Apoptotic-Related MiRNAs Correlated with Functional and Flow Cytometric Parameters in Asthenozoospermic Holstein Bulls After Freeze-Thaw Process.

Many cellular processes in spermatozoa, including apoptosis and motility, are regulated by miRNA. Different miRNAs and molecular pathways are involved in asthenozoospermia (AS) conditions, which are thought to be one of the causes of infertility with reduced sperm motility. Thirty-two semen samples from four Holstein bulls with normozoospermia (NS), total motility ≥ 70%, and progressive motility ≥ 60%, and 32 semen samples from four bulls with AS, total motility ≤ 40%, and progressive motility ≤ 32% were used to investigate the function of apoptosis-related miRNAs in the AS group. Samples were then aspirated into a 0.5 mL straw after dilution with a Tris-egg yolk extender and frozen at -196°C. After freezing, semen samples were thawed for 2 weeks at 37°C and sperm kinematic parameters, plasma membrane integrity, acrosome integrity, DNA fragmentation, apoptosis status, and expression of apoptosis-related miRNAs (miR-2114, miR-296-3p, miR-455-3p, and miR345-3p) were evaluated. Our results showed that the functional and flow cytometric parameters of the NS group were significantly better than those of the AS group. In the NS group, miR-455-3pp and miR-2412 were upregulated, while miR-345-3p was downregulated compared with the AS group. In the AS group, miR-296-39, miR-2412, and miR-345-3p levels were strongly correlated with membrane integrity, DNA fragmentation, and apoptosis status. The findings demonstrated that the selected miRNAs based on bioinformatic analysis in AS and NS samples had a substantial association with functional and flow cytometry indicators and may be involved in regulating apoptosis and motility in AS samples.

Engineered exosome as a biological nanoplatform for drug delivery of Rosmarinic acid to improve implantation in mice with induced endometritis.

Endometritis is an inflammatory and histopathologic disease in uterine tissues that interferes with the proper decidualization and implantation of the embryo. In this study, rosmarinic acid (RA) is used as an anti-inflammatory agent that encapsulates in exosomes and is used to attenuate lipopolysaccharide (LPS)-induced endometritis and improve implantation. For this purpose, exosomes were loaded with RA and then administrated into the animal groups, including RA, exosome, RA plus exosome (RA + Exo), and RA-loaded exosomes (RALExo) groups. The concentrations of RA or exosomes used in this study were 10 mg/kg, and the compounds were injected into the uterine horn 24 h following the induction of endometritis. Upon the presence of inflammation detected by the histopathological method, the most proper groups were mated with male mice. The effect of the treatment group on the implantation rate, progesterone levels, and gene expressions were assessed by Chicago Blue staining, enzyme-linked immunosorbent assay (ELISA), and Quantitative PCR (qPCR), respectively. Results showed RALExo10 and RA10 + Exo10 groups improved pathological alterations, enhanced progesterone levels, increased implantation rate, as well as heightened expression levels of Leukemia inhibitory factor (LIF) and Mucin-16 (MUC-16) genes. Besides, the expression levels of inflammatory cytokines, including Transforming growth factor-ß (TGF-ß), Interlukine-10 (IL-10), Interlukine-15 (IL-15), and Interlukine-18 (IL-18), were regulated. Our findings indicated that the expression of LIF, Muc-16 genes as well as IL-18, were significantly correlated with serum progesterone concentrations and the implantation rate in the treatment groups. The RALExo10 and RA10 + Exo10 groups showed ameliorated implantation rates in experimental groups.

Enhanced anti-inflammatory effect of Rosmarinic acid by encapsulation and combination with the exosome in mice with LPS-induced endometritis through suppressing the TLR4-NLRP3 signaling pathway.

The TLR4-NLRP3 signaling pathway plays an essential role in the development of inflammation and especially endometritis. Rosmarinic acid (RA) can have potent anti-inflammatory effects in the drug-loading system. The purpose of this was to evaluate the anti-inflammatory effects of RA loaded to exosomes (RLE) on lipopolysaccharide (LPS)-induced endometritis in mice. RA was loaded into serum-derived exosome, using sonication methods. Animals in the treatment groups were subjected to uterine horn injection of RA, exosome, RA combination with exosome (R+E), and RA loaded to exosome (RLE) in uterine horn by two dosages in each group (5 and 10 mg/kg of RA or exosome), 24 h after inducing endometritis. Histopathological analysis, MPO production, immunohistochemistry, and qPCR were used to determine whether the treatment groups were adequate in controlling inflammation. The results showed that treatment groups, and mainly RLE10 and R10 +E10 groups, could modulate pathological changes, inhibit myeloperoxidase (MPO) activity, and significantly reduce the gene and protein expression of TLR4, NLRP3, inflammatory cytokines such as IL-1ß, IL-18, and TNF-α, and lastly, GSDM-D as a pyroptosis factor. In conclusion, RA loaded and combination with exosomes at a dosage of 10 mg/kg (RLE10 and R10 +E10) improved endometritis in mice through a suppressing TLR4-NLRP3 signaling pathway.

Effects of vitamin D supplementation in extender on sperm kinematics and apoptosis following the freeze-thaw process in normozoospermic and asthenozoospermic Holstein bulls.

BACKGROUND: Asthenozoospermia is a usual male infertility factor, characterized by decreased semen quality. It has been revealed that antioxidants improve sperm function, enhance endogenous antioxidant activities, and protect spermatozoa against oxidative damage during cryopreservation. This aimed to evaluate the effects of vitamin D on sperm kinematics and apoptosis in the semen of bulls with normozoospermia and asthenozoospermia after the freeze-thaw process. For this purpose, 32 semen samples of four Holstein bulls (normozoospermic, progressive motility > 70 %) and 32 semen samples of four bull (asthenozoospermic progressive motility < 40 %) were collected and pooled separately (normozoospermic and asthenozoospermic). Samples were then diluted into four equal aliquots of extender containing different vitamin D concentrations (0, 5, 10, and 50 ng/mL) and aspirated into a 0.5 mL straw. RESULTS: The percentages of sperm progressive motility and viability were significantly higher (P < 0.05) in 50 ng/mL of vitamin D in normozoospermic group. Sperm kinematics parameters including curvilinear velocity (VCL), straight-line velocity (VSL), and average path velocity (VAP) were significantly higher in the high dose (50 ng/mL) vitamin D-treated group compared to the low dose vitamin D-treated group (5ng/mL) in normozoospermic bull semen samples. The supplementation of the semen extender with different concentrations of vitamin D could not increase the rate of acrosome integrity in normozoospermic bulls compared to the control group (P < 0.05). In the asthenozoospermic group, 10 ng/mL vitamin D-treated group could increase the rate of plasma membrane integrity compared to 5 ng/mL vitamin D-treated group (P < 0.05). The percentages of early-apoptosis (P = 0.049) and late-apoptosis (P = 0.005) were significantly higher in the asthenozoospermic than the normozoospermic group. CONCLUSIONS: The present study revealed that a high dose (50 ng/mL) of vitamin D protected normozoospermic bulls' sperms from the freezing procedure and lead to higher quality of frozen-thawed bull sperm.

RéSUMé: CONTEXTE: L'asthénozoospermie est un facteur courant d'infertilité masculine, caractérisé par une diminution de la qualité du sperme. Il a été montré que les anti-oxydants amélioraient la fonction des spermatozoïdes, augmentaient les activités anti-oxydantes endogènes, et protégeaient les spermatozoïdes contre les dommages oxydatifs lors de la cryoconservation. Cette étude visait à évaluer les effets de la vitamine D sur la cinématique et l'apoptose des spermatozoïdes dans le sperme de taureaux qui présentaient une normozoospermie ou une asthénozoospermie après le processus de congélation-décongélation. À cette fin, 32 échantillons de sperme de quatre taureaux Holstein (normozoospermiques, mobilité progressive >70 %) et 32 échantillons de sperme de quatre taureaux (asthénozoospermiques ; mobilité progressive <40 %) ont été recueillis et regroupés séparément (normozoospermiques et asthénozoospermiques). Les échantillons ont ensuite été dilués en quatre aliquotes égales dans un milieu contenant différentes concentrations de vitamine D (0, 5, 10 et 50 ng/mL), puis aspirés dans une paille de 0.5 mL. RéSULTATS: Les pourcentages de mobilité progressive et de viabilité des spermatozoïdes étaient significativement plus élevés (p<0.05) avec 50 ng/mL de vitamine D dans le groupe normozoospermique. Dans les échantillons de sperme de taureaux normozoospermiques, les paramètres cinématiques des spermatozoïdes, incluant la vitesse curvilinéaire (VCL), la vitesse en ligne droite (VSL), et la vitesse moyenne du trajet (VAP), étaient significativement plus élevés dans le groupe traité par vitamine D à dose élevée (50 ng/mL) que dans le groupe traité par vitamine D à faible dose (5ng/mL). La supplémentation du milieu avec différentes concentrations de vitamine D n'a pas pu augmenter le taux d'intégrité de l'acrosome chez les taureaux normozoospermiques comparés au groupe témoin (p<0.05). Dans les échantillons de sperme de taureaux asthénozoospermiques, le groupe traité par vitamine D à la dose de 10 ng/mL a augmenté le taux d'intégrité de la membrane plasmique par comparaison au groupe traité par vitamine D à la dose de 5 ng/mL (p<0.05). Les pourcentages d'apoptose précoce (p=0.049) et d'apoptose tardive (p=0.005) étaient significativement plus élevés dans le groupe asthénozoospermique que le groupe normozoospermique. CONCLUSIONS: La présente étude a montré qu'une dose élevée (50 ng/mL) de vitamine D protégeait les spermatozoïdes des taureaux normozoospermiques lors de la procédure de congélation, et menait à une meilleure qualité des spermatozoïdes congelés-décongelés chez ces taureaux.