Fuko Matsuda

Development of novel neurokinin 3 receptor (NK3R) selective agonists with resistance to proteolytic degradation.

Neurokinin B (NKB) regulates the release of gonadotropin-releasing hormone (GnRH) via activation of the neurokinin-3 receptor (NK3R). We evaluated the biological stability of NK3R selective agonists to develop novel NK3R agonists to regulate reproductive functions. On the basis of degradation profiles, several peptidomimetic derivatives were designed. The modification of senktide with (E)-alkene dipeptide isostere generated a novel potent NK3R agonist with high stability and prolonged bioactivity.

The luteinising hormone surge-generating system is functional in male goats as in females: involvement of kisspeptin neurones in the medial preoptic area.

A luteinising hormone (LH) surge is fundamental to the induction of ovulation in mammalian females. The administration of a preovulatory level of oestrogen evokes an LH surge in ovariectomised females, whereas the response to oestrogen in castrated males differs among species; namely, the LH surge‐generating system is sexually differentiated in some species (e.g. rodents and sheep) but not in others (e.g. primates). In the present study, we aimed to determine whether there is a functional LH surge‐generating system in male goats, and whether hypothalamic kisspeptin neurones in male goats are involved in the regulation of surge‐like LH secretion. By i.v. infusion of oestradiol (E2; 6 μg/h) for 16 h, a surge‐like LH increase occurred in both castrated male and ovariectomised female goats, although the mean peak LH concentration was lower and the mean peak of the LH surge was later in males compared to females. Dual staining with KISS1 in situ hybridisation and c‐Fos immunohistochemistry revealed that E2 treatment significantly increased c‐Fos expression in the medial preoptic area (mPOA) KISS1 cells in castrated males, as well as ovariectomised females. By contrast, dual‐labelled cells were scarcely detected in the arcuate nucleus (ARC) after E2 treatment in both sexes. These data suggest that kisspeptin neurones in the mPOA, but not those in the ARC, are involved in the induction of surge‐like LH secretion in both male and female goats. In summary, our data show that the mechanism that initiates the LH surge in response to oestrogen, the mPOA kisspeptin neurones, is functional in male goats. Thus, sexual differentiation of the LH surge‐generating system would not be applicable to goats.

Effects of full-length kisspeptin administration on follicular development in Japanese Black beef cows.

Abstract Kisspeptin is a key molecule that stimulates gonadotropin secretion via release of gonadotropin-releasing hormone (GnRH). In the present study, our aim was to investigate whether kisspeptin has stimulatory effects on follicular development via GnRH/gonadotropin secretion in cows. Japanese Black beef cows were intravenously injected with full-length bovine kisspeptin [Kp-53 (0.2 or 2 nmol/kg)] or vehicle 5 days after they exhibited standing estrus (Day 0). In cows injected with Kp-53 at 2 nmol/kg, the follicular sizes of the first dominant follicles increased on Day 6 and thereafter. Ovulation of the first dominant follicle occurred in 1 out of 4 cows treated with Kp-53 at 2 nmol/kg. Injection of Kp-53 at 2 nmol/kg increased the concentration of plasma luteinizing hormone (LH) but not follicle-stimulating hormone, over a 4-h period following injection in all cows. The present study suggests that administration of full-length kisspeptin causes LH secretion, which is sustained for a few hours, and it is capable of stimulating follicular development and/or ovulation.

Molecular and Epigenetic Mechanism Regulating Hypothalamic Kiss1 Gene Expression in Mammals.

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.

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.

KISS1 Gene Expression in the Developing Brain of Female Pigs in Pre- and Peripubertal Periods

Puberty is associated with an increase in gonadotropin secretion as a result of an increase in gonadotropin-releasing hormone (GnRH) secretion. Kisspeptin is considered to play a key role in puberty onset in many mammalian species, including rodents, ruminants and primates. The present study aimed to determine if changes in hypothalamic expression of the KISS1 gene, encoding kisspeptin, are associated with the onset of puberty in pigs. The animals (n=4 in each group) were perfused with 4% paraformaldehyde at 0, 1, 2, 3 and 4 months old, as prepubertal stages, and at 5 months old, as the peripubertal stage, following each blood sampling. KISS1 gene expressions in coronal sections of brains were visualized by in situ hybridization. Plasma luteinizing hormone (LH) was measured by radioimmunoassay. KISS1 mRNA signals were observed in the arcuate nucleus (ARC) at all ages examined without any significant difference in the number of KISS1-expressing cells, indicating that the KISS1 gene is constantly expressed in the ARC throughout pubertal development in pigs. The plasma LH concentration was the highest in 0-month-old piglets and significantly decreased in the 1- and 2 month-old groups (P<0.05), suggesting a developing negative feedback mechanism affecting gonadotropin release during the prepubertal period. Considering the potent stimulating effect of kisspeptin on gonadotropin release in prepubertal pigs, kisspeptin secretion rather than kisspeptin synthesis may be responsible for the onset of puberty in pigs.

Structure-activity relationship study on senktide for development of novel potent neurokinin-3 receptor selective agonists†

Neurokinin B (NKB) regulates the secretion of gonadotropin-releasing hormone (GnRH) in the hypothalamus via activation of the cognate neurokinin-3 receptor (NK3R). The stimulatory effect of NKB and the derivatives on gonadotropin secretion can potentially be used for development of novel regulatory and therapeutic agents for reproductive dysfunctions. Here, we report a comprehensive structure–activity relationship study on the NK3R-selective agonist peptide, senktide. Substitution of the N-terminal succinyl-Asp substructure in senktide with oxalyl-Glu, oxalyl-D-Glu or oxalyl-L-2-aminoadipic acid (Aad) increased receptor binding and NK3R activation. Among these modifications, the oxalyl-D-Glu substructure prevented neutral endopeptidase (NEP) 24.11-mediated degradation, thus providing a novel NK3R agonist peptide with favourable biological and stability properties.

Development of novel NK3 receptor antagonists with reduced environmental impact.

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.

Mouse quiescin sulfhydryl oxidases exhibit distinct epididymal luminal distribution with segment-specific sperm surface associations†

Abstract Sulfhydryl oxidation is part of the sperm maturation process essential for the acquisition of sperm fertilization competency and its structural stabilization; however, the specific sulfhydryl oxidases that fulfill these roles have yet to be identified. In this study, we investigate the potential involvement of one atypical thiol oxidase family called quiescin Q6/sulfhydryl oxidase (QSOX) using the mouse epididymis as our model system. With multidisciplinary approaches, we show that QSOX isoform 1 and 2 exhibit complementary distribution throughout the epididymal duct, but that each variant possesses distinct subcellular localization within the epididymal principal cells. While QSOX2 was exclusively present in the Golgi apparatus of the caput and corpus epididymis, QSOX1c, the most profusely express QSOX1 variant, was abundantly present in the cauda luminal fluids. Moreover, immunohistochemistry studies together with proteomic identification in isolated epididymosomes provided evidence substantiating the release of QSOX2, but not QSOX1c, via an apocrine secretory pathway. Furthermore, we demonstrate for the first time, distinct association of QSOX1c and QSOX2 with the sperm acrosome and implantation fossa, during different stages of their epididymal maturation. In conclusion, our study provides the first comprehensive comparisons between QSOX1 and QSOX2 in the mouse epididymis, revealing their distinct epididymal distribution, cellular localization, mechanisms of secretion and sperm membrane association. Together, these data suggest that QSOX1 and QSOX2 have discrete biological functions in male germ cell development. Summary Sentence Mouse epididymal sulfhydryl oxidases exhibit complementary tissue distribution with disparate cellular localization, the distinct secretory mechanism and sperm membrane association suggesting their participation in different sperm maturation processes.

Evaluation of heat stress response in crossbred dairy cows under tropical climate by analysis of heart rate variability

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.