Shuichi Matsuyama

Gonadotrophin‐Releasing Hormone Pulse Generator Activity in the Hypothalamus of the Goat

Pulsatile release of gonadotrophin‐releasing hormone (GnRH) is indispensable to maintain normal gonadotrophin secretion. The pulsatile secretion of GnRH is associated with synchronised electrical activity in the mediobasal hypothalamus (i.e. multiple unit activity; MUA), which is considered to reflect the rhythmic oscillations in the activity of the neuronal network that drives pulsatile GnRH secretion. However, the cellular source of this ultradian rhythm in GnRH activity is unknown. Direct input from kisspeptin neurones in the arcuate nucleus (ARC) to GnRH cell bodies in the medial preoptic area or their terminals in the median eminence could be the intrinsic source for driving the GnRH pulse generator. To determine whether kisspeptin signalling could be responsible for producing pulsatile GnRH secretion, we studied goats, measured plasma levels of luteinising hormone (LH) and recorded MUA in the posterior ARC, where the majority of kisspeptin neuronal cell bodies are located. Rhythmic volleys of MUA were found to be accompanied by LH pulses with regular intervals in the ARC, where kisspeptin neuronal cell bodies were found. Exogenous administration of kisspeptin stimulated a sustained increase in LH secretion, without influencing MUA, suggesting that the GnRH pulse generator, as reflected by MUA, originated from outside of the network of GnRH neurones, and could plausibly reflect the pacemaker activity of kisspeptin neurones, whose projections reach the median eminence where GnRH fibres project. These observations suggest that the kisspeptin neurones in the ARC may be the intrinsic source of the GnRH pulse generator.

Morphological Evidence for Direct Interaction between Kisspeptin and Gonadotropin-Releasing Hormone Neurons at the Median Eminence of the Male Goat: An Immunoelectron Microscopic Study

Kisspeptin has been thought to play pivotal roles in the control of both pulse and surge modes of gonadotropin-releasing hormone (GnRH) secretion. To clarify loci of kisspeptin action on GnRH neurons, the present study examined the morphology of the kisspeptin system and the associations between kisspeptin and GnRH systems in gonadally intact and castrated male goats. Kisspeptin-immunoreactive (ir) and Kiss1-positive neurons were found in the medial preoptic area of intact but not castrated goats. Kisspeptin-ir cell bodies and fibers in the arcuate nucleus (ARC) and median eminence (ME) were fewer in intact male goats compared with castrated animals. Apposition of kisspeptin-ir fibers on GnRH-ir cell bodies was very rare in both intact and castrated goats, whereas the intimate association of kisspeptin-ir fibers with GnRH-ir nerve terminals was observed in the ME of castrated animals. Neurokinin B immunoreactivity colocalized not only in kisspeptin-ir cell bodies in the ARC but also in kisspeptin-ir fibers in the ME, suggesting that a majority of kisspeptin-ir fibers projecting to the ME originates from the ARC. A dual immunoelectron microscopic examination revealed that nerve terminals containing kisspeptin-ir vesicles made direct contact with GnRH-ir nerve terminals at the ME of castrated goats. There was no evidence for the existence of the typical synaptic structure between kisspeptin- and GnRH-ir fibers. The present results suggest that the ARC kisspeptin neurons act on GnRH neurons at the ME to control (possibly the pulse mode of) GnRH secretion in males.

Relationship between quantity of IFNT estimated by IFN-stimulated gene expression in peripheral blood mononuclear cells and bovine embryonic mortality after AI or ET

BackgroundInterferon tau (IFNT), which is secreted into the uterine cavity during the maternal recognition period (MRP), is a key factor for establishment of pregnancy. The present study aims to clarify the relationship between the ability of a bovine conceptus to produce IFNT during the MRP and the conceptuss ability to establish pregnancy.MethodsIn the first experiment, IFNT (0, 500, or 1000 micrograms) was administered into the uterine horn ipsilateral to the CL 16 or 17 d after standing estrus, and mRNA levels of IFN-stimulated gene 15-kDa protein (ISG15) and Mx2 in peripheral blood mononuclear cells (PBMCs) were determined. In the second experiment, we investigated ISG15 mRNA expression in PBMCs during the MRP in cattle after either artificial insemination (AI) or embryo transfer (ET).ResultsIntrauterine administration of IFNT stimulated ISG15 and Mx2 gene expressions in PBMCs in cattle, and there was a positive correlation between the expressions of peripheral markers and the quantity of IFNT administered. In pregnant and normal interestrous interval (< 25 d) cattle (nIEI cattle), expression levels of the ISG15 gene showed similar patterns after AI and ET, and ISG15 mRNA expression was increased in pregnant cattle but unchanged in nIEI cattle. In contrast, ISG15 gene expression in extended interestrous interval (greater than or equal to 25 d) cattle (eIEI cattle) differed after ET compared with AI. In eIEI cattle after ET, ISG15 gene expression increased, such that the value on day 18 was intermediate between those of pregnant and nIEI cattle. In eIEI cattle after AI, ISG15 gene expression did not increase throughout the observation period.ConclusionsThe results of the current study indicate that the quantity of conceptus-derived IFNT can be estimated by measuring ISG15 mRNA levels in PBMCs from cattle. Using this approach, we demonstrate that ISG15 gene expression during the MRP in eIEI cattle differed after ET compared with AI. In addition, the modest increase in ISG15 gene expression in eIEI cattle after ET suggests that late embryo losses were due to delayed or insufficient growth of the conceptus during the MRP in cattle.

Possible involvement of IFNT in lymphangiogenesis in the corpus luteum during the maternal recognition period in the cow

The corpus luteum (CL), which secretes large amounts of progesterone and is thus essential for establishing pregnancy, contains various types of immune cells that may play essential roles in CL function by generating immune responses. The lymphatic system is the second circulation system and is necessary for immune function, but the lymphatic system of the bovine CL has not been characterized in detail. We collected bovine CLs on days 12 and 16 of the estrous cycle (C12 and C16) and days 16 and 40 of early pregnancy (P16 and P40). Lymphatic endothelial hyaluronan receptor 1 (LYVE1) protein was detected in the CL by immunohistochemistry and western blotting and increased at P40 compared with C16. The mRNA expression levels of lymphangiogenic factors, such as vascular endothelial growth factor-C (VEGFC), VEGFD, and their common receptor VEGFR3, as well as the lymphatic endothelial cell (LyEC) marker podoplanin, increased in P16 and P40 CLs. Thus, it is suggested that the lymphatic system of the bovine CL reconstitutes during early pregnancy. Interferon tau (IFNT) from the conceptus in the uterus is a candidate for activating luteal lymphangiogenesis during the maternal recognition period (MRP). We found that treatment of LyECs isolated from internal iliac lymphatic vessels with IFNT stimulated LyEC proliferation and significantly increased mRNA expression of VEGFC and IFN-stimulated gene 15. Moreover, both IFNT and VEGFC induced LyECs to form capillary-like tubes in vitro. In conclusion, it is suggested that new lymphangiogenesis in the bovine CL begins during the MRP and that IFNT may mediate this novel phenomenon.

Generation of Naïve Bovine Induced Pluripotent Stem Cells Using PiggyBac Transposition of Doxycycline-Inducible Transcription Factors

Generation of pluripotent stem cells (PSCs) in large domestic animals has achieved only limited success; most of the PSCs obtained to date have been classified as primed PSCs, which possess very little capacity to produce chimeric offspring. By contrast, mouse PSCs have been classified as naïve PSCs that can contribute to most of the tissues of chimeras, including germ cells. Here, we describe the generation of two different types of bovine induced pluripotent stem cells (biPSCs) from amnion cells, achieved through introduction of piggyBac vectors containing doxycycline-inducible transcription factors (Oct3/4, Sox2, Klf4, and c-Myc). One type of biPSCs, cultured in medium supplemented with knockout serum replacement (KSR), FGF2, and bovine leukemia inhibitory factor (bLIF), had a flattened morphology like human PSCs; these were classified as primed-type. The other type biPSCs, cultured in KSR, bLIF, Mek/Erk inhibitor, GSK3 inhibitor and forskolin, had a compact morphology like mouse PSCs; these were classified as naïve-type. Cells could easily be switched between these two types of biPSCs by changing the culture conditions. Both types of biPSCs had strong alkaline phosphatase activity, expressed pluripotent markers (OCT3/4, NANOG, REX1, ESRRβ, STELLA, and SOCS3), and formed embryoid bodies that gave rise to differentiated cells from all three embryonic germ layers. However, only naïve-type biPSCs showed the hallmarks of naïve mouse PSCs, such as LIF-dependent proliferation, lack of FGF5 expression, and active XIST expression with two active X chromosomes. Furthermore, naïve-type biPSCs could contribute to the inner cell mass (ICM) of host blastocysts and most tissues within chimeric embryos. This is the first report of generation of biPSCs with several characteristics similar to those of naïve mouse PSCs and a demonstrated potential to contribute to chimeras.

Interferon-Tau Attenuates Uptake of Nanoparticles and Secretion of Interleukin-1β in Macrophages

Background Type I interferons (IFNs), including IFN-alpha (IFNA) and IFN-beta (IFNB), have anti-inflammatory properties and are used to treat patients with autoimmune and inflammatory disorders. However, little is known of the role of IFN-tau (IFNT), a type I IFN produced by ruminant animals for inflammation. Because IFNB has recently been shown to inhibit nucleotide-binding oligomerization domain-like receptor, pyrin domain-containing 3 (NLRP3) inflammasome activation and subsequent secretion of the potent inflammatory cytokine interleukin (IL)-1β, we examined the effects of ruminant IFNT on NLRP3 inflammasome-mediated IL-1β secretion in human THP-1 macrophages. Methods and Results IFNT dose-dependently inhibited IL-1β secretion induced by nano-silica, a well-known activators of NLRP3 inflammasomes, in human macrophages primed with lipopolysaccharide (LPS, TLR4 agonist) and Pam3CSK4 (TLR1/2 agonist). IFNT also suppressed phagocytosis of nano-silica and reactive oxygen species (ROS) generation. Western blot analysis showed that IFNT inhibited both pro-IL-1β and mature IL-1β. In addition, real-time RT-PCR analysis showed that IFNT suppressed IL-1β mRNA expression induced by LPS and Pam3CSK4. Although nano-silica particles did not induce IL-10 secretion, IFNT induced IL-10 secretion in a dose-dependent manner. Furthermore, IFNT-suppressed IL-1β secretion was restored by anti-IL-10 neutralizing antibody. Conclusions Ruminant IFNT inhibits NLRP3 inflammasome-driven IL-1β secretion in human macrophages via multiple pathways, including the uptake of nano-silica particles, generation of ROS, and IL-10-mediated inhibition of pro-IL-1β induction. It may be a therapeutic alternative to IFNA and IFNB.

Characteristics of Bovine Inner Cell Mass-Derived Cell Lines and Their Fate in Chimeric Conceptuses

ABSTRACT Bovine embryonic stem (ES) cells have the potential to provide significant benefits in a range of agricultural and biomedical applications. Here, we employed a combination of conventional methods using glycogen synthase kinase 3 and mitogen-activated protein kinase inhibitors to establish ES cell lines from in vitro fertilization (IVF) and somatic cell nuclear transfer (SCNT) bovine embryos. Five male cell lines were established from IVF embryos, and two female and three male cell lines from SCNT blastocysts; we named these lines bovine ES cell-like cells (bESLCs). The lines exhibited dome-shaped colonies, stained positively for alkaline phosphatase, and expressed pluripotent stem cell markers such as POU5F1, SOX2, and SSEA-1. The expression levels of these markers, especially for NANOG, varied among the cell lines. A DNA methylation assay showed the POU5F1 promoter region was hypomethylated compared to fibroblast cells. An in vitro differentiation assay showed that endoderm and ectoderm marker genes, but not mesoderm markers, were upregulated in differentiating bESLCs. To examine bESLCs in later embryonic stages, we created 22 chimeric blastocysts with a male bESLC line carrying a GFP marker gene and transferred these to a recipient cow. Four chimeric embryos were subsequently retrieved on Day 13 and retransferred to two recipient cows. One living fetus was obtained at Day 62. GFP signals were not identified in fetal cells by fluorescence microscopy; however, genomic PCR analysis detected the GFP gene in major organs. Clusters of GFP-positive cells were observed in amniotic membranes, suggesting that bESLCs can be categorized as a novel type of ICM-derived cells that can potentially differentiate into epiblast and hypoblast lineages.

Activation of melanocortin receptors accelerates the gonadotropin-releasing hormone pulse generator activity in goats

The present study aims to elucidate whether the central melanocortin receptors [melanocortin-3 and -4 receptors (MC3/4-R)] are involved in regulating GnRH pulse generator activity in female goats. The GnRH pulse generator activity was electrophysiologically assessed at the intervals of characteristic increases in multiple-unit activity (MUA volleys) in the mediobasal hypothalamus. In ovariectomized goats, all doses (0.02, 0.2 and 2 nmol) of MT II, an MC3/4-R agonist, injected into the lateral ventricle significantly shortened MUA volley intervals. The duration of the period during which MT II accelerated MUA volleys was positively correlated with the dose of MT II injected. The stimulatory effect of MT II on the GnRH pulse generator activity was attenuated in the presence of estrogen. Intracerebroventricular injection of SHU9119, an MC3/4-R antagonist, significantly prolonged MUA volley intervals at 1 nmol. MT II (0.2 nmol)-induced acceleration of MUA volleys was partially blocked by the antagonism of MC3/4-R with pre-administered SHU9119 (1 nmol). The present findings demonstrate that MC3/4-R are involved in maintaining GnRH pulse generator activity in goats.

Central Cholecystokinin‐Octapeptide Accelerates the Activity of the Hypothalamic Gonadotropin‐Releasing Hormone Pulse Generator in Goats

To clarify central actions of cholecystokinin‐octapeptide (CCK‐8) on reproduction, effects of an intracerebroventricular (i.c.v.) administration of CCK‐8 on the activity of the gonadotropin‐releasing hormone (GnRH) pulse generator were examined in ovariectomized (OVX) goats in the absence or presence of oestradiol. Goats were chronically fitted with recording electrodes in the mediobasal hypothalamus, and electrophysiological manifestations of the GnRH pulse generator were monitored as characteristic increases in the multiple‐unit activity (MUA volleys). In OVX goats, a bolus i.c.v. injection of as little as 0.01 nmol of CCK‐8 induced a MUA volley with a short latency, which resulted in a significant decrease in the post‐treatment volley interval compared to that in the saline injected control. Administration of higher doses of CCK‐8 (0.1 and 2 nmol) did not further accelerate the occurrence of the MUA volley, but stimulatory effects were observed for a longer period than that after the 0.01 nmol injection. When goats were treated with oestradiol, while a bolus i.c.v. injection of 0.01 nmol CCK‐8 had no effect, an injection of 0.1 nmol of the peptide significantly decreased the post‐treatment volley interval. On continuous i.c.v. infusion of CCK‐8 at 3 nmol per 200 µl/h for 3 h, MUA volleys with shorter intervals than those in the control were successively induced without any apparent change in basal plasma luteinizing hormone levels in OVX goats. These results demonstrate that central CCK‐8 strongly accelerates the activity of the GnRH pulse generator in goats.

Immunohistochemical characterization of the arcuate kisspeptin/neurokinin B/dynorphin (KNDy) and preoptic kisspeptin neuronal populations in the hypothalamus during the estrous cycle in heifers.

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.