Knockout of glutathione peroxidase 5 down-regulates the piRNAs in the caput epididymidis of aged mice.

The mammalian epididymis not only plays a fundamental role in the maturation of spermatozoa, but also provides protection against various stressors. The foremost among these is the threat posed by oxidative stress, which arises from an imbalance in reactive oxygen species and can elicit damage to cellular lipids, proteins, and nucleic acids. In mice, the risk of oxidative damage to spermatozoa is mitigated through the expression and secretion of glutathione peroxidase 5 (GPX5) as a major luminal scavenger in the proximal caput epididymidal segment. Accordingly, the loss of GPX5-mediated protection leads to impaired DNA integrity in the spermatozoa of aged Gpx5-/- mice. To explore the underlying mechanism, we have conducted transcriptomic analysis of caput epididymidal epithelial cells from aged (13 months old) Gpx5-/- mice. This analysis revealed the dysregulation of several thousand epididymal mRNA transcripts, including the downregulation of a subgroup of piRNA pathway genes, in aged Gpx5-/- mice. In agreement with these findings, we also observed the loss of piRNAs, which potentially bind to the P-element-induced wimpy testis (PIWI)-like proteins PIWIL1 and PIWIL2. The absence of these piRNAs was correlated with the elevated mRNA levels of their putative gene targets in the caput epididymidis of Gpx5-/- mice. Importantly, the oxidative stress response genes tend to have more targeting piRNAs, and many of them were among the top increased genes upon the loss of GPX5. Taken together, our findings suggest the existence of a previously uncharacterized somatic piRNA pathway in the mammalian epididymis and its possible involvement in the aging and oxidative stress-mediated responses.

Mechanistic target of rapamycin kinase (Mtor) is required for spermatogonial proliferation and differentiation in mice.

Spermatogonial development is a vital prerequisite for spermatogenesis and male fertility. However, the exact mechanisms underlying the behavior of spermatogonia, including spermatogonial stem cell (SSC) self-renewal and spermatogonial proliferation and differentiation, are not fully understood. Recent studies demonstrated that the mTOR complex 1 (mTORC1) signaling pathway plays a crucial role in spermatogonial development, but whether MTOR itself was also involved in any specific process of spermatogonial development remained undetermined. In this study, we specifically deleted Mtor in male germ cells of mice using Stra8-Cre and assessed its effect on the function of spermatogonia. The Mtor knockout (KO) mice exhibited an age-dependent perturbation of testicular development and progressively lost germ cells and fertility with age. These age-related phenotypes were likely caused by a delayed initiation of Mtor deletion driven by Stra8-Cre. Further examination revealed a reduction of differentiating spermatogonia in Mtor KO mice, suggesting that spermatogonial differentiation was inhibited. Spermatogonial proliferation was also impaired in Mtor KO mice, leading to a diminished spermatogonial pool and total germ cell population. Our results show that MTOR plays a pivotal role in male fertility and is required for spermatogonial proliferation and differentiation.

An epididymis-specific carboxyl esterase CES5A is required for sperm capacitation and male fertility in the rat.

Despite the fact that the phenomenon of capacitation was discovered over half century ago and much progress has been made in identifying sperm events involved in capacitation, few specific molecules of epididymal origin have been identified as being directly involved in this process in vivo . Previously, our group cloned and characterized a carboxyl esterase gene Ces5a in the rat epididymis. The CES5A protein is mainly expressed in the corpus and cauda epididymidis and secreted into the corresponding lumens. Here, we report the function of CES5A in sperm maturation. By local injection of Lentivirus -mediated siRNA in the CES5A -expressing region of the rat epididymis, Ces5a -knockdown animal models were created. These animals exhibited an inhibited sperm capacitation and a reduction in male fertility. These results suggest that CES5A plays an important role in sperm maturation and male fertility.

Antimicrobial host defence peptide, LL-37, as a potential vaginal contraceptive.

STUDY QUESTION: Does antimicrobial peptide, LL-37, inhibit sperm fertilizing ability? SUMMARY ANSWER: Our results indicate that LL-37 inhibits mouse and human sperm fertilizing ability. WHAT IS KNOWN ALREADY: LL-37, a cationic antimicrobial peptide, exerts its microbicidal effects through the disruption of microbial cytoplasmic membranes following its interaction with microbial surface anionic phospholipids. ALL-38 (an LL-37 close analogue: LL-37 + Ala at the N-terminus) is produced in the vagina 2-6 h post-intercourse from its precursor hCAP-18, a seminal plasma component. At this time, motile sperm have already swum into the uterine cavity, thus unexposed to ALL-38. Since sperm contain a substantial amount of acidic sulfogalactosylglycerolipid (SGG) on their surface, treatment of sperm with LL-37 may cause their membrane disruption in an analogous manner to that occurring on microbial membranes. STUDY DESIGN, SIZE AND DURATION: Mouse/human sperm treated (2-30 min) with LL-37 in a physiological concentration range (up to 10.8 µM) were assessed for SGG-dependent LL-37 binding, and parameters relevant to fertilizing ability, namely motility and intactness of the sperm acrosome and plasma membrane. Ability of mouse sperm to fertilize eggs in vitro was also evaluated. Each study was performed with greater than or equal to three different sperm samples. The efficacy of LL-37 to inhibit sperm fertilizing ability in vivo was determined in female mice (n = 26 each for LL-37 treatment and no treatment), using sperm retrieved from 26 males. PARTICIPANTS/MATERIALS, SETTING, METHODS: Human sperm samples were donated by fertile men. LL-37 was chemically synthesized and was biotinylated for sperm binding studies. Sperm motility was assessed by videomicroscopy and the acrosomal status by Coomassie blue staining of acrosome-intact mouse sperm or the exposure of CD46, an inner acrosomal membrane protein, of acrosome reacted human sperm. Sperm membrane permeabilization/disruption was assessed by the loss of hypo-osmotic swelling response, an incorporation of Sytox Green (a membrane impermeable fluorescent DNA dye), and electron microscopy. Mouse IVF was scored by the presence of two pronuclei in eggs 6 h post-insemination. Ability of mouse sperm to fertilize eggs in vivo was determined by the pregnancy outcome of female mice injected transcervically with sperm with or without LL-37. MAIN RESULTS AND THE ROLE OF CHANCE: Biotinylated LL-37 bound to both mouse and human sperm and the binding was partially dependent on sperm surface SGG. Mouse and human sperm became immotile and underwent a premature acrosome reaction upon treatment with LL-37 at 3.6 and 10.8 µM, respectively. The initial action of LL-37 on both mouse and human sperm appeared to be through permeabilization/disruption of sperm surface membranes evidenced by the loss of hypo-osmotic swelling response, Sytox Green staining and electron microscopy revealing ultrastructural damage. Mouse sperm treated with 3.6 µM LL-37 lost the ability to fertilize eggs both in vitro and in vivo. All 26 female mice inseminated with sperm and LL-37 did not become pregnant. No apparent damage to the reproductive tract was observed as revealed by histological characterization in LL-37-inseminated mice and these females resumed fecundity following mating with fertile males. LIMITATIONS, REASONS FOR CAUTION: Direct demonstration that LL-37 treated human sperm fail to fertilize eggs was limited by legal restrictions on obtaining human eggs for such use. WIDER IMPLICATIONS OF THE FINDINGS: Our results reveal selective inhibitory effects of LL-37 on sperm fertilizing ability in mice without apparent impairment to the female reproductive tract. LL-37 is therefore a promising candidate to be developed into a vaginal contraceptive with microbicidal activity. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by Grand Challenges Explorations grant from the Bill & Melinda Gates Foundation (OPP1024509), Canadian Institutes of Health Research (MOP119438 & CCI82413) and International Collaboration and Exchanges NSFC of China (No.30611120525). There are no competing interests to declare.

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.

Molecular cloning and identification of mouse epididymis-specific gene mHong1, the homologue of rat HongrES1.

Previous studies have shown that rat epididymis-specific gene HongrES1 plays important roles in sperm capacitation and fertility. In this study, we cloned the mouse homologue gene by sequence alignment and RT-PCR methods and designated it as mHong1. The mHong1 gene is located on chromosome 12p14, spanning five exons. The cDNA sequence consists of 1257 nucleotides and encodes a 419 amino-acid protein with a predicted N-terminal signal peptide of 20 amino acids. The mHong1 mRNA shows similarity with HongrES1 in the expression patterns: (i) specific expression in epididymal tissue, especially in the cauda region; and (ii) androgen-dependence but testicular fluid factor independence. Its protein product shows 71% similarity with HongrES1 and contains a classical serpin domain as does HongrES1. A polyclonal antibody against mHong1 with high specificity and sensitivity was raised. Like HongrES1, the mHong1 protein shows a checker-board expression pattern in the epididymal epithelium and is secreted into the epididymal lumen. The mHong1 protein shows higher glycosylation than HongrES1. Although both of them are deposited onto the sperm head surface, mHong1 is localized to the equatorial segment, which is different from that of HongrES1. The mHong1 protein can be removed from the sperm membrane by high ionic strength and therefore can be classed as an extrinsic membrane protein. Collectively, we conclude that mHong1 is the homologue of HongrES1 and the present work paves the way for establishing animal models to elucidate the precise functions of HongrES1 and mHong1.

Treatment of cancer cachexia in mice by combination of dsRNA-dependent protein kinase inhibitor and medroxyprogesterone acetate.

Inhibitor of dsRNA-dependent protein kinase (PKRI) and medroxyprogesterone acetate (MPA) improve cancer cachexia via different mechanisms. We aimed to compare these two drugs, alone or in combination, in cancer cachexia in mice. Forty male BABL/c mice aged 6-8 weeks were randomly divided into PKRI, MPA, PKRI+MPA, placebo, and healthy control groups. The first 4 groups were injected with colon-26 adenocarcinoma and fed for 12 days and then treated with PKRI and MPA alone or in combination for 7 days. Body weight, tumor volume, wet weight of gastrocnemius muscle, serum levels of nutritional markers and cytokines were measured. The tumor growth (volume and weight) of mice treated with PKRI, MPA alone or PKRI+MPA was slower than that of placebo group. Wet weight of gastrocnemius muscle was significantly higher in PKRI and PKRI+MPA-treated than in placebo animals (P<0.01). All tumor-bearing mice had a significantly lower level of blood glucose, higher level of serum triglyceride and lower level of serum albumin compared with healthy control (P<0.001). However, PKRI, MPA and PKRI+MPA groups had a significant higher level of blood glucose and lower level of serum triglyceride compared with placebo group (P<0.001). All tumor bearing mice had a significant higher level of serum TNF-α, IL-1 and IL-6 compared with healthy control (P<0.001). Serum level of TNF-α and IL-6 was significantly lower in PKRI and PKRI+MPA-treated than in placebo animals (P<0.01). PKRI alone and combination therapy with PKRI and MPA reduce tumor growth and may alleviate cachexia.

Identification and characterization of a novel non-coding RNA involved in sperm maturation.

A long and ever-expanding roster of small (∼20-30 nucleotides) RNAs has emerged during the last decade, and most can be subsumed under the three main headings of microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs), and short interfering RNAs (siRNAs). Among the three categories, miRNAs is the most quickly expanded group. The most recent number of identified miRNAs is 16,772 (Sanger miRbase, April 2011). However, there are insufficient publications on their primary forms, and no tissue-specific small RNAs precursors have been reported in the epididymis. Here, we report the identification in rats of an epididymis-specific, chimeric, noncoding RNA that is spliced from two different chromosomes (chromosomes 5 and 19), which we named HongrES2. HongrES2 is a 1.6 kb mRNA-like precursor that gives rise to a new microRNA-like small RNA (mil-HongrES2) in rat epididymis. The generation of mil-HongrES2 is stimulated during epididymitis. An epididymis-specific carboxylesterase named CES7 had 100% cDNA sequence homology at the 3'end with HongrES2 and its protein product could be downregulated by HongrES2 via mil-HongrES2. This was confirmed in vivo by initiating mil-HongrES2 over-expression in rats and observing an effect on sperm capacitation.

Identification of microRNAs and application of RNA interference for gene targeting in vivo in the rat epididymis.

Identification, detection, and use of small-ribonucleic acid (RNA) molecules have been paid increasing attention in the past decades. Here we show studies of two interrelated lines of this research in a sperm maturation-related organ, the epididymis. First, by using microarray and small-RNA library screening systems, a series of spatially and temporally regulated known and novel small regulatory RNA molecules, referred to as microRNAs, has been identified and characterized. Second, small double-stranded RNAs, called small interfering RNAs, emerged as a powerful tool to knock down gene expression by a pathway known as RNA interference. It has been successfully used to silence epididymal gene expression in vivo to elucidate the function of several sperm motility- and capacitation-related proteins in the rat epididymis.

Rat recombinant ß-defensin 22 is a heparin-binding protein with antimicrobial activity.

Approximately 40-50 ß-defensins are predominantly expressed in the male reproductive system of mammals. This selective expression raises the question as to the roles of these molecules in innate immunity and fertility in the male reproductive tract. Rat ß-defensin 22 is an epididymis-specific ß-defensin expressed in segments 12-14 of the epididymis. This protein contains both ß-defensin and lectin signature sequences, yet its antimicrobial activity and carbohydrate-binding ability have not been shown. We herein demonstrated the antimicrobial activity of recombinant rat ß-defensin 22 against Escherichia coli and Candida albicans. Its lectin-like activity was also investigated by demonstrating its binding ability with heparin beads. This heparin-binding activity implies some potential roles for this defensin in determining the fertilisation capabilities of sperm.

Insulin receptor substrate-1 suppresses transforming growth factor-beta1-mediated epithelial-mesenchymal transition.

We investigated the regulatory effect of insulin receptor substrate-1 (IRS-1) on transforming growth factor-beta1 (TGF-beta1)-induced epithelial-mesenchymal transition (EMT). TGF-beta1-induced EMT and cell migration in A549 cells are associated with a decrease in IRS-1 tyrosine phosphorylation and protein levels. Tissue microarray analysis of human lung carcinoma shows a correlation between IRS-1 protein levels and E-cadherin protein levels. High IRS-1 levels coexist with high E-cadherin levels, whereas low IRS-1 levels coexist with low E-cadherin levels, implying a possibility that IRS-1 protein levels may be linked with EMT. Surprisingly, overexpression of IRS-1 in A549 cells completely blocked TGF-beta1-induced EMT and cell migration, inhibited TGF-beta1-mediated expression of snail and slug genes, and abolished TGF-beta1-mediated repression of E-cadherin promoter activity. In contrast, IRS-1 knockdown by RNAi increased the expression of snail and slug genes and induced EMT. Inhibition of protein tyrosine phosphatase with sodium vanadate, which greatly increased the levels of tyrosine-phosphorylated IRS-1, suppressed TGF-beta1-induced actin remodeling and cell morphologic changes. These results show for the first time that TGF-beta1 induces EMT through mechanisms involving the modulation of IRS-1 signaling, and that IRS-1 functions as a critical EMT suppressor that suppresses TGF-beta1-induced EMT via inhibition of snail and slug expression.

The novel epididymis-specific beta-galactosidase-like gene Glb1l4 is essential in epididymal development and sperm maturation in rats.

We describe a novel epididymis-specific cDNA named Glb1l4, which was isolated from rat epididymis by differential display of mRNAs. Glb1l4 cDNA contains 2607 nucleotides and encodes a 637-amino acid protein with 50% similarity to mouse beta-galactosidase. The gene is located on chromosome 8q13, spanning 21 exons. Northern blot analysis reveals that Glb1l4 is specifically expressed in the caput region of epididymis and upregulated by androgen. A specific polyclonal antiserum against the N-terminal peptide of GLB1L4 has been produced. Western blot analysis and immunohistochemistry assay reveal that GLB1L4 is specifically expressed in the principal cells of the caput epididymis. Interestingly, its expression peaks at Postnatal Day 45 in mRNA level and at Postnatal Day 60 in protein level while the epididymis column cells undergo differentiation. Moreover, within this very period this secretory protein is confined inside the cell with a change of subcellular distribution pattern, which implies its important roles in the cell differentiation process. Only after the epididymal epithelium differentiation is completed and the spermatozoa enter the epididymal lumen is the GLB1L4 secreted into the luminal fluid and bound on the sperm head. Our results suggest that GLB1L4 may play various roles in principal cell differentiation and sperm maturation.

Transcriptome analysis of a cDNA library from adult human epididymis.

Mammalian Gene Collection (MGC) verified over 9,000 human full-ORF genes and FLJ Program reported 21,243 cDNAs of which 14,409 were unique ones and 5,416 seemed to be protein-coding. The pity is that epididymis cDNA library was missing in their sequencing target list. Epididymis is a very important male accessory sex organ for sperm maturation and storage. Fully differentiated spermatozoa left from testis acquire their motility and capacity for fertilization via interactions with the epididymal epithelium duct lumen during passage through this convoluted duct. Here, we report that 20,000 clones from a healthy male epididymis cDNA library have been sequenced. The sequencing data provided 8,234 known sequences and 650 unknown cDNA fragments. Hundred and six of 650 unknown cDNA clone inserts were randomly selected for fully sequencing. There were 25 unknown unique sequences and 19 released but unreported sequences came out. By northern blot analysis, four sequences randomly selected from the 19 released sequences with no known function showed positive mRNA signals in epididymis and testis. The signals for three of six from those unknown group showed as epididymis abundant in a region-specific manner but not in the testis and other tissues tested. All the sequencing data will be available on the website www.sdscli.com.

Male germ cell-specific expression of a novel Patched-domain containing gene Ptchd3.

The Hedgehog (Hh) signaling pathway plays an important role in various biological processes, including pattern formation, cell fate determination, proliferation, and differentiation. Hh function is mediated through its membrane receptor Patched. Herein, we have characterized a novel Patched-domain containing gene Ptchd3 in mouse. Messenger RNA of Ptchd3 was exclusively detected in the testis, and existed in two isoforms Ptchd3a and Ptchd3b. The expression of these two mRNA isoforms was shown to be developmentally regulated in testes, and specifically found in male germ cells. Further analysis revealed that the Ptchd3 protein was located on the midpiece of mouse, rat and human sperm. Collectively, these results indicate that Ptchd3 is a novel male germ cell-specific gene and may be involved in the Hh signaling to regulate sperm development and/or sperm function.

RNase9, an androgen-dependent member of the RNase A family, is specifically expressed in the rat epididymis.

Members of the RNase superfamily participate in a diverse array of biological processes, including RNA degradation, antipathogen activities, angiogenesis, and digestion. In the present study, we cloned the rat RNase9 gene by in silico methods and genome walking based on homology to the Macaca mulatta (rhesus monkey) epididymal RNase9. The gene is located on chromosome 15p14, spanning two exons, and is clustered with other members of the RNase A superfamily. It contains 1279 bp and encodes 182 amino acids, including a 24-amino acid signal peptide, and it has unique features known from other RNases. Unlike those other members, the rat RNase9 mRNA was specifically expressed in the epididymis, especially in the caput and corpus, and exhibited an androgen-dependent expression pattern but was downregulated in an epididymitis animal model. The RNASE9 was expressed in a principal cell-specific pattern. Interestingly, most of the principal cells in the caput expressed the RNASE9; however, in the distal caput, the principal cells showed a checkerboard-like pattern of immunoreactivity. We also observed that the RNASE9 was bound on the acrosomal domain of sperm. Its potential roles in sperm maturation are discussed.

Genome-wide profiling of segmental-regulated transcriptomes in human epididymis using oligo microarray.

Sperm maturation during passage through the epididymis depends on regionalized gene expression which maintains the progressively changing environment within the epididymal tubule. Towards defining the genes that drive the sequential maturation of spermatozoa, we profiled regionally regulated gene expression pattern in the epididymis of a fertile young male donor using Affymetrix human genome U133 plus 2.0 microarray representing approximately the whole human genome. Over 15000 transcripts, almost one-third of the total on the array were identified in whole epididymis. Among them, 65% were detected in all three regions of the epididymis, 410 or 2.6% were present only in one region and the remaining 32.4% were distributed in two regions. Region-specific transcripts observed in caput (264), corpus (61) and cauda (81) epididymides were further classified as empirically determined reported genes or ESTs. This study revealed for the first time, the expression in human epididymis of a number of region-specific genes. The original data will be made publicly available on the Shanghai Science and Technology Database (http://www.scbit.org/human_epididymis_transcriptomes).

An effective method for raising antisera against beta-defensins: double-copy protein expression of mBin1b in E. coli.

Bin1b is a rat epididymis specific beta-defensin which may have fertility related functions in addition to its antimicrobial activity. beta-defensins are cysteine-rich cationic antimicrobial peptides that have their important implications in innate and adaptive immunity. Though considerable numbers of new beta-defensins have been discovered, few corresponding antibodies have been reported. The small peptide with special structure and antimicrobial nature of beta-defensins make them very difficult to express in prokaryotic system. Here we adopted a double-copy protein expression scheme based on which not only the mBin1b protein was successfully expressed but also the immunity of the antigen was enhanced. The validity of the antisera was verified by using Western blotting and immunohistochemical analyses. It will be a useful tool for deeply investigating the roles of Bin1b and also provide a simple but effective method in raising antisera against other members of the beta-defensin gene family.

An epididymis-specific beta-defensin is important for the initiation of sperm maturation.

Although the role of the epididymis, a male accessory sex organ, in sperm maturation has been established for nearly four decades, the maturation process itself has not been linked to a specific molecule of epididymal origin. Here we show that Bin1b, a rat epididymis-specific beta-defensin with antimicrobial activity, can bind to the sperm head in different regions of the epididymis with varied binding patterns. In addition, Bin1b-expressing cells, either of epididymal origin or from a Bin1b-transfected cell line, can induce progressive sperm motility in immotile immature sperm. This induction of motility is mediated by the Bin1b-induced uptake of Ca(2+), a mechanism that has a less prominent role in maintaining motility in mature sperm. In vivo antisense experiments show that suppressed expression of Bin1b results in reduced binding of Bin1b to caput sperm and in considerable attenuation of sperm motility and progressive movement. Thus, beta-defensin is important for the acquisition of sperm motility and the initiation of sperm maturation.

Spatial and temporal expression of germ cell nuclear factor in murine epididymis.

AIM: To investigate the spatial and temporal expression of germ cell nuclear factor (GCNF) in mouse and rat epididymis during postnatal period. METHODS: The epididymal sections from different postnatal days were stained for GCNF by the indirect immunofluorescence technique and digital photographs were taken by a Carl Zeiss confocal microscope. RESULTS: GCNF was first detected on day 12 in mouse epididymis and day 14 in rat epididymis. The highest expression of GCNF was observed on day 35 in both mouse and rat epididymis. In adults, GCNF exhibited a region-specific expression pattern, i.e., it was expressed predominantly in the initial segment, caput and proximal corpus of rat epididymis and was abundant in the proximal corpus of mouse epididymis. GCNF could be found in the nuclei of the principal, apical, narrow, clear and halo cells. CONCLUSION: GCNF may play an important role in epididymal differentiation and development and in sperm maturation.