Copper transporter 1 (CTR1) expression by mouse testicular germ cells, but not Sertoli cells, is essential for functional spermatogenesis.

An imbalance in copper (Cu) tissue homeostasis has a degenerative effect on spermatogenesis and male fertility. The high-affinity Cu transporter 1 (CTR1; SLC31A1) is the major protein responsible for Cu acquisition in eukaryotes and is highly expressed in mouse testes. Studies on yeast and Drosophila have demonstrated the conserved essential function of Cu and CTR1 for meiosis and fertility, implying that CTR1 may play an essential function in mammalian spermatogenesis. In mice, spermatogenesis takes place within the seminiferous epithelium, where tight junctions between somatic Sertoli cells (SCs) create a specialized microenvironment for the development of meiotic germ cells (GCs) by tightly regulating the free transport of metabolites and ions to reach these cells. Here, it is demonstrated that within the seminiferous epithelium, CTR1 is expressed on the membrane of primary pachytene spermatocytes and SCs. To examine the physiological significance of CTR1 in spermatogenesis, mice with a GC-specific (Ctr1ΔGC) and SC-specific (Ctr1ΔSC) disruption of the Ctr1 gene were generated. The testis of Ctr1ΔGC mice exhibits a severe progressive loss of GCs starting at postnatal day (PND) 28 leading to testis hypoplasia by adulthood. No spermatogenic recovery was observed in Ctr1ΔGC testis beyond PND 41, despite the presence of FOXO-1 expressing undifferentiated spermatogonial cells. However, Ctr1ΔSC mice displayed functional spermatogenesis and were fertile, even though testicular Cu levels and Cu-dependent cellular activities were significantly reduced. These results reveal, for the first time, the importance of CTR1 expression by GCs for maintaining functional spermatogenesis.

Regulation of germ cell development by ARI1 family ubiquitin ligases in C. elegans.

RING-between-RING (RBR) E3 ubiquitin ligases are implicated in various developmental processes, and mutations in genes encoding RBR proteins HHARI/ARIH1 and Parkin are associated with human diseases. Here we show by phylogenetic analysis that the ARI1 family has undergone a dramatic expansion within the Caenorhabditis clade in recent history, a characteristic shared by some genes involved in germline development. We then examined the effects of deleting all ARI1 family members in the nematode Caenorhabditis elegans, which to our knowledge represents the first complete knockout of ARI1 function in a metazoan. Hermaphrodites that lacked or had strongly reduced ARI1 activity had low fecundity and were partially defective in initiation of oocyte differentiation. We provide evidence that the C. elegans ARI1s likely function downstream or in parallel to FBF-1 and FBF-2, two closely related RNA-binding proteins that are required for the switch from spermatogenesis to oogenesis during late larval development. Previous studies have shown that the E2 enzymes UBC-18/UBCH7 and UBC-3/CDC34 can functionally collaborate with ARI1 family members. Our data indicated that UBC-18, but not UBC-3, specifically cooperates with the ARI1s in germline development. These findings provide new insights into the functions of RING-between-RING proteins and Ariadne E3s during development.

Two functionally distinct E2/E3 pairs coordinate sequential ubiquitination of a common substrate in Caenorhabditis elegans development.

Ubiquitination, the crucial posttranslational modification that regulates the eukaryotic proteome, is carried out by a trio of enzymes, known as E1 [ubiquitin (Ub)-activating enzyme], E2 (Ub-conjugating enzyme), and E3 (Ub ligase). Although most E2s can work with any of the three mechanistically distinct classes of E3s, the E2 UBCH7 is unable to function with really interesting new gene (RING)-type E3s, thereby restricting it to homologous to E6AP C-terminus (HECT) and RING-in-between-RING (RBR) E3s. The Caenorhabditis elegans UBCH7 homolog, UBC-18, plays a critical role in developmental processes through its cooperation with the RBR E3 ARI-1 (HHARI in humans). We discovered that another E2, ubc-3, interacts genetically with ubc-18 in an unbiased genome-wide RNAi screen in C. elegans These two E2s have nonoverlapping biochemical activities, and each is dedicated to distinct classes of E3s. UBC-3 is the ortholog of CDC34 that functions specifically with Cullin-RING E3 ligases, such as SCF (Skp1-Cullin-F-box). Our genetic and biochemical studies show that UBCH7 (UBC-18) and the RBR E3 HHARI (ARI-1) coordinate with CDC34 (UBC-3) and an SCF E3 complex to ubiquitinate a common substrate, a SKP1-related protein. We show that UBCH7/HHARI primes the substrate with a single Ub in the presence of CUL-1, and that CDC34 is required to build chains onto the Ub-primed substrate. Our study reveals that the association and coordination of two distinct E2/E3 pairs play essential roles in a developmental pathway and suggests that cooperative action among E3s is a conserved feature from worms to humans.

Transdermal Bioavailability in Rats of Lidocaine in the Forms of Ionic Liquids, Salts, and Deep Eutectic.

Tuning the bioavailability of lidocaine was explored by its incorporation into the ionic liquid lidocainium docusate ([Lid][Doc]) and the deep eutectic Lidocaine·Ibuprofen (Lid·Ibu) and comparing the transdermal absorption of these with the crystalline salt lidocainium chloride ([Lid]Cl). Each form of lidocaine was dissolved in a vehicle cream and topically applied to Sprague-Dawley rats. The concentrations of the active pharmaceutical ingredients (APIs) in blood plasma were monitored over time as an indication of systemic absorption. The concentration of lidocaine in plasma varied between applied API-based creams, with faster and higher systemic absorption of the hydrogen bonded deep eutectic Lid·Ibu than the absorption of the salts [Lid]Cl or [Lid][Doc]. Interestingly, a differential transdermal absorption was observed between lidocaine and ibuprofen when Lid·Ibu was applied, possibly indicating different interactions with the tissue components.

Mono-(2-ethylhexyl) phthalate-induced Sertoli cell injury stimulates the production of pro-inflammatory cytokines in Fischer 344 rats.

Exposure of rodents to the Sertoli cell (SC) toxicant mono-(2-ethylhexyl) phthalate (MEHP) has been reported to trigger an infiltration of macrophages into the testis in an age- and species-dependent manner. Here we challenge the hypothesis that the peripubertal rat-specific infiltration of macrophages after MEHP exposure is due, in part, to an increase in SC-specific inflammatory cytokine expression. To rule out that germ cell(GC) apoptosis itself is responsible for macrophage recruitment, rats were exposed to a direct GC toxicant, methoxyacetic acid (MAA), but no infiltration of macrophages was observed. Next, mRNA levels of inflammatory cytokines were evaluated after MEHP exposure. IL-1α, IL-6, and MCP-1 expression were increased in vivo and correlated with macrophage infiltration in a species-specific manner. Additionally, IL-6 and MCP-1 expression was increased in SC-GC co-cultures and ASC-17D SCs. These results indicate that MEHP-injury in pubertal rats specifically stimulates secretion of pro-inflammatory cytokines and alters the immune microenvironment.

Female mice with loss-of-function ITCH display an altered reproductive phenotype.

Major progress in deciphering the role of the E3 ligase, ITCH, in animal physiology has come from the generation and identification of Itch loss-of-function mutant mice (itchy). Mutant mice display an autoimmune-like phenotype characterized by chronic dermatitis, which has been attributed to increased levels of ITCH target proteins (e.g. transcription factors JUNB and CJUN) in T cells. Autoimmune disorders also exist in humans with Itch frameshift mutations resulting in loss of functional ITCH protein. Recent phenotypic analysis of male itchy mice revealed reduced sperm production, although cross breeding experiments showed no difference in litter size when male itchy mice were bred to wild type females. However, a reduction in litter sizes did occur when itchy females were bred to wild type males. Based on these results, characterization of female reproductive function in itchy mice was performed. Developmental analysis of fetuses at gestational day 18.5, cytological evaluation of estrous cyclicity, histopathological analysis of ovaries, and protein analysis were used to investigate the itchy reproductive phenotype. Gross skeletal and soft tissue analysis of gestational day 18.5 itchy fetuses indicated no gross developmental deformities. Itchy females had reduced implantation sites, decreased corpora lutea, and increased estrous cycle length due to increased number of days in estrus compared to controls. Alterations in the expression of prototypical ITCH targets in the ovaries were not indicated, suggesting that an alteration in an as yet defined ovary-specific ITCH substrate or interaction with the altered immune system likely accounts for the disruption of female reproduction. This report indicates the importance of the E3 ligase, ITCH, in female reproduction.

Short- and Long-Term Effects of Prenatal Exposure to Iron Oxide Nanoparticles: Influence of Surface Charge and Dose on Developmental and Reproductive Toxicity.

Iron oxide nanoparticles (NPs) are commonly utilized for biomedical, industrial, and commercial applications due to their unique properties and potential biocompatibility. However, little is known about how exposure to iron oxide NPs may affect susceptible populations such as pregnant women and developing fetuses. To examine the influence of NP surface-charge and dose on the developmental toxicity of iron oxide NPs, Crl:CD1(ICR) (CD-1) mice were exposed to a single, low (10 mg/kg) or high (100 mg/kg) dose of positively-charged polyethyleneimine-Fe2O3-NPs (PEI-NPs), or negatively-charged poly(acrylic acid)-Fe2O3-NPs (PAA-NPs) during critical windows of organogenesis (gestation day (GD) 8, 9, or 10). A low dose of NPs, regardless of charge, did not induce toxicity. However, a high exposure led to charge-dependent fetal loss as well as morphological alterations of the uteri (both charges) and testes (positive only) of surviving offspring. Positively-charged PEI-NPs given later in organogenesis resulted in a combination of short-term fetal loss (42%) and long-term alterations in reproduction, including increased fetal loss for second generation matings (mice exposed in utero). Alternatively, negatively-charged PAA-NPs induced fetal loss (22%) earlier in organogenesis to a lesser degree than PEI-NPs with only mild alterations in offspring uterine histology observed in the long-term.

Surface charge and dosage dependent potential developmental toxicity and biodistribution of iron oxide nanoparticles in pregnant CD-1 mice.

Iron oxide nanoparticles have attracted much attention because of their potential applications, such as drug delivery, biomedical imaging, and photocatalysis. Due to their small size and the potential to cross the placental barrier, the risk to pregnant women and the developing fetus from exposure to nanoparticles is of great concern. The developmental toxicity and biodistribution of a single dose versus multiple doses of iron oxide nanoparticles with positive or negative surface charges were investigated in vivo. Multiple doses of positively-charged nanoparticles given over several days resulted in significantly increased fetal deaths and accumulation of iron in the fetal liver and placenta. These results indicate both positively and negatively charged iron oxide nanoparticles have the ability to cross the placenta and accumulate in the fetus, though greater bioaccumulation and toxicity was observed with a positively-charged surface coating.

Urinary chromium excretion in response to an insulin challenge is not a biomarker for chromium status.

Over 50 years ago, chromium (Cr) was proposed to be an essential trace element; however, recent studies indicate that this status should be removed as the effects of Cr supplementation appear to be pharmacological rather than nutritional. The pharmacological basis for Cr's effects can explain the inability of investigators to discover a biomarker for Cr status. One potential biomarker has not been examined to date. Cr is known to be mobilized in the body in response to insulin (or insulin release in response to a glucose challenge), resulting in an increase in urinary Cr excretion. The magnitude of increase in urinary Cr loss as a function of dietary Cr intake was tested as a potential biomarker for Cr. Zucker lean rats housed in carefully controlled metal-free conditions were provided a series of purified diets containing variable Cr contents (from 16 µg/kg diet to 2,000 µg/kg) for 23 weeks. The 16 µg/kg diet contained less Cr than any diet examined to date. Urine samples were collected before and after insulin and glucose challenges (0, 2, 6, and 12 h postinjection). Urinary Cr levels were analyzed by the standard method of addition using graphite furnace atomic absorption. The rate of urinary Cr loss after a glucose or insulin challenge was found to not be dependent on the Cr content of the rats' diets. Blood iron levels of the rats were also measured to determine if the addition of Cr to the diet altered iron status. The Cr content of the diet was found to have no affect on blood iron levels. Overall, the study demonstrated that insulin-stimulated urinary Cr excretion cannot be used as a biomarker for Cr status.

Comparison of tissue metal concentrations in Zucker lean, Zucker obese, and Zucker diabetic fatty rats and the effects of chromium supplementation on tissue metal concentrations.

Diabetes results in several metabolic changes, including alterations in the transport, distribution, excretion, and accumulation of metals. While changes have been examined in several rat models of insulin resistance and diabetes, the metal ion concentrations in the tissues of Zucker lean, Zucker obese (an insulin resistance and early stage diabetes model), and Zucker diabetic fatty (ZDF, a type 2 diabetes model) have not previously been examined in detail. The concentration of Cu, Zn, Fe, Mg, and Ca were examined in the liver, kidney, heart and spleen, and Cr concentration in the liver and kidney of these rats were examined. Zucker obese rats have a reduction in the concentration of Cu, Zn, Fe, Mg in the liver compared to ZDF and/or lean Zucker rats, presumably as a result of the increased fat content of the liver of the obese rats. ZDF rats have increased concentrations of kidney Cu compared to the lean rats, while kidney Ca concentrations are increased in the Zucker obese rats. Spleen Fe concentrations are decreased in Zucker obese rats compared to the lean rats. No effects on metal concentrations in the heart were observed between the lean, obese, and ZDF rats, and no effects on Cr concentrations were identified. Cr(III) complexes have previously been shown to have beneficial effects on the signs of insulin resistance in Zucker obese and ZDF rats. The effects of daily gavage administration of chromium picolinate ([Cr(pic)(3)]) (1 mg Cr/kg body mass), CrCl(3) (1 mg Cr/kg body mass), and Cr3 ([Cr(3)O(propionate)(6)(H(2)O)(3)](+)) (33 µg and 1 mg Cr/kg body mass) on metal concentrations in these tissues were examined. Treatment with CrCl(3) and Cr3, but not [Cr(pic)(3)], at 1 mg Cr/kg resulted in a statistically significant accumulation of Cr in the kidney of lean and obese but not ZDF rats but resulted in lowering the elevated levels of kidney Cu in ZDF rats, suggesting a beneficial effect on this symptom of type 2 diabetes.

Potential of chromium(III) picolinate for reproductive or developmental toxicity following exposure of male CD-1 mice prior to mating.

Chromium(III) picolinate, [Cr(pic)(3)], is a commonly used nutritional supplement in humans, which has also been approved for use in animals. Health concerns have arisen over the use of [Cr(pic)(3)]. At high [Cr(pic)(3)] doses, developmental toxicity tests in female mice have shown a higher litter incidence of split cervical arch in exposed fetuses, but this was not consistently reproducible. In the current study, male CD-1 mice were used to further assess the potential for reproductive or developmental toxicity. Four weeks prior to mating, the males were fed a diet providing 200 mg/kg/day [Cr(pic)(3)] for comparison with untreated controls. Females were not treated. Each male was mated with two females, which were sacrificed on gestation day 17, and their litters were examined for adverse effects. Mating and fertility indices were not significantly altered by treatment. Male exposure to [Cr(pic)(3)] also had no effect on prenatal mortality, fetal weight, or gross or skeletal morphology. These results suggest that paternal dietary exposure to chromium(III) picolinate has little potential for adverse reproductive effects, even at exposure levels considerably higher than expected human exposures from nutritional supplements (1 mg of Cr per day or less).

Chromium is not an essential trace element for mammals: effects of a "low-chromium" diet.

Chromium was proposed to be an essential trace element over 50 years ago and has been accepted as an essential element for over 30 years. However, the studies on which chromium's status are based are methodologically flawed. Whether chromium is an essential element has been examined for the first time in carefully controlled metal-free conditions using a series of purified diets containing various chromium contents. Male Zucker lean rats were housed in specially designed metal-free cages for 6 months and fed the AIN-93G diet with no added chromium in the mineral mix component of the diet, the standard AIN-93G diet, the standard AIN-93G diet supplemented with 200 µg Cr/kg, or the standard AIN-93G diet supplemented with 1,000 µg Cr/kg. The chromium content of the diet had no effect on body mass or food intake. Similarly, the chromium content of the diet had no effect on glucose levels in glucose tolerance or insulin tolerance tests. However, a distinct trend toward lower insulin levels under the curve after a glucose challenge was observed with increasing chromium content in the diet; rats on the supplemented AIN-93G diets had significantly lower areas (P < 0.05) than rats on the low-chromium diet. The studies reveal that a diet with as little chromium as reasonably possible had no effect on body composition, glucose metabolism, or insulin sensitivity compared with a chromium-"sufficient" diet. Together with the results of other recent studies, these results clearly indicate that chromium can no longer be considered an essential element.

A comparison of the effects of prenatal exposure of CD-1 mice to three imidazolium-based ionic liquids.

BACKGROUND: Ionic liquids (ILs; salts with melting points below 100 degrees C) exhibit wide liquid ranges, non-flammability, and thermal stability among other properties. These unique salts are best known as "green" alternatives to traditional volatile organic solvents, which are utilized in both academia and industry. Our current study compares the developmental toxicity potential of three representative ionic liquids, with various chain lengths: 1-ethyl-3-methylimidazolium chloride ([C(2)mim]Cl), 1-butyl-3-methylimidazolium chloride ([C(4)mim]Cl), and 1-decyl-3methylimidazolium chloride ([C(10)mim]Cl). METHODS: From gestation days (GD) 6-16, mated CD-1 mice were orally dosed with one of the following: 1,000, 2,000, or 3,000 mg/kg/day [C(2)mim]Cl; 113, 169, or 225 mg/kg/day [C(4)mim]Cl; 50, 75, or 100 mg/kg/day [C(10)mim]Cl; or the vehicle only. Dams were sacrificed on GD 17, and their litters were examined for adverse effects. RESULTS: Fetal weight was significantly decreased in the two highest dosage groups exposed to [C(4)mim]Cl and [C(10)mim]Cl in comparison with their controls, but the [C(2)mim]Cl treated groups were not affected. An apparent teratogenic effect was associated with both [C(4)mim]Cl and [C(10)mim]Cl, as the offspring exhibited certain uncommon morphological defects. However, the incidences of malformations were low and no correlation between incidence and dosage could be made. No morphological defects were observed in any of the [C(2)mim]Cl-treated groups, despite maternal morbidity at the highest dosage level. CONCLUSIONS: This study indicates that [C(4)mim]Cl and [C(10)mim]Cl may have adverse effects on development at high maternal exposures and strongly supports the supposition that the toxicity of imidazolium-based ILs is influenced by alkyl chain length.

Urinary chromium loss associated with diabetes is offset by increases in absorption.

The results of the current study indicate that diabetic rats have increased urinary Cr loss as a result of their diabetes; however, this increased urinary Cr loss is offset by increased absorption of Cr. Insulin resistant, obese rats have alterations in the rates of Cr transport and distribution compared to lean rats but have similar levels of urinary Cr loss and Cr absorption. Thus, any increases in urinary Cr loss associated with insulin resistance or diabetes are offset by increased absorption. Given that dietary chromium is normally absorbed with only approximately 1% efficiency, suitable Cr exists in the diet so that a standard diet possesses sufficient chromium to allow for the increases in absorption associated with diabetes. Consequently, supplementing the diet with nutritionally relevant quantities of chromium is not anticipated to have any beneficial effects. Similarly, beneficial effects on plasma variables, such as cholesterol, triglycerides, and insulin concentrations, from supra-nutritional doses of Cr(III) complexes should not arise from alleviation of chromium deficiency. These beneficial effects must arise from pharmacological effects of high dose Cr(III) administration.