17β-estradiol and xenoestrogens reveal synergistic effect on mitochondria of human sperm

Objectives: The aim of the study was to investigate the influence of 17β-estradiol (main endogenous estrogen) and selected xenoestrogens (genistein, bisphenol-A), individually and in combination, on the mitochondrial function of human sper­matozoa. In natural environment, human beings are exposed to multiple xenoestrogens, so their impact is combined with endogenous steroids. Material and methods: The effects of ligands on human spermatozoa were assessed regarding the following phenomena: spermatozoa vitality (propidium iodide staining), phosphatidylserine membrane translocation (staining with annexin V marked with fluorescein), mitochondrial membrane potential (using JC-1 fluorochrome), and production of superoxide anion in mitochondria (using MitoSOX RED dye). Results: Two-hour incubation of spermatozoa with 17β-estradiol, genistein, and bisphenol-A neither altered cell vitality nor stimulated phosphatidylserine membrane translocation. Incubation of spermatozoa with 17β-estradiol or bisphenol-A sepa­rately, as well as incubation with the three ligands simultaneously, resulted in altered mitochondrial membrane potential. Spermatozoa incubation with the three ligands significantly increased the mitochondrial superoxide anion level. Conclusions: It seems safe to conclude that human spermatozoa mitochondria are target cell structures for both, 17β-estradiol and xenoestrogens. The reaction to the 17β-estradiol and xenoestrogens mixture suggests a synergistic mechanism of action. Xenoestrogens may increase the sensitivity of spermatozoa to 17β-estradiol

Hydroxyflutamide alters the characteristics of live boar spermatozoa

Our previous study revealed that in vitro incubation of boar ejaculates with hydroxyflutamide (OH-Flu) causes changes in sperm plasma membrane integrity and its stability and sperm mitochondrial oxidative capability. To broaden the knowledge of cellular physiology of spermatozoa, we investigated direct effects of OH-Flu administered for 2 and 24 hours at concentrations of 5, 50, and 100 µg/mL, on sperm mitochondrial membrane potential and mitochondrial superoxide anion production using JC-1 dye and MitoSOX Red fluorescent probe, respectively. We further measured phosphatidylserine membrane translocation (PST) from the inner to the outer layer of the sperm plasma membrane using an annexin-V binding assay. To provide new information of direct effects of OH-Flu on cell signaling pathway, we measured sperm intracellular calcium ion dynamics using Fluo-3. Finally, we assessed sperm motility using a computer-assisted spermatozoa analysis system. Motile sperm were highlighted using the "C-Ruch" computer program for detailed analysis of the straight line velocity distribution. For each functional test, boar spermatozoa were examined and analyzed by flow cytometry and/or confocal microscopy. The results revealed a significant decrease (P < 0.05) in sperm mitochondrial membrane potential and a concomitant increase (P < 0.05) in mitochondrial superoxide anion production after a 2-hour incubation with 50 mu g OH-Flu compared with the respective controls and other doses used (P < 0.05). The adverse effects of OH-Flu become strengthened over time (P < 0.05). Notably, 50 and 100 µg OH-Flu appeared to be effective in decreasing sperm motility. Hydroxyflutamide significantly decreased (P < 0.05) the fast sperm subpopulation percentage after 15 minutes and reduced the straight line velocity distribution (P < 0.05). An assessment of PST revealed an increase in the percentage of PST-positive spermatozoa (P < 0.05)only after exposure to OH-Flu for 24 hours. Moreover, OH-Flu at all concentrations induced a rapid increase in sperm intracellular calcium ion concentration. Altogether, the altered in vitro characteristics of live boar spermatozoa provide new insight into direct effects of OH-Flu on sperm mitochondrial membrane potential, superoxide anion production, translocation of membrane phosphatidylserine, free calcium ion dynamics, and sperm motility

Analysis of bone osteometry, mineralization, mechanical and histomorphometrical properties of tibiotarsus in broiler chickens demonstrates a influence of dietary chickpea seeds (Cicer arietinum L.) inclusion as a primary protein source.

This study was focused on analyzing the effects of dietary inclusion of raw chickpea seed as a replacement of soybean meal as a primary protein source on bone structure in broiler chickens. Broiler chickens (n = 160) received in their diet either soybean meal (SBM) or raw chickpea seeds (CPS) as a primary protein source throughout the whole rearing period (n = 80 in each group). On the 42th day randomly selected chickens from each group (n = 8) were slaughtered. Collected tibiotarsus were subjected to examination of the biomechanical characteristics of bone mid-diaphysis, microstructure of the growth plate and articular cartilages; the analysis of mineral content and crystallinity of mineral phase, and the measurements of thermal stability of collagen in hyaline cartilage were also carried out. The inclusion of chickpea seeds resulted in increase of bone osteometric parameters (weight, length and mid-diaphysis cross-sectional area) and mechanical endurance (yield load, ultimate load, stiffness, Young modulus). However, when loads were adjusted to bone shape (yield and ultimate stress) both groups did not differ. Mineral density determined by means of densitometric measurements did not differ between groups, however the detailed analysis revealed the differences in the macro- and microelements composition. The results of FT-IR and XRD analyses showed no effect of diet type on mineral phase crystallinity and hydroxyapatite nanocrystallites size. In trabecular bone, the increase of real bone volume (BV/TV) and number of trabeculae was observed in the CPS group. Total thickness of articular cartilage was the same in both groups, save the transitional zone, which was thicker in the SBM group. The total thickness of the growth plate cartilage was significantly increased in the CPS group. The area of the most intense presence of proteoglycans was wider in the SBM group. The structural analysis of fibrous components of bone revealed the increase of fraction of thin, immature collagen content in articular cartilage, trabeculae and compact bone in the CPS group. The dietary inclusion of CPS affected the thermal stability of collagen, as decrease of net denaturation enthalpy was observed. This study showed a beneficial effect of CPS on the skeletal development, improving the overall bone development and the microarchitecture of cancellous bone. It suggests that CPS can be a promising replacement for SBM in broilers feeding in the aspect of animal welfare related to the development of the skeletal system