Cytoplasmic Droplets Research Articles

DGAT2 partially compensates for lipid-induced ER stress in human DGAT1-deficient intestinal stem cells

Dietary lipids are taken up as FAs by the intestinal epithelium and converted by diacylglycerol acyltransferase (DGAT) enzymes into triglycerides, which are packaged in chylomicrons or stored in cytoplasmic lipid droplets (LDs). DGAT1-deficient patients suffer from vomiting, diarrhea, and protein losing enteropathy, illustrating the importance of this process to intestinal homeostasis. Previously, we have shown that DGAT1 deficiency causes decreased LD formation and resistance to unsaturated FA lipotoxicity in patient-derived intestinal organoids. However, LD formation was not completely abolished in patient-derived organoids, suggesting the presence of an alternative mechanism for LD formation. Here, we show an unexpected role for DGAT2 in lipid metabolism, as DGAT2 partially compensates for LD formation and lipotoxicity in DGAT1-deficient intestinal stem cells. Furthermore, we show that (un)saturated FA-induced lipotoxicity is mediated by ER stress. More importantly, we demonstrate that overexpression of DGAT2 fully compensates for the loss of DGAT1 in organoids, indicating that induced DGAT2 expression in patient cells may serve as a therapeutic target in the future.

Hexokinase is necessary for glucose-mediated photosynthesis repression and lipid accumulation in a green alga

Global primary production is driven largely by oxygenic photosynthesis, with algae as major contributors. The green alga Chromochloris zofingiensis reversibly switches off photosynthesis in the presence of glucose in the light and augments production of biofuel precursors (triacylglycerols) and the high-value antioxidant astaxanthin. Here we used forward genetics to reveal that this photosynthetic and metabolic switch is mediated by the glycolytic enzyme hexokinase (CzHXK1). In contrast to wild-type, glucose-treated hxk1 mutants do not shut off photosynthesis or accumulate astaxanthin, triacylglycerols, or cytoplasmic lipid droplets. We show that CzHXK1 is critical for the regulation of genes related to photosynthesis, ketocarotenoid synthesis and fatty acid biosynthesis. Sugars play fundamental regulatory roles in gene expression, physiology, metabolism, and growth in plants and animals, and we introduce a relatively simple, emerging model system to investigate conserved eukaryotic sugar sensing and signaling at the base of the green lineage.

Analysis of the Expression of Genes Related to Lipidic and Glycogenic Metabolism in Pleomorphic Adenomas and Carcinomas Ex Pleomorphic Adenomas

Abstract Introduction The carcinoma ex pleomorphic adenoma (CXPA) arises from a pleomorphic adenoma (PA) and is supposed to gain lipogenesis and glycogenesis during its malignant transformation. Increased lipogenesis is a characteristic of cancer cells, consisting of increased fatty acid synthesis by the enzyme fatty acid synthase (FASN) and by the accumulation of cytoplasmic lipid droplets, which express adipophilin. The increase in glycolytic metabolism has been associated with the activity of glycolytic enzymes and glucose transporters (GLUTs), whose expression is induced by HIF-1 (inducing factor of hypoxia 1). Objectives To analyze the gene expression of FASN, adipophilin, HIF-1α, and GLUT-1 in PA and CXPA samples. Methodology Gene expression analysis of the FASN, adipophilin, HIF1-α, and GLUT-1 genes was performed by the real-time PCR (qPCR) method. Fourteen cases of PA and 14 cases of CXAP were evaluated. Results FASN, adipophilin, HIF-1α, and GLUT-1 were more expressed in CXAP than in AP, although there was no significant difference between levels of adipophilin expression in CXPA and PA. Conclusion Increased expression of FASN, HIF1-α, and GLUT-1 in CXAP may be associated with malignant transformation of PA, while additional studies are needed to understand the role of adipophilin in PA carcinogenesis. Moreover, this is the first study to investigate the role of these genes in the malignant transformation of PA.

A modified in vitro tool for isolation and characterization of rat quiescent islet stellate cells

BackgroundIslet stellate cells (ISCs) play a critical role in islet fibrosis, contributing to the progression of pancreatic diseases. Previous studies have focused on fibrosis-associated activated ISCs obtained by standard islet explant techniques. However, in vitro models of quiescent ISCs (qISCs) are lacking. This study aims to develop a method to isolate qISCs and analyze their phenotype during activation. MethodsImmunofluorescence staining was applied to localize ISCs in normal human, rat, and mouse islets. qISCs were isolated from rat islets using density gradient centrifugation (DGC) method. qRT-PCR, immunoblotting, proliferation, and migration assays were employed for their characterization. ResultsDesmin-positive ISCs were detected in normal human, rat, and mouse islets. Freshly isolated qISCs, obtained by density gradient centrifugation, displayed a polygonal appearance with refringent cytoplasmic lipid droplets and expressed transcriptional markers indicating a low activation/quiescent state. With increasing culture time, the marker expression pattern changed, reflecting ISC activation. qISCs contained more lipid droplets and exhibited lower proliferation and migration abilities compared to spindle-shaped ISCs obtained by traditional explant techniques. ConclusionsThis study describes a new method for efficient isolation of qISCs from rat islets, representing a useful in vitro tool to study the biology of ISCs in more physiological conditions.

Expression and significance of urinary microRNA in patients with chronic hepatitis B.

The aim of this study was to investigate the alterations of urinary microRNA (miRNA) expression and explore its clinical significance in patients with chronic hepatitis B (CHB).The expression levels of urinary miRNA were detected by miRNA microarray and quantitative reverse transcription polymerase chain reaction (qRT-PCR) from 106 CHB and 40 healthy controls (Ctrl) subjects. The correlation between the levels of miRNA expression and clinical characteristics were analyzed. Receiver-operator characteristic (ROC) curves were generated to determine the specificity and sensitivity of each individual miRNA. MiRNAs expression were further measured by PCR from exosomes, which were isolated from urine samples. LX2 cells were transfected with miRNA inhibitor and accumulation of cytoplasmic lipid droplets was analyzed by Oil Red O staining.miRNA expression profile analysis showed that 22 miRNAs were upregulated and 55 miRNAs were downregulated in CHB patients compared with Ctrl subjects (fold-change>1.5 and P < .05). miR-92b-3p, miR-770-5p, miR-5196-5p, and miR-7855-5p were significantly higher (P < .0001) in CHB subjects than in Ctrl subjects. ROC curve analysis showed that these four miRNAs were sensitive and specific enough to distinguish CHB and Ctrl subjects. The levels of miR-92b-3p expression were negatively correlated with total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and APOA-1. Moreover, in vitro experiments indicated that inhibition of miR-92b-3p increased lipid droplet formation in LX2 cells.Aberrant expression of miRNAs has been observed in urine of CHB patients. Our findings may provide novel insights into the pathogenesis of CHB and may assist in the diagnosis of patients with CHB.

Role of Lipid Droplets in the Development of Oocytes and Preimplantation Embryos in Mammals

Lipids are one of the most common and important cell components. Particularly, cytoplasmic lipid droplets (LDs) play a crucial role in cellular energy metabolism. The consideration of the methods for studying intracellular lipids and their composition and role during the oocyte maturation and early embryonic development are the main directions of this review. Recent studies demonstrate that LDs in oocytes and embryos are not only repositories of the energy substrate but also active intracellular structures that perform many other functions.

Certain morphological characteristics of the spermatozoon used for intracytoplasmic sperm injection (ICSI) are associated with a higher likelihood of developmental arrest and embryo degeneration

Around 25% of in-vitro cultured human embryos arrest their development and degenerate during the first 5 days. The functional and morphological characteristics of the spermatozoa, selected for the intracytoplasmic sperm injection (ICSI) could account for the subsequent embryo development failure. The objective of the present study was to assess the relationship between the morphological characteristics of the spermatozoon used for ICSI and the increased risk of developmental arrest and embryo degeneration. Prospective observational study. A total of 83 spermatozoa from 68 men used in ICSI procedures were studied. Inclusion criteria were: 1) patient age < 39 years, 2) patient body mass index (BMI) < 25 kg/m2, 3) excellent quality oocytes. To find out which sperm morphological features could be responsible for the embryo degeneration the morphology of each individual spermatozoon used for ICSI was recorded. The morphology of all individual spermatozoa was evaluated using high magnification (×6100) immediately before the injection. Seven sperm morphological parameters and the presence of sixteen head, neck and tail abnormalities were evaluated. All 83 embryos were cultured in-vitro in Global one-step medium. The frequencies of occurrence of sperm morphological defects were compared between the resulted viable and degenerated embryos. Data was analyzed using the IBM SPSS software, v.21.0. Among 83 ICSI performed, 63 embryos (75.9%) had successful development and 20 embryos (24.1%) arrest their development before day 5. The incidence of pyriform head, thick midpiece and cytoplasmic residues among the spermatozoa that resulted in embryos with developmental arrest were significantly higher in comparison to spermatozoa that led to successfully developed embryos (20% vs. 13%, 40% vs. 21% and 20% vs. 6%, respectively, p<0.05, Chi-square test). In addition, the spermatozoa resulted in embryos with successful development had significantly shorter heads and tails in comparison with those that resulted in embryo development arrest (4.46 vs. 4.63 μm and 41.42 vs. 43.65 μm, p<0.05, Student t-test, respectively). When receiver operating curve (ROC) analysis was performed to find the optimum cut-off value to predict embryo development sperm head length had the best AUC (AUC = 0.66, CI 95%: 0.51–0.76). The optimum cut-off value for head length was 4.25 μm (sensitivity 89.5%, specificity 60.3%). The recorded spermatozoa tail length had lower AUC (0.63, CI 95%: 0.49–0.77) and the optimal cut-off value of 42.5 μm (sensitivity 63.2%, specificity 42.5%). Specific sperm morphological characteristics, such as head length and tail length and the presence of certain sperm abnormalities (pyriform head, thick midpiece and residual cytoplasmic droplets) are associated with an increased risk of developmental arrest and embryo degeneration. Selection of human spermatozoa with head length < 4.25 μm, tail length < 42.5 μm and without pyriform head, thick midpiece and cytoplasmic residues significantly increase the chance for successful embryo development.

The possible protective role of vitamin C against Monosodium glutamate induced structural changes of adrenal cortex of the adult male albino rat: Histological and immunohistochemical study

Introduction: Monosodium glutamate (MSG) is a popularly used taste enhancer. MSG proved to cause many degenerative diseases and oxidative damages. Vitamin C has an antioxidant property.Aim: This work aimed to study the possible protective role of vitamin C against Monosodium glutamate induced structural changes of the adrenal cortex.Material and Methods: Forty-five adult male albino rats were used in this study, aged from 4-6 months and weighting 180-200 Kg. Animals were divided into three equal groups. Group I: It was composed of fifteen rats and was further subdivided into three equal subgroups; Subgroup IA: included 5 rats that kept as negative control. Subgroup IB: included 5 rats that received 1ml of distilled water for 8 weeks and Subgroup IC included 5 rats that received vitamin C in a dose of 0.3 mg/Kg body weight /day for 8 weeks. Group II: It included fifteen adult male rats that received 4mg/Kg body weight/ day of MSG for 8 weeks. Group III: It included fifteen rats that received MSG as in group II and vitamin C in a dose of 0.3 mg/Kg body weight /day for 8 weeks.Results: The present study revealed that MSG induced histological changes of adrenal cortex in the form of disorganized structure of zona glomerulosa and zona fasciculata with increased accumulation of the cytoplasmic droplets and thinning of the capsule. Group III that received vitamin C revealed evident improvement of these changes.Conclusion: Monosodium glutamate led to histoarchitectural changes of adrenal cortex. However, vitamin C greatly improved such changes.

HADHA overexpression disrupts lipid metabolism and inhibits tumor growth in clear cell renal cell carcinoma

Hydroxyacyl-CoA dehydrogenase alpha subunit (HADHA) is a key lipid metabolic enzyme with a novel role in carcinogenesis. We previously reported that HADHA, a prognostic marker, was downregulated in clear cell renal cell carcinoma (ccRCC). Herein, the tumor inhibitory role of HADHA overexpression in ccRCC was investigated further. The quantitative proteomic analysis displayed that a total of 1293 and 1293 proteins were identified in HADHA overexpressed 786-O-hadha and vector-transfected control 786-O-vc cells, respectively, and 206 proteins were found to be up- or downregulated. PANTHER, OmicsNet, STRING, and DAVID tools were utilized on the dysregulated proteins in order to elucidate multiple metabolic pathways (especial lipid metabolism) and lipid metabolism-related proteins (e.g. ACAT1, ACLY). The dysregulation of the lipid metabolic enzymes, ACAT1, ACLY, CYB5R3 and FASN, were confirmed by Western blotting. Further assays demonstrated that HADHA overexpression significantly inhibited cell growth, induced cell apoptosis, and decreased the formation of cytoplasmic lipid droplets (LDs); moreover, it also inhibited tumor growth and lessened the formation of LDs in xenografted mouse. Collectively, these data revealed that HADHA overexpression disrupted lipid metabolism and inhibited tumor growth, which shed light on HADHA as a potential therapeutic target for clinical intervention of ccRCC.

Neutral Lipids Are Not a Source of Arachidonic Acid for Lipid Mediator Signaling in Human Foamy Monocytes

Human monocytes exposed to free arachidonic acid (AA), a secretory product of endothelial cells, acquire a foamy phenotype which is due to the accumulation of cytoplasmic lipid droplets with high AA content. Recruitment of foamy monocytes to the inflamed endothelium contributes to the development of atherosclerotic lesions. In this work, we investigated the potential role of AA stored in the neutral lipids of foamy monocytes to be cleaved by lipases and contribute to lipid mediator signaling. To this end, we used mass spectrometry-based lipidomic approaches combined with strategies to generate monocytes with different concentrations of AA. Results from our experiments indicate that the phospholipid AA pool in monocytes is stable and does not change upon exposure of the cells to the external AA. On the contrary, the AA pool in triacylglycerol is expandable and can accommodate relatively large amounts of fatty acid. Stimulation of the cells with opsonized zymosan results in the expected decreases of cellular AA. Under all conditions examined, all of the AA decreases observed in stimulated cells were accounted for by decreases in the phospholipid pool; we failed to detect any contribution of the triacylglycerol pool to the response. Experiments utilizing selective inhibitors of phospholipid or triacylglyerol hydrolysis confirmed that the phospholipid pool is the sole contributor of the AA liberated by stimulated cells. Thus, the AA in the triacylglycerol is not a source of free AA for the lipid mediator signaling during stimulation of human foamy monocytes and may be used for other cellular functions.

Effect of Pomegranate Seed Oil against Hepatotoxicity- Induced by Sodium Fluoride in Adult Female Rats (Part II).

This experiment was designed to evaluate the hepatoprotective effects of pomegranate seed oil against toxicity- induced by sodium fluoride and in normal rats. Twenty adult female Wistar rats were divided into four equal group and treated daily for 40 days as follows: Group C administered tap water and served as control , group T1 : received sodium fluoride 120ppm in drinking tap water, group T2: received both sodium fluoride 120ppm in drinking water and administered orally pometone (pomegranate seed oil) 30mg/kg B.W. and group T3 : administered pomegranate seed oil as in group T2 orally. Fasting blood samples were collected at 0, 20 and 40 days to estimation of some biochemical parameters and oxidative stress biomarkers . In addition, sections from liver were taken at the end of the experiment for histopathological study. The results revealed that SF (group T1) caused a significant increase in serum aminotransferases (serum alanine aminotransferase and aspartate aminotransferase) activities, total cholesterol ,total bilirubin and peroxynitrite radical concentrations, while GSH concentration was a significantly decrease. PSO caused an alleviation to the hepatic dysfunction induced by sodium fluoride (group T2) manifested through significant elevation of GSH concentration, in addition, a significant reduction in serum transaminases activity, total cholesterol, peroxynitrite radical and total bilirubin concentrations. In contrast, administration of pomegranate seed oil (group T3) showed no alterations in most of these parameters. Furthermore histopathological examination of liver tissues of group T1 manifested aggregation of mononucleated cells, proliferation of hepatocyte, cytoplasmic fat droplet and granulomatous lesion consists of aggregation of macrophage and lymphocyte. All these alteration in liver histology were modified by treatment of rats with pomegranate seed oil (group T2) and no pathological lesion was reported in group T3. On conclusion, this study documented the beneficial effect of pomegranate seed oil against the deleterious effects of SF on liver functions of adult female rats.

Use of testicular tone and echogenicity in identifying boars with low sperm quality

ABSTRACT This study aimed to evaluate the use of some testicular traits to identify boars with low sperm morphological quality. The consistency (scores from 1 to 5), tone with tonometry (mm), parenchyma echogenicity and heterogeneity with ultrasound (pixel) were assessed in 402 mature boars (18.5 months on average). Sperm abnormality thresholds (≤ 25% of total sperm abnormalities, ≤ 5% of heads, acrosome, neck or midpiece defects, ≤ 10% tail defects, and 15% cytoplasmic droplets) were used to classify boars as approved or disapproved. Three classes of testicular traits were formed (extremely low and high values, approximately 15% each, were kept in separated classes). When the traits were individually evaluated, fewer boars were approved if the echogenicity or heterogeneity were high, or if the tone was rigid. When evaluated in combination, the interaction between heterogeneity and tone, and between heterogeneity and echogenicity were significant. The high heterogeneity combined with soft tone or with hypo-echogenicity resulted in lower approval of boars. Tonometry and ultrasonography have a moderate potential to be included in breeding soundness examination of boars. When combined, they provide more reliable information about the impact of testicular parenchymal alterations on morphology of sperm cells.

Advances in understanding mechanisms of long-term sperm storage-the soft-shelled turtle model.

Long-term sperm storage is a special reproductive strategy, which can extend the time window between mating and fertilization in some animal species. Spermatozoa of the soft-shelled turtle, Pelodiscus sinensis, can be stored in the epididymis and oviduct for at least six months and one year, respectively. How spermatozoa can be stored in vivo for such a prolonged period is yet to be explained. We analyze the mechanisms that contribute to long-term sperm storage in P. sinensis, and compare them with other species from three different perspectives: the spermatozoon itself, the storage microenvironment and the interaction between the spermatozoon and microenvironment. Characteristics of soft-shelled turtle spermatozoa itself, such as the huge cytoplasmic droplet with its content of several large lipid droplets (LDs) and onion-like mitochondira, facilitate long-term sperm storage. The microenvironment of reproductive tract, involving in the secretions, structural barriers, exosomes, androgen receptors, Toll-like receptors and survival factor Bcl-2, are important for the maintenance of spermatozoa long-term storage. Sperm heads are always embedded among the oviductal cilia and even intercalate into the apical hollowness of the ciliated cells, indicating that the ciliated cells support the stored spermatozoa. RNA seq is firstly used to detect the molecular mechanism of sperm storage, which shows that autophagy, apoptosis and immune take part in the long-term sperm storage in this species.

Abstract 897: Lipid metabolism is involved in mitotic slippage-induced SASP upon treatment with anti-mitotic drugs

Abstract Aberrant lipid accumulation is a hallmark of cancer. However, there is little known regarding mechanisms linking lipid metabolism to chemotherapy-induced cell fates. Here, we describe lipid accumulation in cells following antimitotic drug-treatment. Cells arrested in mitosis, as well as cells that escaped mitotic arrest and underwent mitotic slippage, showed elevated cytoplasmic lipid droplets. Interestingly, we found that TOFA, an inhibitor that targets acetyl-CoA carboxylase (ACC) and blocks lipid accumulation enhanced survival of antimitotic-treated cells. Our work previously revealed that cells that survive slippage become senescent and confer pro-tumourigenic effects through non-cell-autonomous effects. Modulating lipid biosynthesis in cells post-slippage by TOFA amplified their inflammatory secretion profiles and accelerated development of tumourigenic behavior. In contrast to TOFA, inhibition of lipid accumulation by C75, a drug targeting fatty acid synthase, significantly reduced the production of pro-tumourigenic factors. This suggests that discrete lipid biosynthesis pathways could contribute differentially to the regulation of pro-tumourigenic inflammation. Note: This abstract was not presented at the meeting. Citation Format: Karen C. Crasta, Alex Wong, Bryan Lim. Lipid metabolism is involved in mitotic slippage-induced SASP upon treatment with anti-mitotic drugs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 897.

Fat, fight, and beyond: The multiple roles of lipid droplets in infections and inflammation.

Increased accumulation of cytoplasmic lipid droplets (LDs) in host nonadipose cells is commonly observed in response to numerous infectious diseases, including bacterial, parasite, and fungal infections. LDs are lipid-enriched, dynamic organelles composed of a core of neutral lipids surrounded by a monolayer of phospholipids associated with a diverse array of proteins that are cell and stimulus regulated. Far beyond being simply a deposit of neutral lipids, LDs have come to be seen as an essential platform for various cellular processes, including metabolic regulation, cell signaling, and the immune response. LD participation in the immune response occurs as sites for compartmentalization of several immunometabolic signaling pathways, production of inflammatory lipid mediators, and regulation of antigen presentation. Infection-driven LD biogenesis is a complexly regulated process that involves innate immune receptors, transcriptional and posttranscriptional regulation, increased lipid uptake, and new lipid synthesis. Accumulating evidence demonstrates that intracellular pathogens are able to exploit LDs as an energy source, a replication site, and/or a mechanism of immune response evasion. Nevertheless, LDs can also act in favor of the host as part of the immune and inflammatory response to pathogens. Here, we review recent findings that explored the new roles of LDs in the context of host-pathogen interactions.

Dark side of the epididymis: tails of sperm maturation.

The Hermes body (HB) previously called the cytoplasmic droplet is a focal distension of the flagellar cytoplasm of epididymal spermatozoa consisting mainly of isolated flattened Golgi cisternae. To define a functional role for the HB of epididymal spermatozoa. Isolated fractions of HBs of epididymal spermatozoa were prepared and by quantitative tandem mass spectrometry revealed 1511 proteins. The glucose transporter GLUT-3 was the most abundant protein followed by hexokinase 1, which along with the presence of all glycolytic enzymes suggested a role for the HB in glycolysis. Several TMED/p24 Golgi trafficking proteins were abundant with TMED7/p27 and TMED2/p24 defining the identity of the flattened cisternae within the HB as Golgi, along with the known Golgi proteins, GBF1, GOLPH3, Man2α1, and ManIIX. The Golgi trafficking protein TMED7/p27 via small 50-nm vesicles emanating from the Golgi cisternae was proposed to transport GLUT-3 to the plasma membrane for ATP production related to sperm motility. The internal membranes revealed abundant proteins not only of Golgi cisternae, but also of endoplasmic reticulum and endosomes. COPI and COPII coats, clathrin, SNAREs, annexins, atlastins, and GTPases were identified for vesicular trafficking and membrane fusion, in addition to ribosomes, stress proteins for protection, proteasome proteins involved in degradation, and cytoskeletal elements for migration of the HB along the flagellum. The biogenesis of the HB occurring at step 19 spermatids of the testis just prior to their release was uncovered as a key step in germ cell differentiation, where several proteins were expressed, some for the first time. As epididymal spermatozoa undergo remodeling of their protein makeup through selective degradation of sperm proteins during epididymal transit, then remodeling as a consequence of new protein synthesis is not excluded by our observations.

Liver X receptor-α activation enhances cholesterol secretion in lactating mammary epithelium.

Liver X receptors (LXRs) are ligand-dependent transcription factors activated by cholesterol metabolites. These receptors induce a suite of target genes required for de novo synthesis of triglycerides and cholesterol transport in many tissues. Two different isoforms, LXRα and LXRβ, have been well characterized in liver, adipocytes, macrophages, and intestinal epithelium among others, but their contribution to cholesterol and fatty acid efflux in the lactating mammary epithelium is poorly understood. We hypothesize that LXR regulates lipogenesis during milk fat production in lactation. Global mRNA analysis of mouse mammary epithelial cells (MECs) revealed multiple LXR/RXR targets upregulated sharply early in lactation compared with midpregnancy. LXRα is the primary isoform, and its protein levels increase throughout lactation in MECs. The LXR agonist GW3965 markedly induced several genes involved in cholesterol transport and lipogenesis and enhanced cytoplasmic lipid droplet accumulation in the HC11 MEC cell line. Importantly, in vivo pharmacological activation of LXR increased the milk cholesterol percentage and induced sterol regulatory element-binding protein 1c (Srebp1c) and ATP-binding cassette transporter a7 (Abca7) expression in MECs. Cumulatively, our findings identify LXRα as an important regulator of cholesterol incorporation into the milk through key nodes of de novo lipogenesis, suggesting a potential therapeutic target in women with difficulty initiating lactation.

Molecular markers of sperm quality

Light microscopic semen evaluation provides useful information about a given sperm sample, but due to its subjective nature has limited prognostic value for the reproductive performance of males or the outcome of assisted fertilization. Cryptic sperm abnormalities (occurring at the molecular level) are not easily detectable by light microscopy, but can be revealed by an array of biomarkers. The latter include fluorescent markers of acrosomal status, fluorochromes detecting altered sperm chromatin or DNA integrity, vital dyes revealing sperm mitochondrial activity, probes detecting apoptotic events, and antibodies detecting proteins that are either up- or down-regulated in defective spermatozoa. Many of the above biomarkers are best tested by flow cytometry, permitting rapid, automated, high throughput, objective measurement of the relative abundance of these biomarkers in semen. This review summarizes a strategy for the identification of novel male fertility/sperm quality biomarkers based on proteomic, biochemical and immunocytochemical analyses of defective spermatozoa. This approach identifies proteins or ligands uniquely associated with defective spermatozoa, regardless of whether they carry gross morphological defects or subtle, but critical hidden defects (e.g. DNA strand breaks) not detected with conventional, light microscopic analysis. Such markers, including ubiquitin, sperm thioredoxin SPTRX3/TXNDC8, 15LOX, and Lewis(y)-terminated N-glycans, are associated with poor semen quality and reduced fertility, warranting a designation of negative markers of fertility. The significance of sperm cytoplasmic droplet, a structure that accumulates several of the discussed biomarker proteins, is also discussed with regard to sperm quality and fertility.

Logistic regression analysis of the related factors in discarded semen of boars in Southern China

This study aimed to investigate factors affecting the discarded semen of boars. A total of 176,368 ejaculates of boars from nine AI centers were collected from January 2013 to May 2016 in Southern China. The criteria for determining whether their semen was abnormal included cytoplasmic droplets, coiling tail, sperm agglutination, impurity, poor motility, oligozoospermia, necrozoospermia, azoospermia, and hemospermia. The cause of discarded semen was evaluated with a Chi-square test, and the effects of housing type, breed, age at collection, season identified in the northern hemisphere, and age at herd entry of the discarded semen of boars were analyzed with a logistic regression model. Results indicated the proportion of the discarded semen (PDS) in the nine AI centers was 13.09%. Chi-square test showed the greatest PDS among all causes was found in semen discarded due to cytoplasmic droplets (31.60%), followed by impurity (25.96%), sperm agglutination (20.31%), coiling tail (17.72%), oligozoospermia (10.86%), and others (6.78%; P < 0.0001). Logistic regression analysis revealed the PDS was affected by all these five factors (P < 0.0001). The PDS of boars raised individually in stalls was greater than that of boars raised individually in pens (OR: 1.657; 95% CI: 1.607 to 1.709). The PDSs of Duroc boars (OR: 1.130; 95% CI: 1.093 to 1.167) and Yorkshire boars (OR: 1.432; 95% CI: 1.380 to 1.486) were greater than that of Landrace boars. The PDSs of adult boars (aged from 13 to 24 mo, from 25 to 36 mo, and more than 37 mo with OR: 0.800, 0.941, and 0.838, respectively; 95% CI: 0.771 to 0.831, 0.902 to 0.983, and 0.790 to 0.889, respectively) were lower than those of young boars (aged less than 12 mo). The PDSs of semen collected in summer (OR: 1.367; 95% CI: 1.314 to 1.422), autumn (OR: 1.185; 95% CI: 1.138 to 1.234), and winter (OR: 1.159; 95% CI: 1.115 to 1.206) were greater than those of semen obtained in spring. The PDSs of boars introduced at ages of 5–7 mo (OR: 1.432; 95% CI: 1.380 to 1.486) and 10–12 mo (OR: 1.432; 95% CI: 1.380 to 1.486) were greater than those of boars introduced at an age of 8 and 9 mo. In conclusion, logistic regression analysis reveals discarded semen is affected by housing type, breed, age at collection, season, and age at herd entry. More importantly, cytoplasmic droplets is the primary reason for discarding boar semen, and 8 months at herd entry is the most suitable age for boar introduction.

The prevalence of ultrasonographic abnormalities and histopathologic lesions is high in testes of yearling tropical hair rams

Ultrasonography is commonly used to examine testes as part of a breeding soundness examination in sheep, especially, in cases of infertility or when gross testicular abnormalities are present. A descriptive, prospective, prevalence study was conducted to characterize the ultrasonographic, histopathologic, and spermatozoal morphology abnormalities present in a group of yearling tropic hair rams on the island of St. Kitts. Hyperechoic and shadowing abnormalities increased over a 6 month study period. Hyperechoic abnormalities were present in one or both testes in 89% (25/28) of yearling rams and 71% (40/56) of testes at castration. Shadowing abnormalities were present in one or both testes in 46% (13/28) of rams and 34% (19/56) of testes at castration. Shadowing was present more with moderate and severe hyperechoic abnormalities, with few testes in the mild category having any shadowing. As hyperechoic and shadowing abnormalities increased in severity, so did the severity of microscopic lesions including increased interstitial cellularity/fibrosis, interstitial mineralization, seminiferous tubules mineralization (hyperechoic only), and chronic lymphoplasmacytic orchitis. There were no spermatozoal morphologic abnormalities other than an increase in distal cytoplasmic droplets. The study findings detail a pathologic event in this group of yearling rams that has an unknown etiology. Potential causes may include scrotal insulation, trauma, infectious causes, immunity alterations, nutritional imbalances, and ingestion of a toxin. Further studies are required to elucidate the causative agent.