Proteomic profiling of cryopreserved Trichormus variabilis using various cryoprotectants.

Algae, which may be unicellular or multicellular, can carry out photosynthesis just like plants as they effectively utilize light energy. They contain various physiologically active substances and are, therefore, widely used commercially to produce healthy food and feed additives, cosmetics, and energy supplements. For useful applications, the cryopreservation technique has been used in various fields. Recently, to develop suitable cryopreservation methods for algal applications, various studies have been performed. However, adequate investigations have not been conducted to understand the mechanism underlying algal cryopreservation at the molecular level. Therefore, this study examined the profile alteration of the proteome using cryopreservation with various cryoprotectants (CPAs). Trichormus variabilis was cultured and then cryopreserved with 10% dimethyl sulfoxide, methanol, and glycerol, after which, proteome profiling was done. Finally, signaling pathway search was performed, and a new signaling pathway was established based on differentially expressed proteins. As a result, the expression levels of 17 proteins were observed. Additionally, it was confirmed that the differentially expressed proteins were related to 16 signaling pathways and that they were capable of interacting with each other. The findings suggest that the differentially expressed proteins may be applied as biomarkers for algal cryopreservation and to understand the mechanism underlying T. variabilis cryopreservation. Moreover, it is anticipated that the results from this study would be useful in selecting suitable CPAs and in upgrading the cryopreservation techniques.

Change of Ruminoreticular Temperature and Body Activity before and after Parturition in Hanwoo (Bos taurus coreanae) Cows.

How do body temperature and activity change before and after parturition in pregnant cows? Changes in body temperature such as ruminal, rectal, and vaginal temperature during the parturition have been reported, but there are no results of the simultaneous observation of body temperature and activity. The aim of this study was to simultaneously confirm changes in the ruminoreticular temperature and body activity before and after parturition using the ruminoreticular bio-capsule sensor every 1 h. The 55 pregnant cows were used for the experiment, the ruminoreticular bio-capsule sensor was inserted and stabilized, and the ruminoreticular temperature and body activity were measured. The ruminoreticular temperature was lower by 0.5° from -24 h to -3 h in parturition compared to 48 h before parturition and then recovered again after parturition. Body activity increased temporarily at the time of parturition and 12 h after parturition. Therefore, the ruminoreticular temperature and body activity before and after parturition was simultaneously confirmed in pregnant cows.

Functional and Proteomic Alterations of F1 Capacitated Spermatozoa of Adult Mice Following Gestational Exposure to Bisphenol A.

Studies regarding bisphenol A (BPA) exposure and male (in)fertility have conventionally focused on modifications in ejaculated spermatozoa function from exposed individuals. However, mammalian spermatozoa are incapable of fertilization prior to achieving capacitation, the penultimate step in maturation. Therefore, it is necessary to investigate BPA-induced changes in capacitated spermatozoa and assess the consequences on subsequent fertilization. Here, we demonstrate the effect of gestational BPA exposure (50 µg/kg bw/day, 5 mg/kg bw/day, and 50 mg/kg bw/day) on the functions, biochemical properties, and proteomic profiles of F1 capacitated spermatozoa from adult mice. The data showed that high concentrations of BPA inhibited motility, motion kinematics, and capacitation of spermatozoa, perhaps because of increased lipid peroxidation and protein tyrosine nitration, and decreased intracellular ATP levels and protein kinase-A activity in spermatozoa. We also found that BPA compromised the rates of fertilization and early embryonic development. Differentially expressed proteins identified between BPA-exposed and control groups play a critical role in energy metabolism, stress responses, and fertility. Protein function abnormalities were responsible for the development of several diseases according to bioinformatics analysis. On the basis of these results, gestational exposure to BPA may alter capacitated spermatozoa function and the proteomic profile, ultimately affecting their fertility potential.

Applications of capacitation status for litter size enhancement in various pig breeds.

OBJECTIVE: Several studies have reported the development of new molecular methods for the prognosis and diagnosis of male fertility based on biomarkers aimed at overcoming the limitations of conventional male fertility analysis tools. However, further studies are needed for the field application of these methods. Therefore, alternative methods based on existing semen analysis methods are required to improve production efficiency in the animal industry. METHODS: we examined the possibility of improving litter size in various pig breeds using combined Hoechst 33258/chlortetracycline fluorescence (H33258/CTC) staining. The correlation between field fertility and capacitation status by combined H33258/CTC staining in different ejaculates spermatozoa (n = 3) from an individual boar (20 Landrace, 20 Yorkshire, and 20 Duroc) was evaluated as well as overall accuracy. RESULTS: The acrosome reacted (AR) pattern after capacitation (%) was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 75%, 75%, and 70% in Landrace, Yorkshire, and Duroc pigs, respectively. The difference (Δ) in AR pattern before and after capacitation was positively correlated with the litter size of Landrace, Yorkshire, and Duroc pigs and the overall accuracy was 80%, 65%, and 55% in Landrace, Yorkshire, and Duroc pigs, respectively. However, the difference (Δ) in capacitated (B) pattern before and after capacitation was negatively correlated with the litter size of Landrace pigs and the overall accuracy was 75%. Moreover, average litter size was significantly altered according to different combined H33258/CTC staining parameters. CONCLUSION: These results show that combined H33258/CTC staining may be used to predict male fertility in various breeds. However, the selection of specific efficiency combined H33258/CTC staining parameters requires further consideration. Taken together, these findings suggest that combined H33258/CTC staining may constitute an alternative method for predicting male fertility until such time as fertility-related biomarkers are further validated.

Sex chromosome-dependent differential viability of human spermatozoa during prolonged incubation.

STUDY QUESTION: Are there significant differences in the ability of X chromosome-bearing (X) spermatozoa and Y chromosome-bearing (Y) spermatozoa to survive incubation under stressful conditions? SUMMARY ANSWER: Y spermatozoa are more vulnerable to stress than their X counterparts depending on culture period and temperature, and show higher expression of apoptotic proteins. WHAT IS KNOWN ALREADY: The primary sex ratio is determined by there being an equal number of spermatozoa carrying X and Y chromosomes. This balance can be skewed by exposure to stressful environmental conditions such as changes in pH, pollutants or endocrine disruptors. However, less is known about the ability of sperm carrying either sex chromosome to withstand environmental stress. STUDY DESIGN, SIZE, DURATION: The difference in survival between X and Y spermatozoa was evaluated by measuring motility, viability and Y:X chromosome ratio during incubation for 5 days, at three temperatures (4, 22 and 37°C), and three pH conditions (6.5, 7.5 and 8.5). To identify the critical factors that determine the survival of X and Y bearing spermatozoa, we analysed the expression levels of apoptosis-related proteins (Bcl, Bax and Caspase-3), as well as the extent of DNA damage under a subset of conditions. PARTICIPANTS/MATERIALS, SETTING, METHODS: Semen samples were obtained by masturbation from normozoospermic donors after 3 days of sexual abstinence. Four samples with >60% motility from different donors were mixed to obtain sufficient semen and eliminate sampling-related bias. Data are presented as mean ± SD of three independent experiments. Mean age of donors was 28.7 ± 3.2 years. MAIN RESULTS AND THE ROLE OF CHANCE: In total, 58 489 spermatozoa were scored. The viability of Y spermatozoa was lower after exposure to different temperatures and culture periods than that of X spermatozoa (P < 0.05). Increased expression of apoptotic proteins in live Y spermatozoa was observed, despite the addition of tocopherol to the culture medium (P < 0.05). LIMITATIONS, REASONS FOR CAUTION: Spermatozoa were cultured in vitro during the treatment period. It is difficult to extrapolate the observed lifespan differences to spermatozoa survival in vivo. The experiments were replicated only three times. WIDER IMPLICATIONS OF THE FINDINGS: The prolonged survival of X spermatozoa under stressful conditions might lead to shifts in the ratio of male-to-female births. STUDY FUNDING/COMPETING INTEREST(S): This study was supported by the National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST) (no. NRF-2014R1A2A2A01002706). The authors declare no competing financial interests.

Prediction of male fertility using capacitation-associated proteins in spermatozoa.

Infertility and subfertility account for huge economic losses in the animal industry; indeed, 50% of animal breeding failure is associated with male infertility. Approximately 70% of cattle and 90% of pig livestock are currently produced by artificial insemination. Therefore, breeding-male selection is extremely important for the genetic benefits of progeny. Although conventional semen analysis provides an initial measure of male fertility, its clinical value is questionable. Proteomics approaches recently identified candidate protein markers in spermatozoa for evaluating male fertility. Fertility-related proteins in capacitated boar spermatozoa were shown to predict boar fertility more precisely then those detected in ejaculated spermatozoa, which motivated the development of more accurate and sensitive tools for the assessment of male fertility in relation to sperm function and fertilization. Although protein markers in spermatozoa are capable of discriminating fertile and infertile males, clinical trials are required to validate their predictive utility. This review outlines recent findings regarding the capacitation-related proteome of spermatozoa, and discusses how these proteins may be utilized to better understand the fertility of domestic animals.

Discovery of predictive biomarkers for litter size in boar spermatozoa.

Conventional semen analysis has been used for prognosis and diagnosis of male fertility. Although this tool is essential for providing initial quantitative information about semen, it remains a subject of debate. Therefore, development of new methods for the prognosis and diagnosis of male fertility should be seriously considered for animal species of economic importance as well as for humans. In the present study, we applied a comprehensive proteomic approach to identify global protein biomarkers in boar spermatozoa in order to increase the precision of male fertility prognoses and diagnoses. We determined that l-amino acid oxidase, mitochondrial malate dehydrogenase 2, NAD (MDH2), cytosolic 5'-nucleotidase 1B, lysozyme-like protein 4, and calmodulin (CALM) were significantly and abundantly expressed in high-litter size spermatozoa. We also found that equatorin, spermadhesin AWN, triosephosphate isomerase (TPI), Ras-related protein Rab-2A (RAB2A), spermadhesin AQN-3, and NADH dehydrogenase [ubiquinone] iron-sulfur protein 2 (NDUFS2) were significantly and abundantly expressed in low-litter size spermatozoa (>3-fold). Moreover, RAB2A, TPI, and NDUFS2 were negatively correlated with litter size, whereas CALM and MDH2 were positively correlated. This study provides novel biomarkers for the prediction of male fertility. To the best of our knowledge, this is the first work that shows significantly increased litter size using male fertility biomarkers in a field trial. Moreover, these protein markers may provide new developmental tools for the selection of superior sires as well as for the prognosis and diagnosis of male fertility.

Proteomic Analysis of One-carbon Metabolism-related Marker in Liver of Rat Offspring.

Maternal food intake has a significant effect on the fetal environment, and an inadequate maternal diet may result in intrauterine growth restriction. Intrauterine growth restriction newborn rat pups nursed by normal diet-fed dams exhibited rapid catch-up growth, which plays a critical role in the risk for metabolic and cardiovascular disease in later life. Specifically, one-carbon metabolism in the liver plays a critical role in placental and fetal growth. Impaired functioning of one-carbon metabolism is associated with increased homocysteine levels. In this study, we applied a comprehensive proteomic approach to identify differential expression of proteins related to one-carbon metabolism in the livers of rat offspring as an effect of maternal food restriction during gestation. Data are available via ProteomeXchange with identifier PXD002578. We determined that betaine-homocysteine S-methyltransferase 1, methylenetetrahydrofolate dehydrogenase 1, and ATP synthase subunit beta mitochondrial (ATP5B) expression levels were significantly reduced in the livers of rat offspring exposed to maternal food restriction during gestation compared with in the offspring of rats fed a normal diet (p < 0.05). Moreover, the expression levels of betaine-homocysteine S-methyltransferase 1, methylenetetrahydrofolate dehydrogenase 1, and ATP synthase subunit beta mitochondrial were negatively correlated with serum homocysteine concentration in male offspring exposed to maternal food restriction during gestation and normal diet during lactation. However, in female offspring only expression levels of methylenetetrahydrofolate dehydrogenase 1 were negatively correlated with homocysteine concentration. This study shows that maternal food restriction during late gestation and normal diet during lactation lead to increased homocysteine concentration through disturbance of one-carbon metabolism in the livers of male offspring. This suggests that male offspring have an increased gender-specific susceptibility to disease in later life through fetal programming.

A novel approach to assessing bisphenol-A hazards using an in vitro model system.

BACKGROUND: Although the toxicological impacts of the xenoestrogen bisphenol-A (BPA) have been studied extensively, but the mechanism of action is poorly understood. Eventually, no standard method exists for evaluating the possible health hazards of BPA exposure. Considering mice spermatozoa as a potential in vitro model, we investigated the effects of BPA exposure (0.0001, 0.01, 1, and 100 µM for 6 h) on spermatozoa and the related mechanisms of action. The same doses were also employed to evaluate protein profiles of spermatozoa as a means to monitor their functional affiliation to diseases. RESULTS: Our results demonstrated that high concentrations of BPA negatively affect sperm motility, viability, mitochondrial functions, and intracellular ATP levels by activating the mitogen-activated protein kinase, phosphatidylinositol 3-kinase, and protein kinase-A pathways. Moreover, short-term exposure of spermatozoa to high concentrations of BPA induced differential expressions of 24 proteins. These effects appeared to be caused by protein degradation and phosphorylation in spermatozoa. Proteins differentially expressed in spermatozoa from BPA treatment groups are putatively involved in the pathogenesis of several diseases, mainly cancer, carcinoma, neoplasm, and infertility. CONCLUSIONS: Based on these results, we propose that BPA adversely affects sperm function by the activation of several kinase pathways in spermatozoa. In addition, BPA-induced changes in the sperm proteome might be partly responsible for the observed effects in spermatozoa, subsequently involve in the pathogenesis of many diseases. Therefore, we anticipated that current strategy might broadly consider for the health hazards assessment of other toxicological agents.

Proteomic analysis of fetal programming-related obesity markers.

The objectives of this study were to analyze fetal programming in rat brain using proteomic analysis and to identify fetal programming-related obesity markers. Sprague-Dawley rats were divided into four feeding groups: (i) the Ad Libitum (AdLib)/AdLib group was given a normal diet during pregnancy and the lactation period; (ii) the AdLib/maternal food restriction group (FR) was subjected to 50% FR during the lactation period; (iii) the FR/AdLib group was subjected to 50% FR during pregnancy; and (iv) the FR/FR group was subjected to 50% FR during pregnancy and the lactation period. Offspring from each group were sacrificed at 3 weeks of age and whole brains were dissected. To obtain a maximum number of protein markers related to obesity, 2DE and Pathway Studio bioinformatics analysis were performed. The identities of the markers among the selected and candidate proteins were confirmed by Western blotting and immunohistochemistry. Proteomic and bioinformatics analyses revealed that expression of ubiquitin carboxy-terminal hydrolase L1 (UCHL1) and Secernin 1 (SCRN1) were significantly different in the FR/AdLib group compared with the AdLib/AdLib group for both male and female offspring. These findings suggest that UCHL1 and SCRN1 may be used as fetal programming-related obesity markers.

Bioinformatics Annotation of Human Y Chromosome-Encoded Protein Pathways and Interactions.

We performed a comprehensive analysis of human Y chromosome-encoded proteins, their pathways, and their interactions using bioinformatics tools. From the NCBI annotation release 107 of human genome, we retrieved a total of 66 proteins encoded on Y chromosome. Most of the retrieved proteins were also matched with the proteins listed in the core databases of the Human Proteome Project including neXtProt, PeptideAtlas, and the Human Protein Atlas. When we examined the pathways of human Y-encoded proteins through KEGG database and Pathway Studio software, many of proteins fall into the categories related to cell signaling pathways. Using the STRING program, we found a total of 49 human Y-encoded proteins showing strong/medium interaction with each other. While using the Pathway studio software, we found that a total of 16 proteins interact with other chromosome-encoded proteins. In particular, the SRY protein interacted with 17 proteins encoded on other chromosomes. Additionally, we aligned the sequences of human Y-encoded proteins with the sequences of chimpanzee and mouse Y-encoded proteins using the NCBI BLAST program. This analysis resulted in a significant number of orthologous proteins between human, chimpanzee, and mouse. Collectively, our findings provide the scientific community with additional information on the human Y chromosome-encoded proteins.

Effects of motor vehicle exhaust on male reproductive function and associated proteins.

Air pollution is consistently associated with various diseases and subsequent death among children, adult, and elderly people worldwide. Motor vehicle exhaust contributes to a large proportion of the air pollution present. The motor vehicle exhaust systems emit a variety of toxic components, including carbon monoxide, nitrogen oxides, volatile organic compounds, ozone, particulate matter, and polycyclic aromatic hydrocarbons. Several epidemiological studies and laboratory studies have demonstrated that these components are potentially mutagenic, carcinogenic, and endocrine disrupting agents. However, their impact on male reproductive function and associated proteins is not very clear. Therefore, a comprehensive review on the effects of motor vehicle exhaust on male reproductive function and associated proteins is needed to better understand the risks of exhaust exposure for men. We found that motor vehicle exhaust can cause harmful effects on male reproductive functions by altering organ weights, reducing the spermatozoa qualities, and inducing oxidative stress. Remarkably, motor vehicle exhaust exposure causes significant changes in the expression patterns of proteins that are key components involved in spermatogenesis and testosterone synthesis. In conclusion, this review helps to describe the risks of vehicle exhaust exposure and its relationship to potential adverse effects on the male reproduction system.

Diagnosis and prognosis of male infertility in mammal: the focusing of tyrosine phosphorylation and phosphotyrosine proteins.

Male infertility refers to the inability of a man to achieve a pregnancy in a fertile female. In more than one-third of cases, infertility arises due to the male factor. Therefore, developing strategies for the diagnosis and prognosis of male infertility is critical. Simultaneously, a satisfactory model for the cellular mechanisms that regulate normal sperm function must be established. In this regard, tyrosine phosphorylation is one of the most common mechanisms through which several signal transduction pathways are adjusted in spermatozoa. It regulates the various aspects of sperm function, for example, motility, hyperactivation, capacitation, the acrosome reaction, fertilization, and beyond. Several recent large-scale studies have identified the proteins that are phosphorylated in spermatozoa to acquire fertilization competence. However, most of these studies are basal and have not presented an overall mechanism through which tyrosine phosphorylation regulates male infertility. In this review, we focus of this mechanism, discussing most of the tyrosine-phosphorylated proteins in spermatozoa that have been identified to date. We categorized tyrosine-phosphorylated proteins in spermatozoa that regulate male infertility using MedScan Reader (v5.0) and Pathway Studio (v9.0).

A comprehensive proteomic approach to identifying capacitation related proteins in boar spermatozoa.

BACKGROUND: Mammalian spermatozoa must undergo capacitation, before becoming competent for fertilization. Despite its importance, the fundamental molecular mechanisms of capacitation are poorly understood. Therefore, in this study, we applied a proteomic approach for identifying capacitation-related proteins in boar spermatozoa in order to elucidate the events more precisely. 2-DE gels were generated from spermatozoa samples in before- and after-capacitation. To validate the 2-DE results, Western blotting and immunocytochemistry were performed with 2 commercially available antibodies. Additionally, the protein-related signaling pathways among identified proteins were detected using Pathway Studio 9.0. RESULT: We identified Ras-related protein Rab-2, Phospholipid hydroperoxide glutathione peroxidase (PHGPx) and Mitochondrial pyruvate dehydrogenase E1 component subunit beta (PDHB) that were enriched before-capacitation, and NADH dehydrogenase 1 beta subcomplex 6, Mitochondrial peroxiredoxin-5, (PRDX5), Apolipoprotein A-I (APOA1), Mitochondrial Succinyl-CoA ligase [ADP-forming] subunit beta (SUCLA2), Acrosin-binding protein, Ropporin-1A, and Spermadhesin AWN that were enriched after-capacitation (>3-fold) by 2-DE and ESI-MS/MS. SUCLA2 and PDHB are involved in the tricarboxylic acid cycle, whereas PHGPx and PRDX5 are involved in glutathione metabolism. SUCLA2, APOA1 and PDHB mediate adipocytokine signaling and insulin action. The differentially expressed proteins following capacitation are putatively related to sperm functions, such as ROS and energy metabolism, motility, hyperactivation, the acrosome reaction, and sperm-egg interaction. CONCLUSION: The results from this study elucidate the proteins involved in capacitation, which may aid in the design of biomarkers that can be used to predict boar sperm quality.

Proteomic analysis of fipronil-induced molecular defects in spermatozoa.

The phenylpyrazole insecticide fipronil has wide-ranging applications from agriculture to public health to control undesirable organisms. However, several studies have reported the residual environmental hazards of fipronil and demonstrated its harmful effects even in mammalian reproduction. Therefore, this study was conducted to demonstrate the mode of action of fipronil on mouse spermatozoa. We treated fipronil to spermatozoa and performed comprehensive function evaluations. Moreover, proteomic analyses were conducted to identify the alteration of protein expression levels in spermatozoa. Most of sperm motility and kinematic parameters and intracellular ATP levels were diminished, and the spontaneous acrosome reaction was promoted after treatment with fipronil. Proteomic analyses revealed altered expression levels of 14 proteins after treatment. These proteins have been reported to be associated with sperm-specific pathways, prominently the cytoskeleton of the sperm, "9 + 2" axoneme composition, metabolism, and fertility. Collectively, our results showed that fipronil alters sperm functional-related proteins and therefore influences male fertility. This study elucidates the possible reproductive toxic hazards associated with male infertility through aberrant suppression of sperm proteins.

Effect of 4-nonylphenol (4-NP) on sperm function: Insights into the PI3K/PDK1/AKT signaling pathway during capacitation.

4-Nonylphenol (4-NP) is an endocrine-disrupting chemical that impairs animal and human reproduction. However, the mechanisms underlying male reproductive dysfunction by 4-NP have not been fully understood. Herein, we demonstrated the effects of 4-NP on boar sperm functions and molecular mechanisms. Spermatozoa were treated with various concentrations of 4-NP (0, 10, 25, 50, 75, and 100 µM) during capacitation. Then, we evaluated sperm motility, capacitation status, intracellular ATP level, and cell viability. Finally, we measured the expression of phosphorylated protein kinase A (PKA), tyrosine phosphorylation, and proteins related to the phosphatidylinositol 3 kinase (PI3K)/phosphoinositide-dependent kinase-1 (PDK1)/protein kinase B (AKT) signaling pathways following exposure to 4-NP. Sperm motility and motion kinematics were reduced by 4-NP, whereas intracellular ATP levels were increased significantly in a dose-dependent manner. Furthermore, the expression levels of p-PI3K, PTEN, p-PDK1, AKT, and p-AKT exhibited a significant dose-dependent increase. Moreover, abnormal activation of PKA and tyrosine phosphorylation were observed. Specifically, the ∼24 kDa p-PKA substrate demonstrated a significant reduction following exposure to 4-Np. In addition, the ∼18 kDa p-PKA substrate and tyrosine-phosphorylated proteins displayed a significant dose-dependent increase after exposure to 4-NP. Our results suggest that 4-NP may induce detrimental effects on sperm functions through abnormal changes in PKA activity and tyrosine phosphorylation during capacitation, possibly through unusual alteration of the PI3K/PDK1/AKT signaling pathway. Therefore, 4-NP must be cautiously used considering its reproductive toxicity.

Bifenthrin Diminishes Male Fertility Potential by Inducing Protein Defects in Mouse Sperm.

A synthetic pyrethroid pesticide, bifenthrin, has been commonly used as an effective exterminator, although the rise in its usage has raised concerns regarding its effects on the environment and public health, including reproduction, globally. The current study investigated the function-related molecular disparities and mechanisms in bifenthrin-exposed sperm cells and the underlying mechanism. Therefore, epididymal spermatozoa were released, and various concentrations of bifenthrin were treated (0.1, 1, 10, and 100 µM) to evaluate their effects on sperm. The findings showed that although bifenthrin had no effect on sperm viability, various other sperm functions (e.g., motility, spontaneous acrosome reaction, and capacitation) related to male fertility were decreased, commencing at a 1 µM treatment. Molecular studies revealed nine differentially expressed sperm proteins that were implicated in motile cilium assembly, sperm structure, and metabolic processes. Furthermore, bifenthrin affected sperm functions through abnormal diminution of the expression of specific sperm proteins. Collectively, these findings provide greater insights into how bifenthrin affects male fertility at the molecular level.

Application of sperm motion kinematics and motility-related proteins for prediction of male fertility.

The selection of superior sires is paramount for enhancing the efficiency of animal production in the livestock industry. However, semen quality assessment still relies on conventional semen analysis techniques in both animals and humans. Despite extensive efforts to develop various biomarkers for more accurate and precise predictions of male fertility potential, more effective physiological indicators and advance potential biomarkers are needed. Herein, we aimed to develop new potential biomarkers related to sperm motion kinematics for male fertility prediction. We first evaluated sperm motion kinematic parameters and expression levels of sperm motility-related proteins of 30 Duroc boars. We then explored the correlation between litter size, sperm motion kinematics parameters, and sperm motility-related proteins. Progressive sperm motility (%), rapid sperm motility (%), slow sperm motility (%), straight-line velocity (µm/s), linearity (%), beat cross frequency (Hz), mean angular displacement (degree), wobble (%) were correlated with litter size. Furthermore, the expression of axonemal dynein light intermediate polypeptide 1 (DNALI1) and radial spoke head protein 9 homolog (RSPH9) correlated with litter size. The overall accuracy exceeded 60% for predicting litter size using these sperm motion parameters and proteins. Notably, our study observed an increase in litter size after predicting litter size using these parameters and proteins. Thus, sperm motion kinematic parameters and protein expression, particularly of DNALI1 and RSPH9, could serve as new biomarkers for male fertility. These results may contribute to improved understanding of the mechanisms underlying sperm motility.

Ritonavir Has Reproductive Toxicity Depending on Disrupting PI3K/PDK1/AKT Signaling Pathway.

Ritonavir (RTV) is an antiviral and a component of COVID-19 treatments. Moreover, RTV demonstrates anti-cancer effects by suppressing AKT. However, RTV has cytotoxicity and suppresses sperm functions by altering AKT activity. Although abnormal AKT activity is known for causing detrimental effects on sperm functions, how RTV alters AKT signaling in spermatozoa remains unknown. Therefore, this study aimed to investigate reproductive toxicity of RTV in spermatozoa through phosphoinositide 3-kinase/phosphoinositide-dependent protein kinase-1/protein kinase B (PI3K/PDK1/AKT) signaling. Duroc spermatozoa were treated with various concentrations of RTV, and capacitation was induced. Sperm functions (sperm motility, motion kinematics, capacitation status, and cell viability) and expression levels of tyrosine-phosphorylated proteins and PI3K/PDK1/AKT pathway-related proteins were evaluated. In the results, RTV significantly suppressed sperm motility, motion kinematics, capacitation, acrosome reactions, and cell viability. Additionally, RTV significantly increased levels of phospho-tyrosine proteins and PI3K/PDK1/AKT pathway-related proteins except for AKT and PI3K. The expression level of AKT was not significantly altered and that of PI3K was significantly decreased. These results suggest RTV may suppress sperm functions by induced alterations of PI3K/PDK1/AKT pathway through abnormally increased tyrosine phosphorylation. Therefore, we suggest people who use or prescribe RTV need to consider its male reproductive toxicity.

Effect of atractylenolide III on zearalenone-induced Snail1-mediated epithelial-mesenchymal transition in porcine intestinal epithelium.

BACKGROUND: The intestinal epithelium performs essential physiological functions, such as nutrient absorption, and acts as a barrier to prevent the entry of harmful substances. Mycotoxins are prevalent contaminants found in animal feed that exert harmful effects on the health of livestock. Zearalenone (ZEA) is produced by the Fusarium genus and induces gastrointestinal dysfunction and disrupts the health and immune system of animals. Here, we evaluated the molecular mechanisms that regulate the effects of ZEA on the porcine intestinal epithelium. RESULTS: Treatment of IPEC-J2 cells with ZEA decreased the expression of E-cadherin and increased the expression of Snai1 and Vimentin, which induced Snail1-mediated epithelial-to-mesenchymal transition (EMT). In addition, ZEA induces Snail-mediated EMT through the activation of TGF-ß signaling. The treatment of IPEC-J2 cells with atractylenolide III, which were exposed to ZEA, alleviated EMT. CONCLUSIONS: Our findings provide insights into the molecular mechanisms of ZEA toxicity in porcine intestinal epithelial cells and ways to mitigate it.