Is seminal nerve growth factor-induced luteinizing hormone release in camelids mediated at the hypothalamus?

In brief: Seminal nerve growth factor induces ovulation in camelids by influencing the secretion of gonadotrophin-releasing hormone (GnRH) into the portal vessels of the pituitary gland. We show that the nerve growth factor-induced release of GnRH is not mediated directly through interaction with hypothalamic neurons. Abstract: Ovulation in camelids is triggered by seminal nerve growth factor (NGF). The mechanism of action of NGF appears to occur via the central nervous system. In this study, we tested the hypothesis that NGF acts in the hypothalamus to induce GnRH release. To determine if NGF-induced ovulation is associated with a rise in NGF concentrations in the cerebrospinal fluid (CSF), llamas were i) mated with an urethrostomized male, ii) mated with intact male, or given intrauterine iii) seminal plasma or i.v.) saline (Experiment 1). To characterize the luteinizing hormone (LH) response after central vs peripheral administration, llamas were treated with saline (negative control) or NGF either by i.v. or intracerebroventricular (ICV) administration (Experiment 2). To determine the role of kisspeptin, the effect of ICV infusion of a kisspeptin receptor antagonist on NGF-induced LH secretion and ovulation was tested in llamas (Experiment 3). In Experiment 1, a surge in circulating concentrations of LH was detected only in llamas mated with an intact male and those given intrauterine seminal plasma, but no changes in CSF concentrations of NGF were detected. In Experiment 2, peripheral administration (i.v.) of NGF induced an LH surge and ovulation, whereas no response was detected after central (ICV) administration. In Experiment 3, the kisspeptin receptor antagonist had no effect on the LH response to NGF. In conclusion, results did not support the hypothesis that NGF-induced ovulation is mediated via a trans-synaptic pathway within the hypothalamus, but rather through a releasing effect on tanycytes at the median eminence.

Effects of the temperature-humidity index on conception rates in Holstein heifers and cows receiving in vitro-produced Japanese Black cattle embryos.

We investigated the effect of the temperature-humidity index (THI) on the conception rate (CR) in Holstein heifers and cows receiving in vitro-produced (IVP) Japanese Black cattle fresh embryos. IVP embryos were transferred to Holstein heifers (n = 1,407) and cows (n = 3,189) on 245 commercial farms. The monthly average ambient temperature (AT) and THI ranged from 4.7 to 29°C and 41 to 81, respectively; both were the highest in August. The monthly CR ranged from 16.3% to 46.7% in cows and 23.8% to 74.1% in heifers. The CR of heifers was unaffected by THI, AT, or the month of embryo transfer. However, these parameters affected the CR of cows. The CR at THI values of 61-65 and 71-75 was greater than that at THI > 75, whereas other THI values had no effect. The CR at temperatures > 25°C was lower (P = 0.008) than that at temperatures of 15-20°C and 20-25°C. Moreover, the CR was lowest (P = 0.003) in July. THI and parity (P = 0.057 and P = 0.001, respectively) and AT and parity (P = 0.019 and P = 0.001, respectively) showed significant effects on CR; however, there was no interaction between these two factors. In conclusion, AT > 25°C and THI > 75 adversely affect the CR outcome in cows but not in heifers.

Neuroanatomical basis of the nerve growth factor ovulation-induction pathway in llamas†.

The objective of the study was to characterize the anatomical framework and sites of action of the nerve growth factor (NGF)-mediated ovulation-inducing system of llamas. The expression patterns of NGF and its receptors in the hypothalamus of llamas (n = 5) were examined using single and double immunohistochemistry/immunofluorescence. We also compare the expression pattern of the P75 receptor in the hypothalamus of llama and a spontaneous ovulator species (sheep, n = 5). Both NGF receptors (TrkA and P75) were highly expressed in the medial septum and diagonal band of Broca, and populations of TrkA cells were observed in the periventricular and dorsal hypothalamus. Unexpectedly, we found NGF immunoreactive cell bodies with widespread distribution in the hypothalamus but not in areas endowed with NGF receptors. The organum vasculosum of the lamina terminalis (OVLT) and the median eminence displayed immunoreactivity for P75. Double immunofluorescence using vimentin, a marker of tanycytes, confirmed that tanycytes were immunoreactive to P75 in the median eminence and in the OVLT. Additionally, tanycytes were in close association with GnRH and kisspeptin in the arcuate nucleus and median eminence of llamas. The choroid plexus of llamas contained TrkA and NGF immunoreactivity but no P75 immunoreactivity. Results of the present study demonstrate sites of action of NGF in the llama hypothalamus, providing support for the hypothesis of a central effect of NGF in the ovulation-inducing mechanism in llamas.

Kisspeptin induces LH release and ovulation in an induced ovulator†.

Kisspeptin has been implicated in the ovulatory process of several species of spontaneous ovulators but in only one induced ovulator. In contrast, NGF in semen is the principal trigger of ovulation in other species of induced ovulators-camelids. We tested the hypotheses that kisspeptin induces luteinizing hormone (LH) secretion in llamas through a hypothalamic mechanism, and kisspeptin neurons are the target of NGF in its ovulation-inducing pathway. In Experiment 1, llamas were given either NGF, kisspeptin, or saline intravenously, and LH secretion and ovulation were compared among groups. All llamas treated with NGF (5/5) or kisspeptin (5/5) had an elevation of LH blood concentrations after treatment and ovulated, whereas none of the saline group did (0/5). In Experiment 2, llamas were either pretreated with a gonadotropin-releasing hormone (GnRH) receptor antagonist or saline and treated 2 h later with kisspeptin. Llamas pretreated with saline had elevated plasma LH concentrations and ovulated (6/6) whereas llamas pretreated with cetrorelix did not (0/6). In Experiment 3, we evaluated the hypothalamic kisspeptin-GnRH neuronal network by immunohistochemistry. Kisspeptin neurons were detected in the arcuate nucleus, the preoptic area, and the anterior hypothalamus, establishing synaptic contacts with GnRH neurons. We found no colocalization between kisspeptin and NGF receptors by double immunofluorescence. Functional and morphological findings support the concept that kisspeptin is a mediator of the LH secretory pathway in llamas; however, the role of kisspeptins in the NGF ovulation-inducing pathway in camelids remains unclear since NGF receptors were not detected in kisspeptin neurons in the hypothalamus.

Regeneration of testis tissue after ectopic implantation of porcine testis cell aggregates in mice: improved consistency of outcomes and in situ monitoring.

Ectopic implantation of donor testis cell aggregates in recipient mice results in de novo formation or regeneration of testis tissue and, as such, provides a unique invivo model for the study of testis development. However, currently the results are inconsistent and the efficiency of the model remains low. This study was designed to: (1) examine several factors that can potentially improve the consistency and efficiency of this model and (2) explore the use of ultrasound biomicroscopy (UBM) for the non-invasive invivo evaluation of implants. Testis cell aggregates, containing ~40% gonocytes, from 1-week-old donor piglets were implanted under the back skin of immunodeficient mice through skin incisions using gel matrices or through subcutaneous injection without using gel matrices. The addition of gel matrices led to inconsistent tissue development; gelatin had the greatest development, followed by collagen, whereas agarose resulted in poor development. The results also depended on the implanted cell numbers since implants with 100×106 cells were larger than those with 50×106 cells. The injection approach for cell implantation was less invasive and resulted in more consistent and efficient testis tissue development. UBM provided promising results as a means of non-invasive monitoring of implants.

Source and localization of ovulation-inducing factor/nerve growth factor in male reproductive tissues among mammalian species.

The objectives of the study were to compare the presence and localization of ovulation-inducing factor (OIF)/nerve growth factor (NGF) in male reproductive organs and determine the abundance in ejaculates of species representative of both spontaneous and induced ovulators. We hypothesized that the protein is a widely conserved component of semen among mammals, but is most abundant in camelids. Immunohistochemical analysis was performed on tissues from the male reproductive system of llamas, rats, cattle, bison, elk, and white-tailed deer (n = 2 males/species), and the abundance of OIF/NGF in the seminal plasma of camelids (llamas and alpacas), cattle, horses, and pigs (n = 69, 53, 24, and 16 ejaculates, respectively) were quantified by radioimmunoassay. Based on immunoreactivity in both the glandular epithelium and glandular lumen, the prostate gland was the main source of seminal OIF/NGF in llamas, the vesicular gland and ampullae in bovids (cattle and bison), and the ampullae and prostate in cervids (elk and white-tailed deer). Camelid and bovine seminal plasma induced dendritic growth in the PC12 differentiation bioassay, but no effect was observed with equine or porcine seminal plasma. The concentration of OIF/NGF was 10 times higher in camelid than bovine seminal plasma (1.2 ± 0.21 vs. 0.10 ± 0.03; P < 0.05); OIF/NGF was not detected in equine or porcine ejaculates by radioimmunoassay. Based on tissue localization, abundance, and bioactivity, we conclude that OIF/NGF is a common protein within the male accessory glands among species, and its abundance in camelids, bovids, and cervids suggests an important role in the mechanisms of ovulation in both induced and spontaneous ovulators.

Short-term culture of adult bovine ovarian tissues: chorioallantoic membrane (CAM) vs. traditional in vitro culture systems.

BACKGROUND: A suitable culture system is important for follicle growth in adult bovine ovarian tissue. This study aimed to assess the avian chorioallantoic membrane (CAM) for short-term culture of adult bovine ovarian tissues compared with a traditional in vitro culture system. METHODS: Ovarian cortical tissues (1-2 mm3), collected from slaughtered adult cows, were randomly assigned to control, CAM or in vitro culture groups. In the control group, ovarian tissues were fixed with paraformaldehyde without culture. In CAM and in vitro culture groups, the ovarian tissues were cultured for up to 5 days and then fixed. Ovarian tissues were examined on culture days 0, 1, 3 and 5 for angiogenesis, follicle morphology and growth. In all groups, primordial and growing (healthy and atretic) follicle densities were determined. RESULTS: In the CAM culture, the avian blood vessel density increased (p < 0.01) over time with a decline (p < 0.001) in the bovine blood vessel density. Healthy primordial, atretic primordial and healthy growing follicle densities were higher (p < 0.05) in CAM-cultured ovarian tissues than in vitro-cultured tissues. Regardless of the culture system, the density of healthy primordial follicles decreased (p < 0.001) over time with an increase in healthy growing follicles on day 3 (p < 0.01) and an increase in atretic (primordial and growing) follicles during the 5-day culture period (p < 0.001). The proportions of healthy primordial and atretic growing follicles were also affected by culture day (p < 0.001). CONCLUSIONS: The CAM culture in chick embryos supported the bovine ovarian tissue grafts for 3 days demonstrating that CAM can be used as a satisfactory short-term culture system to assess ovarian tissue health, and to study follicle activation and development.

The relationship between gonadotropin releasing hormone and ovulation inducing factor/nerve growth factor receptors in the hypothalamus of the llama.

BACKGROUND: A molecule identical to nerve growth factor, with ovulation-inducing properties has been discovered in the seminal plasma of South American camelids (ovulation-inducing factor/nerve growth factor; OIF/NGF). We hypothesize that the ovulatory effect of OIF/NGF is initiated at the level of the hypothalamus, presumably by GnRH neurons. The objective of the present study was to determine the structural relationship between GnRH neurons and neurons expressing high- and low-affinity receptors for NGF (i.e., TrkA and p75, respectively) in the hypothalamus. METHODS: Mature llamas (n = 4) were euthanized and their hypothalamic tissue was fixed, sectioned, and processed for immunohistochemistry on free-floating sections. Ten equidistant sections per brain were double stained for immunofluorescence detection of TrkA and GnRH, or p75 and GnRH. RESULTS: Cells immunoreactive to TrkA were detected in most hypothalamic areas, but the majority of cells were detected in the diagonal band of Broca (part of the ventral forebrain) and the supraoptic nuclei and periventricular area. The number of cells immunoreactive to p75 was highest in the diagonal band of Broca and lateral preoptic areas and least in more caudal areas of the hypothalamus (p < 0.05) in a pattern similar to that of TrkA. A low proportion of GnRH neurons were immunoreactive to TrkA (2.5% of total GnRH cells), and no co-localization between GnRH and p75 was detected. GnRH neuron fibers were detected only occasionally in proximity to TrkA immunopositive neurons. CONCLUSIONS: Results do not support the hypothesis that the effect of OIF/NGF is driven by a direct interaction with GnRH neurons, but rather provide rationale for the hypothesis that interneurons exist in the hypothalamus that mediate OIF/NGF-induced ovulation.

Distribution and morphology of gonadotropin-releasing hormone neurons in the hypothalamus of an induced ovulator - The llama (Lama glama).

Gonadotropin-releasing hormone (GnRH) is a decapeptide involved in the regulation of reproduction in all mammals, but the distribution of GnRH neurons within the brain varies widely among species. The objective of the present study was to characterize the number and distribution of GnRH neurons in the hypothalamus and preoptic area of llamas, an induced ovulator. The brains of female llamas (n = 4) were fixed, frozen and sectioned serially every 50 µm in the transverse (coronal) plane. Every 10th section was stained for immunohistochemical detection of GnRH-positive neuron cell bodies and fibers by incubation with 3,3'-diaminobenzidine. The number of counted immunoreactive cells ranged from 222 to 250 (≈241 ±â€¯13 cells in the preoptic area and hypothalamus per animal) and were localized in the medio-basal hypothalamus (44.3%), anterior hypothalamus (27%), preoptic area (14.9%), diagonal band of Broca/medial septum (13.4%), and mammillary area (0.5%). The immunoreactive cells were not localized in specific hypothalamic nuclei, but rather appeared to be distributed diffusely. The highest concentration of immunoreactive neuron fibers was in the median eminence (P < 0.05), but fibers were identified in most of the areas analyzed, including the neurohypophysis. The GnRH neurons within the hypothalamus displayed monopolar (33%), bipolar (39%), and multipolar (28%) morphologies. The bipolar type was most common in the medio-basal region (40%; P < 0.05). We conclude that GnRH neurons and fibers form a network within the anterior and medio-basal hypothalamus of llamas, suggesting the central location of mechanisms controlling reproductive processes in llamas (i.e., induced ovulation).

Private troubles to public issue: empowering communities to reduce alcohol-related harm in Sabah, Malaysia.

Summary: Alcohol is the number three contributor to the burden of disease worldwide so must remain a priority health promotion issue internationally. Malaysia is a Muslim country and alcohol-related harm was not seen as a priority until recently, because it only affects a minority of the population. Sabah has more than 30 different ethnic groups, and alcohol has a traditional role in the cultural practices of many of these groups. In 2009, the Intervention Group for Alcohol Misuse (IGAM) was formed, under the umbrella of Mercy Malaysia by a group of healthcare workers, academics, members of the Clergy and people who were previously alcohol-dependent concerned about the harmful effects of excessive alcohol consumption. IGAM in collaboration with other bodies have organized public seminars, visited villages and schools, encouraged the formation of a support group and trained healthcare professionals in health promotion intervention. The focus later changed to empowering communities to find solutions to alcohol-related harm in their community in a way which is sensitive to their culture. A standard tool-kit was developed using WHO materials as a guide. Village committees were formed and adapted the toolkit according to their needs. This strategy has been shown to be effective, in that 90% of the 20 committees formed are actively and successfully involved in health promotion to reduce alcohol-related harm in their communities.

Comparative analyses of prediction models for inactivation of Escherichia coli in carrot juice by means of pulsed electric fields.

This paper reports the prediction capacity of various microbial inactivation models to prefigure the bactericidal effect using pulsed electric field (PEF) on liquid food. The aim of study was to compare the various inactivation models based on accuracy and bias factor to find out the most accurate inactivation model for Escherichia coli present in carrot juice treated with PEF. In this study, E. coli suspended in carrot juice was treated with varying pulsed electric field strength for different intervals. The obtained data were utilized for the evaluation of parameters of Bigelow, Peleg, Hülsheger and Weibull inactivation models. Furthermore, secondary models were developed for Hülsheger and Weibull to predict the microbial inactivation at any level of field strength and treatment time. The secondary model for Hülsheger exhibits 5.8% error as compared to the Weibull model having 8.5% error in prediction of death kinetics of E. coli suspended in carrot juice by means of PEF. The comparative analysis of secondary models to forecast the unknown data set unveiled the superior functioning of Hülsheger model.

Meta-analysis of gene expression profiles in granulosa cells during folliculogenesis.

Folliculogenesis involves coordinated profound changes in different follicular compartments and significant modifications of their gene expression patterns, particularly in granulosa cells. Huge datasets have accumulated from the analyses of granulosa cell transcriptomic signatures in predefined physiological contexts using different technological platforms. However, no comprehensive overview of folliculogenesis is available. This would require integration of datasets from numerous individual studies. A prerequisite for such integration would be the use of comparable platforms and experimental conditions. The EmbryoGENE program was created to study bovine granulosa cell transcriptomics under different physiological conditions using the same platform. Based on the data thus generated so far, we present here an interactive web interface called GranulosaIMAGE (Integrative Meta-Analysis of Gene Expression), which provides dynamic expression profiles of any gene of interest and all isoforms thereof in granulosa cells at different stages of folliculogenesis. GranulosaIMAGE features two kinds of expression profiles: gene expression kinetics during bovine folliculogenesis from small (6 mm) to pre-ovulatory follicles under different hormonal and physiological conditions and expression profiles of granulosa cells of dominant follicles from post-partum cows in different metabolic states. This article provides selected examples of expression patterns along with suggestions for users to access and generate their own patterns using GranulosaIMAGE. The possibility of analysing gene expression dynamics during the late stages of folliculogenesis in a mono-ovulatory species such as bovine should provide a new and enriched perspective on ovarian physiology.

The dynamics of trkA expression in the bovine ovary are associated with a luteotrophic effect of ovulation-inducing factor/nerve growth factor (OIF/NGF).

BACKGROUND: Ovulation-inducing factor in semen (OIF/NGF) influences ovulation and CL form and function in camelids and, remarkably, in cows. To test the hypothesis that the luteotrophic effect of OIF/NGF is mediated by an increase in trkA receptors in the ovulatory follicle and early CL, a study was designed to characterize the spatial and temporal distribution of trkA in ovarian follicles and CL at known stages of the bovine estrous cycle. METHODS: Sexually mature cattle (n = 14) were examined daily by transrectal ultrasonography to determine the day of ovulation (Day 0), and assigned randomly to be unilaterally ovariectomized on Day 2, 4, 6 or in the pre-ovulatory period just before or after exogenous LH treatment. After a complete interovulatory interval, the cows were re-assigned to a different day-group on which the remaining ovary was removed. Sections of ovarian tissue representing the dominant follicle, largest subordinate follicle, and the CL were processed for immunofluorescent detection and quantification of trkA receptor. RESULTS: TrkA immuno-fluorescence in ovarian tissues was restricted to follicles and the CL (no reaction in stroma or vessels), and was restricted to the cytoplasm (no nuclear staining). The trkA staining intensity, area of staining, and proportion of cells stained was greater in both theca and granulosa layers of dominant follicles than in that of subordinate follicles (P ≤ 0.05) in all day-groups except the Pre-LH group. Among dominant follicles, a progressive reduction in the immuno-positive reaction was detected from Day 2 to Day 6. Among subordinate follicles, immuno-reactivity remained low and unchanged except a rise in the Pre-LH group. The number of immuno-positive cells was greater in early developing CL (Days 2 and 4 combined) than in mature or regressing stage CL (Day 6, Pre- and Post-LH combined; P = 0.01). The intracellular distribution of trkA was more diffuse and widespread in dominant than subordinate follicles, particularly on Day 2 and Post-LH (P < 0.05). CONCLUSIONS: Distinct differences in trkA expression between dominant and subordinate follicles, particularly when circulating progesterone is minimal (early luteal development and after luteolysis) is consistent with a local role of OIF/NGF in follicle selection and early luteogenesis.

Both high and low plasma levels of 25-hydroxy vitamin D increase blood pressure in a normal rat model.

The lower threshold plasma 25-hydroxy vitamin D (25(OH)D) level for optimal cardiovascular health is unclear, whereas the toxicity threshold is less clear. The aim of this study was to examine the cardiovascular-vitamin D dose-response curve in a normal rat model. Doses of cholecalciferol ranged from deficiency to toxic levels (equivalent to human doses of 0, 0·015, 0·25 and 3·75mg/d) for 4 weeks, and then cardiovascular health was examined using blood pressure telemetry and high-resolution ultrasound in normal male rats (n 16/group, 64 rats total). After 1 month, only the 0·25mg/d group had plasma 25(OH)D that was within current recommended range (100-125 nmol/l), and all groups failed to change plasma Ca or phosphate. Systolic blood pressure increased significantly (10-15 mmHg) in the rat groups with plasma 25(OH)D levels at both 30 and 561 nmol/l (groups fed 0 and 3·75mg/d) compared with the group fed the equivalent to 0·015mg/d (43 nmol/l 25(OH)D). Although not significant, the group fed the equivalent to 0·25mg/d (108 nmol/l 25(OH)D) also showed a 10 mmHg increase in systolic blood pressure. Carotid artery diameter was significantly smaller and wall thickness was larger, leading to higher peak carotid systolic blood velocity in these two groups. Despite these vascular changes, cardiac function did not differ among treatment groups. The key finding in this study is that arterial stiffness and systolic blood pressure both showed a U-shaped dose-response for vitamin D, with lowest values (best cardiovascular health) observed when plasma 25(OH)D levels were 43 nmol/l in normal male rats.

Stable reference genes in granulosa cells of bovine dominant follicles during follicular growth, FSH stimulation and maternal aging.

The aim of the present study was to determine a set of reference genes in granulosa cells of dominant follicles that are suitable for relative gene expression analyses during maternal and follicular aging. Granulosa cells of growing and preovulatory dominant follicles were collected from aged and young cows (maternal aging study) and from FSH-stimulated follicles developing under different durations of FSH treatment (follicular aging study). The mRNA levels of the two commonly used reference genes (GAPDH, ACTB) and four novel genes (UBE2D2, EIF2B2, SF3A1, RNF20) were analysed using cycle threshold values. Results revealed that mRNA levels of GAPDH, ACTB, EIF2B2, RNF20, SF3A1 and UBE2D2 were similar (P>0.05) between dominant follicle type, age and among follicles obtained after FSH-stimulation, but differed (P=0.005) due to mRNA processing (i.e. with versus without amplification). The stability of reference genes was analysed using GeNorm, DeltaCT and NormFinder programs and comprehensive ranking order was determined using RefFinder. The mRNA levels of GAPDH and ACTB were less stable than those of UBE2D2 and EIF2B2. The geometric mean of multiple genes (UBE2D2, EIF2B2, GAPDH and SF3A1) is a more appropriate reference control than the use of a single reference gene to compare relative gene expression among dominant and FSH-stimulated follicles during maternal and/or follicular aging studies.

Assessment of freeware programs for the reconstruction of tomography datasets obtained with a monochromatic synchrotron-based X-ray source.

Synchrotron-based in-line phase-contrast computed tomography (PC-CT) allows soft tissue to be imaged with sub-gross resolution and has potential to be used as a diagnostic tool. The reconstruction and processing of in-line PC-CT datasets is a computationally demanding task; thus, an efficient and user-friendly software program is desirable. Four freeware programs (NRecon, PITRE, H-PITRE and Athabasca Recon) were compared for the availability of features such as dark- and flat-field calibration, beam power normalization, ring artifact removal, and alignment tools for optimizing image quality. An in-line PC-CT projection dataset (3751 projections, 180° rotation, 10.13 mm × 0.54 mm) was collected from a formalin-fixed canine prostate at the Biomedical Imaging and Therapy Bending Magnet (BMIT-BM) beamline of the Canadian Light Source. This dataset was processed with each of the four software programs and usability of the program was evaluated. Efficiency was assessed by how each program maximized computer processing power during computation. Athabasca Recon had the least-efficient memory usage, least user-friendly interface, and lacked a ring artifact removal feature. NRecon, PITRE and H-PITRE produced similar quality images, but the Athabasca Recon reconstruction suffered from the lack of a native ring remover algorithm. The 64-bit version of NRecon uses GPU (graphics processor unit) memory for accelerated processing and is user-friendly, but does not provide necessary parameters for in-line PC-CT data, such as dark-field and flat-field correction and beam power normalization. PITRE has many helpful features and tools, but lacks a comprehensive user manual and help section. H-PITRE is a condensed version of PITRE and maximizes computer memory for efficiency. To conclude, NRecon has fewer imaging processing tools than PITRE and H-PITRE, but is ideal for less experienced users due to a simple user interface. Based on the quality of reconstructed images, efficient use of computer memory and parameter availability, H-PITRE was the preferred of the four programs compared.

Organelle reorganization in bovine oocytes during dominant follicle growth and regression.

BACKGROUND: We tested the hypothesis that organelles in bovine oocytes undergo changes in number and spatial distribution in a manner specific for phase of follicle development. METHODS: Cumulus-oocyte-complexes were collected from Hereford heifers by ultrasound-guided follicle aspiration from dominant follicles in the growing phase (n = 5; Day 0 = ovulation), static phase (n = 5), regressing phase (n = 7) of Wave 1 and from preovulatory follicles (n = 5). Oocytes were processed and transmission electron micrographs of ooplasm representing peripheral, perinuclear and central regions were evaluated using standard stereological methods. RESULTS: The number of mitochondria and volume occupied by lipid droplets was higher (P < 0.03) in oocytes from regressing follicles (193.0 ± 10.4/1000 µm(3) and 3.5 ± 0.7 %) than growing and preovulatory stages (118.7 ± 14.4/1000 µm(3) and 1.1 ± 0.3 %; 150.5 ± 28.7/1000 µm(3) and 1.6 ± 0.2 %, respectively). Oocytes from growing, static and preovulatory follicles had >70 % mitochondria in the peripheral regions whereas oocytes from regressing follicles had an even distribution. Oocytes from growing follicles had more lipid droplets in peripheral region than in central region (86.9 vs. 13.1 %). Percent surface area of mitochondria in contact with lipid droplets increased from growing (2.3 %) to static, regressing or preovulatory follicle stage (8.9, 6.1 and 6.2 %). The amount, size and distribution of other organelles did not differ among phases (P > 0.11). CONCLUSIONS: Our hypothesis was supported in that mitochondrial number increased and translocation occurred from a peripheral to an even distribution as follicles entered the regressing phase. In addition, lipid droplets underwent spatial reorganization from a peripheral to an even distribution during the growing phase and mitochondria-lipid contact area increased with follicle maturation.

Bovine adenovirus 3 core protein precursor pVII localizes to mitochondria, and modulates ATP synthesis, mitochondrial Ca2+ and mitochondrial membrane potential.

Viruses modulate the functions of mitochondria by translocating viral proteins to the mitochondria. Subcellular fractionation and sensitivity to proteinase K/Triton X-100 treatment of mitochondrial fractions of bovine adenovirus (BAdV)-3-infected/transfected cells suggested that core protein pVII localizes to the mitochondria and contains a functional mitochondrial localization signal. Moreover, mitochondrial localization of BAdV-3 pVII appears to help in the retention of mitochondrial Ca(2+), inducing a significant increase in the levels of ATP and maintaining the mitochondrial membrane potential (MMP) in transfected cells. In contrast, mitochondrial localization of BAdV-3 pVII has no significant effect on the levels of cytoplasmic Ca(2+) and reactive oxygen species production in the transfected cells. Consistent with these results, expression of pVII in transfected cells treated with staurosporine decreased significantly the activation of caspase-3. Our results suggested that BAdV-3 pVII localizes to mitochondria, and interferes with apoptosis by inhibiting loss of the MMP and by increasing mitochondrial Ca(2+) and ATP production.

Effect of bovine adenovirus 3 on mitochondria.

Viruses alter the structure and the function of mitochondria for survival. Electron microscopy analysis of the cells infected with bovine adenovirus 3 revealed extensive damage to the inner mitochondrial membrane characterized by dissolution of the cristae and amorphous appearance of mitochondrial matrix with little or no damage to the outer mitochondrial membrane. There were fewer cristae with altered morphology. Potential patches of protein synthesis machinary around mitochondria could be observed at 12 hours post infection (hpi). At 24 hpi, the multi vascular bodies were evident throughout the infected cell. ATP production, mitochondrial Ca2+ and mitochondrial membrane potential (MMP) peaked at 18 hpi but decreased significantly at 24 hpi. This decrease coincided with the increased production of superoxide (SO) and reactive oxygen species (ROS), at 24 hpi indicating acute oxidative stress in the cells and suggesting a complete failure of the cellular homeostatic machinary. The results reveal an intericate relationship between Ca2+ homeostasis, the ATP generation ability of cells, SO and ROS production, and regulation of MMP following infection by bovine adenovirus 3.

Evaluation of ecological risk of metal contamination in river Gomti, India: a biomonitoring approach.

The aim of this study was to evaluate the extent of heavy metal pollution in river Gomti and associated ecological risk. River water, sediments and locally abundant mollusk (Viviparus (V.) bengalensis) were sampled from six different sites and analyzed for seven metals: Cadmium (Cd), Chromium (Cr), Copper (Cu), Manganese (Mn), Nickel (Ni), Lead (Pb) and Zinc (Zn). Mean metal concentrations (mg/l) in river water were 0.024 for Cd, 0.063 for Cr, 0.022 for Cr, 0.029 for Mn, 0.044 for Ni, 0.018 for Pb and 0.067 for Zn. In river sediments, the concentrations (mg/kg dry wt) were 5.0 for Cd, 16.2 for Cr, 23.2 for Cr, 203.2 for Mn, 23.9 for Ni, 46.2 for Pb and 76.3 for Zn, while in V. bengalensis mean metal concentrations (mg/kg, dry wt) were 0.57 for Cd, 12.0 for Cr, 30.7 for Cu, 29.9 for Mn, 8.8 for Ni, 3.6 for Pb and 48.3 for Zn. Results indicated elevated concentrations of Cu, Zn and Mn in V. bengalensis as compared to other non-essential elements. Potential ecological risk (RI) in sediments showed high to very high metal contamination. Cluster analysis indicated that Pb, Zn, Cd and Ni in sediments may have anthropogenic sources. The findings thus suggest heavy metal contamination of river water and sediments have reached alarming levels, which is well corroborated by elevated level of metal accumulation in V. bengalensis.