Genomic profiling of bovine corpus luteum maturation
. PLOS ONE 2018
, e0194456 -. Abstract
To unveil novel global changes associated with corpus luteum (CL) maturation, we analyzed transcriptome data for the bovine CL on days 4 and 11, representing the developing vs. mature gland. Our analyses revealed 681 differentially expressed genes (363 and 318 on day 4 and 11, respectively), with ≥2 fold change and FDR of <5%. Different gene ontology (GO) categories were represented prominently in transcriptome data at these stages (e.g. days 4: cell cycle, chromosome, DNA metabolic process and replication and on day 11: immune response; lipid metabolic process and complement activation). Based on bioinformatic analyses, select genes expression in day 4 and 11 CL was validated with quantitative real-time PCR. Cell specific expression was also determined in enriched luteal endothelial and steroidogenic cells. Genes related to the angiogenic process such as NOS3, which maintains dilated vessels and MMP9, matrix degrading enzyme, were higher on day 4. Importantly, our data suggests day 11 CL acquire mechanisms to prevent blood vessel sprouting and promote their maturation by expressing NOTCH4 and JAG1, greatly enriched in luteal endothelial cells. Another endothelial specific gene, CD300LG, was identified here in the CL for the first time. CD300LG is an adhesion molecule enabling lymphocyte migration, its higher levels at mid cycle are expected to support the transmigration of immune cells into the CL at this stage. Together with steroidogenic genes, most of the genes regulating de-novo cholesterol biosynthetic pathway (e.g HMGCS, HMGCR) and cholesterol uptake from plasma (LDLR, APOD and APOE) were upregulated in the mature CL. These findings provide new insight of the processes involved in CL maturation including blood vessel growth and stabilization, leucocyte transmigration as well as progesterone synthesis as the CL matures.
miR-210 and GPD1L regulate EDN2 in primary and immortalized human granulosa-lutein cells
. Reproduction 2018
Endothelin-2 (EDN2), expressed at a narrow window during the periovulatory period, critically affects ovulation and corpus luteum (CL) formation. LH (acting mainly via cAMP) and hypoxia are implicated in CL formation; therefore, we aimed to elucidate how these signals regulate using human primary (hGLCs) and immortalized (SVOG) granulosa-lutein cells. The hypoxiamiR, microRNA-210 (miR-210) was identified as a new essential player in expression. Hypoxia (either mimetic compound-CoCl, or low O) elevated hypoxia-inducible factor 1A (HIF1A), miR-210 and Hypoxia-induced miR-210 was suppressed in HIF1A-silenced SVOG cells, suggesting that miR-210 is HIF1A dependent. Elevated miR-210 levels in hypoxia or by miR-210 overexpression, increased Conversely, miR-210 inhibition reduced levels, even in the presence of CoCl, indicating the importance of miR-210 in the hypoxic induction of A molecule that destabilizes HIF1A protein, glycerol-3-phosphate dehydrogenase 1-like gene-, was established as a miR-210 target in both cell types. It was decreased by miR-210-mimic and was increased by miR-inhibitor. Furthermore, reducing by endogenously elevated miR-210 (in hypoxia), miR-210-mimic or by siRNA resulted in elevated HIF1A protein and levels, implying a vital role for in the hypoxic induction of Under normoxic conditions, forskolin (adenylyl cyclase activator) triggered changes typical of hypoxia. It elevated , and miR-210 while inhibiting Furthermore, HIF1A silencing greatly reduced forskolin's ability to elevate and miR-210. This study highlights the novel regulatory roles of miR-210 and its gene target, GPD1L, in hypoxia and cAMP-induced by human granulosa-lutein cells.
Cloning and characterization of a second lamprey pituitary glycoprotein hormone, thyrostimulin (GpA2/GpB5)
. General and Comparative Endocrinology 2018
, 16 - 27. Abstract
A novel heterodimeric glycoprotein hormone (GpH) comprised of alpha (GpA2) and beta (GpB5) subunits was discovered in 2002 and called thyrostimulin for its ability to activate the TSH receptor in mammals, but its central function in vertebrates has not been firmly established. We report here the cloning and expression of lamprey (l)GpB5, and its ability to heterodimerize with lGpA2 to form a functional l-thyrostimulin. The full-length cDNA of lGpB5 encodes 174 amino acids with ten conserved cysteine residues and one glycosylation site that is conserved with other vertebrate GpB5 sequences. Phylogenetic and synteny analyses support that lGpB5 belongs to the vertebrate GpB5 clade. Heterodimerization of lGpB5 and lGpA2 was shown by nickel pull-down of histidine-tagged recombinant subunits. RNA transcripts of lGpB5 were detected in the pituitary of lampreys during both parasitic and adult life stages. Intraperitoneal injection with lGnRH-III (100 μg/kg) increased pituitary lGpA2, lGpB5, and lGpHβ mRNA expression in sexually mature, adult female lampreys. A recombinant l-thyrostimulin produced by expression of a fusion gene in Pichia pastoris activated lamprey GpH receptors I and II as measured by cAMP enzymeimmunoassay. In contrast to jawed vertebrates that have pituitary LH, FSH, and TSH, our data support that lampreys only have two functional pituitary GpHs, lGpH and l-thyrostimulin, which consist of lGpA2 and unique beta subunits. It is hypothesized that lGpH and l-thyrostimulin differentially regulate reproductive and thyroid activities in some unknown way(s) in lampreys.
Characterization of carp gonadotropins: Structure, annual profile, and carp and zebrafish pituitary topographic organization
. General and Comparative Endocrinology 2018
, 28 - 38. Abstract
Two gonadotropins, follicle stimulating hormone (FSH) and luteinizing hormone (LH), are important players in the hypothalamic-pituitary-gonadal axis of vertebrates. In the present work, we describe the construction of recombinant (r) common carp (Cyprinus carpio; c) FSH (rcFSH) and LH (rcLH) using the Pichia pastoris system, the generation of specific antibodies against their respective β subunits, and their use in the development and validation of specific ELISAs. We produced carp rLH and rFSH as single-chain polypeptides, wherein the GTH subunit α was joined with either cLHβ or cFSHβ mature protein-coding sequences to form a fusion gene that encodes a yoked polypeptide, in which the GTH β-subunit forms the N-terminal part and the α-subunit forms the C-terminal part. Competitive ELISAs were developed, using primary antibodies against rcLHβ or rcFSHβ, respectively, and rcLHβα or rcFSHβα for the standard curves. The standard curves for cLH paralleled those of pituitary extracts of the homologous fish and also those of other cyprinids species like the black carp (Mylopharyngodon piceus), goldfish (Carassius auratus), silver carp (Hypophthalmichthys molitrix), and grass carp (Ctenopharyngodon idella). We used the specific antibodies raised against cFSH and cLH to study the specific localization of the different GTH cells in the pituitary of carp and its taxonomic relative species – the zebrafish. Both FSH and LH cells are localized in the center of the proximal pars distalis enveloping both sides of the neurohypophysis. LH cells form a continuous population throughout the PPD, while FSH cells are more loosely distributed throughout the same area and form small aggregations. Marked annual changes were encountered in gonadosomatic index (GSI), follicle diameter, mRNA levels and protein levels of FSH and LH. From September to November, all fish had low GSI, and the ovary contained previtellogenic follicles. From December, the GSI level increased and remained high until March, the follicular diameter reached its maximum in January, where the ovary contained large fully grown follicles. Thereafter, spawning occurred through March and April and ended in May, and GSI level and follicle diameter increased again; and the ovary contained mid-vitellogenic follicles. LH pituitary content and mRNA levels were low at pre- and early vitellogenesis, increasing gradually during this process to reach a peak of LH mRNA levels in mid vitellogenic ovary and a peak of LH content in fully grown ovarian follicles. However, no significant change occurred in FSH pituitary content and mRNA levels in vitellogenic fish and in fish during final maturation stages. A dramatic difference was found in the total content of each gonadotropin in the pituitary, with higher LH than FSH. Moreover, follicle diameter was positively and significantly correlated with LH pituitary content and its transcript levels – but not with the pituitary content or mRNA levels of FSH. Taken together, these results indicate that in carp, LH alone is sufficient to regulate both vitellogenesis and final oocyte maturation while FSH may have another, yet undefined role.
Identity domains in complex behavior: Toward a biology of personality
. bioRxiv 2018
, 395111. Abstract
Personality traits offer considerable insight into the biological basis of individual differences. However, existing approaches toward understanding personality across species rely on subjective criteria and limited sets of behavioral readouts, resulting in noisy and often inconsistent outcomes. Here, we introduce a mathematical framework for studying individual differences along dimensions with maximum consistency and discriminative power. We validate this framework in mice, using data from a system for high-throughput longitudinal monitoring of group-housed mice that yields a variety of readouts from all across an individual’s behavioral repertoire. We describe a set of stable traits that capture variability in behavior and gene expression in the brain, allowing for better informed mechanistic investigations into the biology of individual differences.
A promotive effect for halofuginone on membrane repair and synaptotagmin-7 levels in muscle cells of dysferlin-null mice
. Hum Mol Genet 2018
In the absence of dysferlin, skeletal muscle cells fail to reseal properly after injury, resulting in slow progress of the dysferlinopathy muscular dystrophy (MD). Halofuginone, a leading agent in preventing fibrosis in MDs, was tested for its effects on membrane resealing post-injury. A hypo-osmotic shock assay on myotubes derived from wild-type (Wt) and dysferlin-null (dysf-/-) mice revealed that pre-treatment with halofuginone reduces the percentage of membrane-ruptured myotubes only in dysf-/- myotubes. In laser-induced injury of isolated myofibers, halofuginone decreased the amount of FM1-43 at the injury site of dysf-/- myofibers while having no effect on Wt myofibers. These results implicate halofuginone in ameliorating muscle-cell membrane integrity in dysf-/- mice. Halofuginone increased lysosome scattering across the cytosol of dysf-/- primary myoblasts, in a protein kinase/extracellular signal-regulated protein kinase and phosphoinositide 3 kinase/Akt-dependent manner, in agreement with an elevation in lysosomal exocytotic activity in these cells. A spatial- and age-dependent synaptotagmin-7 (Syt-7) expression pattern was shown in dysf-/- versus Wt mice, suggesting that these pattern alterations are related to the disease progress and that sytnaptotagmin-7 may be compensating for the lack of dysferlin at least with regard to membrane resealing post-injury. While halofuginone did not affect patch-repair-complex key proteins, it further enhanced Syt-7 levels and its spread across the cytosol in dysf-/- myofibers and muscle tissue, and increased its co-localization with lysosomes. Together, the data imply a novel role for halofuginone in membrane-resealing events with Syt-7 possibly taking part in these events.
Effects of low oxygen during chorioallantoic membrane development on post-hatch growing performance of broiler chickens
. Poult Sci 2018
The prenatal circulatory system is adaptive and capable of plasticity designed for the needs of the growing tissue. When a broiler embryo is faced with hypoxic stress, the process of angiogenesis in tissues begins. Exposure to hypoxic conditions of 17% oxygen during the chorioallantoic membrane (CAM) development (E5 to E12) affected the circulatory system and contributed to an increase in the blood oxygen carrying capacity. The present study aimed to evaluate the effects of hypoxic exposure during CAM development on post-hatch performance of broilers and to examine whether hypoxic exposure improved sustainability of birds exposed to acute heat stress.Two consecutive trials, with male broilers from each of the incubation treatments-optimal conditions and exposure to hypoxia of 15 or 17% oxygen, for 12 h/day, during CAM development-were conducted. In experiment 1, 60 male chicks from each group were raised in individual cages. In experiment 2, 160 male chicks from each group were raised in 40-chick pens until marketing. On d 35, 20 birds from each group were transferred to individual cages kept at a temperature of 23°C for 72 h, and then birds were exposed to 35°C for 5 hours. Body temperatures were measured at 0, 2, and 5 h of the heat exposure. In both experiments BW, feed intake, and FCR were recorded. At marketing, chicks were slaughtered, and relative weights of breast muscle, abdominal fat pad, heart, and liver were calculated.Hypoxia treatment resulted in a FCR advantage. Food intake was similar in all treatments, but groups exposed to hypoxia grew better than controls until the age of 35 days. Hypoxia-treated groups had higher relative breast, heart, and liver weights than controls. Body temperatures of hypoxia-treated chickens remained lower during heat stress exposure, and their mortality rate was lower as well. Intermittent exposure to moderate hypoxia during CAM development confers advantages to broilers in feed utilization efficiency and in coping with heat stress. It may be considered as a mitigating step in incubation to facilitate broilers in achieving their full growth potential.
Detection of tilapia lake virus (TiLV) infection by PCR in farmed and wild Nile tilapia (Oreochromis niloticus) from Lake Victoria
. Journal of Fish Diseases 2018
, 1181 - 1189. Abstract
Abstract Tilapia lake virus disease (TiLVD) has emerged to be an important viral disease of farmed Nile tilapia (Oreochromis niloticus) having the potential to impede expansion of aquaculture production. There is a need for rapid diagnostic tools to identify infected fish to limit the spread in individual farms. We report the first detection of TiLV infection by PCR in farmed and wild Nile tilapia from Lake Victoria. There was no difference in prevalence between farmed and wild fish samples (p = .65), and of the 442 samples examined from 191 fish, 28 were positive for TiLV by PCR. In terms of tissue distribution, the head kidney (7.69%, N = 65) and spleen (10.99%, N = 191), samples had the highest prevalence (p < .0028) followed by heart samples (3.45%, N = 29). Conversely, the prevalence was low in the liver (0.71%, N = 140) and absent in brain samples (0.0%, N = 17), which have previously been shown to be target organs during acute infections. Phylogenetic analysis showed homology between our sequences and those from recent outbreaks in Israel and Thailand. Given that these findings were based on nucleic acid detection by PCR, future studies should seek to isolate the virus from fish in Lake Victoria and show its ability to cause disease and virulence in susceptible fish.
Historical and recent reductions in genetic variation of the Sarotherodon galilaeus population in the Sea of Galilee
. Conservation Genetics 2018
, 1323 - 1333. Abstract
The Sea of Galilee has great significance as a natural habitat and a freshwater source for Israel. Anthropogenic impacts have been placing significant pressure on the species inhabiting this lake, among which is Sarotherodon galilaeus, an omnivorous fish with a relatively large population and significance for commercial fishing. An alarming decline in annual catch towards 2008 suggested that this unique population might be at risk. With that in mind, we characterized the current genetic variation of this species in Israel with reference to fish from Ghana, based on D-loop and microsatellite markers. Genetic variation and differentiation were found mostly among fish from Ghanaian localities and between fish from Israel and Ghana, whereas fish from all Israeli localities had uniform and limited variation, a signature compatible with historical founder effect followed by local adaptations. Such historical processes could leave a population vulnerable as reflected in the sudden and recent population decline. Comparing genetic variation between archived 30 year-old scales and modern lake fish revealed further reduction in genetic variation coincident with the recent population decline. Thus, a recently occurring genetic bottleneck had placed this unique and isolated population at an even higher risk. We carefully discuss the events leading to the current risk status for S. galilaeus in Israel and highlight the need for vigilant monitoring and active management to support a more sustainable future for this and other fish communities in this important habitat.
Resistance of Leishmania infantum to allopurinol is associated with chromosome and gene copy number variations including decrease in the S-adenosylmethionine synthetase (METK) gene copy number
. Includes articles from the scientific meeting: "Anthelmintics: From Discovery to Resistance III", pp. 494-628 2018
403 - 410. Abstract
Leishmania infantum is one of the causative agents of visceral leishmaniasis (VL), a widespread, life-threatening disease. This parasite is responsible for the majority of human VL cases in Brazil, the Middle East, China, Central Asia and the Mediterranean basin. Its main reservoir are domestic dogs which, similar to human patients, may develop severe visceral disease and die if not treated. The drug allopurinol is used for the long-term maintenance of dogs with canine leishmaniasis. Following our report of allopurinol resistance in treated relapsed dogs, we investigated the mechanisms and markers of resistance to this drug. Whole genome sequencing (WGS) of clinical resistant and susceptible strains, and laboratory induced resistant parasites, was carried out in order to detect genetic changes associated with resistance. Significant gene copy number variation (CNV) was found between resistant and susceptible isolates at several loci, including a locus on chromosome 30 containing the genes LinJ.30.3550 through LinJ.30.3580. A reduction in copy number for LinJ.30.3560, encoding the S-adenosylmethionine synthetase (METK) gene, was found in two resistant clinical isolates and four induced resistant clonal strains. Using quantitative real time PCR, this reduction in METK copy number was also found in three additional resistant clinical isolates. Furthermore, inhibition of S-adenosylmethionine synthetase encoded by the METK gene in allopurinol susceptible strains resulted in increased allopurinol resistance, confirming its role in resistance to allopurinol. In conclusion, this study identified genetic changes associated with L. infantum resistance to allopurinol and the reduction in METK copy number identified may serve as a marker for resistance in dogs, and reduced protein activity correlated with increased allopurinol resistance.
Comparison of the immune responses associated with experimental bovine mastitis caused by different strains of Escherichia coli
. Journal of Dairy Research 2017
, 190-197. Abstract
We studied the mammary immune response to different mammary pathogenic Escherichia coli (MPEC) strains in cows, hypothesising that the dynamics of response would differ. E. coli is a major aetiologic agent of acute clinical bovine mastitis of various degrees of severity with specific strains being associated with persistent infections. We compared challenge with three distinct pathogenic MPEC strains (VL2874, VL2732 and P4), isolated from different forms of mastitis (per-acute, persistent and acute, respectively). A secondary objective was to verify the lack of mammary pathogenicity of an environmental isolate (K71) that is used for comparison against MPEC in genomic and phenotypic studies. Twelve cows were challenged by intra-mammary infusion with one of the strains. Cellular and chemokine responses and bacterial culture follow-up were performed for 35 d. All cows challenged by any of the MPEC strains developed clinical mastitis. Differences were found in the intensity and duration of response, in somatic cell count, secreted cytokines (TNF-α, IL-6 and IL-17) and levels of milk leucocyte membrane Toll-like receptor 4 (TLR4). A sharp decrease of TLR4 on leucocytes was observed concomitantly to peak bacterial counts in milk. Intra-mammary infusion of strain K71 did not elicit inflammation and bacteria were not recovered from milk. Results suggest some differences in the mammary immune response to distinct MPEC strains that could be correlated to their previously observed pathogenic traits. This is also the first report of an E. coli strain that is non-pathogenic to the bovine mammary gland. © Proprietors of Journal of Dairy Research 2017.
Use of computer-assisted sperm analysis and flow cytometry to detect seasonal variations of bovine semen quality
. Theriogenology 2017
, 79 - 90. Abstract
Seasonal fluctuations of climate are considered a major factor affecting spermatogenesis and semen quality in the bovine. Our study aimed to investigate the effect of season on functional parameters of frozen-thawed bovine semen using computer-assisted sperm analysis (CASA) and flow cytometry. For this purpose, 86 ejaculates were collected from five mature Holstein-Friesian bulls kept under subtropical conditions during summer (August to September; n = 43) and winter (December to January; n = 43) months. Semen was diluted with a Tris-egg yolk-based extender and frozen at −196 °C. Computer-assisted sperm analysis was performed immediately after thawing (0h) and after 3 hours of incubation (3h) to evaluate the percentage (%) of total motile, progressively motile, and rapidly motile sperm. In addition, the average path, curvilinear, and straight-line velocities as well as the amplitude of lateral head displacement of sperm were determined. The percentages of sperm with intact plasma membrane and acrosome (PMAI, %), with high mitochondrial membrane potential (HMMP, %), with low intracellular Ca+2 levels (LOW-Ca+2, %), and with high DNA fragmentation index (DFI%, %) were flow cytometrically determined at 0 and 3h. The survival rate of sperm under hypotonic conditions (HYPO-SURV, %) and the percentage of sperm with inducible acrosome reaction (IAR, %) were assessed using flow cytometry at 0 and 3h, respectively. The fixed effect of season (winter vs. summer) on the quality parameters of sperm was explored by applying linear mixed-effects models. The results showed an improvement of all CASA parameters, except for the straight-line velocity (P > 0.05) in winter compared with summer for both unincubated and incubated sperm (P < 0.01 in all cases). Ejaculates collected in summer had lower values of IAR (P < 0.001) as well as PMAI, HMMP, and LOW-Ca+2 at 0 and 3h (P < 0.01 in all cases). On the contrary, HYPO-SURV and DFI% (at 0 and 3h) were not affected by season (P > 0.05 in all cases). Concluding, the employment of CASA and flow cytometry revealed season-related alterations in the functional status of cryopreserved bovine sperm, which suggest an adverse effect of summer heat stress on motility, plasma membrane and acrosome integrity, inducibility of acrosome reaction, mitochondrial function and intracellular Ca+2 content, but not on the DNA integrity of sperm after freezing–thawing.
Effect of the herbicide atrazine and its metabolite DACT on bovine sperm quality
. Reproductive Toxicology 2017
, 15 - 25. Abstract
Atrazine (ATZ), one of the most extensively used herbicides, is considered a ubiquitous environmental contaminant. ATZ is a known endocrine disruptor, and deleterious effects on reproductive function have been shown, even at low, ecologically relevant doses (0.1–3μg/L). Once it enters the body, ATZ is metabolized to various metabolites, which are further detected in the urine, serum and tissues. In mammals, the major ATZ metabolite is diaminochlorotriazine (DACT). The current study focuses on direct effects of low doses of ATZ and DACT on bovine sperm isolated from ejaculates or epididymis compartments (head, body and tail). Sperm were incubated under capacitation conditions with or without 0.1–10μM ATZ or 1–100μM DACT. The integrity and functionality of sperm membranes (plasma, acrosomal and mitochondrial) were examined simultaneously by fluorescence staining at 0, 2 and 4h of incubation. Acrosome reaction (AR) was induced by Ca++ ionophore, after capacitation. The findings indicated that both ATZ and DACT adversely affect sperm, expressed by damaged sperm membranes. ATZ had a prominent effect on epididymal-tail sperm, expressed as disruption of all examined membranes, mostly at low (0.1 or 1μM) concentrations; pseudo-AR and that induced by Ca++ ionophore were both affected by exposure to 0.1μM ATZ (P<0.05 and P<0.00004, respectively). A similar pattern was documented for sperm isolated from ejaculates (P<0.002 and P<0.001, respectively). ΔYm was affected by ATZ in sperm isolated from the epididymis tail (1μM, P<0.0009), but not in that isolated from ejaculates. DACT reduced sperm viability at all examined concentrations and in all fractions. DACT at 1μM impaired ΔΨm in sperm isolated from the epididymis tail and ejaculate (P<0.005). DACT at 100μM did not induce pseudo-AR in sperm isolated from the ejaculate, but did in sperm isolated from the epididymis tail (P<0.05). Induction of AR by Ca++ ionophore was impaired in sperm isolated from ejaculate and exposed to 10 or 100μM DACT (P<0.05) and in sperm isolated from the epididymis tail and exposed to 1, 10 or 100μM DACT (P<0.0004). These findings reveal the harmful effect of exposure to ATZ and DACT, mainly at low ecologically relevant doses, on sperm viability, AR and mitochondrial function. We conclude that sperm at advanced stages of spermatogenesis, through its passage and storage in the epididymis compartments as well as in the ejaculate, is sensitive to herbicide. The results suggest that ATZ- or DACT-induced disruptions of sperm membranes might impair sperm fertilization competence.
Effect of Heat Stress on Reproduction in Dairy Cows: Insights into the Cellular and Molecular Responses of the Oocyte
. Annual Review of Animal BiosciencesAnnual Review of Animal Biosciences 2017
151 - 170. Abstract
Among the components of the female reproductive tract, the ovarian pool of follicles and their enclosed oocytes are highly sensitive to hyperthermia. Heat-induced alterations in small antral follicles can be expressed later as compromised maturation and developmental capacity of the ovulating oocyte. This review summarizes the most up-to-date information on the effects of heat stress on the oocyte with an emphasis on unclear points and open questions, some of which might involve new research directions, for instance, whether preantral follicles are heat resistant. The review focuses on the follicle-enclosed oocytes, provides new insights into the cellular and molecular responses of the oocyte to elevated temperature, points out the role of the follicle microenvironment, and discusses some mechanisms that might underlie oocyte impairment. Mechanisms include nuclear and cytoplasmic maturation, mitochondrial function, apoptotic pathways, and oxidative stress. Understanding the mechanism by which heat stress compromises fertility might enable development of new strategies to mitigate its effects.Among the components of the female reproductive tract, the ovarian pool of follicles and their enclosed oocytes are highly sensitive to hyperthermia. Heat-induced alterations in small antral follicles can be expressed later as compromised maturation and developmental capacity of the ovulating oocyte. This review summarizes the most up-to-date information on the effects of heat stress on the oocyte with an emphasis on unclear points and open questions, some of which might involve new research directions, for instance, whether preantral follicles are heat resistant. The review focuses on the follicle-enclosed oocytes, provides new insights into the cellular and molecular responses of the oocyte to elevated temperature, points out the role of the follicle microenvironment, and discusses some mechanisms that might underlie oocyte impairment. Mechanisms include nuclear and cytoplasmic maturation, mitochondrial function, apoptotic pathways, and oxidative stress. Understanding the mechanism by which heat stress compromises fertility might enable development of new strategies to mitigate its effects.
Low level of mono(2-ethylhexyl) phthalate reduces oocyte developmental competence in association with impaired gene expression
. Toxicology 2017
, 38 - 48. Abstract
Di-(2-ethylhexyl) phthalate (DEHP) and its metabolite, mono-(2-ethylhexyl) phthalate (MEHP), are reproductive toxicants. However, disruptive effects of MEHP at low concentrations on the oocyte and developing blastocyst are unknown. Previously, we detected low levels of MEHP in follicular fluid aspirated from DEHP-treated cows associated with reduced estradiol levels. Moreover, the MEHP concentrations found were similar to those reported for follicular fluid aspirated from women who have undergone IVF cycles. In the current study, we used an in vitro embryo production model to examine the effect of MEHP at low levels on oocyte developmental competence. We set up several experiments to mimic the follicular fluid content, i.e., low MEHP level and low estradiol. For all experiments, cumulus oocyte complexes (COCs) were aspirated from bovine ovaries, then matured in vitro in standard oocyte maturation medium (OMM) supplemented with: MEHP at a range levels (20–1000nM) or with estradiol at a range levels (0–2000ng/ml). Then, oocytes were fertilized and cultured for an additional 7days to allow blastocyst development. Findings revealed that MEHP at low levels impairs oocyte developmental competence in a dose-dependent manner (P<0.05) and that estradiol by itself does not impair it. Accordingly, in another set of experiments, COCs were matured in vitro with MEHP at two choosen concentrations (20 or 1000nM) with or without estradiol, fertilized and cultured for 7days. Samples of mature oocytes and their derived blastocysts were subjected to quantitative real-time PCR to examine the profiles of selected genes (CYC1, MT-CO1, ATP5B, POU5F1, SOX2 and DNMT3b). Maturation of COCs with MEHP (20 or 1000nM) affected gene expression in the mature oocyte. Maturation of COCs with MEHP (20 or 1000nM) in the absence of estradiol reduced oocyte developmental competence (P<0.05). A differential carryover effect on transcript abundance was recorded in blastocysts developed from MEHP-treated oocytes. In the presence of estradiol, increased expression was recorded for CYC1, ATP5B, SOX2 and DNMT3b. In the absence of estradiol, decreased expression was recorded, with a significant effect for 1000nM MEHP (P<0.05). Taken together, the findings suggest that low levels of phthalate must be taken into consideration in risk assessments.
Host cell attachment elicits posttranscriptional regulation in infecting enteropathogenic bacteria
. Science 2017
, 735–739. Abstract
Escherichia coli is transformed from a commensal organism into a pathogen by acquisition of genetic elements called pathogenicity islands (PAIs). Katsowich et al. investigated how the PAI virulence genes of enteropathogenic E. coli (EPEC) respond when the bacterium attaches to a host gut cell. EPEC first sticks to the host by means of pili and then uses a PAI-encoded type 3 secretion system (T3SS) to inject multiple effectors into the host cell. But not all virulence mediators are injected. For example, CesT, a bacterial chaperone, delivers virulence effectors into the T3SS apparatus. Then, within the bacterial cytoplasm, it interacts with a gene repressor called CsrA, which reprograms bacterial gene expression to help the bacteria to adapt to epithelial cell–associated life.Science, this issue p. 735The mechanisms by which pathogens sense the host and respond by remodeling gene expression are poorly understood. Enteropathogenic Escherichia coli (EPEC), the cause of severe intestinal infection, employs a type III secretion system (T3SS) to inject effector proteins into intestinal epithelial cells. These effectors subvert host cell processes to promote bacterial colonization. We show that the T3SS also functions to sense the host cell and to trigger in response posttranscriptional remodeling of gene expression in the bacteria. We further show that upon effector injection, the effector-bound chaperone (CesT), which remains in the EPEC cytoplasm, antagonizes the posttranscriptional regulator CsrA. The CesT-CsrA interaction provokes reprogramming of expression of virulence and metabolic genes. This regulation is likely required for the pathogen’s adaptation to life on the epithelium surface.
Breaking the population barrier by single cell analysis: one host against one pathogen
. Current Opinion in Microbiology 2017
, 69 - 75. Abstract
Most of our understanding of the host–bacterium interaction has come from studies of bulk populations. In reality, highly adaptable and dynamic host cells and bacteria engage in complex, diverse interactions. This complexity necessarily limits the depth of understanding that can be gained with bulk population measurements. Here, we will review the merit of single cell analysis to characterize this diversity that can trigger heterogeneous outcomes. We will discuss heterogeneity of bacterial and host populations, differences in host microenvironments, technological advances that facilitate the analysis of rare subpopulations, and the potential relevance of these subpopulations to infection outcomes. We focus our discussion on intracellular bacterial pathogens and on methods that characterize and quantify RNA in single cells, aiming to highlight how novel methodologies have the potential to characterize the multidimensional process of infection and to provide answers to some of the most fundamental questions in the field.