Progesterone (P 4 ) secreted by the corpus luteum (CL) is critical for in utero embryo survival and development, although CL proteins are key regulatory factors during the luteal phase. We, therefore, characterised protein expression patterns in ovine CL of pregnancy (days 12, 16 and 20) compared with those of controls, CL of oestrous cycle (days 12 and 16), using two-dimensional gel electrophoresis (2DE) gel-based proteomics. Proteins in 24 significantly altered spots were identified by tandem mass spectroscopy. At the time of embryo implantation (day 16), 77 spots were up-regulated and 101 spots were down-regulated in CL of pregnancy compared with regressed CL. Vimentin, lamin A/C (LMNA), [Mn] superoxide dismutase (SOD2), isocitrate dehydrogenase 1, annexin A1 and elongation factor Tu, mitochondrial (TUFM) altered during CL regression, whereas glutathione S-transferase A1, apolipoprotein A-1, myxovirus resistance protein 1, ornithine aminotransferase and enoyl-CoA hydratase, mitochondrial (ECHS1) tended to be altered during CL maintenance. biliverdin reductase B (BLVRB), FDXR, guanine nucleotide-binding protein G(I)/G(S)/G(T) subunit beta-2 (GNB2) and cytochrome bc1 complex subunit 1, mitochondrial (UQCRC1) showed divergent expression during CL regression and maintenance. The expression of two representative proteins, SOD2 and BLVRB, by western blot increased in CL of non-pregnant ewes on day 16 compared with that on day 12. SOD2 and BLVRB were localised in the large and small luteal cells and endothelial cells of CL over peri-implantation periods. 2DE gel and mass spectrometry have been used, for the first time, to study ovine CL function. We have identified proteins involved in key pathways, including oxidative stress, steroidogenesis, signal transduction and apoptosis, which have not previously been associated with changes occurring in the CL during the peri-implantation period. These proteins are most likely involved with mechanisms allowing the CL to produce P 4 during early pregnancy.

Formaldehyde is a common agent in our surrounding environment and can adversely affect the male reproductive system. In this study, the effectiveness of Matricaria chamomilla (MC) extract as an antioxidant was investigated in rats treated with formaldehyde. Thirty‐two male Wistar rats were randomly divided into six groups: F (10 mg/kg formaldehyde), M200 (200 mg/kg MC extract), M500 (500 mg/kg MC extract), FM200 (10 mg/kg formaldehyde and 200 mg/kg MC extract), FM500 (10 mg/kg formaldehyde and 500 mg/kg MC extract) and control group (0.9% normal saline). Formaldehyde and MC extract were administered daily for 30 consecutive days via intraperitoneal injection. Hormonal status, sperm parameters, testis tissue histology, germinal cells apoptosis and stereological analyses of testis tissue were investigated. Testosterone and LH levels were significantly increased in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). Sperm count, motility and viability were significantly enhanced in FM200, FM500, F200 and F500 groups compared to F group (p ≤ 0.05). A decrease in the number of apoptotic germ cells in FM200, FM500, M200 and M500 groups (p ≤ 0.05) was evident. In particular, the MC extract in dose 500 mg/kg is seen to reduce the adverse effects of formaldehyde on the reproductive system of male rats.

The expression and regulation of endometrial proteins are crucial for conceptus implantation and development. However, little is known about site-specific proteome profiles of the mammalian endometrium during the peri-implantation period. We utilised a two-dimensional gel electrophoresis/mass spectrometry-based proteomics approach to compare and identify differentially expressed proteins in sheep endometrium. Caruncular and intercaruncular endometrium were collected on days 12 (C12) and 16 (C16) of the oestrous cycle and at three stages of pregnancy corresponding to conceptus pre-attachment (P12), implantation (P16) and post-implantation (P20). Abundance and localisation changes in differentially expressed proteins were determined by western blot and immunohistochemistry. In caruncular endometrium, 45 protein spots (5% of total spots) altered between day 12 of pregnancy (P12) and P16 while 85 protein spots (10% of total spots) were differentially expressed between P16 and C16. In intercaruncular endometrium, 31 protein spots (2% of total spots) were different between P12 and P16 while 44 protein spots (4% of total spots) showed differential expression between C12 and C16. The pattern of protein changes between caruncle and intercaruncle sites was markedly different. Among the protein spots with implantation-related changes in volume, 11 proteins in the caruncular endometrium and six proteins in the intercaruncular endometrium, with different functions such as protein synthesis and degradation, antioxidant defence, cell structural integrity, adhesion and signal transduction, were identified. Our findings highlight the different but important roles of the caruncular and intercaruncular proteins during early pregnancy.

Context. Heat Shock Protein 60 (HSP60) is a chaperone protein which is involved in proteins transfer and re-folding of proteins. Objective. Importance of HSP60 in sperm capacitation and facility of sperm-oocyte membrane binding was confirmed, therefore in this study the effect of HSP60 on the rate of in vitro fertilization and the cleavage rate in mouse embryo was investigated. Design. Ten male mice and twenty five female mice were involved to collect sperms and oocytes required for this study. Subjects and Methods. Sperms were collected from the epididymis of male mouse and oocytes were collected from the oviduct of female mouse following ovarian hyperstimulation. Then, capacitated sperms and oocytes were placed together in fertilization medium in four groups in the presence of different concentrations of HSP60 (10, 50 and 100 ng/mL) and in the absence of HSP60. After calculation of the fertilization rate, zygotes were transformed into the other medium for development and the cleavage rate was monitored to blastocyst stage. Results. There was not a significant difference in the rate of fertilization between 10 ng/mL HSP60 group and the control group. The rate of fertilization and two-cell embryo development decreased significantly (P≤0.05) in 100 ng/mL HSP60 compared to other experimental and control groups. Further, the rate of two-cell embryo development increased significantly (P≤0.05) in 10 ng/mL HSP60 compared to other experimental and control groups. Conclusions. The present study demonstrated that HSP60 in low dose had a positive effect on two-cell embryo development, however it did not have any significant effect on the fertilization rate. Conversely, HSP60 had adverse effects on the fertilization and cleavage rates at higher doses.

Recently, gel-based proteomics has been increasingly applied to investigate proteins involved in female reproductive tract in healthy and disease states. Gel-based proteomics coupled by mass spectrometry (MS) facilitate the identification of new proteins playing roles in cellular and molecular interactions underlying female reproductive biology and it is a useful method to identify novel biomarkers of diseases by studying thousands of proteins simultaneously involved in female reproductive tract in healthy state compared to disease state. This review will discuss the best studies areas contributed to female reproductive biology in which gel-based proteomics coupled by MS has been applied to generate proteome of female reproductive tract in a healthy state.

The stage-specific expression of functional proteins within the endometrium, and their regulation by conceptus-derived signals, are crucial for conceptus development and successful establishment of pregnancy. Accurate knowledge of endometrium-conceptus interactions is key for the development of effective strategies to improve conceptus implantation rates both following natural conception and/or assisted reproductive technologies. The unilateral pregnant ewe provides a powerful experimental model for the study of endometrial function in the presence or absence of conceptuses during the peri-implantation period. Two-dimensional gel electrophoresis and mass spectrometry-based proteomics were used to compare and identify differentially expressed proteins in caruncular endometrium collected from the gravid uterine horns and the non-gravid uterine horns at the time of conceptus attachment (day 16 of pregnancy) and early post-implantation period (day 20 of pregnancy). Fifty seven protein spots were up-regulated in the gravid horn at day 16 of pregnancy and twenty seven protein spots were up-regulated in the gravid horn at day 20 of pregnancy. Sixteen proteins with different functions such as protein metabolism, cholesterol and ion transport and cell adhesion were identified. In conclusion, the use of the unilaterally pregnant ewe model provides evidence that the early implantation and post-implanting conceptus-derived signals up-regulate caruncle endometrial proteins, including carbonic anhydrase 2 (CA-II) and apolipoprotein A-1 (APOA1) and down-regulate caruncle endometrial proteins, including adenosylhomocysteinase (AHCY) and heat shock 60kDa protein 1 (HSP60). These regulated proteins are likely involved in providing a suitable intra-uterine environment required for conceptus attachment, implantation, early post-implantation development and the successful establishment of pregnancy in sheep.

Conceptus-derived paracrine signals play crucial roles in the preparation of a uterine environment capable of supporting implantation and development of the conceptus. However, little is known about the regulation of endometrial tryptophanyl tRNA synthetase (WARS) and manganese superoxide dismutase (SOD2) protein expression by the implanting and post-implanting conceptus. We hypothesized that the conceptus-derived signals favourably influences uterine environment for implantation through regulation of WARS and SOD2 expression in ovine caruncular endometrium. To test this hypothesis, WARS and SOD2 protein and mRNA expression was determined in caruncular endometrial tissues of unilaterally pregnant ewes at implantation (day 16) and post-implantation (day 20) periods. WARS protein expression increased in caruncular tissues of the gravid uterine horns compared with the non-gravid uterine horns on days 16 and 20 of pregnancy. There were no changes in SOD2 protein expression between the gravid and non-gravid uterine horns, irrespective of the day of pregnancy. On day 16 of pregnancy, there were no differences in WARS and SOD2 mRNA expression between the gravid and non-gravid uterine horns but expression of both genes was higher in the gravid uterine horns when compared with the non-gravid uterine horns on day 20 of pregnancy. In conclusion, the use of the unilaterally pregnant ewe model provides for the first time firm evidence that the early implantation and post-implanting conceptus-derived signals up-regulate WARS protein expression within the caruncular endometrium. Further studies are necessary to identify these signalling molecules and to understand mechanisms whereby they exert paracrine action within the endometrium.

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