mouse-models-epididymitis-induced-pathogen-associated-molecular

Mouse Models of Epididymitis Induced by Pathogen-Associated Molecular Patterns

Epididymitis is a highly prevalent disease in the outpatient urologic clinic and a relevant factor in male infertility. Bacterial infections, including ...

São Paulo State University

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 (pEC = 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  -adrenoceptor ( -AR) antagonist prazosin (p  ≅ 8.9), 5-HT antagonists ketanserin (p  ≅ 9.4) and fluoxetine (p  ≅ 7.4), and 5-HT ligands WAY 100635 (p  ≅ 8.9) and buspirone (p  ≅ 7.3). Reverse transcriptase polymerase chain reaction analysis demonstrated that 5-HT and 5-HT transcripts are highly abundant in the cauda epididymis, whereas 5-HT transcript was not found. Altogether, our results reveal that contractions of the CE duct to 5-HT encompasses at least activation of  -ARs and 5-HT and 5-HT receptors, providing new insights into the roles of 5-HT on the epididymal function.

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

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.

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

The epididymis is a tubular structure connecting the vas deferens to the testis. This organ consists of three main regions-caput, corpus, and cauda-that face opposing immunological tasks. A means of combating invading pathogens is required in the distally located cauda, where there is a risk of ascending bacterial infections originating from the urethra. Meanwhile, immune tolerance is necessary at the caput, where spermatozoa with immunogenic neo-antigens originate from the testis. Consistently, when challenged with live bacteria or inflammatory stimuli, the cauda elicits a much stronger immune response and inflammatory-inflicted damage than the caput. At the cellular level, a role for diverse and strategically positioned mononuclear phagocytes is emerging. At the mechanistic level, differential expression of immunoprotective and immunomodulatory mediators has been detected between the three main regions of the epididymis. In this review, we summarize the current state of knowledge about region-specific immunological characteristics and unveil possible underlying mechanisms on cellular and molecular levels. Improved understanding of the different immunological microenvironments is the basis for an improved therapy and counseling of patients with epididymal infections.

Differential Immune Response to Infection and Acute Inflammation Along the Epididymis.

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.

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

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.

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

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.

Advances in non-hormonal male contraception targeting sperm motility.

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.

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

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 SEMG1, 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.

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

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.

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

 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.  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.  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.  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.

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

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.

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

Maternal endothelial dysfunction in pregnancy hypertension is related to impairment of nitric oxide (NO) formation. However, NO levels and hemodynamic repercussions on the female offspring remain unclear. Therefore, this study hypothesized that maternal pregnancy hypertension reduces circulating NO metabolites and increases arterial blood pressure in first-generation offspring female rats.

Maternal pregnancy hypertension impairs nitric oxide formation and results in increased arterial blood pressure in first-generation offspring female rats.

Rheumatoid arthritis (RA) is an inflammatory disease triggered by chronic and systemic activation of the immune system, with consequences for male fertility. However, the influence of RA on sexual accessory glands remains poorly investigated.

Article	Year
Contractile Effects of Serotonin (5-HT) in the Rat Cauda Epididymis: Expression and Functional Characterization of 5-HT Receptors. The Journal of pharmacology and experimental therapeutics\| PubMed ID: 30728250	2019
Epididymal protease inhibitor (EPPIN) is a protein hub for seminal vesicle-secreted protein SVS2 binding in mouse spermatozoa. Molecular and cellular endocrinology\| PubMed ID: 32044375	2020
Differential Immune Response to Infection and Acute Inflammation Along the Epididymis. Frontiers in immunology\| PubMed ID: 33329594	2020
Dissecting EPPIN protease inhibitor domains in sperm motility and fertilizing ability: repercussions for male contraceptive development. Molecular human reproduction\| PubMed ID: 34792600	2021
Low dose of cyclosporine A disrupts sperm parameters and testosterone levels reversibly in mice. Toxicology and applied pharmacology\| PubMed ID: 36634874	2023
Advances in non-hormonal male contraception targeting sperm motility. Human reproduction update\| PubMed ID: 37141450	2023
Pregnancy hypertension-associated endothelial dysfunction is attenuated by isoflurane anesthesia: Evidence of protective effect related to increases in nitric oxide. Life sciences\| PubMed ID: 37648198	2023
Interactions of the male contraceptive target EPPIN with semenogelin-1 and small organic ligands. Scientific reports\| PubMed ID: 37658081	2023
Early-life origin of prostate cancer through deregulation of miR-206 networks in maternally malnourished offspring rats. Scientific reports\| PubMed ID: 37907720	2023
Insights Into Antioxidant Strategies to Counteract Radiation-Induced Male Infertility. Antioxidants & redox signaling\| PubMed ID: 37917108	2024
Regional modulation of toll-like receptor signaling pathway genes in acute epididymitis in mice. Andrology\| PubMed ID: 38497291	2024
Maternal pregnancy hypertension impairs nitric oxide formation and results in increased arterial blood pressure in first-generation offspring female rats. Pregnancy hypertension\| PubMed ID: 38805888	2024
Adjuvant-induced arthritis induces epithelial proliferation and differential expression of SVS2 and SVS3 in the seminal vesicles. Andrology\| PubMed ID: 40622002	2025

Erick J. R. Silva

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