Material and Method: In the study, 40 female Balb/c mice aged 5-6 months with an average weight of 35-40 grams were used. All female mice were cycled by vaginal smear with an average of 3 oestrus cycles. Total of 21 animals with regular cycles were selected and divided into 3 groups according to estrus phases (diestrus, proestrus, estrus and groups, n =7). Animals were decapitated without administering any anesthetic and then brain tissues were frozen on dry ice. Sections were taken from the brain tissue and fluorescent staining steps were applied. At the end of the study, kisspeptin density in ARC, PVN, DMN and VMN nuclei of the hypothalamus was calculated by immunofluorescence method.

Results: It was found that the intensity of kisspeptin higher significantly in the oestrus group compared to the diestrus group in ARC, DMN and VMN. When the intensity of kisspeptin in the PVN was statistically evaluated, insignificant difference was found among the groups. In the DMN and VMN regions, when the estrus and proestrus groups were compared to the diestrus group, the intensity of kisspeptin higher significantly.

Conclusion: It was determined that kisspeptin intensity was different in hypothalamic ARC, DMN, VMN and PVN regions at estrous, diestrus and proestrus stages of female mice.

The GPR54 receptor was first discovered in rats in 1999. While this receptor is highly expressed in the hypothalamus, preoptic area (POA), hippocampus, midbrain, amygdala and medulla, limbic system and basal ganglia in the brain^1,9,10, it is expressed in placenta, pituitary, pancreas, heart, muscle, kidney, liver, lung, thymus, intestine and testicles¹¹. Hypothalamus located in the ventral part of the forebrain plays an important role in the regulation of energy homeostasis, fluid balance, stress, growth, reproductive behavior, emotion, and circadian rhythms ¹². In the hypothalamus, there are several important regions with well-known anatomical and physiological features such as paraventricular nucleus (PVN), dorsomedial nucleus (DMN), arcuate nucleus (ARC), ventromedial nucleus (VMN)¹³. These nuclei have important roles in the regulation of food intake and appetite, hunger-satiety states, stress, body's energy metabolism and homeostat-ic regulation of body weight¹³.

The neuroanatomical distribution of the kisspeptin-expressing cell populations is conserved among mam-malian species. Kisspeptin-expressing neurons are mainly located in two different hypothalamic regions in the brain, namely the rostral periventricular region (RP3V) of the third ventricle and the ARC ^14-16. Expression of kisspeptin in ARC has also been demon-strated in different species such as mouse^16-18, rat^19, hamster²⁰ and human²¹.

The second largest kisspeptin-positive cell population is localized in RP3V (RP3V, anterior ventral periventricular region (AVPV) and periventricular nucleus in mice^16,18,22, rats^15,19, hamsters^20, and humans²¹. (PeN). These two hypothalamic regions are regulated differently by testosterone and estradiol, both during the growth period and in adult-hood^17,22.

Kisspeptin immunoreactive fibers are also found in DMN, ARC and PVN and send projections to preoptic areas^23,24. It has been shown that GPR54 is synthe-sized in the medial preoptic area (MPOA), DMN, ARC and lateral hypothalamic region by the in situ hybridization method^9,24,25. However, changes in kis-speptin expression in female mice at different stages of the sexual cycle are not well known. For this purpose, in this study, it was aimed to investigate the expression intensity of kisspeptin in the brain regions that have important roles in the regulation of reproduction, energy balance and metabolism in different stages of estrus cycles of female mice by immunofluorescence method.

The mice were housed in standard cages in rooms that were kept constant at temperature (22-25 ° C) and relative humidity (40-55%) in Firat University Experimental Research Center, ventilated and a 12-hour light/dark cycle was applied. The photoperiod change was made with an automatic time adjuster at 07:00 in the morning in a bright phase. The mice were fed with special rat feeds in pellet form (Korkutelim Yem Gıda San. Tic. AŞ., Korkuteli/ANTALYA). The water requirement of the animals was met with tap water in bottles placed in special sections in the cages with droppers at the ends.

The cycles of the female mice were followed by vaginal smear method for 15 days (average 3 estrus cycles) between 08:00 and 12:00 every day. Cycle follow-up was started from the day the vaginal opening was seen. The mice that showed regularity periodically during the cycles were divided into 3 groups with 7 animals in each group:

Diestrus (n = 7)
Proestrus (n =7)
Estrus (n =7)

The metaestrus group was not included in the study group because it was defined as a transition period in the first period of the first day of diestrus (diestrus 1).

Immunfluorecent Analysis
Taking Brain Sections
Mice in all three groups were decapitated during the specified sexual periods without any chemical agent application. Brain tissues were quickly removed and frozen in dry ice. 18 micrometer sections were taken from the frozen brains in the cyrostat device (Leica). Sections were taken using the mouse brain atlas²⁶ to cover the areas of interest. The sections taken were glued to the slides and the slides were dried on a heating plate at 40 ± 5oC for 30 minutes.

Fluorescent Imaging
Brain sections were fixed in ethanol at -20°C for 15 minutes. The brains whose fixation was completed were shaken for 3x5 minutes in Phosphate Buffer Solution (PBS) at 80 RPM on a shaker. Blocking Solution prepared with 5% Bovine Serum Albumin (BSA) (Sigma) and PBS-T was added to each brain at 30 microliters (μl) and shaken at 80 RPM for 1 hour at room temperature. Then primary antibody (Pro-teintech) was added and incubated at 80 RPM over-night (approximately 12-16 hours) at 4°C.

The next day, the sections were washed in PBS for 3x5 min at 80 RPM on the shaker. Secondary antibody (Jackson Immuno Research) was added and incubated at 80 RPM for 1 hour at room temperature. After secondary antibody, sections were washed 3x5 min with PBS on a shaker. Core dye (DAPI, 4’,6 Diamidine-2-Phenylindole Dihydrochloride) was added and treated in the dark for 5 minutes. After core staining, sections were washed with PBS for 5 min at 80 RPM. After the sections were dried on the slide in the dark, they were covered with Gel / Mount fluid preservative and covered with coverslip. Images were examined and photographs taken at 10X magnification under the ZEISS fluorescence microscope. Density analysis of the relevant regions was made.

Statistical Analysis
Statistical analysis of the results of the study was performed using the SPSS 23.0 for Windows program. Data are given as Mean ± Standard error (Mean ± SEM). Kruskal-Wallis test was used to evaluate the data. In all analyses, p-value was less than 0.05 were considered statistically significant.

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Table 1: Mean ± SEM values of kisspeptin concentration in hypothalamic nuclei in diestrus, proestrus and estrus groups.

Arcuate Nucleus
The mean ± SEM values of kisspeptin intensity between groups in the arcuate nucleus are shown in table 1. It is observed that kisspeptin intensity higher significantly in estrus group compared to diestrus group (p <0.01). Although there is no statistically significant difference between diestrus and proestrus groups, it is seen that the intensity of kisspeptin in proestrus is high (Figure 1, 2). Similarly, although there is no statistically significant difference between estrus and proestrus groups, it is seen that the intensity of kisspeptin in estrus is higher than that of prooestrus (Figure 1, 2).

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Figure 1: Kisspeptin density in the arcuate nucleus in the diestrus, proestrus and estrus groups. ** p <0.01 compared to the diestrus group.

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Figure 2: A, D-Diestrus; Kisspeptin immunoreactivity in Arcuate nucleus in B, E-proestrus and C, F-estrus groups. Magnification A-E; 10X. Kisspeptin immunoreactivity (Red fluorescent, Cy3), DAPI: Blue Fluorescent, 3V: Third Ventricle.

Paraventricular Nucleus
The mean ± SEM values of kisspeptin density between groups in the paraventricular nucleus are shown in table 1. Although there is no statistically significant difference in kisspeptin density in PVN compared to the groups, it is seen that it is the lowest in the proestrus group, and it is similar in the diestrus and estrus groups (Figure 3, 4).

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Figure 3: Kisspeptin density in the diestrus, proestrus and estrus groups in the paraventricular nucleus.

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Fgure 4: A, D-Diestrus; Immunoreactivity of kisspeptin in paraventricular nucleus in B, E-proestrus and C, F-estrus groups. Magni-fication A-E; 10X. Kisspeptin immunoreactivity (Red fluorescent, Cy3), DAPI: Blue Fluorescent, 3V: Third Ventricle.

Dorsomedial Nucleus
The mean ± SEM values of kisspeptin intensity between groups in the dorsomedial nucleus are shown in table 1. Kisspeptin density higher significantly in the estrus group compared to diestrus (p <0.001), similarly, kisspeptin intensity higher significantly in the proestrus group compared to the diestrus group (p <0.05). There was no statistically significant difference between the proestus and estrus groups (Figure 5, 6).

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Figure 5: Kisspeptin density in the diestrus, proestrus and estrus groups in the dorsomedial nucleus. (DMN; Dorsomedial nucleus). * p <0.05 ve *** p <0.001, compared to the diestrus group.

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Figure 6: A, D-Diestrus; Immunoreactivity of kisspeptin in dorsomedial nucleus in B, E-proestrus and C, F-estrus groups. Magnification A-E; 10X. Kisspeptin immunoreactivity (Red fluorescent, Cy3), DAPI: Blue Fluorescent, 3V: Third Ventricle.

Ventromedial Nucleus
The mean ± SEM values of kisspeptin intensity between groups in the ventromedial nucleus are shown in table 1. Kisspeptin intensity higher significantly in both proestrus and estrus groups compared to diestrus group (p <0.05) (Figure 7, 8).

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Figure 7: Kisspeptin density in the diestrus, proestrus, estrus groups in the ventromedial nucleus. * p <0.05, compared to the diestrus group.

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Figure 8: A, D-Diestrus; Immunoreactivity of kisspeptin in ventro-medial nucleus in B, E-proestrus and C, F-estrus groups. Magnification A-E; 10X. Kisspeptin immunoreactivity (Red fluorescent, Cy3), DAPI: Blue Fluorescent, 3V: Third Ventricle.

The arcuate nucleus is an important brain region in the basomedial hypothalamus that regulates food intake and energy consumption, where kisspeptin expression is most common²⁷. It has been shown that kisspeptin expression in ARC is necessary for pulsatile release of GnRH, and it has been observed that the application of kisspeptin antagonist to ARC decreases the LH pulse frequency ²⁸ and LH release is stimulated when kisspeptin is applied to ARC²⁹. Studies show that kisspeptin neurons in ARC can be a neuronal pace-maker source that provides pulsatile release of GnRH^16,30. In this study, it was observed that the intensity of kisspeptin was significantly higher in the estrus group in female mice compared to the proestrus and diestrus groups in ARC. The high estrogen levels during the proestrus and oestrus periods of the female mice and low estrogen levels explain the changes in the intensity of kisspeptin in this study. It can be said that estrogen higher gradually starting from estrus to proestrus and estrus causes an higher in kisspeptin density in ARC in parallel. In addition, the higher in kisspeptin may have triggered the formation of the LH peak, leading to ovulation. In parallel with this study, it was observed that maximal responses to kisspeptin occurred in estrus³¹.

In the study conducted on Spraque Dawley female rats, it has been shown that the expression of Kiss1 in ARC is regulated in the opposite way in AVPV. In ARC, Kiss1 mRNA peaks towards the end of the diestrus, while it is lowest in the proestrus and estrus stages ¹⁴. These studies yielded results contrary to our findings. The reason for this may be that the study is done in different types and with different methods. As a matter of fact, when looking at the literature, kisspeptin expression also varies according to species. For example, the demonstration of less peptide storage in cell bodies in rats compared to mice may reflect the different dynamics between both species in kisspeptin release³². Another difference between mice and rats has been demonstrated in the kisspeptinergic cell population in the DMN. Unlike mice, no kisspeptin immu-noreactive cells could be detected in the DMN region in rats in proestrus^32,33. Whether this reflects a true species difference, a particular physiological arrangement or methodological differences between studies need to be investigated.

As shown in this study in ARC, which is an important brain region where food intake and energy balance are regulated, the change in the density of kisspeptin at different cycle stages suggests that kisspeptin may have a role in the change of food intake and energy con-sumption at these stages. However, food intake and energy regulation have not been investigated since it is not one of the main objectives of this study. However, in our study, it can be said that the changes that occur in the brain regions related to food intake may change in different estrus cycles, and this may be through leptin, kisspeptin and other orexigenic and anorexigenic hormones.

Kisspeptin fibers extend from cell bodies in RP3V and ARC to major GnRH neurons and different hypothalamic areas. Among them, PVN is one of the main targets of this system^34,35. In different studies, it has been shown that PVN is intensely innervated with kisspeptin fibers in rodents^23,33,35. Within the hypothalamus, PVN is a region with different neuronal populations that play important roles in neuroendocrine / autonomic regulation and control of energy balance, and is a critical regulatory center for energy homeostasis^36,37. Although PVN is not a very important region in the regulation of the HHG axis, it is thought that following the application of kisspeptin-54 to PVN, the stimulation of the HHG axis is mediated by inter-neurons extending to preoptic GnRH neurons. It has been reported that this region shows much less neuronal activation in response to peripheral kisspeptin compared to other regions of the hypothalamus, especially the preoptic nucleus³⁸. Further studies are needed to identify kisspeptin-sensitive neurons in PVN and investigate their relationship with GnRH neurons in other areas of the hypothalamus. Although GPR54 expression is not specifically reported in PVN, it is known that kisspeptin immunoreactive fibers and cell bodies and GnRH immunoreactive neurons are present in this region^23,39. In the present study, no significant difference was observed in the intensity of kis-speptin in the different phases of the estrus cycle in the PVN region. The fact that the difference is not seen in accordance with the literature supports that it does not have an important role in the HHG axis. However, the presence of kisspeptin positive fibers spread through-out the PVN suggests that kisspeptin may play a role in the regulation of many physiological activities other than reproduction controlled by PVN. However, the presence of kisspeptin positive fibers spread through-out the PVN suggests that kisspeptin may play a role in the regulation of many physiological activities other than reproduction controlled by PVN.

It has been shown in an in situ hybridization study that Kiss1r is co-expressed in subpopulations of oxytocin (OT) neurons in the medial part of the PVN in female rats in diestrus³⁶. Oxytocin neurons in PVN have been shown to be projected from kisspeptin neurons in RP3V where kisspeptin expression is high in proestrus^2,36,40,41 and oxytocin higher lordosis behavior in rats⁴² and there is a significant higher in PVN Kiss1r expression in proestrus. These effects can higher female receptive behavior. Oxytocin higher lordosis behavior in rats⁴² and the presence of kisspeptin fibers in PVN in proestrus⁴⁰ suggest that kisspeptin may be associated with receptive behavior in female rats through modulation of oxytocin secretion³⁶. Although there is no significant difference in the intensity of kisspeptin in the PVN region in this study, more studies are needed to reveal whether kisspeptin release and kisspeptin amplify the effects of oxytocin in the oxytocin-producing regions of PVN.

In a study conducted in female rats, higher expression of kisspeptin in estrus was observed in PVN¹⁴. Similar to this study, higher kisspeptin concentration in estrus was observed in PVN in this study. The higher concentration of kisspeptin in estrus suggests that it may lead to receptive behavior in the female associated with oxytocin neurons in this area.

Another hypothalamic core that plays an important role in the regulation of energy homeostasis and stress is DMN. Although direct application of kisspeptin-10 to DMN does not cause any change in circulating LH and testosterone, this indicates that kisspeptin, its input to this core, does not play a role in the activity of the HHG axis²⁹.

Kisspeptin immunoreactivity was found in some cells in DMN in a study conducted on female mice in diestrus^33,43. In this study, significant differences were observed in the intensity of kisspeptin in different estrus stages in the DMN region, especially between prooestrus and diestrus stages and between diestrus and estrus stages. Seeing these differences suggests that DMN may be related to different functions. Especially in estrus, higher kisspeptin concentration compared to other stages may be related to stress and energy metabolism in animals. Unlike mice, no kisspeptin immuno-reactive reaction was observed in DMN in a study in female rats in proestrus. The relatively high level of GPR54 mRNA in this region indicates that kisspeptin may have other roles independent of reproduction⁴³. A subgroup of neurons expressing neuronal nitric oxide synthase (nNOS) in the ventrolateral part of the ventromedial hypothalamus and communicating with kis-speptin neurons is shown. In accordance with the knowledge that nitric oxide (NO) is an important neu-rotransmitter in the effects of kisspeptin neurons, a significant decrease in lordosis behavior has been detected in female mice with impairment in nNOS. The results obtained in the study show that kisspeptin manages both mate preference and sexual motivation in female mice, and sexual behavior and ovulation are coordinated by the same neuropeptide ⁴⁴. In the current study, kisspeptin density in the ventromedial nucleus, proestrus and estrus groups was found to be higher than in the diestrus group.

Consistent with the study mentioned in the literature, the higher in the intensity of kisspeptin in VMN, especially in the estrus phase of the cycle, suggests that kisspeptin neurons may have a central function that regulates sexual behavior in the female mouse brain, as it is known to affect nitric oxide-synthesizing neurons in the ventromedial hypothalamus.

Consequently, in this study, the intensity of kisspeptin expression in the brain regions that have important roles in the regulation of reproduction, energy balance and metabolism in different stages of the estrus cycles of female mice was demonstrated by immunofluores-cence method. Based on the data obtained from the study, the expression of kisspeptin in important nuclei in the hypothalamus shows that it may affect many physiological mechanisms apart from the reproductive axis.

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