Slovenian Veterinary Research 2024 | Vol 61 No 2 | 115 Effects of Irisin on the Reproductive System of Obese Female Rats Induced by a High-fat Diet Key words irisin; obesity; hormones; ovary; apoptosis; female reproduction Nazife Ulker Ertugrul1*, Ahmet Yardimci2, Nalan Kaya Tektemur3, Ferah Bulut4, Mete Ozcan4, Haluk Kelestimur5, Sinan Canpolat2 1Department of Physiology, Faculty of Medicine, Samsun University, Samsun, 2Department of Physiology, Faculty of Medicine, Firat University, Elazig, 3Department of Histology and Embryology, Faculty of Medicine, Firat University, Elazig, 4Department of Biophysics, Faculty of Medicine, Firat University, Elazig, 5Department of Physiology, Faculty of Medicine, Istanbul Okan University, Istanbul, Turkey *Corresponding author: nazife.ulker@samsun.edu.tr Abstract: Obesity is becoming more common all across the world, causing a variety of health problems, including reproductive disruption. Although the novel, exercise-induced hormone irisin may affect the hypothalamus-pituitary-gonadal axis and reproductive function control, its impact on obesity-induced damage to the female reproductive sys- tem is not fully known. Hence, this study aimed to investigate the potential effects of irisin on reproductive hormones and reproductive organs in female rats with obesity induced by a high-fat diet. Forty female rats were divided into four groups: control, irisin, obese, and obese+irisin (n = 10 in each group). After simulating a high-fat diet-induced obesity model (via 60% kcal fat for 12 weeks) in the obese and obese+irisin groups, irisin (100 ng/kg/day via mini-osmotic pumps for about 28 days) was administered subcuta- neously to the irisin and obese+irisin groups. Results showed that subcutaneous irisin perfusion increased serum luteinizing hormone (LH), the LH to follicle-stimulating hor- mone (FSH) ratio (LH/FSH), and progesterone levels while decreasing the histopatho- logical damage in the ovaries of obese rats. On the other hand, endogenous irisin serum concentrations were similar in lean female rats and obese female rats with reproductive disorders. These results suggest that irisin may affect the reproductive axis in obese female rats. An increase in serum LH levels, which trigger ovarian steroidogenesis, and reducing histopathological changes in ovarian tissue could contribute to this effect. Received: 28 February 2023 Accepted: 18 April 2023 Slov Vet Res DOI 10.26873/SVR-1754-2023 UDC 57.017.5:581.133.8:599.323.41:641.1:614.874.25 Pages: 115–25 Original Research Article Introduction The prevalence of obesity has rapidly increased worldwide, and obesity causes several health problems, including re- production. Obesity directly or indirectly negatively affects the hypothalamus-pituitary-ovarian (HPO) axis, resulting in various reproductive disorders by causing hormone im- balances and ovulatory dysfunction. Additionally, it is also known that obesity may have a direct effect on ovarian function, independent of the HPO axis (1, 2). Studies in hu- mans and rodents have shown that obesity-related ovarian dysfunction includes abnormalities in folliculogenesis and ovulation, irregular estrous cyclicity, and depletion of ovar- ian reserve (3). Lifestyle modifications, including a healthy diet and exer- cise, are successful options for treating women with repro- ductive dysfunctions. Specifically, exercise has a protective effect against obesity-induced impairment in the female re- productive system. Exercise, for example, has been shown to improve menstrual cyclicity, ovulation, and pregnancy rates in obese anovulatory women (4, 5). Similarly, in obese polycystic ovary syndrome (PCOS) rats fed a high-fat diet, swimming training also appeared to recover ovarian mor- phology indexes such as the numbers of antral follicles and corpora lutea (6). 116 | Slovenian Veterinary Research 2024 | Vol 61 No 2 Irisin was recently discovered as a new hormone-like myo- kine released into the circulation in response to physical exercise. It is produced by cleavage of its precursor, fibro- nectin type III domain containing 5 (FNDC5). Following se- cretion, irisin promotes the browning of white adipocytes and the expression of uncoupling protein 1 (UCP1), lead- ing to enhanced UCP-1-mediated thermogenesis and in- creased energy expenditure (7, 8). Irisin has a potential role in mammalian growth and regulation of the reproductive axis (9-11). It is documented that irisin deficiency is associ- ated with disordered endocrine metabolism, poor growth, and decreased fertility in female mice (9). Interestingly, it is known that irisin can cause different effects on reproduc- tive function depending on gender in the rat model, such as exhibiting androgenic activity in males and causing re- productive disorders in females (12). Furthermore, recent studies have shown that irisin improves antidepressant-in- duced sexual dysfunction and obesity-related reproductive disorders. Accordingly, it has been revealed that irisin may mediate the effects of exercise on the reproductive system and that irisin and exercise may also have similar effects on reproductive potential (13-16). However, there are not enough studies on the possible potent effects of the irisin hormone on reproductive system disorders caused by obe- sity in female rats. This study was performed to investigate the effects of ex- ogenous irisin on serum levels of reproductive hormones and insulin and on the reproductive organs in obese female rats. Furthermore, endogenous irisin levels were measured in obese female rats to determine whether irisin levels are associated with obesity-induced changes in sex hormones and reproductive organs. Material and Methods Animals and diets All animal experiments were carried out in accordance with the governmental guidelines for the care and use of laboratory animals at Firat University and approved by the Animal Experimental Ethics Committee of Firat University (31.01.2018, number 19). Sprague-Dawley female rats (2-3 months old, 200-250 g) were obtained from the Firat University Experimental Research Unit (Elazig, Turkey). Animals were housed 3-4 per cage and kept in a 12 h light/12 h dark cycle (light on 07:00-19:00), under standard conditions (21 ± 1 °C temperature and 50-60% humidity), with ad libitum access to water and food. Forty rats were randomly allocated to four treatment groups as follows: control (control group with rats subjected to vehicle treat- ment), irisin (irisin-treated rats), obese (obesity-induced rats), and obese+irisin (irisin-treated obesity-induced rats) (n = 10 rats per group). All rats in the control and irisin groups were fed with standard commercial rat food (Korkuteli Yem Gıda Santic A.Ş., Antalya, Turkey), while all rats in the obese and obese+irisin groups were fed a high-fat diet (D12492, Research Diets, 60% kcal fat) for 16 weeks. After 12 weeks of high-fat diet exposure, the induction of obesity in both obesity-induced treatment groups was confirmed by mea- suring the Lee index, which is used for experimental valida- tion of obesity as described previously (16-18). Continuous administration of irisin After 12 weeks of diet exposure (i.e., after the establish- ment of obesity due to the high-fat diet), all rats in the control, irisin, and obese+irisin groups were anesthetized with a mixture of ketamine (6 mg/kg) and xylazine (5 mg/ kg) anesthetics, then a mini-osmotic pump (Alzet, Model 2004; Durect Corp., Cupertino, CA) was subcutaneously implanted between the scapulae of each animal under ster- ile conditions. Alzet mini-osmotic pumps were filled with either deionized water (vehicle) for the control group or iri- sin (SRP8039, Sigma-Aldrich; dissolved in deionized water to deliver at a dose of 100 ng/kg/day) for both irisin and obese+irisin groups as previously described (18). The Alzet model 2004, with a reservoir volume of 200 µL, infused de- ionized water or irisin at a flow rate of 0.25 µL/h for about 28 days. Sample collections The serum hormone levels of female rats fluctuate great- ly depending on the estrous cycle, and therefore, all rats were in the same estrous cycle phase (diestrus) to deter- mine the comparability of hormone levels at the end of the experiment. On days 25-28 of deionized water or irisin perfusion, all rats in the diestrus phase as determined by vaginal smears were sacrificed by decapitation at the light phase between 17:00 h and 19:00 h. After decapitation, trunk blood was immediately collected for a serum-based enzyme-linked immunosorbent assay (ELISA) and centri- fuged (4500 rpm; 4 oC; 5 min) to obtain serum samples, which were stored at -20 oC. In addition, the uterine horns and ovaries were immediately excised and cleaned of fat, and their wet weights were measured and expressed as mg/100 g body weight (BW). The final body weight of each animal was measured just before decapitation. The mid- portion of the uterine horns and all of the ovaries were fixed in a 10% formaldehyde solution for histopathological inves- tigations. The serum and reproductive organs used in this study belonged to the animals used in the previous study (18). Hormone measurements Commercial rat ELISA kits were purchased from Elabscience Biotechnology Inc. (Texas, USA) and Enzo Life Sciences (Switzerland). They were used to assess in duplicate the follicle-stimulating hormone (FSH; E-EL-R0391), luteinizing hormone (LH; ENZ-KIT107), 17β-estradiol (E2; ADI-900- 008), progesterone (ADI-900-011), testosterone (ADI-900- 065), insulin (E-EL-R3034), and irisin (FNDC5, E-EL-R1104) serum levels according to the manufacturer’s instructions Slovenian Veterinary Research 2024 | Vol 61 No 2 | 117 by using an ELISA microplate reader (Multiskan FC, Thermo Scientific, USA). Serum FSH, LH, E2, progesterone, testos- terone, and insulin levels were quantified in all groups. On the other hand, serum irisin levels were measured only in the control and obese groups to determine whether obe- sity-induced damage to the female reproductive system is related to irisin levels. Histopathological examinations For light microscopic evaluation, formalin-fixed uterus and ovary tissues were dehydrated through an increasing alco- hol series (70%, 80%, 96%, and 100%) and then embedded in paraffin wax. Afterward, sections with a thickness of 5 µm were cut from uterine and ovarian tissues and stained with hematoxylin and eosin (H&E) and/or Masson trichrome staining. All of the uterine and ovarian tissue sections were evaluated and photographed by a blinded examiner using a Leica DM500 light microscope (DFC295; Leica, Wetzlar, Germany). At 20x magnification, twenty random fields from a section of each ovary and uterus were examined and/or scored. The follicles (primordial, primary, secondary, and Graaf fol- licles) and corpus luteum in twenty random fields of each ovarian section were counted. Ovarian follicles were clas- sified according to the morphologic criteria as described by Artaş et al. (2018) (19). Histopathological examination of the uterine and ovarian tissue damage was carried out by a thorough qualitative histologic examination, and fibrosis pathology was semi-quantitatively scored (0, no fibrosis; 1, low fibrosis; 2, intermediate fibrosis; 3, severe fibrosis) (12, 20). TUNEL Assay The apoptosis in ovarian tissues was evaluated by the terminal deoxynucleotidyl transferase-mediated deoxy- uridine-biotin nick end labeling (TUNEL) method. For the detection of apoptotic cells, the ApopTagPlus Peroxidase in Situ Apoptosis Detection Kit (Chemicon, Lot: 3006560, USA) was used according to the manufacturer’s instruc- tions. In the evaluation of the TUNEL assay, the nuclei of healthy cells were blue, and the cells with stained brown nuclei were considered apoptotic cells. A total of 200 cells were counted in randomly selected areas under light mi- croscopy at 20x magnification. Accordingly, the apoptot- ic index (%) was calculated as the ratio of the number of TUNEL-positive cells to the total number of cells (21). Statistical analysis All data are presented as mean ± standard error of the mean (SEM) and were analyzed using the SPSS 22.0 soft- ware. Before analysis, the normality of all data was verified using the Shapiro-Wilk test. For multiple comparisons be- tween groups, a one-way analysis of variance (ANOVA) fol- lowed by Tukey’s post-hoc test was utilized. Comparisons between the control and obese groups were evaluated by an unpaired student t-test (irisin levels). p<0.05 was consid- ered statistically significant. Results Serum sex hormone levels Serum FSH, LH, LH/FSH ratio, E2, progesterone, and tes- tosterone levels in all experimental groups are shown in Fig. 1. When compared to the control group, serum FSH levels decreased significantly in the irisin group (p<0.05) although they remained the same in both the obese and obese+irisin groups (Fig. 1A). Serum LH levels were found to increase significantly in the obese and obese+irisin groups due to the high-fat diet (p<0.001), but no obvious changes were observed in the irisin group. Additionally, compared to the obese group, serum LH levels in the obese+irisin group significantly increased depending on subcutaneous ad- ministration of irisin (p<0.001) (Fig. 1B). The LH/FSH ratio was significantly increased depending on the decreased serum FSH levels and unchanged serum LH levels in the irisin group (p<0.01, Fig. 1C). As a result of increased se- rum LH concentration due to the high-fat diet, it was de- termined that LH/FSH ratios increased in both obese and obese+irisin groups (p<0.05 and p<0.001, respectively, Fig 1C). Moreover, compared with the obese group, the LH/ FSH ratio of rats in the obese+irisin group was significantly increased depending on irisin exposure (p<0.001, Fig. 1C). As seen in Fig. 1D, serum E2 levels were reduced in obese rats when compared to control rats (p<0.05). Serum pro- gesterone levels were significantly increased due to subcu- taneous irisin perfusion in the irisin and obese+irisin groups as compared to the control group (p<0.01). This increase in the obese+irisin group was also found to be significant compared to the obese group (p<0.01) (Fig. 1E). In compari- son with the control group, serum testosterone levels were substantially increased in the obese+irisin group (p<0.01). On the other hand, it was determined that serum testoster- one levels were not affected by subcutaneous irisin perfu- sion or high-fat diet exposure alone (Fig. 1F). Serum insulin and irisin levels Continuous administration of irisin, high-fat diet exposure, or both continuous administration of irisin and high-fat diet exposure in female rats did not cause a change in serum insulin levels compared with female rats treated with a ve- hicle (Fig. 2A). As shown in Fig. 2B, there was no significant difference in the serum irisin levels in female rats exposed to a high-fat diet in comparison to the vehicle-treated fe- male rats. 118 | Slovenian Veterinary Research 2024 | Vol 61 No 2 Reproductive organ weights Irisin perfusion had no significant effect on body weight in both lean and obese rats, as determined in our previ- ous study (18). As shown in Table 1, there was a significant reduction in the uterine wet weights normalized for body weights in both irisin and obese+irisin groups compared to the control group (p<0.05 and p<0.001, respectively). When compared to the control group, high-fat diet exposure sig- nificantly decreased reproductive organ weights in female rats (p<0.05 for ovarian wet weights normalized to body weights and p<0.01 for uterine wet weights normalized to body weights). Also, it was determined that subcutaneous irisin perfusion had a statistically insignificant effect on the Figure 1: Effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on serum reproductive hormones in female rats. A) Serum FSH levels. B) Serum LH levels. C) LH/FSH ratio. D) Serum 17β-estradiol levels. E) Serum progesterone levels. F) Serum testosterone levels. Data were expressed as mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001 (one-way ANOVA followed by the Tukey’s post-hoc test, n = 10 in each group). FSH: follicle- stimulating hormone, and LH: luteinizing hormone Slovenian Veterinary Research 2024 | Vol 61 No 2 | 119 decreased reproductive organ weights of high-fat diet-in- duced obese rats in the obese+irisin group (Table 1). Uterine histopathology As shown in Fig. 3, the uterine epithelium, uterine glands in the endometrium, and endometrial connective tissue fibers showed normal histological structure in the control, irisin, and obese+irisin groups. Unlike other groups, epithelial degeneration was determined to be more common in the uterine sections of high-fat diet-induced obese rats (Fig. 3). Ovarian histopathology Histological features of ovarian sections are shown in Fig. 4. Tissue sections from the ovary of vehicle-treated rats show common ovary histology with germinal epithelium consist- ing of single-layered cubic cells, numerous corpus luteum, Figure 2: Effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on A) serum insulin levels and B) serum FNDC5 levels in female rats. Data were expressed as mean ± SEM (one-way ANOVA followed by Tukey’s post-hoc test or Student’s t-test, n = 10 in each group) Table 1: Effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on reproductive organ weights in female rats. Data were expressed as mean ± SEM. *p<0.05, **p<0.01, and ***p<0.001 compared with the control group (one-way ANOVA followed by the Tukey’s post-hoc test, n = 10 in each group). BW: body weight Groups Normalized Ovarian Weight(mg/100 g BW) Normalized Uterine Weight (mg/100 g BW) Control 46.01 ± 2.39 165.21 ± 8.61 Irisin 48.47 ± 1.87 132.18 ± 8.38* Obese 36.25 ± 1.94* 118.89 ± 8.75** Obese+irisin 41.18 ± 2.09 113.22 ± 5.96*** Table 2: Effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on the number of ovarian follicles and the histopathological score of fibrosis in female rats. Data were expressed as mean ± SEM. *p<0.05 compared with the control group (one-way ANOVA followed by the Tukey’s post-hoc test, n = 6 in each group) Variable Control Irisin Obese Obese+irisin Primordial follicle 9 ± 1.22 8.6 ± 0.74 6.2 ± 1.06 8 ± 0.44 Primary follicle 10 ± 1 7.8 ± 1.77 6.6 ± 1.6 9.6 ± 0.97 Secondary follicle 9.6 ± 1.43 10.2 ± 1.15 8.6 ± 0.5 8.6 ± 0.67 Tertiary follicle 1.6 ± 0.4 1.6 ± 0.4 0.6 ± 0.24 1.6 ± 0.24 Corpus luteum 10.8 ± 1.31 12.2 ± 1.31 8.6 ± 1.32 9.4 ± 0.5 Fibrosis 0.33 ± 0.21 0.66 ± 0.33 1.83 ± 0.40* 1 ± 0.25 120 | Slovenian Veterinary Research 2024 | Vol 61 No 2 and ovarian follicles (primordial, primary, secondary, and tertiary follicles) during different stages of development in the cortex. Similar to the control group, many corpus luteum and ovarian follicles at different developmental stages were detected in the irisin, obese, and obese+irisin groups (Fig. 4 and Table 2). However, unlike the control group, extreme vascular dilatation and congestion in the ovarian cortical stroma and ovarian germinal epithelium degeneration were observed in the high-fat diet-induced obese rats (Fig. 4). Along with all these histopathological changes in the obese group, a significant increase in fibrosis was also found in the obese group compared to the control group (p<0.05, Table 2). On the other hand, when compared to the obese group, a decrease was observed in vascular congestion and germinal epithelial degeneration in the obese+irisin group depending on irisin exposure (Fig. 4). Apoptosis in the ovary Fig. 5A shows that TUNEL-positive granulosa cells were observed in the secondary and graff follicles, while apop- tosis was not detected in the primordial and primary fol- licles in all experimental groups. Accordingly, we found that the apoptotic index (%) was significantly higher in the irisin, obese, and obese+irisin groups compared with the con- trol group (control group: 1.71 ± 0.28%, irisin group: 5.42 ± 0.57%, obese group: 5.42 ± 0.61%, and obese+irisin group: 5.14 ± 0.59%; p<0.05 for irisin, obese, and obese+irisin groups, Fig. 5B). Discussion IIn this study, it was revealed for the first time that irisin hormone affects reproductive hormones and ovarian his- topathology in high-fat diet-induced obese female rats. Accordingly, it was observed that irisin exposure increased serum LH, LH/FSH ratio, and progesterone levels in obese female rats, and the histopathological changes in the ovari- an tissues of obese rats decreased with irisin exposure (i.e., vascular congestion and germinal epithelial degeneration). Figure 3: Represents the effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on uterine tissue. A) Control group. B) Irisin group. C) Obese group: epithelial degeneration (arrow). D) Obese+irisin group. Stained with hematoxylin and eosin (H&E), Bar: 200 µm Figure 4: Represents the effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on ovary histology. Obese group: vascular dilatation and congestion (arrowhead), fibrosis (star), and germinal epithelial degeneration (arrow). Stained with hematoxylin and eosin (H&E) and Masson trichrome staining, Bar: 200 µm. CL: corpus luteum; GE: germinal epithelium; PF: primordial follicle (in the control group, arrow); SF: secondary follicle Slovenian Veterinary Research 2024 | Vol 61 No 2 | 121 In addition, it was determined that endogenous irisin serum concentration did not change in the obese female rats, and exogenous irisin administration induced apoptosis in the lean rat ovary. It has been shown in the literature that irisin/FNDC5 is most likely involved in the reproductive system. In female mice, it has been determined that there are various disturbances in the components of the female reproductive system due to the deletion of FNDC5. In some studies conducted in rat models, it was suggested that irisin had a beneficial effect on uterine receptivity or caused disorders in the reproduc- tive system (9, 12, 22). Recent studies have shown that iri- sin has a potentially positive role against obesity-induced reproductive dysfunctions in male rats (15, 16). The ‘obesity epidemic’ in many countries is a serious threat to public health, and reproduction is one of the major health hazards induced by obesity (23). It has been reported that obese women may have three times more reproductive disorders due to disruptions in the HPO axis, resulting in anovulatory cycles, irregular menstruation, and infertility, which are associated with PCOS (24, 25). In previous stud- ies, it has been observed that anterior pituitary gonadotro- pins (FSH and LH) levels, which are critical regulators of ovarian function and female fertility, can increase in obese female rats. For example, Akamine et al. (2010) found that serum FSH levels did not change but serum LH levels were increased in obese female rats subjected to 120 days of high-fat diet treatment (26). Similar results on the secretion of gonadotropins were also found in female rats exposed to a 16-week high-fat diet in our study. Taken together, our data strongly suggest that obesity increases serum LH levels in female rats and thus causes an adverse effect on the gonadal axis of female rats. Irisin either stimulates the expression of FSH and LH or inhibits the secretion of FSH or LH by competing with the gonadotropin-releasing hormone. These dual effects of iri- sin occur simultaneously and interact with each other, re- sulting in variations in circulating hormone levels when one activity is dominant (10, 12, 27). In the current study, it was determined that irisin decreased FSH levels in lean female rats by acting at the central inhibitory effect. In our previ- ous study using female rats, it was determined that irisin administration changed serum levels of gonadotropins, re- sulting in reduced serum FSH levels and elevated serum LH levels (12). Similarly, in the present study, irisin was found to decrease serum FSH levels. However, the unchanged LH levels in the present study are thought to may be related to the application time and method of the irisin (i.e., 4 weeks versus 10 weeks and subcutaneous versus intraperitoneal administration). On the other hand, when the effects of irisin on the secre- tion of gonadotropins, which changed due to obesity, were examined, it was shown that FSH levels did not change but LH levels and the LH/FSH ratio increased by irisin exposure in high-fat diet-induced obese female rats in this study. These results are supported by a study of the exogenous administration of irisin in obese female mice induced by a high-fat diet (13). Another possible condition in which LH levels and the LH/FSH ratio can increase in obese women is known to be PCOS (28, 29). Because of this, depending on the results of this study, it is speculated that irisin may have a triggering effect on the formation of PCOS in obesity. Figure 5: Effects of subcutaneous irisin perfusion and/or high-fat diet-induced obesity on the apoptosis of ovarian granulosa cells in rats. A) Representative images of TUNEL staining show apoptotic granulosa cells in the control, irisin, obese, and obese+irisin groups (arrow), Bar: 100 µm. B) The apoptotic index of each group. Data were expressed as mean ± SEM. *p<0.05, compared with the control group (one-way ANOVA followed by the Tukey’s post-hoc test, n = 6 in each group) 122 | Slovenian Veterinary Research 2024 | Vol 61 No 2 Serum leptin levels are high in obesity, and an increase in leptin levels impairs ovulation and causes infertility. Moreover, it has been reported that serum leptin levels are negatively correlated with serum estradiol levels in obese female rats, and increased serum leptin levels may reduce estradiol synthesis by direct action on the ovary (30). Our previous study demonstrated that serum leptin levels were increased in obese female rats, and irisin administration de- creased the elevated serum leptin levels in these rats (18). Therefore, in the present study, although serum leptin lev- els were not measured, it is speculated that the possible obesity-related increase in serum leptin levels may affect the ovary in obese rats and cause a decrease in E2 synthe- sis. A previous study reported that serum estradiol concen- tration decreased and serum progesterone concentration did not change in cafeteria diet-induced obese female rats (24). Consistently, similar results were also found in obese female rats induced by a high-fat diet in the present study. On the other hand, it was determined that subcutaneous iri- sin perfusion increased serum progesterone levels in both lean and obese rats. In addition to the increase in progester- one levels, it was determined that there was also an increase in serum testosterone levels in the obese+irisin group. It is already known that LH is the major factor in ovarian ste- roidogenesis and triggers steroid production, including estradiol, progesterone, and testosterone (31). Therefore, considering the increasing effect of irisin on the LH levels in obese female rats as stated above, our data suggest that irisin-induced LH increment, independent of insulin, may increase steroidogenic capacity in the obese+irisin group. In our study, serum insulin levels were unchanged in female rats fed a high-fat diet for 16 weeks compared to vehicle- treated female rats. Parallel to our study, Lu et al. (2016) reported that serum insulin levels did not change in Wistar male rats fed a high-fat diet for 24 weeks (32). Insulin plays an important role in the increase in ovarian steroidogen- esis and the process of follicular development (26, 33). Especially, it has been revealed that there is an increase in theca-cell steroidogenesis as a result of the synergistic in- teraction of LH and insulin (34). In the present study, it was assumed that the changes in ovarian steroidogenesis in all experimental groups (i.e., increased progesterone levels in the irisin group, decreased E2 levels in the obese group, and increased progesterone and testosterone levels in the obese+irisin group) may have occurred independently of in- sulin. Furthermore, it is the first time, to our knowledge, that obesity-related reproductive dysfunction in a female rat model was found to be independent of serum irisin levels, considering the unchanged serum irisin levels in obesity in the current study. Different exercise intensities, known to elicit an irisin re- sponse, induce different effects on the reproductive sys- tem in females. For example, it was observed that there is no change in serum FSH, LH, or estradiol levels as a result of short-time exercise or aerobic exercise in young women (35, 36), while there is a decline in the plasma lev- els of FSH, LH, estradiol, and progesterone in women who engage in regular high intensity exercise (37). Depending on the results of the present study and our previous study (12), it was determined that irisin administration at different durations and ways has different effects on reproductive hormones as well as ovarian histology and reproductive or- gan weights in female rats. In light of the above-mentioned information, our data from our studies suggested that the irisin hormone released into circulation due to exercise may play a pivotal role in the relationship between exercise vari- ables and the reproductive system. Gaspar et al. (2016) and Benevides et al. (2019) reported that both the weights of the uterus and ovarian tissues were reduced due to obesity in rats (38, 39). Hence, the marked reduction in reproductive organ weights seen in high-fat diet-induced female rats in the present study is consistent with previous literature and provides evidence of the direct adverse effects of obesity on reproductive organs. Besides, for the first time in the current study, it was revealed that irisin did not affect the weight of reproductive organs in obese female rats. In rodent models, it is also known that obesity causes dif- ferent histopathological changes in ovarian tissue as well as negative effects on the weight of reproductive organs. Akamine et al. (2010) showed that rats fed a high-fat diet for 180 days had significantly abnormal ovarian morphology (26). In addition, Atteia et al. (2020) revealed that there are several histopathological features such as stromal edema, congestion, and a high degree of fibrosis in the ovarian cor- tices of the obese group (40). Our findings agree with the aforementioned studies: obese rats showed histopatholog- ical changes in the ovaries like extreme vascular dilatation and congestion in the ovarian cortical stroma, ovarian ger- minal epithelium degeneration, and fibrosis. With regards to the ovarian follicle reserve, obesity did not affect ovar- ian follicle development in the current study. Benevides et al. (2019) and Hussain et al. (2016) found similar results: obesity did not promote a significant change in the count of ovarian follicles or corpora lutea (39, 41). In particular, similar follicular development in all experimental groups is an expected result due to the unchanged serum levels of insulin involved in follicular development in this study. It is noteworthy that irisin exposure reduced the changes in ovarian pathology (i.e., vascular congestion and germinal epithelial degeneration) in obese rats, while it did not cause any effect on the ovarian tissues of lean rats in the present study. Accordingly, in this study, it could be speculated that irisin exhibited a curative effect on obesity-induced patho- logical changes in the ovarian tissues. Taken together, our data suggested that irisin can act directly on the ovaries in obese rats, regardless of the HPO axis. In the present study, the effects of irisin on ovarian function in rats fed a high-fat diet were also assessed by apoptosis Slovenian Veterinary Research 2024 | Vol 61 No 2 | 123 in addition to hormone assays and histopathological ex- aminations. Our data showed that apoptosis in the ovary was increased, which contributes to ovarian function fail- ure, by irisin exposure alone or obesity alone in rats, but the administration of irisin did not affect granulosa cell apop- tosis in obese rats. This effect of obesity on apoptosis of ovarian follicles shown in this study is supported by recent studies demonstrating that diet-induced obesity is known to increase apoptotic ovarian follicles in rodents (13, 24, 42). Irisin has also been demonstrated to increase apopto- sis in ovarian cancer cells and breast cancer cells (43, 44). Similarly, to our knowledge, our findings have shown for the first time that irisin induces apoptosis in the ovarian tissue of lean rats. In conclusion, we have demonstrated that re- productive impairments in irisin-exposure rats in the pres- ent study may be associated with decreased serum FSH levels, increased serum progesterone levels, decreased uterine weights, and increased granulosa cell apoptosis. In conclusion, our study revealed that irisin increased se- rum LH levels in high-fat diet-induced obese female rats, and irisin-induced LH increment may cause an increase in ovarian steroidogenesis in these obese rats. In addition, it was shown that irisin exposure could alleviate ovarian tis- sue damage in high-fat diet-induced obese rats. Based on these results, we suggest that the irisin hormone may modulate the HPO axis of obese rats at both central neuro- endocrine and gonadal levels. Conclusions It was determined that λ-cyhalothrin has a toxic effect on D. polymorpha and this toxic effect increases depending on the temperature. In our study, it was concluded that the D. polymorpha model organism and some of its biochemical parameters (AChE, CAT, SOD, GSH and MDA) are suitable biomarkers for revealing the effect of temperature variable on toxic response. It has also been shown that biochemi- cal parameters such as SOD, CAT, AChE activities and GSH, TBARS levels used are suitable biomarkers for the evalua- tion of the toxic effects of λ-cyhalothrin. Acknowledgements This work was supported by The Scientific and Technological Research Council of Turkey (TUBITAK, Project No: 118S519). Declaration of competing interest: The authors declare no competing interests. Author contributions: SC and HK conceived and designed the project. NUE, AY, and FB performed all animal experi- ments. NUE performed all hormone measurements and wrote the manuscript. NKT carried out all histological studies and the TUNEL assay. SC and MO analyzed and interpreted the data. The final version of the manuscript was read and approved by all authors. References 1. Goldsammler M, Merhi Z, Buyuk, E. Role of hormonal and inflammato- ry alterations in obesity-related reproductive dysfunction at the level of the hypothalamic-pituitary-ovarian axis. Reprod Biol Endocrinol 2018; 16(1): 45. doi: 10.1186/s12958-018-0366-6 2. Talmor A, Dunphy B. Female obesity and infertility. Best Pract Res Clin Obstet Gynaecol 2015; 29(4): 498–506. doi: 10.1016/j. bpobgyn.2014.10.014 3. Snider AP, Wood JR. Obesity induces ovarian inflammation and reduc- es oocyte quality. Reproduction 2019; 158(3): R79–R90. doi: 10.1530/ REP-18-0583 4. Ferreira RC, Halpern G, Figueira Rde C, et al. Physical activity, obesi- ty and eating habits can influence assisted reproduction outcomes. Women’s Health (Lond) 2010; 6(4): 517–24. doi: 10.2217/whe.10.40 5. Moran LJ, Dodd J, Nisenblat V, Norman RJ. Obesity and reproductive dysfunction in women. Endocrinol Metab Clin North Am 2011; 40(4): 895–906. doi: 10.1016/j.ecl.2011.08.006 6. Wu C, Lin F, Qiu S, Jiang Z. The characterization of obese polycystic ovary syndrome rat model suitable for exercise intervention. PloS One 2014; 9(6): e99155. doi: 10.1371/journal.pone.0099155 7. Boström P, Wu J, Jedrychowski MP, et al. A PGC1-α-dependent myo- kine that drives brown-fat-like development of white fat and thermo- genesis. Nature 2012; 481(7382): 463–8. doi: 10.1038/nature10777 8. Lee P, Linderman JD, Smith S, et al. Irisin and FGF21 are cold-induced endocrine activators of brown fat function in humans. Cell Metab 2014; 19(2): 302–9. doi: 10.1016/j.cmet.2013.12.017 9. Luo Y, Qiao X, Ma Y, Deng H, Xu CC, Xu L. Irisin deletion induces a de- crease in growth and fertility in mice. Reprod Biol Endocrinol 2021; 19(1): 22.doi: 10.1186/s12958-021-00702-7 10. Luo Y, Qiao X, Xu L, Huang G. Irisin: circulating levels in serum and its relation to gonadal axis. Endocrine 2022; 75(3): 663–71. doi: 10.1007/ s12020-022-02981-5 11. Poretsky L, Islam J, Avtanski D, et al. Reproductive effects of irisin: initial in vitro studies. Reprod Biol 2017; 17(3): 285–8. doi: 10.1016/j. repbio.2017.05.011 12. Ulker N, Yardimci A, Tektemur NK, et al. Irisin may have a role in pu- bertal development and regulation of reproductive function in rats. Reproduction 2020; 160(2): 281–92. doi: 10.1530/REP-20-0072 13. Bastu E, Zeybek U, Gurevin EG, et al. Effects of irisin and exercise on metabolic parameters and reproductive hormone levels in high-fat diet-induced obese female mice. Reprod Sci 2018; 25(2): 281–91. doi: 10.1177/1933719117711264 14. Canpolat S, Ulker N, Yardimci A, et al. Irisin ameliorates male sexual dys- function in paroxetine-treated male rats. Psychoneuroendocrinology 2022; 136: 105597. doi: 10.1016/j.psyneuen.2021.105597 15. Ibrahim RH, El-Malkey NF. Role of irisin administration in modulating testicular function in adult obese albino rats. Med J Cairo Univ 2018; 86(8): 4647–55. 16. Yardimci A, Ulker N, Bulmus O, et al. Irisin improves high-fat diet-in- duced sexual dysfunction in obese male rats. Neuroendocrinology 2022; 112(11): 1087. doi: 10.1159/000523689 124 | Slovenian Veterinary Research 2024 | Vol 61 No 2 17. Bernardis LL, Patterson BD. Correlation between'Lee index'and car- cass fat content in weanling and adult female rats with hypothalamic lesions. J Endocrinol 1968; 40(4): 527–8. doi: 10.1677/joe.0.0400527 18. Ozcan S, Ulker N, Bulmus O, Yardimci A, Ozcan M, Canpolat S. The modulatory effects of irisin on asprosin, leptin, glucose levels and lipid profile in healthy and obese male and female rats. Arch Physiol Biochem 2020; 128(3): 724–31. doi: 10.1080/13813455.2020.1722706 19. Artaş G, Pala Ş, Kuloğlu T, Atilgan R, Yavuzkir Ş, Akyol A. The effects of sclerotherapy with 5% trichloroacetic acid on the cyst diameter and ovarian tissue in the rat ovarian cyst model. J Obstet Gynaecol 2018; 38(5): 686–92. doi: 10.1080/01443615.2017.1399991 20. Mazaud S, Guigon CJ, Lozach A, et al. Establishment of the reproduc- tive function and transient fertility of female rats lacking primordial fol- licle stock after fetal γ-irradiation. Endocrinology 2002; 143(12): 4775– 87. doi: 10.1210/en.2002-220464 21. Erdem Guzel E, Kaya Tektemur N, Tektemur A, Etem Önalan E. Carbamazepine-induced renal toxicity may be associated with oxida- tive stress and apoptosis in male rat. Drug Chem Toxicol 2023; 46(1): 136–43. doi: 10.1080/01480545.2021.2014859 22. Daudon M, Bigot Y, Dupont J, Price CA. Irisin and the fibronectin type III domain-containing family: structure, signaling and role in fe- male reproduction. Reproduction 2022; 164(1): R1–R9. doi: 10.1530/ REP-22-0037 23. Ramlau-Hansen CH, Thulstrup AM, Nohr EA, Bonde JP, Sørensen TIA, Olsen J. Subfecundity in overweight and obese couples. Hum Reprod 2007; 22(6): 1634–7. doi: 10.1093/humrep/dem035 24. Bazzano MV, Torelli C, Pustovrh MC, Paz DA, Elia EM. Obesity induced by cafeteria diet disrupts fertility in the rat by affecting multiple ovar- ian targets. Reprod Biomed Online 2015; 31(5): 655–67. doi: 10.1016/j. rbmo.2015.08.004 25. Brewer CJ, Balen AH. The adverse effects of obesity on conception and implantation. Reproduction 2010; 140(3): 347–64. doi: 10.1530/ REP-09-0568 26. Akamine EH, Marçal AC, Camporez JP, et al. Obesity induced by high- fat diet promotes insulin resistance in the ovary. J Endocrinol 2010; 206(1): 65–74. doi: 10.1677/JOE-09-0461 27. Wahab F, Khan IU, Polo IR, et al. Irisin in the primate hypothalamus and its effect on GnRH in vitro. J Endocrinol 2019; 241(3): 175–87. doi: 10.1530/JOE-18-0574 28. Messinis IE, Messini CI, Anifandis G, Dafopoulos K. Polycystic ovaries and obesity. Best Pract Res Clin Obstet Gynaecol 2015; 29(4): 479–88. doi: 10.1016/j.bpobgyn.2014.11.001 29. Yang Y, Yang L, Qi C, et al. Cryptotanshinone alleviates polycystic ovary syndrome in rats by regulating the HMGB1/TLR4/NF-κB sig- naling pathway. Mol Med Rep 2020; 22(5): 3851–61. doi: 10.3892/ mmr.2020.11469 30. Balasubramanian P, Jagannathan L, Mahaley RE, et al. High fat diet affects reproductive functions in female diet‐induced obese and dietary resistant rats. J Neuroendocrinol 2012; 24(5): 748–55. doi: 10.1111/j.1365-2826.2011.02276.x 31. Kannan S, Srinivasan D, Raghupathy PB, Bhaskaran RS. Association between duration of obesity and severity of ovarian dysfunction in rat-cafeteria diet approach. J Nutr Biochem 2019; 71: 132–43. doi: 10.1016/j.jnutbio.2019.05.012 32. Lu Y, Li H, Shen SW, et al. Swimming exercise increases serum irisin level and reduces body fat mass in high-fat-diet fed Wistar rats. Lipids Health Dis 2016; 15: 93. doi: 10.1186/s12944-016-0263-y 33. Sliwowska JH, Fergani C, Gawałek M, Skowronska B, Fichna P, Lehman MN. Insulin: its role in the central control of reproduction. Physiol Behav 2014; 133: 197–206. doi: 10.1016/j.physbeh.2014.05.021 34. Zhang G, Garmey JC, Veldhuis JD. Interactive stimulation by luteiniz- ing hormone and insulin of the steroidogenic acute regulatory (StAR) protein and 17α-hydroxylase/17, 20-lyase (CYP17) genes in porcine theca cells. Endocrinology 2000; 141(8): 2735–42. doi: 10.1210/ endo.141.8.7595 35. Bonen A, Ling WY, Maclntyre KP, Neil R, McGrail JC, Belcastro AN. Effects of exercise on the serum concentrations of FSH, LH, proges- terone, and estradiol. Eur J Appl Physiol Occup Physiol 1979; 42(1): 15–23. doi: 10.1007/BF00421100 36. Smith AJ, Phipps WR, Thomas W, Schmitz KH, Kurzer MS. The effects of aerobic exercise on estrogen metabolism in healthy premenopausal women. Cancer Epidemiol Biomarkers Prev 2013; 22(5): 756–64. doi: 10.1158/1055-9965.EPI-12-1325 37. Arena B, Maffulli N, Maffulli F, Morleo MA. Reproductive hormones and menstrual changes with exercise in female athletes. Sports Med 1995; 19(4): 278–87. doi: 10.2165/00007256-199519040-00005 38. Gaspar RS, Benevides RO, Fontelles JL, et al. Reproductive alterations in hyperinsulinemic but normoandrogenic MSG obese female rats. J Endocrinol 2016; 229(2): 61–72. doi: 10.1530/JOE-15-0453 39. Benevides ROA, Vale CC, Fontelles JLL, et al. Syzygium cumini (L.) Skeels improves metabolic and ovarian parameters in female obese rats with malfunctioning hypothalamus-pituitary-gonadal axis. J Ovarian Res 2019; 12(1): 13. doi: 10.1186/s13048-019-0490-8 40. Atteia HH, Alzahrani S, El-Sherbeeny NA, et al. Evening primrose oil ameliorates hyperleptinemia and reproductive hormone disturbanc- es in obese female rats: Impact on estrus cyclicity. Front Endocrinol (Lausanne) 2020; 10: 942. doi: 10.3389/fendo.2019.00942 41. Hussain MA, Abogresha NM, Hassan R, Tamany DA, Lotfy M. Effect of feeding a high-fat diet independently of caloric intake on reproductive function in diet-induced obese female rats. Arch Med Sci 2016; 12(4): 906–14. doi: 10.5114/aoms.2016.59790 42. Jungheim ES, Schoeller EL, Marquard KL, Louden ED, Schaffer JE, Moley KH. Diet-induced obesity model: abnormal oocytes and persis- tent growth abnormalities in the offspring. Endocrinology 2010; 151(8): 4039–46. doi: 10.1210/en.2010-0098 43. Gannon NP, Vaughan RA, Garcia-Smith R, Bisoffi M, Trujillo KA. Effects of the exercise‐inducible myokine irisin on malignant and non‐malig- nant breast epithelial cell behavior in vitro. Int J Cancer 2015; 136(4): E197–202. doi: 10.1002/ijc.29142 44. Zarei MA, Hosseini ES, Kashani HH, Ahmad E, Nikzad H. Effects of the exercise-inducible myokine irisin on proliferation and malignant prop- erties of ovarian cancer cells through the HIF-1 α signaling pathway. Sci Rep 2023; 13(1): 170. doi: 10.1038/s41598-022-26700-2 Slovenian Veterinary Research 2024 | Vol 61 No 2 | 125 Učinki irisina na reproduktivni sistem debelih samic podgan, povzročeni s prehrano z visoko vsebnostjo maščob N. U. Ertugrul, A. Yardimci, N. K. Tektemur, F. Bulut, M. Ozcan, H. Kelestimur, S. Canpolat Izvleček: Debelost je vse pogostejša po vsem svetu in povzroča različne zdravstvene težave, vključno z motnjami re- produkcije. Čeprav hormon irisin, ki se izloča med vadbo, lahko vpliva na hipotalamično-hipofizno-gonadno os in re- produktivno funkcijo, njegov vpliv na z debelostjo povezane poškodbe ženskega reproduktivnega sistema ni povsem znan. Zato je bil namen te študije raziskati morebitne učinke irisina na reproduktivne hormone in reproduktivne organe pri samicah podgan z debelostjo, povzročeno s prehrano z visoko vsebnostjo maščob. Štirideset samic podgan smo razdel- ili v štiri skupine: kontrola, irisin, debelost, debelost+irisin (n=10 v vsaki skupini). Po 12 tednih simulacije modela debelosti, povzročene s prehrano z visoko vsebnostjo maščob (60 % kcal maščobe), smo v skupinah debelost in debelostjo+irisin podganam podkožno dajali irisin (100 ng/kg/dan prek mini-osmotskih črpalk približno 28 dni). Podkožna aplikacija irisina je povečala serumski luteinizirajoči hormon (LH), razmerje med LH in folikle stimulirajočim hormonom (FSH) (LH/FSH) in raven progesterona, hkrati pa zmanjšala histopatološke poškodbe v jajčnikih debelih podgan. Vendar pa so bile kon- centracije endogenega irisina v serumu vitkih in debelih podgan z reproduktivnimi motnjami podobne. Rezultati kažejo, da bi irisin lahko vplival na reproduktivno os debelih podgan. K temu učinku bi lahko prispevala povečanje serumske koncentracije LH, kar sproža steroidogenezo jajčnikov, ter zmanjšanje histopatoloških sprememb tkiva jajčnikov. Ključne besede: : irisin; debelost; hormoni; jajčnik; apoptoza; reprodukcija pri samicah