担当区分：最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biology of Reproduction  

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.

DOI： 10.1093/biolre/ioab004

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担当区分：最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Domestic Animal Endocrinology  

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.

DOI： 10.1016/j.domaniend.2020.106522

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Asian-Australasian Journal of Animal Sciences  

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.

DOI： 10.5713/ajas.19.0893

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Neuroscience Letters  

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.

DOI： 10.1016/j.neulet.2020.135276

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Reproduction and Development  

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.

DOI： 10.1262/jrd.2019-145

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Neuroendocrinology  

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.

DOI： 10.1111/jne.12857

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Reproduction and Development  

Negative energy balance in domestic animals suppresses their reproductive function. These animals commonly use long-chain fatty acids (LCFAs) from adipocytes as an energy source under states of malnutrition. The G-protein coupled receptor, GPR120, is a specific receptor for LCFAs, but its role in reproductive function remains unknown in domestic animals. The purpose of this study was to examine whether GPR120 is involved in the reproductive system of cattle. GPR120 mRNA expression was evaluated in brain, pituitary, and ovarian tissue samples by RT-PCR. GPR120 gene expression was detected with high intensity only in the anterior pituitary sample, and GPR120-immunoreactive cells were found in the anterior pituitary gland. Double immunohistochemistry of GPR120 in the anterior pituitary hormone-producing cells, such as gonadotropes, thyrotropes, lactotropes, somatotropes, and corticotropes, was performed to clarify the distribution of GPR120 in the anterior pituitary gland of ovariectomized heifers. Luteinizing hormone Β subunit (LHΒ)- and folliclestimulating hormone Β subunit (FSHΒ)-immunoreactive cells demonstrated GPR120 immunoreactivity at 80.7% and 85.9%, respectively. Thyrotropes, lactotropes, somatotropes, and corticotropes coexpressed GPR120 at 21.1%, 5.4%, 13.6%, and 14.5%, respectively. In conclusion, the present study suggests that GPR120 in the anterior pituitary gland might mediate LCFA signaling to regulate gonadotrope functions, such as hormone secretion or production, in cattle.

DOI： 10.1262/jrd.2019-111

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記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Reproduction and Development  

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 longterm measurement of ovarian temperature could prove useful in understanding the mechanisms underlying the temperaturedependent 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.

DOI： 10.1262/jrd.2019-097

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記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Reproduction and Development  

Prediction of parturition is essential for sustainable production in beef and dairy cattle, yet the present methods are limited by their high invasiveness and low utility. Here we compared prepartum changes in ventral tail base surface temperature (ST) with changes in vaginal temperature (VT) and behavioral indices. We analyzed 22 parturitions from 22 beef cows. Changes in daily values of ST, VT, and behavioral indices over the 7 days before parturition were investigated. Hourly values were calculated as the actual values minus the mean values for the same hour over a 3-day period, and the changes in hourly values over the 48 h before parturition were investigated. To test the effect of ambient temperature, tested cows were assigned to two season-groups based on the ambient temperature to which they were exposed (warm: N = 13; cool: N = 9), and the daily and hourly values of the indices were compared between seasons. A decrease in ST occurred approximately 30 h before parturition, which was similar to the time of the decrease in VT and earlier than the increase of behavioral indices. In addition, a unique fluctuation of ST observed in the last few hours before parturition indicates that ST could provide a sign for parturition not only in the long-term like VT, but also in the short-term like behavioral indices. Although ST was more sensitive to ambient temperature than VT or the behavioral indices, the day of parturition could be predicted from ST in both the warm and cool seasons.

DOI： 10.1262/jrd.2019-087

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記述言語：日本語   出版者・発行元：動物臨床医学会  

記述言語：日本語   出版者・発行元：(公社)日本獣医学会  

記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Reproduction, Fertility and Development  

In order to help elucidate the process of epiblast and trophoblast cell differentiation in bovine embryos in vitro, we attempted to develop a suitable culture medium to allow extended embryo culture. Day 7 bovine blastocysts developed in conventional medium were cultured further in embryonic stem cell medium with or without leukaemia inhibitory factor (LIF) until Day 23. At Day 14, the expression of octamer-binding transcription factor 3/4 (OCT3/4) and VIMENTIN was significantly higher in embryos cultured with than without LIF, but embryonic disc formation was not observed. Although expression of SRY (sex determining region Y)-box 17 (SOX17) mRNA was significantly lower in Day 14 embryos cultured with and without LIF than in in vivo embryos, hypoblast cells formed just inside the trophoblast cells of the in vitro-cultured embryos. On Day 23, expression of placental lactogen (PL) and prolactin-related protein 1 (PRP1) was not affected by LIF in in vitro-cultured embryos, levels of both genes were significantly lower in the in vitro than in vivo embryos. Similar to in vivo embryos, binucleate cell clusters seen in Day 23 in vitro-cultured embryos were composed of PL-negative and -positive cells. These results suggest that our culture system partially reproduced the differentiation process of trophoblast cells in vivo.

DOI： 10.1071/RD18343

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記述言語：日本語   出版者・発行元：(公社)日本畜産学会  

記述言語：英語   出版者・発行元：(公社)日本畜産学会  

記述言語：英語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Reproduction in Domestic Animals  

Decreased fertility associated with maternal ageing is a well-known critical problem, and progesterone (P4) concentration decreases during the menopause transition in women. The corpus luteum (CL) secretes P4, thereby supporting the implantation and maintenance of pregnancy. It is proposed that a bovine model is suitable for studying age-associated decline of fertility in women because the physiology of cows is similar to that of women and cows have a greater longevity compared with other animal models. Thus, we investigated the age-dependent qualitative changes and inflammatory responses in the bovine CL. In vivo experiment: Cows were divided into three groups, namely, young (mean age: 34.8 months), middle (80.1 months) and aged (188.9 months). Blood samples were collected on days 7 and 12 during the estrous cycle. In vitro experiments: Cows were divided into young (mean age: 27.6 months) and aged (183.1 months). The CL tissues of these groups were collected from a local slaughterhouse and used for tissue culture experiments. An in vivo experiment, plasma P4 concentration in aged cows was significantly lower than that in young cows, whereas no difference was found regarding the area of CL. An in vitro examination in the bovine CL tissues showed that the luteal P4 concentration, P4 secretion, and mRNA expression of StAR and 3β-HSD were lower in aged cows compared with young cows, especially in the early luteal phase. However, no differences were detected in the mRNA expression of inflammation- and senescence-related factors and inflammatory responses to lipopolysaccharides between the CL tissues from young and aged cows, indicating that an age-dependent increase in inflammation is not involved in the luteal function. P4 production and secretion from the bovine CL diminish in old cows, especially during the early luteal phase, suggesting that senescence may affect the luteal function in cows.

DOI： 10.1111/rda.13303

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担当区分：最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Journal of Animal Science  

Short-chain fatty acids (SCFAs) are the main source of energy for postweaning ruminants. The monocarboxylic acid transporters, MCT1 and MCT4, are thought to contribute to the absorption of SCFAs from the surface of the rumen following weaning. The present study measured changes in MCT1 and MCT4 expression in ruminal epithelial cells isolated from male preweaning (22 to 34 d old, n = 6) and postweaning (55 to 58 d old, n = 8) calves after euthanasia and sought to examine whether SCFAs stimulate the expression of these transporters. In the current study, cluster of differentiation 147 (CD147) gene expression in the rumen was also investigated since CD147 has been considered to act as ancillary protein for MCT1 and MCT4 to express their correct function. The gene expression levels of MCT1, MCT4, and CD147 in the rumen were found to be significantly higher in postweaning calves than in preweaning calves. Strong MCT1 immunoreactivity was detected in both the stratum basale (SB) and the stratum spinosum (SS) in postweaning ruminal epithelium. Expression of MCT1 in preweaning calves was localized to a specific region of the SB and of the SS. MCT4-immunopositive cells were detected in the stratum corneum (SC) of the ruminal epithelium in postweaning calves. However, only a low level of signal was detected in the SC of preweaning animals. Furthermore, in vitro experiments, ruminal epithelial cells were incubated for 24 h with acetate (0.04, 0.4, and 4 mM), propionate (0.2, 2, and 20 mM), butyrate (0.1, 1, and 10 mM), or β-hydroxybutyrate (BHBA; 0.1, 1, and 10 mM), respectively. Both propionate and butyrate induced an increase in the gene expression levels of MCT4 and CD147, but did not affect MCT1 gene expression. There are no significant effects of acetate and BHBA treatment on these gene expressions. Taken together, these results suggest that an increase in MCT4 and CD147 gene expression in the ruminal epithelium of postweaning calves is likely to be due to the effects of propionate and butyrate derived from a solid-based diet, which may contribute to ruminal development following weaning.

DOI： 10.1093/jas/sky334

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-018-19725-z

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41598-017-17968-w

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1262/jrd.2017-025

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1089/jir.2017.0057

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記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1186/s12958-017-0284-z

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担当区分：最終著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）   出版者・発行元：Biology of Reproduction  

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.

DOI： 10.1093/biolre/iox068

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記述言語：日本語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者,　責任著者   記述言語：日本語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者,　責任著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

担当区分：筆頭著者   記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）  

記述言語：英語   掲載種別：研究論文（学術雑誌）