[Aims] Corpus luteum (CL) regression is required for subsequent estrus cycle in most mammals including cattle. This study aimed to evaluate the autophagic and lysosomal activities associated with apoptotic activity during bovine CL regression. [Material and Methods] Mid and late CL were collected from a local abattoir. Autophagic status was evaluated by measuring expression of autophagy-related genes (LC3α, LC3β, Atg3 and Atg7) by qRT-PCR and detecting LC3 protein by immunohistochemistry and western blotting. Next, activities of lysosome and cathepsin B were measured by commercial kits. Moreover expression of cathepsin-related genes (CTSB, CTSD, and CTSZ), that are members of lysosomal protease were performed in addition to cathepsin B protein was detected by immunohistochemistry. Apoptotic status was evaluated by measuring expression of CASP3 and detection of caspase 3 activity with commercial kit. [Results and Discussion] Expressions of LC3α, LC3β, Atg3 and Atg7 and the level of LC3 protein especially LC3II showed a significant increase in late CL than mid CL. Lysosomal activity showed a significant increase in late CL than mid CL. Moreover expressions of CTSB, CTSD, and CTSZ were significantly higher in late CL than those in mid CL. Furthermore higher level of activity and expression of cathepsin B protein were also observed in late CL than those in mid CL. A significantly higher CASP3 expression and higher activity of caspase 3 were observed in late CL than mid CL. These results suggest that autophagy and lysosomal activities have a role in bovine CL regression through expressions of autophagy-related genes and protein in relation to activities of lysosome and cathepsins.

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Identification of sex has a crucial role for poultry industries, birder and ecologist. In this study, we measured the hormonal content in eggshells during the post hatching to check whether it make possible to identify sex or not. If there is a difference in the amounts of sex hormones, it will be an alternative way to estimate sex ratio in a habitat especially for precocial birds. [Materials and methods] Eggshells were collected, dried, pulverized and mixed with 80% methanol. After centrifugation at 3000 rpm for 10 min, the supernatant samples were refrigerated at –20ºC for hormonal measurements by RIAs. [Results] In the present study, testosterone (T), estradiol (E2) and corticosterone (CORT) were measured. T and CORT concentration were detected at all stages in the eggshell and had contrary relationship to each other. T level was significantly high (p<0.01) in fresh eggshell and low at the 15^th d incubation and post hatched. Unlike to T result, CORT level was low after laying and gradually increased till hatching. During hatching CORT level was significantly different (p<0.01) between male and female eggshell. E2 was undetected since after laying, unless it was high at the 15^th d incubation and significantly high in female embryo (p<0.05). [Conclusion] Hatching time is kind of stressful condition for chicks. At this time chicks need to overcome this situation by increasing the stress hormone to expense more energy to survive. High CORT level may suppress the reproductive hormones. Further study is needed to verify the CORT level in male and female chicks’ eggshell and its effect on progesterone level.

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4-nitrophenol (PNP), a chemical material, is generally regarded as an environmental endocrine disruptor. Phytosterin (PS), a new feed additive, possesses the highly efficient antioxidant activities. Transcription factor nuclear factor-erythroid 2-related factor 2 (Nrf2) is an important regulator of cellular oxidative stress. This study examined the PNP-induced testicular oxidative damage and the PS-mediated protective actions in rats. The generation of MDA and H₂O₂ upon PNP and PS treatment was lower than that of PNP alone. These effects were accompanied by partially reversed levels of SOD, CAT, GSH and GSH-Px, though there was no return to normal. Moreover, PNP significantly reduced the caudal epididymal sperm counts and serum testosterone levels. Typical morphological changes were also observed in the testes of PNP-treated animals. PNP reduced the transcriptional level of Nrf2 as evaluated by RT-PCR, but promoted the dissociation from the Nrf2 complex, stabilization and translocation into the nucleus as evaluated by immunohistochemistry and western blot. Moreover, there was also an increased nuclear translocation upon PS treatment alone. In addition, PNP enhanced Nrf2-dependent gene expression of heme oxygenase-1 (HO-1) and Glutamate-cysteine ligase catalytic subunit (GCLC), however, simultaneous supplementation with PS restored these parameters near to the control. These results suggest that the Nrf2 pathway play an important role in PNP-induced oxidative damage and PS possesses attenuating effects on PNP-induced oxidative damage in rat testes.

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The oviduct is classically described as a sterile milieu, though pathogens and endotoxins could invade its mucosal surfaces via the uterus, peritoneal cavity and follicular fluid. We have recently shown that polymorphonuclear neutrophils (PMNs), the first line of defense against microorganisms, are present in the bovine oviduct fluid during preovulatory stages. We further suggested that the bovine oviduct provides a PGE-rich microenvironment to protect sperm from phagocytosis by PMNs that they possibly face in vivo, thereby supporting sperm survival in the oviduct. Alpha 1-acid glycoprotein (AGP) is a major acute-phase protein produced mainly in the liver, having an immunomodulatory functions. In this study, 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. AGP gene was expressed in cultured bovine oviduct epithelial cells (BOECs) and AGP protein was detected in oviduct fluid. Pre-exposure 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 secrete PGE2, and AGP and PGE2 at local levels additively inhibited sperm phagocytosis. The results provide evidence that locally produced AGP may be involved in protecting sperm from phagocytosis by PMNs in the bovine oviduct.

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Compromised Deoxyribonucleic acid (DNA) of sperm results in fertilization failure. Better detection of DNA intactness sperm would help to improve the protocols of cryopreservation. The present study was aimed to optimize the acridine orange staining to detect fragmented DNA of buffalo bull sperm. Semen was collected through artificial vagina from three buffalo (Bubalis bualis) bulls, pooled, diluted with Tris based extender and frozen using standard procedure. Two straws were thawed per replicate, pooled and divided into three aliquots to conduct three protocols of staining. This procedure involved smear formation of either washed or unwashed sperm (Protocol I). It was dried and fixed in carnoy’s solution (methanol and acetic acid in 3:1) for 2hr. Smear was dried and incubated in tampon solution (0.1M citric acid + 0.3M Na₂HPO4 in 16:1) either with heating or without heating (Protocol II). It was rinsed with water and stained with five concentrations of stain i.e. 1, 5, 200, 500 and 1000 µg/ml (Protocol III). In each protocol, positive control of fragmented DNA was used. Washing was found useful to remove stained debris. Heated incubation at 60^oC for 5–7 min in tempon solution proved better staining (50%) of damaged DNA. Higher concentrations (500 and 1000 µg/ml) stained the highly fragmented DNA (red color) but 500 µg/ml did not stain the moderately damaged DNA (yellow to orange color). It is concluded that buffalo sperm should be stained through washing, heated incubation in tempon solution and stained with higher concentration of acridine orange stain.

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Toxic effects induced by exposure to endocrine disruptors during fetal and neonatal periods could be irreversible and last for the whole life. Previous study showed rats neonatal exposed to estrogenic compound 17α-ethynyl estradiol (EE) within 24 hours after birth (postnatal day 0, PND0) had the shorter reproductive lifespan and reduced primordial follicle number in adulthood. To identify key target genes in neonatal EE exposed ovary, microarray analysis was conducted. It was resulted that Hrk, one of apoptosis activator, was down-regulated at PND1. To confirm the microarray data, ovaries were collected from PND1, 3, 7, 14 and 21 after exposing EE or sesame oil and the expressions of apoptosis related factors were analyzed by real time PCR. In addition to Hrk, the expression of puma, another apoptosis activator, was reduced at PND3 in EE exposed ovaries. The immunohistochemical analysis shows Hrk protein was localized in oocytes at PND1 ovaries. To investigate apoptosis in the ovary, TUNEL staining shows stronger positive staining in control ovaries than EE exposed ovaries at PND1. Furthermore, the reduction of primordial follicle number was observed at PND8 of EE exposed ovary in follicular composition analysis. To confirm the direct effect of EE on ovary, ovaries were collected from intact rats at PND0 and cultured with or without EE for 1 day, and then the gene expressions of apoptosis related factors were investigated. It was resulted that the expressions of Hrk and Puma were reduced in the ovaries cultured with EE. Taken together, we conclude that EE may directly inhibit apoptosis in neonatal ovary and disrupt primordial follicle formation via decreasing the expression of Hrk and Puma.

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We have shown that the bovine oviduct protects sperm against phagocytosis by polymorphonuclear neutrophils (PMNs) through PGE2 action. We also showed before that PGE2 with PGF2alpha, and vasoactive peptides, angiotensin II (Ang II) and endothelin-1 (EDN1) regulate bovine oviductal contractions. Thus, we investigated the possible effects of Ang II and EDN1 on the phagocytic activity of PMNs towards sperm. Ang II dose-dependently (10–8 to 10–11 M) increased the phagocytic activity of PMNs towards capacitated sperm by 135% of untreated control (100%) PMNs. This stimulatory effect of Ang II was partially eliminated by losartan, AT1 receptor antagonist. The gene expression of angiotensin-converting enzyme (ACE), and Ang II receptors AT1 and AT2 were detected in PMNs. Additionally, Ang II suppressed the ACE mRNA expression in PMNs as determined by real-time PCR. In contrast, EDN1 suppressed dose-dependently (10–8 to 10–11 M) the phagocytic activity of PMNs towards sperm by 60–90% of control. Moreover, this suppression was partially eliminated by ETB receptor antagonist (BQ-788). The gene expression of EDN1 and ETA and ETB receptors were detected in PMNs. The stimulation with EDN1 suppressed ETB receptors mRNA expression in PMNs. Furthermore, a stimulation of PMNs with both Ang II and EDN1 at concentrations detected in the oviduct (10–10 M) canceled the effects of both peptides on the phagocytic activity. The combinations of oviductal concentrations of Ang II, EDN1 and PGE2 resulted in the suppression of the phagocytic activity of PMNs towards sperm. The results give evidence that the angiotensin-endothelin-PGE2 system may be involved in the fine regulation of sperm survival in the bovine oviduct.

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