Increased expression of HOXA11-AS attenuates endometrial decidualization in recurrent implantation failure patients.

Endometrial decidualization is a prerequisite for implantation, and impaired decidualization is associated with recurrent implantation failure (RIF). Coding genes of the HOX family have been clarified as critical regulators in endometrial decidualization, but the role of long non-coding RNAs (lncRNAs) in the HOX gene family has yet to be determined. The aim of this study was to clarify the possible roles of lncRNAs in the HOX gene family in decidualization. In this study, we identified that HOXA11-AS was the most reduced lncRNA in the HOX family in the human endometrium during the window of implantation, and it was elevated in RIF patients. Mechanistically, HOXA11-AS negatively regulated decidualization through competitive interaction with PTBP1, an RNA-binding protein. Binding of PTBP1 to HOXA11-AS limited PTBP1 availability to regulate PKM1/2 alternative splicing, resulting in enhanced PKM1 and diminished PKM2 expression, thus attenuating decidualization. The pattern of high HOXA11-AS expression and impaired PKM2 splicing was consistently observed in RIF patients. Collectively, our study indicates that the increase of HOXA11-AS is detrimental to endometrial decidualization, likely contributing to RIF. Our study may shed light on the pathogenesis and treatment of RIF.

Identification, characterization and expression analysis of rLcn13, an epididymal lipocalin in rats.

As the essential tissue for sperm maturation and storage, the epididymis secretes a number of tissue-specific proteins to exert its functions. Among these proteins, epididymal lipocalins have been intensively studied because of their epididymis-specific expression pattern and clustered genomic organization. In this study, rLcn13, a member of the rat epididymal lipocalin family, is identified and elaborately characterized. The cDNA sequence of rLcn13 consists of 719 nucleotides and encodes a 176 amino-acid protein with a predicted N-terminal signal peptide of 19 amino acids. rLcn13 shares a similar genomic structure and predicted 3D protein structure with other lipocalin family members. A recombinant rLCN13 mature peptide of 157 amino acids is expressed and purified, which is used to raise a polyclonal antibody against rLCN13 with high specificity and sensitivity. Northern blot, western blot, and immunohistochemistry assays reveal that rLcn13 is an epididymis-specific gene which is expressed predominantly in the initial segment and proximal caput epididymis and influenced by androgen. The rLCN13 protein is modified by N-glycosylation and secreted into the epididymal lumen, and then binds to the acrosome region of the sperm. Our data demonstrate that rLcn13 exhibits a specific temporospatial expression pattern and androgen dependence, indicating its potential roles in sperm maturation.

Comprehensive study of the occurrence and characteristics of organic matter, nitrogen, and phosphorus in sediments and riparian soils of a large drinking water reservoir.

The contamination of nutrients has caused considerable worry about the environment, resource value, and ecological worth of drinking water reservoirs. Therefore, we comprehensively studied the abundance, sources, distribution, and environmental behavior of carbon, nitrogen, and phosphorus in Fengshuba Reservoir (FSBR) (a large drinking reservoir, China). A graded leaching technique (introduced in 2003), the European Standard, Measurement and Testing (SMT) protocol, and spectrometry combined with parallel factor analysis (EEMs-PARAFAC) were used to assess nitrogen, phosphorus forms, and spectra data in the sediment and soil phases, respectively. The study demonstrates that seasonal hydrological variation had no significant effect on the nutrient abundance and nutrient structure composition of the FSBR, while different environmental media (e.g., sediment and soil phase) exhibited considerable differences in nutrient abundance, composition, and environmental behavior. The abundance of colored dissolved organic matter (CDOM), as well as molecular weight, aromatization degree, and humification degree of dissolved organic matter (DOM) were all lower in sediments than in soils, whereas the authigenic component was greater than in soils. Microbial-derived humus (C1), terrestrial-derived humus (C2), and protein-like tryptophan (C3) were identified as the three primary fluorescence components. Principal component analysis indicated that three components were closely associated with phosphorus in the sediment phase, whereas nitrogen and phosphorus in the soil phase were mainly related to C1 and C2. In summary, soil media (drawdown area) must be carefully considered in the management and control of water environment nutrients in reservoirs.

Identification of HOXA10 target genes in human endometrial stromal cells by RNA-seq analysis.

Homeobox A10 (HOXA10) is a transcription factor belonging to the homeobox gene family. It is highly expressed in endometrial stromal cells (ESCs) and plays essential roles in the proliferation and differentiation of endometrium, the establishment of endometrial receptivity and embryo implantation. However, little is known about the target genes and signaling pathways regulated by HOXA10 in ESCs. In this study, we identified 1830 transcripts regulated by HOXA10 in ESCs by RNA interference (RNAi) and RNA-sequencing (RNA-seq) analysis, of which 980 were positively regulated by HOXA10 and 850 were negatively regulated by HOXA10. Interestingly, matrix metallopeptidase-11 was downregulated by HOXA10 in stromal cells verified by quantitative real-time polymerase chain reaction and western blot analysis. Pathway analysis demonstrated that the target genes were enriched in various pathways, including cellular metabolism, DNA replication and repair, cell junction, and lysosome and signal transduction. The results of the present study provide novel insights into the mechanism underlying HOXA10 regulation in ESCs and may identify novel targets for the diagnosis and treatment of endometrium-related infertility.

Emerging roles for PIWI proteins in cancer.

It is generally accepted that PIWI proteins are predominately expressed in the germline but absent in somatic tissues. Their best-characterized role is to suppress transposon expression, which ensures genomic stability in the germline. However, increasing evidence has suggested that PIWI proteins are linked to the hallmarks of cancer defined by Weinberg and Hanahan, such as cell proliferation, anti-apoptosis, genomic instability, invasion and metastasis. This provides new possibilities for anticancer therapies through the targeting of PIWI proteins, which may have fewer side effects due to their potential classification as a CTA (cancer/testis antigen). Furthermore, PIWI has been proposed to act as a diagnostic and prognostic marker for many types of cancer, and even to differentiate early- and late-stage cancers. We herein summarize the latest progress in this exciting field, hoping to encourage new investigations of PIWIs in cancer biology that will help to develop new therapeutics for clinical application.

Spink13, an epididymis-specific gene of the Kazal-type serine protease inhibitor (SPINK) family, is essential for the acrosomal integrity and male fertility.

Sperm maturation involves numerous surface modifications by a variety of secreted proteins from epididymal epithelia. The sperm surface architecture depends on correct localization of its components and highlights the importance of the sequence of the proteolytic processing of the sperm surface in the epididymal duct. The presence of several protease inhibitors from different families is consistent with the hypothesis that correctly timed epididymal protein processing is essential for proper sperm maturation. Here we show that the rat (Rattus norvegicus) epididymis-specific gene Spink13, an androgen-responsive serine protease inhibitor, could bind to the sperm acrosome region. Furthermore, knockdown of Spink13 in vivo dramatically enhanced the acrosomal exocytosis during the process of capacitation and thus led to a significant reduction in male fertility, indicating that Spink13 was essential for sperm maturation. We conclude that blockade of SPINK13 may provide a new putative target for post-testicular male contraceptives.

An androgen receptor-microrna-29a regulatory circuitry in mouse epididymis.

MicroRNAs are involved in a number of cellular processes; thus, their deregulation is usually apt to the occurrence of diverse diseases. Previous studies indicate that abnormally up-regulated miR-29a is associated with several diseases, such as human acute myeloid leukemia and diabetes; therefore, the proper level of miR-29a is critical for homeostasis. Herein, we observed that miR-29a was repressed by androgen/androgen receptor signaling in mouse epididymis by targeting a conserved androgen response element located 8 kb upstream of miR-29b1a loci. It is well known that multiple regulatory programs often form a complicated network. Here, we found that miR-29a reversibly suppressed androgen receptor and its target genes by targeting IGF1 and p53 pathways. miR-29b1a-overexpressing transgenic mice displayed epididymis hypoplasia partially similar to the phenotype of those mice with an impaired androgen-androgen receptor signal system. Taken together, the results demonstrated that there is a regulatory circuitry between the androgen signaling pathway and miR-29a in mouse epididymis that may be vital for epididymal development and functions.

Androgenic regulation of beta-defensins in the mouse epididymis.

BACKGROUND: The majority of beta-defensin family members are exclusively expressed in the epididymis, and some members have been shown to play essential roles in sperm maturation and fertility in rats, mice and humans. Therefore, beta-defensins are hypothesized to be potential targets for contraception and infertility diagnosis and treatment. Clarifying the regulatory mechanisms for the expression of these genes is necessary. Androgen/androgen receptor (AR) signaling plays an important regulatory role in epididymal structure and function. However, very little is known about the androgenic regulation on the production and secretion of the epididymal beta-defensins. METHODS: The expression of beta-defensins was detected by quantitative RT-PCR. The androgen dependence of beta-defensins was determined by bilateral orchiectomy and androgen supplementation. The androgen response elements (AREs) in the promoters of beta-defensins were identified using the MatInspector software. The binding of AR to AREs was assayed by ChIP-PCR/qPCR. RESULTS: We demonstrated that 23 mouse caput epididymal beta-defensins were differentially regulated by androgen/androgen receptor. Six genes, Defb18, 19, 20, 39, 41, and 42, showed full regulation by androgens. Ten genes, Defb15, 30, 34, 37, 40, 45, 51, 52, 22 and Spag11a, were partially regulated by androgens. Defb15, 18, 19, 20, 30, 34, 37, 39, 41, 42, 22 and Spag11a were associated with androgen receptor binding sites in their promoter or intronic regions, indicating direct regulation of AR. Six genes, Defb1, 12, 13, 29, 35, and spag11b/c, exhibited an androgen-independent expression pattern. One gene, Defb25, was highly dependent on testicular factors rather on androgens. CONCLUSIONS: The present study provides novel insights into the mechanisms of androgen regulation on epididymal beta-defensins, enabling a better understanding of the function of beta-defensins in sperm maturation and fertility.

Ovarian ferroptosis induced by androgen is involved in pathogenesis of PCOS.

STUDY QUESTION: Does ovarian ferroptosis play an active role in the development of polycystic ovary syndrome (PCOS)? SUMMARY ANSWER: Increased ovarian ferroptosis was present in PCOS ovaries and the inhibition of ferroptosis with ferrostatin-1 (Fer-1) ameliorated polycystic ovary morphology and anovulation. WHAT IS KNOWN ALREADY: Programmed cell death plays a fundamental role in ovarian follicle development. However, the types and mechanisms of cell death involved in the ovary are yet to be elucidated. Ferroptosis is a recently discovered iron-dependent programmed cell death. Impaired iron metabolism and cell death have been observed in women with PCOS, the main cause of anovulatory infertility. Additionally, previous studies reported that an abnormal expression of noncoding RNA may promote ferroptosis in immortalized ovarian granulosa cell lines. However, little is known about whether ovarian ferroptosis is increased in PCOS, and there is insufficient direct evidence for a role of ferroptosis in PCOS, and the underlying mechanism. Moreover, the effect of the inhibition of ferroptosis with Fer-1 in PCOS remains unclear. STUDY DESIGN SIZE DURATION: Ferroptosis was evaluated in human granulosa cells (hGCs) from non-PCOS (n = 6-16) and PCOS (n = 7-18) patients. The experimental study was completed in vitro using primary hGCs from women undergoing IVF. Improvements in PCOS indicators following ferroptosis inhibition with Fer-1 were investigated in a dehydroepiandrosterone (DHEA)-induced PCOS rat model (n = 8 per group). PARTICIPANTS/MATERIALS SETTING METHODS: Ovarian ferroptosis was evaluated in the following ways: by detecting iron concentrations via ELISA and fluorescent probes; measuring malondialdehyde (MDA) concentrations via ELISA; assessing ferroptosis-related protein abundance with western blotting; observing mitochondrial morphology with transmission electron microscopy; and determining cell viability. Primary hGCs were collected from women undergoing IVF. They were treated with dihydrotestosterone (DHT) for 24 h. The effect of DHT on ferroptosis was examined in the presence or absence of small interfering RNA-mediated knockdown of the putative receptor coregulator for signaling molecules. The role of ovarian ferroptosis in PCOS progression was explored in vivo in rats. The DHEA-induced PCOS rat model was treated with the ferroptosis inhibitor, Fer-1, and the oocytes and metaphase II oocytes were counted after ovarian stimulation. Additionally, rats were treated with the ferroptosis inducer, RSL3, to further explore the effect of ferroptosis. The concentrations of testosterone, FSH, and LH were assessed. MAIN RESULTS AND THE ROLE OF CHANCE: Increased ferroptosis was detected in the ovaries of patients with PCOS and in rats with DHEA-induced PCOS. Increased concentrations of Fe2+ (P < 0.05) and MDA (P < 0.05), and upregulated nuclear receptor coactivator 4 protein levels, and downregulated ferritin heavy chain 1 (FTH1) and glutathione peroxidase 4 (GPX4) proteins were observed in the hGCs in patients with PCOS and ovaries of PCOS rats (P < 0.05 versus control). DHT was shown to induce ferroptosis via activation of NOCA4-dependent ferritinophagy. The inhibition of ferroptosis with Fer-1 in rats ameliorated a cluster of PCOS traits including impaired glucose tolerance, irregular estrous cycles, reproductive hormone dysfunction, hyperandrogenism, polycystic ovaries, anovulation, and oocyte quality (P < 0.05). Treating rats with RSL3 resulted in polycystic ovaries and hyperandrogenism (P < 0.05). LARGE-SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: Although ovarian-targeted ferroptosis inhibition may be a more targeted treatment for PCOS, the underlying mechanisms in the cycle between ferroptosis and hyperandrogenism require further exploration. Additionally, since PCOS shows high heterogeneity, it is important to investigate whether ferroptosis increases are present in all patients with PCOS. WIDER IMPLICATIONS OF THE FINDINGS: Androgen-induced ovarian ferroptosis appears to play a role in the pathogenesis of PCOS, which potentially makes it a promising treatment target in PCOS. STUDY FUNDING/COMPETING INTERESTS: This study was supported by the National Key R&D Program of China (2023YFC2705500, 2023YFC2705505, 2019YFA0802604), National Natural Science Foundation of China (No. 82130046, 82320108009, 82101708, 82101747, and 82001517), Shanghai leading talent program, Innovative research team of high-level local universities in Shanghai (No. SHSMU-ZLCX20210201, No. SSMU-ZLCX20180401), Shanghai Jiaotong University School of Medicine, Affiliated Renji Hospital Clinical Research Innovation Cultivation Fund Program (RJPY-DZX-003) and Shanghai Municipal Education Commission-Gaofeng Clinical Medicine Grant Support (No. 20161413), Shanghai's Top Priority Research Center Construction Project (2023ZZ02002), and Three-Year Action Plan for Strengthening the Construction of the Public Health System in Shanghai (GWVI-11.1-36). The authors report no competing interests.

MicroRNA-29a inhibited epididymal epithelial cell proliferation by targeting nuclear autoantigenic sperm protein (NASP).

Cell proliferation often decreases gradually during postnatal development of some organs. However, the underlying molecular mechanisms remain unclear. Epididymis, playing important roles in sperm maturation, is a typical organ of this type, which displays a decreased proliferation during postnatal development and even ceased at the adult stage. Here, epididymis was employed as a model to explore the underlying mechanisms. We profiled the microRNA and mRNA expression of newborn (1 day) and adult (90 day) rat epididymis by microarray analysis, and found that the level of miR-29a was dramatically up-regulated during postnatal development of rat epididymis. Subsequent investigations demonstrated that overexpression of miR-29a inhibited the proliferation of epididymal epithelial cells in vitro. The nuclear autoantigenic sperm protein (NASP), a novel target of miR-29a, was significantly down-regulated during postnatal development of rat epididymis. Further analysis showed that silence of NASP mimicked the anti-proliferation effect of miR-29a, whereas overexpression of this protein attenuated the effect of miR-29a. As in rat epididymis, miR-29a was up-regulated and Nasp was down-regulated during postnatal development of mouse epididymis, heart, liver, and lung. Moreover, miR-29a can also inhibit the proliferation of cancer cells by targeting Nasp. Thus, an increase of miR-29a, and hence decrease of Nasp, may contribute to inhibit cell proliferation during postnatal organ development.

Different effects of androgen on the expression of Fut1, Fut2, Fut4 and Fut9 in male mouse reproductive tract.

The α-(1,2) fucosyltransferases (Fut1 and Fut2) and α-(1,3) fucosyltransferases (Fut4, Fut9) are responsible for the synthesis of Lewis X (LeX) and Lewis Y (LeY) conjugated to glycoproteins. We recently reported that these fucosyltransferases were differentially expressed in the reproductive tract of male mouse. Here, we studied the effect of androgen on fucosyltransferase expression through the use of mouse castration models. We found that Fut1 mRNA and Fut4 mRNA were upregulated, while Fut2 mRNA and Fut9 mRNA were downregulated by androgen in the caput epididymis. However, in the vas deferens and prostate, only Fut4 mRNA and Fut2 mRNA were respectively upregulated following exposure to androgen. In the seminal vesicle, all fucosyltransferases, with the exception of Fut9, were upregulated. We identified the androgen receptor binding sites (ARBSs) of Fut2, Fut4 and Fut9 in the caput epididymis. Luciferase assay for these ARBSs is able to provide an indication as to why Fut4 and Fut9 are differently expressed and regulated by androgen, although they catalyze the same α-(1,3) fucose linkage. Our study showed that androgen could differentially regulate the expression of these fucosyltransferases and provided an insight into the characteristic distribution of each fucosyltransferase responsible for LeX/LeY biosynthesis in the male reproductive tract.

[Androgen/androgen receptor directly regulates the expression of Caveolin-1 in mouse epididymides].

OBJECTIVE: To investigate the mechanisms of androgen/androgen receptor (AR) regulating the expression of Caveolin-1 in the mouse epididymis. METHODS: The AR binding sites associated with the Caveolin-1 gene were identified by searching the database of genomewide AR binding sites in mouse epididymides obtained from chromatin immunoprecipitation-sequencing (ChIP-seq). Total RNA was extracted from the epididymal tissues of normal and castrated mice and those castrated but supplemented with testosterone propionate, and the expression of Caveolin-1 mRNA was detected by RT-PCR and RT-qPCR. ChIP was performed with AR antibodies, and ChIP-PCR and ChIP-qPCR were used to determine the in vivo AR occupancies on the two sites associated with Caveolin-1. RESULTS: Two AR binding sites associated with Caveolin-1 were found in the database, both located in the second intron region. After castration, the expression of Caveolin-1 was significantly increased, 1.8 +/- 0.17 times that of the control group (P < 0.05), and the fold enrichments of the two AR binding sites were dramatically reduced from 13.5 +/- 1.47 and 10.5 +/- 1.03 to 1.05 +/- 0.17 and 1.4 +/- 0.14, respectively (P < 0.01). After androgen supplement, however, the expression of Caveolin-1 was decreased to normal (P < 0.05), and the fold enrichments of the two AR binding sites significantly increased to 16.4 +/- 2.6 and 10 +/- 0.92, respectively (P < 0.01). CONCLUSION: Caveolin-1 is a bona fide AR direct target gene in the mouse epididymis, and its expression is negatively regulated by androgen. These findings have provided a new insight into the androgen/AR regulatory network in mouse epididymides.

iTRAQ-based Proteomic Analysis Unveils ACSL4 as a Novel Potential Regulator of Human Endometrial Receptivity.

Competent endometrial receptivity is a prerequisite for successful embryo implantation. Identification of novel key molecules involved in endometrial receptivity is essential to better interpret human implantation and improve pregnancy rates in assisted reproduction treatment. Isobaric tags for relative and absolute quantitation (iTRAQ)-based proteomics was performed to profile the proteomes of the prereceptive (luteinizing hormone [LH] + 2, n = 4) and receptive (LH + 7, n = 4) endometrial tissues. A total of 173 differentially expressed proteins (DEPs) between LH + 2 and LH + 7 endometrial samples were identified. Integrated analysis of the proteomic data and published transcriptomic data was performed to identify the concordant DEPs with differential expression at both the messenger RNA and protein levels. Protein-protein interaction (PPI) network analysis was performed on concordant DEPs. We first identified 63 novel concordant DEPs and 5 hub proteins (ACSL4, ACSL5, COL1A1, PTGS1, and PLA2G4F) between LH + 2 and LH + 7 endometrial samples. ACSL4 was predominantly expressed in endometrial epithelial cells and its expression was significantly upregulated by progesterone in the LH + 7 endometrium and significantly downregulated in repeated implantation failure patients. Knockdown of ACSL4 in endometrial epithelial cells induced the downregulation of endometrial receptivity markers (HOXA10, COX2, and LIF) and the significant decrease of implantation rate during in vitro implantation analysis. This study provides the first gel-independent quantitative proteomes of the LH + 2 and LH + 7 human endometrium using iTRAQ technology. The identified concordant DEPs and hub proteins open a new avenue for future studies aimed at elucidating the underlying mechanisms governing endometrial receptivity. ACSL4 was identified as a novel regulatory molecule in the establishment of endometrial receptivity and might play important roles during implantation.

Novel mutations in PLCZ1 lead to early embryonic arrest as a male factor.

Early embryonic arrest is one of the causes of assist reproduction technology (ART) failure. We have previously reported that the first sperm-derived genetic factor, ACTL7a mutations, could lead to early embryonic arrest. However, whether there are other male genetic factors associated with early embryonic arrest remains elusive. Here, we reported bi-allelic mutations in PLCZ1, a well-known causal gene of total fertilization failure, in four infertile males. Among these mutations, p.403_404del, p.I489S, and p.W536X were newly reported in this study. Histological and Western blotting analysis of the patients' sperm indicated these variants as loss-of-function mutations. These patients manifested normal conventional semen parameters and ultra-structures in sperm heads. However, among four in vitro fertilization (IVF) cycles, 81.8% (18/22) of the oocytes were polyspermic fertilized, which was rarely reported in PLCZ1-related male patients. In the following six ICSI cycles, artificial oocyte activation (AOA) was applied and successfully rescued the fertilization failure and polyspermy phenotypes, with 31.3% (15/48) of the MII oocytes normally fertilized. However, 60.0% (9/15) of these normally fertilized zygotes were arrested at 2-5-cell stage, with one failing to cleave, indicating that PLCZ1 was not only necessary for fertilization, but also crucial for early embryonic development. However, these rescued zygotes showed a lower potential in developing into blastocysts when cultured in vitro. Thus, fresh cleavage transfer was tried and two live births were successfully achieved thereafter. In conclusion, this study provided novel mutations in PLCZ1 gene to expand the pathogenic mutational spectrum in male infertility and demonstrated that PLCZ1 was a crucial sperm-related genetic factor for early embryonic arrest. We also proposed that cleavage transfer after ICSI and AOA treatment could be a potential treatment method for male patients carrying bi-allelic mutations in PLCZ1.

Chronic stress induces meiotic arrest failure and ovarian reserve decline via the cAMP signaling pathway.

Chronic stress is suspected to be a causal factor of female subfertility; however, the underlying mechanisms remain unclear. Here, we found that chronic stress inhibited the cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway, leading to ovarian reserve decline in mice. A chronic stress model was constructed using restraint stress for 8 weeks. An elongated estrous cycle and a significant increase in the number of atretic follicles were observed in the stress group. We identified a significant increase in meiotic arrest failure (MAF) in oocytes in the stress group, characterized by condensed metaphase chromosomes, assembled spindles, or polar bodies in the oocytes. Whole-mount ovarian reserve estimation at the single-oocyte level using the CUBIC method (clear, unobstructed brain/body imaging cocktails and computational analysis) revealed a significant decrease in quiescent oocytes from 2,261/ovary in the control group to 1,373/ovary in the stress group. The number of growing oocytes also significantly decreased from 220/ovary in the control group to 150/ovary in the stress group. Real-time quantitative polymerase chain reaction (RT-qPCR) analysis of the meiotic arrest maintenance pathways revealed significant downregulation of Gpr3, Nppc, and Npr2 in the stress group. These results indicate that blocking cAMP production contributes to MAF and a decline in ovarian reserve. Overall, we present new insights into the mechanisms underlying chronic-stress-induced oocyte loss and potential targets for ovarian reserve preservation.

The epididymis-specific antimicrobial peptide ß-defensin 15 is required for sperm motility and male fertility in the rat (Rattus norvegicus).

Spermatozoa acquire forward motility and fertilizing capacity during their transit through the epididymis. The maturation process involves modifications of the sperm surface by different proteins that are secreted by a series of specialized regions in the epididymal epithelium. Here we show that the rat epididymis-specific ß-defensin 15 (Defb15) exhibits an androgen-dependent expression pattern, and it can bind to the acrosomal region of caput sperm. Coculture of caput spermatozoa with Defb15 antibody in vitro resulted in a significant decline in sperm motility. Moreover, the total and progressive motility of the spermatozoa dramatically decreased in rats when Defb15 was downregulated by lentivirus-mediated RNAi in vivo. Remarkably, knock down of Defb15 led to a reduction in fertility and embryonic development failure. In addition, the recombinant Defb15 showed antimicrobial activity in a dose-dependent fashion. These results suggest that Defb15 plays a dual role in both sperm maturation and pathogen defense in rat epididymis.

Cloning and primary characterizations of rLcn9, a new member of epididymal lipocalins in rat.

Lipocalins are a structurally conserved and diversely functional family of proteins that are of potential importance in epididymis functions. The rat Lcn9 gene was cloned by in silico methods and genome walking based on homology to the rhesus monkey epididymal ESC513 and its polyclonal antisera were prepared. The rat Lcn9 gene is located on chromosome 3p13 spanning 7 exons, contains 2.3 kb and encodes 179 amino acids with a 17-amino acid signal peptide. Northern blot, western blot, and immunohistochemical staining analysis revealed that rat Lcn9 was a novel epididymis-specific gene, expressed selectively in the proximal caput region, influenced by luminal fluid testicular factors. Moreover, Lcn9 protein was modified by N-glycosylation and bound on the postacrosomal domain of caput sperm. In conclusion, the rat Lcn9 exhibited tissue-, region-, and temporal-specific expression patterns and its expression was regulated by luminal testicular factors. Its potential roles in sperm maturation are discussed.