Proteomic and phosphoproteomic characterisation of primary mouse embryonic fibroblasts.

Fibroblasts are the most common cell type in stroma and function in the support and repair of most tissues. Mouse embryonic fibroblasts (MEFs) are amenable to isolation and rapid growth in culture. MEFs are therefore widely used as a standard model for functional characterisation of gene knockouts, and can also be used in co-cultures, commonly to support embryonic stem cell cultures. To facilitate their use as a research tool, we have performed a comprehensive proteomic and phosphoproteomic characterisation of wild-type primary MEFs from C57BL/6 mice. EIF2/4 and MTOR signalling pathways were abundant in both the proteome and phosphoproteome, along with extracellular matrix (ECM) and cytoskeleton associated pathways. Consistent with this, kinase enrichment analysis identified activation of P38A, P90RSK, P70S6K, and MTOR. Cell surface markers and matrisome proteins were also annotated. Data are available via ProteomeXchange with identifier PXD043244. This provides a comprehensive catalogue of the wild-type MEF proteome and phosphoproteome which can be utilised by the field to guide future work.

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.

Oocyte and cumulus cell cooperativity and metabolic plasticity under the direction of oocyte paracrine factors.

Mammalian oocytes develop and mature in a mutually dependent relationship with surrounding cumulus cells. The oocyte actively regulates cumulus cell differentiation and function by secreting soluble paracrine oocyte-secreted factors (OSFs). We characterized the molecular mechanisms by which two model OSFs, cumulin and BMP15, regulate oocyte maturation and cumulus-oocyte cooperativity. Exposure to these OSFs during mouse oocyte maturation in vitro altered the proteomic and multispectral autofluorescence profiles of both the oocyte and cumulus cells. In oocytes, cumulin significantly upregulated proteins involved in nuclear function. In cumulus cells, both OSFs elicited marked upregulation of a variety of metabolic processes (mostly anabolic), including lipid, nucleotide, and carbohydrate metabolism, whereas mitochondrial metabolic processes were downregulated. The mitochondrial changes were validated by functional assays confirming altered mitochondrial morphology, respiration, and content while maintaining ATP homeostasis. Collectively, these data demonstrate that cumulin and BMP15 remodel cumulus cell metabolism, instructing them to upregulate their anabolic metabolic processes, while routine cellular functions are minimized in the oocyte during maturation, in preparation for ensuing embryonic development.NEW & NOTEWORTHY Oocyte-secreted factors (OSFs) promote oocyte and cumulus cell cooperativity by altering the molecular composition of both cell types. OSFs downregulate protein catabolic processes and upregulate processes associated with DNA binding, translation, and ribosome assembly in oocytes. In cumulus cells, OSFs alter mitochondrial number, morphology, and function, and enhance metabolic plasticity by upregulating anabolic pathways. Hence, the oocyte via OSFs, instructs cumulus cells to increase metabolic processes on its behalf, thereby subduing oocyte metabolism.

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.

Elucidation of the protein composition of mouse seminal vesicle fluid.

The seminal vesicles are male accessory sex glands that contribute the major portion of the seminal plasma in which mammalian spermatozoa are bathed during ejaculation. In addition to conveying sperm through the ejaculatory duct, seminal vesicle secretions support sperm survival after ejaculation, and influence the female reproductive tract to promote receptivity to pregnancy. Analysis of seminal vesicle fluid (SVF) composition by proteomics has proven challenging, due to its highly biased protein signature with a small subset of dominant proteins and the difficulty of solubilizing this viscous fluid. As such, publicly available proteomic datasets identify only 85 SVF proteins in total. To address this limitation, we report a new preparative methodology involving sequential solubilization of mouse SVF in guanidine hydrochloride, acetone precipitation, and analysis by label-free mass spectrometry. Using this strategy, we identified 126 SVF proteins, including 83 previously undetected in SVF. Members of the seminal vesicle secretory protein family were the most abundant, accounting for 79% of all peptide spectrum matches. Functional analysis identified inflammation and formation of the vaginal plug as the two most prominent biological processes. Other notable processes included modulation of sperm function and regulation of the female reproductive tract immune environment. Together, these findings provide a robust methodological framework for future SVF studies and identify novel proteins with potential to influence both male and female reproductive physiology.

Proteomic analysis of spermatozoa reveals caseins play a pivotal role in preventing short-term periods of subfertility in stallions†.

Stallions experience transient fluctuations in fertility throughout the breeding season. Considering pregnancy diagnoses cannot be ascertained until ~14 days postbreeding, the timely detection of decreases in stallion fertility would enhance industry economic and welfare outcomes. Therefore, this study aimed to identify the proteomic signatures reflective of short-term fertility fluctuations and to determine the biological mechanisms governing such differences. Using liquid chromatography-mass spectrometry (LC-MS/MS), we compared the proteomic profile of semen samples collected from commercially "fertile" stallions, during high- and low-fertility periods. A total of 1702 proteins were identified, of which, 38 showed a significant change in abundance (P ≤ 0.05). Assessment of intra- and interstallion variability revealed that caseins (namely κ-, α-S1-, and α-S2-casein) were significantly more abundant during "high-fertility" periods, while several epididymal, and seminal plasma proteins (chiefly, epididymal sperm binding protein 1 [ELSPbP1], horse seminal plasma protein 1 [HSP-1], and clusterin), were significantly more abundant during "low-fertility" periods. We hypothesized that an increased abundance of caseins offers greater protection from potentially harmful seminal plasma proteins, thereby preserving cell functionality and fertility. In vitro exposure of spermatozoa to casein resulted in decreased levels of lipid scrambling (Merocyanine 540), higher abundance of sperm-bound caseins (α-S1-, α-S2-, and κ-casein), and lower abundance of sperm-bound HSP-1 (P ≤ 0.05). This study demonstrates key pathways governing short-term fertility fluctuations in the stallion, thereby providing a platform to develop robust, fertility assessment strategies into the future.

Phospho-heavy-labeled-spiketide FAIMS stepped-CV DDA (pHASED) provides real-time phosphoproteomics data to aid in cancer drug selection.

Global high-throughput phosphoproteomic profiling is increasingly being applied to cancer specimens to identify the oncogenic signaling cascades responsible for promoting disease initiation and disease progression; pathways that are often invisible to genomics analysis. Hence, phosphoproteomic profiling has enormous potential to inform and improve individualized anti-cancer treatment strategies. However, to achieve the adequate phosphoproteomic depth and coverage necessary to identify the activated, and hence, targetable kinases responsible for driving oncogenic signaling pathways, affinity phosphopeptide enrichment techniques are required and often coupled with offline high-pressure liquid chromatographic (HPLC) separation prior to nanoflow liquid chromatography-tandem mass spectrometry (nLC-MS/MS). These complex and time-consuming procedures, limit the utility of phosphoproteomics for the analysis of individual cancer patient specimens in real-time, and restrict phosphoproteomics to specialized laboratories often outside of the clinical setting. To address these limitations, here we have optimized a new protocol, phospho-heavy-labeled-spiketide FAIMS Stepped-CV DDA (pHASED), that employs online phosphoproteome deconvolution using high-field asymmetric waveform ion mobility spectrometry (FAIMS) and internal phosphopeptide standards to provide accurate label-free quantitation (LFQ) data in real-time. Compared with traditional single-shot LFQ phosphoproteomics workflows, pHASED provided increased phosphoproteomic depth and coverage (phosphopeptides = 4617 pHASED, 2789 LFQ), whilst eliminating the variability associated with offline prefractionation. pHASED was optimized using tyrosine kinase inhibitor (sorafenib) resistant isogenic FLT3-mutant acute myeloid leukemia (AML) cell line models. Bioinformatic analysis identified differential activation of the serine/threonine protein kinase ataxia-telangiectasia mutated (ATM) pathway, responsible for sensing and repairing DNA damage in sorafenib-resistant AML cell line models, thereby uncovering a potential therapeutic opportunity. Herein, we have optimized a rapid, reproducible, and flexible protocol for the characterization of complex cancer phosphoproteomes in real-time, a step towards the implementation of phosphoproteomics in the clinic to aid in the selection of anti-cancer therapies for patients.

Reproductive biology research down under: highlights from the Australian and New Zealand Annual Meeting of the Society for Reproductive Biology, 2021.

Against the backdrop of a global pandemic, the Society for Reproductive Biology (SRB) 2021 meeting reunited the Australian and New Zealand reproductive research community for the first time since 2019 and was the first virtual SRB meeting. Despite the recent global research disruptions, the conference revealed significant advancements in reproductive research, the importance of which span human health, agriculture, and conservation. A core theme was novel technologies, including the use of medical microrobots for therapeutic and sperm delivery, diagnostic hyperspectral imaging, and hydrogel condoms with potential beyond contraception. The importance of challenging the contraceptive status quo was further highlighted with innovations in gene therapies, non-hormonal female contraceptives, epigenetic semen analysis, and in applying evolutionary theory to suppress pest population reproduction. How best to support pregnancies, particularly in the context of global trends of increasing maternal age, was also discussed, with several promising therapies for improved outcomes in assisted reproductive technology, pre-eclampsia, and pre-term birth prevention. The unique insights gained via non-model species was another key focus and presented research emphasised the importance of studying diverse systems to understand fundamental aspects of reproductive biology and evolution. Finally, the meeting highlighted how to effectively translate reproductive research into policy and industry practice.

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.

Proteomic analysis of koala (phascolarctos cinereus) spermatozoa and prostatic bodies.

The aims of this study were to investigate the proteome of koala spermatozoa and that of the prostatic bodies with which they interact during ejaculation. For this purpose, spermatozoa and prostatic bodies were fractionated from the semen of four male koalas and analysed by HPLC MS/MS. This strategy identified 744 sperm and 1297 prostatic body proteins, which were subsequently attributed to 482 and 776 unique gene products, respectively. Gene ontology curation of the sperm proteome revealed an abundance of proteins mapping to the canonical sirtuin and 14-3-3 signalling pathways. By contrast, protein ubiquitination and unfolded protein response pathways dominated the equivalent analysis of proteins uniquely identified in prostatic bodies. Koala sperm proteins featured an enrichment of those mapping to the functional categories of cellular compromise/inflammatory response, whilst those of the prostatic body revealed an over-representation of molecular chaperone and stress-related proteins. Cross-species comparisons demonstrated that the koala sperm proteome displays greater conservation with that of eutherians (human; 93%) as opposed to reptile (crocodile; 39%) and avian (rooster; 27%) spermatozoa. Together, this work contributes to our overall understanding of the core sperm proteome and has identified biomarkers that may contribute to the exceptional longevity of koala spermatozoa during ex vivo storage.

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.

Platelet activating factor receptor acts to limit colitis-induced liver inflammation.

Liver inflammation is a common extraintestinal manifestation in inflammatory bowel disease (IBD), yet, the mechanisms driving gut-liver axis inflammation remain poorly understood. IBD leads to a breakdown in the integrity of the intestinal barrier causing an increase in portal and systemic gut-derived antigens, which challenge the liver. Here, we examined the role of platelet activating factor receptor (PAFR) in colitis-associated liver damage using dextran sulfate sodium (DSS) and anti-CD40-induced colitis models. Both DSS and anti-CD40 models exhibited liver inflammation associated with colitis. Colitis reduced global PAFR protein expression in mouse livers causing an exclusive re-localization of PAFR to the portal triad. The global decrease in liver PAFR was associated with increased sirtuin 1 while relocalized PAFR expression was limited to Kupffer cells (KCs) and co-localized with toll-like receptor 4. DSS activated the NLRP3-inflammasome and increased interleukin (IL)-1ß in the liver. Antagonism of PAFR amplified the inflammasome response by increasing NLRP3, caspase-1, and IL-1ß protein levels in the liver. LPS also increased NLRP3 response in human hepatocytes, however, overexpression of PAFR restored the levels of NLPR3 and caspase-1 proteins. Interestingly, KCs depletion also increased IL-1ß protein in mouse liver after DSS challenge. These data suggest a protective role for PAFR-expressing KCs during colitis and that regulation of PAFR is important for gut-liver axis homeostasis.

Post-testicular sperm maturation in the saltwater crocodile Crocodylus porosus: assessing the temporal acquisition of sperm motility.

Conservation efforts to secure the long-term survival of crocodilian species would benefit from the establishment of a frozen sperm bank in concert with artificial breeding technologies to maintain genetic diversity among captive assurance populations. Working towards this goal, our research has focused on the saltwater crocodile Crocodylus porosus as a tractable model for understanding crocodilian sperm physiology. In extending our systematic characterisation of saltwater crocodile spermatozoa, in this study we examined the development of motility during sperm transport through the excurrent duct system of the male crocodile. The results show that approximately 20% of crocodile testicular spermatozoa are immediately motile but experience a gradient of increasing motility (percentage motile and rate of movement) as they transit the male reproductive tract (epididymis). Moreover, we confirmed that, as in ejaculated crocodile spermatozoa, increased intracellular cAMP levels promoted a significant and sustained enhancement of sperm motility regardless of whether the cells were isolated from the testis or epididymis. Along with the development of artificial reproductive technologies, this research paves the way for the opportunistic recovery, storage and potential utilisation of post-mortem spermatozoa from genetically valuable animals.

Gross and microanatomy of the male reproductive duct system of the saltwater crocodile Crocodylus porosus.

Information on the morphology and histology of the male reproductive system of the Crocodylia species is necessary to determine the role of these tissues in the production of functional spermatozoa. Accordingly, in this study we examined the gross morphology and microanatomy of the testis and the male excurrent duct system through which spermatozoa pass before ejaculation. The data demonstrate that the reproductive system in male saltwater crocodiles comprises paired testes, which convey spermatozoa distally via the rete testis into an excurrent duct system comprising ductuli efferentes, ductuli epididymides, ductus epididymidis and ductus deferens. The epithelium delineating the male tract was dominated by non-ciliated and ciliated cells structured into a simple columnar lining of the ductuli efferentes and ductuli epididymides, through to the high pseudostratified columnar epithelium of the ductus epididymidis and ductus deferens. The morphology and histochemical staining of these ducts suggest their involvement in seminal fluid production and/or its modification, which likely contributes to the nourishment, protection and/or storage of crocodile spermatozoa. As a reflection of their common Archosaurs ancestry, the overall structural characteristics we describe for the crocodile male excurrent duct system share closer similarities to those of the Aves than other clades within the Reptilia class or Mammalia.

Time-resolved proteomic profiling of cigarette smoke-induced experimental chronic obstructive pulmonary disease.

BACKGROUND AND OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is the third leading cause of illness and death worldwide. Current treatments aim to control symptoms with none able to reverse disease or stop its progression. We explored the major molecular changes in COPD pathogenesis. METHODS: We employed quantitative label-based proteomics to map the changes in the lung tissue proteome of cigarette smoke-induced experimental COPD that is induced over 8 weeks and progresses over 12 weeks. RESULTS: Quantification of 7324 proteins enabled the tracking of changes to the proteome. Alterations in protein expression profiles occurred in the induction phase, with 18 and 16 protein changes at 4- and 6-week time points, compared to age-matched controls, respectively. Strikingly, 269 proteins had altered expression after 8 weeks when the hallmark pathological features of human COPD emerge, but this dropped to 27 changes at 12 weeks with disease progression. Differentially expressed proteins were validated using other mouse and human COPD bronchial biopsy samples. Major changes in RNA biosynthesis (heterogeneous nuclear ribonucleoproteins C1/C2 [HNRNPC] and RNA-binding protein Musashi homologue 2 [MSI2]) and modulators of inflammatory responses (S100A1) were notable. Mitochondrial dysfunction and changes in oxidative stress proteins also occurred. CONCLUSION: We provide a detailed proteomic profile, identifying proteins associated with the pathogenesis and disease progression of COPD establishing a platform to develop effective new treatment strategies.

Modification of Crocodile Spermatozoa Refutes the Tenet That Post-testicular Sperm Maturation Is Restricted To Mammals.

Competition to achieve paternity has contributed to the development of a multitude of elaborate male reproductive strategies. In one of the most well-studied examples, the spermatozoa of all mammalian species must undergo a series of physiological changes, termed capacitation, in the female reproductive tract before realizing their potential to fertilize an ovum. However, the evolutionary origin and adaptive advantage afforded by capacitation remains obscure. Here, we report the use of comparative and quantitative proteomics to explore the biological significance of capacitation in an ancient reptilian species, the Australian saltwater crocodile (Crocodylus porosus,). Our data reveal that exposure of crocodile spermatozoa to capacitation stimuli elicits a cascade of physiological responses that are analogous to those implicated in the functional activation of their mammalian counterparts. Indeed, among a total of 1119 proteins identified in this study, we detected 126 that were differentially phosphorylated (± 1.2 fold-change) in capacitated versus, noncapacitated crocodile spermatozoa. Notably, this subset of phosphorylated proteins shared substantial evolutionary overlap with those documented in mammalian spermatozoa, and included key elements of signal transduction, metabolic and cellular remodeling pathways. Unlike mammalian sperm, however, we noted a distinct bias for differential phosphorylation of serine (as opposed to tyrosine) residues, with this amino acid featuring as the target for ∼80% of all changes detected in capacitated spermatozoa. Overall, these results indicate that the phenomenon of sperm capacitation is unlikely to be restricted to mammals and provide a framework for understanding the molecular changes in sperm physiology necessary for fertilization.

Proteomic Profiling of Mouse Epididymosomes Reveals their Contributions to Post-testicular Sperm Maturation.

The functional maturation of spermatozoa that is necessary to achieve fertilization occurs as these cells transit through the epididymis, a highly specialized region of the male reproductive tract. A defining feature of this maturation process is that it occurs in the complete absence of nuclear gene transcription or de novo, protein translation in the spermatozoa. Rather, it is driven by sequential interactions between spermatozoa and the complex external milieu in which they are bathed within lumen of the epididymal tubule. A feature of this dynamic microenvironment are epididymosomes, small membrane encapsulated vesicles that are secreted from the epididymal soma. Herein, we report comparative proteomic profiling of epididymosomes isolated from different segments of the mouse epididymis using multiplexed tandem mass tag (TMT) based quantification coupled with high resolution LC-MS/MS. A total of 1640 epididymosome proteins were identified and quantified via this proteomic method. Notably, this analysis revealed pronounced segment-to-segment variation in the encapsulated epididymosome proteome. Thus, 146 proteins were identified as being differentially accumulated between caput and corpus epididymosomes, and a further 344 were differentially accumulated between corpus and cauda epididymosomes (i.e., fold change of ≤ -1.5 or ≥ 1.5; p, < 0.05). Application of gene ontology annotation revealed a substantial portion of the epididymosome proteins mapped to the cellular component of extracellular exosome and to the biological processes of transport, oxidation-reduction, and metabolism. Additional annotation of the subset of epididymosome proteins that have not previously been identified in exosomes revealed enrichment of categories associated with the acquisition of sperm function (e.g., fertilization and binding to the zona pellucida). In tandem with our demonstration that epididymosomes are able to convey protein cargo to the head of maturing spermatozoa, these data emphasize the fundamental importance of epididymosomes as key elements of the epididymal microenvironment responsible for coordinating post-testicular sperm maturation.

Mass spectrometry reveals distinct proteomic profiles in high- and low-quality stallion spermatozoa.

The horse breeding industry relies upon optimal stallion fertility. Conventional sperm assessments provide limited information regarding ejaculate quality and are not individually predictive of fertilizing potential. The aim of this study was to harness mass spectrometry to compare the proteomic profiles of high- and low-quality stallion spermatozoa, with the ultimate goal of identifying fertility biomarker candidates. Extended stallion semen (n = 12) was fractionated using Percoll density gradients to isolate low-quality and high-quality sperm populations. Motility and morphological assessments were carried out, and proteomic analyses was conducted using UHPLC-MS/MS. High-quality spermatozoa recorded higher total (95.2 ± 0.52% vs 70.6 ± 4.20%; P ≤ 0.001) and progressive motilities (43.4 ± 3.42% vs 27.3 ± 4.32%; P ≤ 0.05), and a higher proportion of morphologically normal cells (50.2 ± 4.34% vs 38.8 ± 2.72%; P ≤ 0.05). In total, 1069 proteins were quantified by UHPLC-MS/MS, of which 22 proteins were significantly more abundant in the high-quality sperm population (P ≤ 0.05). A-kinase anchor protein 4 (AKAP4) and Hexokinase 1 (HK1) were considered possible biomarker candidates and their differential expression was confirmed by immunoblot. Protein expression was significantly correlated with total (AKAP4 R2 = 0.38, P ≤ 0.01; HK1 R2 = 0.46, P ≤ 0.001) and progressive motilities (AKAP4 R 2 = 0.51, P ≤ 0.001; HK1 R2 = 0.55, P ≤ 0.01), percentage rapid (AKAP4 R2 = 0.29, P ≤ 0.05; HK1 R2 = 0.58, P ≤ 0.001), straight-line velocity (HK1 R2 = 0.50, P ≤ 0.01) and straightness (HK1 R2 = 0.40, P ≤ 0.01). Furthermore, AKAP4 was highly susceptible to adduction by 4-hydroxynonenal (4HNE), which resulted in a global reduction in the phosphorylation profiles following capacitation. In conclusion, the proteomic profiles of high- and low-quality stallion spermatozoa differ substantially, and proteins such as AKAP4 and HK1 could serve as biomarkers of ejaculate quality.

Building an Understanding of Proteostasis in Reproductive Cells: The Impact of Reactive Carbonyl Species on Protein Fate.

Significance: Stringent regulation of protein homeostasis pathways, under both physiological and pathological conditions, is necessary for the maintenance of proteome fidelity and optimal cell functioning. However, when challenged by endogenous or exogenous stressors, these proteostasis pathways can become dysregulated with detrimental consequences for protein fate, cell survival, and overall organism health. Most notably, there are numerous somatic pathologies associated with a loss of proteostatic regulation, including neurodegenerative disorders, type 2 diabetes, and some cancers. Recent Advances: Lipid oxidation-derived reactive carbonyl species (RCS), such as 4-hydroxynonenal (4HNE) and malondialdehyde, are relatively underappreciated purveyors of proteostatic dysregulation, which elicit their effects via the nonenzymatic post-translational modification of proteins. Emerging evidence suggests that a subset of germline proteins can serve as substrates for 4HNE modification. Among these, prevalent targets include succinate dehydrogenase, heat shock protein A2 and A-kinase anchor protein 4, all of which are intrinsically associated with fertility. Critical Issues: Despite growing knowledge in this field, the RCS adductomes of spermatozoa and oocytes are yet to be comprehensively investigated. Furthermore, the manner by which RCS-mediated adduction impacts protein fate and drives cellular responses, such as protein aggregation, requires further examination in the germline. Given that RCS-protein adduction has been attributed a role in infertility, there has been sparked research investment into strategies to prevent lipid peroxidation in germ cells. Future Directions: An increased depth of knowledge regarding the mechanisms and substrates of RCS-mediated protein modification in reproductive cells may reveal important targets for the development of novel therapies to improve fertility and pregnancy outcomes for future generations.