Combined effects of obesity and di-(2-ethylhexyl) phthalate on testosterone levels and kisspeptin/GPR54 expression in hypothalamus and testes of male mice.

BACKGROUND: This study evaluated whether obese male mice exposed to di-(2-ethylhexyl) phthalate (DEHP) showed synergistic effects on testosterone levels and the potential underlying mechanism. METHODS: Forty-eight male mice were assigned to six groups for 12-week treatments as follows: normal, DEHP100, diet-induced obesity (DIO), DIO + DEHP30, DIO + DEHP100, and DIO + DEHP300. Serum hormone levels, including testosterone (T), luteinizing hormone (LH), and leptin, were detected by ELISA. The levels of Ob-R, kisspeptin, and GPR54 protein expression in hypothalamus and testicular tissues were measured by western blot. RESULTS: There were significantly lower levels of serum T and LH, higher levels of serum leptin and Ob-R, and kisspeptin and GPR54 protein expression were reduced in hypothalamus and testicular tissues in the DIO and DEHP groups compared with controls. Moreover, serum T and leptin levels were more severe in the combined DIO and DEHP exposure group than in the single exposure groups. Serum LH levels and GPR54 expression in the testis were significantly decreased in DIO + DEHP300 mice compared with DIO mice (p < 0.05). CONCLUSION: Obesity- and DEHP-only exposure had adverse effects on testosterone levels in mice, which may be due to high leptin levels and decreased Ob-R, kisspeptin, and GPR54 expression. Obesity combined with DEHP exposure had an additive adverse effect on testosterone levels in mice. One of the potential mechanisms is higher leptin levels and decreased GPR54 expression in the testes.

Kiss1R Identification and Biodistribution Analysis Employing a Western Ligand Blot and Ligand-Derivative Stain with a FITC-Kisspeptin Derivative.

It is not always easy to establish specific antibodies against receptors. Most receptors are hydrophobic and have complicated three-dimensional structures, making them difficult to use as immunogens. Thus, we developed receptor detection methods with a fluorescein-labeled ligand as an antibody alternative, which we referred to as a western ligand blot (WLB) and ligand derivative stain (LDS). Kisspeptin receptor (Kiss1R) was detected by its ligand. Kiss1R expression was confirmed in eight human cell lines by the WLB and in four pathological tissues by the LDS. Next, Kiss1R was stained by LDS in organs, revealing Kiss1R expression by [67 Ga]Ga-DOTA-kisspeptin 10 accumulation. As a result, Kiss1R-expressing cells in each organ could be stained with fluorescein-labeled kisspeptin 14 instead of an antibody and observed by light microscopy. The combination of the WLB and LDS allows identification of receptors in tissues, which can be readily applied to target receptor detection by a synthetic ligand derivative.

Alanine scanning and characterization of core peptides in Scombridae fish family for construction of Kiss1 super analog.

Chronic Kiss1 administration strongly promotes gonadal development in immature chub mackerel (cm) (Scomber japonicus). Here, we performed an Alanine scanning (Ala-scanning) of Kiss1 to determine its key residues. Additionally, we examined functional peptides from 16 Scombridae species to develop maturation-inducing super-analogs that can be used universally in Scombridae species. In the Ala-scanning of Kiss1-15 (QDMSSYNFNSFGLRY), substitution of Gln1 and Asp2 did not affect agonistic activity. This suggests that peptides could be downsized. Furthermore, it is possible that Phe8 can be substituted by unnatural amino acids that are difficult to degrade. In molecular cloning, only Scomber showed a 16-residue form as a putative mature peptide. The other genera, did not have a His residue at the N-terminal, which indicated that the functional peptide was 15 residues and the second and third residues from the N-terminal showed variation between interspecies. Next, we examined the binding affinity of various synthetic Kiss1 core peptides in Scombridae interspecies using an SRE-Luc reporter system. We cloned Kiss1 receptors (KissR1) from bluefin tuna (bft) (Thunnus orientalis) and Japanese Spanish mackerel (jsm) (Scomberomorus niphonius) for the first time. In binding affinity with cmKissR1, bftKissR1, and jsmKissR1, the species specificity of the second residue from the N-terminus in each ligand could be ignored, but the difference in the third residue strongly affected receptor binding. Scombridae species possess the same Kiss1 system but the structure of the functional peptide might be species-specific.

Region-specific changes in brain kisspeptin receptor expression during estrogen depletion and the estrous cycle.

Kisspeptin acts as a potent neuropeptide regulator of reproduction through modulation of the hypothalamic-pituitary-gonadal axis. Previous studies revealed sex differences in brain expression patterns as well as regulation of expression by estrogen. Alternatively, sex differences and estrogen regulation of the kisspeptin receptor (encoded by Kiss1r) have not been examined at cellular resolution. In the current study, we examined whether Kiss1r mRNA expression also exhibits estrogen sensitivity and sex-dependent differences using in situ hybridization. We compared Kiss1r mRNA expression between ovariectomized (OVX) rats and estradiol (E2)-replenished OVX rats to examine estrogen sensitivity, and compared expression between gonadally intact male rats and female rats in diestrus or proestrus to examine sex differences. In OVX rats, E2 replenishment significantly reduced Kiss1r expression specifically in the hypothalamic arcuate nucleus (ARC). A difference in Kiss1r expression was also observed between diestrus and proestrus rats in the hypothalamic paraventricular nucleus (PVN), but not in the ARC. Thus, estrogen appears to have region- and context-specific effects on Kiss1r expression. However, immunostaining revealed minimal colocalization of estrogen receptor alpha (ERα) in Kiss1r-expressing neuronal populations of ARC and PVN, indicating indirect or ERα-independent regulation of Kiss1r expression. Surprisingly, unlike the kisspeptin ligand, no sexual dimorphisms were observed in either the brain distribution of Kiss1r expression or in the number of Kiss1r-expressing neurons within enriched brain nuclei. The current study reveals marked differences in regulation between kisspeptin and kisspeptin receptor, and provides an essential foundation for further study of kisspeptin signaling and function in reproduction.

The expression of kisspeptin and its receptor in the domestic cat ovary and uterus in different stages of the ovarian cycle.

Kisspeptin is well known for its indispensable role in the regulation of reproduction, mainly through controlling the release of GnRH at the hypothalamic level. Recent studies have shown that kisspeptin and the kisspeptin receptor are expressed in the ovary and uterus, indicating an additional local function in reproduction at the extra-hypothalamic level. In this study, we aimed: (1) to investigate the localization pattern of kisspeptin and its receptor in the domestic cat ovary and uterus throughout the ovarian cycle using immunohistochemistry; and (2) to compare the relative expression of ovarian Kiss1 mRNA levels at different ovarian stages with qPCR analysis. Ovaries and uteri were collected and classified into three ovarian stages (inactive, follicular and luteal stages (n = 7 in each stage)) according to the ovarian morphology, vaginal cytology and serum progesterone. Kisspeptin immunoreactivity (Kp-IR) and kisspeptin receptor immunoreactivity (KpR-IR) were present in the ovaries and uteri at all ovarian stages, with no notable differences in the localization patterns between the ovarian stages. In the ovary, Kp-IR and KpR-IR were present in various ovarian compartments, including the follicles at all classes and the corpus luteum (CL). In the follicles, Kp-IR and KpR-IR were present in the oocytes, granulosa cells and theca cells. Kp-IR was also detected in the follicular fluid of antral follicles. In CL, a strong intensity of Kp-IR was present in the periphery CL of development/maintenance, with a relatively fainter intensity in the central CL. By contrast, KpR-IR was present in both peripheral and central CL at the same intensity. In the uterus, Kp-IR and KpR-IR were present in the uterine glands, myometrium and perimetrium. The relative ovarian Kiss1 mRNA level was higher in the follicular stage than in the luteal stage (P < 0.05). We concluded that kisspeptin and its receptor are present in the cat ovary and uterus, suggesting possible local functions of kisspeptin at the extra-hypothalamic level, such as folliculogenesis, oocyte survival and uterine adenogenesis.

Presence and function of kisspeptin/KISS1R system in swine ovarian follicles.

Kisspeptin and its receptor KISS1R are involved in the neuroendocrine regulation of mammalian reproduction and their role on follicular development and function can be hypothesized. The present work was designed to confirm the immunopresence of kisspeptin and its receptor in the ovary of swine and to study the effects of kisspeptin 10 and its antagonist, kisspeptin 234, on main functional parameters of granulosa cells (i.e. cell proliferation, steroid production, and redox status) as well as their modulatory action on angiogenesis. The immunopresence of kisspeptin and KISS1R were detected in granulosa cells. Kisspeptin 10 stimulated progesterone in vitro production, thus indirectly suggesting that it can have a role in the luteinization process of granulosa cells. Kisspeptin 10 displayed potentiating effects on non-enzymatic scavenging activity, thus supporting its involvement in the control of the antioxidant defense system of ovarian follicles. In addition, results from the angiogenesis bioassay suggest that kisspeptin may have a role in the physiological development of new ovarian vessels. Additional studies are needed to confirm the functional significance of the kisspeptin/KISS1R system within the swine ovary.

Mapping of Kisspeptin Receptor mRNA in the Whole Rat Brain and its Co-Localisation with Oxytocin in the Paraventricular Nucleus.

The neuropeptide kisspeptin and its receptor play an essential role in reproduction as a potent modulator of the gonadotrophin-releasing hormone (GnRH) neurone. In addition to its reproductive function, kisspeptin signalling is also involved in extra-hypothalamic-pituitary-gonadal (HPG) axis systems, including oxytocin and arginine vasopressin (AVP) secretion. By contrast to the accumulating information for kisspeptin neurones and kisspeptin fibres, the histological distribution and function of the kisspeptin receptor in the rat brain remain poorly characterised. Using in situ hybridisation combined with immunofluorescence, the present study aimed to determine the whole brain map of Kiss1r mRNA (encoding the kisspeptin receptor), and to examine whether oxytocin or AVP neurones express Kiss1r. Neurones with strong Kiss1r expression were observed in several rostral brain areas, including the olfactory bulb, medial septum, diagonal band of Broca and throughout the preoptic area, with the most concentrated population being around 0.5 mm rostral to the bregma. Co-immunofluorescence staining revealed that, in these rostral brain areas, the vast majority of the Kiss1r-expressing neurones co-expressed GnRH. Moderate levels of Kiss1r mRNA were also noted in the rostral periventricular area, paraventricular nucleus (PVN), and throughout the arcuate nucleus. Relatively weak Kiss1r expression was observed in the supraoptic nucleus and supramammillary nuclei. Moderate to weak expression of Kiss1r was also observed in several regions in the midbrain, including the periaqueductal gray and dorsal raphe nucleus. We also examined whether oxytocin and AVP neurones in the PVN co-express Kiss1r. Immunofluorescence revealed the co-expression of Kiss1r in a subset of the oxytocin neurones but not in the AVP neurones in the PVN. The present study provides a fundamental anatomical basis for further examination of the kisspeptin signalling system in the extra-HPG axis, as well as in reproductive function.