Role In Sperm Maturation Research Articles

DEFB126 2-nt Deletion (rs11467417) as a Potential Risk Factor for Chlamydia Trachomatis Infection and Subsequent Infertility in Iranian Men

Background: Chlamydia trachomatis (CT) is one of the most prevalent sexually transmitted infections, causing genital tract infections and infertility. Defensins have an immunomodulatory function and play an important role in sperm maturation, motility, and fertilization. DEFB126 is present on ejaculated spermatozoa and is essential for them to pass through the female reproductive tract. The purpose of the study was to determine the frequency of the 2-nt deletion of the DEFB126 (rs11467417) in Iranian infertile males with a recurrent history of CT.
 Methods: Semen samples of 1080 subfertile males were investigated. Among patients who had CT-positive results, sperm DNA from 50 symptomatic and 50 asymptomatic patients were collected for the DEFB126 genotype analysis. Additionally, a control group comprising 100 DNA samples from individuals with normal spermogram and testing negative for CT was included in the study. The PCR-sequencing for detecting the 2-nt deletion of the second exon of the DEFB126 was performed.
 Results: The Chi-squared test comparing all three groups revealed no significant difference across the different genotypes. Moreover, no significant difference between the symptomatic and asymptomatic groups was seen. However, analysis within CT-positive patients and controls demonstrated significant difference between the frequencies of homozygous del/del.
 Conclusion: The higher frequency of the 2-nt deletion of the DEFB126 in CT- positive patients suggests that the occurrence of mutations in the DEFB-126 may cause the impairment of the antimicrobial activity of the DEFB126 protein and consequently makes individuals more susceptible to infections such as CT.

Extracellular vesicle-coupled miRNA profiles of chicken seminal plasma and their potential interaction with recipient cells

The presence of EVs in seminal plasma (SPEVs) suggests their involvement on fertility via transmitting information between the original cells and recipient cells. SPEVs-coupled miRNAs have been shown to affect sperm motility, maturation, and capacitation in mammals, but rarely in poultry species. The present study aims to reveal the profile of SPEVs miRNAs and their potential effect on sperm storage and function in poultry. The SPEVs was successfully isolated from 4 different chicken breeds by ultracentrifugation and verified. Deep sequencing of SPEVs small RNA library of each breed identified 1077 miRNAs in total and 563 shared ones. The top 10 abundant miRNAs (such as miR-10-5p, miR-100-5p, and miR-10a-5p etc.) accounted for around 60% of total SPEVs miRNA reads and are highly conserved across species, predisposing their functional significance. Target genes prediction and functional enrichment analysis indicated that the most abundantly expressed miRNAs may regulate pathways like ubiquitin-mediated proteolysis, endocytosis, mitophagy, glycosphingolipid biosynthesis, fatty acid metabolism, and fatty acid elongation. The high abundant SPEVs-coupled miRNAs were found to target 107 and 64 functionally important mRNAs in the potential recipient cells, sperm and sperm storage tubules (SST) cells, respectively. The pathways that enriched by target mRNAs revealed that the SPEVs-coupled miRNA may rule the fertility by affecting the sperm maturation and regulating the female's immune response and lipid metabolism. In summary, this study presents the distinctive repertoire of SPEVs-coupled miRNAs, and extends our understanding about their potential roles in sperm maturation, capacitation, storage, and fertility, and may help to develop new therapeutic strategies for male infertility and sperm storage.

Progesterone receptors in extratesticular ducts of the Amazonian stingray Potamotrygon wallacei: A potential role in sperm maturation and aggregate formation

In cururu stingray (Potamotrygon wallacei Carvalho, Rosa and Araújo 2016) males, plasma progesterone (P4) levels appear to be associated with spermiation events. However, the specific contribution of P4 in sperm maturation via extratesticular ducts in this stingray species is unknown. With the aim of filling this knowledge gap, this study examined the morphology and the presence of progesterone receptors (PR) in the ducts, and analyzed the relationship of progesterone (P4) with sperm maturation and formation of aggregates. Morphological analysis showed that a columnar pseudostratified epithelium with stereocilia lined all the attached ducts. In active males, the secretory cells of the epididymis and the Leydig glands presented PR; however, these receptors were not found in the distal region of the epididymis (essential for nurturing and capacitation events) of regressing males. In the seminal vesicles of active males, the spermatozoa are parallelly aligned and embedded in a matrix to form the spermatozeugmata. The matrixes are formed by proteins secreted by the ducts and Sertoli cell cytoplasts. These structures presented PR, which suggests that P4 engages in sperm metabolism during storage. Our findings allude to the potential role of P4 in regulating the development and function of the attached ducts in different reproductive phases. Furthermore, P4 seems to be an essential component for regulating sperm progress, protein secretion, aggregate formation, and maintenance of sperm during storage in this freshwater stingray.

Measuring free radicals with relaxometry: Pioneering steps for measurements in human semen

A possible biological mechanism for unexplained male infertility is due to the effect of oxidative stress (OS), defined by the imbalance of reactive oxygen species (ROS) production, and the capacity of the antioxidant defence system to counteract it. In physiological concentrations, ROS and especially free radicals play an essential role in sperm maturation and fertilization, while an overabundance could lead to OS-induced damage to spermatozoa. To date, there are no direct detection techniques available that can measure the total amount of free radicals real time and identify where and when free radicals are generated. This study applies a quantum sensing technique using fluorescent nanodiamonds (FNDs), called T1 relaxometry, which is uniquely sensitive and specific for free radicals allowing measurements of the current radical load for nanoscale detection in living cells and body fluids. This proof-of-principle study investigates if we can use this technique to detect the free radical generation in human whole and separated, using density gradient centrifugation, semen. This method could be potentially used as new diagnostic measure for unexplained infertility or to track the effect of therapeutic interventions such as lifestyle changes. We adapted the existing relaxometry technique to measure free radicals in semen. The measured relaxation time (T1 time) was correlated to sperm concentration and progressive motility. Additionally, we explored the influence of the oxidative trigger hydrogen peroxide and the antioxidant glutathione on the free radical concentration measured. No significant correlations were found, which indicates that measurements in more proximity of the sperm cell are required to use relaxometry as a potential diagnostic tool for unexplained male infertility.

Adhesion G protein-coupled receptor G2 is dispensable for lumicrine signaling regulating epididymal initial segment differentiation and gene expression†.

The mammalian epididymis is the organ for functional sperm maturation. In rodents, the initial segment, the most proximal region of the epididymis, plays a critical role in sperm maturation. The luminal epithelial differentiation and the following gene expression of the initial segment are regulated by the lumicrine signaling, a testis-epididymis transluminal secreted signaling. Adhesion G protein-coupled receptor G2 (ADGRG2) is expressed in the efferent duct and the initial segment epididymis. In the preceding study, Adgrg2 ablation decreased the expression of several genes expressed in the initial segment. Such downregulated genes include those known to be regulated by lumicrine signaling, suggesting the involvement of ADGRG2 in lumicrine signaling. The present study examined whether ADGRG2 is associated with the lumicrine signaling regulating epididymal initial segment differentiation and gene expression. Adgrg2-null mice were generated by CRISPR/CAS9-mediated genome editing. The postnatal differentiation of the Adgrg2-null male epididymal initial segment was histologically comparable with that of control wild-type animals. The RNA-seq of Adgrg2-null mice was performed together with those of efferent duct-ligated and W/Wv mice in both of which lumicrine signaling is defective. The comparative transcriptome analyses clarified that the expressions of genes expressed in the initial segment and regulated by lumicrine signaling were decreased by Adgrg2 nullification. However, the extent of such downregulations observed in Adgrg2-null epididymis was not so prominent compared with those of lumicrine signaling deficient Nell2-/-, efferent duct-ligated, or W/Wv mice. Collectively, these findings indicate that ADGRG2 is dispensable for the lumicrine regulation of epididymal initial segment differentiation.

Collective cell dynamics and luminal fluid flow in the epididymis: A mechanobiological perspective.

The mammalian epididymis is a specialized duct system that serves a critical role in sperm maturation and storage. Its distinctive, highly-coiled tissue morphology provides a unique opportunity to investigate the link between form and function in reproductive biology. While recent genetic studies have identified key genes and signaling pathways involved in the development and physiological functions of the epididymis, there has been limited discussion about the underlying dynamic and mechanical processes that govern these phenomena. In this review, we aim to address this gap by examining two key aspects of the epididymis across its developmental and physiological phases. First, we discuss how the complex morphology of the Wolffian/epididymal duct emerges through collective cell dynamics, including duct elongation, cell proliferation, and arrangement during embryonic development. Second, we highlight dynamic aspects of luminal fluid flow in the epididymis, essential for regulating the microenvironment for sperm maturation and motility, and discuss how this phenomenon emerges and interplays with epididymal epithelial cells. This review not only aims to summarize current knowledge but also to provide a starting point for further exploration of mechanobiological aspects related to the cellular and extracellular fluid dynamics in the epididymis. This article is protected by copyright. All rights reserved.

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.

Microfluidics facilitating the use of small extracellular vesicles in innovative approaches to male infertility.

Sperm are transcriptionally and translationally quiescent and, therefore, rely on the seminal plasma microenvironment for function, survival and fertilization of the oocyte in the oviduct. The male reproductive system influences sperm function via the binding and fusion of secreted epididymal (epididymosomes) and prostatic (prostasomes) small extracellular vesicles (S-EVs) that facilitate the transfer of proteins, lipids and nucleic acids to sperm. Seminal plasma S-EVs have important roles in sperm maturation, immune and oxidative stress protection, capacitation, fertilization and endometrial implantation and receptivity. Supplementing asthenozoospermic samples with normospermic-derived S-EVs can improve sperm motility and S-EV microRNAs can be used to predict non-obstructive azoospermia. Thus, S-EV influence on sperm physiology might have both therapeutic and diagnostic potential; however, the isolation of pure populations of S-EVs from bodily fluids with current conventional methods presents a substantial hurdle. Many conventional techniques lack accuracy, effectiveness, and practicality; yet microfluidic technology has the potential to simplify and improve S-EV isolation and detection.

P-111 Spermiogenic maturation in relation to sperm genomic integrity throughout the epididymis

Abstract Study question Which portion of the epididymis yields spermatozoa with the highest chromatin integrity and embryo developmental competence? Summary answer Spermatozoa retrieved from the distal portion of the epididymis retains the highest chromatin integrity, characterized by higher fertilization, implantation, and clinical pregnancy rates. What is known already Surgical sperm retrieval from the proximal male reproductive tract has proven to be an effective treatment for couples with recurrent pregnancy loss due to high DNA fragmentation in the ejaculate. Although spermatozoa retrieved directly from the germinal epithelium displays the highest genomic integrity, epididymal spermatozoa maintain a similar DNA integrity profile but yield higher fertilization and pregnancy rates. Although the caput is the preferred site for sperm aspiration procedures in the epididymis, it is unclear which epididymal region yields the best-quality gametes. Study design, size, duration Over a 10-year period, we identified 51 obstructive azoospermic (OA) men who underwent surgical sperm retrieval. These men were grouped according to the different epididymal regions from which their spermatozoa were obtained. Sperm chromatin fragmentation (SCF) was assessed and compared among these groups. The epididymal spermatozoa were then used for ICSI cycles. Fertilization and clinical outcomes were compared among the epididymal regions. All female partners (≤37 years old) had negative infertility workups. Participants/materials, setting, methods Epididymal spermatozoa were retrieved from the caput (n = 39), corpus (n = 8), and cauda (n = 4) regions. SCF was assessed by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay, with a normal threshold of ≤ 15%. Unpaired t and Fisher’s exact tests were used to compare the SCF as well as ICSI and pregnancy outcomes according to the epididymal sperm source. P < 0.05 was considered statistically significant. Main results and the role of chance A total of 51 men were categorized based on the site of spermatozoa retrieval: caput, corpus, and cauda. Maternal age was comparable among all three groups (37.9±5, 37.3±4, and 38±3, respectively). Sperm concentration was 23.9±30x106/ml (caput), 24.3±30x106/ml (corpus), and 29.3±36x106/ml (cauda), with 10.8±15% (caput), 14.3±20%(corpus), and 15.2±13% motility (cauda). Average oocytes injected per group was 13.3, 12.3, and 13.2, respectively. Average SCF scores were 19.8%, 9.2% (P < 0.05), and 8.4% (P < 0.05), respectively. The fertilization rates rose from 69.0% in the caput to 78.6% in the corpus and 86.2% (P < 0.05) in the cauda. Implantation rates followed a similar trend: 35.3% in the caput and 44.4% and 50.1% in the corpus and cauda, respectively. In cycles using caput epididymal spermatozoa, there was a 52.9% delivery rate with a 5.5% pregnancy loss rate. Cycles using corpus epididymal sperm showed a 75.0% delivery rate with no pregnancy loss. Finally, cycles using spermatozoa from the cauda resulted in a 100% delivery rate. Limitations, reasons for caution Although spermatozoa retrieved from the cauda epididymis has superior genomic integrity and yielded satisfactory clinical outcomes, this approach may be restricted to patients with obstructive azoospermia. While we attempted to control for an eventual confounding female factor, it cannot be entirely ruled out. Wider implications of the findings The epididymis plays an important role in sperm maturation; epididymal spermatozoa retain adequate chromatin integrity as demonstrated by the fertilization and pregnancy rates achieved with sperm retrieved from the cauda region. Confirmation of these findings in larger studies may help determine the most favorable site for epididymal sperm retrieval. Trial registration number Not applicable

Heat shock protein family D member 1 in boar spermatozoa is strongly related to the litter size of inseminated sows

BackgroundSperm quality evaluation is the logical first step in increasing field fertility. Spermatozoa contain cytoplasmic organelles and biomolecules known as sperm-intrinsic factors, which play key roles in sperm maturation, sperm-oocyte fusion, and embryo development. In particular, sperm membrane proteins [e.g., arginine vasopressin receptor 2, beta-actin, prohibitin, and heat shock protein family D member 1 (HSPD1)] and RNA could be used as functional indicators of male fertility. We sought to clarify the effects of differential mRNA expression of selected genes on several fertilisation parameters, including sperm motility, motion kinematics, capacitation, and litter size, in a porcine model.ResultsOur results demonstrated that HSPD1 expression was significantly correlated with male fertility, as measured by the litter size of inseminated sows. The expression of HSPD1 mRNA was linked to sperm motility and other motion kinematic characteristics. Furthermore, HSPD1 had a 66.7% overall accuracy in detecting male fertility, and the high-litter size group which was selected with the HSPD1 marker had a 1.34 greater litter size than the low-litter size group.ConclusionsOur findings indicate that HSPD1 might be a helpful biomarker for superior boar selection for artificial insemination, which could boost field fertility.

The effects of selenium on GPX4-mediated lipid peroxidation and apoptosis in germ cells.

Emerging evidence suggests that selenium plays an essential role in sperm maturation. However, the specific signaling pathway by which selenium exerts effect has not been elucidated. To evaluate the effect of selenium on GPX4-mediated lipid peroxidation and apoptosis in germ cells, selenium deficiency was modeled by culturing GC2-spd cells in serum-free medium. Treatment with 0.5-μM sodium selenite (NaSe) or 5.0-μM selenomethionine (SeMet) significantly improved the proliferation rate and GPX4 protein expression after selenium deficiency. Moreover, NaSe and SeMet decreased the MDA content and lipid peroxidation. When adenovirus was used to knockdown the expression of the GPX4 gene (shRNA-GPX4), the early apoptosis rate of the shRNA-GPX4 cells was significantly higher than that of the EGFP cells. Increased expression of Caspase3 and Bax, as well as MDA content were observed in the shRNA-GPX4 cells compared with EGFP cells. In further, overexpression of the GPX4 gene (ORF-GPX4) cells exhibited increased cell proliferation and decreased MDA content. However, there was no significant difference in 12/15-lox expression both in ORF-GPX4 cells and shRNA-GPX4 cells. Conclusively, GPX4 was involved in the regulation of lipid peroxidation and apoptosis in GC2-spd cells. Selenium played a role in promoting cell proliferation by mediating GPX4. The regulation of GPX4 may occur independently of 12/15-Lox. These findings confirmed the effect of selenium on spermatogenesis and offered a potential target for treating abnormal semen quality in men.

Physiology and modulation factors of ovulation in rabbit reproduction management

Rabbit is an induced ovulatory species, so ovulation takes place after mating. Traditionally, exogenous and synthetic hormonal factors (administered by intramuscular and intravaginal route) such as GnRH and analogues, or different physical procedures (i.e. stimulation by intravaginal cannula) have been used to induce ovulation in females when artificial insemination is applied in rabbit farms. Restriction and public rejection of the use of hormones is leading to the study of the seminal plasma components with potential action on ovulation induction. The aim of the present review is to collect and summarise the strategies used in recent years to trigger ovulation and improve rabbit fertility management with respect to more animal-friendly manipulation methods. Furthermore, special attention has been paid to the use of a semen component (as endogen molecule) such as beta nerve growth factor (β-NGF) in male and female rabbit reproductive physiology. This neurotrophin and its receptors (TrKA and p75NTR) are abundantly distributed in both male and female rabbit reproductive tracts, and it seems to have an important physiological role in sperm maturation and behaviour (velocity, apoptosis and capacitation), as well as a modulatory factor of ovulation. Endogen β-NGF is diluted in the seminal doses with the extenders; hence it could be considered an innovative and alternative strategy to avoid the current exogenous (by intramuscular route) and stressful hormonal treatments used in ovulation induction. Their addition in seminal dose could be more physiological and improve animal welfare in rabbit farms.

Epididymal mRNA and miRNA transcriptome analyses reveal important genes and miRNAs related to sperm motility in roosters

Sperm motility is a crucial trait in chicken production, and the epididymis is an essential organ in the reproductive system. Currently, the molecular mechanisms underlying sperm motility in the epididymis are unclear. In this study, 8 cDNA libraries and eight miRNA libraries were constructed from roosters (4 chickens per group) with diverse sperm motility. After a comparative analysis of epididymal transcriptomes, we detected 84 differentially expressed genes (DEGs) using the edgeR package. Integrated interpretation of DEGs indicated that MMP9, SLN, WT1, PLIN1, and LRRIQ1 are the most promising candidate genes affecting sperm motility in the epididymis of roosters. MiR-146a, mir-135b, and mir-205 could play important regulatory roles in sperm maturation, capacitation, and motility. Additionally, a comprehensive analysis of the mRNA and miRNAs transcriptomes in silico identified a promising gene-miRNA pair miR-135b-HPS5, which may be a vital regulator of sperm motility in the epididymis. Our findings provide novel integrated information of miRNAs and genes that shed light on the regulatory mechanisms of fertility in roosters.

Sperm-borne sncRNAs: potential biomarkers for semen fertility?

Semen infertility or sub-fertility, whether in humans or livestock species, remains a major concern for clinicians and technicians involved in reproduction. Indeed, they can cause tragedies in human relationships or have a dramatic overall negative impact on the sustainability of livestock breeding. Understanding and predicting semen fertility issues is therefore crucial and quality control procedures as well as biomarkers have been proposed to ensure sperm fertility. However, their predictive values appeared to be too limited and additional relevant biomarkers are still required to diagnose sub-fertility efficiently. During the last decade, the study of molecular mechanisms involved in spermatogenesis and sperm maturation highlighted the regulatory role of a variety of small non-coding RNAs (sncRNAs) and led to the discovery that sperm sncRNAs comprise both remnants from spermatogenesis and post-testicular sncRNAs acquired through interactions with extracellular vesicles along epididymis. This has led to the hypothesis that sncRNAs may be a source of relevant biomarkers, associated either with sperm functionality or embryo development. This review aims at providing a synthetic overview of the current state of knowledge regarding implication of sncRNA in spermatogenesis defects and their putative roles in sperm maturation and embryo development, as well as exploring their use as fertility biomarkers.

Morphological and morphometric changes and epithelial apoptosis are induced in the rat epididymis by long-term letrozole treatment.

The epididymis is an organ that plays a key role in sperm maturation. The aim of this study was to examine the association between the chronic treatment of mature male rats with letrozole and morphological evaluation and morphometric values of epididymis as well as changes in the number of apoptotic cells in epididymal epithelium. Adult rats were treated with letrozole for 6 months and the epididymis weight, morphology, morphometric values and the number of apoptotic cells in the epithelium were examined. Long-term aromatase inhibition resulted in presence of intraepithelial clear vacuoles, hyperplasia of clear cells and a hyperplastic alteration in the epithelium known as a cribriform change. Moreover, changes in diameters of the epididymal duct and the epididymal lumen and changes in the epididymal epithelium height were observed. The number of apoptotic epithelial cells was increased in letrozole-treated group. It can be indicated that chronic treatment with letrozole can affect morphology, morphometric values and apoptosis in the epididymis of adult male rats. Observed changes are similar to that observed in the aging processes and may also be important for patients treated with aromatase inhibitors.

P–026 Seminal plasma exosomes: a promising source of biomarkers for fertility evaluation

Abstract Study question Do exosomes from seminal plasma have a role in male fertility? Summary answer Exosomes isolated from seminal plasma have a pivotal role during spermatogenesis and sperm maturation and may represent eligible biomarkers for male fertility/infertility. What is known already During their journey along the male reproductive tract, exosomes contained in seminal fluid are involved in the transfer of several molecules to the maturing sperm. Exosomes are extracellular vesicles (EVs) released by all the cells; they carry a cargo of nucleic acids, proteins and lipids. In the male genital tract, they are released at various levels and their composition differs between men of proven fertility and infertile male patients. Recent studies reported the proteomic profile of exosomes, revealing the presence of several proteins with a well know role in sperm maturation and fertilizing ability acquiring. Study design, size, duration This prospective study consisted of 36 Caucasian men; according to seminal parameters (WHO 2010) they were divided in normozoospermic (N; n = 12), oligoasthenoteratozoospermic (OAT: n = 12) and azoospermic (A; n = 12). Semen samples were collected between October 2020 and January 2021 at the Assisted Reproductive Unit, Siena University Hospital (Italy) after institutional ethical approval and signed written consent from all the participants. Participants/materials, setting, methods Ejaculated sperm were analyzed according to WHO–2010 criteria and divided into the three groups: N, OAT and A. Exosomes were isolated by an in-house modified ExoGAG®-polymer precipitation-based protocol and characterized for size and ultrastructure by Nanoparticle tracking analysis (NTA) and transmission electron microscopy (TEM). The exosomal proteins were extracted and analyzed by 2D-electrophoresis and the identified profiles were examined by applying bioinformatic tools. The expression of selected genes was evaluated by digital droplets PCR (ddPCR). Main results and the role of chance The present work is readily providing an improvement of the standard ExoGAG® protocol and underlines its advantages over more conventional EVs isolation protocols used to date for recovery from seminal fluid: the number of recovered EVs and their size were finely included in the range of exosomes. This isolation protocol provides samples suitable for proteomic analyses, representing the first 2D-electrophoresis reference map of exosome-pay loaded proteins in N respect to OAT/A groups and providing an innovative and comprehensive functional overview of its proteins. Moreover, the STRING protein-protein interaction analysis revealed the deregulation of specific pathways (e.g. signaling proteins, chromatin packaging and/or remodeling, protein folding and apoptosis) in A and OAT in comparison with N group. Gene expression by ddPCR analysis highlighted that most of the analyzed genes are modulated in according to seminal parameters, in particular: GAPDHS (Glyceraldehyde–3-Phosphate Dehydrogenase, Spermatogenic); SPAM1 (Sperm Adhesion Molecule–1) encoding a members of hyaluronidase family; ADAM2 (ADAM Metallopeptidase Domain–2) that plays an important role in sperm-egg interactions; CRISP1,2,3 (Cysteine Rich Secretory Protein 1,2,3) expressed in the epididymis and secreted into the epididymal lumen; CLGN (Calmegin) encoding a testis-specific chaperone protein and PGK2 (Phosphoglycerate Kinase–2) expressed in the later stages of spermatogenesis. Limitations, reasons for caution This study represents a preliminary experiment. We suggest further comparative studies in larger study cohorts. Wider implications of the findings: This pilot study, demonstrating the unique proteomic and transcriptomic pattern of exosomes in N/OAT/A groups, supports the importance of exosomes in sperm production and maturation. This methodological set-up is expected to open new ways for advancement in the use of exosomes as fertility biomarkers, making possible personalized approaches in ART. Trial registration number Not applicable

Assessment of progesterone synthesis and its regulation role on dihydrotestosterone secretion in sheep epididymis.

Progesterone (P4) is an anti-androgen compound whose role in sperm maturation and functionality remains unclear in sheep. Here, we aimed to investigate the regulation mechanism of P4 on the epididymal secretion of dihydrotestosterone (DHT). To this end, we performed enzyme-linked immunosorbent assays, immunohistochemical staining, western blotting, and quantitative real-time polymerase chain reaction to detect P4 concentration as well as StAR, P450scc, and 3β-HSD expression in sheep epididymis. Besides, cauda epithelial cells were cultured at different concentrations of P4 (10-9-10-5 g ml-1) as well as with or without the P4 receptor (PGR) inhibitor RU486 (10-7 M) or the PI3K-AKT inhibitor LY294006 (10-7 M) to explore the effect of P4 on DHT secretion and the underlying regulatory mechanism. The results showed that the caput, corpus, and cauda of sheep epididymis could synthesize P4 but had different synthesis ability. The PGR expression levels were the highest in the cauda, followed by the corpus. In vitro cell culture showed that P4 inhibition of DHT secretion and 5α-reductase 1 and 2 expression in epididymal epithelial cells could be moderately mitigated by RU486 but not by LY294002. Our results indicated that the paracrine and autocrine P4 could affect the secretion of DHT in epididymal cells through PGR. Overall, this study provides new data regarding the involvement of P4 in sperm maturation and functionality in sheep.

Palmitoylated GLB1L4 transfers via exosomes to maintain sperm function in rat epididymis.

Epididymal specific proteins play a crucial role in sperm maturation. Some of the post-translational modified proteins are transported from the caput to the cauda of the epididymis through exosomes which regulate the function of sperm in cauda epididymis. Rat beta-galactosidase-1-like protein 4 (GLB1L4) expressed specifically in the caput epididymis, localizes on the sperm; however, the regulatory ways in which GLB1L4 protein interacts with sperm to maintain sperm function are unclear. In this study, knockdown of rat GLB1L4 could inhibit in vitro capacitation of sperm in cauda epididymis and reduce the fertility of the male rats by injection of special lentivirus-shRNA into caput epididymis. Moreover, a considerable proportion of GLB1L4 proteins from rat caput epididymis were loaded on exosomes. The exosomes loaded GLB1L4 from in vitro primary rat caput epididymal epithelial cells could bind with spermatozoa in cauda epididymis. Further, the palmitoylation status of cysteine residues at the 12th and 15th sites of the protein molecule could significantly affect cellular localization of GLB1L4 protein. It was identified that most of GLB1L4 was palmitoylated in the presence of exosomes from primary caput epididymal cells and the level of palmitoylated GLB1L4 in the exosomes could be inhibited by 2-bromopalmitate (2-BP). These results suggested that the palmitoylated GLB1L4 from rat caput epididymis could be transported to the cauda epididymis to regulate the sperm function by exosomes.

Mouse defensin beta 20 (Defb20) is expressed specifically in the caput region of the epididymis and regulated by androgen and testicular factors

Sperm cells undergo maturation during their transit throughout the epididymis. This process takes place in region-specific manner in which sperm are battered by proteins secreted by epithelium lining the epididymal duct. Most of the genes that encode for the proteins involved in the sperm maturation remain uncharacterized. Previous studies showed that family of β-defensins preferentially eaxpressed in male reproductive tracts and play an important role in both innate immunity and sperm fertility. In this study we characterized Defb20 to gain insight on its role in sperm maturation. Bioinformatic tools were used to analyzed functional domains and signal peptide. qRT-PCR analyses were used to analyzed tissue distribution, dependency on androgen and testicular factors and developmental-regulated expression analysis. Defb20 sequence contains important domains such as N-myristoilation and kinase binding sites which are putatively involved in the protein activation and protein-plasma membrane interaction. Moreover, DEFB20 contains a signal peptide indicating characteristic of secretory proteins. Defb20 was expressed exclusively in the epididymis with the highest expression in the caput region and was down-regulated by gonadectomy. Defb20 was also regulated by testicular factors in which the expression was down-regulated after efferent duct ligation (EDL). The dependency on the androgen was further confirmed by postnatal expression analysis in which Defb20 began to express at day-20 postnatal indicating specific stage of expression after initial development of the testis. In conclusion, Defb20 have a potential to be involved in the epididymal sperm maturation process.

Circulating sex hormone levels in relation to male sperm quality

BackgroundAlthough sex hormones play critical roles in spermatogenesis and sperm maturation, it remains inconclusive whether circulating sex hormones can serve as non-invasive biomarkers to improve the assessment of sperm quality.MethodsWe systematically evaluated the association of various sex hormones in serum with sperm quality among 338 men in subfertile couples. Concentrations of luteinizing hormone (LH), follicle-stimulating hormone (FSH), total testosterone (TT), total estradiol (E2), and sex hormone-binding globulin (SHBG) were detected by chemiluminescent immunoassay. Free testosterone and estradiol were calculated using a validated algorithm. A generalized liner regression model controlling for lifestyle factors was used to evaluate the associations with sperm count, concentration, motility, and morphology.ResultsAfter adjusting for age, body mass index, current smoking and alcohol drinking, LH, FSH, and TT levels were all inversely associated with sperm motility (all P for trend < 0.05); however, in mutual adjustment analysis, only LH remained an inverse association with sperm motility after adjusting for FSH and TT levels (P for trend = 0.04). Higher concentrations of LH were also associated with lower sperm progressive motility (P for trend = 0.04). Moreover, LH and FSH levels were both inversely associated with normal sperm morphology (P for trend = 0.04 and 0.02, respectively).ConclusionsIncreased levels of LH are associated with poor sperm motility and morphology, suggesting that LH may play a central role in sperm maturation. Future studies are warranted to assess potential clinical utility of LH for risk stratification and tailed prevention of male infertility.