Mohamed Ali Marey

An acute-phase protein as a regulator of sperm survival in the bovine oviduct: alpha 1-acid-glycoprotein impairs neutrophil phagocytosis of sperm in vitro.

We have previously shown that polymorphonuclear neutrophils (PMNs) are present in bovine oviduct fluid under physiological conditions, and that the oviduct provides a microenvironment that protects sperm from phagocytosis by PMNs. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver that has immunomodulatory functions. AGP mRNA is expressed in extrahepatic organs, such as the lung, kidney, spleen, lymph node, uterus, and ovary. Therefore, in this study, we investigated, 1) the local production of AGP in the bovine oviduct, 2) the effect of AGP on the phagocytic activity of PMNs for sperm and superoxide production and 3) the impact of AGP desialylation on the PMN phagocytosis of sperm. The AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Preexposure of PMNs to AGP at physiological levels impaired PMN phagocytosis for sperm and superoxide generation. The desialylation of AGP eliminated these suppressive effects of AGP on PMN. Scanning electron microscopy revealed that AGP drastically reduced the formation of DNA-based neutrophil extracellular traps (NETs) for sperm entanglement. Additionally, AGP dose-dependently stimulated BOECs to produce prostaglandin E2 (PGE2) which has been shown to partially contribute to the regulation of sperm phagocytosis in the bovine oviduct. AGP and PGE2 at concentrations detected in the oviducts additively suppressed sperm phagocytosis by PMNs. These results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

Evidence for a novel, local acute-phase response in the bovine oviduct: Progesterone and lipopolysaccharide up-regulate alpha 1-acid-glycoprotein expression in epithelial cells in vitro

Little is known about the local production and function of alpha 1‐acid glycoprotein (AGP), an acute‐phase protein, in the female reproductive tract. This study aimed to investigate the regulation and immune function of AGP in cultured bovine oviduct epithelial cells. Analysis by Western blotting and immunohistochemistry revealed that bovine oviduct tissue expresses AGP protein in epithelial cells and the smooth muscle layer. Stimulation of bovine oviduct epithelial cells in culture with either progesterone (1 ng/ml) or lipopolysaccharide (LPS, 10 ng/ml) induced both mRNA expression and secretion of AGP. Estradiol (1 ng/ml), progesterone (1 ng/ml), and luteinizing hormone (10 ng/ml), which are observed during the peri‐ovulatory period in oviduct tissues (steroids) or in circulation (luteinizing hormone), suppressed LPS‐induced expression and secretion of AGP, which in turn induced the expression of Toll‐like receptor‐4 (TLR‐4) and interleukin‐1β (IL‐1B), but suppressed TLR‐2 and tumor necrosis factor‐α (TNFA) expression. AGP also inhibited LPS‐induced TLR‐2 and TNFA expression, but had no effect on LPS‐induced TLR‐4 and IL‐1B expression. These findings suggest that oviductal epithelial cells can participate in antimicrobial processes through the secretion of AGP, which is partly regulated by ovarian steroids. Moreover, oviductal AGP may regulate the response of epithelial cells, thereby reducing the expression of the acute pro‐inflammatory cytokine TNFA, which could contribute to the local homeostasis during the acute response to endotoxin release in the oviducts anti‐infection process. Mol. Reprod. Dev. 81: 861–870, 2014.

An autoregressive logistic model to predict the reciprocal effects of oviductal fluid components on in vitro spermophagy by neutrophils in cattle

After intercourse/insemination, large numbers of sperm are deposited in the female reproductive tract (FRT), triggering a massive recruitment of neutrophils (PMNs) into the FRT, possibly to eliminate excessive sperm via phagocytosis. Some bovine oviductal fluid components (BOFCs) have been shown to regulate in vitro sperm phagocytosis (spermophagy) by PMNs. The modeling approach-based logistic regression (LR) and autoregressive logistic regression (ALR) can be used to predict the behavior of complex biological systems. We, first, compared the LR and ALR models using in vitro data to find which of them provides a better prediction of in vitro spermophagy in bovine. Then, the best model was used to identify and classify the reciprocal effects of BOFCs in regulating spermophagy. The ALR model was calibrated using an iterative procedure with a dynamical search direction. The superoxide production data were used to illustrate the accuracy in validating logit model-based ALR and LR. The ALR model was more accurate than the LR model. Based on in vitro data, the ALR predicted that the regulation of spermophagy by PMNs in bovine oviduct is more sensitive to alpha-1 acid glycoprotein (AGP), PGE2, bovine serum albumin (BSA), and to the combination of AGP or BSA with other BOFCs.

Zearalenone (ZEN) disrupts the anti-inflammatory response of bovine oviductal epithelial cells to sperm in vitro

Dietary contamination by Zearalenone (ZEN) has a detrimental effect on bovine fertility. Recently, we showed a novel anti-inflammatory response of bovine oviductal epithelial cells (BOEC) to active sperm cells in vitro. The aim of the present study was to investigate the effect of ZEN exposure of BOEC on the immune-related cytokine expression in response to bovine sperm. At concentrations of 100 and 1000ng/mL, ZEN induced the expression of TNF and IL1B (pro-inflammatory cytokines) as well as IL8 (chemokine) in BOEC in a dose-dependent manner. Furthermore, ZEN induced PTGES expression and PGE2 secretion in BOEC. Sperm co-culture induced an anti-inflammatory response in BOEC with upregulation of TGFB, secretion of PGE2 and downregulation of TNF. Most importantly, ZEN at 1-1000ng/mL eliminated the response of BOEC to sperm. Estradiol-17β (5ng/mL) treatment did not produce the same effects as ZEN, suggesting that the response of BOEC to ZEN is, at least in part, not mediated by estrogen receptors. Taken together, ZEN can produce inflammatory effects on BOEC by stimulating the expressions of pro-inflammatory cytokines and disrupt the normal interaction between sperm and BOEC at the level of cytokine expressions and PGE2 production. Thus, exposure of the bovine oviduct to ZEN may negatively affect sperm survival and reduce fertility.

Angiotensin II increases sperm phagocytosis by neutrophils in vitro: A possible physiological role in the bovine oviduct.

This study aimed to investigate the possible effects of the vasoactive peptide angiotensin II (ANG II), secreted by bovine oviduct epithelial cells, on the in vitro phagocytic activity of polymorphonuclear leukocytes, specifically neutrophils, towards sperm. The measured concentrations of ANG II in oviduct flushes and conditioned medium from primary bovine oviduct epithelial culture ranged from 10−10 to 10−11 M. In our experiments, neutrophils were incubated for 2 hr with ANG II (0, 10−11, 10−10, 10−9, and 10−8 M). Phagocytosis and superoxide production were then assessed by co‐incubation of these neutrophils with sperm pretreated to induce capacitation, revealing a dose‐dependent increase in both metrics by ANG II. This stimulatory effect of ANG II was eliminated by losartan, an angiotensin receptor type 1 (AGTR1) antagonist. ANG II also suppressed neutrophil transcription of angiotensin converting enzyme‐1 (ACE) and AGTR1, but not AGTR2, suggesting the involvement of the AGTR1 receptor‐mediated pathway in the response to sperm. Scanning electron microscopy further revealed that incubation of neutrophils with ANG II stimulated the formation of DNA‐based extracellular traps for sperm entanglement. The addition of prostaglandin E2 at concentrations found in the oviduct suppressed the ANG II‐stimulated phagocytic activity of neutrophils towards sperm. Thus the physiological levels of ANG II stimulate neutrophil phagocytosis of sperm in vitro, and suggest that an angiotensin/prostaglandin E2 system may fine‐tune the local immune response that fosters sperm survival in the bovine oviduct. Mol. Reprod. Dev. 83: 630–639, 2016.

Short communication: Urea induces T helper 2 (Th2) type environment at transcriptional level and prostaglandin E2 secretion in bovine oviduct epithelial cells in culture.

Excess dietary protein intake in early lactation dairy cows resulting in blood urea nitrogen of greater than 19 to 20mg/dL is associated with decreased fertility. Little is known about the local interference of urea in the normal immunological environment of the oviduct that provides optimal conditions for early reproductive events. A bovine oviduct epithelial cell (BOEC) culture was used to determine how urea influences immune environment. The BOEC monolayer was supplemented with low (20mg/dL) and high (40mg/dL) concentrations of urea together with ovarian steroids, estradiol (1ng/mL) and progesterone (1ng/mL), and LH (10ng/mL) at concentrations observed during the preovulatory period. The urea values used in this study were equivalent to 9.3 and 18.7mg/dL of blood urea nitrogen, which are typically common in lactating dairy cows with low or high protein intake, respectively. Stimulation of BOEC with 40mg/dL of urea induced gene expression of IL10 and IL4, epithelial-derived T helper type 2-driving (anti-inflammatory) cytokines as well as mPGES-1 expression and prostaglandin E2 (PGE2) secretion. However, urea concentrations of both 20 and 40mg/dL failed to alter the expression of IL1B and TNFA, Th1-driving cytokines, and the gene expression of TLR4. However, a concentration of 40mg/dL of urea stimulated α 1-acid glycoprotein expression, an acute phase protein. Data from this in vitro study suggest that urea, at least in part, contributes to influence the expression of some immune-related genes toward T helper type 2 type and prostaglandin E2 secretion, leading to disruption in local environment for fertilization and early embryonic development.