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The growing zone (GZ) of the unicellular coenocytic sporangiophore of Phycomyces blakesleeanus represents the site of stimulus reception (light, gravity, gas) and stimulus response, i.e., local modulations of the elongation growth, which may result, in dependence of the stimulus direction, in tropic bending. Until now, evidence for a possible participation of the columella in sensory reception is absent. We confirm with light microscopy earlier studies that show that the GZ and the columella are not separated by a membrane or cell wall, but rather form a spatial continuum that allows free exchange of cytoplasm and organelle transport. Evidence is presented that the columella is responsive to external stimuli. Columellae, from which spores and sporangial cell wall had been removed, respond to exogenous auxin with a local depolarization of the membrane potential and an increased growth rate of the GZ. In contrast, auxin applied to the GZ causes a decrease of the growth rate irrespective of the presence or absence of sporangia. The response pattern is specific and relevant for the sensory reception of Phycomyces, because the light-insensitive mutant C148carAmadC, which lacks the RAS-GAP protein MADC, displays abnormal IAA sensitivity and membrane depolarization. We argue that the traditional concept of the GZ as the only stimulus-sensitive zone should be abandoned in favor of a model in which GZ and columella operate as a single entity capable to orchestrate a multitude of stimulus inputs, including auxin, to modulate the membrane potential and elongation growth of the GZ.

Metabolism of metals in microalgae and adaptation to metal excess are of significant environmental importance. We report a three-step mechanism that the green microalga Chlorella sorokiniana activates during the acquisition of and adaptation to manganese (Mn), which is both an essential trace metal and a pollutant of waters. In the early stage, Mn2+ was mainly bound to membrane phospholipids and phosphates in released mucilage. The outer cell wall was reorganized and lipids were accumulated, with a relative increase in lipid saturation. Intracellular redox settings were rapidly altered in the presence of Mn excess, with increased production of reactive oxygen species that resulted in lipid peroxidation and a decrease in the concentration of thiols. In the later stage, Mn2+ was chelated by polyphosphates and accumulated in the cells. The structure of the inner cell wall was modified and the redox milieu established a new balance. Polyphosphates serve as a transient Mn2+ storage ligand, as proposed previously. In the final stage, Mn was stored in multivalent Mn clusters that resemble the structure of the tetramanganese–calcium core of the oxygen-evolving complex. The present findings elucidate the bioinorganic chemistry and metabolism of Mn in microalgae, and may shed new light on water-splitting Mn clusters.

Background: Prostasomes, extracellular vesicles (EVs) abundantly present in seminal plasma, express distinct tetraspanins (TS) and galectin-3 (gal-3), which are supposed to shape their surface by an assembly of different molecular complexes. In this study, detergent-sensitivity patterns of membrane-associated prostasomal proteins were determined aiming at the solubilization signature as an intrinsic multimolecular marker and a new parameter suitable as a reference for the comparison of EVs populations in health and disease. Methods: Prostasomes were disrupted by Triton X-100 and analyzed by gel filtration under conditions that maintained complete solubilization. Redistribution of TS (CD63, CD9, and CD81), gal-3, gamma-glutamyltransferase (GGT), and distinct N-glycans was monitored using solid-phase lectin-binding assays, transmission electron microscopy, electrophoresis, and lectin blot. Results: Comparative data on prostasomes under normal physiology and conditions of low sperm count revealed similarity regarding the redistribution of distinct N-glycans and GGT, all presumed to be mainly part of the vesicle coat. In contrast to this, a greater difference was found in the redistribution of integral membrane proteins, exemplified by TS and gal-3. Accordingly, they were grouped into two molecular patterns mainly consisting of overlapped CD9/gal-3/wheat germ agglutinin-reactive glycoproteins and CD63/GGT/concanavalin A-reactive glycoproteins. Conclusions: Solubilization signature can be considered as an all-inclusive distinction factor regarding the surface properties of a particular vesicle since it reflects the status of the parent cell and the extracellular environment, both of which contribute to the composition of spatial membrane arrangements.

The growing zone (GZ) of the unicellular coenocytic sporangiophore of Phycomyces blakesleeanus represents the site of stimulus reception (light, gravity, gas) and stimulus response, i.e., local modulations of the elongation growth, which may result, in dependence of the stimulus direction, in tropic bending. Until now, evidence for a possible participation of the columella in sensory reception is absent. We confirm with light microscopy earlier studies that show that the GZ and the columella are not separated by a membrane or cell wall, but rather form a spatial continuum that allows free exchange of cytoplasm and organelle transport. Evidence is presented that the columella is responsive to external stimuli. Columellae, from which spores and sporangial cell wall had been removed, respond to exogenous auxin with a local depolarization of the membrane potential and an increased growth rate of the GZ. In contrast, auxin applied to the GZ causes a decrease of the growth rate irrespective of the presence or absence of sporangia. The response pattern is specific and relevant for the sensory reception of Phycomyces, because the light-insensitive mutant C148carAmadC, which lacks the RAS-GAP protein MADC, displays abnormal IAA sensitivity and membrane depolarization. We argue that the traditional concept of the GZ as the only stimulus-sensitive zone should be abandoned in favor of a model in which GZ and columella operate as a single entity capable to orchestrate a multitude of stimulus inputs, including auxin, to modulate the membrane potential and elongation growth of the GZ.

Background: Human seminal prostasomes are intrinsically heterogeneous extracellular vesicles (EVs) whose composition is, additionally, influenced by different physiological conditions. Aiming at the molecular properties of the prostasomal surface exemplified by glycan compositions as a possible distinction factor, we applied lectin-affinity chromatography (LAC) as a new tool for their separation. Since glycans, generally, exhibit various biological activities, introduction of glyco-parameters as reference could upgrade standardization of EVs isolated by different methods and intended for use in biomedicine. Methods: Preparations of seminal prostasomes from normozoospermic (sPro-N) and oligozoospermic (sPro-O) men were subjected to LAC on concanavalin A (Con A) and wheat germ agglutinin (WGA) columns. Prostasomes recovered in LAC-separated fractions were characterized according to the distribution of selected markers: gamma-glutamyl transferase (GGT), alkaline phosphatase (ALP), tetraspanin CD63, and total protein/glycoprotein composition. Results: Two CD63-immunoreactive populations exhibiting prostasome signature bands but differing in GGT activity and surface glycans were separated on the WGA column. Additional populations having distinct profiles of total glycoproteins and which can be tracked down by ALP activity were enriched on the Con A column. WGA-separated populations were similar in sPro-N and sPro-O, whereas Con A-separated ones were strikingly different. Conclusions: Membrane-associated gamma-glutamyl transferase and alkaline phosphatase in the context of Con A- and WGA-reactive glycans mark seminal prostasomes populations from normozoospermic and oligozoospermic men.

Overt and subclinical maternal hypothyroidism affects cartilage and bone extracellular matrix formation during fetal endochondral bone development. Less is known about the effect maternal hypothyroidism has on cell proliferation and death of growth plate chondrocytes. This study aimed to evaluate the expression of proliferation (Ki67), autophagy (LC3) and apoptosis (caspase 3) markers in the epiphyseal cartilage of the tibia in pups from subclinical and overt hypothyroid does, in their early postnatal development.Hypothyroidism was induced with propylthiouracil using low (1.5 mg/L) for subclinical (H1) and high dose (150 mg/L) for overt (H2) form. Propylthiouracil was administered through drinking water in pregnant Albino Oxford does from the first day of gravidity and during lactation. Control (C) group was not treated. Six, seven-day-old male pups from each group were euthanized. Histological examinations were performed on paraffin sections of the proximal tibial growth plate. Immunohistochemistry was used to assess the expression of Ki67, LC3 and caspase 3. The number of Ki67 positive chondrocytes was evaluated in the resting and proliferating zone using Image J. The expression of LC3 was performed by counting the number of positive dots in chondrocytes.The number of Ki67 positive chondrocytes was higher in both hypothyroid groups, while the number of LC3 positive dots per cell was lower indicating that the chondrocyte cell cycle in hypothyroid animals is faster and that the basal level of autophagy is reduced due to frequent mitosis. Compared to controls, caspase 3 expression in the terminal hypertrophic chondrocytes was reduced in H1 and completely absent in H2 pups which might indicate impaired differentiation.We conclude that both forms of maternal hypothyroidism in rats lead to accelerated proliferation, slowed autophagy and compromised apoptosis of terminal hypertrophic chondrocytes in the early infantile period and a delay in cartilage to bone transition.

Microalgae have evolved mechanisms to respond to changes in copper ion availability, which are very important for normal cellular function, to tolerate metal pollution of aquatic ecosystems, and for modulation of copper bioavailability and toxicity to other organisms. Knowledge and application of these mechanisms will benefit the use of microalgae in wastewater processing and biomass production, and the use of copper compounds in the suppression of harmful algal blooms. Here, using electron microscopy, synchrotron radiation-based Fourier transform infrared spectroscopy, electron paramagnetic resonance spectroscopy, and X-ray absorption fine structure spectroscopy, we show that the microalga Chlorella sorokiniana responds promptly to Cu2+ at high non-toxic concentration, by mucilage release, alterations in the architecture of the outer cell wall layer and lipid structures, and polyphosphate accumulation within mucilage matrix. The main route of copper detoxification is by Cu2+ coordination to polyphosphates in penta-coordinated geometry. The sequestrated Cu2+ was accessible and could be released by extracellular chelating agents. Finally, the reduction in Cu2+ to Cu1+ appears also to take place. These findings reveal the biochemical basis of the capacity of microalgae to adapt to high external copper concentrations and to serve as both, sinks and pools of environmental copper.

Introduction. The ovarian surface epithelium (OSE) undergoes intensive regeneration and remodeling after each ovulation during the whole reproductive period. This process increases the risk of one of the most common ovarian tumors in women and the female dog. Considering the fact that maternal hypothyroidism highly impacts cell proliferation and cell death during folliculogenesis in the early neonatal period, we aimed to analyze its effect on OSE morphology and dynamics. Materials and Methods. The study was performed on newborn (24-h-old) and neonatal (4-day-old) female rats, a randomized trial between the control and hypothyroid groups, born under controlled circumstances and hypothyroid mothers, respectively. Their ovaries were analyzed histologically and processed to determine the OSE cell height as an average value of four measurement points. Also, the immunopositivity of the proliferating cell nuclear antigen (PCNA) and caspase-3 were assessed semiquantitatively. Results and Conclusions. No major structural differences of OSE were found between groups within the given ages except for a slight increment of OSE cell height and incompleteness of apical cell membrane with cytoplasmic projections in hypothyroid animals. PCNA immunopositivity of the OSE cells was higher in ovaries of hypothyroid animals of both ages in comparison to the controls. Moreover, only scarce OSE cells were caspase-3 positive in both groups and ages, with no difference in immunopositivity. Our study confirms the impact of hypothyroidism in the early postnatal period on morphology and proliferation rate of OSE cells, with no effect on caspase-3 dependent cell removal, which may serve as a premise for future investigation of potential carcinogenesis, in terms of prevention and treatment of ovarian cancer. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. 175061]

The impact of ionizing radiation on microorganisms such as microalgae is a topic of increasing importance for understanding the dynamics of aquatic ecosystems in response to environmental radiation, and for the development of efficient approaches for bioremediation of mining and nuclear power plants wastewaters. Currently, nothing is known about the effects of ionizing radiation on the microalgal cell wall, which represents the first line of defence against chemical and physical environmental stresses. Using various microscopy, spectroscopy and biochemical techniques we show that the unicellular alga Chlorella sorokiniana elicits a fast response to ionizing radiation. Within one day after irradiation with doses of 1–5 Gy, the fibrilar layer of the cell wall became thicker, the fraction of uronic acids was higher, and the capacity to remove the main reactive product of water radiolysis increased. In addition, the isolated cell wall fraction showed significant binding capacity for Cu 2+, Mn 2+, and Cr 3+. The irradiation further increased the binding capacity for Cu 2+, which appears to be mainly bound to glucosamine moieties within a chitosan-like polymer in the outer rigid layer of the wall. These results imply that the cell wall represents a dynamic structure that is involved in the protective response of microalgae to ionizing radiation. It appears that microalgae may exhibit a significant control of metal mobility in aquatic ecosystems via biosorption by the cell wall matrix.

The purpose of this work was to investigate the influence of hypothyroidism on spleen tissue morphology and immune cell density in fourteen-day-old juvenile rats. Hypothyroidism in pups (n=10) was induced by administration of propylthiouracil (PTU) in drinking water (1.5 mg/L) to their mothers during pregnancy and period of lactation. Fourteen-day-old pups were sacrificed and the thyroid-stimulating hormone (TSH) serum concentration and thyroid activation index (Ia) were determined. Increased serum level of TSH and increased Ia showed that pups from PTU treated mothers were hypothyroid. White and red spleen pulp, marginal zone and connective tissue volume density has been assessed by using the stereological method. Using immunohistochemistry, the present CD3+ T lymphocytes, CD45RA+ B lymphocytes and CD68+ macrophages were quantified. A significant reduction of volume density of the periarteriolar lymphocyte sheath (VvPALS) and lymphatic follicles (Vvf) due to depletion of T and B lymphocytes respectively, was observed in the spleens of hypothyroid pups compared to controls. The volume density of the red pulp (Vvrp), marginal zone (Vvmz) and connective tissue (Vvct) was increased, as well as the number of CD68+ macrophages in the spleens of hypothyroid pups compared to controls. These results indicate that thyroid hormones might be important for normal development of both, specific and innate immune cells in the spleen during prenatal and early postnatal period.

Induced prenatal hypothyroidism in rat pups leads to accelerated primordial follicle assembly and premature follicular atresia with ovary failure. This work investigates the influence of maternal hypothyroidism induced with 6-n-propyl-2-thyouracil (PTU) on the number and morphology of oogonia/oocytes in newborn rat pups with light and transmission electron microscopy. Expression of apoptosis and autophagy markers in oogonia/oocytes were examined using immunohistochemistry. Hypothyroid newborn pups had a decreased number of mitotic and resting oogonia, while the number of altered oogonia/oocytes was increased. Ultrastructural observations revealed the increased presence of degenerated pachytene oocytes (Z-cells) and oogonia/oocytes undergoing autophagy, apoptosis and combined apoptosis and autophagy, in this group. The most abundant altered oogonia/oocytes in the hypothyroid group were those with morphological features of advanced autophagy and Z-cells. The percentage of TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labeling) positive oogonia/oocytes was significantly lower in the hypothyroid group. No significant difference was recorded in the expression of caspase-3, ATG7 and LC3 possibly reflecting that these proteins were not involved in the oogonia/oocyte alteration process during prenatal rat hypothyroidism. The obtained results indicate that developmental hypothyroidism in the offspring enhances the number of Z-cells and oogonia/oocytes altered with the advanced process of autophagy.

Thyroid hormones (TH) are one of the key factors for normal prenatal development in mammals. Previously, we showed that subclinical maternal hypothyroidism leads to premature atresia of ovarian follicles in female rat offspring in the pre-pubertal and pubertal periods. The influence of decreased concentration of TH on primordial follicles pool formation during neonatal and early infantile period of rat pups was not investigated previously. Maternal hypothyroidism during pregnancy has irreversible negative influence on primordial follicles pool formation and population of resting oocytes in female rat offspring. The study was done on neonatal and early infantile control (n-10) and hypothyroid (n-10) female rat pups derived from control (n-6) and propylthiouracil (PTU) treated pregnant dams (n-6), respectively. Ovaries of all pups were removed and processed for light and transmission electron microscopy (TEM). Number of nests, oogonia and oocytes per nest, primordial, primary, secondary and preantral follicles were determined. Screening for overall calcium presence in ovarian tissue was done using Alizarin red staining. Morphology and volume density of nucleus, mitochondria and smooth endoplasmic reticulum (sER) in the oocytes in primordial follicles was also assessed. Caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL), both markers for apoptosis, and proliferating cell nuclear antigen (PCNA) for proliferation were determined in oocytes and granulosa cells in different type of follicles. In neonatal period, ovaries of hypothyroid pups had a decreased number of oogonia, oocytes and nests, an increased number of primordial follicles and a decreased number of primary and secondary follicles, while in early infantile period, increased number of primary, secondary and preantral follicles were found. Alizarin red staining was intense in hypothyroid neonatal rats that also had the highest content of dilated sER. Number of mitochondria with altered morphology in both groups of hypothyroid pups was increased. Apoptosis markers have not shown significant difference between groups but PCNA had an increased expression in the oocytes and granulosa cells in primordial follicles of hypothyroid rats. Light and electron microscopy analysis indicate that previously detected premature ovarian follicular atresia in pre-pubertal and pubertal hypothyroid rats is preceded with premature formation of primordial follicles followed by slight changes on sER and mitochondria in examined oocytes, and increased expression of PCNA. This is the peer-reviewed version of the article: Danilović Luković, J.; Korać, A.; Milošević, I.; Lužajić, T.; Puškaš, N.; Kovačević Filipović, M.; Radovanović, A. Altered State of Primordial Follicles in Neonatal and Early Infantile Rats Due to Maternal Hypothyroidism: Light and Electron Microscopy Approach. Micron 2016, 90, 33–42. [https://doi.org/10.1016/j.micron.2016.08.007]