Thrombospondin-1 at the crossroads of corpus luteum fate decisions
. Reproduction 2018
The multimodular matricellular protein thrombospondin-1 (THBS1) was among the first identified endogenous antiangiogenic molecules. Recent studies have shown THBS1-mediated suppression of angiogenesis and other critical activities for corpus luteum (CL) regression. THBS1 is specifically induced by prostaglandin F2alpha in mature CL undergoing regression, whereas luteinizing signals such as luteinizing hormone and insulin reduced its expression. THBS1 interacts both synergistically and antagonistically with other essential luteal factors, such as fibroblast growth factor 2, transforming growth factor beta1, and serpin family E member 1, to promote vascular instability, apoptosis, and matrix remodeling during luteal regression. Expression of THBS1 is also downregulated by pregnancy recognition signals to maintain the CL during early pregnancy. This dynamic pattern of luteal expression, the extensive interactivity with other luteal factors, and strong antiangiogenic and proapoptotic activities indicate that THBS1 is a major determinant of CL fate.
Innate immune functions of avian intestinal epithelial cells: Response to bacterial stimuli and localization of responding cells in the developing avian digestive tract
. PLoS One 2018
Intestinal epithelial cells are multi-tasked cells that participate in digestion and absorption as well as in protection of the digestive tract. While information on the physiology and immune functions of intestinal epithelial cells in mammals is abundant, little is known of their immune function in birds and other species. Our main objectives were to study the development of anti-bacterial innate immune functions in the rapidly developing gut of the pre- and post-hatch chick and to determine the functional diversity of epithelial cells. After establishing primary intestinal epithelial cell cultures, we demonstrated their capacity to uptake and process bacteria. The response to bacterial products, LPS and LTA, induced expression of pro-inflammatory cytokine genes (IL-6, IL-18) as well as the expression of the acute phase proteins avidin, lysozyme and the secretory component derived from the polymeric immunoglobulin receptor. These proteins were then localized in gut sections, and the goblet cell was shown to store avidin, lysozyme as well as secretory component. Lysozyme staining was also located in a novel rod-shaped intestinal cell, situated at different loci along the villus, thus deviating from the classical Paneth cell in the mammal, that is restricted to crypts. Thus, in the chicken, the intestinal epithelium, and particularly goblet cells, are committed to innate immune protection. The unique role of the goblet cell in chicken intestinal immunity, as well as the unique distribution of lysozyme-positive cells highlight alternative solutions of gut protection in the bird.
Plasma Lipoprotein Particle Subclasses in Preterm Infants
. Am J Perinatol 2018
OBJECTIVE: A pilot study to determine lipoprotein classes and subclasses in premature infants and examine associations with nutritional intake, gestational age (GA), and morbidity.
STUDY DESIGN: Plasma lipoprotein particle concentrations were analyzed in a cohort of 15 premature infants in the first 5 days of life and again at 2 weeks. Breast milk samples were analyzed for fatty acid content. Associations between lipoprotein particle subclasses and GA, breast milk intake, milk fatty acid intake, and chronic lung disease (CLD) were determined.
RESULTS: At 2 weeks of age, more premature infants had higher concentrations of total very low-density lipoprotein and lower concentrations of total high-density lipoprotein (HDL) and large HDL particles (similar to profiles seen in adults and children with infectious disease, cardiometabolic disease, and diabetes). Lower total HDL, large HDL, and medium HDL and a higher small HDL:total HDL ratio at 2 weeks were each associated with CLD with GA a likely confounder. Intake of human milk C18 and C20 fatty acids was inversely correlated with plasma total LDL concentration at 2 weeks of age.
CONCLUSION: Dyslipidemia was common in extremely premature infants and was associated with CLD and with lower intake of specific long chain fatty acids.
The Effect of Iodophor Post-Milking Teat Disinfection on Iodine Content in Goat Milk
. Israel Journal of Veterinary Medicine 2018
, 14 - 22.Abstract
Iodine intake is important for thyroid function and human health. Goat milk can be an important source of iodine for human nutrition. However, data regarding the effect of iodophor post-milking teat disinfection on iodine content in goat milk is lacking. Our aim was to assess the iodine concentrations in raw milk of dairy goats and to investigate the effect of post-milking teat-dipping iodophor practice on iodine content in goat milk. Two groups of dairy goats (n=6 in each) were treated with different post-milking teat-dipping disinfection: iodine-free solution (iodine-free group) and iodine-based solution (4,000 μg/L) (iodophors group). Treatments were carried out for 19 experimental days, following a 14-day pre-experimental period, in which only iodine-free sanitizer was used for both groups. The results showed that Iodine concentrations in milk of all goats were 49 ± 23, 49, 17-86 μg/100g (mean ± SD, median, range) at days-3,-2,-1 and 45 ± 26, 42, 14-96 μg/100g (mean ± SD, median, range) at days 17, 18, 19 of treatment. Iodine concentration increased by 7 μg/100g (mean) in the iodophors group while iodine concentration decreased by 15 μg/100g by day 17-19 of treatment in the iodine free group. It was concluded that relatively high iodine concentrations were found in raw milk of dairy goats whose teats were dipped post-milking in disinfectants with or without iodine. Post-milking teat-dipping iodophor practice may increase iodine content in goat milk within an average period of 20 days. This information can help in controlling iodine content in goat milk and iodine intake in the public.
Fluorimetric Techniques for the Assessment of Sperm Membranes
. JoVE 2018
, e58622. Publisher's VersionAbstract
Standard spermiograms describing sperm quality are mostly based on the physiological and visual parameters, such as ejaculate volume and concentration, motility and progressive motility, and sperm morphology and viability. However, none of these assessments is good enough to predict the semen quality. Given that maintenance of sperm viability and fertilization potential depends on membrane integrity and intracellular functionality, evaluation of these parameters might enable a better prediction of sperm fertilization competence. Here, we describe three feasible methods to evaluate sperm quality using specific fluorescent probes combined with fluorescence microscopy or flow cytometry analyses. Analyses assessed plasma membrane integrity using 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI), acrosomal membrane integrity using fluorescein isothiocyanate-conjugated Pisum sativum agglutinin (FITC-PSA) and mitochondrial membrane integrity using 5,5',6,6'-tetra-chloro-1,1',3,3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1). Combinations of these methods are also presented. For instance, use of annexin V combined with PI fluorochromes enables assessing apoptosis and calculating the proportion of apoptotic sperm (apoptotic index). We believe that these methodologies, which are based on examining spermatozoon membranes, are very useful for the evaluation of sperm quality.
Stress-induced alterations in oocyte transcripts are further expressed in the developing blastocyst
. Molecular Reproduction and Development 2018
, 821 - 835. Publisher's VersionAbstract
The oocyte achieves its developmental competence through the lengthy process of folliculogenesis. It can therefore potentially be exposed to various stressors while enclosed in the follicle. Oocyte maturation relies mainly on maternal sources. These include nuclear, cytoplasmic, and molecular maturation, which involve DNA and RNA organization. Maternal transcripts are dominant through the first embryonic cleavages, up until embryonic genome activation. Thus, it is suggested that any perturbations during oocyte storage, in particular of the maternal transcripts, might lead to genetic and/or epigenetic changes, which might be further expressed in the developing embryo. The review discusses the effects of three representative stressors?environmental heat stress, endocrine-disrupting compounds (phthalates), and inflammatory stress (mastitis)?shown to be involved in reduced fertility. The review highlights the carryover response from the oocyte to the developing embryo; it includes intracellular and molecular disruptive mechanisms with an emphasis on maternal transcripts. The review provides insights into the oocyte?s cellular and molecular responses with an emphasis on the effects of various stressors on the maternal (nuclear and mitochondrial) transcripts and the association with embryonic development. A comparison between stressors might clarify, at least in part, a few open questions. For instance, (a) whether stress-induced alterations share the same mechanism and if so (b) whether this mechanism involves alterations of maternal transcripts; (c) whether stress-induced alterations in the maternal transcript are further expressed at the developing blastocyst stage, that is, after embryonic genome activation.
Symposium review: Reduction in oocyte developmental competence by stress is associated with alterations in mitochondrial function1
. Journal of Dairy Science 2018
, 3642 - 3654. Publisher's VersionAbstract
ABSTRACTStress can affect reproductive performance of lactating cows by targeting the ovarian pool of follicles and their enclosed oocytes. Among the documented stressors are heat stress (i.e., high temperature-humidity index) as well as environmental and food toxins. Oocytes collected during the hot season are of lower quality than those collected in the winter, expressed by reduced oocyte maturation and developmental competence. A similar pattern has been reported for oocytes exposed to endocrine-disrupting chemicals. Whereas the underlying mechanism might differ among stressors, accumulating evidence suggests that stress-induced impairment of oocyte developmental competence involves alterations in mitochondrial functioning. Within the oocyte, mitochondria are involved in ATP generation, calcium homeostasis, regulation of cytoplasmic reduction–oxidation, signal transduction, and apoptosis. Summer heat stress is strongly associated with alterations in mitochondrial distribution and alterations in mitochondria membrane potential. Heat stress impairs the expression of mitochondrion-associated genes, in particular those related to mitochondrial DNA transcription and replication and encoding oxidative phosphorylation complexes for ATP production. Reduction of ATP levels below the required threshold is suggested to compromise the progression of oocyte maturation and, subsequently, embryonic development. Another mechanism associated with mitochondrial function is the increase in reactive oxygen species (ROS), which has been documented in oocytes exposed to heat stress or environmental toxicants. Oxidative phosphorylation in mitochondria is the major source of ROS. Under physiological conditions, ROS are essential for nuclear maturation; however, disequilibrium between ROS production and antioxidative capacity might lead to DNA damage and apoptosis. The current review provides new insights into the oocyte's cellular and molecular responses to stress with an emphasis on the mitochondria. It discusses some strategies to mitigate the effects of stress on the mitochondria, such as incorporation of coenzyme Q10—a key component of the mitochondrial respiratory chain—administration of antioxidants, and injection of healthy mitochondria. Exploring the oocyte's cellular and molecular responses, in particular that of the mitochondria, might lead to the development of new strategies to mitigate the effects of various stressors on fertility.
Aflatoxin B1 impairs sperm quality and fertilization competence
. Toxicology 2018
, 42 - 50. Publisher's VersionAbstract
Aflatoxins are poisonous byproducts of the soilborne fungus Aspergillus, involved in the decomposition of plant materials. Aflatoxins can be found in various food products, such as maize, sorghum, millet, rice and wheat. AFB1 is the most toxic of these, classified as a carcinogen and mutagen for both humans and animals. AFB1 has been detected in human cord blood and placenta; however, its toxic effect on sperm is less known. The current study examines sperm responses associated with AFB1 exposure. These included acrosome integrity and function, mitochondrial polarity, DNA fragmentation, fertilization competence and early embryonic development. Spermatozoa were obtained from bull ejaculate and epididymis and capacitated in vitro for 4h with 0, 0.1, 1, 10 and 100μM AFB1. Following capacitation, acrosome reaction (AR) was induced by Ca2+ ionophore. The integrity and functionality of sperm were examined simultaneously by florescent staining. A Halosperm DNA fragmentation kit was used to evaluate DNA integrity. An in-vitro culture system was used to evaluate fertilization competence and blastocyst formation rate, using bovine oocytes. Findings indicate dose-responsive variation among compartments to AFB1 exposure. Sperm viability, expressed by integrity of the plasma membrane, was lower in sperm isolated from ejaculate or epididymis after culturing with AFB1. Exposure to AFB1 reduced the proportion of sperm from the epididymis tail undergoing acrosome reaction induced by Ca2+ ionophore. AFB1 impaired mitochondrial membrane potential (ΔYm) in sperm isolated from ejaculate and the epididymis tail. Exposing ejaculated sperm to AFB1 increased the proportion of sperm with fragmented DNA and reduced the proportion of embryos that cleaved to the 2- to 4-cell stage, 42h postfertilization, however, the proportion of embryos that developed to blastocysts, 7days postfertilization, did not differ among groups. The findings explore the harmful effects of AFB1 on sperm viability, ΔΨm and DNA integrity associated with fertility competence. We postulate that AFB1-induced fragmentation in paternal DNA might have a carryover effect on the quality of developing embryos. Further evaluation for the quality of blastocysts derived from sperm exposed to AFB1 is warranted.
The effects of replacing eggs with chicks on mesotocin, dopamine, and prolactin in the native Thai hen
. General and Comparative Endocrinology 2018
, 32 - 42. Publisher's VersionAbstract
The mesotocinergic (MTergic) and dopaminergic (DAergic) systems have been documented to play pivotal roles in maternal behaviors in native Thai chickens. In native Thai chickens, plasma prolactin (PRL) concentrations are associated with maternal behaviors, which are also controlled by the DAergic system. However, the role of MT in conjunction with the roles of DA and PRL on the neuroendocrine regulation of the transition from incubating to rearing behavior has never been studied. Therefore, the aim of this study was to investigate the association of MT, DA, and PRL during the transition from incubating to rearing behavior in native Thai hens. Using an immunohistochemistry technique, the numbers of MT-immunoreactive (-ir) and tyrosine hydroxylase-ir (TH-ir, a DA marker) neurons were compared between incubating hens (INC; n = 6) and hens for which the incubated eggs were replaced with 3 newly hatched chicks for 3 days after 6, 10, and 14 days of incubation (REC; n = 6). Plasma PRL concentrations were determined by enzyme-linked immunosorbent assay. The results revealed that the numbers of MT-ir neurons within the nucleus supraopticus, pars ventralis (SOv), nucleus preopticus medialis (POM), and nucleus paraventricularis magnocellularis (PVN) increased in the REC hens when compared with those of the INC hens at 3 different time points (at days 9, 13, and 17). On the other hand, the number of TH-ir neurons in the nucleus intramedialis (nI) decreased in the REC13 and REC17 hens when compared with those of the INC hens. However, the number of TH-ir neurons in the nucleus mamillaris lateralis (ML) only decreased in the REC13 hens when compared with the INC13 hens. The decrease in the numbers of TH-ir neurons within the nI and ML is associated with the decrease in the levels of plasma PRL. This study suggests that the presence of either eggs or chicks is the key factor regulating the MTergic system within the SOv, POM, and PVN and the DAergic system within the nI and ML during the transition from incubating to rearing behavior in native Thai chickens. The results further indicate that these two systems play pivotal roles in the transition from incubating to rearing behavior in this equatorial species.
Hormonal levels of estradiol, testosterone, and progesterone at entry into lay of year 1980 vs. 2000 broiler breeder females under fast and slow release from feed restriction
. Poultry Science 2018
, 3728 - 3735. Publisher's VersionAbstract
In the mid-1960s egg production, fertility, and hatchability of broiler breeder females dropped precipitously. Due to disrupted follicle hierarchies and development of the erratic oviposition and defective eggs (EODES) syndrome. EODES was controlled by restricting feed. In the 1990s, another set of problems arose at entry of broiler breeders into lay and characterized by high mortality followed by lower peak lay and reduction in egg and chick production. These problems are induced by even slight over-feeding, and hence we termed it the “Over Feeding Complex” (OFC). We have speculated that OFC is a quasi-EODES condition, induced by the intense selection for increased breast proportion. To test this, we compared, under fast (FF) and slow (SF) release from feed restriction, body composition and reproductive performance of a broiler breeder from year 1980 (B1980) and kept without selection for performance traits since then, to a line hatched in 2000 (B2000). During the first 16 d of lay, feeding treatment had little effect on egg mass or Laying % for the B1980 birds, while for the B2000 birds, SF treatment resulted in significantly greater egg mass and Laying % compared to FF, showing that the OFC indeed manifested in this experiment. However, contrary to hypothesis, follicle hierarchies were normal for both lines under both feeding treatments. To gain further insight into the OFC syndrome, we here report levels of estradiol, testosterone, and progesterone for these line and treatment groups in the time period leading up to and into lay. A significant line × feeding treatment interaction effect was found for estradiol and testosterone, to a lesser extent for progesterone. For all 3 hormones, for B1980 levels 2 to 3 wk post entry into lay were similar and intermediate under FF and SF, but differed significantly for B2000, being much greater under SF than under FF. Thus, the hormonal effects were parallel and may explain the egg mass and Laying % effects of FF and SF in the 2 genetic types.
In-ovo green light photostimulation during different embryonic stages affect somatotropic axis
. Poultry Science 2018
, 1998 - 2004. Publisher's VersionAbstract
Previous studies demonstrated that in-ovo photostimulation with monochromatic green light increased the somatotropic axis expression in broilers embryos. The objective of the current study was to detect the critical period for in-ovo GL photostimulation, in order to find the optimal targeted photostimulation period during the incubation process. Three hundred thirty-six fertile broiler eggs were divided into 4 groups. The first group was incubated under dark conditions as a negative control. The second incubated under intermittent monochromatic green light using light-emitting diode (LED) lamps with an intensity of 0.1 W\m2 at shell level from d 0 of the incubation as a positive control. The third group incubated under intermittent monochromatic green light from d 10 of the incubation. The last group incubated under intermittent monochromatic green light from d 15 of the incubation. In-ovo green light photostimulation from embryonic d 0 (ED0) increased plasma growth hormone (GH), as well as hypothalamic growth hormone releasing hormone (GHRH) and liver growth hormone receptor (GHR) and insulin-like growth factor-1 (IGF-1) mRNA levels. In-ovo green light photostimulation from ED10 increased the GH plasma levels compared to the negative control group, without affecting somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1. In-ovo green light photostimulation from ED15 caused an increase in both the plasma GH levels and the somatotropic axis mRNA genes expressions of GHRH, GHR, and IGF-1, compared to the negative control group. These results suggest that the critical period of somatotropic axis acceleration by GL photostimulation start at 15 d of incubation.
Mammary core clock gene expression is impacted by photoperiod exposure during the dry period in goats
. Journal of Applied Animal Research 2018
, 1214 - 1219. Publisher's VersionAbstract
ABSTRACTShort-day photoperiod (SDPP) during the dry period increases milk production compared to long-day photoperiod (LDPP) in goats. Photoperiod information is sent to the master clock in the suprachiasmatic nuclei (SCN), which send temporal information to peripheral clocks located in every tissue of the body. We hypothesized photoperiod effects on milk production are mediated in part by changes in mammary clocks. Our objective was to determine the effect of photoperiod manipulation during the dry period in goats on core clock genes expression in mammary gland. Multiparous goats (n?=?6) were blocked at dry off into two treatments: LDPP and SDPP. Serial mammary biopsies were taken over a 24?h period during three weeks prepartum. Total RNA was isolated, and q-PCR analysis of the core clock genes CLOCK, ARNTL, PER1, CRY1, and CRY2 found exposure to LDPP significantly increased ARNTL (P?
Low Iodine Intake from Dairy Foods Despite High Milk Iodine Content in Israel
. Thyroid 2018
, 1042 - 1051. Publisher's VersionAbstract
Background: Milk is a major source of iodine in human nutrition. Because both iodine content and the consumption of milk and dairy vary widely over time and populations, their contribution to iodine intake must be evaluated regularly. A recent national iodine survey found Israel's population to be mildly iodine deficient, possibly due to unmonitored changes in the food content of dietary iodine. Accounting for dairy iodine content can help guide efforts to prevent iodine deficiency. Objectives: This study aimed to determine the iodine concentration of dairy products typically consumed in the Israeli diet, and to estimate iodine intake from dairy products among Israeli adults. Methods: Iodine was analyzed in 33 selected dairy products that account for 89% of the total population's dairy intake according to the ?MABAT? Israeli National Health and Nutrition survey. Based on these data, the distribution of iodine intake from milk, dairy, and dairy-based foods in the adult population was calculated. Results: Israeli milk is rich in iodine, with a mean concentration of 22??g/100?g. However, due to low dairy consumption, the mean iodine intake from milk and dairy was only 34??g/day (median 23??g/day; range: 0?337??g/day) or 22% of the recommended daily allowance. Self-reported intake among poor, male, and Arab subgroups was even lower. Conclusions: Because Israeli milk and dairy products are iodine rich, their contribution to the population's iodine intake would increase if they were consumed in greater amounts, particularly by high-risk groups. Dairy's potential contribution to iodine nutrition should be considered in recommendations for dairy consumption and iodine prophylaxis.Background: Milk is a major source of iodine in human nutrition. Because both iodine content and the consumption of milk and dairy vary widely over time and populations, their contribution to iodine intake must be evaluated regularly. A recent national iodine survey found Israel's population to be mildly iodine deficient, possibly due to unmonitored changes in the food content of dietary iodine. Accounting for dairy iodine content can help guide efforts to prevent iodine deficiency. Objectives: This study aimed to determine the iodine concentration of dairy products typically consumed in the Israeli diet, and to estimate iodine intake from dairy products among Israeli adults. Methods: Iodine was analyzed in 33 selected dairy products that account for 89% of the total population's dairy intake according to the ?MABAT? Israeli National Health and Nutrition survey. Based on these data, the distribution of iodine intake from milk, dairy, and dairy-based foods in the adult population was calculated. Results: Israeli milk is rich in iodine, with a mean concentration of 22??g/100?g. However, due to low dairy consumption, the mean iodine intake from milk and dairy was only 34??g/day (median 23??g/day; range: 0?337??g/day) or 22% of the recommended daily allowance. Self-reported intake among poor, male, and Arab subgroups was even lower. Conclusions: Because Israeli milk and dairy products are iodine rich, their contribution to the population's iodine intake would increase if they were consumed in greater amounts, particularly by high-risk groups. Dairy's potential contribution to iodine nutrition should be considered in recommendations for dairy consumption and iodine prophylaxis.
The cAMP-EPAC Pathway Mediates PGE2-Induced FGF2 in Bovine Granulosa Cells
. Endocrinologyendo 2018
, 3482 - 3491. Publisher's VersionAbstract
During the periovulatory period, the profile of fibroblast growth factor 2 (FGF2) coincides with elevated prostaglandin E2 (PGE2) levels. We investigated whether PGE2 can directly stimulate FGF2 production in bovine granulosa cells and, if so, which prostaglandin E2 receptor (PTGER) type and signaling cascades are involved. PGE2 temporally stimulated FGF2. Accordingly, endoperoxide-synthase2–silenced cells, exhibiting low endogenous PGE2 levels, had reduced FGF2. Furthermore, elevation of viable granulosa cell numbers by PGE2 was abolished with FGF2 receptor 1 inhibitor, suggesting that FGF2 mediates this action of PGE2. Epiregulin (EREG), a known PGE2-inducible gene, was studied alongside FGF2. PTGER2 agonist elevated cAMP as well as FGF2 and EREG levels. However, a marked difference between cAMP-induced downstream signaling was observed for FGF2 and EREG. Whereas FGF2 upregulated by PGE2, PTGER2 agonist, or forskolin was unaffected by the protein kinase A (PKA) inhibitor H89, EREG was significantly inhibited. FGF2 was dose-dependently stimulated by the exchange protein directly activated by cAMP (EPAC) activator; a similar induction was observed for EREG. However, forskolin-stimulated FGF2, but not EREG, was inhibited in EPAC1-silenced cells. These findings ascribe a novel autocrine role for PGE2, namely, elevating FGF2 production in granulosa cells. This study also reveals that cAMP-activated EPAC1, rather than PKA, mediates the effect of PGE2/PTGER2 on the expression of FGF2. Stimulation of EREG by PGE2 is also mediated by PTGER2 but, in contrast to FGF2, EREG was found to be PKA sensitive. PGE2-stimulated FGF2 can act to maintain granulosa cell survival; it can also act on ovarian endothelial cells to promote angiogenesis.
Genomic profiling of bovine corpus luteum maturation
. PLOS ONE 2018
, e0194456 -. Publisher's VersionAbstract
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. Publisher's VersionAbstract
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. Publisher's VersionAbstract
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.