Neonatal Aromatase Inhibition Blocked Defeminization of AVPV Kiss1 Neurons and LH Surge-Generating System in Male Rats.

The neuroendocrine system that controls the preovulatory surge of gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH), which triggers ovulation in female mammals, is sexually differentiated in rodents. A transient increase in circulating testosterone levels in male rats within a few hours of birth is primarily responsible for the defeminization of anteroventral periventricular nucleus (AVPV) kisspeptin neurons, which are critical regulators of the GnRH/LH surge. The present study aimed to determine whether neonatal estradiol-17ß (E2) converted from testosterone by aromatase primarily causes the defeminization of AVPV kisspeptin neurons and the surge of GnRH/LH in male rodents. The results of the present study showed that the neonatal administration of letrozole (LET), a nonsteroidal aromatase inhibitor, within 2 hours of birth rescued AVPV Kiss1 expression and the LH surge in adult male rats, while the neonatal administration of testosterone propionate (TP) irreversibly attenuated AVPV Kiss1 expression and the LH surge in adult female rats. Furthermore, the neonatal LET-treated Kiss1-Cre-activated tdTomato reporter males exhibited a comparable number of AVPV Kiss1-Cre-activated tdTomato-expressing cells to that of vehicle-treated female rats, while neonatal TP-treated females showed fewer AVPV Kiss1-Cre-activated tdTomato-expressing cells than vehicle-treated females. Moreover, neonatal TP administration significantly decreased the number of arcuate Kiss1-expressing and Kiss1-Cre-activated tdTomato-positive cells and suppressed LH pulses in adult gonadectomized female rats; however, neonatal LET administration failed to affect them. These results suggest that E2 converted from neonatal testosterone is primarily responsible for the defeminization of AVPV kisspeptin neurons and the subsequent GnRH/LH surge generation in male rats.

Assessing Molecular Localization of Symbiont Microalgae in Coral Branches Through Mass Spectrometry Imaging.

Reef-building corals are a fundamental pillar of coral reef ecosystems in tropical and subtropical shallow environments. Corals harbor symbiotic dinoflagellates belonging to the family Symbiodiniaceae, commonly known as zooxanthellae. Extensive research has been conducted on this symbiotic relationship, yet the fundamental information about the distribution and localization of Symbiodiniaceae cells in corals is still limited. This information is crucial to understanding the mechanism underlying the metabolite exchange between corals and their algal symbionts, as well as the metabolic flow within holobionts. To examine the distribution of Symbiodiniaceae cells within corals, in this study, we used fluorescence imaging and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry imaging (MS-Imaging) on branches of the Acropora tenuis coral. We successfully prepared frozen sections of the coral for molecular imaging without fixing or decalcifying the coral branches. By combining the results of MS-Imaging with that of the fluorescence imaging, we determined that the algal Symbiodiniaceae symbionts were not only localized in the tentacle and surface region of the coral branches but also inhabited the in inner parts. Therefore, the molecular imaging technique used in this study could be valuable to further investigate the molecular dynamics between corals and their symbionts.

A lipidomics approach reveals novel phospholipid changes in palmitate-treated C2C12 myotubes.

Type 2 diabetes mellitus (T2DM) is a highly prevalent metabolic disorder. Insulin resistance and oxidative stress are associated with T2DM development. The hypothesis that patients with T2DM show excess accumulation of lipids, such as ceramides (Cers) and diacylglycerols (DAGs), in their skeletal muscles has been widely supported; however, detailed lipidomic data at the molecular species level are limited. Therefore, in this study, we aimed to investigate the in vitro dynamics of total lipids, including phospholipids (PLs), sphingolipids, and neutral lipids, in palmitic acid-induced insulin-resistant C2C12 skeletal muscle cells. Our data demonstrated that the profiles of not only Cers and DAGs but also those of PLs showed considerably differences after palmitate treatment. We found that PL synthesis reduced and PL degradation increased after palmitate treatment. These findings may aid in the development of treatments to ameliorate muscle dysfunction caused by lipid accumulation in muscles.

Impact of genetic polymorphism on personalized diet and exercise program for steatotic liver disease.

AIMS: The effects of genetic polymorphism on a personalized diet and exercise program for steatotic liver disease (SLD) are still unclear. METHODS: Participants of this retrospective cohort study were 203 Japanese patients with SLD diagnosed by abdominal ultrasonography. All of them were introduced the personalized diet and exercise treatment. A diet of 25-30 kcal/kg multiplied by ideal body weight (BW) daily and aerobic and resistance exercise (exercise intensity of 4-5 metabolic equivalents daily, respectively) were performed for 6 days. Treatment efficacy was evaluated in terms of the rate of decrease of liver function tests, glycolipid metabolism markers, physical findings, image findings, and cardiovascular disease (CVD) risk score at 6 months compared with baseline. Furthermore, the impact of genetic polymorphism was also investigated. RESULTS: At 6 months compared with baseline, liver function tests (AST, ALT, γGTP), glycolipid metabolism markers (hemoglobin A1c, triglycerides [TG], low-density lipoprotein cholesterol), physical findings (BW, body mass index), image finding (liver stiffness measurement), and CVD risk score (Suita score) improved significantly. There was no significant difference in treatment efficacy, except for the rates of decrease of TG, according to genotype PNPLA3 rs738409, TM6SF2 rs58542926, and HSD17B13 rs6834314. The rates of decrease of TG with TM6SF2 CT were significantly higher than those with CC or TT, and the rates of TG with HSD17B13 AA were significantly higher than those with AG by multiple comparisons. CONCLUSION: Personalized diet and exercise program for SLD improved liver function tests, physical findings, glycolipid metabolism markers, and CVD risk score. Genetic polymorphism might partially affect treatment efficacy. Further studies should be performed to develop an individualized program for SLD, considering genetic polymorphism.

Conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons caused estrogen-dependent LH pulse disruption and LH surge attenuation in female rats.

The gonadotropin-releasing hormone (GnRH) pulse and surge are considered to be generated by arcuate kisspeptin/neurokinin B/dynorphin A (KNDy) neurons and anteroventral periventricular nucleus (AVPV) kisspeptin neurons, respectively, in female rodents. The majority of KNDy and AVPV kisspeptin neurons express κ-opioid receptors (KORs, encoded by Oprk1) in female rodents. Thus, this study aimed to investigate the effect of a conditional Oprk1-dependent Kiss1 deletion in kisspeptin neurons on the luteinizing hormone (LH) pulse/surge and fertility using Kiss1-floxed/Oprk1-Cre rats, in which Kiss1 was deleted in cells expressing or once expressed the Oprk1/Cre. The Kiss1-floxed/Oprk1-Cre female rats, with Kiss1 deleted in a majority of KNDy neurons, showed normal puberty while having a one-day longer estrous cycle and fewer pups than Kiss1-floxed controls. Notably, ovariectomized (OVX) Kiss1-floxed/Oprk1-Cre rats showed profound disruption of LH pulses in the presence of a diestrous level of estrogen but showed apparent LH pulses without estrogen treatment. Furthermore, Kiss1-floxed/Oprk1-Cre rats, with Kiss1 deleted in approximately half of AVPV kisspeptin neurons, showed a lower peak of the estrogen-induced LH surge than controls. These results suggest that arcuate and AVPV kisspeptin neurons expressing or having expressed Oprk1 have a role in maintaining normal GnRH pulse and surge generation, the normal length of the estrous cycle, and the normal offspring number in female rats.

Sex difference in developmental changes in visualized Kiss1 neurons in newly generated Kiss1-Cre rats.

Hypothalamic kisspeptin neurons are master regulators of mammalian reproduction via direct stimulation of gonadotropin-releasing hormone and consequent gonadotropin release. Here, we generated novel Kiss1 (kisspeptin gene)-Cre rats and investigated the developmental changes and sex differences in visualized Kiss1 neurons of Kiss1-Cre-activated tdTomato reporter rats. First, we validated Kiss1-Cre rats by generating Kiss1-expressing cell-specific Kiss1 knockout (Kiss1-KpKO) rats, which were obtained by crossing the current Kiss1-Cre rats with Kiss1-floxed rats. The resulting male Kiss1-KpKO rats lacked Kiss1 expression in the brain and exhibited hypogonadotropic hypogonadism, similar to the hypogonadal phenotype of global Kiss1 KO rats. Histological analysis of Kiss1 neurons in Kiss1-Cre-activated tdTomato reporter rats revealed that tdTomato signals in the anteroventral periventricular nucleus (AVPV) and arcuate nucleus (ARC) were not affected by estrogen, and that tdTomato signals in the ARC, AVPV, and medial amygdala (MeA) were sexually dimorphic. Notably, neonatal AVPV tdTomato signals were detected only in males, but a larger number of tdTomato-expressing cells were detected in the AVPV and ARC, and a smaller number of cells in the MeA was detected in females than in males at postpuberty. These findings suggest that Kiss1-visualized rats can be used to examine the effect of estrogen feedback mechanisms on Kiss1 expression in the AVPV and ARC. Moreover, the Kiss1-Cre and Kiss1-visualized rats could be valuable tools for further detailed analyses of sexual differentiation in the brain and the physiological role of kisspeptin neurons across the brain in rats.

Smart utilization of betaine lipids in the giant clam Tridacna crocea.

The giant clam Tridacna crocea thrives in poorly nourished coral reef water by forming a holobiont with zooxanthellae and utilizing photosynthetic products of the symbiont. However, detailed metabolic crosstalk between clams and symbionts is elusive. Here, we discovered that the nonphosphorous microalgal betaine lipid DGCC (diacylglycerylcarboxy-hydroxymethylcholine) and its deacylated derivative GCC are present in all tissues and organs, including algae-free sperm and eggs, and are metabolized. Colocalization of DGCC and PC (phosphatidylcholine) evidenced by MS imaging suggested that DGCC functions as a PC substitute. The high content of GCC in digestive diverticula (DD) suggests that the algal DGCC was digested in DD for further utilization. Lipidomics analysis showing the organ-specific distribution pattern of DGCC species suggests active utilization of DGCC as membrane lipids in the clam. Thus, the utilization of zooxanthellal DGCC in animal cells is a unique evolutionary outcome in phosphorous-deficient coral reef waters.

Novel predator-induced phenotypic plasticity by hemoglobin and physiological changes in the brain of Xenopus tropicalis.

Organisms adapt to changes in their environment to survive. The emergence of predators is an example of environmental change, and organisms try to change their external phenotypic systems and physiological mechanisms to adapt to such changes. In general, prey exhibit different phenotypes to predators owing to historically long-term prey-predator interactions. However, when presented with a novel predator, the extent and rate of phenotypic plasticity in prey are largely unknown. Therefore, exploring the physiological adaptive response of organisms to novel predators is a crucial topic in physiology and evolutionary biology. Counterintuitively, Xenopus tropicalis tadpoles do not exhibit distinct external phenotypes when exposed to new predation threats. Accordingly, we examined the brains of X. tropicalis tadpoles to understand their response to novel predation pressure in the absence of apparent external morphological adaptations. Principal component analysis of fifteen external morphological parameters showed that each external morphological site varied nonlinearly with predator exposure time. However, the overall percentage change in principal components during the predation threat (24 h) was shown to significantly (p < 0.05) alter tadpole morphology compared with that during control or 5-day out treatment (5 days of exposure to predation followed by 5 days of no exposure). However, the adaptive strategy of the altered sites was unknown because the changes were not specific to a particular site but were rather nonlinear in various sites. Therefore, RNA-seq, metabolomic, Ingenuity Pathway Analysis, and Kyoto Encyclopedia of Genes and Genomes analyses were performed on the entire brain to investigate physiological changes in the brain, finding that glycolysis-driven ATP production was enhanced and ß-oxidation and the tricarboxylic acid cycle were downregulated in response to predation stress. Superoxide dismutase was upregulated after 6 h of exposure to new predation pressure, and radical production was reduced. Hemoglobin was also increased in the brain, forming oxyhemoglobin, which is known to scavenge hydroxyl radicals in the midbrain and hindbrain. These suggest that X. tropicalis tadpoles do not develop external morphological adaptations that are positively correlated with predation pressure, such as tail elongation, in response to novel predators; however, they improve their brain functionality when exposed to a novel predator.

Enkephalin-δ opioid receptor signaling partly mediates suppression of LH release during early lactation in rats.

Gonadal function is often suppressed during lactation in mammals including rodents, ruminants, and primates. This suppression is thought to be mostly due to the inhibition of the tonic (pulsatile) release of gonadotropin-releasing hormone (GnRH) and consequent gonadotropin. Accumulating evidence suggests that kisspeptin neurons in the arcuate nucleus (ARC) play a critical role in the regulation of pulsatile GnRH/gonadotropin release, and kisspeptin mRNA (Kiss1) and/or kisspeptin expression in the ARC are strongly suppressed by the suckling stimuli in lactating rats. This study aimed to examine whether the central enkephalin-δ-opioid receptor (DOR) signaling mediates the suckling-induced suppression of luteinizing hormone (LH) release in lactating rats. Central administration of a selective DOR antagonist increased the mean plasma LH levels and baseline of LH pulses in ovariectomized lactating mother rats compared to vehicle-injected control dams on day 8 of lactation without affecting the number of Kiss1-expressing cells and the intensity of Kiss1 mRNA signals in the ARC. Furthermore, the suckling stimuli significantly increased the number of enkephalin mRNA (Penk)-expressing cells and the intensity of Penk mRNA signals in the ARC compared to non-lactating control rats. Collectively, these results suggest that central DOR signaling, at least in part, mediates the suppression of LH release induced by suckling stimuli in lactating rats via indirect and/or direct inhibition of ARC kisspeptin neurons.

Kisspeptin and lactational anestrus: Current understanding and future prospects.

Lactational anestrus, characterized by the suppression of pulsatile gonadotropin-releasing hormone (GnRH)/luteinizing hormone (LH) release, would be a strategic adaptation to ensure survival by avoiding pregnancy during lactation in mammals. In the present article, we first provide a current understanding of the central regulation of reproduction in mammals, i.e., a fundamental role of arcuate kisspeptin neurons in mammalian reproduction by driving GnRH/LH pulses. Second, we discuss the central mechanism inhibiting arcuate Kiss1 (encoding kisspeptin) expression and GnRH/LH pulses during lactation with a focus on suckling stimulus, negative energy balance due to milk production, and the role of circulating estrogen in rats. We also discuss upper regulators that control arcuate kisspeptin neurons in rats during the early and late lactation periods based on the findings obtained by a lactating rat model. Finally, we discuss potential reproductive technology for the improvement of reproductive performance in milking cows.

LPGAT1/LPLAT7 regulates acyl chain profiles at the sn-1 position of phospholipids in murine skeletal muscles.

Skeletal muscle consists of both fast- and slow-twitch fibers. Phospholipids are important structural components of cellular membranes, and the diversity of their fatty acid composition affects membrane characteristics. Although some studies have shown that acyl chain species in phospholipids differ among various muscle fiber types, the mechanisms underlying these differences are unclear. To investigate this, we analyzed phosphatidylcholine (PC) and phosphatidylethanolamine (PE) molecules in the murine extensor digitorum longus (EDL; fast-twitch) and soleus (slow-twitch) muscles. In the EDL muscle, the vast majority (93.6%) of PC molecules was palmitate-containing PC (16:0-PC), whereas in the soleus muscle, in addition to 16:0-PC, 27.9% of PC molecules was stearate-containing PC (18:0-PC). Most palmitate and stearate were bound at the sn-1 position of 16:0- and 18:0-PC, respectively, and 18:0-PC was found in type I and IIa fibers. The amount of 18:0-PE was higher in the soleus than in the EDL muscle. Peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) increased the amount of 18:0-PC in the EDL. Lysophosphatidylglycerol acyltransferase 1 (LPGAT1) was highly expressed in the soleus compared with that in the EDL muscle and was upregulated by PGC-1α. LPGAT1 knockout decreased the incorporation of stearate into PC and PE in vitro and ex vivo and the amount of 18:0-PC and 18:0-PE in murine skeletal muscle with an increase in the level of 16:0-PC and 16:0-PE. Moreover, knocking out LPGAT1 decreased the amount of stearate-containing phosphatidylserine (18:0-PS), suggesting that LPGAT1 regulated the acyl chain profiles of phospholipids, namely, PC, PE, and PS, in the skeletal muscle.

Stable isotope-labeled carnitine reveals its rapid transport into muscle cells and acetylation during contraction.

Carnitine plays multiple roles in skeletal muscle metabolism, including fatty acid transport and buffering of excess acetyl-CoA in the mitochondria. The skeletal muscle cannot synthesize carnitine; therefore, carnitine must be taken up from the blood into the cytoplasm. Carnitine metabolism, its uptake into cells, and the subsequent reactions of carnitine are accelerated by muscle contraction. Isotope tracing enables the marking of target molecules and monitoring of tissue distribution. In this study, stable isotope-labeled carnitine tracing was combined with matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) imaging to determine carnitine distribution in mouse skeletal muscle tissues. Deuterium-labeled carnitine (d3-carnitine) was intravenously injected into the mice and diffused to the skeletal muscles for 30 and 60 min. To examine whether muscle contraction changes the distribution of carnitine and its derivatives, unilateral in situ muscle contraction was performed; 60 min muscle contraction showed increased d3-carnitine and its derivative d3-acetylcarnitine in the muscle, indicating that carnitine uptake in cells is promptly converted to acetylcarnitine, consequently, buffering accumulated acetyl-CoA. While the endogenous carnitine was localized in the slow type fibers rather than fast type, the contraction-induced distributions of d3-carnitine and acetylcarnitine were not necessarily associated with muscle fiber type. In conclusion, the combination of isotope tracing and MALDI-MS imaging can reveal carnitine flux during muscle contraction and show the significance of carnitine in skeletal muscles.

Treatment efficacy of diet and exercise program for fatty liver and pretreatment predictors.

AIMS: Both diet and exercise counseling are recommended for patients with fatty liver, including nonalcoholic fatty liver disease (NAFLD), to achieve weight loss goals. However, data evaluating treatment efficacy are limited. METHODS: The subjects of this retrospective cohort study were 186 consecutive Japanese cases with fatty liver diagnosed by abdominal ultrasonography. Treatment efficacy and predictive factors of "Hospitalization Program for Improvement Purpose for Fatty Liver" as a combined diet and aerobic and resistance exercise program were evaluated according to the hospitalization group (153 cases) or the no hospitalization group (33 cases). To balance the confounding biases, treatment efficacy was evaluated using propensity score-matched analysis. In the hospitalization group, a diet of 25-30 kcal/kg multiplied by ideal body weight (BW) daily and aerobic and resistance exercise (exercise intensity of 4-5 metabolic equivalents daily, respectively) were performed for 6 days. RESULTS: In liver function tests and BW at 6 months compared with baseline, the rates of decrease of the hospitalization group (24 cases) were significantly higher than those of the no hospitalization group (24 cases), using propensity score-matched analysis. In markers of glycolipid metabolism and ferritin levels, the rates of the hospitalization group were not different from those of the no hospitalization group. In the hospitalization group (153 cases), multivariate regression analysis identified the etiology of non-NAFLD, the presence of diabetes mellitus, and large waist circumference as independent predictors of decreased rates of hemoglobin A1c levels. CONCLUSION: The diet and exercise program for fatty liver improved liver function tests and BW. Further study should be performed to develop a feasible and suitable program.

Hindbrain Adenosine 5-Triphosphate (ATP)-Purinergic Signaling Triggers LH Surge and Ovulation via Activation of AVPV Kisspeptin Neurons in Rats.

Ovulation disorders are a serious problem for humans and livestock. In female rodents, kisspeptin neurons in the anteroventral periventricular nucleus (AVPV) are responsible for generating a luteinizing hormone (LH) surge and consequent ovulation. Here, we report that adenosine 5-triphosphate (ATP), a purinergic receptor ligand, is a possible neurotransmitter that stimulates AVPV kisspeptin neurons to induce an LH surge and consequent ovulation in rodents. Administration of an ATP receptor antagonist (PPADS) into the AVPV blocked the LH surge in ovariectomized (OVX) rats treated with a proestrous level of estrogen (OVX + high E2) and significantly reduced the ovulation rate in proestrous ovary-intact rats. AVPV ATP administration induced a surge-like LH increase in OVX + high E2 rats in the morning. Importantly, AVPV ATP administration could not induce the LH increase in Kiss1 KO rats. Furthermore, ATP significantly increased intracellular Ca2+ levels in immortalized kisspeptin neuronal cell line, and coadministration of PPADS blocked the ATP-induced Ca2+ increase. Histologic analysis revealed that the proestrous level of estrogen significantly increased the number of P2X2 receptor (an ATP receptor)-immunopositive AVPV kisspeptin neurons visualized by tdTomato in Kiss1-tdTomato rats. The proestrous level of estrogen significantly increased varicosity-like vesicular nucleotide transporter (a purinergic marker)-immunopositive fibers projecting to the vicinity of AVPV kisspeptin neurons. Furthermore, we found that some hindbrain vesicular nucleotide transporter-positive neurons projected to the AVPV and expressed estrogen receptor α, and the neurons were activated by the high E2 treatment. These results suggest that hindbrain ATP-purinergic signaling triggers ovulation via activation of AVPV kisspeptin neurons.SIGNIFICANCE STATEMENT Ovulation disorders, which cause infertility and low pregnancy rates, are a serious problem for humans and livestock. The present study provides evidence that adenosine 5-triphosphate, acting as a neurotransmitter in the brain, stimulates kisspeptin neurons in the anteroventral periventricular nucleus, known as the gonadotropin-releasing hormone surge generator, via purinergic receptors to induce the gonadotropin-releasing hormone/luteinizing hormone surge and ovulation in rats. In addition, histologic analyses indicate that adenosine 5-triphosphate is likely to be originated from the purinergic neurons in the A1 and A2 of the hindbrain. These findings may contribute to new therapeutic controls for hypothalamic ovulation disorders in humans and livestock.

Enkephalin-δ Opioid Receptor Signaling Mediates Glucoprivic Suppression of LH Pulse and Gluconeogenesis in Female Rats.

Energy availability is an important regulator of reproductive function at various reproductive phases in mammals. Glucoprivation induced by 2-deoxy-D-glucose (2DG), an inhibitor of glucose utilization, as an experimental model of malnutrition suppresses the pulsatile release of GnRH/LH and induces gluconeogenesis. The present study was performed with the aim of examining whether enkephalin-δ-opioid receptor (DOR) signaling mediates the suppression of pulsatile GnRH/LH release and gluconeogenesis during malnutrition. The administration of naltrindole hydrochloride (NTI), a selective DOR antagonist, into the third ventricle blocked the suppression of LH pulses and part of gluconeogenesis induced by IV 2DG administration in ovariectomized rats treated with a negative feedback level of estradiol-17â€…ß (OVX + low E2). The IV 2DG administration significantly increased the number of Penk (enkephalin gene)-positive cells coexpressing fos (neuronal activation marker gene) in the paraventricular nucleus (PVN), but not in the arcuate nucleus (ARC) in OVX + low E2 rats. Furthermore, double in situ hybridization for Penk/Pdyn (dynorphin gene) in the PVN revealed that approximately 35% of the PVN Penk-expressing cells coexpressed Pdyn. Double in situ hybridization for Penk/Crh (corticotropin-releasing hormone gene) in the PVN and Penk/Kiss1 (kisspeptin gene) in the ARC revealed that few Penk-expressing cells coexpressed Crh and Kiss1. Taken together, these results suggest that central enkephalin-DOR signaling mediates the suppression of pulsatile LH release during malnutrition. Moreover, the current study suggests that central enkephalin-DOR signaling is also involved in gluconeogenesis during malnutrition in female rats.

Changes in Tissue Distribution of Tetrodotoxin and Its Analogues in Association with Maturation in the Toxic Flatworm, Planocera multitentaculata.

The toxic flatworm, Planocera multitentaculata, possesses highly concentrated tetrodotoxin (TTX), also known as pufferfish toxin, throughout its life cycle, including the egg and larval stages. Additionally, TTX analogues, 5,6,11-trideoxyTTX and 11-norTTX-6(S)-ol, have also been detected in the flatworm. The high concentration of TTX in the eggs and larvae appears to be for protection against predation, and 11-norTTX-6(S)-ol in the pharyngeal tissue in the adults is likely used to sedate or kill prey during predation. However, information on the role of 5,6,11-trideoxyTTX, a potential important biosynthetic intermediate of TTX, in the toxic flatworm is lacking. Here, we aimed to determine the region of localization of TTX and its analogues in the flatworm body, understand their pharmacokinetics during maturation, and speculate on their function. Flatworm specimens in four stages of maturity, namely juvenile, mating, spawning, and late spawning, were subjected to LC-MS/MS analysis, using the pharyngeal tissue, oocytes in seminal receptacle, sperm, and tissue from 12 other sites. Although TTX was consistently high in the pharyngeal tissue throughout maturation, it was extremely high in the oocytes during the spawning period. Meanwhile, 5,6,11-trideoxyTTX was almost undetectable in the pharyngeal part throughout the maturation but was very abundant in the oocytes during spawning. 11-norTTX-6(S)-ol consistently localized in the pharyngeal tissue. Although the localization of TTX and its analogues was approximately consistent with the MS imaging data, TTX and 11-norTTX-6(S)-ol were found to be highly localized in the parenchyma surrounding the pharynx, which suggests the parenchyma is involved in the accumulation and production of TTXs.

Possibility of Multiple Drug-Drug Interactions in Patients Treated with Statins: Analysis of Data from the Japanese Adverse Drug Event Report (JADER) Database and Verification by Animal Experiments.

Adverse drug events due to drug-drug interactions can be prevented by avoiding concomitant use of causative drugs; therefore, it is important to understand drug combinations that cause drug-drug interactions. Although many attempts to identify drug-drug interactions from real-world databases such as spontaneous reporting systems have been performed, little is known about drug-drug interactions caused by three or more drugs in polypharmacy, i.e., multiple drug-drug interactions. Therefore, we attempted to detect multiple drug-drug interactions using decision tree analysis using the Japanese Adverse Drug Event Report (JADER) database, a Japanese spontaneous reporting system. First, we used decision tree analysis to detect drug combinations that increase the risk of rhabdomyolysis in cases registered in the JADER database that used six statins. Next, the risk of three or more drug combinations that significantly increased the risk of rhabdomyolysis was validated with in vivo experiments in rats. The analysis identified a multiple drug-drug interaction signal only for pitavastatin. The reporting rate of rhabdomyolysis for pitavastatin in the JADER database was 0.09, and it increased to 0.16 in combination with allopurinol. Furthermore, the rate was even higher (0.40) in combination with valsartan. Additionally, necrosis of leg muscles was observed in some rats simultaneously treated with these three drugs, and their creatine kinase and myoglobin levels were elevated. The combination of pitavastatin, allopurinol, and valsartan should be treated with caution as a multiple drug-drug interaction. Since multiple drug-drug interactions were detected with decision tree analysis and the increased risk was verified in animal experiments, decision tree analysis is considered to be an effective method for detecting multiple drug-drug interactions.

Dynorphin-κ-opioid receptor signaling, but not µ-opioid receptor signaling, partly mediates the suppression of luteinizing hormone release during late lactation in rats.

Follicular development and ovulation are profoundly suppressed during lactation. This suppression is suggested to be due to the suckling-induced inhibition of the kisspeptin gene (the master regulator of reproduction) in the arcuate nucleus (ARC) and subsequent inhibition of pulsatile gonadotropin-releasing hormone (GnRH)/gonadotropin release. The present study examined whether hypothalamic κ-opioid receptor (KOR) or µ-opioid receptor (MOR) signaling mediates the suppression of luteinizing hormone (LH) release induced by suckling stimulus during late lactation in rats. Central administration of a selective KOR antagonist blocked the suppression of LH release on Day 16 of lactation; however, central administration of a selective MOR antagonist failed to block the suppression. The suckling stimulus significantly increased the number of fos (a marker for neural activation)-positive Pdyn (dynorphin gene)-expressing cells in the paraventricular nucleus (PVN) and supraoptic nucleus (SON) but not in the ARC. Taken together, these results suggest that central KOR signaling, but not MOR signaling, at least partly, mediates the suppression of LH release induced by suckling stimulus during late lactation, and PVN and SON Dyn neurons may be involved in the suppression in rats.

Establishment of embryo transfer in the musk shrew (Suncus murinus).

The present study established techniques to induce pseudopregnancy, in vitro oocyte cultures from pronuclear to 2- to 4-cell stages, and embryo transfer in musk shrews, a reflex ovulator. Offspring were subsequently obtained by transferring in vivo-developed or in vitro-cultured embryos. Female musk shrews received human chronic gonadotropin (hCG), with or without mating stimuli, from vasectomized males to produce pseudopregnant recipients. Embryos at the 2- to 4-cell stage were collected 44-48 h after mating. Another set of embryos was collected 26-27 h after mating and then cultured for 20 h from the pronuclear to 2- to 4-cell stages. Subsequently, embryos were transferred into the oviducts of pseudopregnant recipients 24 or 48 h after the induction of pseudopregnancy. Offsprings were successfully obtained from recipients that received hCG 24 h before embryo transfer, regardless of mating stimuli. These techniques may be valuable for producing transgenic musk shrews.

Opioidergic pathways and kisspeptin in the regulation of female reproduction in mammals.

Endogenous opioid peptides have attracted attention as critical neuropeptides in the central mechanism regulating female reproduction ever since the discovery that arcuate dynorphin neurons that coexpress kisspeptin and neurokinin B (NKB), which are also known as kisspeptin/neurokinin B/dynorphin (KNDy) neurons, play a role as a master regulator of pulsatile gonadotropin-releasing hormone (GnRH) release in mammals. In this study, we first focus on the role of dynorphin released by KNDy neurons in the GnRH pulse generation. Second, we provide a historical overview of studies on endogenous opioid peptides. Third, we discuss how endogenous opioid peptides modulate tonic GnRH/gonadotropin release in female mammals as a mediator of inhibitory internal and external cues, such as ovarian steroids, nutritional status, or stress, on reproduction. Then, we discuss the role of endogenous opioid peptides in GnRH surge generation in female mammals.