Three-Dimensional Cell Culture of Epididymal Basal Cells and Organoids: A Novel Tool for Toxicology.

Spermatozoa are formed in the testis but must transit through the epididymis to acquire motility and the ability to fertilize. The epididymis is a single convoluted tubule comprising several anatomically and physiologically distinct regions. The pseudostratified epithelium consists of multiple cell types, including principal cells, clear cells, narrow cells, and apical cells, that line the lumen of the epididymis. Basal cells are present at the base of the epithelium, and halo cells, which includes macrophages/monocytes, mononuclear phagocytes, and T lymphocytes, are also present in the epithelium. Several aspects of this complex spermatozoan maturation process are well established, but a great deal remains poorly understood. Given that dysfunction of the epididymis has been associated with male infertility, in vitro tools to study epididymal function and epididymal sperm maturation are required. Our lab and others have previously developed human, rat, and mouse epithelial principal cell lines, which have been used to address certain questions, such as about the regulation of junctional proteins in the epididymis, as well as the toxicity of nonylphenols. Given that the epididymal epithelium comprises multiple cell types, however, a 3D in vitro model provides a more comprehensive and realistic tool that can be used to study and elucidate the multiple aspects of epididymal function. The purpose of this article is to provide detailed information regarding the preparation, maintenance, passaging, and immunofluorescent staining of rat epididymal organoids derived from adult basal cells, which we have demonstrated to be a type of adult stem cell in the rat epididymis. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Isolation of epididymal cells Basic Protocol 2: Magnetic activated cell sorting and isolation of basal cells Basic Protocol 3: Preparation and culture of epididymal basal cell organoids Basic Protocol 4: Passage of epididymal basal cell organoids Basic Protocol 5: Freezing and thawing of epididymal basal cell organoids Basic Protocol 6: Immunofluorescent staining of epididymal basal cell organoids.

A screen for MeCP2-TBL1 interaction inhibitors using a luminescence-based assay.

Understanding the molecular pathology of neurodevelopmental disorders should aid the development of therapies for these conditions. In MeCP2 duplication syndrome (MDS)-a severe autism spectrum disorder-neuronal dysfunction is caused by increased levels of MeCP2. MeCP2 is a nuclear protein that binds to methylated DNA and recruits the nuclear co-repressor (NCoR) complex to chromatin via an interaction with the WD repeat-containing proteins TBL1 and TBLR1. The peptide motif in MeCP2 that binds to TBL1/TBLR1 is essential for the toxicity of excess MeCP2 in animal models of MDS, suggesting that small molecules capable of disrupting this interaction might be useful therapeutically. To facilitate the search for such compounds, we devised a simple and scalable NanoLuc luciferase complementation assay for measuring the interaction of MeCP2 with TBL1/TBLR1. The assay allowed excellent separation between positive and negative controls, and had low signal variance (Z-factor = 0.85). We interrogated compound libraries using this assay in combination with a counter-screen based on luciferase complementation by the two subunits of protein kinase A (PKA). Using this dual screening approach, we identified candidate inhibitors of the interaction between MeCP2 and TBL1/TBLR1. This work demonstrates the feasibility of future screens of large compound collections, which we anticipate will enable the development of small molecule therapeutics to ameliorate MDS.

Arl13b controls basal cell stemness properties and Hedgehog signaling in the mouse epididymis.

Epithelial cells orchestrate a series of intercellular signaling events in response to tissue damage. While the epididymis is composed of a pseudostratified epithelium that controls the acquisition of male fertility, the maintenance of its integrity in the context of tissue damage or inflammation remains largely unknown. Basal cells of the epididymis contain a primary cilium, an organelle that controls cellular differentiation in response to Hedgehog signaling cues. Hypothesizing its contribution to epithelial homeostasis, we knocked out the ciliary component ARL13B in keratin 5-positive basal cells. In this model, the reduced size of basal cell primary cilia was associated with impaired Hedgehog signaling and the loss of KRT5, KRT14, and P63 basal cell markers. When subjected to tissue injury, the epididymal epithelium from knock-out mice displayed imbalanced rates of cell proliferation/apoptosis and failed to properly regenerate in vivo. This response was associated with changes in the transcriptomic landscape related to immune response, cell differentiation, cell adhesion, and triggered severe hypoplasia of the epithelium. Together our results indicate that the ciliary GTPase, ARL13B, participates in the transduction of the Hedgehog signaling pathway to maintain basal cell stemness needed for tissue regeneration. These findings provide new insights into the role of basal cell primary cilia as safeguards of pseudostratified epithelia.

Discovery and Structural Characterization of Small Molecule Binders of the Human CTLH E3 Ligase Subunit GID4.

Targeted protein degradation (TPD) strategies exploit bivalent small molecules to bridge substrate proteins to an E3 ubiquitin ligase to induce substrate degradation. Few E3s have been explored as degradation effectors due to a dearth of E3-binding small molecules. We show that genetically induced recruitment to the GID4 subunit of the CTLH E3 complex induces protein degradation. An NMR-based fragment screen followed by structure-guided analog elaboration identified two binders of GID4, 16 and 67, with Kd values of 110 and 17 µM in vitro. A parallel DNA-encoded library (DEL) screen identified five binders of GID4, the best of which, 88, had a Kd of 5.6 µM in vitro and an EC50 of 558 nM in cells with strong selectivity for GID4. X-ray co-structure determination revealed the basis for GID4-small molecule interactions. These results position GID4-CTLH as an E3 for TPD and provide candidate scaffolds for high-affinity moieties that bind GID4.

Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides.

Genetically-encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution-phase macrocyclization of side chain-protected linear peptides obtained from standard solid-phase peptide synthesis. Cyclic peptide targets, including cyclo-[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically-encoded counterparts. Synthetic products were characterized by tandem high-resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation-induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost-effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.

Emerging organoid models to study the epididymis in male reproductive toxicology.

The importance of the epididymis on sperm maturation and consequently male fertility has been well documented. The pseudostratified epithelium of the epididymis is comprised of multiple cell types, including principal cells, which are the most abundant, and basal cells. The role of basal cells has been unclear and has been a source of discussion in the literature. However, the recent demonstration that these cells are multipotent or adult stem cells has opened new areas of research in epididymal biology. One such avenue is to understand the regulation of these stem cells, and to exploit their properties to develop tools for toxicological studies to elucidate the effects of chemicals on cell differentiation and epididymal function in vitro. Studies in both rat and mouse have shown that purified single epididymal basal cells cultured under 3D conditions can proliferate and differentiate to form organoids, or mini organs. Furthermore, these epididymal basal stem cells can self-renew and differentiate into other epididymal cell types. It is known that during epididymal development, basal cells are derived from undifferentiated columnar cells, which have been reported to share common properties to stem cells. Like basal cells, these undifferentiated columnar cells can also form organoids under 3D culture conditions and can differentiate into basal, principal and clear cells. Organoids derived from either basal cells or columnar cells offer unique models for toxicology studies and represent an exciting and emerging approach to understand the epididymis.

Differential gene expression and hallmarks of stemness in epithelial cells of the developing rat epididymis.

Epididymal development can be subdivided into three phases: undifferentiated, a period of differentiation, and expansion. The objectives of this study were (1) to assess gene expression profiles in epididymides, (2) predict signaling pathways, and (3) develop a novel 3D cell culture method to assess the regulation of epididymal development in vitro. Microarray analyses indicate that the largest changes in differential gene expression occurred between the 7- to 18-day period, in which 1452 genes were differentially expressed, while 671 differentially expressed genes were noted between days 18 and 28, and there were 560 differentially expressed genes between days 28 and 60. Multiple signaling pathways were predicted at different phases of development. Pathway associations indicated that in epididymides of 7- to 18-day old rats, there was a significant association of regulated genes implicated in stem cells, estrogens, thyroid hormones, and kidney development, while androgen- and estrogen-related pathways were enriched at other phases of development. Organoids were derived from CD49f + columnar cells from 7-day old rats, while no organoids developed from CD49f- cells. Cells cultured in an epididymal basal cell organoid medium versus a commercial kidney differentiation medium supplemented with DHT revealed that irrespective of the culture medium, cells within differentiating organoids expressed p63, AQP9, and V-ATPase after 14 days of culture. The commercial kidney medium resulted in an increase in the number of organoids positive for p63, AQP9, and V-ATPase. Together, these data indicate that columnar cells represent an epididymal stem/progenitor cell population.

Identification and optimization of molecular glue compounds that inhibit a noncovalent E2 enzyme-ubiquitin complex.

Pharmacological control of the ubiquitin-proteasome system (UPS) is of intense interest in drug discovery. Here, we report the development of chemical inhibitors of the ubiquitin-conjugating (E2) enzyme CDC34A (also known as UBE2R1), which donates activated ubiquitin to the cullin-RING ligase (CRL) family of ubiquitin ligase (E3) enzymes. A FRET-based interaction assay was used to screen for novel compounds that stabilize the noncovalent complex between CDC34A and ubiquitin, and thereby inhibit the CDC34A catalytic cycle. An isonipecotamide hit compound was elaborated into analogs with ~1000-fold increased potency in stabilizing the CDC34A-ubiquitin complex. These analogs specifically inhibited CDC34A-dependent ubiquitination in vitro and stabilized an E2~ubiquitin thioester reaction intermediate in cells. The x-ray crystal structure of a CDC34A-ubiquitin-inhibitor complex uncovered the basis for analog structure-activity relationships. The development of chemical stabilizers of the CDC34A-ubiquitin complex illustrates a general strategy for de novo discovery of molecular glue compounds that stabilize weak protein interactions.

Human and animal fertility studies in cystinosis reveal signs of obstructive azoospermia, an altered blood-testis barrier and a subtherapeutic effect of cysteamine in testis.

Cystinosis is an inherited metabolic disorder caused by autosomal recessive mutations in the CTNS gene leading to lysosomal cystine accumulation. The disease primarily affects the kidneys followed by extra-renal organ involvement later in life. Azoospermia is one of the unclarified complications which are not improved by cysteamine, which is the only available disease-modifying treatment. We aimed at unraveling the origin of azoospermia in cysteamine-treated cystinosis by confirming or excluding an obstructive factor, and investigating the effect of cysteamine on fertility in the Ctns-/- mouse model compared with wild type. Azoospermia was present in the vast majority of infantile type cystinosis patients. While spermatogenesis was intact, an enlarged caput epididymis and reduced levels of seminal markers for obstruction neutral α-glucosidase (NAG) and extracellular matrix protein 1 (ECM1) pointed towards an epididymal obstruction. Histopathological examination in human and mouse testis revealed a disturbed blood-testis barrier characterized by an altered zonula occludens-1 (ZO-1) protein expression. Animal studies ruled out a negative effect of cysteamine on fertility, but showed that cystine accumulation in the testis is irresponsive to regular cysteamine treatment. We conclude that the azoospermia in infantile cystinosis is due to an obstruction related to epididymal dysfunction, irrespective of the severity of an evolving primary hypogonadism. Regular cysteamine treatment does not affect fertility but has subtherapeutic effects on cystine accumulation in testis.

Self-renewal and differentiation of rat epididymal basal cells using a novel in vitro organoid model†.

The epididymis is composed of a pseudostratified epithelium that is comprised of various cell types. Studies have shown that rat basal cells share common properties with adult stem cells and begin to differentiate in vitro in response to fibroblast growth factor and 5α-dihydrotestosterone. The characterization of rat basal cells is therefore necessary to fully understand the role of these cells. The objectives of this study were to assess the ability of single basal cells to develop organoids and to assess their ability to self-renew and differentiate in vitro. We isolated basal cells from the rat epididymis and established three-dimensional cell cultures from the basal and nonbasal cell fractions. Organoids were formed by single adult epididymal basal cells. Organoids were dissociated into single basal cells, which were able to reform new organoids, and were maintained over 10 generations. Long-term culture of organoids revealed that these cells could be differentiated into cells expressing the principal cell markers aquaporin 9 and cystic fibrosis transmembrane conductance regulator. Electron microscopy demonstrated that organoids were composed of several polarized cell types displaying microvilli and the ability to form tight junctions. Additionally, organoids could be formed by basal cells from either the proximal or distal region of the epididymis and are able to secrete clusterin, a protein implicated in the maturation of spermatozoa. These data indicate that rat basal cells can be used to derive epididymal organoids and further support that notion that these may represent a stem cell population in the epididymis.

Hedgehog signaling pathway regulates gene expression profile of epididymal principal cells through the primary cilium.

Primary cilia (PC) are organelles that sense and respond to dynamic changes of the extracellular milieu through the regulation of target genes. By using the epididymis as a model system, we determined the contribution of primary cilia in the regulation of epithelial cell functions through the transduction of the Hedgehog (Hh) signaling pathway. Both Sonic (SHH) and Indian Hedgehog (IHH) ligands were detected in epididymal epithelial cells by confocal microscopy and found secreted in the extracellular space. Gene expression profiling preformed on ciliated epithelial cells indicated that 153 and 1052 genes were differentially expressed following treatment with the Hh agonist SAG or the Hh antagonist cyclopamine (Cyclo), respectively. Strikingly, gene ontology analysis indicated that genes associated with immune response were the most affected following Hh modulation. The contribution of epididymal PC to canonical Hh pathway transduction was validated by ciliobrevin D treatment, which induced a significant decrease in PC length and a reduction in the expression Hh signaling targets. Such findings bring us closer to a molecular understanding of the subtle immune balance observed in some epithelia, including the epididymis and the intestine, which are organs featuring both tolerance toward autoimmune spermatozoa (or commensal bacteria) and defense against pathogens.

Beta-defensin126 is correlated with sperm motility in fertile and infertile men†.

A crucial function of the epididymis is providing a surface glycocalyx that is important for sperm maturation and capacitation. Defensins are antimicrobial peptides expressed in the epididymis. In the macaque epididymis, defensin beta 126 (DEFB126) is important for sperm motility, however, it is not known whether this is the case in humans. The objectives were to determine: (1) if DEFB126 on human ejaculated sperm was correlated with sperm motility in fertile and infertile men, (2) that recombinant DEFB126 could induce immature sperm motility in vitro. Immunofluorescence staining indicated that the proportion of DEFB126-positive sperm was significantly higher in motile sperm. Furthermore, the proportion of DEFB126-labeled sperm was positively correlated with sperm motility and normal morphology. Additional studies indicated that the proportion of DEFB126-positive spermatozoa in fertile volunteers was significantly higher than in volunteers with varicocele, and in infertile volunteers with semen deficiencies. To determine the role of DEFB126 on sperm motility, the DEFB126 gene was cloned and used to generate recombinant DEFB126 in H9C2 cells (rat embryonic heart myoblast cells). Deletion mutations were created into two regions of the protein, which have been linked to male infertility. Immotile testicular spermatozoa were incubated with cells expressing the different forms of DEFB126. Full-length DEFB126 significantly increased motility of co-cultured spermatozoa. However, no increase in sperm motility was observed with the mutated forms of DEFB126. In conclusion, these results support the notion that DEFB126 is important in human sperm maturation and the potential use of DEFB126 for in vitro sperm maturation.

Probing Anti-inflammatory Properties Independent of NF-κB Through Conformational Constraint of Peptide-Based Interleukin-1 Receptor Biased Ligands.

Interleukin-1ß (IL-1ß) binds to the IL-1 receptor (IL-1R) and is a key cytokine mediator of inflammasome activation. IL-1ß signaling leads to parturition in preterm birth (PTB) and contributes to the retinal vaso-obliteration characteristic of oxygen-induced retinopathy (OIR) of premature infants. Therapeutics targeting IL-1ß and IL-1R are approved to treat rheumatoid arthritis; however, all are large proteins with clinical limitations including immunosuppression, due in part to inhibition of NF-κB signaling, which is required for immuno-vigilance and cytoprotection. The all-D-amino acid peptide 1 (101.10, H-d-Arg-d-Tyr-d-Thr-d-Val-d-Glu-d-Leu-d-Ala-NH2) is an allosteric IL-1R modulator, which exhibits functional selectivity and conserves NF-κB signaling while inhibiting other IL-1-activated pathways. Peptide 1 has proven effective in experimental models of PTB and OIR. Seeking understanding of the structural requirements for the activity and biased signaling of 1, a panel of twelve derivatives was synthesized employing the various stereochemical isomers of α-amino-γ-lactam (Agl) and α-amino-ß-hydroxy-γ-lactam (Hgl) residues to constrain the D-Thr-D-Val dipeptide residue. Using circular dichroism spectroscopy, the peptide conformation in solution was observed to be contingent on Agl, Hgl, and Val stereochemistry. Moreover, the lactam mimic structure and configuration influenced biased IL-1 signaling in an in vitro panel of cellular assays as well as in vivo activity in murine models of PTB and OIR. Remarkably, all Agl and Hgl analogs of peptide 1 did not inhibit NF-κB signaling but blocked other pathways, such as JNK and ROCK2 phosphorylation contingent on structure and configuration. Efficacy in preventing preterm labor correlated with a capacity to block IL-1ß-induced IL-1ß synthesis. Furthermore, the importance of inhibition of JNK and ROCK2 phosphorylation for enhanced activity was highlighted for prevention of vaso-obliteration in the OIR model. Taken together, lactam mimic structure and stereochemistry strongly influenced conformation and biased signaling. Selective modulation of IL-1 signaling was proven to be particularly beneficial for curbing inflammation in models of preterm labor and retinopathy of prematurity (ROP). A class of biased ligands has been created with potential to serve as selective probes for studying IL-1 signaling in disease. Moreover, the small peptide mimic prototypes are promising leads for developing immunomodulatory therapies with easier administration and maintenance of beneficial effects of NF-κB signaling.

Effects of prostaglandin E2 on gap junction protein alpha 1 in the rat epididymis.

Gap junctions are responsible for intercellular communication. In the adult mammalian epididymis, gap junction protein alpha 1 (GJA1) is localized between basal and either principal or clear cells. GJA1 levels and localization change during the differentiation of basal cells. The present objective was to determine the role of basal cells and prostaglandin E2 (PGE2) on GJA1 in the rat epididymis. Prior to basal cell differentiation, GJA1 is colocalized with TJP1 at the apical lateral margins between adjacent epithelial cells. When basal cells are present, GJA1 becomes associated between basal and principal cells, where it is primarily immunolocalized until adulthood. Basal cells express TP63, differentiate from epithelial cells, and produce prostaglandin-endoperoxide synthase 1 by 21 days of age. Prior to day 21, GJA1and TP63 are not strongly associated at the apical region. However, by day 28, TP63-positive basal cells migrate to the base of the epithelium, and also express GJA1. To assess effects of PGE2 on GJA1, rat caput epididymal (RCE) cells were exposed to PGE2 (50 µM) for 3 h. PGE2 increased levels of Gja1 mRNA in RCE cells, while levels of Gjb1, Gjb2, Gjb4, and GjB5 were unaltered. Furthermore, PGE2 increased protein levels of GJA1, phospho-GJA1, phospho-AKT, CTNNB1, and phospho-CTNNB1. Total AKT and the tight junction protein claudin1 were also not altered by PGE2. Data suggest that development of the epididymal epithelium and differentiation of epididymal basal cells regulate the targeting of GJA1, and that this appears to be mediated by PGE2.

Operational modifications for the development of nitrifying bacteria in a large-scale biological aerated filter and its impact on wastewater treatment.

To develop a better understanding for fixed biomass processes, the development of a nitrifying bacterial biofilm, as well as the performance of treatment during modifications to operational conditions of a full-scale submerged biological filter were examined. The development of the nitrifying biofilm was investigated at four depth levels (1, 2, 4 and 5 feet). The result of bacterial subpopulations analyzed by qPCR relative to the physico-chemical parameters of the wastewater during the various tests (sustained aeration, modified backwash parameters and inflow restriction) revealed an increase of the relative presence of nitrifying microorganisms throughout the biofilm (especially for nitrite oxidizing bacteria (NOB)), but this was not necessarily accompanied by a better nitrification rate. The highest observed nitrification rate was 49% of removal in the test cell during backwashing conditions, whereas the relative ammonia oxidizing bacteria (AOB) population was 0.032% and NOB was 0.008% of the total biomass collected. The highest percentage of nitrifying bacteria observed (0.034% AOB and 0.18% NOB) resulted in a nitrification rate of 21%. The treatment of organic matter determined by measuring the chemical and biochemical oxygen demand (COD, CBOD5) was improved.

Cellular junctions in the epididymis, a critical parameter for understanding male reproductive toxicology.

Epididymal sperm maturation is a critical aspect of male reproduction in which sperm acquire motility and the ability to fertilize an ovum. Sperm maturation is dependent on the creation of a specific environment that changes along the epididymis and which enables the maturation process. The blood-epididymis barrier creates a unique luminal micro-environment, different from blood, by limiting paracellular transport and forcing receptor-mediated transport of macromolecules across the epididymal epithelium. Direct cellular communication between cells allows coordinated function of the epithelium. A limited number of studies have directly examined the effects of toxicants on junctional proteins and barrier function in the epididymis. Effects on the integrity of the blood-epididymis barrier have resulted in decreased fertility and, in some cases, the development of sperm granulomas. Studies have shown that in addition to tight junctions, proteins implicated in the maintenance of adherens junctions and gap junctions alter epididymal functions. This review will provide an overview of the types and roles of cellular junctions in the epididymis, and how these are targeted by different toxicants.