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Conditional kisspeptin neuron-specific Kiss1 knockout with newly generated Kiss1-floxed and Kiss1-Cre mice replicates a hypogonadal phenotype of global Kiss1 knockout mice.

ABSTRACT: The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 KO mice showed pubertal failure along with a suppression of gonadotropin secretion and ovarian atrophy. These results indicate that newly-created hypothalamic Kiss1 KO mice obtained by the Cre-loxP system recapitulated the infertility of global Kiss1 KO models, suggesting that hypothalamic kisspeptin, but not peripheral kisspeptin, is critical for reproduction. Importantly, these Kiss1-floxed mice are now available and will be a valuable tool for detailed analyses of roles of each population of kisspeptin neurons in the brain and peripheral kisspeptin-producing cells by the spatiotemporal-specific manipulation of Cre expression.

SUBMITTER: Ikegami K

PROVIDER: S-EPMC7470906 | biostudies-literature | 2020 Aug

REPOSITORIES: biostudies-literature

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Conditional kisspeptin neuron-specific Kiss1 knockout with newly generated Kiss1-floxed and Kiss1-Cre mice replicates a hypogonadal phenotype of global Kiss1 knockout mice.

Ikegami Kana K Goto Teppei T Nakamura Sho S Watanabe Youki Y Sugimoto Arisa A Majarune Sutisa S Horihata Kei K Nagae Mayuko M Tomikawa Junko J Imamura Takuya T Sanbo Makoto M Hirabayashi Masumi M Inoue Naoko N Maeda Kei-Ichiro KI Tsukamura Hiroko H Uenoyama Yoshihisa Y

The Journal of reproduction and development 20200420 4

The present study aimed to evaluate whether novel conditional kisspeptin neuron-specific Kiss1 knockout (KO) mice utilizing the Cre-loxP system could recapitulate the infertility of global Kiss1 KO models, thereby providing further evidence for the fundamental role of hypothalamic kisspeptin neurons in regulating mammalian reproduction. We generated Kiss1-floxed mice and hypothalamic kisspeptin neuron-specific Cre-expressing transgenic mice and then crossed these two lines. The conditional Kiss1 ...[more]

PMID: 32307336

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Project description:The kisspeptin/Gpr54 signalling pathway plays a critical role in reproduction by stimulating the secretion of gonadotrophin-releasing hormone (GnRH), yet mice carrying mutations in Kiss1 (which encodes kisspeptin) or Gpr54 exhibit partial sexual maturation. For example, a proportion of female Kiss1(-/-) and Gpr54(-/-) mice exhibit vaginal oestrus, and some male Kiss1(-/-) and Gpr54(-/-) mice exhibit spermatogenesis. To characterise this partial sexual maturation, we examined the vaginal cytology of female Kiss1(-/-) and Gpr54(-/-) mice over time. Almost all mutant mice eventually enter oestrus, and then spontaneously transition from oestrus to dioestrus and back to oestrus again. These transitions are not associated with ovulation, and the frequency of these transitions increases with age. The oestrus exhibited by female Kiss1(-/-) and Gpr54(-/-) mice was disrupted by the administration of the competitive GnRH antagonist acyline, which also resulted in lower uterine weights and, in Kiss1(-/-) mice, lower serum follicle-stimulating hormone (FSH) and luteinising hormone (LH) concentrations. Similarly, male Kiss1(-/-) and Gpr54(-/-) mice treated with acyline had smaller testicular sizes and an absence of mature sperm. In addition to examining intact Kiss1(-/-) and Gpr54(-/-) mice, we also assessed the effects of acyline on gonadotrophin concentrations in gonadectomised mice. Gonadectomy resulted in a significant increase in serum FSH concentrations in male Gpr54(-/-) and Kiss1(-/-) mice. Acyline administration to gonadectomised Kiss1(-/-) and Gpr54(-/-) male mice lowered serum FSH and LH concentrations significantly. By contrast to males, gonadectomy did not result in significant gonadotrophin changes in female Kiss1(-/-) and Gpr54(-/-) mice, but acyline administration was followed by a decrease in LH concentrations. These results demonstrate that, although kisspeptin signalling is critical for the high levels of GnRH activity required for normal sexual maturation and for ovulation, Kiss1(-/-) and Gpr54(-/-) mice retain some degree of GnRH activity. This GnRH activity is sufficient to produce significant effects on vaginal cytology and uterine weights in female mice and on spermatogenesis and testicular weights in male mice.

Project description:Kisspeptin neuropeptides are encoded by the Kiss1 gene and play a critical role in the regulation of the mammalian reproductive axis. Kiss1 neurones are found in two locations in the rodent hypothalamus: one in the arcuate nucleus (ARC) and another in the RP3V region, which includes the anteroventral periventricular nucleus (AVPV). Detailed mapping of the fibre distribution of Kiss1 neurones will help with our understanding of the action of these neurones in other regions of the brain. We have generated a transgenic mouse in which the Kiss1 coding region is disrupted by a CRE-GFP transgene so that expression of the CRE recombinase protein is driven from the Kiss1 promoter. As expected, mutant mice of both sexes are sterile with hypogonadotrophic hypogonadism and do not show the normal rise in luteinising hormone after gonadectomy. Mutant female mice do not develop mature Graafian follicles or form corpora lutea consistent with ovulatory failure. Mutant male mice have low blood testosterone levels and impaired spermatogenesis beyond the meiosis stage. Breeding Kiss-CRE heterozygous mice with CRE-activated tdTomato reporter mice allows fluorescence visualisation of Kiss1 neurones in brain slices. Approximately 80-90% of tdTomato positive neurones in the ARC were co-labelled with kisspeptin and expression of tdTomato in the AVPV region was sexually dimorphic, with higher expression in females than males. A small number of tdTomato-labelled neurones was also found in other locations, including the lateral septum, the anterodorsal preoptic nucleus, the amygdala, the dorsomedial and ventromedial hypothalamic nuclei, the periaquaductal grey, and the mammillary nucleus. Three dimensional visualisation of Kiss1 neurones and fibres by CLARITY processing of whole brains showed an increase in ARC expression during puberty and higher numbers of Kiss1 neurones in the caudal region of the ARC compared to the rostral region. ARC Kiss1 neurones sent fibre projections to several hypothalamic regions, including rostrally to the periventricular and pre-optic areas and to the lateral hypothalamus.

Project description:Kisspeptin is a potent activator of GnRH-induced gonadotropin secretion and is a proposed central regulator of pubertal onset. In mice, there is a neuroanatomical separation of two discrete kisspeptin neuronal populations, which are sexually dimorphic and are believed to make distinct contributions to reproductive physiology. Within these kisspeptin neuron populations, Kiss1 expression is directly regulated by sex hormones, thereby confounding the roles of sex differences and early activational events that drive the establishment of kisspeptin neurons. In order to better understand sex steroid hormone-dependent and -independent effects on the maturation of kisspeptin neurons, hypogonadal (hpg) mice deficient in GnRH and its downstream effectors were used to determine changes in the developmental kisspeptin expression. In hpg mice, sex differences in Kiss1 mRNA levels and kisspeptin immunoreactivity, typically present at 30 days of age, were absent in the anteroventral periventricular nucleus (AVPV). Although immunoreactive kisspeptin increased from 10 to 30 days of age to levels intermediate between wild type (WT) females and males, corresponding increases in Kiss1 mRNA were not detected. In contrast, the hpg arcuate nucleus (ARC) demonstrated a 10-fold increase in Kiss1 mRNA between 10 and 30 days in both females and males, suggesting that the ARC is a significant center for sex steroid-independent pubertal kisspeptin expression. Interestingly, the normal positive feedback response of AVPV kisspeptin neurons to estrogen observed in WT mice was lost in hpg females, suggesting that exposure to reproductive hormones during development may contribute to the establishment of the ovulatory gonadotropin surge mechanism. Overall, these studies suggest that the onset of pubertal kisspeptin expression is not dependent on reproductive hormones, but that gonadal sex steroids critically shape the hypothalamic kisspeptin neuronal subpopulations to make distinct contributions to the activation and control of the reproductive hormone cascade at the time of puberty.

Project description:The Cre-loxP strategy for tissue-specific gene inactivation has become a widely employed tool in several research studies. Conversely, inadequate breeding and genotyping without considering the potential for non-specific Cre-recombinase expression may lead to misinterpretations of results. Nestin-Cre transgenic mice, widely used for the selective deletion of genes in neurons, have been observed to have an incidence of Cre-line germline recombination. In this study, we attempted to generate neuron-specific Glucagon-like peptide 1 receptor (Glp1r) knock-out mice by crossing mice harboring the Nestin-Cre transgene with mice harboring the Glp1r gene modified with loxP insertion, in order to elucidate the role of Glp1r signaling in the nervous system. Surprisingly, during this breeding process, we discovered that the null allele emerged in the offspring irrespective of the presence or absence of the Nestin-Cre transgene, with a high probability of occurrence (93.6%). To elucidate the cause of this null allele, we conducted breeding experiments between mice carrying the heterozygous Glp1r null allele but lacking the Nestin-Cre transgene. We confirmed that the null allele was inherited by the offspring independently of the Nestin-Cre transgene. Furthermore, we assessed the gene expression, protein expression, and phenotype of mice carrying the homozygous Glp1r null allele generated from the aforementioned breeding, thereby confirming that the null allele indeed caused a global knock-out of Glp1r. These findings suggest that the null allele in the NestinCre-Glp1r floxed breeding arose due to germline recombination. Moreover, we demonstrated the possibility that germline recombination may occur not only during the spermatogenesis at testis but also during epididymal sperm maturation. The striking frequency of germline recombination in the Nestin-Cre driver underscores the necessity for caution when implementing precise breeding strategies and employing suitable genotyping methods.

Dataset Information

Conditional kisspeptin neuron-specific Kiss1 knockout with newly generated Kiss1-floxed and Kiss1-Cre mice replicates a hypogonadal phenotype of global Kiss1 knockout mice.

Publications

Conditional kisspeptin neuron-specific Kiss1 knockout with newly generated Kiss1-floxed and Kiss1-Cre mice replicates a hypogonadal phenotype of global Kiss1 knockout mice.

Similar Datasets

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