Prenatal kisspeptin antagonist exposure prevents polycystic ovary syndrome development in prenatally-androgenized rats in adulthood: An experimental study

Abstract Background Increased levels of kisspeptin are associated with hypothalamus-pituitary-ovary axis dysfunction. It may lead to the development of polycystic ovary syndrome (PCOS). Objective We aimed to investigate the effect of prenatal kisspeptin antagonist exposure on the development of PCOS in prenatally androgenized rats in adulthood. Materials and Methods In this experimental study, pregnant rats were injected with free testosterone (T, 5 mg/day) or T+P271 (kisspeptin antagonist) on the 20 th day of the pregnancy period (n = 5 in each group), while rats in the control group received solvent. Female offspring were examined in terms of anogenital distance (AGD), anovaginal distance (AVD), vaginal opening, serum total testosterone (TT) levels, ovarian follicles, and the regularity of estrous cycles in adulthood. AGD and AVD were measured using a vernier caliper. TT levels were measured using the enzyme-linked immunosorbent assay method. Ovaries were fixed in 10% formalin, tissue processing was done by a standard protocol, and then ovaries embedded in paraffin. 5 μm-thickness ovarian sections mounted on a glass slide, deparaffinized, and stained using Harris's Hematoxylin and Eosin Y. Results AGD, AVD (p < 0.001), TT levels (p = 0.02), and the numbers of preantral and antral follicles (p < 0.001) in the ovaries were significantly decreased in prenatally T-P271-exposed rats compared to prenatally T-exposed rats. The age of vaginal opening was early in T-P271-exposed rats compared to prenatally T-exposed rats (p < 0.001). The number of corpora lutea was significantly increased in T-P271-exposed rats (p < 0.001). No cystic follicles were observed in the ovaries of prenatally T-P271-exposed rats. Prenatally T-P271-exposed rats had regular estrous cycles compared to prenatally T-exposed rats. Conclusion Prenatal exposure to kisspeptin antagonist can prevent PCOS development in prenatally androgenized rats in adulthood.


Introduction
Polycystic ovary syndrome (PCOS), one of the most common endocrine disorders, affects 5-20% of reproductive-age women worldwide (1,2). PCOS is associated with reproductive, metabolic, and public health disorders such as luteinizing hormone (LH) hypersecretion, hyperandrogenism (clinical and/or biochemical), ovarian cysts, oligo/anovulation, obesity, insulin resistance, and physical and emotional morbidities in those affected (3,4). can be associated with increased activity of gonadotropin-releasing hormone (GnRH) neurons and LH hypersecretion; leading to androgen excess as one of the main endocrine abnormalities in PCOS subjects (7,8).
Kisspeptin, a recently discovered neuropeptide acts upstream of GnRH neurons and is the key regulator of the hypothalamic-pituitary-ovary (HPO) axis. The kisspeptin neural system plays an important role in the maturation and function of the reproductive system (9,10).
A previous study reported that LH levels were directly related to kisspeptin levels (11).
Women with PCOS have also had high levels of kisspeptin (12). PCOS can be described as the disturbed organization of the hypothalamic kisspeptin system, possibly due to exposure to abnormal levels of sex steroid hormones, such as androgens, during early life (12,13). Previous studies have shown that kisspeptin mRNA levels and the number of kisspeptin neurons in the arcuate nucleus region of the hypothalamus were elevated in animal models of PCOS (14,15). kisspeptin is considered as one of the activator factors for GnRH neurons, therefore the increasing in the number of kisspeptin-producing cells or the levels of kisspeptin mRNA could be a potential cause of increased GnRH neuron activity and LH secretion leading to PCOS development (16,17). It has been shown that kisspeptin knockout in healthy mice causes LH pulse disruption, which is followed by irregular estrous cycles and defects in ovarian folliculogenesis (18).
Despite the prevalence of PCOS and its effects on health, there is no definitive curative option for this syndrome, and all the available treatment modalities relieve symptoms (19,20 (21). Female offspring of androgenized rats (group 2) were considered as the prenatally-androgenized rat model of PCOS (20).
After weaning, female offspring of 4 groups, including prenatally T-P271-exposed rats (group 1'), non-exposed PCOS rats (group 2'), prenatally P271-exposed control rats (group 3'), and nonexposed control rats (group 4') were kept in groups of 4 per cage with free access to food and water. All female offspring were assessed in terms of body weight, morphological parameters, serum TT levels, ovarian tissue, and the regularity of estrous cycles in later life (in adulthood). The selection process of rats is presented in figure 1.

Determination of body weights (BWs)
The BWs of the female offspring of all study groups (n = 16 in each group) were measured

Evaluation of estrous cycle
Microscopic observations of vaginal smears were performed to assess our study rats' regularity or irregularity of the estrous cycles.

Blood collection
At the estrus phase of the sexual cycle, adult female offspring (85-95 days of age, n = 8 in each group) were anesthetized with i.p. injection of a mixture of 50 mg/kg of 10% ketamine and 10 mg/kg of 2% xylazine (Alfasan, Woerden, Holland). After deep anesthesia, blood samples were taken from the heart. Blood samples were centrifuged at 6000 g for 5 min at 4°C.
The sera were stored at -80°C for subsequent measurement of TT levels (20).
The sensitivity of the kit was 0.02-15.0 ng/ml. Intra-assay coefficients of variation for TT were < 10%.

Ovarian histological examination
After the blood collection, rats were killed by heart incision, and the ovaries were immediately removed from the body cavity and prepared for histological studies using the method described

BWs
A comparison of the BWs of prenatally T-P271exposed and nonexposed PCOS rats compared to controls at different ages is presented in figure   2. No significant differences were observed in the BWs of female offspring of all 4 study groups at birth, 15, and 30 days of age (p > 0.05). While BWs in group 1' were significantly lower than compared to group 2' at 45, 60, and 75 days of age (p < 0.05), approximately reaching these values that were observed in control rats (groups 3' and 4') ( Figure 2). There were statistically significant lower than in AGD and AVD in group 1' compared to group 2'

VO
Timing of VO, as a marker of puberty onset, was determined for all rats during 30-45 days of age.
Based on our records, the age of VO was earlier in T-P271-exposed rats compared to prenatally T-

Observation of vaginal smears daily for 15
consecutive days demonstrated that rats in group 1', had regular estrous cycles similar to those observed in control rats (groups 3' and 4'), while estrous cycles were irregular in rats of group 2' .

Discussion
In the present study, we found that prenatallyandrogenized rats exposed to a single dose of consequently, androgen excess that is one of the main endocrine abnormalities in PCOS subjects (20,21). The underlying mechanisms that link the hyperandrogenic state with disturbances in GnRH pulse generator activity in the hypothalamus are incompletely understood; however, several assumed pathways have been reported so far.
In a study conducted on female sheep exposed to androgens during the critical periods of development, a reduction in synaptic contact with GnRH neurons was observed (26); indicating changes in synaptic connectivity following exposure to androgens; leading to alterations in GnRH pulsation. Another study suggested that androgen receptor activation may cause changes in the movement of GABA-releasing neurons to GnRH neurons (27). Moreover, exposure to the supraphysiologic doses of androgens during fetal life may lead to the desensitization of GnRH neurons to the negative feedback of sex steroids (28). Reduced negative feedback of sex steroids has been reported in prenatally androgenexposed female monkeys (29) and in women with PCOS (30). It may lead to increased pulsatile LH secretion and, subsequently, androgen excess.
Furthermore, it was suggested that prenatal exposure to androgens may affect gonadotropic sensitivity to the GnRH stimulation (31). In the present study, PCOS rats showed an irregular estrous cycle, a finding in agreement with previous studies (20,34). This could be due to the irregularities and changes that occurred in the hypothalamic-pituitary-gonadal axis following prenatal exposure to androgens (35). AGD is an anthropometric biomarker of the androgenic environment during the development of the reproductive system in fetal life and reveals reproductive health (36,37). In the present study, AGD and AVD were increased in the prenatallyandrogenized rat model of PCOS, indicating a male-like morphology, as reported in the previous study (20). This may be due to the presence and increase of androgen receptors before birth and their sensitivity to exogenous androgen before the final development of the reproductive tract.
In the present study, in agreement with previous studies, a significant increase in body weight of the rat model of PCOS compared to other groups was observed (20,38). The relationship between obesity and PCOS has previously been reported (39). This increase may be due to increased insulin resistance in the PCOS group, which is due to decreased insulin binding to its receptor or defective in receptor autophosphorylation due to insulin receptor mutation (40).
Kisspeptin, a hypothalamic peptide encoded by the kiss1 gene (41) as a regulator of the HPO axis, plays an important role in the onset of puberty and the maintenance of reproductive function (9,15). It has been reported that kisspeptin through binding to its receptor controls GnRH secretion. Therefore, kisspeptin can involve in the pathophysiology of the HPO axis. A previous study proposed that exposure to elevated levels of androgens during early life can negatively affect the hypothalamic-kisspeptin system (13).
Subsequently, it may lead to the development of PCOS. Studies in animals and humans have reported increased hypothalamic expression of kisspeptin and GnRH in PCOS conditions (12,13,42).
An experimental study indicated that kisspeptin antagonist decreases LH pulse frequency and amplitude (43). As a result, kisspeptin antagonist through deceasing in the activity of GnRH neurons may improve GnRH/LH pulse frequency in PCOS subjects (12,21). In line with this evidence, our study results revealed that prenatal exposure to kisspeptin antagonist during the development of the HPO axis can prevent the appearance of PCOS phenotype (irregular sexual cycles, androgen excess, ovulation dysfunction, and ovarian cysts) in adulthood, despite exposure to androgens during fetal life. In our previous study conducted on prenatally-androgenized rats, decreases in GnRH mRNA expression, levels of sex steroid hormone, and gonadotropins were observed in prenatally-kisspeptin antagonistexposed rats. Additionally, in agreement with a previous study, in the present study, kisspeptinantagonist-treated rats showed regular estrous cycles, probably due to the inhibition of increased gonadotropin secretion and the inhibition of neuron GnRH activity following exposure to kisspeptin antagonist (21).

Conclusion
Prenatal exposure to kisspeptin antagonist can prevent PCOS development in adult life, despite the exposure to androgens during fetal life.
However, further studies are needed to confirm and expand our findings.