Effect made by the growth of multiple primary malignant tumors on the hypothalamicpituitary-gonadal axis in BALB/C nude mice of both sexes

Oleg I. Kit, Elena M. Frantsiyants, Valeriya A. Bandovkina*, Irina V. Kaplieva, Alla I. Shikhlyarova, Ekaterina I. Surikova, Irina V. Neskubina, natalia D. Cheryarina, Yulia A. Pogorelova, Lidia K. Trepitaki, Inga M. Kotieva, Lyudmila Y. Rozenko, Marina A. Arzamastseva, Elena I. Agarkova, Marina M. Sergeeva. Effect made by the growth of multiple primary malignant tumors on the hypothalamic-pituitary-gonadal axis in BALB/c Nude mice of both sexes. Cardiometry; Issue No. 27; May 2023; p. 26-32; DOI: 10.18137/cardiometry.2023.27.2632; Available from:

In vertebrates, the endocrine system includes the hypothalamic-pituitary-adrenal (HPA), hypothalamic-pituitary-gonadal (HPG), and hypothalamic-pituitary-thyroid (HGT) axes [1]. Although each axis regulates the relevant physiological processes with the help of its own specific hormones, cross-interactions are known that occur both in the central links: the hypothalamus and pituitary gland, and in the peripheral ones, in particular, due to common binding sites at the nuclear receptors of thyroid and steroid hormones [2]. In addition, the mechanisms and reactivity of the interaction of the regulatory axes are gender-specific, and the HPG axis modulates how men and women respond to various stressors [3; 4].

HPG covers the hypothalamic gonadotropin-releasing hormone (GnRH), which stimulates the secretion of the anterior pituitary hormones, luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which are ultimately responsible for control of the sex steroid production and gametogenesis [5].

Sex steroid hormones are formed from cholesterol and include estrogens: 17β-estradiol, estriol and estrone, progestogens: progesterone, and androgens: testosterone [6]. While sex hormones are primarily produced by the tissues of the female ovaries and the male testicles, there are some other tissues, including the adipose tissue, the adrenal glands, the skin, the brain, and the bones, which also contribute to and promote the production of sex hormones [6]. Both sexes produce all types of sex hormones, but in different amounts, and their biological effects depend on their systemic concentrations, the expression of their nuclear receptors, and their specific interaction with target organs [6;7]. Based on current epidemiological evidence, estrogens and/or progesterone may play a role both in the onset and prevention of certain types of cancer [8].

It is known that both the growth of experimental B16/F10 melanoma and concomitant comorbid pathologies have a sex-dependent effect on the regulatory axes of the body [9;10].

Previously, we have shown that the simultaneous growth of two malignant tumors (MPMTs) B16/F10 melanoma and Lewis carcinoma (LLC) has its own gender-specific characteristics, resulting in a decrease in life expectancy against the background of changes in the average tumor volumes in animals of both sexes [11]. In connection with the above, the aim of our research was to study changes in the functioning of the HPG axis in BALB/c Nude mice of both sexes with MPMT.

Materials and methods

The studies were carried out in 84 BALB/c Nude mice of both sexes, aged 8–9 weeks, weighing 21–22 g, delivered by the Andreevka Scientific Center for Biomedical Technologies at the Federal Medical and Biological Agency (Moscow Region). The animals were kept under the same conditions in standard plastic boxes, 5 animals in each box, under natural light conditions, at an ambient air temperature 22–26°C, with free access to water and food. The mice received daily standard food and drinking water. The animals were constantly monitored with regular examination and body mass weighing.

All studies were completed in accordance with the requirements and conditions stipulated by 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. Our research work with animals was carried out 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). Reference: Record No. 7/111 dated 31.03.2021 prepared by our Ethics Committee.

We used murine B16/F10 melanoma metastasizing to the lungs and Lewis carcinoma (LLC) supplied by the Russian Cancer Research Center named after N.N. Blokhin at the RAMS (Moscow). The tumor strains were inoculated into females and males by subcutaneous injection of 0.5 ml of a suspension of tumor tissue in Hank’s solution or medium 199 (1:10).

The animals were divided into groups as follows: intact males and females of the BALB/c Nude line (n=14), mice of both sexes with standard subcutaneous inoculation of B16/F10 melanoma (n=14), mice with reproduction of the model of primary multiple tumor growth (MPMT) (n=14). To reproduce MPMT, B16/F10 melanoma and Lewis carcinoma were inoculated into the mice sequentially subcutaneously on two sides: one on the left side and the other on the right side, respectively.

GnRH, LH, and FSH were determined by ELISA using standard kits in 1% homogenates of the hypothalamic and pituitary tissues, and the content of estradiol (E2), testosterone (T) and progesterone (P4) was determined in 10% ovarian and testicular homogenates, as well as in blood serum using standard RIA kits (Immunotech, Czech Republic).

Statistical processing of the obtained results was carried out using the parametric Student’s test with a personal computer employing the STATISTI-CA 10.0 software and the nonparametric Wilcoxon-Mann-Whitney test. All results obtained were checked for their compliance with the law of normal distribution. Some indicators corresponded to the law, while the others were found not to be in compliance with the latter. For those indicators, which corresponded to the normal distribution, we used parametric statistics, and for the other indicators, the distribution of which did not correspond to the normal distribution, we used non-parametric statistics. Differences between the two samples were considered statistically significant at p<0.05.

Results

The malignant growth in its independent variant and in that with MPMT, compared with the intact ani- mals, induced in the females an increase in the level of GnRH in the hypothalamus by 4 and 2.5 times, respectively, while in the males, on the contrary, it initiated a decrease therein by 3.3 times and 3 times, respectively (see Table 1 herein).

In the pituitary gland of the females, the growth of melanoma alone and in combination with Lewis carcinoma caused an increase in the LH content by 1.4 times and 1.3 times (p<0.05), respectively, but a decrease in FSH by 7.8 times and 13.7 times as compared with the intact animals. In the males, the level of LH in the pituitary gland increased by 1.6 times (p<0.05) only with the independent growth of melanoma; in case of PMSO that did not differ from the indices in the intact mice. The content of FSH in the pituitary gland of males decreased by 1.5–1.7 times (p<0.05), in both variants of malignant growth, as compared with the intact animals. As a result, in the mice of both sexes, in response to any of the studied variants of the tumor growth, the LH/FSH ratio increased as follows: under B16/F10 in the males by 2.4 times, and in the females by 11 times; under MPMT in the males by 1.8 times (p<0.05), and in the females by 17 times, as compared with the intact animals.

Next, we studied the level of hormones in the gonads in response to the growth of B16/F10 melanoma in its independent variant or in the combined variant with Lewis carcinoma (see Table 2 herein).

In the females, in response to the growth of B16/ F10 melanoma, both in the independent melanoma

Table 1

Indicators of the central links of the HPG axis in mice of both sexes

Indicators	Intact mice	Mice with В16	Mice with В16+ LLC (MPMT)
Hypothalamus in females
GnRH (pg/g tissue)	0,2±0,018	0,8±0,091	0,5±0,071
Hypothalamus in males
GnRH (pg/g tissue)	0,6±0,08	0,18±0,031	0,2±0,0331
Pituitary gland in females
LH (mIU/g tissue)	67,13±7,2	92,6±8,41	84,7±9,61
FSH (mIU/g tissue)	43,75±5,7	5,6±0,81	3,2±0,31
LH/FSH	1,5±0,17	16,5±1,91	26±2,81
Pituitary gland in males
LH (mIU/g tissue)	60,5±6,4	96,1±8,61	63,5±7,1
FSH (mIU/g tissue)	1,9±0,2	1,3±0,161	1,1±0,141
LH/FSH	31,9±3,6	75,0±7,31	56,2±5,91

Note: significant differences: 1compared with intact animals p<0.05

Table 2

The content of sex steroids in the gonads in mice with different variants of the growth of melanoma B16/F10

Indicators	Intact mice	Mice with В16	Mice with В16+ LLC (MPMT)
Ovaries in females
Е2 (pg/g tissue)	43,6±4,7	70,0±8,3 Р1=0,000000	89,3±9,4 Р1=0,000001
Т (pg/g tissue)	1200±116,2	700±64,1 Р1=0,000070	900±83,5 Р1=0,003363
Р4 (ng/g tissue)	170,8±18,2	101,6±11,4 Р1=0,000000	97,9±8,6 Р1=0,000000
Testes in males
Е2 (pg/mL)	96,6±10,3	77,9±8,5	79,6±8,8
Т (pg/mL)	70,8±8,1	87,9±9,3	61,1±6,4
Р4 (ng/mL)	3,3±0,4	40,0±3,8 Р1=0,000000	4,5±0,5 Р1=0,003363

Note: significant differences: 1 – compared with intact animals 28 | Cardiometry | Issue 27. May 2023

and in the MPMT variants, the level of E2 in the ovaries increased by 1.6 times and 2 times, respectively, but T decreased by 1.7 times and 1.3 times, respectively, and P4 by an average of 1.7 times compared with the indices in the ovaries of the intact animals.

In the males, in the testes, an increase in the level of P4 with the independent growth of melanoma by 12

times and with MPMT by 1.4 times was found, compared with the indices recorded in the intact animals. The other sex steroids in the testes did not have significant differences from the parameters reported in the intact animals in any of the variants of melanoma growth.

Further, we conducted a study of the level of hormones in blood, and it turned out that in the female

Table 3

The content of hormones in blood serum of mice with different variants of the growth of melanoma B16/F10

Indicators	Intact mice	Mice with В16	Mice with В16+ LLC (MPMT)
Blood serum, females
LH (mIU/mL)	49,3±4,7	0,6±0,05 р=0,000000	0,7±0,08 р=0,000000
FSH (mIU/mL)	2,1±0,3	2,6±0,3	3,3±0,4 р=0,000002
LH/FSH	23,4±2,5	0,2±0,016 р=0,000000	0,2±0,022 р=0,000000
Е2 (pg/mL)	153,9±14,8	292,9±31,6 р=0,000000	256,3±32,1 р=0,000001
Т (pg/mL)	340±36,2	380±41,4	900±84,6 р=0,000002
Е2/Т	0,45±0,06	0,77±0,08 р=0,000000	0,28±0,03 р=0,000000
Р4 (ng/mL)	6,5±0,7	12,3±1,4 р=0,000000	5,0±0,8
Е1 (pg/mL)	15,1±1,7	22,5±2,4 р=0,000024	274,9±30,6 р=0,000000
Т fr. (pg/mL)	0,06±0,008	0,18±0,03 р=0,000000	6,4±0,7 р=0,000000
PRL (ng/mL)	0,5±0,06	0,05±0,007 р=0,000018	0,4±0,05
Blood serum, males
LH (mIU/mL)	64,3±7,1	3,2±0,4 р=0,000000	0,5±0,07 р=0,000000
FSH (mIU/mL)	3,2±0,42	2,4±0,3 р=0,000114	0,5±0,06 р=0,000000
LH/FSH	20,0±1,9	1,3±0,17 р=0,000000	1,0±0,13 р=0,000000
Е2 (pg/mL)	169,3±17,2	350,7±31,9 р=0,000000	288,0±30,6 р=0,000000
Т (ng/mL)	0,49±0,06	5,2±0,7 р=0,000000	0,4±0,05
Т/Е2	2,9±0,3	14,8±1,7 р=0,000000	1,4±0,16 р=0,000000
Р4 (ng/mL)	0,3±0,04	13,4±1,5 р=0,000000	21,7±2,3 р=0,000000
Е1 (pg/mL)	9,1±1,1	16,9±1,9 р=0,000000	10,0±1,2
Т fr. (pg/mL)	0,24±0,03	17,7±1,8 р=0,000000	0,08±0,009 р=0,000000
PRL (ng/mL)	0,5±0,07	0,14±0,02 р=0,000002	0,08±0,007 р=0,000000

Note: significant differences compared with intact animals

mice with melanoma in its independent variant and in the variant with MPMT, the content of LH in blood, despite the elevated content of that hormone in the pituitary gland, was considerably reduced: in case of B16 by 82.2 times, and in the mice with B16+ LLC by 70.4 times as compared with the intact mice. At the same time, the level of FSH, despite its decrease in the pituitary gland, in the blood serum of the animals with B16+LLC, on the contrary, was recorded to be increased by 1.6 times (р<0.05). In addition, it was found that in blood of the females E2 increased by 1.9 times with B16 / F10, and by 1.7 times with MPMT; the content of T was 2.6 times higher than the normal value only with MPMT, and P4 was 1.9 times greater with the independent growth of melanoma only.

In the males, despite the elevation of the level of LH in the pituitary gland with the independent growth of melanoma and the absence of changes in MPMT, the content of tropic hormone in blood was sharply reduced: by 20.1 times and 128.6 times, respectively, compared with the intact animals. The decrease in the concentration of FSH in the pituitary gland in the males coincided with a decrease in its level in blood: by 1.3 times with the independent growth of melanoma and by 6.4 times with PMSO compared with the intact animals. The absence of changes in the concentrations of E2 and T in the testes was accompanied by an increase in E2 in blood as follows: by 2.1 times with the independent growth of B16/F10 and by 1.7 times with MPMT, respectively, and in the T content by 10.6 times with the independent growth of melanoma only. An increase in the synthesis of P4 by the testes was reflected in blood parameters, where the level of the hormone increased by 44.7 times with B16/F10, and by 72.3 times with PMSO, as against the intact animals.

Discussion

Previously, we reported that in the females, B16 and LCC developed both in an independent variant and in a combined one B16 + LCC. In the males, only B16 developed in its independent variant, and MPMT B16 + LCC also took place, while LCC in its independent variant could not be reproduced in the males [11].

It is now known that the body’s response to strong acting stimuli is a complex composition of interactions between the autonomic nervous system, the immune system and the endocrine system. It is assumed that this response is aimed at ensuring survival, despite 30 | Cardiometry | Issue 27. May 2023

adverse conditions. The development and growth of a tumor in the body can be classified as the strong acting stimulation.

One of the obvious facts of the development of tumors in the males is a decrease in GnRH in the hypothalamus, while in the females, on the contrary, all variants of the tumor growth lead to an increase in the level of GnRH in their hypothalamus.

GnRH is responsible for the control of the pituitary-gonadal axis. Embryological studies in various vertebrate species have shown that the GnRH-pro-ducing neuroendocrine cells migrate from the medial part of the nasal epithelium to the forebrain [12]. In mammals, they migrate in close association with fibers of the vomeronasal and terminal nerves. Having reached the base of the telencephalon, they penetrate into the brain together with the central processes of the terminal nerves, slightly caudal to the olfactory bulb anlage, and move along the medial wall of the cerebral hemisphere into the preoptic/hypothalamic region [13]. From here, the cells extend to their final locations in the arcuate nucleus and preoptic area of the hypothalamus. The cells then lengthen their axons to the median eminence, where they can interact with the portal vessels of the hypothalamic-pituitary gland [12]. After secretion, GnRH is released into the portal vessels of the hypothalamic-pituitary gland and moves to receptors located at the gonadotropic cells of the anterior pituitary gland. Finally, it binds to receptors and leads to the secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), and to subsequent stimulation of various gonadal functions, including the production of sex hormones [14].

We have shown that in the pituitary gland of the females, the LH level increases in all tumor growth variants (B16, LCC, and B16+LCC), while in the males, LH increases only with B16, but not in the B16+LCC variant. At the same time, the level of FSH in the pituitary gland of animals of both sexes in all variants of the tumor growth decreases quite sharply that leads to an increase in the LH/FSH ratio that is recorded to be much more pronounced in the females.

The production and activity of LH and FSH depend on numerous elements of the hypothalamic-pituitary-gonadal axis, the functioning of which is regulated by positive and negative feedback loops. The action of LH and FSH is determined by many factors, such as the occurrence rate and amplitude of the GnRH peaks, different isoforms of LH and FSH, polymorphism of

FSH and LH and their receptors, and intracellular signal transmission [15]. More and more evidence data indicate that the anti-Müllerian hormone plays a role in the neuroendocrine control of reproduction and in the action of gonadotropins [16]. Anti-Müllerian hormone receptors have been found in the GnRH hypothalamic neurons and in some cell lines derived from gonadotrophs [17]. In vivo and in vitro experiments have shown that the anti-Müllerian hormone acts on the GnRH neurons, thereby increasing the LH pulsation and secretion, and that it interacts both with the hypothalamic and pituitary cells to facilitate gonadotropin secretion [18].

In the tissue of the gonads in the females, the level of LH increased, while the level of FSH remained unchanged. That was accompanied by a decrease in the level of T and an increase in the content of E2. At the same time, the level of progesterone remained unchanged. In general, there was a state of hyperestro-genism.

Abnormalities in the secretion regimen of gonadotropin-releasing hormone (GnRH) lead to a relative increase in the LH release compared with the FSH release, and an increased LH/PS ratio results in stimulation of the E2 production in the ovary. Ovarian estrogen initiates an abnormal feedback mechanism that causes an LH surge [19]. It is known that in the ovarian theca cells, LH stimulates the secretion of androgens, which are transferred to granulosa cells for their conversion into estradiol (E 2) by the aromatase enzyme. A deficiency in the production or in the action of LH and FSH threatens gametogenesis and the production of gonadal steroids [15].

Probably, in the ovaries of the females, the function of stimulating of the production of gonadal steroids was completely controlled by LH. In the males, the level of both gonadotropins in the testes was reduced, and the content of sex steroids did not have significant differences from those recorded in the intact males. At the same time, the level of progesterone with B16 significantly exceeded the relevant reference values. Estradiol (E2) and prolactin are also involved in the control of the male reproductive function. E2, produced both by the testicles and via the peripheral conversion of androgen precursors, is a powerful inhibitor of LH and FSH [20].

As to the blood serum, first of all, attention is drawn to a sharp drop in the LH / FSH ratio, leading to an increase in the level of estrogens, both in the females and the males in all variants of the tumor growth in question. Perhaps this state of absolute or relative hy-perestrogenism is a necessary condition for the tumor growth under immunodeficiency.

Thus, it was found that in mice both with transplanted B16/F10 melanoma and MPMT, the GnRH secretion in the hypothalamus changed depending on the sex: it decreased in the males, while it increased in the females. At the same time, the recorded changes in the content of tropic hormones in the pituitary gland turned out to be unidirectional: LH increased, and FSH decreased, and as a result, the LH/FSH ratio increased in the animals that indicated abnormalities in the central regulation and a lack of coordination in the performance of the hypothalamus and the pituitary gland. In the animals of both sexes, the growth of malignant tumors, both in their independent and combined variants, led to an increase in the LH/FSH ratio in the gonads, but at the same time to a decrease in that indicator in blood, that was found to be more pronounced in the females. At the same time, in the gonads of the females, both with the independent growth of B16 and with MPMT, hyperestrogenism and progesterone deficiency were produced, while in the males, only with the growth of melanoma, hyper-androgenism and an excess of P4 were revealed.

In case of the development of MPMT in the females, hyperandrogenism, a progesterone deficiency, an imbalance between LH/FSH towards the prevalence of FSH, and the absence of connections with the central regulatory systems and the gonads were recorded in terms of blood parameters. In the males with MPMT, hyperestrogenism, an excess of P4, an imbalance of LH/FSH towards the prevalence of FSH, and a disordering in connections both with the hypothalamus and the pituitary gland, and with the gonads were detected. From the results obtained, it can be concluded that the abnormalities in the central hypothalamic-pituitary regulation, as well as in the hormonal status of the body, along with primary immunodeficiency, plays a decisive role in overcoming antitumor protection in case of transplantation of strains of malignant tumors that are not characteristic for this type of experimental animals. The unification of hormonal changes is a confirmation of this assumption.