Differences in the functioning of the hypothalamic-pituitarygonadal axis of regulation in male rats at one (liver) site and two (liver and lungs) sites of metastasis of sarcoma 45 in the experiment

Oleg I. Kit, Irina V. Kaplieva, Elena M. Frantsiyants, Lidia K. Trep-itaki, Valeriya A. Bandovkina, Yulia A. Pogorelova, Irina A. Goro-shinskaya, Ekaterina I. Surikova, Irina V. Neskubina, Galina V. Zhukova, Alla I. Shikhlyarova, Marina A. Engibaryan, Tatiana V. Ausheva, Viktoria L. Volkova, Marina A. Gusareva, Polina S. Kachesova. Differences in the functioning of the hypothalamic-pituitary-gonadal axis of regulation in male rats at one (liver) site and two (liver and lungs) sites of metastasis of sarcoma 45 in the experiment. Cardiometry; Issue No. 27; May 2023; p. 5462; DOI: 10.18137/cardiometry.2023.27.5462; Available from:

The hypothalamic-pituitary-gonadal axis, along with the hypothalamic-pituitary-adrenal and hypothalamic-pituitary-thyroid axes, is one of the three main regulatory hormonal systems of the body. It has a central regulatory link, which includes the hypothalamus and the pituitary gland, a peripheral regulatory link, consisting of the gonads, and an effector link, represented by tissues, which are furnished with receptors for sex steroids or peptides and where ectopic synthesis of sex hormones occurs [1, 2 ]. Gonadotropin-releasing hormone (GnRH) is a decapeptide secreted by the hypothalamic neurons of the arcuate nucleus. It is transported through the axoplasm of nerve endings and flows through the pituitary portal veins to the anterior pituitary gland, where it is supplied by blood to the pituitary cells that secrete luteinizing (LH) and follicle-stimulating (FSH) hormones. These latter, by regulating the level of sex hormones, make their influence on the formation of gametes and the endocrine system of the gonads [3,4]. In turn, circulating levels of testosterone, estradiol, and some of their metabolites modulate the pulse secretion of GnRH by the hypothalamus directly into the pituitary portal vein system, despite the regulatory effects of GnRH extending to the other organs of HPGA [5].

FSH and LH are tropic pituitary hormones encoded by the same single copying gene [6]. Gonadotropins can exert a carcinogenic effect through a process that has been termed “enhanced hormonal stimulation” of target tissues. It is possible that the procarcinogen-ic effect made by some peptide hormones is realized with the mediating role of some peripheral hormones controlled by them. Normally, in males, the LH and FSH receptors are mainly limited to gonadal cells [7]. However, under the conditions of malignant pathology, the FSH receptors are found in intratumoral vessels both of the primary tumors (the lungs, the breast, the prostate, the colon, the kidneys, leiomyosarcoma) and metastatic tumors (metastases of the bones, the liver, the lymph nodes, the brain, the lungs, the pleura), and their density differs in different tumors [8].

It is known that sex steroids are the initiators and promoters of carcinogenesis of tumors commonly referred to as hormone-dependent: the tumors of the ovaries, the uterus, the prostate, and the breast. At the same time, it has been established that the pathogenesis of some malignant tumors, which were not previously classified as hormone-sensitive ones, involves the HPGA regulation in colorectal cancer [9], skin melanoma [10] and in some other cases. There are some studies devoted to the research of the functioning of the gonadal axis of the regulation under metastasizing of malignant tumors [11]. However, most works on this subject study the content of individual hormones either in blood or in tumors. Fragmentary information does not allow us to put together a complete picture of the functioning of HPGA under the conditions of onco-pathology. It is known that the liver is considered one of the most frequent sites of metastasis of various solid tumors [12]. It is also known that the liver is involved in the metabolism of steroid hormones, which, in turn, make their impact on the metabolic processes in the liver [13]. Based on the above, it seems relevant to study the features of the functioning of HPGA at the stages of metastasizing of malignant tumors to the liver.

The aim of our research work was to study the features of the functioning of the hypothalamic-pituitary-gonadal axis of the regulation in male rats at the stages of metastasizing to the liver.

Material and methods

The experimental study was carried out in white outbred male rats weighing 180-250 grams (n=30), which were delivered by the breeding facility at the National Medical Research Center of Oncology at the Ministry of Health of Russia, and which were kept under the natural light conditions with free access to water and food. Work with animals was conducted in accordance with the rules of the European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Directive 86/609/EEC), as well as in compliance with the International Guiding Principles for Biomedical Research Involving Animals and Order No. 267 “Approval of the rules of laboratory practice” dated June 19, 2003, issued by the Ministry of Health of Russia.

The rats were divided into groups as listed below: group 1 (reference) where the spleen was positioned under the skin (n=10); group 2 (MTS5) – 5 weeks after the introduction of sarcoma 45 (S45) cells into the spleen (n=11); group 3 (MTS7) – 7 weeks after the introduction of S45 cells into the spleen (n=9). The third group was divided into 2 subgroups as follows: MTS7A covering the rats with metastasis in the liver only (n=6) and MTS7B covering the rats with liver and lung metastasis (n=3). An experimental model of the liver metastases in rats was developed by us earlier [14]. The choice of the timing of the study is due to the fact that week 5 is a stage characterized by the presence of a formed primary tumor in the spleen, preceding the visual appearance of metastases in the liver, while week 7 is a stage characterized by the presence of formed metastases in the liver. Moreover, in some animals, the liver metastases were combined with the lung metastases, which at the 7th week of the development of the malignant process were represented by many miliary nodules located through the thickness layer of the lung parenchyma, while the liver metastases looked like large tumor nodules 18.7–34.6 cm3 in size, with one-two focal growth.

We used the S45 strain supplied by the Federal State Budgetary Institution “The Russian Cancer Research Center named after N.N. Blokhin” at the Ministry of Health of Russia. Material for transplantation

Issue 27. May 2023 | Cardiometry | 55

was obtained from donor rats on days 16-17 of the tumor growth. 3-4 weeks after the positioning of the spleen under the skin, healing of the surgical wound and recovery of the animal, 0.1 ml of a suspension of the S45 tumor cells (at a dilution of 1x106 in physiological saline) was injected intralienally.

The rats were decapitated, trunk blood was collected in dry sterile test tubes without preservatives, and serum was isolated by centrifugation. Homogenates were obtained from the internal organs as given below herein: 2% homogenate (the hypothalamus, the pituitary gland, the prostate) and 10% homogenate (the testes, the liver tissue in rats from the MTS5 group, the liver metastases and the liver tissue surrounding metastases – the parametastatic zone (PZ), in rats from the MTS7A and MTS7B groups). Work with tissues was carried out on ice. In the obtained samples of biological material, the following was studied: by radioimmunoassay (RIA) the content of LH, FSH, estradiol (E2), total testosterone (Ttot), progesterone (P4) (Immunotech, Czech Republic); by the ELISA method the concentrations of GnRH, free testosterone (Tfr), estrone (E1) and sex steroid binding globulin (SSBG) (Casabio, China). Statistical processing of the obtained results was completed using the parametric Student’s test with a personal computer employing the STATISTICA 12.0 software and the non-parametric Wilcoxon-Mann-Whitney test, having previously checked the data sets on normality of distribution of the indicators (Shapiro-Wilk test for small samples). Table data are presented herein as M±m, where M is the arithmetic mean, m is the standard error of the mean. Differences between two samples were considered statistically significant at p<0.05.

Results

It was found that in the rats with the model of metastatic liver damage, compared with the reference, the content of GnRH in the hypothalamus decreased in the MTS5 group by 2.0 times, in the MTS7A group by 2.2 times, and in the MTS7B group by 2.6 times (see Table 1 herein).

The dynamics of concentrations of tropic hormones in the pituitary gland was multidirectional: the level of LH at the stages of metastasis decreased by 1.61.7 times compared with the reference, while the content of FSH, on the contrary, increased in the MTS5 group by 6.3 times, in the MTS7A group by 6.5 times, and in the MTS7B group by 6.8 times, respectively (see Table 1 herein). It should be noted that, unlike the pituitary gland, the concentrations of LH and FSH in blood depended on the stage of the study and the presence of metastases in the lungs. Thus, the content of LH in blood of the rats from the MTS5 group did not differ from the reference, while at the stage of metastasis it increased in the MTS7A group by 4.6 times, and in the MTS7B group by 2.1 times compared with the reference and the pre-metastatic stage; the difference between the groups of the rats with formed liver metastases, but with different status of lung metastases, was 2.2 times (see Table 1 herein). The level of FSH in blood, on the contrary, decreased in the MTS5 and

Table 1

The content of GnRH in the hypothalamus, FSH and LH in the pituitary gland and blood serum in male rats at the stages of metastasizing

Indicator	Reference	МТS5	МТS7
			А	B
GnRH in hypothalamus (ng/g tissue)	0,041 ±0,004	0,020+ ±0,004	0,019+ ±0,003	0,016+ ±0,003
LH in pituitary gland (IU/g tissue)	0,019 ±0,000	0,012+ ±0,002	0,012+ ±0,002	0,011+ ±0,001
LH in blood serum (IU/L)	0,18 ±0,05	0,18 ±0,02	0,83+,5 ±0,05	0,38 +,5,7 ±0,02
FSH in pituitary gland (IU/g tissue)	0,0010 ±0,000	0,0063+ ±0,000	0,0065+ ±0,0002	0,0068+ ±0,0003
FSH in blood serum (IU/L)	8,83 ±0,65	2,74+ ±0,46	8,325 ±0,65	5,98+,5,7 ±0,44

Notes: Statistically significant differences from the following data: + from the reference; 5 from the MTS5 data; 7 from the MTS7A data.

MTS7B groups by 3.2 times and 1.5 times, respectively, while in the MTS7A group it did not statistically significantly differ from the reference; the difference between the MTS7A and MTS7B groups was 1.4 times (see Table 1 given herein).

The content of P4 in the liver of the rats in the MTS5 group decreased compared with reference values by 3.2 times (see Table 2 herein). In the rats in the MTS7A group, the level of the hormone in the metastasis and PZ corresponded to the reference values. In the rats in the MTS7B group, the content of P4 increased sharply and became greater than in the reference group: in the metastasis by 3.6 times and in PZ by 11.3 times, correspondingly, and compared with the values of the MTS5 group it increased in the metastasis by 11.4 times and in PZ by 35.8 times, respectively; as compared with the corresponding tissues of the rats in the MTS7A group in the metastasis by 5.2 times and in PZ by 17.4 times, correspondingly (see Table 2 herein).

The content of E1 in the liver of the rats in the MTS5 group did not statistically significantly differ from the reference values, while in the rats at the stage of the formed liver metastases it increased, reaching greater values in the metastases than it was the case with the surrounding tissues, especially in the rats in the MTS7B group (see Table 2 herein). Thus, the concentration of E1 in the liver metastasis was higher than that measured in the liver tissue in the reference rats and the rats from the MTS5 group: in the MTS7A group by 6.0 times and 8.3 times, respectively, and in the MTS7B group by 13.2 times and 18.1 times, respectively; the difference in the content of the above hormone in the liver metastases in the rats with dif- ferent types of metastasis was 2.2 times (see Table 2 herein). The level of E1 in PZ was the same in all rats: less than in the corresponding metastases, namely, in the rats in the MTS7A group by 3.7 times and in the rats in the MTS7B group by 8.1 times, but it was 1.6 times higher than in the reference group and 2.2 times higher than it was the case with the MTS5 group.

The level of E2 increased in comparison with the reference values by 1.1 times in the liver tissue only in the rats in the MTS5 and MTS7A groups (see Table 2 herein). The content of Tfr became higher than the reference values in the liver in the rats in the MTS5 group by 3.4 times, as well as in metastasis and PZ by 1.8 times in the rats in the MTS7B group. The difference in the level of Tfr between the MTS7A and MTS7B rats in their metastases was 1.8 times and that recorded in PZ was 2.0 times (see Table 2 given herein). The content of Ttot in the liver tissues increased at all stages of metastasizing: in the rats in the MTS5 group by 15.0 times, in the rats in the MTS7A and MTS7B groups by more than 200.0 times as against the corresponding reference indicator. As a result, the level of Ttot in the malignant and surrounding liver tissue was more than 13.0 times higher than at the previous stage of the study (see Table 2 given herein).

In the reproductive organs at the stage of the formed metastases, the content of P4 increased as compared with the reference as follows: in the MTS7A group only in the testes by 20.1 times; in the MTS7B group and in the testes by 10.0 times, and in the prostate by 6.5 times (see Table 3 herein). It should be noted that in the prostate in 60% of the rats from the MTS5 group, the level of P4 decreased by 22.0 times, and in 40% of the cases it increased by 2.4 times com-

Table 2

The content of sex steroids in the liver (per gram of tissue) at the stages of metastasizing