Dynamic changes in sumoylation related proteins SUMO1, SENP1, and UBC9 during the peri implantation period in mice.

SUMO1 showed cytoplasmic and nuclear localization, and its expression intensity was different between the experimental groups. The highest SUMO1 expression in the luminal and glandular epithelium of all experimental groups was observed on the 5th day of pregnancy. SUMO1 expression was decreased in the luminal epithelium ( p  < 0,0001) and glandular epithelium ( p  < 0,001) on the 6th day of pregnancy compared to the 5th day of pregnancy, with a statistically significant difference. In the stroma, SUMO1 expression was highest on the 5th and 6th days of pregnancy compared to all experimental groups. We observed a statistically significant decrease in SUMO1 expression at the implantation sites on the 8th day of pregnancy ( p  < 0,0001) compared to the implantation sites on the 5th and 6th days of pregnancy. SUMO1 expression was low in the primary decidual zone (PDZ) on day 6 and intense in the secondary decidual zone (SDZ) on both the 6th and 8th days of pregnancy (Fig.  1 ). Fig. 1 SUMO1 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. SUMO1 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. SENP1 exhibited both cytoplasmic and nuclear localization, with varying expression levels among the experimental groups. In the luminal epithelium, SENP1 expression was significantly higher on the 4th and 5th days of pregnancy ( p  < 0,05) and inter-implantation sites ( p  < 0,001) compared to the 1st day of pregnancy. In the glandular epithelium, the highest expression was on the 5th day of pregnancy, followed by a significant decrease on the 6th day of pregnancy ( p  < 0.0001). In the stroma, SENP1 expression was highest on the 4th day of pregnancy compared to the other groups ( p  < 0,001). On the 6th day of pregnancy, SENP1 expression was low in the PDZ and intense in the SDZ. SENP1 expression was intense in the SDZ on the 8th day of pregnancy (Fig.  2 ). Fig. 2 SENP1 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. SENP1 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. The experimental groups exhibited varying expression intensities for UBC9, and UBC9 was localized in both the cytoplasmic and nuclear compartments. UBC9 expression in the luminal epithelium was at the highest level on the 4th day of pregnancy and gradually decreased afterwards. UBC9 expression in the glandular epithelium was observed to be high at the uteri on the 1st day of pregnancy and implantation sites on the 5th day of pregnancy compared to other groups ( p  < 0,0001). In the stroma, UBC9 expression was higher on the 4th day of pregnancy and implantation sites on the 5th, 6th, and 8th days of pregnancy compared to the 1st day of pregnancy ( p  < 0,0001). UBC9 expression was higher in SDZ compared to PDZ on the 6th day of pregnancy. We observed intense UBC9 expression in the SDZ on the 8th day of pregnancy (Fig.  3 ). Fig. 3 UBC9 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. UBC9 immunohistochemistry in the luminal epithelium, the glandular epithelium, and the stroma of the uterus, implantation, and inter-implantation sites of the experimental groups. le Luminal epithelium, ge Glandular epithelium, s Stroma, e Embryo, pdz Primary decidual zone, sdz Secondary decidual zone. For figures LE, GE, and S; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy. Scale bars: 1st line 150 μm, second, third, and fourth lines 50 μm. SUMO1 expression showed a gradual increase from the 5th day of pregnancy, followed by a gradual decrease. Except for the 8th day of pregnancy, SUMO1 expression was at its lowest level on the 1st day of pregnancy with a statistically significant difference ( p  < 0.0001). SUMO1 expression was at the highest level on the 5th day of pregnancy compared to the 6th and 8th days of pregnancy ( p  < 0.0001) (Fig.  4 ). Fig. 4 SUMO1 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. SUMO1 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. SENP1 expression was similar between the 1st, 5th, and 6th days of pregnancy. The lowest and highest expressions of SENP1 were observed on the 8th and 4th days of pregnancy, respectively. On the 4th day, SENP1 expression was higher than all other experimental groups with a statistically significant difference ( p  < 0.0001) (Fig.  5 ). Fig. 5 SENP1 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. SENP1 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy, d: inter-implantation sites of the 5th day of pregnancy, e: 6th day of pregnancy. UBC9 expression was similar between the experimental groups. The lowest UBC9 expression was observed on the 1st day of pregnancy. UBC9 expression in the uteri on the 1st day of pregnancy was lower compared to other groups ( p  < 0.0001), except the 4th day of pregnancy group. The highest UBC9 expression was observed on the 5th day of pregnancy compared to all experimental groups with a statistically significant difference ( p  < 0.0001) (Fig.  6 ). Fig. 6 UBC9 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy. UBC9 Western Blot in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy. The highest estrogen level was observed on the 4th day of pregnancy, which was higher than in all experimental groups and showed a statistically significant difference ( p  < 0.0001). The lowest estrogen level was observed on the 8th day of pregnancy, it was lower than all experimental groups but it was only statistically significant compared to the 1st and 4th days of pregnancy ( p  < 0.0001) (Fig.  7 ). Fig. 7 Estrogen levels in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy. Estrogen levels in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy. Progesterone levels showed a gradual increase from the 1st day of pregnancy towards the 8th day of pregnancy. The highest progesterone level was observed on the 8th day of pregnancy. It was higher than the 1st, 4th, and 5th days of pregnancy with a statistically significant difference ( p  < 0.0001). Additionally, progesterone levels on the 6th day of pregnancy were higher than on the 1st and 4th days of pregnancy, with a statistically significant difference ( p  < 0.0001) (Fig.  8 ). Fig. 8 Progesterone levels in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy. Progesterone levels in the uterus and implantation sites of all experimental groups and analysis; different letters indicate a significant difference compared to that group; a: 1st day of pregnancy, b: 4th day of pregnancy, c: implantation sites of the 5th day of pregnancy.

Female Balb/C mice, aged 6–8 weeks ( n  = 50), and male Balb/C mice, aged 12 weeks ( n  = 10), were obtained from the Akdeniz University Animal Research Unit in Antalya, Turkey. Mice were maintained at room temperature (22–24 °C) with a 12-hour light/dark cycle and had free access to both mouse chow and tap water ad libitum. All methods were performed in accordance with the ARRIVE guidelines ( https://arriveguidelines.org/ ). Female mice were mated with males overnight. The presence of a vaginal plug was examined the next morning, indicating the first day of pregnancy for female mice 26 . Pregnant mice were randomly divided into five groups based on their stage of pregnancy: D1 ( n  = 10), D4 ( n  = 10), D5 ( n  = 10), D6 ( n  = 10), and D8 ( n  = 10). To determine the number of mice to be used in the experiments, a power analysis was performed using the G*Power 3.1.9.4 program. Cohen’s d coefficient was taken as 0.8 (large effect size) for sample calculation. When the number of mice in each experimental group was equal, it was concluded that 6 mice were required for each group at an alpha 0.05 significance level and a 0.85 power level. However, to better account for biological variability and obtain more robust statistical results, 10 mice were used per group. On the 1st day of pregnancy, oviducts were dissected, and when 1 single-cell embryo (zygote) was detected, the uterus was obtained. On the 4th day of pregnancy, one of the uterine horns was flushed with PBS, and when a blastocyst was observed, the unflushed uterine horn was collected. To visualize implantation sites, a 1% Chicago Blue dye solution (#C8679, Sigma Aldrich) was intravenously injected before sacrification on days 5 and 6 of pregnancy. Thus, the implantation sites became visible. From the 8th day of pregnancy onwards, no injection is required; the implantation sites are visible 27 . The mice were anesthetized using a Ketamine-Xylazine Mix (10 ml/kg, intraperitoneally), and implantation sites were collected. Subsequently, euthanasia was performed via cervical dislocation (Table  1 ). Table 1 Establishment of experimental groups. Experimental Groups Days of pregnancy 1 4 5 6 8 D1 ( n  = 10) Vaginal plug checked; Recovering embryos from the oviducts. Uterus were collected. D4 ( n  = 10) Vaginal plug checked Recovering embryos from the uterus. Uterus were collected. D5 ( n  = 10) Vaginal plug checked Chicago blue injection; Implantation sites were demarcated by discrete blue bands. Implantation and inter-implantation sites were collected. D6 ( n  = 10) Vaginal plug checked Chicago blue injection; Implantation sites were demarcated by discrete blue bands. Implantation sites were collected. D8 ( n  = 10) Vaginal plug checked Implantation sites were visible Implantation sites were collected. Female mice with a vaginal plug were admitted on the 1st day of pregnancy, thereby forming the experimental groups; D1 1st day of pregnancy, D4 4th day of pregnancy, D5 5th day of pregnancy, D6 6th day of pregnancy, D8 8th day of pregnancy. Establishment of experimental groups. Vaginal plug checked; Recovering embryos from the oviducts. Uterus were collected. Recovering embryos from the uterus. Uterus were collected. Chicago blue injection; Implantation sites were demarcated by discrete blue bands. Implantation and inter-implantation sites were collected. Chicago blue injection; Implantation sites were demarcated by discrete blue bands. Implantation sites were collected. Implantation sites were visible Implantation sites were collected. Female mice with a vaginal plug were admitted on the 1st day of pregnancy, thereby forming the experimental groups; D1 1st day of pregnancy, D4 4th day of pregnancy, D5 5th day of pregnancy, D6 6th day of pregnancy, D8 8th day of pregnancy. Similar to our previous study, we followed the same protocol 28 . We used the primary antibodies at a dilution of 1:25 for rabbit polyclonal anti-SUMO1 (Cell Signaling #4930), 1:400 for rabbit polyclonal anti-SENP1 (Abcam #ab236094), and 1:600 for rabbit polyclonal anti-UBC9 (Abcam #ab33044). We used 1:1000, 1:100, and 1:600 dilutions of rabbit IgG for isotype controls, respectively (Isotype control; Rabbit IgG, Sigma, I5006). Then, the samples were incubated in a biotinylated anti-rabbit secondary antibody at a dilution of 1:500 (#BA-1000, Vector Laboratories, Burlingame, USA). After that, sections were incubated at room temperature for 20 min with HRP-conjugated streptavidin complex (#TS125HR, Thermo Scientific, Fremont, CA, USA). Di Amino Benzidine (DAB) chromogen (#D4168, Sigma Aldrich, St. Louis, MO, USA) was used to signalize the reaction on sections. Mayer’s Hematoxylin (#109249 Merck, Darmstadt, Germany) was used for counterstaining. Entellan medium (#107961; Merck, Darmstadt, Germany) was used to seal and preserve the sections. The Nikon Eclipse E200 microscope was used to examine and photograph them. The ImageJ analysis program was used to evaluate the immunohistochemistry results for SUMO1, SENP1, and UBC9. Photographs were taken at six different sites of the sections of all experimental groups. DAB, hematoxylin, and background images were obtained after “color deconvolution” was made on the X400 magnification photographs of each section. The mathematical values of DAB intensities of all experimental groups were compared statistically. The uteri, implantation sites, and inter-implantation sites were frozen in liquid nitrogen and thawed. The proteins were then isolated. Proteins were extracted, and immunoblot analysis was carried out following previously described methods 29 . Antibodies were applied to the membranes as rabbit polyclonal anti-SUMO1 (Cell Signaling #4930) at 1:1000 dilution, rabbit polyclonal anti-SENP1 (Abcam #ab236094) at 1:2000 dilution, and rabbit polyclonal anti-UBC9 (Abcam #ab33044) at 1:1000 dilution. The incubation was performed overnight at + 4ºC. The membranes were treated with the Super Signal Chemiluminesans (CL)-HRP substrate system for five minutes (#34080, Thermo Fisher Scientific, Rockford, USA). Then, protein bands were observed using the Azure C280 imaging device. This led to the determination of the quantity of SUMO1, SENP1, and UBC9 protein expressions. To confirm that the samples were equally loaded, a rabbit monoclonal anti-beta actin antibody (#4970, Cell Signaling Technology, Danvers, MA, USA) was labeled to the membranes using the same protocol and at a dilution of 1:5000, and beta actin served as an internal control. NIH image analysis software was used to quantify the bands. (Image J Version 1st36b; National Institutes of Health, MD, USA). First, a one-way ANOVA was conducted, followed by a post hoc Tukey HSD test for statistical analysis. P-values less than 0.05 were considered significant, and values are presented as mean ± SEM (standard error of the mean). Before sacrification, approximately 1 ml of blood was drawn from the tail vein of mice. Blood obtained in single-use endotoxin-free blood tubes was allowed to coagulate at room temperature for approximately two hours. Then, the samples were centrifuged at 5000 rpm at 4 °C for 15 min in a Sigma 2–16 K centrifuge. The supernatants (serum) were stored at -80 °C after centrifugation. Estrogen (#E-EL-0150, Elabscience Biotechnology) and progesterone (#E-EL-0154, Elabscience Biotechnology) levels were detected in mouse serum samples. ELISA was performed according to the procedure specified in the datasheet. Each standard and sample was studied in duplicate, and the results were presented as an average. The log-log graph of the logistic curve was drawn using the Curve Expert 1.4 program, with standard concentration on the x-axis and OD values on the y-axis. The mathematical values obtained as a result of immunohistochemistry, Western blot, and ELISA techniques were compared statistically. We calculated the median values, standard deviations, and mean + standard error (SEM) of the obtained data. To determine whether the distribution of the obtained data was normal, we used Kolmogorov-Smirnov and Shapiro-Wilk tests. For normally distributed data, we performed one-way ANOVA followed by Tukey’s HSD test for post hoc comparisons, as it controls for Type I error in multiple comparisons while maintaining statistical power. Effect sizes were also calculated where appropriate: partial eta squared (η²p) was reported for ANOVA results to quantify the magnitude of group differences. Probability values of less than 0.05 were considered significant; values are presented as mean ± SEM (standard error of the mean). Statistical analyses were performed with GraphPad Prism version 9.

Endometrial cells proliferate and differentiate under the influence of molecules regulated by steroid hormones. Besides the healthy development of the embryo, several factors are pivotal for a successful pregnancy, including synchronous receptivity of the uterus, embryo-uterus crosstalk, stromal cell differentiation into decidua cells, angiogenesis, and placental development. This study scrutinized the expression and localization patterns of SUMO1, SENP1, and UBC9, components of the sumoylation mechanism, in mouse uteri and implantation sites during the peri-implantation period. As a result, it is possible to say that the sumoylation mechanism may have roles during early pregnancy (Fig.  9 ). Our investigation represents the pioneering exploration of the potential roles of the sumoylation mechanism in the peri-implantation mouse uterus and implantation sites in vivo. The sumoylation mechanism showed dynamic changes in the uterus and implantation sites on different days of pregnancy. Elucidation of this post-translational mechanism will be crucial for understanding the molecular basis of pregnancy success. Understanding the sumoylation mechanism may lead to an increase in pregnancy success in women suffering from recurrent pregnancy losses of unknown cause or spontaneous abortions. Fig. 9 Schematic illustration of the interaction between SUMO1, SENP1, and UBC9 proteins during the peri-implantation period in the mouse uterus. The diagram integrates hormonal fluctuations (E 2 and P 4 ), protein expression dynamics across implantation days (1st, 4th, 5th, 6th and 8th ), and their potential roles in implantation, decidualization, and angiogenesis. Red arrows represent inhibition expression; T-bars represent decreased expression. Schematic illustration of the interaction between SUMO1, SENP1, and UBC9 proteins during the peri-implantation period in the mouse uterus. The diagram integrates hormonal fluctuations (E 2 and P 4 ), protein expression dynamics across implantation days (1st, 4th, 5th, 6th and 8th ), and their potential roles in implantation, decidualization, and angiogenesis. Red arrows represent inhibition expression; T-bars represent decreased expression.

The implantation of the embryo takes place when the uterus is receptive. Morphological and molecular changes in the endometrium are crucial for achieving endometrial receptivity. The period when the uterus is receptive is called “the implantation window’’ and ovarian steroid hormones play a vital role in this process. For implantation to be successful, the development of the embryo to the blastocyst stage and the uterus to the receptive stage must occur in a synchronized manner 30 . SUMO (small ubiquitin-like modifier) proteins expressed in eukaryotic cells are involved in post-translational modification 6 . Whether the sumoylation mechanism is involved in the implantation and/or decidualization processes in mice has not been investigated in the literature. In this study, we investigated the expression and localization of the members of the sumoylation mechanism, including SUMO-1, SENP-1, and UBC-9, in the uterus and implantation sites of mice at the peri-implantation stages. We also determined hormone-dependent changes in the expressions of SUMO1, SENP1, and UBC9. Luminal and glandular epithelium and stromal cells in the uterus respond differently to E 2 and P 4 . A coexistence of E 2 and P 4 regulates the proliferation and differentiation of uterine cells. In mice, uterine epithelial cells proliferate on the 1st day of pregnancy in response to pre-implantation E 2 . Proliferation in the stroma is stimulated by P 4 secreted from the corpus luteum, and it is amplified by the ovarian E 2 secreted on the morning of the 4th day of pregnancy. Also, the proliferation of epithelial cells stops, and differentiation begins 31 . The E 2 levels are high in the morning and return to the basal level in the afternoon in mice in the estrous phase 32 . In a study conducted by Liu et al., the regulation of protein expression involved in sumoylation in the mouse endometrium was investigated throughout the estrous cycle. SUMO1 was essentially detected in the luminal and glandular epithelial cells of the endometrium, and SUMO2/3 was detected in the stroma. UBC9 (E2 binding enzyme) showed intense expression throughout the estrous cycle. SENP1 was highly expressed in the stroma in the estrous phase, gradually decreasing during the metestrous and diestrous phases, resulted in the outcome that sumoylation plays a crucial role in remodeling uterine tissue during the estrous cycle 22 . Moreover, it was reported that deletion of the SUMO-specific protease SENP1 caused enhanced ERα sumoylation, promoting its transcriptional activity and driving endometrial epithelial proliferation 33 . Additionally, a previous study demonstrated that E 2 upregulates H4 sumoylation and promotes the proliferation of Ishikawa cells 25 . It is known that increased sumoylation in correlation with high estrogen levels may induce proliferation and cause endometriosis 34 . According to our ELISA findings, E 2 levels were higher on the 1st and 4th days of pregnancy compared to other groups. The relatively high expression of SENP1, as determined by our immunohistochemistry and Western blot results, led us to consider that SENP1 may play a role in the maturation of SUMO 2, 3, or 4 on the first day of pregnancy. Our Western Blot findings made us think that high UBC9 expression on the 1st day of pregnancy may be involved in the proliferation of the estrogen-influenced luminal epithelium, conjugated with SUMO 2/3. The adhesion, which is the second phase of implantation, takes place on the night of the 4th day of pregnancy in mice 35 . Also, vascular permeability increases at implantation sites during this time 36 . The mechanism of angiogenesis, which begins in the early stages of implantation, is supported by angiogenic factors and hormones. Estrogen is known to increase uterine vascular permeability and inhibit angiogenesis, whereas progesterone has a minimal effect on vascular permeability but stimulates angiogenesis 37 , 38 . Vascular endothelial growth factor (VEGF), an angiogenic factor, begins to be expressed in the uterine luminal epithelium on the first day of pregnancy in mice and increases during implantation. VEGF is expressed in uterine stromal cells on the 3rd day of pregnancy and in trophoblast cells on the 8th day of pregnancy 39 , 40 . In a study, it was demonstrated that the specific deletion of SENP1 in vascular endothelial cells resulted in increased sumoylation levels of VEGFR2 and decreased VEGFR2-mediated angiogenesis 41 . Our ELISA findings showed that estrogen levels were at their highest on the 4th day of pregnancy. According to our results, the presence of similar and high expressions of SUMO1, SENP1, and UBC9 on the 4th day of pregnancy suggests that the sumoylation and de-sumoylation mechanisms are active on this day, potentially regulating angiogenesis and contributing to estrogen-mediated vascular permeability. Our immunohistochemistry results showed that the expression levels of SUMO1, UBC9, and SENP1 were similar in uterine stromal cells on the 4th day of pregnancy. In a study using the mouse menstruation model, deletion of SENP1 in uterine stromal cells was shown to induce ERα sumoylation, which increases the transcriptional activity and proliferation effect of estrogen receptor alpha (ERα) 33 . It is known that steroid hormone receptors regulate the paracrine interaction between the epithelial and stromal cells of the uterus and are critical in regulating uterine receptivity necessary for a successful pregnancy 42 . The presence of ERα in uterine epithelial and stromal cells during pregnancy has been demonstrated in mice. ERα mRNA was observed to be localized in the luminal and glandular epithelium on the 1st and 2nd days of pregnancy and in stromal cells on the 3rd and 4th days of pregnancy during the peri-implantation period in mice. In addition, ERα was shown to be down-regulated in the decidual cells surrounding the blastocyst on the 8th day of pregnancy 43 . Decidualization does not occur when the Esr1 gene is deleted in the uterine epithelial and stromal cells of mice. Ki67-positive stromal cell count was performed to determine how ERα affects stromal cell proliferation. Ki67-positive cells in uterine stromal cells with the Esr1 gene deletion were significantly reduced. These results show that Esr1 coordinates a triple molecular communication between the uterine glandular epithelium, luminal epithelium, and stroma an regulates their function 44 . In addition, stromal Esr1 has been reported to play a role in the synthesis of stroma-derived growth factors such as epidermal growth factor (EGF), insulin-like growth factors 1 and 2 (IGF1 and IGF2), which play a role in the proliferative effect of estrogen on the epithelium 45 – 48 . Together with these studies, our immunohistochemistry results suggest that SUMO1-mediated sumoylation may play a role in the proliferation of the stroma cells on the 4th day of pregnancy. In addition, SUMO1 expression was less than UBC9 expression according to our Western blot results on the 4th day of pregnancy. For this reason, we think that UBC9 may be conjugated with SUMO 2/3 and participate in stromal cell proliferation by increasing the transcriptional activity of ERα on the 4th day of pregnancy. Many molecules, such as integrins, cytokines, and growth modulators, are known to be involved in uterine receptivity 49 . HOXA10, one of the factors having a role in the process of preparation for the receptive phase, has been reported to be high in the middle-secretory phase, which corresponds to the time of implantation in the human uterus. It is well established that the deletion of the Hoxa10 gene in mice results in impaired uterine receptivity and infertility 50 . In addition, HOXA10 is expressed in the stroma during the receptive phase, and its expression is further increased during decidualization in mice. The weak proliferation of stromal cells leads to implantation failure in Hoxa10 -/- mice 51 , 52 . SUMO1-mediated sumoylation has been reported to inhibit endometrial receptivity and embryo implantation by reducing the stability and transcriptional activity of the HOXA10 protein 53 . In this study, aberrantly high levels of sumoylated HOXA10 were observed in mid-secretory endometrial samples from women with recurrent implantation failure (RIF). Besides, the sumoylation of HOXA10 inhibited the BeWo spheroid and mouse embryo attachment to Ishikawa cells. As a result, it is suggested that the sumoylation of HOXA10 impairs embryo implantation in vitro and contributes to the development of RIF 53 . According to our results, the expression of SUMO1 in the luminal epithelium on the 4th day of pregnancy was lower than that of UBC9 and SENP1. This suggests that a decrease in SUMO1-mediated sumoylation may support embryo implantation on the fourth day of pregnancy. Histotroph is a unique secretion of the uterine gland epithelial cells that includes ions, amino acids, carbohydrates, proteins, and lipids 54 . It has been known for many years that uterine glands and their secretions play a role in uterine receptivity and embryo implantation in mice and humans 54 , 55 . In mice, the leukemia inhibitor factor (LIF) is specifically expressed by the uterine glands in response to the increased level of estrogen on the 4th day of pregnancy 56 – 58 . Embryo implantation does not occur in mice with the deletion of the Lif gene, a member of the interleukin-6 cytokine family, and LIF expression is necessary for uterine receptivity and embryo implantation 57 . In our study, SUMO1 expression in the uterine gland epithelium on the 4th day of pregnancy was observed to be higher than SENP1 and UBC9 expressions. This suggests that the sumoylation mechanism may be active in the glandular epithelium on the 4th day of pregnancy. Decidualization, defined as the differentiation of stromal cells into decidual cells, occurs after the attachment of the blastocyst to the uterine luminal epithelium in mice. This progesterone-mediated cellular differentiation process is a prerequisite for a successful pregnancy 59 . The primary decidual zone (PDZ) begins to form in the afternoon of the 5th day of pregnancy and is completely formed on the 6th day of pregnancy. PDZ disappears as the secondary decidual zone (SDZ) begins to form. SDZ is a layer of stromal cells that proliferate and differentiate around PDZ and is completely formed on the 8th day of pregnancy 60 . Sumoylation significantly modulates the function of many different target proteins by including their stability, protein-protein interactions, and cellular localization, including that of transcription factors, such as PR 61 – 63 . The sumoylation mechanism was investigated in an in vitro decidualization model utilizing human endometrial stromal cells (HESCs). It has been reported that the transcription of PR-A was increased, there was free SUMO-1 accumulation, and Ubc9 expression was low. In addition, it has been reported that SENP1 expression was decreased and SENP2 and SENP6 expressions were increased in decidualized cells. It was concluded that, cAMP induces PR-A hyposumoylation to increase P 4 -dependent transcription activation in HESCs, and it is involved in the differentiation of HESCs into decidual cells by regulating SENP expression in the SUMO cycle 20 . A study revealed that EHD1 overexpression in endometrial stromal cells of RIF patients impairs decidualization and progesterone responsiveness, while the SUMO-specific protease SENP1 reduced EHD1’s effects, restoring PRB activity 21 . Another study reported that reactive oxygen species (ROS) increased sumoylation in human endometrial stromal cells (HESCs), which led to enhanced modification and transcriptional inhibition of the progesterone receptor (PR) 64 . Overexpression of MAP kinase phosphatase 1 (MKP1) reduced JNK signaling, prevented hypersumoylation, and maintained PR activity in undifferentiated HESCs exposed to ROS. These findings demonstrated that the sumoylation pathway, influenced by ROS and JNK signaling, contributes to early pregnancy maintenance, and hypersumoylation response is absent in decidual cells for PR activity and cellular homeostasis during oxidative stress conditions. Based on our findings, we believe that the sumoylation mechanism may influence the proliferation of PDZ cells, resulting from the conjugation of SUMO1 with UBC9 under the influence of P 4 on the 5th day of pregnancy. According to our Western blot results, UBC9 expression was higher than SUMO1 and SENP1 expressions on the 6th day of pregnancy. Also, our immunohistochemistry results showed that SUMO1 and UBC9 expressions were high, while SENP1 expression was low in the stroma on the 6th day of pregnancy. SUMO1 may support the proliferation of the SDZ that starts to form on the 6th day of pregnancy, conjugating with UBC9 in parallel with P 4 increase, as shown by our ELISA results. On the 6th day of pregnancy, it is expected that there will be no proliferation in the luminal epithelium because the epithelial cells are removed by apoptosis and entosis on the evening of this day 30 . Studies have shown that ubiquitin-like proteins are involved in many regulatory processes such as cell cycle, cell signaling, immune recognition, apoptosis, cell proliferation, differentiation, inflammation, and DNA damage repair as well as post-translational modifications 65 – 67 . Daxx (death domain-associated protein) regulates gene expression by interacting with multiple DNA-binding transcription factors and chromatin-associated proteins as a transcriptional co-inhibitor or coactivator and mediates apoptosis through exogenous death receptor pathways 68 , 69 . In addition, Daxx has been reported to contain SUMO-interacting motifs (SIM). In a study, it was reported that at least one SIM mediated the interaction of Daxx with Ubc9 70 . Our detection of high SUMO1 expression and low SENP1 and UBC9 expressions in the luminal epithelium on the 6th day of pregnancy suggests that the factors involved in apoptosis may undergo a SIM-mediated sumoylation mechanism. SUMO1e3 and sumo-protein conjugate and UBC9 levels were found to be higher in the placenta of women with severe preeclampsia compared to the control group. Based on these results, it was inferred that hypersumoylation may contribute to placental pathogenesis 71 . In another study, the human placenta was shown to express all four SUMO isoforms, and these isoforms were expressed at higher levels in preeclampsia. These results suggest that the sumoylation mechanism may play a role in the placentation, and high expression might be associated with underlying placental dysfunction 72 . According to our Western blot analysis, SUMO1 and SENP1 expressions were low, and UBC9 expression was high on the 8th day of pregnancy. These results suggest that hyposumoylation may occur during normal placental development or that UBC9 may perform the sumoylation mechanism, conjugating with SUMO2/3. While previous studies have explored sumoylation in the context of human endometrial function and related conditions, such as endometriosis and recurrent implantation failure, they often identified similar roles for SUMO1, UBC9, and SENP1. This study, however, offers a comprehensive temporal and spatial analysis of these proteins during murine peri-implantation. To further illustrate the molecular interactions and hormonal regulation observed in this study, a schematic diagram is included (Fig.  9 ). This flowchart visually summarizes the interaction between the key components of the sumoylation pathways (SUMO1, SENP1, and UBC9), their temporal expression profiles, and their regulation by steroid hormones (E 2 and P 4 ) during the peri-implantation period. It also maps these molecular changes to uterine structures such as the luminal epithelium, stromal cells, and decidual zones, emphasizing how coordinated expression and localization of these proteins may influence implantation and decidualization. This visual representation facilitates an understanding of the dynamic nature of post-translational regulation during early pregnancy. For example, although prior research in human endometrial stromal cells has shown altered expression of SENP1 and hypersumoylation of transcription factors like PR and HOXA10 under pathological conditions, our results indicate that SUMO1 and UBC9 undergo dynamic changes during normal murine implantation 53 , 64 . Additionally, Liu et al. reported sumoylation-dependent remodeling of the endometrium during the estrous cycle in mice 22 . Still, they did not assess these proteins in the context of pregnancy or implantation. These comparisons underscore the importance of investigating sumoylation mechanisms across various reproductive models. Our findings contribute to bridging this gap by demonstrating hormone-responsive and cell-specific patterns of SUMO-related protein expression during critical stages of implantation and decidualization in a well-established murine model. The real-world relevance of our findings can be illustrated through recurrent implantation failure (RIF), a clinical condition in which high-quality embryos fail to implant despite repeated assisted reproductive technology (ART) attempts. Previous human studies have reported abnormal SUMO1-mediated sumoylation of HOXA10 and progesterone receptor (PR) in endometrial stromal cells of RIF patients, leading to impaired decidualization and hormone resistance. Our mouse model revealed dynamic regulation of SUMO1, SENP1, and UBC9 during early pregnancy stages, suggesting that temporal misregulation of the SUMO pathway could similarly disrupt uterine receptivity in humans. For example, our finding of high SENP1 and UBC9 expression on Day 4 in mice, which coincides with the implantation window, highlights a potential regulatory checkpoint that, if disrupted, may contribute to implantation failure. Thus, mapping this dynamic pathway in mouse models provides translational insight into the molecular mechanisms that potentially underlie unexplained infertility or RIF in women. The dynamic sumoylation mechanism is involved in cell cycle progression, nuclear organization, proteotoxic stress, pluripotency, immunity, and virtually every nuclear process 73 . A comparative overview of related studies and our in vivo work is provided in Table  2 . This table highlights the uniqueness of our study in providing temporal and spatial sumoylation protein expression data in direct relation to hormonal dynamics during the peri-implantation period. Our findings suggest a tightly regulated pattern of sumoylation-related protein expression in the peri-implantation uterus, which may reflect the dynamic modulation of cellular functions essential for embryo implantation and early development. While these observations are based on a mouse model, considering their relevance to other mammals, particularly humans, is important. Although the core components of the sumoylation mechanism, such as SUMO1, SENP1, and UBC9, are evolutionarily conserved, significant interspecies differences in endometrial biology must be acknowledged. Variations in endometrial responsiveness and hormonal regulation may influence sumoylation’s spatial and temporal dynamics. Therefore, while our data provide valuable insights into potential regulatory mechanisms, further investigations in human endometrial tissues or organoid systems are necessary to evaluate the conservation and translational potential of these findings across species. Table 2 Comparative summary of related studies and the present study in terms of methodology, tools, biological models, and outcomes. Study Model/system Techniques used Evaluation tools/metrics Advantages Limitations Jones et al. (2006) 20 Human Endometrial Stromal Cells (HESCs) In vitro decidualization, SUMO pathway manipulation RT-PCR, Western blot, Immunocytochemistry Showed SUMO1’s role in decidualization In vitro model, not in vivo relevance Jiang et al. (2017) 53 Human endometrium (RIF patients) HOXA10 sumoylation assays Cell adhesion, protein stability assays Demonstrated HOXA10 dysfunction via SUMO Limited to one protein, no systemic view Liu et al. (2020) 22 Mouse uterus (estrous cycle) Hormonal treatment, protein localization Immunohistochemistry SUMO1 and UBC9 localization in endometrial phases Not pregnancy-specific, no hormonal profile Zhang et al. (2024) 21 HESCs from RIF patients EHD1 knockdown, SENP1 overexpression Gene expression assays, functional assays Linked sumoylation to PRB activity Patient-specific variability, lacks time-course data Present study Mouse uterus (peri-implantation days 1–8) In vivo time-course, Immunohistochemistry, Western blot, ELISA Quantitative protein expression and localization, hormonal assays First to show dynamic, hormone-dependent changes of SUMO1, SENP1, and UBC9 in peri-implantation; integrates hormonal, spatial and temporal data Species-specific, does not address human uterine environment directly Comparative summary of related studies and the present study in terms of methodology, tools, biological models, and outcomes.

Although the success of embryo implantation depends on uterine receptivity and decidualization, the molecular mechanisms underlying these events remain incompletely understood 1 . In particular, post-translational modifications such as sumoylation are emerging as key regulators but have not been comprehensively studied in vivo during early pregnancy. The concept of uterine receptivity involves three stages: pre-receptive, receptive, and non-receptive (refractory) 2 . For implantation to occur, the uterus must be in the receptive phase 3 . Implantation is divided into three stages: apposition, adhesion (attachment), and penetration. Trophectoderm cells of the blastocyst are positioned opposite the luminal epithelium during apposition. During the adhesion phase, trophoectoderm cells attach to the luminal epithelium of the receptive uterus. In the penetration phase, the trophoectoderm invades the luminal epithelium 4 . The adhesion phase occurs at night on day 4 of pregnancy in mice. Estrogen (E 2 ) and progesterone (P 4 ) are two crucial ovarian steroid hormones. They prepare the uterus for the development and implantation of an embryo. Preovulatory ovarian E 2 induces luminal and glandular epithelial cell proliferation during the first two days of pregnancy in mice. P 4 secretion from the corpora lutea induces stromal cell proliferation on day 3 of pregnancy. The proliferation is superimposed on the preimplantation ovarian E 2 secretion, which occurs on the 4th day of pregnancy, known as the day of implantation. The primary decidual zone (PDZ) is avascular and forms in the stroma cells surrounding the blastocyst on day 5 of pregnancy. On the 6th day of pregnancy, the secondary decidual zone (SDZ) is formed around the primary decidual zone (PDZ). The PDZ degenerates by day 8 of pregnancy 5 . Understanding the mechanism of implantation and decidualization processes is crucial for new treatment strategies for implantation failure. SUMO (small ubiquitin-like modifier) proteins play a role in post-translational modification in eukaryotic cells 6 . Attaching SUMO proteins covalently to the lysine groups of target proteins is known as sumoylation. They regulate protein-protein and protein-DNA interactions by binding to target proteins. They change the intracellular localization of proteins and protect the cell from ubiquitin-related degradation 7 . Four SUMO homologs have been identified in mammals. These are classified as SUMO1, SUMO-2, SUMO-3, and SUMO-4. SUMO-2 and SUMO-3 are 95% similar, and SUMO1 and SUMO-2/3 have only 50% similarity 8 . SUMO1 is present in the cells in the conjugated form with proteins 9 . Many different substrates can also be conjugated with SUMO2-3 10 – 12 . SUMO1 and SUMO-2/3 have been shown to be functionally somewhat different from each other. In terms of SUMO-4 proteins, little information is available. SUMO-4 protein shows 86% similarity with SUMO-2 protein 9 . The sumoylation cycle consists of two phases: conjugation and deconjugation. In the sumoylation cycle, E1 (SAE1/2 [SUMO-activating enzyme subunit 1/2]), E2 (SUMO-specific conjugating enzyme Ubc9), and E3 (SUMO-ligases) are involved 13 , 14 . The binding process of the SUMO cycle in mammals begins with the E1 enzyme activating SUMO proteins in an ATP-dependent manner. During this activation, a thiol ester linkage is formed between the glycine residue in the C-terminus of the SUMO protein and the cysteine residue in the SAE2 active site of the E1 enzyme. Then, the SUMO protein binds to the active cysteine site of the binding enzyme, Ubc9. E3 ligase enzymes act as bridges between SUMO-linked Ubc9 and substrate proteins, and they also retain the SUMO-Ubc9 thiol ester linkage to provide the transfer of SUMO proteins. In addition, two prerequisites determine the sumoylation of a protein. A protein-specific E3 ligase may recognize the protein or directly interact with the Ubc9-SUMO thioester linkage. In either case, an acceptor lysine must access the Ubc9-SUMO thioester linkage. Three different mechanisms that determine lysine selection are currently known. These include direct sumoylation from the SUMO consensus site, sumoylation via the SUMO-interacting motif (SIM), and E3 ligase-dependent sumoylation. SUMO-interacting motif (SIM) sumoylation involves both hydrophobic and hydrophilic interactions and includes lysine residues in SUMO and acidic or serine residues surrounding the hydrophobic SIM core 15 . SUMO1 modification is not only regulated by conjugation. The sumoylation mechanism can be reversed by SENPs known as Sumo-specific proteases (de-sumoylation) 16 . In mammals, 6 isoforms of SENP (SENP1, SENP2, SENP3, SENP5, SENP6, and SENP7) have been defined 17 . As a primary regulator of Sumo protein maturation, SENP1/2 plays a key role, and also participates in the de-conjugation of both SUMO1 and SUMO-2/3 from substrates 18 , 19 . SENP1 localization is affected by the expression and localization of SUMO1 target proteins in the cell 16 . SENP2 is known to be endogenously localized in the nuclear membrane. Correct localization of SENP2 is important for its regulation. The mislocalization of SENP2 leads to the incorrect de-conjugation of SUMO1 18 . For the de-conjugation of SUMO1/2/3, SENP1 and SENP2 have broad specificity. Other SENP isoforms (SENP 3, 5, 6, and 7) prefer SUMO-2/3 over SUMO1 for de-conjugation 17 . The mechanism of sumoylation was investigated in differentiating human endometrial stromal cells (HESCS). Hyposumoylation was observed to influence many proteins, including the progesterone receptor, and it was concluded that changes in the SUMO cycle might play a role in the decidualization of HESCs 20 . Another study revealed that EHD1 (EH domain-containing protein 1) is overexpressed in the endometrial stromal cells of patients with recurrent implantation failure (RIF), leading to impaired decidualization and reduced progesterone responsiveness 21 . In this study, it was shown that EHD1 suppressed the transcriptional activity of progesterone receptor B (PRB) and promoted its degradation through enhanced sumoylation and ubiquitination. The SUMO-specific protease SENP1 was shown to mitigate the suppressive effects of EHD1, thereby restoring PRB activity. Moreover, the knockdown of EHD1 in stromal cells from RIF patients significantly increased the expression of decidualization markers including prolactin and insulin-like growth factor-binding protein 1 (IGFBP1). These findings highlight that elevated EHD1 contributes to progesterone resistance in RIF by enhancing PRB sumoylation and degradation. The expression of some members of the sumoylation pathway was determined during the estrous cycle in the mouse endometrium. During the estrous cycle, UBC9 exhibited intense expression, and SENP1 was highly expressed in the stroma. Based on these findings, sumoylation was considered crucial for the remodeling of the uterus during the estrous cycle 22 . Embryos of Ubc9 knock-out mice die in the post-implantation period 23 . A recent study using a mouse model of menstruation showed that activation and differentiation of stromal SM22α+-derived CD34 + KLF4 + stem cells were associated with increased SUMOylation of estrogen receptor alpha (ERα) 24 . It was reported that deletion of the SUMO-specific protease SENP1 caused enhanced ERα sumoylation, promoting its transcriptional activity and driving epithelial proliferation. These findings highlight the role of sumoylation in endometrial regeneration and suggest that SENP1 is a key regulator of estrogen signaling in stromal stem cells. Moreover, another study demonstrated the role of SUMO1 in regulating cell proliferation and apoptosis in Ishikawa endometrial cancer cells 25 . It was reported that progesterone receptor (PR) expression and histone H4 SUMOylation were downregulated via SUMO-1 siRNA, which induced apoptosis. Additionally, E 2 was demonstrated to upregulate H4 sumoylation and promote cell cycle progression through the S and G2/M phases in Ishikawa cells. These findings highlight the critical role of SUMO-1 in regulating endometrial cell proliferation and apoptosis, particularly through histone H4 sumoylation. Despite previous studies on the role of sumoylation in the estrous cycle, endometrial cells and decidualization of human endometrial stromal cells, the precise function of sumoylation-related proteins during early pregnancy remains unclear. This is the first in vivo study to investigate the spatial and temporal expression of SUMO1, SENP1, and UBC9 in the mouse uterus and implantation sites across peri-implantation days. We provide a comprehensive profile correlating protein expression with steroid hormone levels (E 2 and P 4 ). Our findings suggest dynamic sumoylation may play a critical role in decidualization and uterine receptivity. Understanding the expression and localization of SUMO1, SENP1, and UBC9 during the peri-implantation period may provide new insights into their role in uterine receptivity and implantation success. Our findings contribute to the understanding of molecular mechanisms underlying uterine receptivity and implantation, which may have implications for fertility research. This knowledge is crucial for developing potential therapeutic strategies for implantation failure and reproductive disorders. In our study, the Materials and Methods section describes the experimental design, including the animal model, tissue collection during the peri-implantation period, and the immunohistochemical, Western blot, and ELISA procedures used to assess protein and hormone expression. The results section presents our findings on the temporal and spatial distribution of SUMO1, SENP1, and UBC9 in uterine tissues and implantation sites, along with the dynamics of hormone levels. The discussion section presents a comprehensive analysis that integrates our findings with those of previous studies and highlights the potential implications. Finally, the Conclusion section concludes with a summary of key contributions and outlines directions for future research in the context of reproductive biology.

Below is the link to the electronic supplementary material. Supplementary Material 1 Supplementary Material 1