Role of resistin in the porcine uterus: effects on endometrial steroidogenesis

Introduction

Many biological processes in the body depend on hormones produced by the adipose tissue. Adipokines, adipocyte-derived hormones, participate in numerous processes, such as maintaining proper energy homeostasis by controlling hunger and satiety, insulin sensitivity, carbohydrate and fat metabolism, and inflammation (Fasshauer and Blüher 2015). The peptide resistin, so named because of its capability to induce insulin resistance and establish a connection between obesity and diabetes, is a member of the adipokines family. Resistin is also known in the literature as adipocyte-specific secretory factor (ADSF) or found in inflammatory zone 3 (FIZZ3). The hormone is a cysteine-rich protein encoded by the RETN gene and belongs to the resistin-like molecule (RELM) family (Steppan et al. 2001). In the human blood, resistin occurs in the form of a dimer through the formation of disulfide bridges between monomers, the formation of which depends on a large number of cysteine residues (Banerjee and Lazar 2001). Although resistin was discovered several years ago, its receptor has still not been identified. Since resistin performs pleiotropic functions, it is considered that there may be more than one receptor type for this adipokine. It has been shown that resistin binds to the isoform of decorin (DCN), the orphan tyrosine kinase-like receptor 1 (ROR1), the adenylyl cyclase-associated protein 1 (CAP1), and Toll-like receptor 4 (TLR4) (Tarkowski et al. 2010; Daquinag et al. 2011; Sánchez-Solana et al. 2012; Lee et al. 2014). McTernan et al. (2002) suggest that resistin may be a link between obesity, insulin resistance, diabetes, and their complications. It is known that resistin can exert its effects by influencing signaling pathways such as protein kinase B (Akt), extracellular signal-regulated kinases (ERK) 1 and 2 (ERK1/2) and p38 mitogen-activated protein kinases (MAPK), as well as signal transducer and activator of transcription 3 (Stat-3), and peroxisome proliferator-activated receptor γ (PPARγ) (Estienne et al. 2019).

Adipocytes and macrophages are the main sources of resistin. Nonetheless, resistin is also expressed in structures of the hypothalamic–pituitary–ovarian (HPG) axis that regulates the reproductive tract. The presence of resistin was reported in the rodent hypothalami (Wilkinson et al. 2005). The adipokine is expressed in the anterior pituitary (AP) cells, and what is more, it modulates luteinising hormone (LH) secretion by AP cells in vitro (Maillard et al. 2017). In the case of ovaries, resistin is expressed in theca (Th) and granulosa (G) cells and oocytes of rodents as well as in the G and luteal (L) cells of cattle (Maillard et al. 2011). Moreover, Maillard et al. (2011) reported that resistin influences basal and insulin-like growth factor (IGF)-1-stimulated steroidogenesis and proliferation of G cells. In porcine ovaries, resistin is expressed in Th and G cells and influences the steroidogenesis and mitosis of ovarian cells (Rak-Mardyła et al. 2013). In the case of tissues related to the reproductive system outside the HPG axis, resistin expression was found in the human and ovine uterus, as well as in the human and rat placenta (Yura et al. 2003; Caja et al. 2005; Oh et al. 2017; Dall’Aglio et al. 2019). In addition, expression of the resistin gene and protein has also been found in syncytiotrophoblast and extravillous trophoblasts in early gestation and the syncytiotrophoblast in late gestation (Yura et al. 2003). Chen et al. (2005) showed that the concentration of resistin in the serum of pregnant women increases in the third trimester and is higher than in non-pregnant ones. Resistin modulates the expression and activity of glucose transporter 1 (GLUT-1), which is involved in the transplacental movement of glucose necessary for placenta growth and consequently for embryo development (Di Simone et al. 2009).

In the reproductive system, in addition to the ovary, the uterus can also be a source of steroid hormones. Since 2008, it has been known that the porcine uterus can produce steroid hormones, such as oestrone (E₁), oestradiol (E₂), androstenedione (A₄), and testosterone (T) (Franczak 2008; Franczak and Kotwica 2008). Our earlier research established the impact of metabolism-linked hormones (adiponectin, chemerin, orexin A and B) on the uterine functioning in pigs including the process of steroid hormones’ production (Smolinska et al. 2016; Kiezun et al. 2017; Kaminski et al. 2018; Kisielewska et al. 2019; Rytelewska et al. 2020, 2021; Gudelska et al. 2022). It has been shown that resistin is involved in the process of ovarian steroidogenesis in pigs (Rak-Mardyła et al. 2014; Kurowska et al. 2021). The above inspired us to investigate if adipokine is present in the uterine tissues and how it affects steroidogenesis in the porcine uterus. Thus, we hypothesise that resistin is expressed in the porcine endometrium and myometrium during the oestrous cycle and early pregnancy and that the expression is influenced by the hormonal status of animals. We further postulated that the adipokine may be a potential uterine- or adipose-derived factor that regulates steroidogenesis in the uterus of pigs. This study aimed to determine the expression of the resistin gene and protein in the porcine endometrium and myometrium and the adipokine concentrations in the blood plasma and uterine luminal flushings (ULF) during the oestrous cycle and early pregnancy, as well as the effect of resistin on progesterone (P₄) and E₂ production and steroidogenic acute regulatory protein (StAR), P450 side-chain cleavage enzyme (P450_SCC), cytochrome P450_C17 (P450_C17), cytochrome P450 aromatase (P450_AROM; CYP19A1), and 3-hydroxysteroid dehydrogenase (3βHSD) protein abundances in the porcine endometrium collected during early pregnancy and the mid-luteal phase of the cycle. Furthermore, we wanted to ascertain whether the hormone’s mode of action involves the Akt signaling pathway.

Materials and methods

Fig. 1 shows the experimental design of this study.

Fig. 1.

The experimental design of this study. Akt, protein kinase B; P₄, progesterone; E₂, oestradiol; StAR, acute regulatory protein; P450_SCC, P450 side-chain cleavage enzyme; 3βHSD, 3β-hydroxysteroid dehydrogenase; P450_C17, cytochrome P450_C17; P450_AROM, cytochrome P450 aromatase. Created in BioRender https://BioRender.com/t00t230.

Results

Gene and protein expression of resistin in the porcine endometrium

During the oestrous cycle, the RETN gene expression was higher on days 14–16 of the cycle compared to days 2–3 and 17–19 (Fig. 2a; P < 0.05). Higher amounts of resistin protein in the endometrial explants were observed on days 2–12 compared to days 14–16 of the cycle (Fig. 2b; P < 0.05).

Fig. 2.

Resistin gene and protein expression in the porcine endometrium during the oestrous cycle and early pregnancy. A comparison of resistin mRNA (quantitative real-time PCR; a, c, e) and protein (western blot; b, d, f) abundances in the porcine endometrium between days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle (a, b), between days 10–11, 12–13, 15–16, and 27–28 of pregnancy (c, d), and between days 10–12 of the oestrous cycle and days 10–11, 12–13, 15–16, and 27–28 of pregnancy (e, f). Upper panels – representative immunoblots; lower panels – densitometric analysis of resistin protein relative content normalised with the actin protein. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05). RETN, resistin.

During early pregnancy, the endometrial expression of the RETN gene was the highest on days 10–11 and the lowest on days 15–16 (Fig. 2c; P < 0.05). The expression of resistin protein in the endometrium of pregnant pigs was higher on days 12–13 of gestation in comparison with other periods of pregnancy (Fig. 2d; P < 0.05).

The comparison of the RETN gene expression during early pregnancy with that observed on days 10–12 of the oestrous cycle shows higher abundances of resistin mRNA on days 10–13 of pregnancy in relation to the mid-luteal phase of the cycle (Fig. 2e; P < 0.05). Significantly higher amounts of resistin protein were noted in the endometrium on days 12–13 of pregnancy compared to days 10–12 of the oestrous cycle (Fig. 2f; P < 0.05).

Gene and protein expression of resistin in the porcine myometrium

In the myometrium during the oestrous cycle, the RETN mRNA content was the highest on days 10–12, lower on days 17–19, and the lowest on days 2–3 and 14–16 of the cycle (Fig. 3a; P < 0.05). The adipokine protein abundance was at the same level throughout the oestrous cycle (Fig. 3b; P < 0.05).

Fig. 3.

Resistin gene and protein expression in the porcine myometrium during the oestrous cycle and early pregnancy. A comparison of resistin mRNA (quantitative real-time PCR; a, c, e) and protein (western blot; b, d, f) abundances in the porcine myometrium between days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle (a, b), between days 10–11, 12–13, 15–16, and 27–28 of pregnancy (c, d), and between days 10–12 of the oestrous cycle and days 10–11, 12–13, 15–16, and 27–28 of pregnancy (e, f). Upper panels – representative immunoblots; lower panels – densitometric analysis of resistin protein relative content normalised with the actin protein. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05). RETN, resistin.

During gestation, the expression of the resistin gene was the highest on days 15–16, lower on days 27–28, and the lowest on days 10–13 of pregnancy (Fig. 3c; P < 0.05). A western blot analysis showed that the expression of the hormone protein was higher on days 10–11 compared to days 12–13 of pregnancy (Fig. 3d; P < 0.05).

Higher expression of the RETN gene was observed on days 10–12 of the oestrous cycle in comparison with all periods of early pregnancy (Fig. 3e; P < 0.05). In the case of resistin protein, there were no differences in the expression between pregnant and non-pregnant animals (Fig. 3f; P < 0.05).

Resistin concentrations in the porcine blood plasma

During the oestrous cycle, resistin concentrations in the porcine blood plasma were on the same level regardless of the cycle phase (Fig. 4a; P < 0.05). During early pregnancy, the highest hormone levels were observed on days 12–13, lower on days 15–16, and the lowest on days 10–11 and 27–28 (Fig. 4b; P < 0.05). A comparison of early pregnancy with the mid-luteal phase of the cycle showed differences in the hormone concentrations between pregnant and non-pregnant pigs. Higher levels of adipokine were noted on days 12–13 of pregnancy compared to days 10–12 of the oestrous cycle. In contrast, concentrations of resistin on days 10–11 and 27–28 of pregnancy were lower than that observed during the mid-luteal phase of the cycle (Fig. 4c; P < 0.05).

Fig. 4.

Resistin concentrations in the porcine blood plasma and uterine luminal flushings during the oestrous cycle and early pregnancy. A comparison of resistin levels (ELISA) in the blood plasma between days 2–3, 10–12, 14–16, and 17–19 of the oestrous cycle (a), between days 10–11, 12–13, 15–16, and 27–28 of pregnancy (b), and between days 10–12 of the oestrous cycle and days 10–11, 12–13, 15–16, and 27–28 of pregnancy (c), as well as in the uterine luminal flushings (ULF) between phases of the oestrous cycle (d), between periods of early pregnancy (e), and between days 10–12 of the oestrous cycle and periods of early pregnancy (f). Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05).

The effect of resistin on P₄ and E₂ secretion by the porcine endometrium

Even though there was no independent effect of resistin on the P₄ secretion, the interaction between both main factors was observed (Supplementary Table S1). Consequently, the post hoc test was used. On days 12–13 and 27–28 of gestation, concentrations of P₄ were higher in media after incubation in the presence of resistin (1 and 0.1 ng/mL, respectively). The secretion of P₄ was decreased under the influence of resistin on days 10–11 (1 ng/mL) and 15–16 (0.1 ng/mL) of gestation (Fig. 5a; Table S1; P < 0.05). Basal secretion of this hormone was lower on days 12–13 of gestation in comparison with other periods of pregnancy and days 10–12 of the cycle (Fig. 5a; Table S1; P < 0.05).

Fig. 5.

The impact of resistin on P₄ and E₂ secretion. The effect of resistin (0.1, 1, and 10 ng/mL) on the secretion of progesterone (P₄; a) and oestradiol (E₂; b) by the in vitro incubated endometrium from pigs during early pregnancy (days 10–11, 12–13, 15–16, and 27–28) and on days 10–12 of the oestrous cycle. Concentrations of P₄ and E₂ in media were measured using radioimmunoassay analysis. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05). C, control; R0.1, resistin 0.1 ng/mL; R1, resistin 1 ng/mL; R10, resistin 10 ng/mL.

In the endometrial tissue explants incubated in the presence of resistin the production of E₂ was increased on days 10–11 (0.1 ng/mL) of gestation. On days 27–28 of pregnancy, a lower secretion of E₂ by endometrial tissue was observed under the impact of resistin (0.1 and 1 ng/mL; Fig. 5b; Table S2; P < 0.05). Basal production of this steroid was higher on days 27–28 of pregnancy compared to the other analysed periods (Fig. 5b; Table S2; P < 0.05).

The effect of resistin on StAR, P450_SCC, 3βHSD, P450_C17, and P450_AROM proteins abundances and Akt protein phosphorylation in the porcine endometrium

On days 12–13 of gestation, as well as on days 10–12 of the cycle, resistin (all doses) decreased the amounts of StAR protein in the endometrium (Fig. 6a; Table S3; P < 0.05). Basal expression of StAR protein was the highest on days 10–12 of the oestrous cycle, lower on days 12–13 of pregnancy, and the lowest on days 10–11 and 15–28 of pregnancy (Fig. 6a; Table S3; P < 0.05).

Fig. 6.

The impact of resistin on StAR, P450_SCC and 3βHSD protein expression. The effect of resistin (0.1, 1, and 10 ng/mL) on protein abundances (western blot) of steroidogenic acute regulatory protein (StAR; a), P450 side-chain cleavage enzyme (P450_SCC; b), and 3β-hydroxysteroid dehydrogenase (3βHSD; c) in the in vitro-incubated endometrium from pigs during early pregnancy (days 10–11, 12–13, 15–16 and 27–28) and on days 10–12 of the oestrous cycle. Upper panels, representative immunoblots; lower panels, densitometric analysis of target proteins’ relative content normalised with the actin protein. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05). C, control; R0.1, resistin 0.1 ng/mL; R1, resistin 1 ng/mL; R10, resistin 10 ng/mL.

Amounts of P450_SCC protein were higher under the impact of resistin (10 ng/mL) on days 15–16 of pregnancy. Expression of P450_SCC protein was decreased by the adipokine in the endometrium harvested on days 10–11 (0.1 ng/mL) and 27–28 (all doses) of pregnancy, as well as on days 10–12 of the oestrous cycle (all doses; Fig. 6b; Table S4; P < 0.05). Basal P450_SCC protein amounts were higher on days 10–12 of the cycle and 27–28 of pregnancy compared to the remaining periods of pregnancy (Fig. 6b; Table S4; P < 0.05).

Despite the absence of an independent effect of resistin on 3βHSD protein amounts, the interaction between both main factors was observed (Table S5), thus the post hoc test was conducted. The adipokine (10 ng/mL) reduced the enzyme protein abundance on days 12–13 of gestation (Fig. 6c; Table S5; P < 0.05). Basal 3βHSD protein expression was the highest on days 12–13 of pregnancy, lower on days 10–12 of the cycle, and the lowest on days 10–11 and 15–28 of gestation (Fig. 6c; Table S5; P < 0.05).

Similarly to P₄ and 3βHSD, no independent effect of resistin on the amount of P450_C17 protein was noted, but an interaction between both main factors was observed (Table S6), therefore a post hoc test was performed. The abundances of the enzyme protein were higher under the influence of resistin (10 ng/mL) on days 15–16 of pregnancy (Fig. 7a; Table S6; P < 0.05). During the mid-luteal phase of the cycle, resistin (all doses) decreased the amounts of P450_C17 protein (Fig. 7a; Table S6; P < 0.05). Basal P450_C17 protein expression was the highest on days 10–12 of the oestrous cycle, lower on days 15–16 of pregnancy, and the lowest during the remaining periods of pregnancy (Fig. 7a; Table S6; P < 0.05).

Fig. 7.

The impact of resistin on P450_C17 and P450_AROM protein expression. The effect of resistin (0.1, 1, and 10 ng/mL) on protein abundances (western blot) of cytochrome P450_C17 (P450_C17; a) and cytochrome P450 aromatase (P450_AROM; b) in the in vitro-incubated endometrium from pigs during early pregnancy (days 10–11, 12–13, 15–16, and 27–28) and on days 10–12 of the oestrous cycle. Upper panels, representative immunoblots; lower panels, densitometric analysis of target proteins’ relative content normalised with the actin protein. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05). C, control; R0.1, resistin 0.1 ng/mL; R1, resistin 1 ng/mL; R10, resistin 10 ng/mL.

Resistin caused a drop in the P450_AROM protein abundance on days 15–16 (0.1 and 1 ng/mL) and 27–28 (0.1 and 10 ng/mL) of gestation as well as during the oestrous cycle (all doses; Fig. 7b; Table S7; P < 0.05). Basal P450_AROM protein abundances were the highest on days 10–12 of the oestrous cycle, whereas the lowest on days 12–13 of pregnancy (Fig. 7b; Table S7; P < 0.05).

Resistin increased the phosphorylation of Akt protein after 2, 5, and 10 min of incubation in comparison with the control group (Fig. 8; P < 0.05).

Fig. 8.

The impact of resistin on the activation of protein kinase B (Akt) signaling pathway. The effect of resistin (1 ng/mL) on phosphorylation of Akt protein in the in vitro-incubated endometrium from pigs on days 10–12 of the oestrous cycle. Upper panels, representative immunoblots; lower panels, densitometric analysis of phospho-Akt protein relative content normalised with the total Akt protein. Results are presented as means ± s.e.m. (n = 5). Statistically significant differences are presented by different superscripts (P < 0.05).

Discussion

The adipose tissue, the primary organ for storing energy, generates several hormones that impact uterine functioning. An overview of the literature on the expression of adipokines and their receptors in the endometrium of rodents, humans, and pigs can be found in Reverchon et al. (2014). To our knowledge, this is the first publication to demonstrate the presence of resistin mRNA and protein in the uterine tissues, as well as the concentrations of adipokine in the blood plasma and ULF of domestic pigs during the oestrous cycle and early pregnancy. For now, a few reports are showing the expression of resistin or its role in the functioning of the uterus. Resistin is an adipokine that has so far been found only in the uterus of women and sheep (Oh et al. 2017; Dall’Aglio et al. 2019). Dall’Aglio et al. (2019) found that the resistin protein abundance in the ovine uterus depends on the nutritional status of these animals, further supporting the relationship between nutritional status and reproductive activity. The presented study noted that the abundance of resistin mRNA and protein in the porcine uterine tissues and the hormone concentrations in the blood plasma and ULF vary during the cycle and early pregnancy. This indicates that the hormonal environment influences the adipokine levels during the cycle and early pregnancy. Rak et al. (2015a) noted that T and E₂ increased RETN expression in the porcine ovarian follicles, whereas P₄ had no impact on the hormone mRNA abundance. Moreover, the authors also observed a stimulatory effect of T, P₄, and E₂ on resistin protein expression in ovarian follicles and the hormone levels in the culture medium (Rak et al. 2015a). Results of this study show that the highest resistin concentrations during the oestrous cycle in ULF were during the follicular phase. This stage of the cycle is characterised by increasing levels of E₂, prostaglandin F_2α (PGF_2α), and follicle-stimulating hormone (FSH) in circulation (Ka et al. 2018), as well as elevated E₂ concentrations in porcine endometrial cytoplasmic extracts (Deaver and Guthrie 1980). Moreover, higher protein expression in the endometrial tissue and concentrations of resistin in blood plasma were observed during the maternal recognition of pregnancy. Concentrations of the hormone in ULF increased on days 12–13 of pregnancy and persisted in the further examined periods. In pigs, this period of pregnancy is characterised by higher P₄, oestrogens, prostaglandins, calcium, and protein levels in ULF (Geisert et al. 1982; Dobrzyn et al. 2017). In addition to E₂, elongating porcine embryos also secrete interleukin 1β2 (IL1B2), interferon δ (IFND), and interferon γ (IFNG) (Ka et al. 2018), which may also influence resistin levels. Increased resistin concentrations when those hormones levels are high supports the consideration that resistin expression is hormonally related. The overlapping pattern of resistin expression in the endometrium with its concentration in circulation and ULF during maternal recognition of pregnancy confirms the statement that the uterus is one of the sources of resistin. Both the oestrous cycle and early pregnancy involve significant immune system activity in the endometrium, with lymphocytes, neutrophils, macrophages, and plasma cells present in varying numbers depending on the phase of the cycle or period of gestation (Bischof et al. 1995; Kaeoket et al. 2002). This suggests that immune cells may serve as an additional source of resistin in the ULF. Previous studies have shown an increased number of endometrial leucocytes in pregnant pigs compared to non-pregnant animals (Bischof et al. 1995). Resistin is known to be secreted by immune cells (Li et al. 2021), including porcine alveolar macrophages (Hua et al. 2023). This could partially explain the observed differences in resistin concentrations in ULF between the oestrous cycle and early pregnancy, as well as between pregnant and non-pregnant animals. Notably, resistin concentrations in ULF during pregnancy were significantly lower than in the mid-luteal phase of the cycle. Since resistin functions as a proinflammatory factor (Estienne et al. 2019), it is plausible that the molecules produced by developing porcine embryos, such as IL1B2, IFND, IFNG, and E₂, suppress resistin secretion to prevent embryonic rejection and ensure successful non-invasive implantation. Further research is necessary to determine if and how resistin expression depends on a pig’s endogenous hormone profile.

In the present study, the expression pattern of the resistin protein differs from the expression of the RETN gene. Other researchers also found variations in gene and corresponding protein expression patterns (Gygi et al. 1999; Gry et al. 2009). Although the exact causes of these differences are unknown, intracellular feedback that controls gene expression, variations in the stability of mRNAs and proteins, and the post-transcriptional processing of mRNA may be the reason (Gygi et al. 1999; Gry et al. 2009; Li et al. 2014). Changes in expression depending on the day of the cycle and/or pregnancy, and alternating high and low expression, particularly of a gene, may be related to the feedback effect of gene expression in response to high mRNA levels (Gry et al. 2009; Li et al. 2014). Gene expression levels indicate the tissue’s capacity to synthesise the protein rather than the absolute amount of protein.

The oestrous cycle and early pregnancy in pigs are characterised by dynamic changes, often occurring very suddenly (Oestrup et al. 2009; Soede et al. 2011). During the oestrous cycle, the uterus undergoes cyclical changes to properly prepare for the reception of embryos. The developing CL generates an increasing amount of P₄ with a peak in concentration during the mid-luteal phase of the cycle (Anderson 2009; Ka et al. 2018) and inhibiting gonadotrophin secretion (Soede et al. 2011). This is all related to CL activity or, if its functioning is not maintained, to its luteolysis. Moreover, it is known that during the oestrous cycle the content of receptors for E₂, P₄, and T changes in both the endometrium and myometrium (Koziorowski et al. 1984). This may also be the reason for the variable influence of, in this case, steroid hormones on resistin expression in the porcine uterus. A similar phenomenon may apply to other hormones, not only steroids. It is possible that the aforementioned hormones can affect resistin mRNA abundances, but this requires more research.

The ability of porcine embryos to synthesise and secrete oestrogens is especially important because mainly E₂ is a signal that informs maternal organisms about conceptuses’ presence in the uterus in pigs (Geisert et al. 1990). Recent studies conducted on CYP19A1 –/– knockout porcine embryos showed sustain of CL functioning and embryo survival until days 27–30 of pregnancy (Meyer et al. 2019). In addition to embryos, the endometrium may also be a compensating source of oestrogens released into the uterine environment (Franczak and Kotwica 2008; Kaczynski et al. 2023) and that may be related to CYP19A1 –/– knockout embryos subsistence pending the end of the implantation process. Resistin caused enhanced secretion of E₂ by the porcine endometrium on days 10–11 of pregnancy, just before maternal recognition of pregnancy. However, on days 15–28 of pregnancy, resistin downregulates protein abundances of P450_AROM, an enzyme catalysing the conversion of T to E₂. This indicates that resistin may be one of the factors involved in the assurance of appropriate conditions for fertilisation by affecting E₂ and P₄ secretion from endometrial cells and by that, supporting pregnancy establishment.

Although the prevalence of uterine steroidogenesis has been established for a long time, its regulation remains unclear. Franczak and Kotwica (2008) demonstrated the ability of both porcine endometrium and myometrium to produce E₁, E₂, A₄, and T (Franczak 2008; Franczak and Kotwica 2008). Production of steroid hormones by the uterus may support embryonic steroidogenesis, and thus uterine receptivity and development of uterine glands, as well as uterine responsiveness to other hormones (Tarleton et al. 1999; Hu et al. 2003; Blitek et al. 2007). In our previous study, we determined the expression of StAR, P450_SCC, 3βHSD, P450_C17, and P450_AROM in the porcine endometrium (Gudelska et al. 2020). However, steroid hormones in the uterus were also demonstrated in other species. Mann et al. (2007) discovered that bovine endometrial tissue stores E₂. The equine endometrium can metabolise P₄ to E₂ (Goff et al. 1993). Schiff et al. (1993) demonstrated that the P450_SCC gene is also expressed in cells of the mouse uterus, the decidua. Furthermore, in the disease-free human endometrium, mRNA levels of aromatase and 17β-hydroxysteroid dehydrogenase (17βHSD) type 1, which catalyses the conversion of E₁ to E₂, were extremely low. In contrast, mRNA levels of enzymes involved in converting E₂ into less active metabolites, such as 17βHSD types 2 and 4, oestrogen sulfotransferase (EST), and steroid sulfatase (STS), were high. Moreover, P₄ enhanced the expression of 17βHSD type 2 and EST during the in vitro study (Dassen et al. 2007). Since resistin is expressed in the uterine tissues of pigs, we investigated the mechanism underlying resistin activity in the endometrium. We found that uterine steroid hormone production changed under the resistin impact. This study shows, for the first time, how resistin affects the secretion of P₄ and E₂, as well as the amount of StAR, P450_SCC, 3βHSD, P450_C17, and P450_AROM proteins in the endometrium of pigs throughout the early pregnancy and mid-luteal phase of the oestrous cycle. In research conducted by Maillard et al. (2011), the adipokine decreased P₄ and E₂ secretion by bovine G cells. Studies by Spicer et al. (2011) showed inhibition of steroidogenesis in Th and G cells from cattle ovarian follicles under the impact of adipokine. Resistin reduced P₄ and E₂ production in human G cells in response to IGF-1 but not FSH (Reverchon et al. 2013). Furthermore, resistin increased basal P₄ secretion in cultured rat G cells but did not affect E₂ production (Maillard et al. 2011). Resistin enhanced P₄, A₄, and T secretion and levels of P450_SCC, 3βHSD, P450_c17, and 17βHSD mRNAs and proteins, whereas the hormone had no effects on E₂ secretion and P450_AROM expression in the prepubertal porcine ovarian follicles (Rak-Mardyła et al. 2013). In the porcine luteal cells, resistin inhibited P₄ and stimulated E₂ secretion by affecting steroidogenic enzymes’ expression (Kurowska et al. 2021). In brief, the effect of resistin on ovarian steroidogenesis seems to be tissue- and species-dependent. The action of resistin on the secretion of E₂ and P₄ may be associated with providing appropriate conditions for fertilisation, and subsequent establishment of pregnancy.

Most of the research shows the inhibitory effect of resistin on E₂ production; however, the influence of the adipokine on P₄ secretion varies between species (Maillard et al. 2011; Spicer et al. 2011; Rak-Mardyła et al. 2013; Reverchon et al. 2013; Kurowska et al. 2021). In the present study, uterine synthesis of P₄ during the transuterine migration of embryos and at the beginning of implantation was reduced under the impact of resistin. In contrast, during the maternal recognition of pregnancy and at the end of implantation, P₄ release was higher in tissues incubated in the presence of the adipokine. The effect of resistin on E₂ production was opposed. The adipokine increased the steroid levels on days 10–11 of gestation, whereas on days 27–28 of pregnancy, E₂ levels were decreased under the impact of resistin. This may depend on differences in the uterine environment that occur during these periods of pregnancy in the pig. Pregnancy is a complex process in which many factors are involved. Disruption in the production of any of them may lead to failure in pregnancy establishment. During the maternal recognition of pregnancy in pigs, the key role is played by E₂ secreted by developing embryos. In the porcine uterus, resistin had no impact on endometrial E₂ release during this stage of gestation. It can imply that resistin does not belong to factors, which can directly affect this very meaningful process. The development of endometrial receptivity for embryo implantation is under the strong influence of P₄. Consistently, the exposition of the endometrium to P₄ has a paradoxical effect of downregulating P₄ receptor (PGR) expression in the luminal (LE) and glandular (GE) epithelium (Geisert et al. 1994). In the present study, we noted that basal production of P₄ by endometrium was the lowest on days 12–13 of pregnancy and that resistin increased the secretion of P₄ on those days, which could be related to supporting the ovarian production of P₄, and by that, to maintaining proper levels of the steroid to prepare the uterus for implantation. Subsequently, on days 15–16 of gestation, resistin inhibited P₄ secretion. This may be intended to prevent too high hormone concentrations because hormonal homeostasis during early pregnancy is crucial for reproductive performance.

Munir et al. (2005) noted that CYP17A1 expression and P450_C17 activity – a measure of ovarian hyperandrogenism in PCOS females – were improved by resistin alone or by resistin, forskolin, and insulin together. In the porcine endometrium, resistin increased the amounts of P450_C17 on days 15–16 of pregnancy and decreased amounts of the enzyme during the mid-luteal phase of the oestrous cycle. We propose that the resistin effect on the production of steroid hormones is species- and tissue-specific based on the findings of this study and earlier published investigations. The existence of distinct resistin isoforms could account for these contradicting results and the functional variety of resistin across species. Neither the resistin receptor nor the mechanism of the hormone activity in the porcine uterus are known. It can be suggested that local production of P₄ and E₂ in the porcine uterus might be inversely correlated, with uterine-derived P₄ potentially providing endometrial protection against the development of pathological conditions in pigs. This may explain the differing effects of resistin on P₄ and E₂ production observed in this experiment. Resistin stimulates the secretion of P₄, possibly by 17βHSD type 2 and EST, which inhibits the formation of E₂. However, such a hypothesis requires more research. Steroid hormones are synthesised on an ongoing basis depending on demand and are not stored in the cell (Bremer and Miller 2014). Therefore, it can be assumed that resistin affects the de novo synthesis of steroid hormones.

In this study, the distinction in resistin effect on steroidogenic enzyme abundances and P₄ and E₂ concentration patterns could be explained by the difference between enzyme protein expression and its activity as well as post-translational modification of proteins. Moreover, increased protein expression does not always reflect its enzymatic activity. The expressed protein may have little or no ability to convert substrate into a suitable product. There is also a feedback loop that inhibits the expression or activity of steroidogenic enzymes when the concentration of the resulting hormone is too high. It is known that the activity of 3βHSD is reduced under the impact of P₄ in rat ovaries (Tanaka et al. 1993) or by androgens in rat testicular Leydig cells (Fanjul et al. 1992). Furthermore, conceptuses could also be a source of other steroid hormones, not only E₂, which may be transformed by enzymes present in the endometrium or they may influence the secretion of others. Kaczynski et al. (2021) determined that intrauterine administration of E₂ provoked intensified levels of P₄ observed in the porcine CLs. To exclude this possibility, research should be conducted on other steroid hormones.

The Akt signaling pathway is known to be involved in the process of steroidogenesis. It was convincingly shown that resistin at the dose of 10 ng/mL promoted the activation of many kinases, including Akt, MAPKs, p38, and ERK1/2, in cultured bovine and rat G cells (Maillard et al. 2011). Resistin (10 ng/mL) increased Stat-3, Akt, and ERK1/2 phosphorylation in cultured porcine ovarian follicular cells (Rak et al. 2015b). Moreover, the adipokine reduced indicators of apoptosis, such as gene expression of pro-apoptotic genes, caspase activity, and DNA fragmentation, most likely through the ERK1/2, JAK/STAT and PI3K signaling pathways (Rak et al. 2015b). The results obtained in this experiment are in agreement with those previously described. Resistin (1 ng/mL) enhanced the abundance of the phosphorylated Akt protein in the porcine endometrium. Although during the mid-luteal phase of the oestrous cycle resistin did not significantly affect the secretion of P₄ and E₂, it inhibited the expression of StAR, P450_SCC, 3βHSD, P450_C17, and P450_AROM proteins. Thus, the influence of resistin on the Akt pathway might be connected to the adipokine’s influence on the steroidogenic enzyme activity or other steroid hormones’ production in the endometrial cells.

Conclusion

This study revealed that resistin is present in the porcine uterus, blood plasma, and ULF during the oestrous cycle and early pregnancy. Moreover, we demonstrated that resistin mRNA and protein abundances and concentrations of the hormone in blood plasma and ULF are dependent on the phase of the oestrous cycle and/or period of early pregnancy. These findings suggest that the hormonal status of pigs may be related to this phenomenon. Furthermore, resistin affects P₄ and E₂ production and expression of key steroidogenic enzymes (StAR, P450_SCC, 3βHSD, P450_C17, and P450_AROM) depending on the examined period of pregnancy. Resistin stimulated Akt signaling pathway activation, suggesting the involvement of this pathway in the mechanism of that adipokine action. Given the circumstances, resistin may be one of the regulators of pregnancy establishment in pigs; however, further studies are necessary to find out more details.

Supplementary material

Supplementary material is available online.