthy controls. We validated three miRNAs that could act as novel biomarkers for diagnosis of LOH. These miRNAs may be involved in the development of LOH. However, further large and functional studies are warranted to confirm our findings.

Association of Alcohol Consumption with Markers of Prostate Health and Reproductive Hormone Profiles: A Multi-Center Study of 4,535 Men in China.

BACKGROUND: The effect of alcohol consumption on prostate health and reproductive hormone profiles has long been investigated and currently, no consensus has been reached. Additionally, large studies focusing on this topic are relatively rare in China. PURPOSE: To investigate the association of alcohol consumption with prostate measurements and reproductive hormone profiles in Chinese population; and to examine the relationship between hormone levels and prostate measurements. METHODS: This cross-sectional study included 4535 men from four representative provinces of China. Demographic details, family history of prostate disease, tobacco and alcohol consumption, as well as International Prostate Symptom Score (I-PSS) were collected through a questionnaire. Total prostate specific antingen (total PSA), free PSA, free PSA/total PSA ratio (f/tPSA), and reproductive hormones were measured in serum. Multi-variable regression models were used to test for association of alcohol consumption with markers of prostate health, used to test for association of alcohol consumption with reproductive hormones, and reproductive hormones with markers of prostate health. RESULTS: Alcohol consumption had no obvious impact on total PSA concentration and I-PSS. Current drinkers had lower level of free PSA (ß = -0.11, p = 0.02) and f/tPSA (ß = -0.03, p = 0.005), former drinkers also had lower level of free PSA (ß = -0.19, p = 0.02) when compared with never drinkers. Lower Luteinizing hormone (LH) (ß = -1.05, p = 0.01), sex hormone-binding globulin (SHBG) (ß = -4.71, p = 0.01) and higher estradiol (ß = 7.81, p = 0.01) was found in current drinkers than never drinkers, whereas higher LH (ß = 1.04, p = 0.04) and free testosterone (FT) (ß = 0.03, p = 0.02) was detected in former drinkers than never drinkers. Furthermore, LH was positively associated with f/tPSA (ß = 0.002, p = 0.006), SHBG was also positively related with free PSA (ß = 0.003, p = 0.003) and f/tPSA (ß = 0.0004, p = 0.01). Both total testosterone (TT) and FT were inversely related with I-PSS (OR = 0.97, 95% CI, 0.95-0.98; OR = 0.23, 95% CI, 0.11-0.45, respectively). CONCLUSIONS: Alcohol consumption could affect serum free PSA concentration and also f/tPSA ratio, and also acts as an endocrine disruptor on the male reproductive hormone profiles. LH and SHBG were positively related with fPSA and f/tPSA, and higher level of TT and FT may be helpful for improving participants' subjective symptoms.

Construction of a catsper1 DNA vaccine and its antifertility effect on male mice.

Cation channel of sperm 1 (CATSPER1) is a unique sperm cation channel protein, and essential for sperm function and male fertility. CATSPER1 exclusively expresses in meiotic and postmeiotic spermatogenic cells, thus belongs to the spermatogenesis-specific antigen that escape central tolerance. We have previously demonstrated the immunocontraceptive potential of its transmembrane domains and pore region, and reported the antifertility effects of its B-cell epitopes on male mice. Aiming to develop DNA vaccine targeting CATSPER1 for male contraception, here the whole open reading frame of mouse Catsper1 was cloned into the plasmid pEGFP-N1 to obtain a DNA vaccine pEGFP-N1-Catsper1. The vaccine was confirmed to be transcribed and translated in mouse N2a cell in vitro and mouse muscle tissue in vivo. Intramuscular injection with the vaccine on male mice induced specific immune reaction and caused significant inhibition on sperm hyperactivated motility and progressive motility (P<0.001 for both), and consequently reduced male fertility. The fertility rate of experimental group was 40.9%, which was significant lower (P=0.012) than control group (81.8%). No significant change in mating behavior, sperm production and histology of testis/epididymis was observed. Given that Catsper1 exhibits a high degree of homology among different species, Catsper1 DNA vaccine might be a good strategy for developing an immunocontraceptive vaccine for human and animal use.

Urokinase-type plasminogen activator: a new target for male contraception?

Urokinase-type plasminogen activator (uPA) is closely related to male reproduction. With the aim of investigating the possibility for uPA as a potential contraceptive target, in the present work, Kunming male mice were immunized by human uPA subcutaneous injection at three separate doses for 3 times. Then the potency of the anti-human uPA antibody in serum was analyzed, and mouse fertility was evaluated. Serum antibody titers for human uPA in immunized groups all reached 1:10,240 or higher levels by enzyme linked immunosorbent assay, and mating experiments revealed that pregnancy rates and the mean number of embryos implanted after mating declined obviously (P < 0.05) when compared with control groups. However, the mating capacity and reproductive organ weights had no obvious change, and histological analysis of the testes and epididymides also showed normal morphology for immunized male mice. Sperm function tests suggested that the sperm concentration, sperm viability, sperm motility, and in vitro fertilization rate for the cauda epididymis sperm in uPA-immunized groups were lower than those in the controls (P < 0.05). Together, these observations indicated that subcutaneous injection human uPA to the male mice could effectively reduce their fertility, and uPA could become a new target for immunocontraception in male contraceptive development.

Expression of Attractin in male reproductive tract of human and mice and its correlation with male reproduction.

The expression of Attractin mRNA and protein in testis and semen of human and male mice was investigated. Human testis and semen samples were all collected from Reproductive Center of Renmin Hospital, Wuhan University in December, 2012. Testis samples were collected from 7 cases of obstructive azoospermias when they were subjected to diagnosed testis biopsy, and 30 normal human semen samples were obtained from those cases of semen analysis. Adult mice testis tissues were obtained from 10 2-month-old male BALB/c mice, and 60 male mice at different ages were classified into 10 groups (day 1, 5, 10, 15, 21, 28, 35, 42, 56, and 120 respectively, n=6 each). The expression of Attractin mRNA and protein in testis was detected by RT-PCR and Western blotting respectively. Human semen samples were centrifuged into sperm plasma (SP) and sperm extract (SE), and mice sperm samples were collected from the epididymis of 10 adult male BALB/c mice. Western blotting was used to determine the Attractin protein expression level. Attractin mRNA and protein were expressed in the testis of both patients with obstructive azoospermias and adult Bcl/B mice. Quantitative RT-PCR revealed that no Attractin mRNA was detectable in day 1 male BALB/c mice group. The Attractin mRNA and protein levels were low on the day 10, and increased with age until day 56. On the day 120, the expression levels of Attractin were decreased. As for human semen samples, Attractin protein was expressed in both SP and SE, but didn't exist in samples from the epididymis of male BALB/c mice. It was suggested that Attractin acted as a novel active substance and was involved in male reproduction in both human and BALB/c mice, but it exerted a different expression profile in different mammal species.