Regulation of uterine function during estrous cycle, anestrus phase and pregnancy by steroids in red deer (Cervus elaphus L.)

Steroid synthesis and production in ruminant uterus is not obvious, especially in seasonally reproduced. We compared steroid production by investigating enzymes involved in red deer uterine steroid metabolism in reproductive seasons. Blood and uteri (endometrium and myometrium) were collected post mortem from hinds on 4th day (N = 8), 13th day of the cycle (N = 8), anestrus (N = 8) and pregnancy (N = 8). The expression of cytochrome P450 aromatase (P450), 3 -beta-hydroxysteroid dehydrogenase (3β-HSD), 17 -beta-hydroxysteroid dehydrogenase (17β-HSD), aldo–keto reductase family 1 C1 (AKR1C1), estrogen receptor alpha (ERα), and progesterone receptors (PRs), were analyzed using real-time-PCR and Western Blotting. Plasma samples were assayed for 17-beta-estradiol (E2), progesterone (P4), luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone (T4) concentrations by EIA. Hinds at the beginning of the estrous cycle, mainly in endometrium, were characterized by a high mRNA expression of 3β-HSD, AKR1C1, PRs and ERα, contrary to the expression in myometrium during pregnancy (P < 0.05). For P4, E2, and FSH, concentration was the highest during the 13th day of the estrous cycle (P < 0.05). Uterine steroid production and output in hinds as a representative seasonally reproduced ruminant occurred mainly during the estrous cycle and sustained in anestrus.


Results
P450, 3β-HSD, 17β-HSD, AKR1C1 enzymes engagement in the estrous cycle, anestrus, and pregnancy. mRNA and protein expressions in endometrium. The mRNA expression for AKR1C1, P450, and 3β-HSD was higher on the 4th day of the estrous cycle than on the 13th day of the estrous cycle, pregnancy and the anestrus groups (P < 0.05; Fig. 1A-C). For 17β-HSD, the mRNA expression increased during the anestrus compared with the 4th day of the estrous cycle (P < 0.05; Fig. 1D).
The protein expression for AKR1C1 was the highest on 4th day of the estrous cycle and differed from 13th day of cycle, the anestrus phase and pregnancy (P < 0.05; Fig. 1E). P450 protein expression was higher in the endometrium on the 13th day than other experimental groups (P < 0.05; Fig. 1F). 3β-HSD and 17β-HSD protein expression was on the similar level throughout the examined experimental periods (P > 0.05; Fig. 1G,H).
mRNA and protein expressions in myometrium. For AKR1C1, P450 and 3β-HSD, the highest mRNA expression occurred in pregnancy and increased compared with the 4th, 13th day of the estrous cycle, and the anestrus (P < 0.05; Fig. 1A-C), whereas 17β-HSD was up-regulated on 13th day of the cycle comparing to 4th day of the cycle (P < 0.05; Fig. 1D).
The highest AKR1C1 protein expression was observed in pregnancy and differed from all examined groups (P < 0.05; Fig. 1E). However, the P450 protein expression was the highest on the 13th day of the estrous cycle and differed from the anestrus, pregnancy, and 4th day of the estrous phase (P < 0.05; Fig. 1F). 3β-HSD and 17β-HSD protein expression was on the similar level throughout the examined experimental periods (P > 0.05; Fig. 1G,H).
Interaction between expression in endometrium and myometrium. Interaction in mRNA expression occurred for three of them (AKR1C1, P450, 3β-HSD) and was higher in endometrium than in myometrium on 4th day of estrous cycle (P < 0.0001; Fig. 1A-C). In addition for AKR1C1 mRNA expression was higher in myometrium in pregnancy group than on 4th day of estrous cycle (P < 0.0001; Fig. 1A). For 17β-HSD, the interaction occurred between all tested groups except the anestrus and was higher in myometrium for all three of them. However, the highest mRNA expression was on the 13th day of the estrous cycle (P < 0.0001), lower in pregnancy group (P < 0.001) and the lowest on 4th day of the estrous cycle in myometrium (P < 0.01; Fig. 1D).
Receptivity of uterus for P4 and E2 during the estrous cycle, anestrus, and pregnancy. mRNA and protein expressions in endometrium. Compared with the anestrus, pregnancy, and 13th day, PRs and ERα mRNA expression increased on the 4th day of the estrous cycle in the endometrium (P < 0.05; Fig. 2A,B). The highest PRs protein expression was in pregnant hinds and differed from the 4th and 13th day of the estrous cycle (P < 0.05; Fig. 2C). ERα protein expression was the lowest in pregnancy, compared with the 4th, and 13th day of the estrous cycle, and the anestrus in the endometrium (P < 0.05; Fig. 2D).
mRNA and protein expression in myometrium. An increase in the mRNA expression was observed for PRs in the myometrium during pregnancy compared with other groups and PRs mRNA expression on 4th day of cycle was higher than on 13th day of the cycle and anestrus (P < 0.05; Fig. 2A). The mRNA expression in myometrium for ERα was lower on the 13th day of the estrous cycle, pregnancy, and the anestrus compared with the 4th day of the estrous cycle (P < 0.05; Fig. 2B). PRs protein expression in pregnancy was up-regulated compared with the  . The lowest statistical significance was P < 0.05. Asterisks indicate statistical differences between endometrium and myometrium (*P < 0.1; **P < 0.01; ***P < 0.001; ****P < 0.0001). Different letters indicate statistical differences (P < 0.05) between the experimental groups throughout endometrium (A, B) and myometrium (a-b) respectively. www.nature.com/scientificreports/ 4th day of the estrous cycle (P < 0.05; Fig. 2C). ERα protein expression was higher on the 4th and, 13th day of the estrous cycle and the anestrus than in pregnancy (P < 0.05; Fig. 2D).

Scientific Reports
Interaction between expression in endometrium and myometrium. For PRs mRNA expression, an interaction between endometrium and myometrium was exhibited during the 4th and 13th day of the estrous cycle (P < 0.05; Fig. 2A) and during pregnancy (P < 0.0001; Fig. 2A). In the case of ERα, the mRNA expression in the estrous cycle (4th and 13th day) confirmed the interaction between the tissues examined and the expression was upregulated in the endometrium than myometrium (P < 0.0001; Fig. 2B). Additionally, an interaction was exhibited during pregnancy and the expression was higher in the endometrium (P < 0.05; Fig. 2B). PRs protein expression in the pregnancy in the endometrium was higher than in the myometrium (P < 0.001; Fig. 2C).
Plasma concentration of P4, E2, T4 LH, and FSH. The plasma P4, E2, and FSH levels were higher on the 13th day of the estrous cycle than other groups (P < 0.05; Fig. 3A,B,E). The level of P4 was the nearest in anestrus comparing to other experimental periods (P < 0.05; Fig. 3A). T4 and LH concentrations were on a similar level, and no significant differences were observed (P > 0.05, Fig. 3C,D).  in uterine tissues (endometrium and myometrium) on 4th and 13th day of estrous cycle, in pregnancy and anestrus phase. Data were normalized against β-actin (ACTB) for proteins expression and against GAPDH for mRNA expression. Each bar represents one experimental group with SEM. Statistical differences were analyzed by two-way analysis of variance (ANOVA) followed by the Bonferroni post hoc test using GraphPad PRISM (Version 8.3.0). The lowest statistical significance was P < 0.05. Asterisks indicate statistical differences between endometrium and myometrium (*P < 0.1; ***P < 0.001; ****P < 0.0001). Different letters indicate statistical differences (P < 0.05) between the experimental groups throughout endometrium (A, B) and myometrium (a-c) respectively. www.nature.com/scientificreports/

Discussion
Most of our understanding of the steroid action in uterus comes from primate research 24 , whereas knowledge about steroid pathways regulating seasonal reproductive cycles in red deer is weak 25,26,28 and comes mainly from research using sheep and goats 29,30 . For the first time, using red deer females as an experimental model, we demonstrated that the steroid pathways regulation is different and varies with the reproductive seasons. The breeding season and anestrus were characterized by maximum endometrial mRNA expression of 3β-HSD, AKR1C1, PRs, ERα, whereas pregnancy by the highest mRNA myometrial expression for P450, 3β-HSD, AKR1C1, 17β-HSD and PRs. The fluctuations in protein expression were not so spectacular but occurred mainly in myometrium for AKR1C1 during pregnancy and for 3β-HSD in anestrus. Changes in reproductive status may result from steroid-feedback mechanisms found in sheep and goats 30 .
P450, 3β-HSD, 17β-HSD, AKR1C1 enzymes engagement in the estrous cycle, the anestrus and pregnancy. According to Rękawiecki et al. 31 , the mRNA expression and the protein for main enzymes involved in steroidogenesis (cytochrome P450scc and 3β-HSD) in the bovine uterus at the beginning of the estrous cycle are high. However, the opposite effect occurred in bovine endometrium on 17-20 days of the estrous cycle. These results suggested that P450 and 3β-HSD expression were reduced in the later estrous cycle phases 32 . We obtained similar results in the hind uterus. At the early luteal stage-4th day of the estrous cycle in the endometrium, the mRNA expression increased for P450 and 3β-HSD compared with the 13th day of the estrous cycle and other experimental groups. However, P450 protein expression was up-regulated on the 13th day of the estrous cycle both in the endometrium and in the myometrium. No much information is available about the expression of 17β-HSD in ruminants' uterus. However, in pigs it has been well understood. Wojciechowicz et al. 33 observed a decrease of 17β-HSD mRNA and protein expression in the pig endometrium on 15-16 days of pregnancy and the ovary on 15-16 days of luteolysis. We received the increase of 17β-HSD mRNA expression in myometrium which may indicate that the process of steroid inactivation occurs during this periods in myometrium, however such up-regulation was not received on the level of protein expression. In turn, Naidansuren et al. 13 examined the mRNA and protein expression for 20α-HSD in the placenta, ovary, and uterus. Because they reported that in the placenta, the expression mRNA and protein for 20α-HSD were higher than in the ovary and uterus, they suggested that deer placenta plays a crucial role in P4 regulation on 30, 60 and 70 days of pregnancy. However, according to Kim et al. 34 , during early pregnancy bovine ovary contributes significantly to steroid hormone regulation because it is the primary source of 20α-HSD mRNA and protein production, although the placenta and endometrium synthesize it as well 34 . This study confirmed 20α-HSD mRNA and the protein expression in the deer uterus as its biological activity in AKR1C1. Therefore, the mRNA expression and the protein were lower in pregnancy and the estrous cycle than in the anestrus in endometrium on the contrary to myometrium, where its expression was the highest during pregnancy. Explanation of such results in endometrium may be the readiness of red deer uterus for the resumption of steroids production necessary for the beginning of the reproductive season and the estrous phase. Whereas production of P4 by myometrial AKR1C1 may support the pregnancy development.
Receptivity of uterus for P4 and E2 during the estrous cycle, anestrus and pregnancy. In the uterus of sheep and sika deer distribution of PRs and ERα differed between endometrium and myometrium. In sheep, staining of ERα decreased on the 11th day of the estrous cycle in uterine tissue and remained weak during pregnancy; although in sika deer, staining of ERα was low during the estrous cycle in the endometrium. High staining was detected during the early pregnancy of sika deer (20-25 days) for PRs in myometrium similar in sheep and cattle 35 . Similar results obtained by Garcίa-Palencia et al. 36 , showed differences in expressions of ERα and PRs in the uterus of sheep after the estrous cycle was synchronized by prostaglandin analogue or by treatment with progestagens. The sheep treated with progestagens showed a decreased ER expression in the superficial uterine compartment (luminal epithelium, glands, and stroma) and myometrium and in the PR expression in all the uterine cell compartments except the myometrium. The reduction of both receptors' expression in the superficial compartments could compromise embryonic implantation due to the significance these steroid receptors' in maternal identification during pregnancy. According to Duan et al. 30 , ERα mRNA expression level was high in the sheep endometrium and myometrium and for PRs' expression in myometrium during the estrous cycle. However, in the estrous cycle, PRs' protein expression in the myometrium was significantly low. These results suggest that both receptors are essential for sheep reproductive function in the uterus, but its expression is changeable in uterus tissues 30 . It is known that estrogens during the follicular phase up-regulate both PRs and ERα in the uterus of most mammalian and P4 during the luteal phase down-regulates them; it should be taken into account that mRNA expressions do not always reflect protein concentration 37 . Our research results in which the mRNA expression for PRs was the highest in pregnancy in myometrium conformed other studies. Protein expression for PRs was also the highest during pregnancy, however, in both uterine tissues. Rekawiecki et al. 38 found that high protein levels for both PRs isoforms at the beginning of the protein expression were the highest during the anestrus phase in both (endometrium and myometrium) tissues examined. In turn, the ERα mRNA expression was the highest on the 4th day of the estrous cycle in the endometrium and the nearest its protein expression was received during pregnancy. Such results indicate that especially during the pregnancy development uterus doesn`t need additional source of estrogens.
Plasma concentration of P4, E2, T4, LH and FSH. An essential finding reported here was the marked seasonal variation of LH, FSH, P4, E2, and T4 concentrations in hinds' blood plasma as described before 39  www.nature.com/scientificreports/ results obtained by Li et al. 9 . In anestrus sheep, the concentration of LH in the serum was lower than during the reproductive season. Opposite LH concentration results during anestrus obtained by Meikle and Fisher 28 in ovariectomized red female deer. The anestrus was characterized by a maximum suppression of LH and minimum suppression of breeding season. These changes were similar to the intact herdmates. This agrees with work using sheep and goats and indicates a change in the steroid-feedback mechanism and changes in reproductive status. Yuthasastrakosol et al. 40 noted the high LH levels in anestrus in ewes, and they suggested an association of these values with the increase in P4 levels. Low levels of P4 during anestrus in goats and high levels of P4 in the luteal phase in sheep, during the middle of reproductive season, were observed by Thimonier 39 . In turn, Li et al. 9 did not observe a luteal P4 peak in sheep during anestrus. In comparison, our results showed a significant increase in P4 concentrations during the 13th day of the estrous cycle, while in the anestrus phase, this hormone's level was the lowest. On the 13th day of the estrous cycle, we noted the highest concentrations for FSH and E2 in the hind plasma. In cattle, low P4 allows FSH to increase from the 16th day of the cycle and the E2 concentration decreases after that, along with the regression of subordinate follicles 41 . In pregnant cows, there are few reports on the peripheral plasma levels of androgens. According to Gaiani et al. 42 , plasma T4 values increased throughout pregnancy. Kanchev et al. 43 reported an increase in plasma T4 concentrations at the onset of luteolysis in non-pregnant cows. In turn, Herriman et al. 44 compared measurements of the plasma concentrations of T4 during the estrous cycle of ewes and heifers. The maximum concentration of T4 was similar in both species and occurred at the onset of luteolysis; in the ewe, the peak concentration of plasma was significantly greater than the concentrations of T4 measured during the remainder of the estrous cycle. According to our research, the T4 concentration in the female red deer was at a similar level in all tested phases, with the tendency to decrease in the anestrus.

Conclusion
The mRNA expression for all examined factors, except 17β-HSD, was the highest on the 4th day of the estrous cycle in the endometrium. Receptivity for P4 is changeable, and PRs mRNA expression is up-regulated in the endometrium during the examined days of the estrous cycle but not during the pregnancy. Whereas in pregnancy, PRs protein expression is increased in the endometrium because it is responsible for maintaining pregnancy. PRs mRNA expression is elevated in pregnancy as if it additionally played a supporting role in its development.
The profile of mRNA, protein expression and immunolocalization of PRS was described by Kowalik et al. 45 in uterine endometrium and myometrium in cows. The authors pointed that in both uterine layers, those receptors are expressed. Moreover the localization of PRS in the endothelial cells of blood vessels in the bovine uterus, according to the mentioned authors, suggests that P4 affects blood flow in this organ through mPRs 45 . Receptivity for ERα and expression of P450 is up-regulated only at the beginning of the cycle, probably for enhancement of follicles' development during the estrous cycle. In the case of hormones concentration, the highest values were on the 13th day of the estrous cycle for P4, E2, and FSH, whereas for T4 and LH, no differences we found between the examined seasons, and further studies are necessary for the detailed recognition of hormone production with more examined days of the estrous cycle and pregnancy in hinds. However, it is evident that steroid regulation in hinds is variable, dependently on the reproductive status, and prevails in the endometrium over myometrium and differ from non-seasonal ruminants. Knowledge of the molecular mechanisms by which steroid hormones regulate gene expression for their receptors is critical for understanding steroid hormone actions during the estrous cycle, anestrus, and pregnancy in the uterus in the female red deer as an example of seasonally reproduced ruminants.

Material and methods
Sample collection. The experimental material (blood samples collected from the heard and uteri) was col- . Moreover informed consent was obtained from the farm owner for the collection of samples. The first experimental group (4th day of the estrous cycle) representing the follicular phase (N = 8), and the second experimental group (13th day of the estrous cycle) representing the luteal phase (N = 8) were obtained on the 17th and 23rd of September 2019 after pharmacological synchronization on the farm in Rudzie. The estrus and ovulation induction hinds during the estrous cycle was performed by applying a double controlled internal drug-release (CIDR) insert (1.38 g of P4; Pfizer Animal Health, New York, US), using a 12-day regimen of intravaginal CIDR devices. For better synchronization, the device was replaced after 7 days to maintain the luteal concentration of P4 until the end of the treatment period. Additionally, 200 IU of human chorionic gonadotropin (hCG; Folligon, Intervet, International B.V., Boxmeer, Holland) was injected intramuscularly on day 12. The estrus was observed 54-56 h after the second CIDR insert removal in the hinds. The day of the estrous cycle was evaluated by macroscopically observing the ovaries and uterus and confirmed by determining E2 and P4 levels in the blood plasma using an Enzyme-Linked Immunosorbent Assay (ELISA) 25,26 . The reasons for culling animals from the herd on the farm were for economic considerations and herd renewal. www.nature.com/scientificreports/ The third group (hinds in the anestrus) were collected from non-pregnant females on the 10th of May, 2019 (N = 8) on the farm in Rudzie. The last experimental group (pregnant hinds) was possessed between the 2nd and 4th of January 2019 (N = 8) from wild individuals in Strzałowo Forestry. Pregnacy was confirmed by the presence of embryo and PAGs concentration determination examined by EIA according to Korzekwa et al. 27 . The uterine horn tissue was immediately placed on ice and transported in liquid nitrogen to the laboratory, where it was subsequently stored at − 80 °C for further procedures. Plasma samples were collected by jugular venipuncture (EDTA-loaded vacuum tubes). Samples were held in ice until centrifugation at 3000 g at 4 °C for 10 min after which the plasma was placed on ice and transported to the laboratory within 30 min and stored at − 20 °C until assayed by ELISA.
Experimental procedure. P450, 3β-HSD, 17β-HSD, and AKR1C1 enzymes engagement in the estrous cycle, anestrus, and pregnancy was determined in the uterine endometrial and myometrial tissue by real-time RT -PCR and Western Blotting.
Receptivity of uterus for P4 and E2 during the estrous cycle, anestrus and pregnancy was determined in the endometrium and myometrium tissue by real-time RT -PCR and Western Blotting.
Concentration of LH, FSH, P4, E2 and T4 involved in enzymes metabolism was assayed by ELISA.   Table 1.

Determinations.
For efficiency evaluation, standard curves consisting of serial dilutions of the cDNA were plotted and the best cDNA concentration was chosen for further analysis. The first stage of the reaction was the initial denaturation of the strand and activation of the polymerase (95 °C for 2 min). The next stage consisted of 45 cycles of successive reactions: denaturation (95 °C for 5 s), primer annealing, and elongation of PCR products (6 °C for 20 s). To ensure the reaction's specificity, the melting curves of the PCR products were analyzed after the amplification was completed. Data obtained were analysed using the Miner program. Control reactions lacking the template or primers were performed to confirm that products were free of primer-dimers and genomic DNA contamination, respectively. PCR products were sequenced and compared with the appropriate genes in NCBI.
Total protein isolation. The uterine tissue (30 mg; separately endometrium and myometrium) was homogenized on ice in a RIPA lysis buffer (5 mM EDTA, 150 mM NaCl, 50 mM TRIS, 0.1% SDS, 1% Triton X-100, 0.5% sodium deoxycholate, and protease inhibitor-Sigma-Aldrich, S8830, pH 7.4). The obtained lysate was centrifuged at 10,000×g for 20 min at 4 °C, and the supernatant was transferred to a fresh tube and sonified. The protein concentration was estimated according to the Bradford's method. The lysate was stored at − 80 °C until further analysis.
Western blot analysis. The expression of P450, 17β-HSD, 3β-HSD, AKR1C1, PRs, and ERα proteins in the uterine tissues was determined by Western Blotting. For each sample, 30 µg of total protein was mixed with 5 µl of SDS gel-loading buffer, heated at 95 °C for 5 min, and separated in 10% SDS-PAGE. Afterward, the proteins were transferred on 0.2 μm nitrocellulose membranes in transfer buffer during the electroblotting for 1.5 h. The membranes were blocked in a 5% solution of skimmed milk with 1xTBS-T for 1.5 h in room temperature (RT), and then incubated overnight at 4 °C with specific primary antibodies for P450 (Abcam, Cambridge, UK, ab28146, 1:1000), 17β-HSD (Thermo Fisher Scientific, PA5-30063, 1:500), 3β-HSD (Gene Tex, Ca, USA, GTX114081, 1:500), AKR1C1 (Thermo Fisher Scientific, PA5-21672, 1:500), PRs (Thermo Fisher Scientific, MA1-410, 1:1000), and ERα (Thermo Fisher Scientific, MA1-310, 1:1000). As a reference the protein β-actin (ACTB, Sigma-Aldrich, A2228, 1:1000) was used. After that, the membranes was washes three times for 10 min in 1xTBS-T solution and incubated with secondary polyclonal anti-rabbit antibodies for P450, 3β-HSD, 17β-HSD, and AKR1C1 enzymes (Sigma-Aldrich, A3687, 1:10,000), and anti-mouse for PRs and ERα (Sigma-Aldrich, A3562, 1:10,000) for 1 ELISA. Plasma concentrations of LH, FSH, T4, P4, and E2 after validation 25 were evaluated using ELISA. LH and FSH concentrations were estimated using a kit for bovine (Cloud-Clone Corp., TX, USA, CEA441Bo for LH and CEA830Bo for FSH). The assay's sensitivity for LH was 137.3 pg/ml and for FSH was 0.93 pg/ml. The mean intra-and inter-assays CVs were 10% and 12%, respectively. A commercial kit (Abbexa, abx574314) designed to measure the concentration of T4 has been assessed for measuring T4 in the plasma. The sensitivity of the assay was 1.95 pg/ml. The intra-and inter-assays CVs were 5.8 and 7.9%, respectively. According to the manufacturer's instructions, the P4 and E2 concentrations were measured in plasma samples using an ELISA kit (Bioassay Technology Laboratory, Shanghai, China, E0018Bo for P4 and E0004Bo for E2). The assay's sensitivities for P4 and E2 were 0.22 ng/ml and 2.53 pg/ml respectively. The mean intra-and inter -assays CVs were found to be 8% and 10%, respectively. Each run was proceeded in duplicate and further the average was considered as a single sample. All samples were detected in duplicate at 450 nm.
Statistical analysis. GraphPad PRISM (Version 8.3.0, San Diego, CA, USA) was used for data analysis. The relationship between the mRNA and protein expression between endometrium and myometrium and between each experimental group throughout endometrium and myometrium was determined using two-way analysis of variance (ANOVA) followed by a Bonferroni post hoc test. One-way ANOVA was used for analysis of hormones` concentration, followed by Tukey's test. All numerical data were expressed as the arithmetic mean ± standard error of mean (SEM). Statistical significance was P < 0.05.