We have recently shown that sperm attachment to bovine endometrial epithelial cells (BEECs) triggers uterine local innate immunity with induction of a pro-inflammatory response in vitro , however details of the mechanism remain unknown. Here, we investigated the involvement of Toll-like receptor 2/4 (TLR2/4) pathway in mediating sperm-BEECs inflammatory process. Immunohistochemistry of the uterine tissue revealed that TLR2 and TLR4 proteins were present in the luminal and glandular epithelia of bovine endometrium. Moreover, BEECs monolayers were treated with TLR2 agonist (Pam; 0, 10, 100, and 1000 ng/ml) or TLR4 agonist (LPS; 0, 0.1, 1, and 10 ng/ml) for 0, 1, 3, or 6 h, followed by evaluating mRNA expression of the pro-inflammatory genes ( TNFA , IL-1B , IL-8 , and PGES ) in BEECs using a real-time PCR. Both Pam and LPS treatments showed a dose-dependent stimulation of mRNA expression of the pro-inflammatory genes. To elucidate the functional role of TLR2/4 in sperm-BEECs interaction, BEECs monolayers were incubated with either TLR2 antagonist or TLR4 antibody for 2 h prior to the co-culture with sperm for 3 h. Importantly, pre-incubation of BEECs with TLR2 antagonist or TLR4 antibody prevented the stimulatory effect of sperm on the transcription of pro-inflammatory genes in BEECs. Furthermore, sperm increased the phosphorylation levels of TLR2/4 downstream targets (p38MAPK and JNK) in BEECs within 1 h of the co-culture. Treatment of BEECs with TLR2 antagonist prior to sperm addition inhibited JNK phosphorylation, while TLR4 antibody inhibited the phosphorylation of both p38MAPK and JNK. In conclusion, the present in vitro findings strongly suggest that bovine endometrial epithelial cells respond to sperm via TLR2/4 signal transduction.

In the cow, cryopreserved semen is inseminated into the uterus, and most of sperm are removed by backflow and phagocytes. Nevertheless, the mechanism responsible for sperm phagocytosis is unclear. Here, we used cultured bovine uterine epithelial cells (BUECs) to investigate the uterine response to sperm and the mechanism that activates polymorphonuclear neutrophils (PMNs). BUEC monolayers were co-cultured with different numbers of washed sperm obtained from cryopreserved semen (10 , 10 , and 10 sperm/ml) for 3 hr. Sperm dose-dependently up-regulated IL8 (Interleukin 8). Sperm at 10 /ml increased mRNA expression of TNFA (Tumor necrosis factor alpha), IL1B (Interleukin 1B), NFKB2 (Nuclear factor kappa B2), and C3 (Complement factor 3), as well as PGES (Prostaglandin E synthase) expression and PGE release. Live sperm, but not dead sperm, attached to BUECs, and dead sperm did not induce an acute inflammatory response. Time-dependent effects were evaluated by co-culture of 10 /ml washed sperm with BUECs for 0, 1, 3, and 6 hr. The number of detached sperm increased gradually toward 6 hr. Maximum mRNA expression of IL8, TNFA, IL1B, and NFKB2 was induced at 3 hr, while C3 continued to increase toward 6 hr. Sperm did not stimulate mRNA expression of anti-inflammatory cytokines TGFB1 (Transforming growth factor beta 1) or IL10 (Interleukin 10). Medium conditioned by sperm co-incubated with BUECs stimulated PMNs phagocytosis of sperm in vitro. Fresh media supplemented with low levels of IL1B, TNFA, and PGE up-regulated sperm phagocytosis by PMNs as well. In conclusion, our findings strongly suggest that the active sperm attach to BUECs and trigger uterine local innate immunity with induction of a pro-inflammatory response that enhances sperm phagocytosis by PMNs.

The current study was conducted to investigate the protective properties of Eucalyptus globulus leaves methanolic extract (EGLME) against diclofenac sodium (DS) induced hepatorenal and testicular toxicity in male rats. A total of 40 rats were equally divided into 4 groups, Control, Diclofenac sodium (DS), EGLME and DS + EGLME groups, respectively. DS and EGLME were administered orally at dose rate 2.5 and 100 mg/kg BW, 4 times/week for 8 weeks, respectively. Administration of DS distorted hepatorenal functions manifested by alteration of serum levels of ALT, AST, total protein and albumin, creatinine and urea with changes of histological architectures. DS caused reproductive toxicity represented by minimized sperm count, individual sperm motility and viability; depleted concentration of reduced glutathione (GSH) in testicular tissue; and decreased testosterone level with alteration in testicular histological features. In contrast, co-treatment of DS intoxicated rats with EGLME protected rats against the adverse effects of DS revealing enhancing properties of EGLME on rats’ liver, kidney and testes. In conclusion, we demonstrated that EGLME had a potent protecting property against DS induced hepatic, renal and testicular toxicity in male rats, with special concern to testicular tissue via modulation of GSH as an oxidant marker. Taxonomy (classification by EVISE): Diclofenac sodium toxicity (hepatorenal and testicular toxicity), co-treatment with natural herbal extract, blood biochemical assays, tissue anti-oxidants assay, histopathology and reproductive indices analyses.

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Sodium nitrite is widely used as a food additive in processed meats and fish products (Jørgen & Marianne, 2011) to prevent the growth of Clostridium botulinum, a major cause of food poisoning (lee et al., 2018). Moreover, sodium nitrites can regulate many physiological processes (Deng, 2006) and it has therapeutic applications for certain illnesses such as myocardial infarction (Webb et al., 2004) and hemorrhagic cerebral vasospasm (Pluta, Dejam, Grimes, Gladwin, & Oldfield, 2005). However, excessive sodium nitrite exerts a toxic effect through generation of free radicals, which causes oxidant/ antioxidant imbalance and results in detrimental biological effects (Jensen, 2007; Naik, Pilgaonkar, & Panda, 2006). For example, nitrite was shown to react with food amines in the stomach acidic media producing carcinogenic

This study examined the protective effect of earthworm extract (EE) on acrylamide (ACR)-induced reproductive dysfunction. Forty male rats were allocated into four groups (n = 10). The G I (control) group received distilled water (D.W.). The G II group received ACR (5 mg kg−1 B.W. in D.W.) 5 days per week, orally, for 3 weeks. The G III group was administered EE (300 mg kg−1 B.W in D.W.) 5 days per week, orally, for 3 weeks. The G IV group was pretreated with EE for 3 weeks and then co-treated with EE and ACR for an additional 3 weeks. ACR decreased the number of sperm, sperm viability, and total motility. However, it increased testosterone levels with no effect on the FSH or LH levels. Moreover, ACR increased the concentrations of malondialdehyde (MDA) and nitric oxide (NO). Meanwhile, it decreased the glutathione (GSH) concentration in testicular tissues. Notably, the expression levels of p53 and Ki-67 were increased in the degenerated spermatogenic cells and in the hyperplastic Leydig cells of the testis of the ACR-treated group, respectively. Acrylamide induced alterations in the testicular tissue architecture. Interestingly, EE restored the sperm parameters and recovered the testicular histological structures and the biochemical alterations induced by ACR. In conclusion, earthworm extract ameliorated ACR-induced reproductive toxicity via restoring the testicular antioxidant balance and suppressing p53 and Ki-67 expressions in testicular tissues.

No abstract

The aim of this study was to perform ultrasonographic imaging of the testes and accessory sex glands in adult Barki rams during the breeding season. The impact of testosterone on the Doppler indices of accessory sex glands was also investigated. Scrotal contents, pelvic urethra and accessory sex glands of twelve mature Barki rams were scanned with multiple imaging of B-mode and colour Doppler ultrasonography. Serum concentrations of testosterone, FSH and LH were determined. The results revealed that the breeding season changed the echogenicity of testicular parenchyma, spermatic cord, epididymal tail, glans penis and echotexture of accessory sex glands. Serum testosterone was 7.27±0.37 ng/mL, FSH was 6.46±0.2 and LH was 5.6±0.28 m IU/mL. The pulsatility index (PI) for the supra-testicular artery (STA), marginal artery (MA) of the testes and epididymal tail was 1.01±0.07, 0.58±0.04 and 0.5±0.04. The resistive index (RI) for the same structures was 0.6±0.04, 0.33±0.04 and 0.3±0.03, respectively. Importantly, testosterone downregulated PI and RI of the ampulla, vesicular gland, prostate gland and bulbourethral gland. In conclusion, the breeding season changed the echogenicity of reproductive organs and accessory genital glands of rams, and testosterone regulated the hemodynamic parameters of the accessory sex glands.