Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice.

BACKGROUND: Region-specific immune environments in the epididymis influence the immune responses to uropathogenic Escherichia coli (UPEC) infection, a relevant cause of epididymitis in men. Toll-like receptors (TLRs) are essential to orchestrate immune responses against bacterial infections. The epididymis displays region-specific inflammatory responses to bacterial-derived TLR agonists, such as lipopolysaccharide (LPS; TLR4 agonist) and lipoteichoic acid (LTA; TLR2/TLR6 agonist), suggesting that TLR-associated signaling pathways could influence the magnitude of inflammatory responses in epididymitis. OBJECTIVES: To investigate the expression and regulation of key genes associated with TLR4 and TLR2/TLR6 signaling pathways during epididymitis induced by UPEC, LPS, and LTA in mice. MATERIAL AND METHODS: Epididymitis was induced in mice using UPEC, ultrapure LPS, or LTA, injected into the interstitial space of the initial segment or the lumen of the vas deferens close to the cauda epididymidis. Samples were harvested after 1, 5, and 10 days for UPEC-treated animals and 6 and 24 h for LPS-/LTA-treated animals. Ex vivo epididymitis was induced by incubating epididymal regions from naive mice with LPS or LTA. RT-qPCR and Western blot assays were conducted. RESULTS: UPEC infection up-regulated Tlr2, Tlr4, and Tlr6 transcripts and their associated signaling molecules Cd14, Ticam1, and Traf6 in the cauda epididymidis but not in the initial segment. In these epididymal regions, LPS and LTA differentially modulated Tlr2, Tlr4, Tlr6, Cd14, Myd88, Ticam1, Traf3, and Traf6 expression levels. NFKB and AP1 activation was required for LPS- and LTA-induced up-regulation of TLR-associated signaling transcripts in the cauda epididymidis and initial segment, respectively. CONCLUSION: The dynamic modulation of TLR4 and TLR2/TLR6 signaling pathways gene expression during epididymitis indicates bacterial-derived antigens elicit an increased tissue sensitivity to combat microbial infection in a spatial manner in the epididymis. Differential activation of TLR-associated signaling pathways may contribute to fine-tuning inflammatory responses along the epididymis.

Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats.

The Developmental Origins of Health and Disease (DOHaD) concept has provided the framework to assess how early life experiences can shape health and disease throughout the life course. While maternal malnutrition has been proposed as a risk factor for the developmental programming of prostate cancer (PCa), the molecular mechanisms remain poorly understood. Using RNA-seq data, we demonstrated deregulation of miR-206-Plasminogen (PLG) network in the ventral prostate (VP) of young maternally malnourished offspring. RT-qPCR confirmed the deregulation of the miR-206-PLG network in the VP of young and old offspring rats. Considering the key role of estrogenic signaling pathways in prostate carcinogenesis, in vitro miRNA mimic studies also revealed a negative correlation between miR-206 and estrogen receptor α (ESR1) expression in PNT2 cells. Together, we demonstrate that early life estrogenization associated with the deregulation of miR-206 networks can contribute to the developmental origins of PCa in maternally malnourished offspring. Understanding the molecular mechanisms by which early life malnutrition affects offspring health can encourage the adoption of a governmental policy for the prevention of non-communicable chronic diseases related to the DOHaD concept.

Insights Into Antioxidant Strategies to Counteract Radiation-Induced Male Infertility.

Significance: Radiotherapy, which employs ionizing radiation to destroy or prevent the multiplication of tumor cells, has been increasingly used in the treatment of neoplastic diseases, especially cancers. However, radiation collaterally leads to prolonged periods of sperm count suppression, presumably due to impaired spermatogenesis by depleting the germ cell pool, which has long-term side effects for male reproduction. Recent Advances: Studies of antioxidant compounds as a potential strategy for male fertility preservation have been performed mainly from animal models, aiming to prevent and restore the male germinal tissue and its function, particularly against the oxidative stress effects of radiation. Evidence in preclinical and clinical trials has shown that inhibitors of the renin-angiotensin system and other drugs, such as statins and metformin, are candidates for ameliorating radiation-induced damage to several tissues, including the testis and prostate. Critical Issues: Research for developing an ideal radioprotective agent is challenging due to toxicity in the normal tissue, tumor radioresistance, cellular response to radiation, costs, regulation, and timeline development. Moreover, male radioprotection experiments in humans, mainly clinical trials, are scarce and use few individuals. This scenario is reflected in the slow progress of innovation in the radioprotection field. Future Directions: Expanding human studies to provide clues on the efficacy and safety of radioprotective compounds in the human reproductive system is necessary. Drug repurposing, frequently used in clinical practice, can be a way to shorten the development pipeline for innovative approaches for radioprotection or radiomitigation of the repercussions of radiotherapy in the male reproductive system.

Interactions of the male contraceptive target EPPIN with semenogelin-1 and small organic ligands.

Novel male contraceptives will promote gender equality in sharing contraceptive responsibility. The sperm-associated protein epididymal protease inhibitor (EPPIN) is a promising target for non-hormonal male contraception. EPPIN interacts with the semen coagulum protein semenogelin-1 (SEMG1) on the sperm surface, leading to transient inhibition of sperm motility after ejaculation. Small organic molecules targeting EPPIN's SEMG1-binding are under development as male contraceptives. Here, we combined computational approaches to uncover key aspects underlying EPPIN binding to SEMG1 and small organic ligands. We generated a human EPPIN model showing a typical arrangement of the WFDC (Whey-acid four disulfide core)-type and Kunitz-type domains, connected by a hinge region. Determining the EPPIN model's intrinsic motion by molecular dynamics simulations and normal mode analysis revealed a conformation, presenting a binding pocket that accommodates SEMG1Glu229-Gln247, EP055, and EP012. EPPIN's residues Phe63 and Lys68 (WFDC domain), Asp71 (hinge region), and Asn113, Asn114, and Asn115 (Kunitz domain) were identified as hot spots for SEMG1, EP055, and EP012 binding. Moreover, hydrophobic and hydrophilic residues in the WFDC and Kunitz domains allow plasma membrane anchoring, orienting the EPPIN binding pocket to the solvent. Targeting EPPIN's essential residues for its biomolecular interactions may improve the rational design of EPPIN ligands as spermiostatic compounds.

Pregnancy hypertension-associated endothelial dysfunction is attenuated by isoflurane anesthesia: Evidence of protective effect related to increases in nitric oxide.

AIMS: Pregnancy hypertension-induced endothelial dysfunction associated with impairment of nitric oxide (NO) bioavailability and hemodynamic derangements is a challenging for urgent procedures requiring maternal anesthesia. The volatile anesthetic isoflurane has demonstrated NO-associated protective effects. However, this isoflurane-induced effect is still unclear in pregnancy hypertension. Therefore, the present study examined the potential protective effects of isoflurane anesthesia on endothelial dysfunction and hemodynamic changes induced by hypertensive pregnancy associated with fetal and placental growth restrictions. MATERIALS AND METHODS: Animals were distributed into four groups: normotensive pregnant rats (Preg), anesthetized pregnant rats (Preg+Iso), hypertensive pregnant rats (HTN-Preg), and anesthetized hypertensive pregnant rats (HTN-Preg+Iso). Systolic and diastolic pressures, mean arterial pressure (MAP), heart rate, fetal and placental weights, vascular contraction, endothelium-derived NO-dependent vasodilation, and NO levels were assessed. The vascular endothelial growth factor (VEGF) levels and endothelial NO synthase (eNOS) Serine (1177) phosphorylation (p-eNOS) expression were also examined. KEY FINDINGS: Isoflurane produced more expressive hypotensive effects in the HTN-Preg+Iso versus Preg+Iso group, with respective reductions in MAP by 50 ± 13 versus 25 ± 4 mmHg (P < 0.05). Also, HTN-Preg+Iso compared to the HTN-Preg group showed (respectively) preventions against the weight loss of the fetuses (4.0 ± 0.6 versus 2.8 ± 0.6 g, P < 0.05) and placentas (0.37 ± 0.06 versus 0.30 ± 0.06 mg, P < 0.05), hyper-reactive vasocontraction response (1.8 ± 0.4 versus 2.8 ± 0.6 g, P < 0.05), impaired endothelium-derived NO-dependent vasodilation (84 ± 8 versus 50 ± 17 %, P < 0.05), reduced VEGF levels (147 ± 46 versus 25 ± 13 pg/mL, P < 0.05), and decreased p-eNOS expression (0.24 ± 0.07 versus 0.09 ± 0.05 arbitrary units, P < 0.05). SIGNIFICANCE: Isoflurane anesthesia protects maternal endothelial function in pregnancy hypertension, and possibly endothelium-derived NO is involved.

Advances in non-hormonal male contraception targeting sperm motility.

BACKGROUND: The high rates of unintended pregnancy and the ever-growing world population impose health, economic, social, and environmental threats to countries. Expanding contraceptive options, including male methods, are urgently needed to tackle these global challenges. Male contraception is limited to condoms and vasectomy, which are unsuitable for many couples. Thus, novel male contraceptive methods may reduce unintended pregnancies, meet the contraceptive needs of couples, and foster gender equality in carrying the contraceptive burden. In this regard, the spermatozoon emerges as a source of druggable targets for on-demand, non-hormonal male contraception based on disrupting sperm motility or fertilization. OBJECTIVE AND RATIONALE: A better understanding of the molecules governing sperm motility can lead to innovative approaches toward safe and effective male contraceptives. This review discusses cutting-edge knowledge on sperm-specific targets for male contraception, focusing on those with crucial roles in sperm motility. We also highlight challenges and opportunities in male contraceptive drug development targeting spermatozoa. SEARCH METHODS: We conducted a literature search in the PubMed database using the following keywords: 'spermatozoa', 'sperm motility', 'male contraception', and 'drug targets' in combination with other related terms to the field. Publications until January 2023 written in English were considered. OUTCOMES: Efforts for developing non-hormonal strategies for male contraception resulted in the identification of candidates specifically expressed or enriched in spermatozoa, including enzymes (PP1γ2, GAPDHS, and sAC), ion channels (CatSper and KSper), transmembrane transporters (sNHE, SLC26A8, and ATP1A4), and surface proteins (EPPIN). These targets are usually located in the sperm flagellum. Their indispensable roles in sperm motility and male fertility were confirmed by genetic or immunological approaches using animal models and gene mutations associated with male infertility due to sperm defects in humans. Their druggability was demonstrated by the identification of drug-like small organic ligands displaying spermiostatic activity in preclinical trials. WIDER IMPLICATIONS: A wide range of sperm-associated proteins has arisen as key regulators of sperm motility, providing compelling druggable candidates for male contraception. Nevertheless, no pharmacological agent has reached clinical developmental stages. One reason is the slow progress in translating the preclinical and drug discovery findings into a drug-like candidate adequate for clinical development. Thus, intense collaboration among academia, private sectors, governments, and regulatory agencies will be crucial to combine expertise for the development of male contraceptives targeting sperm function by (i) improving target structural characterization and the design of highly selective ligands, (ii) conducting long-term preclinical safety, efficacy, and reversibility evaluation, and (iii) establishing rigorous guidelines and endpoints for clinical trials and regulatory evaluation, thus allowing their testing in humans.

Low dose of cyclosporine A disrupts sperm parameters and testosterone levels reversibly in mice.

The prevalence of autoimmune diseases has increased worldwide, including in men of reproductive age. Cyclosporine A (CsA) is an immunosuppressive drug commonly used for long periods in the prophylaxis and treatment of autoimmune dysfunction and transplant rejection. Owing to CsA toxicity, most clinical settings use lower CsA doses. Therefore, we evaluated whether a low dose (10 mg/kg) of CsA affects sperm parameters (daily sperm production, motility, morphology, mitochondrial activity, and acrosomal integrity), plasma testosterone levels, and fertility after short-term (10 days) and long-term (50 days) treatments in mice. Short-term CsA treatment partially affected sperm parameters and fertility, as shown by the reduction in sperm hyperactivation and gestational rate 10 days after the interruption of short-term CsA treatment. Long-term CsA treatment impairs sperm count, hyperactivated motility, and acrosomal integrity. This treatment regimen further decreased plasma testosterone concentrations but did not affect reproductive outcomes in mating trials. These outcomes were reversed 50 days after the interruption of long-term CsA treatment. We conclude that a low CsA dose differentially impairs sperm parameters and testicular steroidogenesis in a time-dependent and mostly reversible manner but does not affect male fertility.

Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development.

EPPIN (epididymal protease inhibitor) is a mammalian conserved sperm-binding protein displaying an N-terminal WFDC (whey-acidic protein four-disulfide core) and a C-terminal Kunitz protease inhibitor domains. EPPIN plays a key role in regulating sperm motility after ejaculation via interaction with the seminal plasma protein SEMG1 (semenogelin-1). EPPIN ligands targeting the SEMG1 binding site in the Kunitz domain are under development as male contraceptive drugs. Nevertheless, the relative contributions of EPPIN WFDC and Kunitz domains to sperm function remain obscure. Here, we evaluated the effects of antibodies targeting specific epitopes in EPPIN's WFDC (Q20E antibody, Gln20-Glu39 epitope) and Kunitz (S21C and F21C antibodies, Ser103-Cys123 and Phe90-C110 epitopes, respectively) domains on mouse sperm motility and fertilizing ability. Computer-assisted sperm analysis showed that sperm co-incubation with S21C antibody (but not F21C antibody) lowered progressive and hyperactivated motilities and impaired kinematic parameters describing progressive (straight-line velocity; VSL, average path velocity; VAP and straightness; STR) and vigorous sperm movements (curvilinear velocity; VCL, amplitude of lateral head movement; ALH, and linearity; LIN) compared with control. Conversely, Q20E antibody-induced milder inhibition of progressive motility and kinematic parameters (VAP, VCL and ALH). Sperm co-incubation with S21C or Q20E antibodies affected in vitro fertilization as revealed by reduced cleavage rates, albeit without changes in capacitation-induced tyrosine phosphorylation. In conclusion, we show that targeting specific epitopes in EPPIN Kunitz and WFDC domains inhibits sperm motility and capacitation-associated events, which decrease their fertilizing ability; nevertheless, similar observations in vivo remain to be demonstrated. Simultaneously targeting residues in S21C and Q20E epitopes is a promising approach for the rational design of EPPIN-based ligands with spermostatic activity.

Lipopolysaccharide-induced epididymitis modifies the transcriptional profile of Wfdc genes in mice†.

Whey-acidic protein four-disulfide core domain (WFDC) genes display putative roles in innate immunity and fertility. In mice, a locus on chromosome 2 contains 5 and 11 Wfdc genes in its centromeric and telomeric subloci, respectively. Although Wfdc genes are highly expressed in the epididymis, their contributions to epididymal function remain elusive. Here, we investigated whether Wfdc genes are regulated in response to lipopolysaccharide (LPS)-induced epididymitis, an inflammatory condition that impairs male fertility. We induced epididymitis in mice via (i) interstitial LPS injection into epididymal initial segment and (ii) intravasal LPS injection into the vas deferens towards cauda epididymis. Interstitial and intravasal LPS induced a differential upregulation of inflammatory mediators (interleukin 1 beta, interleukin 6, tumor necrosis factor, interferon gamma, and interleukin 10) in the initial segment and cauda epididymis within 72 h post-treatment. These changes were accompanied by a time-dependent endotoxin clearance from the epididymis. In the initial segment, interstitial LPS upregulated all centromeric (Slpi, Wfdc5, Wfdc12, Wfdc15a, and Wfdc15b) and five telomeric (Wfdc2, Wfdc3, Wfdc6b, Wfdc10, and Wfdc13) Wfdc transcripts at 24 and 72 h. In the cauda epididymis, intravasal LPS upregulated Wfdc5 and Wfdc2 transcripts at 24 h, followed by a downregulation of Wfdc15b and three telomeric (Wfdc6a, Wfdc11, and Wfdc16) gene transcripts at 72 h. Pharmacological inhibition of nuclear factor kappa B activation prevented LPS-induced upregulation of centromeric and telomeric Wfdc genes depending on the epididymal region. We show that LPS-induced inflammation differentially regulated the Wfdc locus in the proximal and distal epididymis, indicating region-specific roles for the Wfdc family in innate immune responses during epididymitis.

Epididymal protease inhibitor (EPPIN) is a protein hub for seminal vesicle-secreted protein SVS2 binding in mouse spermatozoa.

EPPIN is a sperm-surface drug target for male contraception. Here we investigated EPPIN-interacting proteins in mouse spermatozoa. We showed that EPPIN is an androgen-dependent gene, expressed in the testis and epididymis, but also present in the vas deferens, seminal vesicle and adrenal gland. Mature spermatozoa presented EPPIN staining on the head and flagellum. Immunoprecipitation of EPPIN from spermatozoa pre-incubated with seminal vesicle fluid (SVF) followed by LC-MS/MS or Western blot revealed the co-immunoprecipitation of SVS2, SVS3A, SVS5 and SVS6. In silico and Far-Western blot approaches demonstrated that EPPIN binds SVS2 in a protein network with other SVS proteins. Immunofluorescence using spermatozoa pre-incubated with SVF or recombinant SVS2 demonstrated the co-localization of EPPIN and SVS2 both on sperm head and flagellum. Our data show that EPPIN's roles in sperm function are conserved between mouse and human, demonstrating that the mouse is a suitable experimental model for translational studies on EPPIN.

Contractile Effects of Serotonin (5-HT) in the Rat Cauda Epididymis: Expression and Functional Characterization of 5-HT Receptors.

Serotonin [5-hydroxytryptamine (5-HT)] exerts multiple central and peripheral functions. High concentrations of 5-HT have been found in the epididymis, a ductal organ that plays pivotal roles in sperm transport and maturation. The contraction of the epididymal smooth muscle is essential for sperm transport and emission during ejaculation. The contributions of the epididymal 5-HT system to these events are poorly understood. Here, we assessed the contractile function of 5-HT in the rat cauda epididymis (CE), pharmacologically targeting the receptor(s) and the reuptake mechanism involved in this system. Segments of CE duct from adult Wistar rats were set up in an organ bath system for isometric tension recordings, and concentration-response curves to 5-HT and norepinephrine were obtained. 5-HT elicited concentration-dependent contractions of the CE duct (pEC50 = 6.5 ± 0.1) that were potentiated with high potency by the norepinephrine transporter (NET) inhibitor desipramine and with low potency by the highly selective serotonin transporter inhibitor paroxetine, indicating that the NET is the major mediator of 5-HT reuptake in vitro. CE contractions to 5-HT were antagonized by the α 1-adrenoceptor (α 1-AR) antagonist prazosin (pA 2 ≅ 8.9), 5-HT2A/2C antagonists ketanserin (pA 2 ≅ 9.4) and fluoxetine (pA 2 ≅ 7.4), and 5-HT1A ligands WAY 100635 (pA 2 ≅ 8.9) and buspirone (pA 2 ≅ 7.3). Reverse transcriptase polymerase chain reaction analysis demonstrated that 5-HT1A and 5-HT2A transcripts are highly abundant in the cauda epididymis, whereas 5-HT2C transcript was not found. Altogether, our results reveal that contractions of the CE duct to 5-HT encompasses at least activation of α 1-ARs and 5-HT1A and 5-HT2A receptors, providing new insights into the roles of 5-HT on the epididymal function.

Lipopolysaccharide and lipotheicoic acid differentially modulate epididymal cytokine and chemokine profiles and sperm parameters in experimental acute epididymitis.

Bacterial infections are the most prevalent etiological factors of epididymitis, a commonly diagnosed inflammatory disease in the investigation of male infertility factors. The influence of early pathogenic mechanisms at play during bacterial epididymitis on reproductive outcomes is little understood. We report here that experimental epididymitis induced in rats by Gram-negative (LPS) and Gram-positive (LTA) bacterial products resulted in differential patterns of acute inflammation in the cauda epididymis. LPS elicited a strong inflammatory reaction, as reflected by upregulation of levels of mRNA for seven inflammatory mediators (Il1b, Tnf, Il6, Ifng, Il10, Nos2 and Nfkbia), and tissue concentration of six cytokines/chemokines (IL1A, IL1B, IL6, IL10, CXCL2 and CCL2) within the first 24 h post-treatment. Conversely, LTA induced downregulation of one (Nfkbia) and upregulation of six (Il1b, Il6, Nos2, Il4 Il10 and Ptgs1) inflammatory gene transcripts, whereas increased the tissue concentration of three cytokines/chemokines (IL10, CXCL2 and CCL2). The stronger acute inflammatory response induced by LPS correlated with a reduction of epididymal sperm count and transit time that occurred at 1, 7, and 15 days post-treatment. Our study provides evidence that early epididymal inflammatory signaling events to bacterial activators of innate immunity may contribute to the detrimental effects of epididymitis upon male fertility.

ß-defensins and the epididymis: contrasting influences of prenatal, postnatal, and adult scenarios.

ß-defensins are components of host defense, with antimicrobial and pleiotropic immuno-modulatory properties. Research over the last 15 years has demonstrated abundant expression of a variety of ß-defensins in the postnatal epididymis of different species. A gradient of region- and cell-specific expression of these proteins is observed in the epithelium of the postnatal epididymis. Their secretion into the luminal fluid and binding to spermatozoa as they travel along the epididymis has suggested their involvement in reproduction-specific tasks. Therefore, continuous attention has been given to various ß-defensins for their role in sperm function and fertility. Although ß-defensins are largely dependent on androgens, the underlying mechanisms regulating their expression and function in the epididymis are not well understood. Recent investigation has pointed out to a new and interesting scenario where ß-defensins emerge with a different expression pattern in the Wolffian duct, the embryonic precursor of the epididymis, as opposed to the adult epididymis, thereby redefining the concept concerning the multifunctional roles of ß-defensins in the developing epididymis. In this review, we summarize some current views of ß-defensins in the epididymis highlighting our most recent data and speculations on their role in the developing epididymis during the prenatal-to-postnatal transition, bringing attention to the many unanswered questions in this research area that may contribute to a better understanding of epididymal biology and male fertility.

Non-hormonal male contraception: A review and development of an Eppin based contraceptive.

Developing a non-hormonal male contraceptive requires identifying and characterizing an appropriate target and demonstrating its essential role in reproduction. Here we review the development of male contraceptive targets and the current therapeutic agents under consideration. In addition, the development of EPPIN as a target for contraception is reviewed. EPPIN is a well characterized surface protein on human spermatozoa that has an essential function in primate reproduction. EPPIN is discussed as an example of target development, testing in non-human primates, and the search for small organic compounds that mimic contraceptive antibodies; binding EPPIN and blocking sperm motility. Although many hurdles remain before the success of a non-hormonal male contraceptive, continued persistence should yield a marketable product.

Dynamic changes in the spatio-temporal expression of the ß-defensin SPAG11C in the developing rat epididymis and its regulation by androgens.

Herein, we characterized the spatio-temporal expression, cellular distribution and regulation by androgens of the ß-defensin SPAG11C, the rat ortholog of the human SPAG11B isoform C, in the developing epididymis by using RT-PCR, in situ hybridization and immunohistochemistry. We observed that Spag11c mRNA was ubiquitously expressed in rat fetuses, but preferentially detected in male reproductive tissues at adulthood. SPAG11C (mRNA and protein) was prenatally mainly detected in the mesenchyme of the Wolffian duct, switching gradually after birth to a predominant localization in the epididymis epithelium during postnatal development. In the adult epididymis, smooth muscle and interstitial cells were also identified as sources of SPAG11C. Furthermore, SPAG11C was differentially immunolocalized on spermatozoa surface during their transit from testis throughout caput and cauda epididymis. Developmental and surgical castration studies suggested that androgens contribute to the epididymal cell type- and region-specific modulation of SPAG11C mRNA levels and immunolocalization. Together our findings provide novel insights into the potential role of ß-defensins in the epididymis.

Interacting proteins on human spermatozoa: adaptive evolution of the binding of semenogelin I to EPPIN.

Semenogelin I (SEMG1) is found in human semen coagulum and on the surface of spermatozoa bound to EPPIN. The physiological significance of the SEMG1/EPPIN interaction on the surface of spermatozoa is its capacity to modulate sperm progressive motility. The present study investigates the hypothesis that the interacting surface of SEMG1 and EPPIN co-evolved within the Hominoidea time scale, as a result of adaptive pressures applied by their roles in sperm protection and reproductive fitness. Our results indicate that some amino acid residues of SEMG1 and EPPIN possess a remarkable deficiency of variation among hominoid primates. We observe a distinct residue change unique to humans within the EPPIN sequence containing a SEMG1 interacting surface, namely His92. In addition, Bayes Empirical Bayes analysis for positive selection indicates that the SEMG1 Cys239 residue underwent positive selection in humans, probably as a consequence of its role in increasing the binding affinity of these interacting proteins. We confirm the critical role of Cys239 residue for SEMG1 binding to EPPIN and inhibition of sperm motility by showing that recombinant SEMG1 mutants in which Cys239 residue was changed to glycine, aspartic acid, histidine, serine or arginine have reduced capacity to interact to EPPIN and to inhibit human sperm motility in vitro. In conclusion, our results indicate that EPPIN and SEMG1 rapidly co-evolved in primates due to their critical role in the modulation of sperm motility in the semen coagulum, providing unique insights into the molecular co-evolution of sperm surface interacting proteins.

Epididymal protease inhibitor (EPPIN) is differentially expressed in the male rat reproductive tract and immunolocalized in maturing spermatozoa.

EPPIN (epididymal protease inhibitor; SPINLW1), an antimicrobial cysteine-rich protein containing both Kunitz and whey acidic protein (WAP)-type four disulfide core protease inhibitor consensus sequences, is a target for male contraception because of its critical role in sperm motility. Here, we characterized EPPIN's expression and cellular distribution in rat tissues and its in vivo regulation by androgens in the epididymis. EPPIN (mRNA and protein) was abundantly expressed in the rat testis and epididymis; we also found that the vas deferens, seminal vesicles, and brain were novel sites of EPPIN expression. PCR studies demonstrated that in addition to Sertoli cells, spermatogenic cells expressed Eppin mRNA. EPPIN was immunolocalized in Sertoli cells and spermatogenic cells (pachytene spermatocytes and round and elongated spermatids) and in epithelial cells and spermatozoa from efferent ductules and epididymis. EPPIN staining was observed on the middle and principal pieces of the flagellum of testicular spermatozoa. Epididymal spermatozoa had more intense EPPIN staining on the flagellum, and the EPPIN staining became apparent on the head and neck regions. This suggested that the EPPIN found on maturing spermatozoa was secreted primarily by the epithelial cells of the epididymis. Surgical castration down-regulated EPPIN expression levels (mRNA and protein) in the caput and cauda epididymis, an effect reversed by testosterone replacement. Altogether, our data suggested that EPPIN expression in rats is more widespread than in humans and mice, and is androgen-dependent in the epididymis. This species could be used as an experimental model to further study EPPIN's role in male fertility.

Characterization of EPPIN's semenogelin I binding site: a contraceptive drug target.

Epididymal protease inhibitor (EPPIN) is found on the surface of spermatozoa and works as a central hub for a sperm surface protein complex (EPPIN protein complex [EPC]) that inhibits sperm motility on the binding of semenogelin I (SEMG1) during ejaculation. Here, we identify EPPIN's amino acids involved in the interactions within the EPC and demonstrate that EPPIN's sequence C102-P133 contains the major binding site for SEMG1. Within the same region, the sequence F117-P133 binds the EPC-associated protein lactotransferrin (LTF). We show that residues Cys102, Tyr107, and Phe117 in the EPPIN C-terminus are required for SEMG1 binding. Additionally, residues Tyr107 and Phe117 are critically involved in the interaction between EPPIN and LTF. Our findings demonstrate that EPPIN is a key player in the protein-protein interactions within the EPC. Target identification is an important step toward the development of a novel male contraceptive, and the functionality of EPPIN's residues Cys102, Tyr107, and Phe117 offers novel opportunities for contraceptive compounds that inhibit sperm motility by targeting this region of the molecule.

Therapeutic ultrasound as a potential male contraceptive: power, frequency and temperature required to deplete rat testes of meiotic cells and epididymides of sperm determined using a commercially available system.

BACKGROUND: Studies published in the 1970s by Mostafa S. Fahim and colleagues showed that a short treatment with ultrasound caused the depletion of germ cells and infertility. The goal of the current study was to determine if a commercially available therapeutic ultrasound generator and transducer could be used as the basis for a male contraceptive. METHODS: Sprague-Dawley rats were anesthetized and their testes were treated with 1 MHz or 3 MHz ultrasound while varying power, duration and temperature of treatment. RESULTS: We found that 3 MHz ultrasound delivered with 2.2 Watt per square cm power for fifteen minutes was necessary to deplete spermatocytes and spermatids from the testis and that this treatment significantly reduced epididymal sperm reserves. 3 MHz ultrasound treatment reduced total epididymal sperm count 10-fold lower than the wet-heat control and decreased motile sperm counts 1,000-fold lower than wet-heat alone. The current treatment regimen provided nominally more energy to the treatment chamber than Fahim's originally reported conditions of 1 MHz ultrasound delivered at 1 Watt per square cm for ten minutes. However, the true spatial average intensity, effective radiating area and power output of the transducers used by Fahim were not reported, making a direct comparison impossible. We found that germ cell depletion was most uniform and effective when we rotated the therapeutic transducer to mitigate non-uniformity of the beam field. The lowest sperm count was achieved when the coupling medium (3% saline) was held at 37 degrees C and two consecutive 15-minute treatments of 3 MHz ultrasound at 2.2 Watt per square cm were separated by 2 days. CONCLUSIONS: The non-invasive nature of ultrasound and its efficacy in reducing sperm count make therapeutic ultrasound a promising candidate for a male contraceptive. However, further studies must be conducted to confirm its efficacy in providing a contraceptive effect, to test the result of repeated use, to verify that the contraceptive effect is reversible and to demonstrate that there are no detrimental, long-term effects from using ultrasound as a method of male contraception.

Glucocorticoid receptor in the rat epididymis: expression, cellular distribution and regulation by steroid hormones.

Glucocorticoids regulate several physiological functions, including reproduction, in mammals. Curiously, little is known about glucocorticoid-induced effects on the epididymis, an androgen-dependent tissue with vital role on sperm maturation. Here, RT-PCR, Western blot and immunohistochemical studies were performed to evaluate expression, cellular distribution and hormonal regulation of glucocorticoid receptor (GR) along rat epididymis. The rat orthologue of human GRalpha (mRNA and protein) was detected in caput, corpus and cauda epididymis and immunolocalized in the nucleus and cytoplasm of different epididymal cells (epithelial, smooth muscle and interstitial cells) and nerve fibers. Changes in plasma glucocorticoid and androgen levels differentially regulated GR expression in caput and cauda epididymis by homologous and heterologous mechanisms. In vivo treatment with dexamethasone significantly changed the expression of glucocorticoid-responsive genes and induced ligand-dependent GR nuclear translocation in epithelial cells from epididymis, indicating that GR is fully active in this tissue. Heterologous regulation of androgen receptor expression by glucocorticoids was also demonstrated in cauda epididymis. Our results demonstrate that the epididymis is under glucocorticoid regulation, opening new insights into the roles of this hormone in male fertility.