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Summary. To detect the major sites of viral replication in immunodeficiency virus-infected individuals, we quantified proviral DNA and infectious viruses using quantitative PCR and a plaque assay, respectively, in various tissues of SHIV KU-2-infected monkeys in the early and AIDS stages of infection. Compared the quantity of infectious virus among PBMC and the lymphoid tissues, the mesenteric lymph node had the largest number of infectious viruses at the AIDS stage more than at the early stage of infection. These results suggested that the gastrointestinal tract was a major site of viral replication. In the brain, proviral DNA was detected at the early and AIDS stage of infection, but infectious viruses were detected at only the AIDS stage. Moreover, we analyzed the nucleotide sequences of the env V3 region in infectious virus clones isolated from each plaque. The viruses in the lymphoid tissues of the monkey that developed AIDS diverged from the inoculated virus and had the same three amino acid substitutions. However, the viruses in the brain were almost identical to the inoculated virus, suggesting that the virus entered the brain early after infection and persisted without replication and genetic diversion until the AIDS stage. [ABSTRACT FROM AUTHOR]

Neurons co-expressing kisspeptin, neurokinin B, and dynorphin A (KNDy neurons), located in the arcuate nucleus (ARC) of the hypothalamus, are indicated to be the gonadotropin-releasing hormone (GnRH) pulse generator. Dynorphin A is reported to suppress GnRH pulse generator activity. Nalfurafine is a selective agonist of the κ-opioid receptor (KOR), a receptor for dynorphin A, clinically used as an anti-pruritic drug. This study aimed to evaluate the effects of nalfurafine on GnRH pulse generator activity and luteinizing hormone (LH) pulses using female goats. Nalfurafine (0, 2, 4, 8, or 16 μg/head) was intravenously injected into ovariectomized Shiba goats. The multiple unit activity (MUA) in the ARC area was recorded, and plasma LH concentrations were measured 2 and 48 h before and after injection, respectively. The MUA volley interval during 0-2 h after injection was significantly increased in the nalfurafine 8 and 16 μg groups compared with the vehicle group. In 0-2 h after injection, the number of LH pulses was significantly decreased in the nalfurafine 8 and 16 μg groups, and the mean and baseline LH were significantly decreased in all nalfurafine-treated groups (2, 4, 8, and 16 μg) compared with the vehicle group. These results suggest that nalfurafine inhibits the activity of the GnRH pulse generator in the ARC, thus suppressing pulsatile LH secretion. Therefore, nalfurafine could be used as a reproductive inhibitor in mammals., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: This study was partly supported by funds donated by Toray Industries, Inc., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Cryopreservation adversely affects embryo quality and viability in vitro.We investigated the effects of cryopreservation solutions supplemented with the antioxidant carnosine on frozen-thawed bovine embryo viability. Bovine blastocysts were produced in vitro and cryopreserved using slow freezing. The rates of re-expanded and hatched blastocysts in the 50 μg/ml carnosine-supplemented group at 4, 24, and 48 h after thawing were higher than those in the control (P< 0.05) group. In frozen-thawed embryos, cryopreservation solution supplemented with carnosine (50 μg/ml) significantly reduced reactive oxygen species (ROS) production(P < 0.05), decreased TUNEL-positive apoptotic cells (P< 0.05), and increased the mRNA expression of BCL2 (P< 0.05), an apoptosis suppressor gene. The expression of translocase of outer mitochondrial membrane 20 (TOMM20), which is involved in protein mitochondrial transport, in the carnosine (50 μg/ml)-treated embryos was significantly higher than that in the control group (P < 0.05). ATP production in frozen-thawed embryos in the 50 μg/ml carnosine-supplemented group was significantly higher than that in the control group (P< 0.05), however no significant difference in the total number of cells per embryo among the groups was observed. These results suggest that supplementing the cryopreservation solution with carnosine can improve the viability of frozen-thawed bovine embryos by reducing oxidative damage.

Dysfunction of central serotonergic neurons is known to cause depressive disorders in humans, who often show reproductive and/or glucose metabolism disorders. This study examined whether dorsal raphe (DR) serotonergic neurons sense high glucose availability to upregulate reproductive function via activating hypothalamic arcuate (ARC) kisspeptin neurons (= KNDy neurons), a dominant stimulator of gonadotropin-releasing hormone (GnRH)/gonadotropin pulses, using female rats and goats. RNA-seq and histological analysis revealed that stimulatory serotonin-2C receptor (5HT2CR) was mainly expressed in the KNDy neurons in female rats. The serotonergic reuptake inhibitor administration into the mediobasal hypothalamus (MBH), including the ARC, significantly blocked glucoprivic suppression of luteinizing hormone (LH) pulses and hyperglycemia induced by intravenous 2-deoxy-D-glucose (2DG) administration in female rats. A local infusion of glucose into the DR significantly increased in vivo serotonin release in the MBH and partly restored LH pulses and hyperglycemia in the 2DG-treated female rats. Furthermore, central administration of serotonin or a 5HT2CR agonist immediately evoked GnRH pulse generator activity, and central 5HT2CR antagonism blocked the serotonin-induced facilitation of GnRH pulse generator activity in ovariectomized goats. These results suggest that DR serotonergic neurons sense high glucose availability to reduce gluconeogenesis and upregulate reproductive function by activating GnRH/LH pulse generator activity in mammals., (© 2024. The Author(s).)

The single nucleotide polymorphism (SNP) rs107856856, located in the tryptophan hydroxylase-2 gene, is associated with the behavioural phenotype for sheep temperament measured at weaning. Here, we tested the association between that SNP and physiological and behavioural responses to stressors in adult sheep. Two groups of adult sheep, one with genotype A/A (calm genotype) and the other with G/G (nervous genotype) in rs107856856, were selected from 160 sheep and were exposed, twice, to an open-field arena and an isolation box test (IBT). During each repeat, the behaviour and physiological responses (cortisol, prolactin, dehydroepiandrosterone [DHEA], brain derived neurotrophic factor [BDNF], characteristics of the response of body temperature, and oxidative stress) were measured. The behavioural and physiological responses of the sheep were compared between genotypes and also between groups classified on their phenotype as assessed by their initial isolation box score ("low responders" and "high responders"). The SNP rs107856856 had some effects on the behavioural phenotype (IBT score) but no effects on the physiological response to stress (cortisol, prolactin, DHEA, BDNF, oxidative stress or changes in body temperature) in the adult sheep, probably because the sheep were exposed, and therefore had adapted, to human contact during their life., (© 2024. The Author(s).)

In villous trophoblasts, DROSHA is a key ribonuclease III enzyme that processes pri-microRNAs (pri-miRNAs) into pre-miRNAs at the placenta-specific, chromosome 19 miRNA cluster (C19MC) locus. However, little is known of its other functions. We performed formaldehyde crosslinking, immunoprecipitation, and sequencing (fCLIP-seq) analysis of terminal chorionic villi to identify DROSHA-binding RNAs in villous trophoblasts. In villous trophoblasts, DROSHA predominantly generated placenta-specific C19MC pre-miRNAs, including antiviral C19MC pre-miRNAs. The fCLIP-seq analysis also identified non-miRNA transcripts with hairpin structures potentially capable of binding to DROSHA (e.g., SNORD100 and VTRNA1-1). Moreover, in vivo immunohistochemical analysis revealed DROSHA in the cytoplasm of villous trophoblasts. DROSHA was abundant in the cytoplasm of villous trophoblasts, particularly in the apical region of syncytiotrophoblast, in the full-term placenta. Furthermore, in BeWo trophoblasts infected with Sindbis virus (SINV), DROSHA translocated to the cytoplasm and recognized the genomic RNA of SINV. Therefore, in trophoblasts, DROSHA not only regulates RNA metabolism, including the biogenesis of placenta-specific miRNAs, but also recognizes viral RNAs. After SINV infection, BeWo DROSHA-binding VTRNA1-1 was significantly upregulated, and cellular VTRNA1-1 was significantly downregulated, suggesting that DROSHA soaks up VTRNA1-1 in response to viral infection. These results suggest that the DROSHA-mediated recognition of RNAs defends against viral infection in villous trophoblasts. Our data provide insight into the antiviral functions of DROSHA in villous trophoblasts of the human placenta., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

TomoTherapy involves image-guided radiation therapy (IGRT) using Mega-voltage CT (MVCT) for each treatment session. The acquired MVCT images can be utilized for the retrospective assessment of dose distribution. The TomoTherapy provides 18 distinct imaging conditions that can be selected based on a combination of algorithms, acquisition pitch, and slice interval. We investigated the accuracy of dose calculation and deformable image registration (DIR) depending on MVCT scan parameters and their effects on adaptive radiation therapy (ART). We acquired image values for density calibration tables (IVDTs) under 18 different MVCT conditions and compared them. The planning CT (pCT) was performed using a thoracic phantom, and an esophageal intensity-modulated radiation therapy (IMRT) plan was created. MVCT images of the thoracic phantom were acquired under each of the 18 conditions, and dose recalculation was performed. DIR was performed on the MVCT images acquired under each condition. The accuracy of DIR, depending on the MVCT scan parameters, was compared using the mean distance to agreement (MDA) and Dice similarity coefficient (DSC). The dose distribution calculated on the MVCT images was deformed using deformed vector fields (DVF). No significant differences were observed in the results of the 18 IVDTs. The esophageal IMRT plan also showed a small dose difference. Regarding verifying the DIR accuracy, the MDA increased, and the DSC decreased as the acquisition pitch and slice interval increased. The difference between the dose distributions after dose mapping was comparable to that before DIR. The MVCT scan parameters had little effect on ART., (© 2024. The Author(s), under exclusive licence to Japanese Society of Radiological Technology and Japan Society of Medical Physics.)

Pre-ovulatory follicles are cooler than the neighboring reproductive organs in cows. Thus, measuring the temperature of reproductive organs could be a useful method for predicting estrus and ovulation in cows, and the establishment of a non-invasive technique is required. In this study, we used infrared thermography (IRT) to measure ocular surface temperature as a potential surrogate for reproductive organ temperature. Five Japanese Black cows with synchronized estrus were subjected to temperature measurements in five regions of the ocular surface, including the nasal conjunctiva, nasal limbus, center cornea, temporal limbus, and temporal conjunctiva, twice a day (0800 h and 1600 h) during the experimental period. The temperatures in the five regions significantly declined in cows from estrus to ovulation. To the best of our knowledge, this study is the first to use IRT to show a temperature decrease in the ocular surface along with estrus to ovulation in Japanese Black cows.

Superovulation (SOV) treatment of cows results in unovulated follicles and inconsistent quality of the recovered embryos. It has been demonstrated that luteinizing hormone (LH) secretion is suppressed during SOV treatment of cows, which may cause insufficient follicle development and variation in the development of recovered embryos and unovulated follicles. Pulsatile gonadotropin-releasing hormone/LH secretion is controlled by the activity of kisspeptin, neurokinin B and dynorphin (KNDy) neurons in the arcuate nucleus in many mammals. As neurokinin B promotes the activity of KNDy neurons, we hypothesized that senktide, a neurokinin B receptor agonist, has the potential as a therapeutic drug to improve the ovulation rate and quality of recovered embryos in SOV-treated cows via stimulation of LH secretion. Senktide was administered intravenously (30 or 300 nmol/min) for 2 h, beginning from 72 h after the start of SOV treatment. LH secretion was examined before and after administration, and embryos were collected 7 d after estrus. Senktide administration increased LH secretion in SOV-treated cows. The ratios of code 1, code 1 and 2, and blastocyst stage embryos to recovered embryos were increased by senktide (300 nmol/min) administration. Moreover, the mRNA levels of MTCO1, COX7C, and MTATP6 were upregulated in recovered embryos of senktide (300 nmol/min)-administered animals. These results indicate that the administration of senktide to SOV-treated cows enhances LH secretion and upregulates the expression of genes involved in mitochondrial metabolism in embryos, thereby improving embryo development and embryo quality., (© The Author(s) 2023. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The isolation of the Koala retrovirus-like virus from Australian megabats and the identification of endogenous retroviruses in the bat genome have raised questions on bat susceptibility to retroviruses in general. To answer this, we studied the susceptibility of 12 cell lines from 11 bat species to four well-studied retroviruses (human and simian immunodeficiency viruses [HIV and SIV] and murine leukemia viruses [B- and N-MLV]). Systematic comparison of retroviral susceptibility among bats revealed that megabat cell lines were overall less susceptible to the four retroviruses than microbat cell lines, particularly to HIV-1 infection, whereas lineage-specific differences were observed for MLV susceptibility. Quantitative PCR of reverse transcription (RT) products, infection in heterokaryon cells, and point mutation analysis of the capsid (CA) revealed that (i) HIV-1 and MLV replication were blocked at the nuclear transport of the pre-integration complexes and before and/or during RT, respectively, and (ii) the observed lineage-specific restriction can be attributed to a dominant cellular factor constrained by specific positions in CA. Investigation of bat homologs of the three previously reported post-entry restriction factors constrained by the same residues in CA, tripartite motif-protein 5α (TRIM5α), myxovirus resistance 2/B (Mx2/MxB), and carboxy terminus-truncated cleavage and polyadenylation factor 6 (CPSF6-358), demonstrated poor anti-HIV-1 activity in megabat cells, whereas megabat TRIM5α restricted MLV infection, suggesting that the major known CA-dependent restriction factors were not dominant in the observed lineage-specific susceptibility to HIV-1 in bat cells. Therefore, HIV-1 susceptibility of megabat cells may be determined in a manner distinct from that of primate cells. IMPORTANCE Recent studies have demonstrated the circulation of gammaretroviruses among megabats in Australia and the bats' resistance to HIV-1 infection; however, the origins of these viruses in megabats and the contribution of bats to retrovirus spread to other mammalian species remains unclear. To determine the intrinsic susceptibility of bat cells to HIV-1 infection, we investigated 12 cell lines isolated from 11 bat species. We report that lineage-specific retrovirus restriction in the bat cell lines can be attributed to CA-dependent factors. However, in the megabat cell lines examined, factors known to bind capsid and block infection in primate cell culture, including homologs of TRIM5α, Mx2/MxB, and CPSF6, failed to exhibit significant anti-HIV-1 activities. These results suggested that the HIV-1 susceptibility of megabat cells occurs in a manner distinct from that of primate cells, where cellular factors, other than major known CA-dependent restriction factors, with lineage-specific functions could recognize retroviral proteins in megabats.

To improve the dairy sector in Cambodia in the future, we aimed to reveal the genetic variation and the milk production in Cambodian crossbred dairy cattle. We calculated the percent (%) milk fat content and the average milk yield per cow (L/day) for two farms (Farm R and M) based on the farmers' records and interviews. The crossbred cows originated from Cambodian local farmers and Thailand breeders in Farm R, whereas the crossbred cows originated in Thailand breeders in Farm M. Then, we performed genetic characterization for 75 individuals from the two farms and an individual Japanese pure Holstein-Friesian cow based on 133,705 single nucleotide polymorphisms (SNPs) obtained by the GRAS-Di method. The milk fat contents in the bulk milk in the dry season and the average milk yield per cow on Farm R were 3.77 ± 0.98% and 7.81 ± 2.66 L/day, respectively, and were higher than those on Farm M (3.35 ± 0.54% and 6.5-7.5 L/day). Cattle originating in Cambodia in Farm R possessed a unique genetic character different from cattle from Thailand in Farm M. The present study suggests that the differences in milk fat content between the two farms might be explained by the genetic differences in crossbred cows.

Dihydroorotate dehydrogenase (DHODH) catalyzes the rate-limiting step in de novo pyrimidine biosynthesis and is a promising cancer treatment target. This study reports the identification of indoluidin D and its derivatives as inhibitors of DHODH. Cell-based phenotypic screening revealed that indoluidin D promoted myeloid differentiation and inhibited the proliferation of acute promyelocytic leukemia HL-60 cells. Indoluidin D also suppressed cell growth in various other types of cancer cells. Cancer cell sensitivity profiling with JFCR39 and proteomic profiling with ChemProteoBase revealed that indoluidin D is a DHODH inhibitor. Indoluidin D inhibited human DHODH activity in vitro ; the DHODH reaction product orotic acid rescued indoluidin D-induced cell differentiation. We synthesized several indoluidin D diastereomer derivatives and demonstrated that stereochemistry was vital to their molecular activity. The indoluidin D derivative indoluidin E showed similar activity to its parent compound and suppressed tumor growth in a murine lung cancer xenograft model. Hence, indoluidin D and its derivatives selectively inhibit DHODH and suppress cancer cell growth.

The mechanism of bovine endometrial regeneration after parturition remains unclear. Here, we hypothesized that bovine endometrial stem/progenitor cells participate in the postpartum regeneration of the endometrium. Flow cytometry analysis identified the presence of side population (SP) cells among endometrial stromal cells. Endometrial SP cells were shown to differentiate into osteoblasts and adipocytes. RNA-seq data showed that the gene expression pattern was different between bovine endometrial SP cells and main population cells. Gene Set Enrichment Analysis identified the enrichment of stemness genes in SP cells. Significantly (false discovery rate < 0.01) upregulated genes in SP cells contained several stem cell marker genes. Gene ontology (GO) analysis of the upregulated genes in SP cells showed enrichment of terms related to RNA metabolic process and transcription. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of upregulated genes in SP cells revealed enrichment of signaling pathways associated with maintenance and differentiation of stem/progenitor cells. The terms involved in TCA cycles were enriched in GO and KEGG pathway analysis of downregulated genes in SP cells. These results support the assumption that bovine endometrial SP cells exhibit characteristics of somatic stem/progenitor cells. The ratio of SP cells to endometrial cells was lowest on days 9-11 after parturition, which gradually increased thereafter. SP cells were shown to differentiate into epithelial cells. Collectively, these results suggest that bovine endometrial SP cells were temporarily reduced immediately after calving possibly due to their differentiation to provide new endometrial cells., (© The Author(s) 2021. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Kisspeptin neurons located in the hypothalamic preoptic area (POA) are suggested to be responsible for the induction of the gonadotropin-releasing hormone (GnRH) surge and the following luteinizing hormone (LH) surge to regulate female mammals' ovulation. Accumulating evidence demonstrates that the preovulatory level of estrogen activates the POA kisspeptin neurons (estrogen positive feedback), which in turn induces a GnRH/LH surge. This study aimed to derive a cell line from goat POA kisspeptin neurons as an in vitro model to analyze the estrogen positive feedback mechanism in ruminants. Neuron-derived cell clones obtained by the immortalization of POA tissue from a female Shiba goat fetus were analyzed for the expression of kisspeptin (KISS1) and estrogen receptor α (ESR1) genes using quantitative real-time reverse transcription-polymerase chain reaction and three cell clones were selected as POA kisspeptin neuron cell line candidates. One cell line (GP64) out of the three clones showed significant increase in the KISS1 level by incubation with estradiol for 24 h, indicating that the GP64 cells mimic endogenous goat POA kisspeptin neurons. The GP64 cells showed immunoreactivities for kisspeptin and estrogen receptor α and retained a stable growth rate throughout three passages. Further, intracellular calcium levels in the GP64 cells were increased by the KCl challenge, indicating their neurosecretory ability. In conclusion, we generated a new KISS1-expressing cell line derived from goat POA. The current GP64 cell line could be a useful model to elucidate the estrogen positive feedback mechanism responsible for the GnRH/LH surge generation in ruminants.

Heat stress disrupts reproductive function in cattle. In summer, high ambient temperature and humidity elevate core body temperature, which is considered to be detrimental to reproductive abilities in cattle. Neurokinin B (NKB) is a factor that generates pulsatile GnRH and subsequent LH secretion in mammals. Recent studies have reported that NKB-neurokinin 3 receptor (NK3R) signaling is associated with heat-defense responses in rodents. The present study aimed to clarify the role of NKB-NK3R signaling in thermoregulation in cattle. We examined the effects of an NK3R-selective agonist, senktide, on vaginal temperature as an indicator of core body temperature in winter and summer. In both seasons, continuous infusion of senktide for 4 h immediately decreased vaginal temperature, and the mean temperature change in the senktide-treated group was significantly lower than that of both vehicle- and GnRH-treated groups. Administration of GnRH induced LH elevation, but there was no significant difference in vaginal temperature change between GnRH- and vehicle-treated groups. Moreover, we investigated the effects of senktide on ovarian temperature. Senktide treatment seemed to suppress the increase in ovarian temperature from 2 h after the beginning of administration, although the difference between groups was not statistically significant. Taken together, these results suggest that senktide infusion caused a decline in the vaginal temperature of cattle, in both winter and summer seasons, and this effect was not due to the gonadotropin-releasing action of senktide. These findings provide new therapeutic options for senktide to support both heat-defense responses and GnRH/LH pulse generation., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Objective: The present study aimed to survey seasonal changes in reproductive performance of local cows receiving artificial insemination (AI) in the Pursat province of Cambodia, a tropical country, to investigate if ambient conditions affect the reproductive performance of cows as to better understand the major problems regarding cattle production., Methods: The number of cows receiving AI, resultant number of calving, and calving rate were analyzed for those receiving the first AI from 2016 to 2017. The year was divided into three seasons: cool/dry (from November to February), hot/dry (from March to June), and wet (from July to October), based on the maximal temperature and rainfall in Pursat, to analyze the relationship between ambient conditions and the reproductive performance of cows. Body condition scores (BCS) and feeding schemes were also analyzed in these seasons., Results: The number of cows receiving AI was significantly higher in the cool/dry season than the wet season. The number of calving and calving rate were significantly higher in cows receiving AI in the cool/dry season compared with the hot/dry and wet seasons. The cows showed higher BCSs in the cool/dry season compared to the hot/dry and wet seasons probably due to the seasonal changes in the feeding schemes: these cows grazed on wild grasses in the cool/dry season but fed with a limited amount of grasses and straw in the hot/dry and wet seasons., Conclusion: The present study suggests that the low number of cows receiving AI, low number of calving, and low calving rate could be mainly due to poor body condition as a result of the poor feeding schemes during the hot/dry and wet seasons. The improvement of body condition by the refinement of feeding schemes may contribute to an increase in the reproductive performance in cows during the hot/dry and wet seasons in Cambodia.

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is essential for regulating reproductive functions in mammals. GnRH pulses are governed by a neural mechanism that is termed the GnRH pulse generator. In the present study, we investigated the role of central calcitonin receptor (CTR) signaling in the regulation of the GnRH pulse generator activity in ovariectomized goats by administering amylin, an endogenous ligand for CTR, into the lateral ventricle. GnRH pulse generator activity was measured using multiple unit activity (MUA) recordings in the mediobasal hypothalamus. We analyzed changes in the interval of characteristic increases in MUA (MUA volleys). The MUA volley interval shortened immediately after amylin administration, followed by prolonged intervals. Double in situ hybridization for KISS1 (kisspeptin gene) and CALCR (CTR gene) revealed that low expression levels of CALCR were found in the arcuate kisspeptin neurons, which is suggested as the main population of neurons, involved in GnRH pulse generator activity. These results suggest that central amylin-CTR signaling has a biphasic role in the regulation of GnRH pulse generator activity by acting on cells other than the arcuate kisspeptin neurons in goats., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Accumulating evidence suggests that KNDy neurons located in the hypothalamic arcuate nucleus (ARC), which are reported to express kisspeptin, neurokinin B, and dynorphin A, are indispensable for the gonadotropin-releasing hormone (GnRH) pulse generation that results in rhythmic GnRH secretion. The aims of the present study were to investigate the effects of peripheral administration of the neurokinin 3 receptor (NK3R/TACR3, a receptor for neurokinin B) antagonist, SB223412, on GnRH pulse-generating activity and pulsatile luteinizing hormone (LH) secretion in ovariectomized Shiba goats treated with luteal phase levels of estrogen. The NK3R antagonist was infused intravenously for 4 h {0.16 or 1.6 mg/(kg body weight [BW]·4 h)} during which multiple unit activity (MUA) in the ARC was recorded, an electrophysiological technique commonly employed to monitor GnRH pulse generator activity. In a separate experiment, the NK3R antagonist (40 or 200 mg/[kg BW·day]) was administered orally for 7 days to determine whether the NK3R antagonist could modulate pulsatile LH secretion when administered via the oral route. Intravenous infusion of the NK3R antagonist significantly increased the interval of episodic bursts of MUA compared with that of the controls. Oral administration of the antagonist for 7 days also significantly prolonged the interpulse interval of LH pulses. The results of this study demonstrate that peripheral administration of an NK3R antagonist suppresses pulsatile LH secretion by acting on the GnRH pulse generator, suggesting that NK3R antagonist administration could be used to modulate reproductive functions in ruminants.

The reproductive performance of cattle can be suppressed by heat stress. Reproductive organ temperature, especially ovarian temperature, may affect follicle development and ovulation. The establishment of a technique for long-term measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperature-dependent changes in follicular development and subsequent ovulation in cows. Here we report a novel method facilitating long-term and continuous recording of ovarian parenchymal temperature in cows. The method revealed that the ovarian temperature in the luteal phase was constantly maintained lower than the vaginal temperature, and that the diurnal temperature variation in the ovary was significantly greater than that in the vagina, suggesting that the ovaries may require a lower temperature than other organs to maintain their functions. This novel method could be used for the further understanding of ovarian functions during estrous cycles in cows.

Kisspeptin plays a critical role in governing gonadotrophin-releasing hormone (GnRH)/gonadotrophin secretion and subsequent reproductive function in mammals. The hypothalamic arcuate nucleus (ARC) kisspeptin neurones, which co-express neurokinin B (NKB) and dynorphin A (Dyn) and are referred to as KNDy neurones, are considered to be involved in GnRH generation. The present study aimed to establish cell lines derived from goat KNDy and GnRH neurones. Primary-cultured cells of female Shiba goat foetal hypothalamic ARC and preoptic area (POA) tissues were immortalised with the infection of lentivirus containing the simian virus 40 large T-antigen gene. Clones of the immortalised cells were selected by the gene expression of a neuronal marker, and then the neurone-derived cell clones were further selected by the gene expression of KNDy or GnRH neurone markers. As a result, we obtained a KNDy neurone cell line (GA28) from the ARC, as well as two GnRH neurone cell lines (GP11 and GP31) from the POA. Immunocytochemistry revealed the expression of kisspeptin, NKB and Dyn in GA28 cells, as well as GnRH in GP11 and GP31 cells. GnRH secretion from GP11 and GP31 cells into the media was confirmed by an enzyme immunoassay. Moreover, kisspeptin challenge increased intracellular Ca2+ levels in subsets of both GP11 and GP31 cells. Kisspeptin mRNA expression in GA28 cells, which expressed the oestrogen receptor alpha gene, was significantly reduced by 17β-oestradiol treatment. Furthermore, the transcriptional core promoter and repressive regions of the goat NKB gene were detected using GA28 cells. In conclusion, we have established goat KNDy and GnRH neurone cell lines that could be used to analyse molecular and cellular mechanisms regulating KNDy and GnRH neurones in vitro, facilitating the clarification of reproductive neuroendocrine mechanisms in ruminants., (© 2020 British Society for Neuroendocrinology.)

Ovulation is an essential phenomenon for reproduction in mammalian females along with follicular growth. It is well established that gonadal function is controlled by the neuroendocrine system called the hypothalamus-pituitary-gonadal (HPG) axis. Gonadotropin-releasing hormone (GnRH) neurons, localized in the hypothalamus, had been considered to be the head in governing the HPG axis for a long time until the discovery of kisspeptin. In females, induction of ovulation and folliculogenesis has been linked to a surge mode and pulse mode of GnRH releases, respectively. The mechanisms of how the two modes of GnRH are differently regulated had long remained elusive. The discovery of kisspeptin neurons, distributed in two hypothalamic nuclei, such as the arcuate nucleus in the caudal hypothalamus and preoptic area or the anteroventral periventricular nucleus in the rostral hypothalamic regions, and analyses of the detailed functions of kisspeptin neurons have led marked progress on the understanding of different mechanisms regulating GnRH surges (ovulation) and GnRH pulses (folliculogenesis). The present review will focus on the role of kisspeptin neurons as the GnRH surge generator, including the sexual differentiation of the surge generation system and factors that regulate the surge generator. Comparative aspects between mammalian species are especially focused on., (© 2019 Japan Society of Obstetrics and Gynecology.)

Pulsatile gonadotropin-releasing hormone (GnRH) secretion is indispensable for reproduction in mammals. Kisspeptin neurons in the hypothalamic arcuate nucleus (ARC), referred to as KNDy neurons because of the coexpression of neurokinin B and dynorphin A, are considered as components of the GnRH pulse generator that produces rhythmic GnRH secretion. The present study aimed to investigate if peripheral administration of PF-4455242, a κ-opioid receptor (KOR, a dynorphin A receptor) antagonist, facilitates pulsatile luteinizing hormone (LH) secretion and GnRH pulse generator activity in estrogen-treated ovariectomized Shiba goats to determine the possibility of using KOR antagonists to artificially control ovarian activities. PF-4455242 was intravenously infused for 4 h (1 or 10 μmol/kg body weight/4 h) or as a single subcutaneous injection (1 or 10 μmol/kg body weight). In a separate experiment, the same KOR antagonist (10 μmol/kg body weight/4 h) was intravenously infused during the recording of multiple unit activity (MUA) in the ARC that reflects the activity of the GnRH pulse generator to test the effects of KOR antagonist administration on GnRH pulse generator activity. Intravenous infusion and single subcutaneous injection of the KOR antagonist significantly increased the frequency of LH pulses compared with controls. Intravenous infusion of KOR antagonist also significantly increased the frequency of episodic bursts in the MUA. The present study demonstrates that peripherally administered KOR antagonist stimulates pulsatile LH secretion by acting on the GnRH pulse generator, and peripheral administration of PF-4455242 can be used to facilitate pulsatile LH secretion, which in turn facilitates ovarian activities in farm animals., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The highly enantioselective Diels-Alder reaction of acetylenic dienophiles is shown to be effectively catalyzed by cationic chiral palladium complexes. Not only the degree but also the sense of enantioselectivity critically depends on the steric demand of ligands. Computational analyses indicate that the steric demand does not affect the endo/exo-selectivity but the enantioface selectivity of dienes., (© 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This study was aimed at evaluating the effects of multi-layered cumulus cells (MCCs) during vitrification and in vitro fertilization (IVF) of mature bovine oocytes and embryogenesis after IVF. The rates of cleavage and blastocyst formation were higher in vitrified and fertilized oocytes with MCCs than in denuded oocytes (P < 0.05), but were comparable to the rates in fresh oocytes with MCCs or without (denuded). When the MCC-enclosed oocytes were denuded before IVF, blastocyst formation rate reduced compared with that in vitrified oocytes with MCCs (P < 0.05). This suggested that the MCCs surrounding the mature bovine oocytes play important roles during cryopreservation: protecting them against freezing and promoting their survival and development post IVF, thereby increasing the success rates of IVF and embryonic development. Herein, we showed for the first time that calves could be produced using only 14-19 vitrified mature oocytes with MCCs from the ovaries of individual cows post slaughter.

The present study aims to examine the effect of tropical temperatures on autonomic nervous activity in Cambodian dairy cattle by analyzing heart rate variability (HRV). Holter-type electrocardiograms were recorded in adult crossbred cows (Cambodian native × Holstein) either in a sheltered area or under direct sunlight. Rectal temperatures and heart rates increased in animals under direct sunlight as compared to those in the shelter. The power spectral analysis of HRV revealed that three out of the five cows studied underwent a decrease in parasympathetic nervous activity under direct sunlight with the remaining two cows showing no apparent change. The HRV analysis would prove to be a useful tool to reveal information about heat tolerance in dairy cows.

Exposure to a ram during spring stimulates luteinizing hormone (LH) secretion and can induce ovulation in sexually quiescent ewes ("ram effect"). Kisspeptin (Kiss) present in the arcuate nucleus (ARC) and the preoptic area (POA) is a potent stimulators of LH secretion. Our aim was to investigate whether Kiss neurons mediate the increase in LH secretion during the ram effect. With double immunofluorescent detection, we identified Kiss neurons (Kiss IR) activated (Fos IR) by exposure to a ram for 2 hours (M2) or 12 hours (M12) or to ewes for 2 hours (C). The density of cells Kiss + Fos IR and the proportion of Kiss IR cells that were also Fos IR cells were higher in M2 and M12 than in C in ARC (P < 0.002) and POA (P < 0.02). In ARC, these parameters were also higher in M12 than in M2 (P < 0.02 and P < 0.05). Kiss antagonist (P234 10-6M) administered by retrodialysis in POA for 3 hours at the time of introduction of the ram reduced the amplitude of the male-induced increase in LH concentration compared with solvent (P < 0.02). In ARC, P234 had a more limited effect (P < 0.038 1 hour after P234) but pulse frequency increased less than after solvent (P = 0.07). In contrast, Kiss antagonist (P271 10-4M) infused in ARC but not POA 6 to 18 hours after introduction of the ram prevented the LH surge in the ewe (0/6 vs 4/5 and 4/6 in C). These results suggest that both populations of Kiss neurons are involved in the ram-induced pulsatile LH secretion and in the LH surge., (Copyright © 2017 Endocrine Society.)

Pulsatile gonadotropin-releasing hormone (GnRH) secretion, which is indispensable for follicular development, is suppressed in lactating dairy and beef cattle. Neurokinin B (NKB) neurons in the arcuate nucleus of the hypothalamus are considered to play an essential role in generating the pulsatile mode of GnRH/luteinizing hormone (LH) secretion. The present study aimed to clarify the role of NKB-neurokinin 3 receptor (NK3R) signaling in the pulsatile pattern of GnRH/gonadotropin secretion in postpartum lactating cattle. We examined the effects of the administration of an NK3R-selective agonist, senktide, on gonadotropin secretion in lactating cattle. The lactating cattle, at approximately 7 days postpartum, were intravenously infused with senktide (30 or 300 nmol/min) or vehicle for 24 h. The administration of 30 or 300 nmol/min senktide significantly increased LH pulse frequency compared to in the control group during 0-4 or 20-24 h after infusion, respectively. Moreover, LH and follicle-stimulating hormone levels were gradually increased by 300 nmol/min administration of senktide during the 0-4-h sampling period. Ultrasonography of the ovaries was performed to identify the first postpartum ovulation in senktide-administered lactating cattle. The interval from calving to first postpartum ovulation was significantly shorter in the 300 nmol/min senktide-administered group than in the control group. Taken together, these findings suggest that senktide infusion elicits an increase in LH pulse frequency that may stimulate follicular development and, in turn, induce the first postpartum ovulation in lactating cattle., (© The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The present study aimed to evaluate hormonal responses and their association with the TAK-683 blood concentrations in goats administered TAK-683 at a low dose, which had been previously determined as the minimally effective dose for luteinizing hormone (LH) stimulation in ovariectomized goats. In Experiment 1, 5 µg of TAK-683 treatment had no significant stimulatory effect on LH secretion in ovariectomized Shiba goats (n = 4). In Experiment 2, cycling goats received the treatment of prostaglandin F 2α and progesterone-releasing controlled internal drug releasing (CIDR) to induce the follicular phase, then they were treated with 5 µg of TAK-683 (hour 0) intravenously (n = 4, IV) or subcutaneously (n = 3, SC) or with vehicle intravenously (n = 4, control) at 12 h after CIDR removal. Blood samples were collected at 10-min (-2-6 h), 2-h (6-24 h), or 6-h (24-48 h) intervals. Ovarian ultrasonographic images were assessed daily to confirm ovulation after the treatment. A surge-like release of LH was immediately observed after injection in all animals in the IV (peak time: 4.2 ± 0.6 h, peak concentration: 73.3 ± 27.5 ng/ml) and SC (peak time: 4.6 ± 0.4 h, peak concentration: 62.6 ± 23.2 ng/ml) groups, but not in the control group. Ovulation was detected within 3 days after TAK-683 injection in all animals in the IV and SC groups, and the interval period from TAK-683 administration to ovulation in the IV group was significantly (P < 0.05) shorter than that of the control group. No significant changes were observed between the IV and SC groups in terms of luteal diameter and blood progesterone levels after ovulation. The present findings suggest that the involvement of one or more ovarian factor(s) is indispensable for a TAK-683-induced LH surge leading to ovulation in goats.

Elucidating the physiological mechanisms that control reproduction is an obvious strategy for improving the fertility of cattle and developing new agents to control reproductive functions. The present study aimed to identify kisspeptin neurons in the bovine hypothalamus, clarifying that a central mechanism is also present in the cattle brain, as kisspeptin is known to play an important role in the stimulation of gonadotropin-releasing hormone (GnRH)/gonadotropin secretion in other mammals. To characterize kisspeptin neurons in the bovine hypothalamus, the co-localizations of kisspeptin and neurokinin B (NKB) or kisspeptin and dynorphin A (Dyn) were examined. Hypothalamic tissue was collected from Japanese Black or Japanese Black × Holstein crossbred cows during the follicular and luteal phases. Brain sections, including the arcuate nucleus (ARC) and the preoptic area (POA), were dual immunostained with kisspeptin and either NKB or Dyn. In the ARC, both NKB and Dyn were co-localized in kisspeptin neurons during both the follicular and luteal phases, demonstrating the presence of kisspeptin/NKB/Dyn-containing neurons, referred to as KNDy neurons, in cows. In the POA, no co-localization of kisspeptin with either NKB or Dyn was detected. Kisspeptin expression in the follicular phase was higher than that in the luteal phase, suggesting that kisspeptin expression in the POA is positively controlled by estrogen in cows. The kisspeptin neuronal populations in the ARC and POA likely play important roles in regulating the GnRH pulse and surge, respectively, in cows.

The neurokinin B (NKB)-neurokinin-3 receptor (NK3R) signaling positively regulates the release of gonadotropin-releasing hormone (GnRH) from the hypothalamus. The NK3R-selective antagonists may suppress the reproductive functions of mammals. For development of novel NK3R antagonists with reduced environmental toxicity, a structure-activity relationship study of an NK3R antagonist, talnetant, was carried out. Among several talnetant derivatives with labile functional groups in the natural environment, 3-mercaptoquinoline 2f exhibited a comparable biological activity to that of the parent talnetant. Additionally, compound 2f was converted into the disulfide 3f or isothiazolone 8 by air-oxidation, both of which showed no binding affinity to NK3R., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

After the discovery of hypothalamic kisspeptin encoded by the Kiss1 gene, the central mechanism regulating gonadotropin-releasing hormone (GnRH) secretion, and hence gonadotropin secretion, is gradually being unraveled. This has increased our understanding of the central mechanism regulating puberty and subsequent reproductive performance in mammals. Recently, emerging evidence has indicated the molecular and epigenetic mechanism regulating hypothalamic Kiss1 gene expression. Here we compile data regarding DNA and histone modifications in the Kiss1 promoter region and provide a hypothetic scheme of the molecular and epigenetic mechanism regulating Kiss1 gene expression in two populations of hypothalamic kisspeptin neurons, which govern puberty and subsequent reproductive performance via GnRH/gonadotropin secretion., (© 2016 S. Karger AG, Basel.)

Langerhans cells (LCs), a subset of dendritic cells (DCs), reside in body surface presenting antigens from various pathogens and activate immune system after migrating to vicinal lymph nodes. We recently demonstrated that the E-cadherin interaction allowed peripheral blood (PB) CD14 + cells to differentiate into LC-like cells that closely resemble primary LCs. Here, with a combination of GM-CSF, TGF-β, and TNF-α, we induced LC-like cells from umbilical cord blood (UCB)derived CD34 + cells and compared them with those induced from PB CD14 + cells. In contrast to PB CD14 + cell-derived LC-like cells with an undetectable surface level of toll-like receptor (TLR)4 and an unresponsiveness feature to bacterial lipopolysaccharide (LPS), CB CD34 + cellsderived LC like cells expressed a low, but apparent, surface level of TLR4 and a reduced level of intracellular TLR3. Consistent with this result, they responded to bacterial LPS, but poorly to poly(I:C) reflecting viral RNA. These findings suggest that LC-precursors from circulating PB CD14 + cells seem to be arranged in the outer barrier of skin, while LC-precursors from local undifferentiated UCB-derived CD34 + cells may be arranged in the inner barrier of mucosal tissues and work together to combat against external pathogens as well as internal malignancies throughout body surface.

This study aimed to evaluate the hormonal and ovarian responses to the administration of a metastin/kisspeptin analog (TAK-683) under the endocrine environments of luteal and follicular phases in goats. Five estrous cycling goats received a prostaglandin F2α injection followed by 10 days of progesterone treatment by CIDR. The TAK-683 (35nmol) was intravenously administered (Hour 0) on 3 days after CIDR insertion (luteal phase condition; LC) and at 12h after CIDR removal (follicular phase condition; FC). Blood samples were collected at 10min (-2 to 6h), 2h (6-24h) or 6h intervals (24-48h). In the LC, small increases in the basal concentrations of LH were observed after TAK-683 administration from 0 to 6h, which were associated with an increase in estradiol concentration, followed by a surge-like release of LH with a peak at 12.5±1.0h (n=4) after TAK-683 administration. In the FC, a surge-like release of LH occurred immediately after TAK-683 administration with a peak at 6.0±3.5h (n=5), which was earlier than that in the LC (P<0.01). The peak concentration of estradiol did not differ between the two conditions, whereas the time interval from TAK-683 treatment to estradiol peak in the LC was longer than that in the FC (12.0±0.0 compared with 6.0±4.2h, P<0.05). These findings suggest that the timing of surge-like release of LH after TAK-683 administration is associated with blood estradiol concentration at the time of treatment., (Copyright © 2015 Elsevier B.V. All rights reserved.)