Immortalized mouse caput epididymal epithelial (mECap18) cell line recapitulates the in-vivo environment.

Residing between the testes and the vas deferens, the epididymis is a highly convoluted tubule whose unique luminal microenvironment is crucial for the functional maturation of spermatozoa. This microenvironment is created by the combined secretory and resorptive activity of the lining epididymal epithelium, including the release of extracellular vesicles (epididymosomes), which encapsulate fertility modulating proteins and a myriad of small non-coding RNAs (sncRNAs) that are destined for delivery to recipient sperm cells. To enable investigation of this intercellular communication nexus, we have previously developed an immortalized mouse caput epididymal epithelial cell line (mECap18). Here, we describe the application of label-free mass spectrometry to characterize the mECap18 cell proteome and compare this to the proteome of native mouse caput epididymal epithelial cells. We report the identification of 5,313 mECap18 proteins, as many as 75.8% of which were also identified in caput epithelial cells wherein they mapped to broadly similar protein classification groupings. Furthermore, key pathways associated with protein synthesis (e.g., EIF2 signaling) and cellular protection in the male reproductive tract (e.g., sirtuin signaling) were enriched in both proteomes. This comparison supports the utility of the mECap18 cell line as a tractable in-vitro model for studying caput epididymal epithelial cell function.

Proteomic Dissection of the Impact of Environmental Exposures on Mouse Seminal Vesicle Function.

Seminal vesicles are an integral part of the male reproductive accessory gland system. They produce a complex array of secretions containing bioactive constituents that support gamete function and promote reproductive success, with emerging evidence suggesting these secretions are influenced by our environment. Despite their significance, the biology of seminal vesicles remains poorly defined. Here, we complete the first proteomic assessment of mouse seminal vesicles and assess the impact of the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or control daily for five consecutive days prior to collecting seminal vesicle tissue. A total of 5013 proteins were identified in the seminal vesicle proteome with bioinformatic analyses identifying cell proliferation, protein synthesis, cellular death, and survival pathways as prominent biological processes. Secreted proteins were among the most abundant, and several proteins are linked with seminal vesicle phenotypes. Analysis of the effect of acrylamide on the seminal vesicle proteome revealed 311 differentially regulated (FC ± 1.5, p ≤ 0.05, 205 up-regulated, 106 downregulated) proteins, orthogonally validated via immunoblotting and immunohistochemistry. Pathways that initiate protein synthesis to promote cellular survival were prominent among the dysregulated pathways, and rapamycin-insensitive companion of mTOR (RICTOR, p = 6.69E-07) was a top-ranked upstream driver. Oxidative stress was implicated as contributing to protein changes, with acrylamide causing an increase in 8-OHdG in seminal vesicle epithelial cells (fivefold increase, p = 0.016) and the surrounding smooth muscle layer (twofold increase, p = 0.043). Additionally, acrylamide treatment caused a reduction in seminal vesicle secretion weight (36% reduction, p = 0.009) and total protein content (25% reduction, p = 0.017). Together these findings support the interpretation that toxicant exposure influences male accessory gland physiology and highlights the need to consider the response of all male reproductive tract tissues when interpreting the impact of environmental stressors on male reproductive function.

A novel role for milk fat globule-EGF factor 8 protein (MFGE8) in the mediation of mouse sperm-extracellular vesicle interactions.

Spermatozoa transition to functional maturity as they are conveyed through the epididymis, a highly specialized region of the male excurrent duct system. Owing to their transcriptionally and translationally inert state, this transformation into fertilization competent cells is driven by complex mechanisms of intercellular communication with the secretory epithelium that delineates the epididymal tubule. Chief among these mechanisms are the release of extracellular vesicles (EV), which have been implicated in the exchange of varied macromolecular cargo with spermatozoa. Here, we describe the optimization of a tractable cell culture model to study the mechanistic basis of sperm-extracellular vesicle interactions. In tandem with receptor inhibition strategies, our data demonstrate the importance of milk fat globule-EGF factor 8 (MFGE8) protein in mediating the efficient exchange of macromolecular EV cargo with mouse spermatozoa; with the MFGE8 integrin-binding Arg-Gly-Asp (RGD) tripeptide motif identified as being of particular importance. Specifically, complementary strategies involving MFGE8 RGD domain ablation, competitive RGD-peptide inhibition and antibody-masking of alpha V integrin receptors, all significantly inhibited the uptake and redistribution of EV-delivered proteins into immature mouse spermatozoa. These collective data implicate the MFGE8 ligand and its cognate integrin receptor in the mediation of the EV interactions that underpin sperm maturation.

Transcriptomic analysis of the seminal vesicle response to the reproductive toxicant acrylamide.

BACKGROUND: The seminal vesicles synthesise bioactive factors that support gamete function, modulate the female reproductive tract to promote implantation, and influence developmental programming of offspring phenotype. Despite the significance of the seminal vesicles in reproduction, their biology remains poorly defined. Here, to advance understanding of seminal vesicle biology, we analyse the mouse seminal vesicle transcriptome under normal physiological conditions and in response to acute exposure to the reproductive toxicant acrylamide. Mice were administered acrylamide (25 mg/kg bw/day) or vehicle control daily for five consecutive days prior to collecting seminal vesicle tissue 72 h following the final injection. RESULTS: A total of 15,304 genes were identified in the seminal vesicles with those encoding secreted proteins amongst the most abundant. In addition to reproductive hormone pathways, functional annotation of the seminal vesicle transcriptome identified cell proliferation, protein synthesis, and cellular death and survival pathways as prominent biological processes. Administration of acrylamide elicited 70 differentially regulated (fold-change ≥1.5 or ≤ 0.67) genes, several of which were orthogonally validated using quantitative PCR. Pathways that initiate gene and protein synthesis to promote cellular survival were prominent amongst the dysregulated pathways. Inflammation was also a key transcriptomic response to acrylamide, with the cytokine, Colony stimulating factor 2 (Csf2) identified as a top-ranked upstream driver and inflammatory mediator associated with recovery of homeostasis. Early growth response (Egr1), C-C motif chemokine ligand 8 (Ccl8), and Collagen, type V, alpha 1 (Col5a1) were also identified amongst the dysregulated genes. Additionally, acrylamide treatment led to subtle changes in the expression of genes that encode proteins secreted by the seminal vesicle, including the complement regulator, Complement factor b (Cfb). CONCLUSIONS: These data add to emerging evidence demonstrating that the seminal vesicles, like other male reproductive tract tissues, are sensitive to environmental insults, and respond in a manner with potential to exert impact on fetal development and later offspring health.

The contribution of epididymosomes to the sperm small RNA profile.

It is now well established that mature spermatozoa harbour a rich and diverse profile of small non-protein-coding regulatory RNAs (sRNAs). There is also growing appreciation that this sRNA profile displays considerable plasticity, being altered in response to paternal exposure to a variety of environmental stressors. Coupled with evidence that upon delivery to the oocyte at the moment of fertilisation, sperm-borne sRNAs are able to influence both early embryonic development and the subsequent health of the offspring, there is now interest in both the timing and degree of change in the composition of the sRNA cargo of sperm. Models in which such epigenetic changes are linked to the spermatogenic cycle are seemingly incompatible with the lack of overt phenotypic changes in the spermatozoa of affected males. Rather, there is mounting consensus that such changes are imposed on sperm during their transit and storage within the epididymis, a protracted developmental window that takes place over several weeks. Notably, since spermatozoa are rendered transcriptionally and translationally silent during their development in the testes, it is most likely that the epididymis-documented alterations to the sperm sRNA profile are driven extrinsically, with a leading candidate being epididymosomes: small membrane enclosed extracellular vesicles that encapsulate a complex macromolecular cargo of proteins and RNAs, including the sRNAs. Here, we review the role of epididymosome-sperm communication in contributing to the establishment of the sperm sRNA profile during their epididymal transit.

Heat exposure induces oxidative stress and DNA damage in the male germ line.

The reproductive consequences of global warming are not currently understood. In order to address this issue, we have examined the reproductive consequences of exposing male mice to a mild heat stress. For this purpose, adult male mice were exposed to an elevated ambient temperature of 35°C under two exposure models. The first involved acute exposure for 24 h, followed by recovery periods between 1 day and 6 weeks. The alternative heating regimen involved a daily exposure of 8 h for periods of 1 or 2 weeks. In our acute model, we identified elevated sperm mitochondrial ROS generation (P < 0.05), increased sperm membrane fluidity (P < 0.05), DNA damage in the form of single-strand breaks (P < 0.001), and oxidative DNA damage (P < 0.05), characteristic of an oxidative stress cascade. This DNA damage was detected in pachytene spermatocytes (P < 0.001) and round spermatids (P < 0.001) isolated from testes after 1 day heat recovery. Despite these lesions, the spermatozoa of heat-treated mice exhibited no differences in their ability to achieve hallmarks of capacitation or to fertilize the oocyte and support development of embryos to the blastocyst stage (all P > 0.05). Collectively, our acute heat stress model supports the existence of heat susceptible stages of germ cell development, with the round spermatids being most perturbed and spermatogonial stem cells exhibiting resistance to this insult. Such findings were complemented by our chronic heat stress model, which further supported the vulnerability of the round spermatid population.

Extracellular vesicles alter trophoblast function in pregnancies complicated by COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and resulting coronavirus disease (COVID-19) causes placental dysfunction, which increases the risk of adverse pregnancy outcomes. While abnormal placental pathology resulting from COVID-19 is common, direct infection of the placenta is rare. This suggests that pathophysiology associated with maternal COVID-19, rather than direct placental infection, is responsible for placental dysfunction and alteration of the placental transcriptome. We hypothesized that maternal circulating extracellular vesicles (EVs), altered by COVID-19 during pregnancy, contribute to placental dysfunction. To examine this hypothesis, we characterized maternal circulating EVs from pregnancies complicated by COVID-19 and tested their effects on trophoblast cell physiology in vitro . We found that the gestational timing of COVID-19 is a major determinant of circulating EV function and cargo. In vitro trophoblast exposure to EVs isolated from patients with an active infection at the time of delivery, but not EVs isolated from Controls, altered key trophoblast functions including hormone production and invasion. Thus, circulating EVs from participants with an active infection, both symptomatic and asymptomatic cases, can disrupt vital trophoblast functions. EV cargo differed between participants with COVID-19 and Controls, which may contribute to the disruption of the placental transcriptome and morphology. Our findings show that COVID-19 can have effects throughout pregnancy on circulating EVs and circulating EVs are likely to participate in placental dysfunction induced by COVID-19.

The microbiota and T cells non-genetically modulate inherited phenotypes transgenerationally.

The host-microbiota relationship has evolved to shape mammalian physiology, including immunity, metabolism, and development. Germ-free models are widely used to study microbial effects on host processes such as immunity. Here, we find that both germ-free and T cell-deficient mice exhibit a robust sebum secretion defect persisting across multiple generations despite microbial colonization and T cell repletion. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that non-genetic information in the gametes is required for microbial-dependent phenotypic transmission. Accordingly, gene expression in early embryos derived from gametes from germ-free or T cell-deficient mice is strikingly and similarly altered. Our findings demonstrate that microbial- and immune-dependent regulation of non-genetic information in the gametes can transmit inherited phenotypes transgenerationally in mice. This mechanism could rapidly generate phenotypic diversity to enhance host adaptation to environmental perturbations.

Phosphoproteomic analysis of the adaption of epididymal epithelial cells to corticosterone challenge.

BACKGROUND: The epididymis has long been of interest owing to its role in promoting the functional maturation of the male germline. More recent evidence has also implicated the epididymis as an important sensory tissue responsible for remodeling of the sperm epigenome, both under physiological conditions and in response to diverse forms of environmental stress. Despite this knowledge, the intricacies of the molecular pathways involved in regulating the adaptation of epididymal tissue to paternal stressors remains to be fully resolved. OBJECTIVE: The overall objective of this study was to investigate the direct impact of corticosterone challenge on a tractable epididymal epithelial cell line (i.e., mECap18 cells), in terms of driving adaptation of the cellular proteome and phosphoproteome signaling networks. MATERIALS AND METHODS: The newly developed phosphoproteomic platform EasyPhos coupled with sequencing via an Orbitrap Exploris 480 mass spectrometer, was applied to survey global changes in the mECap18 cell (phospho)proteome resulting from sub-chronic (10-day) corticosterone challenge. RESULTS: The imposed corticosterone exposure regimen elicited relatively subtle modifications of the global mECap18 proteome (i.e., only 73 out of 4171 [∼1.8%] proteins displayed altered abundance). By contrast, ∼15% of the mECap18 phosphoproteome was substantially altered following corticosterone challenge. In silico analysis of the corresponding parent proteins revealed an activation of pathways linked to DNA damage repair and oxidative stress responses as well as a reciprocal inhibition of pathways associated with organismal death. Corticosterone challenge also induced the phosphorylation of several proteins linked to the biogenesis of microRNAs. Accordingly, orthogonal validation strategies confirmed an increase in DNA damage, which was ameliorated upon selective kinase inhibition, and an altered abundance profile of a subset of microRNAs in corticosterone-treated cells. CONCLUSIONS: Together, these data confirm that epididymal epithelial cells are reactive to corticosterone challenge, and that their response is tightly coupled to the opposing action of cellular kinases and phosphatases.

A ligation-independent sequencing method reveals tRNA-derived RNAs with blocked 3' termini.

Despite the numerous sequencing methods available, the vast diversity in size and chemical modifications of RNA molecules makes the capture of the full spectrum of cellular RNAs a difficult task. By combining quasi-random hexamer priming with a custom template switching strategy, we developed a method to construct sequencing libraries from RNA molecules of any length and with any type of 3' terminal modification, allowing the sequencing and analysis of virtually all RNA species. Ligation-independent detection of all types of RNA (LIDAR) is a simple, effective tool to comprehensively characterize changes in small non-coding RNAs and mRNAs simultaneously, with performance comparable to separate dedicated methods. With LIDAR, we comprehensively characterized the coding and non-coding transcriptome of mouse embryonic stem cells, neural progenitor cells, and sperm. LIDAR detected a much larger variety of tRNA-derived RNAs (tDRs) compared to traditional ligation-dependent sequencing methods, and uncovered the presence of tDRs with blocked 3' ends that had previously escaped detection. Our findings highlight the potential of LIDAR to systematically detect all RNAs in a sample and uncover new RNA species with potential regulatory functions.

The microbiota and immune system non-genetically affect offspring phenotypes transgenerationally.

The host-microbiota relationship has evolved to shape mammalian processes, including immunity, metabolism, and development 1-3 . Host phenotypes change in direct response to microbial exposures by the individual. Here we show that the microbiota induces phenotypic change not only in the individual but also in their succeeding generations of progeny. We found that germ-free mice exhibit a robust sebum secretion defect and transcriptional changes in various organs, persisting across multiple generations despite microbial colonization and breeding with conventional mice. Host-microbe interactions could be involved in this process, since T cell-deficient mice, which display defective sebum secretion 4 , also transgenerationally transmit their phenotype to progeny. These phenotypes are inherited by progeny conceived during in vitro fertilization using germ-free sperm and eggs, demonstrating that epigenetic information in the gametes is required for phenotypic transmission. Accordingly, small non-coding RNAs that can regulate embryonic gene expression 5 were strikingly and similarly altered in gametes of germ-free and T cell-deficient mice. Thus, we have uncovered a novel mechanism whereby the microbiota and immune system induce phenotypic changes in successive generations of offspring. This epigenetic form of inheritance could be advantageous for host adaptation to environmental perturbation, where phenotypic diversity can be introduced more rapidly than by genetic mutation.

Cloning and Sequencing Eukaryotic Small RNAs.

Small RNAs are ubiquitous regulators of gene expression that participate in nearly all aspects of physiology in a wide range of organisms. There are many different classes of eukaryotic small RNAs that play regulatory roles at every level of gene expression, including transcription, RNA stability, and translation. While eukaryotic small RNAs display diverse functions across and within classes, they are generally grouped functionally based on the machinery required for their biogenesis, the effector proteins they associate with, and their molecular characteristics. The development of techniques to clone and sequence small RNAs has been critical for their identification, yet the ligation-dependent addition of RNA adapters and the use of reverse transcriptase to generate cDNA in traditional library preparation protocols can be unsuitable to detect certain small RNA subtypes. In particular, 3' or 5' chemical modifications that are characteristic of specific types of small RNAs can impede the ligation-dependent addition of RNA adapters, while internal RNA modifications can interfere with accurate reverse transcription. The inability to clone certain small RNA subtypes with traditional protocols results in an inaccurate assessment of small RNA abundance and diversity, where some RNAs appear over-represented and others are not detected. This overview aims to guide users on how to design small RNA cloning workflows in eukaryotes to more accurately capture specific small RNAs of interest. Hence, we discuss the molecular biology underlying the identification and quantitation of small RNAs, explore the limitations of commonly used protocols, and detail the alternative approaches that can be used to enrich specific small RNA classes. © 2022 Wiley Periodicals LLC.

Quantitative proteomic dataset of mouse caput epididymal epithelial cells exposed to acrylamide in vivo.

This article reports the proteomic legacy of in vivo exposure to the xenobiotic, acrylamide, on the epithelial cell population of the proximal segments of the mouse epididymis. Specifically, adult male mice were administered acrylamide (25 mg/kg bw/day) or vehicle control for five consecutive days before dissection of the epididymis. Epididymal epithelial cells were isolated from the proximal (caput) epididymal segment and subjected to quantitative proteomic analysis using multiplexed tandem mass tag (TMT) labeling coupled to mass spectrometry. Here, we report the data generated by this strategy, including the identification of 4405 caput epididymal epithelial cell proteins, approximately 6.8% of which displayed altered expression in response to acrylamide challenge. Our interpretation and discussion of these data features in the article "Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development".

Roles of male reproductive tract extracellular vesicles in reproduction.

Extracellular vesicles (EVs) are secreted cell-derived membrane structures present in all organisms across animal, bacterial, and plant phyla. These vesicles play important roles in cell-cell communication in many processes integral to health and disease. Recent studies demonstrate that EVs and their cargo have influential and conserved roles in male reproduction. While EVs have been isolated from virtually all specialized tissues comprising the male reproductive tract, they are best characterized in the epididymis (epididymosomes) and seminal fluid (seminal fluid extracellular vesicles or prostasomes). Broadly speaking, EVs promote reproductive success through supporting sperm development and function, as well as influencing the physiology of female reproductive tract cells after mating. In this review, we present current knowledge on the composition and function of male reproductive tract EV populations in both normal physiology and pathology, and argue that their functions identify them as critical regulators of fertility and fecundity.

Acrylamide modulates the mouse epididymal proteome to drive alterations in the sperm small non-coding RNA profile and dysregulate embryo development.

Paternal exposure to environmental stressors elicits distinct changes to the sperm sncRNA profile, modifications that have significant post-fertilization consequences. Despite this knowledge, there remains limited mechanistic understanding of how paternal exposures modify the sperm sncRNA landscape. Here, we report the acute sensitivity of the sperm sncRNA profile to the reproductive toxicant acrylamide. Furthermore, we trace the differential accumulation of acrylamide-responsive sncRNAs to coincide with sperm transit of the proximal (caput) segment of the epididymis, wherein acrylamide exposure alters the abundance of several transcription factors implicated in the expression of acrylamide-sensitive sncRNAs. We also identify extracellular vesicles secreted from the caput epithelium in relaying altered sncRNA profiles to maturing spermatozoa and dysregulated gene expression during early embryonic development following fertilization by acrylamide-exposed spermatozoa. These data provide mechanistic links to account for how environmental insults can alter the sperm epigenome and compromise the transcriptomic profile of early embryos.

A novel approach to nonsurgical sterilization; application of menadione-modified gonocyte-targeting M13 bacteriophage for germ cell ablation in utero.

There remains a compelling need for the development of nonsurgical sterilizing agents to expand the fertility management options for both domestic and feral animal species. We hypothesize that an efficacious sterilization approach would be to selectively ablate nonrenewable cell types that are essential for reproduction, such as the undifferentiated gonocytes within the embryonic gonad. Here, we report a novel strategy to achieve this goal centered on the use of a chemically modified M13 bacteriophage to effect the targeted delivery of menadione, a redox-cycling naphthoquinone, to mouse gonocytes. Panning of the M13 random peptide 'phage display library proved effective in the isolation of gonocyte-specific targeting clones. One such clone was modified via N-succinimidyl-S-acetylthioacetate (SATA) linkage to the N-terminus of the major PVIII capsid protein. Subsequent deacetylation of the SATA was undertaken to expose a thiol group capable of reacting with menadione through Michael addition. This chemical modification was confirmed using UV spectrophotometry. In proof-of-concept experiments we applied the modified 'phage to primary cultures of fetal germ cells and induced, an approximately, 60% reduction in the viability of the target cell population. These studies pave the way for in vivo application of chemically modified M13 bacteriophage in order to achieve the selective ablation of nonrenewable cell types in the reproductive system, thereby providing a novel nonsurgical approach the regulation of fertility in target species.

A Kinase Anchor Protein 4 Is Vulnerable to Oxidative Adduction in Male Germ Cells.

Oxidative stress is a leading causative agent in the defective sperm function associated with male infertility. Such stress commonly manifests via the accumulation of pathological levels of the electrophilic aldehyde, 4-hydroxynonenal (4HNE), generated as a result of lipid peroxidation. This highly reactive lipid aldehyde elicits a spectrum of cytotoxic lesions owing to its propensity to form stable adducts with biomolecules. Notably however, not all elements of the sperm proteome appear to display an equivalent vulnerability to 4HNE modification, with only a small number of putative targets having been identified to date. Here, we validate one such target of 4HNE adduction, A-Kinase Anchor Protein 4 (AKAP4); a major component of the sperm fibrous sheath responsible for regulating the signal transduction and metabolic pathways that support sperm motility and capacitation. Our data confirm that both the precursor (proAKAP4), and mature form of AKAP4, are conserved targets of 4HNE adduction in primary cultures of post-meiotic male germ cells (round spermatids) and in mature mouse and human spermatozoa. We further demonstrate that 4HNE treatment of round spermatids and mature spermatozoa results in a substantial reduction in the levels of both proAKAP4 and AKAP4 proteins. This response proved refractory to pharmacological inhibition of proteolysis, but coincided with an apparent increase in the degree of protein aggregation. Further, we demonstrate that 4HNE-mediated protein degradation and/or aggregation culminates in reduced levels of capacitation-associated phosphorylation in mature human spermatozoa, possibly due to dysregulation of the signaling framework assembled around the AKAP4 scaffold. Together, these findings suggest that AKAP4 plays an important role in the pathophysiological responses to 4HNE, thus strengthening the importance of AKAP4 as a biomarker of sperm quality, and providing the impetus for the design of an efficacious antioxidant-based intervention strategy to alleviate sperm dysfunction.