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Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and P4-PR signaling in mouse

Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and... Background Taurine performs multiple physiological functions, and the maintenance of taurine level for most mam- mals relies on active uptake from diet and endogenous taurine synthesis through its synthesis enzymes, including cysteine dioxygenase (CDO). In addition, uterus tissue and uterus fluid are rich in taurine, and taurine synthesis is regulated by estrogen (E ) and progesterone (P ), the key hormones priming embryo-uterine crosstalk during embryo 2 4 implantation, but the functional interactions and mechanisms among which are largely unknown. The present study was thus proposed to identify the effects of CDO and taurine on embryo implantation and related mechanisms by using Cdo knockout (KO) and ovariectomy (OVX) mouse models. Results The uterine CDO expression was assayed from the first day of plugging (d 1) to d 8 and the results showed that CDO expression level increased from d 1 to d 4, followed by a significant decline on d 5 and persisted to d 8, which was highly correlated with serum and uterine taurine levels, and serum P concentration. Next, Cdo KO mouse was established by CRISPER/Cas9. It was showed that Cdo deletion sharply decreased the taurine levels both in serum and uterus tissue, causing implantation defects and severe subfertility. However, the implantation defects in Cdo KO mice were partly rescued by the taurine supplementation. In addition, Cdo deletion led to a sharp decrease in the expressions of P receptor (PR) and its responsive genes Ihh, Hoxa10 and Hand2. Although the expression of uterine estrogen receptor (ERα) had no significant change, the levels of ERα induced genes (Muc1, Ltf) during the implanta- tion window were upregulated after Cdo deletion. These accompanied by the suppression of stroma cell proliferation. Meanwhile, E inhibited CDO expression through ERα and P upregulated CDO expression through PR. 2 4 Conclusion The present study firstly demonstrates that taurine and CDO play prominent roles in uterine receptiv- ity and embryo implantation by involving in E -ERα and P -PR signaling. These are crucial for our understanding the 2 4 mechanism of embryo implantation, and infer that taurine is a potential agent for improving reproductive efficiency of livestock industry and reproductive medicine. Keywords CDO, E , Embryo implantation, P , Taurine 2 4 Di Zhang and Zhijuan Wang contributed equally to this work. Yangzhou 225009, People’s Republic of China State Key Laboratory of Agrobiotechnology, College of Biological *Correspondence: Sciences, China Agricultural University, 100193 Beijing, People’s Republic Sheng Cui of China cuisheng@yzu.edu.cn Institute of Reproduction and Metabolism, Yangzhou University, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009 Jiangsu, People’s Republic of China 225009, People’s Republic of China Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 2 of 14 as leukemia inhibitory factor (LIF), ions, sugars, lipids, Background proteins and amino acids [20–24], among which taurine High rate of embryo loss in early pregnancy is a major is included [25]. It is much interested that taurine con- constraint both in livestock industry and human repro- centration in the uterine luminal fluid (UFL) of mouse duction, whereas much pregnancy wastage is caused by is much higher during the implantation [25]. But as we the failure of embryo implantation [1–3]. The implan - know, there is no direct evidence about the functional tation of the blastocyst into the maternal uterus is relations of taurine with uterine receptivity and embryo a crucial step in mammalian reproduction [4, 5]. It is implantation, although it is reported that Cdo knockout generally accepted that embryo implantation depends (KO) mice exhibit impaired reproductive capacity [26]. on blastocyst quality, endometrial receptivity, and the Taurine is one of the most abundant non-essential synchronization of their development [6]. In mice, amino acids in most mammals [27], and performs embryo implantation consists of apposition between numerous physiological functions, including bile salts the trophectoderm layer of blastocyst and the luminal synthesis and hepatoprotection [28], energy metabolism epithelium (LE), attachment and final invasion into the 2+ [29], maintenance of Ca homeostasis [30], anti-oxida- LE [7]. Upon embryo invasion, the uterine stromal cells tive, osmoregulation, anti-inflammatory and anti-apop - are rapidly remodeled in the process of decidualization, totic [31–33]. While the maintenance of the body taurine which is characterized by morphological and func- level mainly relays active uptake from diet and the endog- tional changes in stromal cells in the form of prolifera- enous taurine synthesis through the sequential actions tion and differentiation into large epithelioid decidual of its synthesis enzymes, including cysteine dioxygenase cells [8]. However, the cell proliferation and differentia - (CDO) [34]. tion of specific uterine cell types in early pregnancy are CDO is a critical enzyme for taurine synthesis and dependent on the coordinated actions of ovarian ster- CDO expression has been detected in liver, adipose tis- oid hormones, including progesterone (P ) and estro- sue, pancreas, kidneys, lungs and reproductive system gen (E ). During mouse pregnancy, an E surge on d 1 2 2 [35]. Cdo KO results in a higher incidence of postnatal stimulates uterine epithelial cell proliferation, and the mortality, retards postnatal growth and damages male decline of E level on d 2 leads to apoptosis of a large fertility [26, 36]. In addition, CDO is highly expressed in number of epithelial cells. P , from the newly formed mouse ovary and uterus [37], but the functions of CDO corpora lutea on d 3 [8], initiates uterine stromal cell in female reproduction remains unclear. Interestingly, proliferation. In conjunction with P , an acute E spike 4 2 uterus tissue and ULF are rich in taurine, and ULF tau- on d 4 further stimulates uterine stromal cell prolif- rine concentration is increased during embryo implan- eration and renders the uterus receptivity for the blas- tation [25]. Furthermore, CDO expression in uterus is tocyst to implant [8–10]. However, it is still largely up-regulated by P , whereas E decreases CDO expres- unknown about the molecular mechanisms of coordi- 4 2 sion [37]. In addition, our recent study shows that E reg- nate proliferative events induced by E and differentia - 2 2 ulates taurine synthesis through E -ERα-CSD signaling tive processes directed by P 4. 2 [38]. These make us to hypothesize that CDO may play Although the receptivity of the uterus during implanta- important roles in E and P primed embryo implanta- tion is primed by E and P , their actions on cell prolifera- 2 4 2 4 tion, possibly through the physiological actions of tau- tion and differentiation are complicated, and the relative rine. The present study was thus proposed to identify the molecular mechanisms have been extensively studied. effects of CDO and taurine on embryo implantation and The related molecular and genetic studies indicate that illustrate the related mechanisms by using Cdo KO and E and P act respectively via estrogen receptor 1 (Esr1, 2 4 ovariectomy (OVX) mouse models. ERα) [11] and progesterone receptor (Pgr, PR) [12] to govern the embryo-uterine crosstalk during peri-implan- Materials and methods tation stage by targeting local transcriptional factors, sig- Animals and treatments nals or paracrine molecules [13], some of which include Eight weeks old ICR mice were used in fertility test and Muc1 [14], Ltf [15], Hox10a [16, 17], Hand2 [17] and IHH other animal experiments. Cdo KO mice were gener- [18, 19]. In addition, uterine epithelium and its secre- ated by using 129 mice (Additional file  1). Mice were tions are essential for uterine receptivity and embryo raised in controlled temperature (25 ± 1 °C) and humidity implantation. Uterine epithelium includes LE and glan- (60%–70%) with a 12 h light, 12 h dark cycle. The animal dular epithelium (GE), which directly synthesize, secrete experiments were approved by the Chinese Association or selectively transport a wide variety of substances from for Laboratory Animal Sciences. Virgin female mice were serum and transudate, collectively termed histotroph mated with sexually matured males to induce pregnancy [20], into uterus lumen. Whereas histotroph is com- (day 1 is the day of vaginal plug checked, d 1). Embryos plex and comprised of many different substances, such Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 3 of 14 were collected from oviducts on d 1. The implantation (ab32063, abcam, Cambridge, UK; 1:2000) overnight at sites (IS) were visualized by intravenous injection of 4 °C. The PVDF membrane was then washed 3 times for 0.1 mL 1% Chicago blue dye (Sangon Biotech, Shanghai, 30  min in TBST (0.1% Tween-20 in TBS) and incubated China) in saline on d 5 [39]. To investigate the ovarian for 2 h with horseradish peroxidase-conjugated goat anti- hormonal influence on uterine CDO expression, wild- rabbit IgG or horseradish peroxidase-conjugated goat type (WT) mice were ovariectomized. Oil, 100 ng/mouse anti-mouse IgG (Zhongshan, Beijing, China). After wash- 17β-estradiol (E ; MedChemExpress, NJ, USA) or 2  mg/ ing for 30  min with 3 changes of TBST, the membrane mouse progesterone (P ; MedChemExpress, NJ, USA) was treated with the ECL kit (Vazyme, Nanjing, China) was injected 7  d later [40]. The treated mice were then and visualized by Tannon gel imager (Tanon, Shanghai, sacrificed at indicated times for further experiments. China). The intensity values pertaining to each group were normalized against the optical density of GAPDH corresponding to the same group. Real‑time quantitative PCR (RT‑qPCR) and common PCR Total RNA of the uterus tissues was isolated using the TRIzol reagent (Takara, Dalian, China), purified by Immunohistochemistry (IHC) and immunofluorescence (IF) DNase I and quantified by spectrophotometry. 1 μg puri - Tissues were fixed in 4% paraformaldehyde, dehydrated fied total RNA was used as a template for cDNA synthesis via graded ethanol solutions, and then embedded in par- using HiScript Reverse Transcriptase (Vazyme, Nanjing, affin to obtain 5  μm thick sections. IHC was performed China) according to the manufacturer’s instructions. as previously described [41]. The sections were incubated RT-qPCR was performed using SYBR Green master with CDO antibody (ab53436, abcam, Cambridge, UK; mix (Vazyme, Nanjing, China) in the StepOnePlus Real- 1:200), PR antibody (ab2765, abcam, Cambridge, UK; Time PCR System (Applied Biosystems, Foster City, CA, 1:200), ERα antibody (ab32063, abcam, Cambridge, UK; USA) and reactions were done in triplicate. RT-qPCR 1:200) or Ki67 antibody (D385, CST, MA, USA; 1:200) conditions were as follows: 95  °C for 2  min, followed by diluted in PBS overnight at 4 °C. After washing with PBS 40 cycles of 95  °C for 15  s and 60  °C for 1  min. Relative for 30 min, the sections were incubated with biotinylated gene expressions were normalized to endogenous con- goat anti-rabbit/mouse IgG (31820/31802, Thermofisher, trol Gapdh. All Primers listed in Table S3 were designed Waltham, MA, USA; 1:200) for 3  h at RT. After wash- using NCBI. ing with PBS for 30  min, the sections were incubated The genotype identification of the Cdo KO mice was with streptavidin peroxidase complex (SA10001, Ther - performed by common PCR using primers as described mofisher, Waltham, MA, USA; 1:200) for 30 min at room in Table S4. Amplifications were carried out on PCR temperature. Finally, the signals were visualized by incu- instrument (Bio-Rad, Hercules, CA, USA) using the fol- bating the sections with 0.05  mol/L Tris–HCL (pH 6.5) lowing protocol: 94  °C for 5  min (one time); 94  °C for containing 0.06% (w/v) diaminobenzidine (DAB, ZSGB- 50  s, 65  °C for 30  s, 72  °C for 30  s (35 times); 72  °C for Bio, Beijing, China) and 0.03% (v/v) H O . For IF stain- 10 min; and holding at 4 °C. 2 2 ing, Hand2 (ab200040, abcam, Cambridge, UK; 1:200), Muc1 (ab15481, abcam, Cambridge, UK; 1:200) primary Western blotting (WB) antibodies, and their respective secondary antibod- The uterus tissues were lysed with RIPA buffer (Beyotime, ies (Jackson Immuno Research, Philadelphia, PA, USA; Shanghai, China) containing 1  mmol/L phenylmeth- 1:200) were used. Nuclear staining was performed using anesulfonyl fluoride (PMSF, Sangon Biotech, Shanghai, 4’,6-diamidino-2-phenylindole (DAPI) dye (0.1  μg/mL, China). The protein concentration of each group was Beyotime, Shanghai, China). The signals were captured determined by using the BCA assay reagent (CoWin Bio- using a microscope (Olympus, Tokyo, Japan). sciences, Jiangsu, China) according to the manufacturer’s recommendations. Equal amounts of 50 μg proteins were electrophoresed on 12% sodium dodecyl sulfate–poly- In vivo production of embryo acrylamide gel (SDS-PAGE), and the bands were trans- Non-superovulated virgin female mice (8–9  weeks) ferred to 0.45  μm polyvinylidene difluoride (PVDF) were mated with adult males. The males and females membrane (Millipore, MA, USA). The membrane was (1:2) were housed overnight and the presence of a vagi- blocked with 5% (w/v) nonfat dry milk in 0.05  mol/L nal plug in the following morning was regarded as suc- pH 7.4 Tris buffered saline (TBS) for 3  h and incubated cessful mating (d 1 is the day of vaginal plug checked). with CDO antibody (ab53436, abcam, Cambridge, UK; Mice were killed by cervical dislocation and oviducts 1:2000), internal control GAPDH antibody (AM4300, were immediately dissected and placed in M2 solu- Ambion, TX, USA; 1:10,000), PR antibody (ab2765, tion (CaCl ·2H O 1.71  mmol/L, Glucose 5.56  mmol/L, abcam, Cambridge, UK; 1:2000) or ERα antibody 2 2 Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 4 of 14 methanol. Flow rate was 1.2  mL/min, and the detection HEPES 20.85  mmol/L, KCl 4.78  mmol/L, MgSO ·7H O 4 2 wavelength was 340 nm. The duration times were 2.3 min 1.19  mmol/L, NaCl 94.66  mmol/L, NaHCO and 4.95 min for the internal standard and taurine. 4.15  mmol/L, Sodium lactate 23.28  mmol/L, Sodium pyruvate 0.33 mmol/L) at 11:00 on d 2. Embryos punched Radioimmunoassay (RIA) out from the ampulla were digested with hyaluronidase Serum P and E were analyzed using RIA reagents pro- (HA) followed by three times washing with M2 and cul- 4 2 vided by the Beijing North Institute Biological Tech- tured in KSOM at 37 °C until the time of embryo transfer. nology (Beijing, China). The minimum detectable concentrations were 2 pg/mL for E and 0.2 ng/mL for P . Embryo transfer 2 4 For each RIA the intra and inter assay coefficients of vari - The uterine horn was exposed by flank laparotomy and ation were respectively less than 15% and 10%. six expanding embryos were transferred with a minimal amount of medium into the uterine cavity of pseudo- Statistical analysis pregnant (d 3) recipients. The uterine horn was then Statistical analysis was performed using GraphPad Prism placed back into the abdominal cavity and the incision 6.0. Data from at least three independent samples were site was closed. The procedure was repeated in the oppo - expressed as mean ± SEM. Two group comparison stud- site flank where another six expanding embryos were ies were performed using Student’s t-test and one-way transferred. The recipients were then placed individually analysis of variance (ANOVA) for data comprising three in clean cages to recover from anesthesia in a warm room or more groups. P < 0.05 was considered to be statistically (28–30 °C). significant. Fertility test Results Cdo KO females and their nesting WT females were Uterine CDO expression and taurine levels during early caged with adult wild-type males (8–9 weeks) at the rate pregnancy of male:female = 1:2. Vaginal plug was examined the In order to evaluate the physiological significance of next morning. Female mice with vaginal plug withdrew CDO and taurine in adult female mouse, we firstly exam - from the experiment, while the none plugged females ined the CDO expression profiles in different organs by were backed to the test after one day’s rest. The plugged RT-qPCR and WB. The results showed that CDO mRNA females were caged alone to observe their pregnancy and and protein were highly expressed in mouse uterus parturition. (Fig.  S1). Next, the uterine CDO mRNA and protein expressions, their relations to the changes of uterine tau- Measurement of taurine rine concentrations and serum steroid hormones were Uterus, liver, and serum taurine contents were measured analyzed in the duration of early pregnancy, from d 1 to by HPLC–UV (HPLC). Firstly, samples were weighed, d 8. RT-qPCR results showed that Cdo mRNA increased homogenized and deproteinized using 0.2  mol/L sulfo- from d 1 to d 4 and reached the maximum on d 4, which salicylic acid. After being centrifuged at 14,000  ×  g for followed by a sharp decline on d 5 and persisted on d 6 20  min, the supernatants were added into a dual-bed and d 8 (Fig. 1 A). WB results showed that CDO protein column containing cation exchange resins to remove levels were highly correlated with Cdo mRNA levels in other amino acids and metabolic precursors of tau- the duration examined (Fig. 1 B). In addition, IHC stain- rine. Secondly, samples were added with 100  μmol/L ing revealed that CDO was located in uterine epithelial glutamine as an internal standard. All samples were cells, including LE and GE, and some stromal cells (Fig. 1 then filtrated through a 0.22-μm PVDF membrane and E). The taurine concentrations in uterus tissue and serum saved in −80  °C refrigerator until use. The samples and were assayed by HPLC and the results indicated that the the standard samples of taurine which were 100, 50, 25, uterine taurine levels exhibited an increasing tendency 10, 5, 2 and 1  μmol/L were derivated with OPA (Sigma- from d 1 to d 4, and reached the maximum on d 4, fol- Aldrich, St. Louis, MO, USA) solution (20  mg OPA, lowed by a steady decline and returned to the similar 2  mL methanol, 80 μL 2-hydroxy-1-etanethiol, 18  mL level of d 1 on d 8 (Fig. 1 C). The serum taurine level also 0.1  mol/L borate buffer (pH 9.6)) for 3  min. Then 20 increased from d 1 to d 4, which significantly declined μL sample was automatically injected into a six-port from d 5 to d 8 (Fig.  1 C). In addition, the dynamic pat- valve to analysis with Waters Symmetry C18 Column terns of the uterine CDO expression and taurine concen- (4.6  μm, 150  mm × 5  mm) (Waters, Milford, MA, USA) tration corresponded to the change of serum P from d on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). 1 to d 6 (Fig.  1 D). These suggest that CDO might play The HPLC conditions were: flow A: 100% methanol, important roles in regulating embryo implantation. flow B: sodium phosphate buffer pH 4.7 containing 50% Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 5 of 14 Fig. 1 Uterine CDO mRNA and protein expressions and their relations to taurine levels in uterus from d 1 to d 8 of pregnancy mice. A, Cdo mRNA expression in mouse uteri detected by RT-qPCR (n = 3). B, CDO protein expression in uteri of d 1 to d 8 pregnancy mice assayed by WB. C, Serum and uterine taurine concentrations assayed by HPLC (n = 3). D, Serum P concentrations assayed by RIA (n = 5). E, CDO IHC staining of uterine cross-sections. Data shown as Mean ± SEM. Different letters represent significant differences (P < 0.05). d, days post coitum; E , estrogen; GE, glandular epithelium; LE, luminal epithelium; P , progesterone; St, stromal cells. Bars: 50 μm Establishment of Cdo KO mouse taurine levels of the Cdo KO females from d 1 to d 8 To detect the functions of CDO in uterus, Cdo KO mice were assayed by HPLC. The results showed that Cdo KO were generated by CRISPR/Cas9 technology. Two pairs of sharply decreased the taurine levels in the serum and single guide RNAs (sgRNAs) (Table S1) targeting exon1 uterus tissue compared with that of WT mice, and there and exon3 of Cdo gene respectively (Fig. 2 A), and SpCas9n were no significant differences from d 1 to d 8 (Fig.  3A, (D10A) mRNA were co-injected into d 1 embryos (Addi- B). However, there was a rising spike on d 4 in WT mice tional file  1). A Cdo KO mutant line with 7489 nucleotide (Fig. 3A, B). deletion from the first to the third exon was used in the fol - Further, the effects of Cdo deletion on the implanta - lowing experiments (Fig. 2 B). The genotypes were identified tion and fertility were examined. Adult WT and Cdo KO by PCR that the 245-base pair (bp) strand represent mutant females were respectively mated with ICR males, the par- type and the 325 bp strand represent wild type (Fig. 2 C). The turition and pups of each litter were then recorded. The knockout efficiency was identified by RT-qPCR, IHC and results showed that the plug-positive WT females exhib- WB, and the results showed that CDO was totally deleted, at ited normal fecundity, but only 38.8% (7/18) of the plug- least in mouse uterus (Fig. 2D–F). As CDO is a key enzyme positive Cdo KO females produced litters (Fig.  3 C), and for taurine synthesis, we detected the changes of taurine lev- the litter size (5.429 ± 0.65 pups/litter, n = 7) was much els after Cdo knocked out using HPLC. The results showed smaller than that of the WT females (11.29 ± 0.68 pups/ that the taurine concentrations in the liver, serum and uterus litter, n = 7) (Fig. 3 D). of Cdo KO mice decreased by 76.24%, 51.20% and 70.33% In order to answer whether the subfertility of Cdo KO respectively than that of WT mice (Fig. 2G–I). These results mice was resulted from the abnormalities in the ovaries demonstrate that CDO is successfully deleted from mouse or embryos, the embryos were flushed out from oviducts genome and the lack of CDO leads to taurine deficiency. of d 2 Cdo KO females, which naturally mated with WT males. The flushed 2-cell embryo numbers did not have Cdo KO causes impairment of embryo implantation significant difference between WT and Cdo KO mice and severe subfertility (Fig.  S2 B), and morphological defect was not observed In order to identify the effects of Cdo deletion on either (Fig. S2 A). In addition, the histological abnormal- the embryo implantation, the serum and uterine ity was not observed in 10 weeks Cdo KO mouse ovaries Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 6 of 14 Fig. 2 Generation of Cdo KO mouse. A, Cas9n target scheme in the first and the third exons of Cdo gene. The blue lines indicate sgRNAs’ spacer and the red lines indicate PAM (protospacer adjacent motif ). B, Sanger sequencing of the target region in F0 mice. The Cdo KO mice get a miss of 7489 nucleotides. C, Genotype identification results using PCR. The 325 bp strand indicates wild type and the 245 bp strand indicates mutant type. D, IHC staining of CDO in WT and Cdo KO mouse uterus. Bars, 50 μm. E, RT-qPCR analysis of Cdo mRNA levels in WT and Cdo KO mouse uteri. F, Representative patterns of WB analysis of CDO expressions. G-I, Taurine levels in WT and Cdo KO mice liver, serum and uterus detected by HPLC. Data are the Mean ± SEM. (n ≥ 3). Different letters above columns indicate significant differences (P < 0.05). GE: glandular epithelium. LE: luminal epithelium. Sm: smooth muscle cells. St: stromal cells. Scale bars: 50 μm (Fig. S2 C). Serum P and E levels did not exhibit signifi - identified by employing the Chicago blue dye assay [39]. 4 2 cant differences between the Cdo KO and WT females on The implantation sites (IS) were clearly observed in WT d 4 (Fig. S2 D and E). females, but only a few or no IS were detected in Cdo Further, in order to dissect the cause of the subfer- KO females (Fig.  3 E, G). However, Cdo KO females tility in Cdo KO females, the effect of Cdo deletion on had blastocysts which could be flushed out from uterus the embryo implantation was examined. The plug posi - (Fig. 3 F). tive mice were euthanized on d 5 and the numbers In order to confirm whether the subfertility of Cdo of embryos implanted to the uterine epithelium were KO females was resulted from the defects of uterus Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 7 of 14 Fig. 3 CDO deletion causes impairment of embryo implantation and severe subfertility. A and B, Serum (A) and uterus (B) taurine concentrations of pregnancy Cdo KO and WT females from d 1 to d 8, assayed by HPLC (n = 3). C, Pregnancy rates of WT and KO females. The number within brackets indicate females with pups over total number of plug-positive females. D, Average litter sizes of WT and Cdo KO females (n = 7). E, Representative images of d 5 pregnant uteri from WT and Cdo KO females. Bars, 1 cm. F, Blastocysts flushed from Cdo KO uterus. Bar, 50 μm. G, Average numbers of implanted embryos represented by implantation sites (n = 5). H, Representative patterns of d 5 WT receptive mice uteri transferred with WT and Cdo KO embryos. I, d 5 KO receptive mice uteri transferred with WT embryos. Arrowheads indicate the location of blastocysts. Bars, 1 cm. K, IS numbers of different treated mice uteri (n = 3). Data are presented as Mean ± SEM. Different letters above columns indicate significant differences (P < 0.05) receptive status, Cdo KO and WT females were sepa- which the WT and Cdo KO embryos were respectively rately mated with Cdo KO and WT males to obtain Cdo transferred (Fig.  3 H, K). However, the IS number in KO and WT embryos. The harvested Cdo KO and WT Cdo KO receptive females was much less than in WT embryos were respectively transferred to WT female receptive females after both of which accepted WT uteri. On d 5, IS numbers were assayed and there was embryos (Fig.  3 I, K). Meanwhile, blastocysts could be no significant difference between the WT females to flushed out from Cdo KO uterus (Fig.  3  J). These infer Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 8 of 14 that the failure of embryo implantation and subfertility uterine PR expression during early stage of pregnancy in Cdo KO females are resulted from the defects of uter- (Fig.  1A–D  and Fig.  3A, B), we further identified the ine receptivity. effects of P and E on uterine CDO expression by using 4 2 OVX mouse model. RT-qPCR results showed that uter- Cdo KO results in the defects of uterine receptivity ine Cdo mRNA levels were decreased by 54.65%, 90% and by dysregulating PR and ER signaling in mouse 93% after 2, 6 and 12 h after 100 ng E treatments in OVX Our results showed that Cdo KO had no significant effects mice (Fig.  5 A). These were further confirmed by the on ovary morphology and functions, including E and P CDO IHC staining results that the CDO staining inten- 2 4 secretions (Fig.  S2C–E). As the window of uterine recep- sity got much weaker after 12  h E treatment (Fig.  5 C). tivity coincides with the P -mediated down-regulation of It was opposite that 2  mg P injection sharply increased 4 4 ERα activity in uterine LE, we thus assayed the effects of uterine CDO mRNA and protein levels, which were sev- Cdo deletion on uterus PR expression on d 4 by RT-qPCR eral folds higher than that of the controls after 12  h P and WB. The results showed that PR mRNA and protein treatment (Fig.  5 B and C). Further, in order to identify levels in Cdo KO uteri respectively decreased by 28.74% whether the regulating effects of P and E on uterine 4 2 and 26.63% compared with that in WT uteri (Fig.  4A–C). CDO expressions were through P -PR and E -ERα sign- 4 2 IHC results showed that PR staining intensity on LE, GE aling, OVX mice were respectively pretreated with PR and stromal cells were much weaker in Cdo KO mice than inhibitor RU486 and ERα inhibitor ICI182780, followed in WT mice (Fig.  4 G). In addition, the uterine mRNA by P or E treatment. The uterine CDO expressions were 4 2 expression levels of the known PR responsive genes Ihh, then assayed and the results showed that RU486 blocked Hoxa10 and Hand2 decreased by 56.33%, 35.49% and the enhancing effect of P on CDO expression (Fig.  5G– 45.38% in KO mice than that of the WT mice (Fig.  4 H). I), and ICI182780 restrained the inhibiting effect of E on Further, Hand2 IF staining was performed and it was CDO expression (Fig. 5D–F). observed that Hand2 was located only in the stroma cells, but its staining intensity was much weaker in Cdo KO mice Taurine supplement partly recues the defects of embryo than that in WT mice (Fig. 4 J), which was consistent with implantation and subfertility caused by Cdo KO PR IHC staining result (Fig. 4 G). As the maintenance of the global taurine level relays on In addition, Cdo deletion had no significant effects on the endogenous taurine synthesis through the action of ERα mRNA and protein levels (Fig. 4D–G). Whereas the CDO and the active uptake from the diet, we thus sup- expression levels of E -responsive genes Muc1 and Ltf plied extra taurine in the regular diet of Cdo KO mice to elevated over 2.5 and 8 times in Cdo KO uterus than that confirm whether the defects of uterine receptivity and of the WT uterus on d 4 (Fig.  4 I). The further IF stain - subfertility caused by Cdo KO could be rescued. Adult ing confirmed the Muc1 expression in LE and GE cells of female Cdo KO mice were supplemented with drink- Cdo KO mouse uteri, but which was not detected in WT ing water containing 0.2% (w/v) taurine. The uterine and mice (Fig. 4 J). serum taurine levels were assayed. The results showed Another important character of uterus receptivity to that taurine supplement markedly elevated taurine lev- embryo is a cessation of epithelial cell proliferation and els in uteri and serum, but all of which were significantly robust proliferation of stroma prior to implantation. We lower than that in wild mice with regular diet (Fig.  6A, thus detected the cell proliferation in the uterine tissues B). In addition, the embryo implantation was detected of Cdo KO and WT mice on d 4 by Ki67 IHC staining. on d 5. The results showed that taurine supplement sig - The results showed that Ki67 was not detected in LE nificantly increased the IS number of Cdo KO females, and GE cells of WT and Cdo KO mice uterus, but Ki67 although it was still lower than that of the WT females staining intensity in the stroma of WT uteri was much (Fig.  6C–E). These results demonstrate that taurine sup - stronger than that of Cdo KO uteri. Moreover, Ki67 posi- plementation can partly rescue the subfertility of Cdo KO tive cells were concentrated around the LE in WT uteri females and suggest that CDO and taurine are essential (Fig.  4  K), but Ki67 positive cells in Cdo KO uteri were factors for embryo implantation. irregularly scattered (Fig.  4  K). These indicate that Cdo KO results in the defects of uterine receptivity by dysreg- Discussion ulating PR and ER signaling in mouse. The present study, for the first time as we know, dem - onstrates that CDO and taurine play important roles for P and  E are involved in regulating uterine CDO embryo implantation in mouse. This is supported by our 4 2 expression results here that uterine CDO expression and serum tau- As the dynamic patterns of uterine CDO expression and rine level during early stage of mouse pregnancy are par- taurine level were parallel to serum P concentration and allel to the uterine taurine level, which sharply increases 4 Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 9 of 14 Fig. 4 Eec ff ts of Cdo deletion on PR and ER signaling transduction and the cell proliferation in the uteri of d 4 pregnancy mice. A, RT-qPCR analysis of relative Pgr mRNA expression levels. B and C, WB detection and analysis of PR protein expression. D, RT-qPCR analysis of relative Esr1 mRNA levels. E and F, WB detection and analysis of ERα protein expression levels. G, IHC staining of PR and ERα. Scale bars: 50 μm. H, Relative mRNA expressions of Ihh, Areg and Hand2. I, Relative mRNA levels of Ltf and Muc1. J, IF staining of Hand2 and Muc1. Scale bars: 50 μm. K, Ki67 (proliferative cell marker) IHC staining. Scale bars: 50 μm. GE: glandular epithelium. LE: luminal epithelium. St: stroma. Data are presented as Mean ± SEM, n ≥ 4. Different letters represent significant differences (P < 0.05) and reaches a peak just on d 4, the window of implan- regulate the embryo implantation [7, 43], behaves a simi- tation [42], followed by a steadily decline under physi- lar dynamic pattern with the uterine CDO expression and ological condition. In addition, serum P , a key factor to taurine concentration in the duration examined. Whereas 4 Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 10 of 14 global Cdo KO markedly decreases uterine taurine level Ueki et al. [26]. Another possible explanation is that dif- and omits the dynamic pattern of uterine taurine, accom- ferent mouse strains have been used in our study and the panied by the decline of serum taurine level, although previous report [26]. Cdo KO does not affect the ovary functions to synthesize In addition, the results presented here demonstrate P and E . These demonstrate that uterus has function to that Cdo deletion impairs embryo implantation by dys- 4 2 synthesize taurine, whereas Cdo deletion causes a severe regulating P and E signaling in mouse uterus. The 4 2 subfertility in female mice. CDO and endogenous taurine related molecular and genetic studies indicate that both synthesis are thus essential for embryo implantation and E and P play functions through their corresponding 2 4 the maintenance of animal reproduction. These are also receptor ERα and PR [48, 49] together with local tran- supported by our results here that taurine supplement scriptional and paracrine factors to govern the compli- to the Cdo KO mice can partly rescue the reproductive cated embryo-uterine crosstalk [11, 12, 43, 50]. It is much capacity. interested that our results here show that CDO involves Another important finding of this study is that Cdo in regulating embryo implantation by affecting P and E 4 2 KO causes defects in uterine epithelium receptivity and signaling. In support, the uterus PR mRNA and protein decline of pregnancy rate. It has been well documented levels in Cdo deleted mice are significantly decreased, that successful embryo implantation depends on the accompanied by decline of Ihh, Hoxa10 and Hand2 intimate connection between the blastocyst and mater- expressions, which are known PR targeting molecules nal endometrial surface [43, 44]. Healthy embryos at the [18, 43, 51]. These are also confirmed by PR and Hand2 blastocyst stage and receptive maternal endometrium are IHC results of this study. However, Cdo deletion does necessary for the implantation [45]. The results of this not affect ERα mRNA and protein levels, but the mRNA study show that Cdo deleted embryos do not have mor- levels of E -ERα responsive genes Lf and Muc1 [14, 15] phological defects, and behave normal implantation as are increased over 5 and 1.5 time in Cdo KO mouse uteri, that of the WT embryos in WT recipients. However, the especially the abnormal expression of Muc1 in Cdo KO implantation and birth rates are dramatically decreased LE and GE cells. In addition, CDO or/and taurine might after the WT embryos are transferred to the Cdo deleted participate in E -ERα signaling by affecting the ERα activ - recipients. These imply that Cdo deleted embryos have ity through ERα Ser 118 site phosphorylation [43, 52], normal capacity to get implantation. Another important although we did not detect it in this study. Collectively, character of uterine receptivity is the cessation in epithe- the results presented here demonstrate that CDO is a lial cell proliferation and robust proliferation in stroma crucial factor to affect uterine receptivity during embryo cells just prior to implantation [46, 47]. The results of implantation by involving in P -PR and E -ERα signaling 4 2 present study are in agreement with reports [44, 46] in mouse uterus, although the relating signaling pathways that the cell proliferation, marked by Ki67 IHC staining, and mechanisms need to be clarified in future study. is undetectable in the uterine epithelium, but the num- Furthermore, the regulating effects of P and E on 4 2 ber of Ki67 positive stroma cells significantly decreases uterine CDO are confirmed by using OVX mouse in Cdo KO mice compared with that of WT mice. These model. The in vivo results show that uterus CDO expres - collective data suggest that Cdo deletion leads to the sion level is decreased over 90% after 12  h E treatment abnormalities of uterine receptivity and embryo implan- in OVX mouse, which is in agreement with the reports tation, and the subfertility of Cdo KO mice is primarily of that E inhibits taurine synthesis through estrogen-ERs- uterine origin. CDO/CSAD signaling in liver and uterus [37, 38]. It is However, it has been reported that Cdo KO females are inversed that uterine CDO expression level is sharply fertile and carry their pregnancies to term [26], whereas elevated after P injection in OVX mouse. In addition, our results here demonstrate that 38.8% of plug-positive it is well known that E and P are key drivers of uter- 2 4 Cdo KO females are fertile, and the IS number and litter ine plasticity throughout the sexual cycle and early stage size of Cdo KO females are much less than that of WT of pregnancy, we thus propose that both E and P may 2 4 females. These discordant statements might be resulted play roles through their respective receptors to regulate from the limited assessment, and no description about uterine CDO expression and taurine synthesis, which the statistics analysis of the IS numbers and birth rates by subsequently affect the uterine receptivity and embryo (See figure on next page.) Fig. 5 The relations of P -PR and E -ERα signaling to uterine CDO expression. A and B, Relative Cdo mRNA levels in OVX mouse uteri after 0 4 2 (control), 2, 6 and 12 h treatments with 100 ng E (A) and 2 mg P (B). C, CDO IHC staining in OVX mouse uteri after 0 (control) and 12 h 100 ng E 2 4 2 or 2 mg P treatments. Scale bars: 50 μm. D, E and F, Relative Cdo mRNA (D) and protein levels (E and F) in OVX mouse uteri treated with E and E 4 2 2 inhibitor ICI182780 (ICI). G, H and I, Relative Cdo mRNA (G) and protein levels (H and I) in OVX mouse uteri treated with 2 mg P and PR inhibitor RU486. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n = 3) Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 11 of 14 Fig. 5 (See legend on previous page.) Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 12 of 14 Fig. 6 Taurine supplement increases the taurine levels in serum and uteri, and partly rescues the defects of embryo implantation in the CDO KO mice. A, Serum taurine concentration. B, Taurine concentration in uterus tissues. C, Representative images of implantation sites. Bars: 1 cm. WT: wide type female mice. KO: Cdo KO female mice. KO + taurine: Cdo KO female mice supplied with taurine. n = 3 for each genotype. D, Blastocysts flushed out from Cdo KO uterus. Bar, 50 μm. E, Numbers of implantation sites. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n ≥ 3) implantation. However, it remains to be elucidated about receptivity and embryo implantation by involving in the interactions or cross-talks among P -PR signaling, E -ERα and P -PR signaling pathways. These are crucial 4 2 4 E -ERα signaling, CDO and taurine in the duration of for our understanding the mechanism of embryo implan- embryo implantation. tation, and infer that taurine is a potential agent for Finally, the maintenance of taurine levels in uterus improving reproductive efficiency of livestock industry and serum relays on the endogenous taurine synthesis and/or for reproductive medicine. But it still remains to through the action of CSAD, CDO, and the active uptake be elucidated about the interactions among P -PR signal- from the diet [34, 35], while Cdo deletion impairs embryo ing, E -ERα signaling, CDO and taurine in the duration implantation and causes severe subfertility as it is showed of embryo implantation. in this study. Whereas taurine supplementation signifi cantly increases the litter size and parturition rate of Cdo Conclusions KO females (Table S2). These provide the in  vivo evi - The present study demonstrates that taurine and CDO dence that CDO and taurine are crucial factors for the play prominent roles for the uterine receptivity and maintenance and improvement of animal production, embryo implantation by involving in E -ERα and P -PR 2 4 and suggest that taurine may be potential agent for ani- signaling in early pregnancy stage. These elucidate a mal production. new mechanism for taurine regulating embryo implan- In conclusion, the present study demonstrates that tation. On the other side, abuse of steroid hormones in taurine and CDO play prominent roles for uterine stockbreeding industry usually gives rise to premature Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 13 of 14 Consent for publication ovary failure and declines animal fertility. The balancing Not applicable. effect of CDO and taurine between P -PR signaling and E -ERα signaling shows that taurine is a potential agent Competing interests The authors declare no competing interests. for improving reproductive efficiency of livestock indus - try and reproductive medicine. Received: 19 July 2022 Accepted: 20 November 2022 Abbreviations CDO Cysteine dioxygenase E Estrogen Esr1 (ERα) Estr ogen receptor 1 References GE Glandular epithelium 1. Boivin J, Bunting L, Collins JA, Nygren KG. 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Cysteine dioxygenase and taurine are essential for embryo implantation by involving in E2-ERα and P4-PR signaling in mouse

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10.1186/s40104-022-00804-1
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Abstract

Background Taurine performs multiple physiological functions, and the maintenance of taurine level for most mam- mals relies on active uptake from diet and endogenous taurine synthesis through its synthesis enzymes, including cysteine dioxygenase (CDO). In addition, uterus tissue and uterus fluid are rich in taurine, and taurine synthesis is regulated by estrogen (E ) and progesterone (P ), the key hormones priming embryo-uterine crosstalk during embryo 2 4 implantation, but the functional interactions and mechanisms among which are largely unknown. The present study was thus proposed to identify the effects of CDO and taurine on embryo implantation and related mechanisms by using Cdo knockout (KO) and ovariectomy (OVX) mouse models. Results The uterine CDO expression was assayed from the first day of plugging (d 1) to d 8 and the results showed that CDO expression level increased from d 1 to d 4, followed by a significant decline on d 5 and persisted to d 8, which was highly correlated with serum and uterine taurine levels, and serum P concentration. Next, Cdo KO mouse was established by CRISPER/Cas9. It was showed that Cdo deletion sharply decreased the taurine levels both in serum and uterus tissue, causing implantation defects and severe subfertility. However, the implantation defects in Cdo KO mice were partly rescued by the taurine supplementation. In addition, Cdo deletion led to a sharp decrease in the expressions of P receptor (PR) and its responsive genes Ihh, Hoxa10 and Hand2. Although the expression of uterine estrogen receptor (ERα) had no significant change, the levels of ERα induced genes (Muc1, Ltf) during the implanta- tion window were upregulated after Cdo deletion. These accompanied by the suppression of stroma cell proliferation. Meanwhile, E inhibited CDO expression through ERα and P upregulated CDO expression through PR. 2 4 Conclusion The present study firstly demonstrates that taurine and CDO play prominent roles in uterine receptiv- ity and embryo implantation by involving in E -ERα and P -PR signaling. These are crucial for our understanding the 2 4 mechanism of embryo implantation, and infer that taurine is a potential agent for improving reproductive efficiency of livestock industry and reproductive medicine. Keywords CDO, E , Embryo implantation, P , Taurine 2 4 Di Zhang and Zhijuan Wang contributed equally to this work. Yangzhou 225009, People’s Republic of China State Key Laboratory of Agrobiotechnology, College of Biological *Correspondence: Sciences, China Agricultural University, 100193 Beijing, People’s Republic Sheng Cui of China cuisheng@yzu.edu.cn Institute of Reproduction and Metabolism, Yangzhou University, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu 225009 Jiangsu, People’s Republic of China 225009, People’s Republic of China Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou University, © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. The Creative Commons Public Domain Dedication waiver (http:// creat iveco mmons. org/ publi cdoma in/ zero/1. 0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 2 of 14 as leukemia inhibitory factor (LIF), ions, sugars, lipids, Background proteins and amino acids [20–24], among which taurine High rate of embryo loss in early pregnancy is a major is included [25]. It is much interested that taurine con- constraint both in livestock industry and human repro- centration in the uterine luminal fluid (UFL) of mouse duction, whereas much pregnancy wastage is caused by is much higher during the implantation [25]. But as we the failure of embryo implantation [1–3]. The implan - know, there is no direct evidence about the functional tation of the blastocyst into the maternal uterus is relations of taurine with uterine receptivity and embryo a crucial step in mammalian reproduction [4, 5]. It is implantation, although it is reported that Cdo knockout generally accepted that embryo implantation depends (KO) mice exhibit impaired reproductive capacity [26]. on blastocyst quality, endometrial receptivity, and the Taurine is one of the most abundant non-essential synchronization of their development [6]. In mice, amino acids in most mammals [27], and performs embryo implantation consists of apposition between numerous physiological functions, including bile salts the trophectoderm layer of blastocyst and the luminal synthesis and hepatoprotection [28], energy metabolism epithelium (LE), attachment and final invasion into the 2+ [29], maintenance of Ca homeostasis [30], anti-oxida- LE [7]. Upon embryo invasion, the uterine stromal cells tive, osmoregulation, anti-inflammatory and anti-apop - are rapidly remodeled in the process of decidualization, totic [31–33]. While the maintenance of the body taurine which is characterized by morphological and func- level mainly relays active uptake from diet and the endog- tional changes in stromal cells in the form of prolifera- enous taurine synthesis through the sequential actions tion and differentiation into large epithelioid decidual of its synthesis enzymes, including cysteine dioxygenase cells [8]. However, the cell proliferation and differentia - (CDO) [34]. tion of specific uterine cell types in early pregnancy are CDO is a critical enzyme for taurine synthesis and dependent on the coordinated actions of ovarian ster- CDO expression has been detected in liver, adipose tis- oid hormones, including progesterone (P ) and estro- sue, pancreas, kidneys, lungs and reproductive system gen (E ). During mouse pregnancy, an E surge on d 1 2 2 [35]. Cdo KO results in a higher incidence of postnatal stimulates uterine epithelial cell proliferation, and the mortality, retards postnatal growth and damages male decline of E level on d 2 leads to apoptosis of a large fertility [26, 36]. In addition, CDO is highly expressed in number of epithelial cells. P , from the newly formed mouse ovary and uterus [37], but the functions of CDO corpora lutea on d 3 [8], initiates uterine stromal cell in female reproduction remains unclear. Interestingly, proliferation. In conjunction with P , an acute E spike 4 2 uterus tissue and ULF are rich in taurine, and ULF tau- on d 4 further stimulates uterine stromal cell prolif- rine concentration is increased during embryo implan- eration and renders the uterus receptivity for the blas- tation [25]. Furthermore, CDO expression in uterus is tocyst to implant [8–10]. However, it is still largely up-regulated by P , whereas E decreases CDO expres- unknown about the molecular mechanisms of coordi- 4 2 sion [37]. In addition, our recent study shows that E reg- nate proliferative events induced by E and differentia - 2 2 ulates taurine synthesis through E -ERα-CSD signaling tive processes directed by P 4. 2 [38]. These make us to hypothesize that CDO may play Although the receptivity of the uterus during implanta- important roles in E and P primed embryo implanta- tion is primed by E and P , their actions on cell prolifera- 2 4 2 4 tion, possibly through the physiological actions of tau- tion and differentiation are complicated, and the relative rine. The present study was thus proposed to identify the molecular mechanisms have been extensively studied. effects of CDO and taurine on embryo implantation and The related molecular and genetic studies indicate that illustrate the related mechanisms by using Cdo KO and E and P act respectively via estrogen receptor 1 (Esr1, 2 4 ovariectomy (OVX) mouse models. ERα) [11] and progesterone receptor (Pgr, PR) [12] to govern the embryo-uterine crosstalk during peri-implan- Materials and methods tation stage by targeting local transcriptional factors, sig- Animals and treatments nals or paracrine molecules [13], some of which include Eight weeks old ICR mice were used in fertility test and Muc1 [14], Ltf [15], Hox10a [16, 17], Hand2 [17] and IHH other animal experiments. Cdo KO mice were gener- [18, 19]. In addition, uterine epithelium and its secre- ated by using 129 mice (Additional file  1). Mice were tions are essential for uterine receptivity and embryo raised in controlled temperature (25 ± 1 °C) and humidity implantation. Uterine epithelium includes LE and glan- (60%–70%) with a 12 h light, 12 h dark cycle. The animal dular epithelium (GE), which directly synthesize, secrete experiments were approved by the Chinese Association or selectively transport a wide variety of substances from for Laboratory Animal Sciences. Virgin female mice were serum and transudate, collectively termed histotroph mated with sexually matured males to induce pregnancy [20], into uterus lumen. Whereas histotroph is com- (day 1 is the day of vaginal plug checked, d 1). Embryos plex and comprised of many different substances, such Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 3 of 14 were collected from oviducts on d 1. The implantation (ab32063, abcam, Cambridge, UK; 1:2000) overnight at sites (IS) were visualized by intravenous injection of 4 °C. The PVDF membrane was then washed 3 times for 0.1 mL 1% Chicago blue dye (Sangon Biotech, Shanghai, 30  min in TBST (0.1% Tween-20 in TBS) and incubated China) in saline on d 5 [39]. To investigate the ovarian for 2 h with horseradish peroxidase-conjugated goat anti- hormonal influence on uterine CDO expression, wild- rabbit IgG or horseradish peroxidase-conjugated goat type (WT) mice were ovariectomized. Oil, 100 ng/mouse anti-mouse IgG (Zhongshan, Beijing, China). After wash- 17β-estradiol (E ; MedChemExpress, NJ, USA) or 2  mg/ ing for 30  min with 3 changes of TBST, the membrane mouse progesterone (P ; MedChemExpress, NJ, USA) was treated with the ECL kit (Vazyme, Nanjing, China) was injected 7  d later [40]. The treated mice were then and visualized by Tannon gel imager (Tanon, Shanghai, sacrificed at indicated times for further experiments. China). The intensity values pertaining to each group were normalized against the optical density of GAPDH corresponding to the same group. Real‑time quantitative PCR (RT‑qPCR) and common PCR Total RNA of the uterus tissues was isolated using the TRIzol reagent (Takara, Dalian, China), purified by Immunohistochemistry (IHC) and immunofluorescence (IF) DNase I and quantified by spectrophotometry. 1 μg puri - Tissues were fixed in 4% paraformaldehyde, dehydrated fied total RNA was used as a template for cDNA synthesis via graded ethanol solutions, and then embedded in par- using HiScript Reverse Transcriptase (Vazyme, Nanjing, affin to obtain 5  μm thick sections. IHC was performed China) according to the manufacturer’s instructions. as previously described [41]. The sections were incubated RT-qPCR was performed using SYBR Green master with CDO antibody (ab53436, abcam, Cambridge, UK; mix (Vazyme, Nanjing, China) in the StepOnePlus Real- 1:200), PR antibody (ab2765, abcam, Cambridge, UK; Time PCR System (Applied Biosystems, Foster City, CA, 1:200), ERα antibody (ab32063, abcam, Cambridge, UK; USA) and reactions were done in triplicate. RT-qPCR 1:200) or Ki67 antibody (D385, CST, MA, USA; 1:200) conditions were as follows: 95  °C for 2  min, followed by diluted in PBS overnight at 4 °C. After washing with PBS 40 cycles of 95  °C for 15  s and 60  °C for 1  min. Relative for 30 min, the sections were incubated with biotinylated gene expressions were normalized to endogenous con- goat anti-rabbit/mouse IgG (31820/31802, Thermofisher, trol Gapdh. All Primers listed in Table S3 were designed Waltham, MA, USA; 1:200) for 3  h at RT. After wash- using NCBI. ing with PBS for 30  min, the sections were incubated The genotype identification of the Cdo KO mice was with streptavidin peroxidase complex (SA10001, Ther - performed by common PCR using primers as described mofisher, Waltham, MA, USA; 1:200) for 30 min at room in Table S4. Amplifications were carried out on PCR temperature. Finally, the signals were visualized by incu- instrument (Bio-Rad, Hercules, CA, USA) using the fol- bating the sections with 0.05  mol/L Tris–HCL (pH 6.5) lowing protocol: 94  °C for 5  min (one time); 94  °C for containing 0.06% (w/v) diaminobenzidine (DAB, ZSGB- 50  s, 65  °C for 30  s, 72  °C for 30  s (35 times); 72  °C for Bio, Beijing, China) and 0.03% (v/v) H O . For IF stain- 10 min; and holding at 4 °C. 2 2 ing, Hand2 (ab200040, abcam, Cambridge, UK; 1:200), Muc1 (ab15481, abcam, Cambridge, UK; 1:200) primary Western blotting (WB) antibodies, and their respective secondary antibod- The uterus tissues were lysed with RIPA buffer (Beyotime, ies (Jackson Immuno Research, Philadelphia, PA, USA; Shanghai, China) containing 1  mmol/L phenylmeth- 1:200) were used. Nuclear staining was performed using anesulfonyl fluoride (PMSF, Sangon Biotech, Shanghai, 4’,6-diamidino-2-phenylindole (DAPI) dye (0.1  μg/mL, China). The protein concentration of each group was Beyotime, Shanghai, China). The signals were captured determined by using the BCA assay reagent (CoWin Bio- using a microscope (Olympus, Tokyo, Japan). sciences, Jiangsu, China) according to the manufacturer’s recommendations. Equal amounts of 50 μg proteins were electrophoresed on 12% sodium dodecyl sulfate–poly- In vivo production of embryo acrylamide gel (SDS-PAGE), and the bands were trans- Non-superovulated virgin female mice (8–9  weeks) ferred to 0.45  μm polyvinylidene difluoride (PVDF) were mated with adult males. The males and females membrane (Millipore, MA, USA). The membrane was (1:2) were housed overnight and the presence of a vagi- blocked with 5% (w/v) nonfat dry milk in 0.05  mol/L nal plug in the following morning was regarded as suc- pH 7.4 Tris buffered saline (TBS) for 3  h and incubated cessful mating (d 1 is the day of vaginal plug checked). with CDO antibody (ab53436, abcam, Cambridge, UK; Mice were killed by cervical dislocation and oviducts 1:2000), internal control GAPDH antibody (AM4300, were immediately dissected and placed in M2 solu- Ambion, TX, USA; 1:10,000), PR antibody (ab2765, tion (CaCl ·2H O 1.71  mmol/L, Glucose 5.56  mmol/L, abcam, Cambridge, UK; 1:2000) or ERα antibody 2 2 Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 4 of 14 methanol. Flow rate was 1.2  mL/min, and the detection HEPES 20.85  mmol/L, KCl 4.78  mmol/L, MgSO ·7H O 4 2 wavelength was 340 nm. The duration times were 2.3 min 1.19  mmol/L, NaCl 94.66  mmol/L, NaHCO and 4.95 min for the internal standard and taurine. 4.15  mmol/L, Sodium lactate 23.28  mmol/L, Sodium pyruvate 0.33 mmol/L) at 11:00 on d 2. Embryos punched Radioimmunoassay (RIA) out from the ampulla were digested with hyaluronidase Serum P and E were analyzed using RIA reagents pro- (HA) followed by three times washing with M2 and cul- 4 2 vided by the Beijing North Institute Biological Tech- tured in KSOM at 37 °C until the time of embryo transfer. nology (Beijing, China). The minimum detectable concentrations were 2 pg/mL for E and 0.2 ng/mL for P . Embryo transfer 2 4 For each RIA the intra and inter assay coefficients of vari - The uterine horn was exposed by flank laparotomy and ation were respectively less than 15% and 10%. six expanding embryos were transferred with a minimal amount of medium into the uterine cavity of pseudo- Statistical analysis pregnant (d 3) recipients. The uterine horn was then Statistical analysis was performed using GraphPad Prism placed back into the abdominal cavity and the incision 6.0. Data from at least three independent samples were site was closed. The procedure was repeated in the oppo - expressed as mean ± SEM. Two group comparison stud- site flank where another six expanding embryos were ies were performed using Student’s t-test and one-way transferred. The recipients were then placed individually analysis of variance (ANOVA) for data comprising three in clean cages to recover from anesthesia in a warm room or more groups. P < 0.05 was considered to be statistically (28–30 °C). significant. Fertility test Results Cdo KO females and their nesting WT females were Uterine CDO expression and taurine levels during early caged with adult wild-type males (8–9 weeks) at the rate pregnancy of male:female = 1:2. Vaginal plug was examined the In order to evaluate the physiological significance of next morning. Female mice with vaginal plug withdrew CDO and taurine in adult female mouse, we firstly exam - from the experiment, while the none plugged females ined the CDO expression profiles in different organs by were backed to the test after one day’s rest. The plugged RT-qPCR and WB. The results showed that CDO mRNA females were caged alone to observe their pregnancy and and protein were highly expressed in mouse uterus parturition. (Fig.  S1). Next, the uterine CDO mRNA and protein expressions, their relations to the changes of uterine tau- Measurement of taurine rine concentrations and serum steroid hormones were Uterus, liver, and serum taurine contents were measured analyzed in the duration of early pregnancy, from d 1 to by HPLC–UV (HPLC). Firstly, samples were weighed, d 8. RT-qPCR results showed that Cdo mRNA increased homogenized and deproteinized using 0.2  mol/L sulfo- from d 1 to d 4 and reached the maximum on d 4, which salicylic acid. After being centrifuged at 14,000  ×  g for followed by a sharp decline on d 5 and persisted on d 6 20  min, the supernatants were added into a dual-bed and d 8 (Fig. 1 A). WB results showed that CDO protein column containing cation exchange resins to remove levels were highly correlated with Cdo mRNA levels in other amino acids and metabolic precursors of tau- the duration examined (Fig. 1 B). In addition, IHC stain- rine. Secondly, samples were added with 100  μmol/L ing revealed that CDO was located in uterine epithelial glutamine as an internal standard. All samples were cells, including LE and GE, and some stromal cells (Fig. 1 then filtrated through a 0.22-μm PVDF membrane and E). The taurine concentrations in uterus tissue and serum saved in −80  °C refrigerator until use. The samples and were assayed by HPLC and the results indicated that the the standard samples of taurine which were 100, 50, 25, uterine taurine levels exhibited an increasing tendency 10, 5, 2 and 1  μmol/L were derivated with OPA (Sigma- from d 1 to d 4, and reached the maximum on d 4, fol- Aldrich, St. Louis, MO, USA) solution (20  mg OPA, lowed by a steady decline and returned to the similar 2  mL methanol, 80 μL 2-hydroxy-1-etanethiol, 18  mL level of d 1 on d 8 (Fig. 1 C). The serum taurine level also 0.1  mol/L borate buffer (pH 9.6)) for 3  min. Then 20 increased from d 1 to d 4, which significantly declined μL sample was automatically injected into a six-port from d 5 to d 8 (Fig.  1 C). In addition, the dynamic pat- valve to analysis with Waters Symmetry C18 Column terns of the uterine CDO expression and taurine concen- (4.6  μm, 150  mm × 5  mm) (Waters, Milford, MA, USA) tration corresponded to the change of serum P from d on a Shimadzu HPLC system (Shimadzu, Kyoto, Japan). 1 to d 6 (Fig.  1 D). These suggest that CDO might play The HPLC conditions were: flow A: 100% methanol, important roles in regulating embryo implantation. flow B: sodium phosphate buffer pH 4.7 containing 50% Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 5 of 14 Fig. 1 Uterine CDO mRNA and protein expressions and their relations to taurine levels in uterus from d 1 to d 8 of pregnancy mice. A, Cdo mRNA expression in mouse uteri detected by RT-qPCR (n = 3). B, CDO protein expression in uteri of d 1 to d 8 pregnancy mice assayed by WB. C, Serum and uterine taurine concentrations assayed by HPLC (n = 3). D, Serum P concentrations assayed by RIA (n = 5). E, CDO IHC staining of uterine cross-sections. Data shown as Mean ± SEM. Different letters represent significant differences (P < 0.05). d, days post coitum; E , estrogen; GE, glandular epithelium; LE, luminal epithelium; P , progesterone; St, stromal cells. Bars: 50 μm Establishment of Cdo KO mouse taurine levels of the Cdo KO females from d 1 to d 8 To detect the functions of CDO in uterus, Cdo KO mice were assayed by HPLC. The results showed that Cdo KO were generated by CRISPR/Cas9 technology. Two pairs of sharply decreased the taurine levels in the serum and single guide RNAs (sgRNAs) (Table S1) targeting exon1 uterus tissue compared with that of WT mice, and there and exon3 of Cdo gene respectively (Fig. 2 A), and SpCas9n were no significant differences from d 1 to d 8 (Fig.  3A, (D10A) mRNA were co-injected into d 1 embryos (Addi- B). However, there was a rising spike on d 4 in WT mice tional file  1). A Cdo KO mutant line with 7489 nucleotide (Fig. 3A, B). deletion from the first to the third exon was used in the fol - Further, the effects of Cdo deletion on the implanta - lowing experiments (Fig. 2 B). The genotypes were identified tion and fertility were examined. Adult WT and Cdo KO by PCR that the 245-base pair (bp) strand represent mutant females were respectively mated with ICR males, the par- type and the 325 bp strand represent wild type (Fig. 2 C). The turition and pups of each litter were then recorded. The knockout efficiency was identified by RT-qPCR, IHC and results showed that the plug-positive WT females exhib- WB, and the results showed that CDO was totally deleted, at ited normal fecundity, but only 38.8% (7/18) of the plug- least in mouse uterus (Fig. 2D–F). As CDO is a key enzyme positive Cdo KO females produced litters (Fig.  3 C), and for taurine synthesis, we detected the changes of taurine lev- the litter size (5.429 ± 0.65 pups/litter, n = 7) was much els after Cdo knocked out using HPLC. The results showed smaller than that of the WT females (11.29 ± 0.68 pups/ that the taurine concentrations in the liver, serum and uterus litter, n = 7) (Fig. 3 D). of Cdo KO mice decreased by 76.24%, 51.20% and 70.33% In order to answer whether the subfertility of Cdo KO respectively than that of WT mice (Fig. 2G–I). These results mice was resulted from the abnormalities in the ovaries demonstrate that CDO is successfully deleted from mouse or embryos, the embryos were flushed out from oviducts genome and the lack of CDO leads to taurine deficiency. of d 2 Cdo KO females, which naturally mated with WT males. The flushed 2-cell embryo numbers did not have Cdo KO causes impairment of embryo implantation significant difference between WT and Cdo KO mice and severe subfertility (Fig.  S2 B), and morphological defect was not observed In order to identify the effects of Cdo deletion on either (Fig. S2 A). In addition, the histological abnormal- the embryo implantation, the serum and uterine ity was not observed in 10 weeks Cdo KO mouse ovaries Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 6 of 14 Fig. 2 Generation of Cdo KO mouse. A, Cas9n target scheme in the first and the third exons of Cdo gene. The blue lines indicate sgRNAs’ spacer and the red lines indicate PAM (protospacer adjacent motif ). B, Sanger sequencing of the target region in F0 mice. The Cdo KO mice get a miss of 7489 nucleotides. C, Genotype identification results using PCR. The 325 bp strand indicates wild type and the 245 bp strand indicates mutant type. D, IHC staining of CDO in WT and Cdo KO mouse uterus. Bars, 50 μm. E, RT-qPCR analysis of Cdo mRNA levels in WT and Cdo KO mouse uteri. F, Representative patterns of WB analysis of CDO expressions. G-I, Taurine levels in WT and Cdo KO mice liver, serum and uterus detected by HPLC. Data are the Mean ± SEM. (n ≥ 3). Different letters above columns indicate significant differences (P < 0.05). GE: glandular epithelium. LE: luminal epithelium. Sm: smooth muscle cells. St: stromal cells. Scale bars: 50 μm (Fig. S2 C). Serum P and E levels did not exhibit signifi - identified by employing the Chicago blue dye assay [39]. 4 2 cant differences between the Cdo KO and WT females on The implantation sites (IS) were clearly observed in WT d 4 (Fig. S2 D and E). females, but only a few or no IS were detected in Cdo Further, in order to dissect the cause of the subfer- KO females (Fig.  3 E, G). However, Cdo KO females tility in Cdo KO females, the effect of Cdo deletion on had blastocysts which could be flushed out from uterus the embryo implantation was examined. The plug posi - (Fig. 3 F). tive mice were euthanized on d 5 and the numbers In order to confirm whether the subfertility of Cdo of embryos implanted to the uterine epithelium were KO females was resulted from the defects of uterus Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 7 of 14 Fig. 3 CDO deletion causes impairment of embryo implantation and severe subfertility. A and B, Serum (A) and uterus (B) taurine concentrations of pregnancy Cdo KO and WT females from d 1 to d 8, assayed by HPLC (n = 3). C, Pregnancy rates of WT and KO females. The number within brackets indicate females with pups over total number of plug-positive females. D, Average litter sizes of WT and Cdo KO females (n = 7). E, Representative images of d 5 pregnant uteri from WT and Cdo KO females. Bars, 1 cm. F, Blastocysts flushed from Cdo KO uterus. Bar, 50 μm. G, Average numbers of implanted embryos represented by implantation sites (n = 5). H, Representative patterns of d 5 WT receptive mice uteri transferred with WT and Cdo KO embryos. I, d 5 KO receptive mice uteri transferred with WT embryos. Arrowheads indicate the location of blastocysts. Bars, 1 cm. K, IS numbers of different treated mice uteri (n = 3). Data are presented as Mean ± SEM. Different letters above columns indicate significant differences (P < 0.05) receptive status, Cdo KO and WT females were sepa- which the WT and Cdo KO embryos were respectively rately mated with Cdo KO and WT males to obtain Cdo transferred (Fig.  3 H, K). However, the IS number in KO and WT embryos. The harvested Cdo KO and WT Cdo KO receptive females was much less than in WT embryos were respectively transferred to WT female receptive females after both of which accepted WT uteri. On d 5, IS numbers were assayed and there was embryos (Fig.  3 I, K). Meanwhile, blastocysts could be no significant difference between the WT females to flushed out from Cdo KO uterus (Fig.  3  J). These infer Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 8 of 14 that the failure of embryo implantation and subfertility uterine PR expression during early stage of pregnancy in Cdo KO females are resulted from the defects of uter- (Fig.  1A–D  and Fig.  3A, B), we further identified the ine receptivity. effects of P and E on uterine CDO expression by using 4 2 OVX mouse model. RT-qPCR results showed that uter- Cdo KO results in the defects of uterine receptivity ine Cdo mRNA levels were decreased by 54.65%, 90% and by dysregulating PR and ER signaling in mouse 93% after 2, 6 and 12 h after 100 ng E treatments in OVX Our results showed that Cdo KO had no significant effects mice (Fig.  5 A). These were further confirmed by the on ovary morphology and functions, including E and P CDO IHC staining results that the CDO staining inten- 2 4 secretions (Fig.  S2C–E). As the window of uterine recep- sity got much weaker after 12  h E treatment (Fig.  5 C). tivity coincides with the P -mediated down-regulation of It was opposite that 2  mg P injection sharply increased 4 4 ERα activity in uterine LE, we thus assayed the effects of uterine CDO mRNA and protein levels, which were sev- Cdo deletion on uterus PR expression on d 4 by RT-qPCR eral folds higher than that of the controls after 12  h P and WB. The results showed that PR mRNA and protein treatment (Fig.  5 B and C). Further, in order to identify levels in Cdo KO uteri respectively decreased by 28.74% whether the regulating effects of P and E on uterine 4 2 and 26.63% compared with that in WT uteri (Fig.  4A–C). CDO expressions were through P -PR and E -ERα sign- 4 2 IHC results showed that PR staining intensity on LE, GE aling, OVX mice were respectively pretreated with PR and stromal cells were much weaker in Cdo KO mice than inhibitor RU486 and ERα inhibitor ICI182780, followed in WT mice (Fig.  4 G). In addition, the uterine mRNA by P or E treatment. The uterine CDO expressions were 4 2 expression levels of the known PR responsive genes Ihh, then assayed and the results showed that RU486 blocked Hoxa10 and Hand2 decreased by 56.33%, 35.49% and the enhancing effect of P on CDO expression (Fig.  5G– 45.38% in KO mice than that of the WT mice (Fig.  4 H). I), and ICI182780 restrained the inhibiting effect of E on Further, Hand2 IF staining was performed and it was CDO expression (Fig. 5D–F). observed that Hand2 was located only in the stroma cells, but its staining intensity was much weaker in Cdo KO mice Taurine supplement partly recues the defects of embryo than that in WT mice (Fig. 4 J), which was consistent with implantation and subfertility caused by Cdo KO PR IHC staining result (Fig. 4 G). As the maintenance of the global taurine level relays on In addition, Cdo deletion had no significant effects on the endogenous taurine synthesis through the action of ERα mRNA and protein levels (Fig. 4D–G). Whereas the CDO and the active uptake from the diet, we thus sup- expression levels of E -responsive genes Muc1 and Ltf plied extra taurine in the regular diet of Cdo KO mice to elevated over 2.5 and 8 times in Cdo KO uterus than that confirm whether the defects of uterine receptivity and of the WT uterus on d 4 (Fig.  4 I). The further IF stain - subfertility caused by Cdo KO could be rescued. Adult ing confirmed the Muc1 expression in LE and GE cells of female Cdo KO mice were supplemented with drink- Cdo KO mouse uteri, but which was not detected in WT ing water containing 0.2% (w/v) taurine. The uterine and mice (Fig. 4 J). serum taurine levels were assayed. The results showed Another important character of uterus receptivity to that taurine supplement markedly elevated taurine lev- embryo is a cessation of epithelial cell proliferation and els in uteri and serum, but all of which were significantly robust proliferation of stroma prior to implantation. We lower than that in wild mice with regular diet (Fig.  6A, thus detected the cell proliferation in the uterine tissues B). In addition, the embryo implantation was detected of Cdo KO and WT mice on d 4 by Ki67 IHC staining. on d 5. The results showed that taurine supplement sig - The results showed that Ki67 was not detected in LE nificantly increased the IS number of Cdo KO females, and GE cells of WT and Cdo KO mice uterus, but Ki67 although it was still lower than that of the WT females staining intensity in the stroma of WT uteri was much (Fig.  6C–E). These results demonstrate that taurine sup - stronger than that of Cdo KO uteri. Moreover, Ki67 posi- plementation can partly rescue the subfertility of Cdo KO tive cells were concentrated around the LE in WT uteri females and suggest that CDO and taurine are essential (Fig.  4  K), but Ki67 positive cells in Cdo KO uteri were factors for embryo implantation. irregularly scattered (Fig.  4  K). These indicate that Cdo KO results in the defects of uterine receptivity by dysreg- Discussion ulating PR and ER signaling in mouse. The present study, for the first time as we know, dem - onstrates that CDO and taurine play important roles for P and  E are involved in regulating uterine CDO embryo implantation in mouse. This is supported by our 4 2 expression results here that uterine CDO expression and serum tau- As the dynamic patterns of uterine CDO expression and rine level during early stage of mouse pregnancy are par- taurine level were parallel to serum P concentration and allel to the uterine taurine level, which sharply increases 4 Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 9 of 14 Fig. 4 Eec ff ts of Cdo deletion on PR and ER signaling transduction and the cell proliferation in the uteri of d 4 pregnancy mice. A, RT-qPCR analysis of relative Pgr mRNA expression levels. B and C, WB detection and analysis of PR protein expression. D, RT-qPCR analysis of relative Esr1 mRNA levels. E and F, WB detection and analysis of ERα protein expression levels. G, IHC staining of PR and ERα. Scale bars: 50 μm. H, Relative mRNA expressions of Ihh, Areg and Hand2. I, Relative mRNA levels of Ltf and Muc1. J, IF staining of Hand2 and Muc1. Scale bars: 50 μm. K, Ki67 (proliferative cell marker) IHC staining. Scale bars: 50 μm. GE: glandular epithelium. LE: luminal epithelium. St: stroma. Data are presented as Mean ± SEM, n ≥ 4. Different letters represent significant differences (P < 0.05) and reaches a peak just on d 4, the window of implan- regulate the embryo implantation [7, 43], behaves a simi- tation [42], followed by a steadily decline under physi- lar dynamic pattern with the uterine CDO expression and ological condition. In addition, serum P , a key factor to taurine concentration in the duration examined. Whereas 4 Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 10 of 14 global Cdo KO markedly decreases uterine taurine level Ueki et al. [26]. Another possible explanation is that dif- and omits the dynamic pattern of uterine taurine, accom- ferent mouse strains have been used in our study and the panied by the decline of serum taurine level, although previous report [26]. Cdo KO does not affect the ovary functions to synthesize In addition, the results presented here demonstrate P and E . These demonstrate that uterus has function to that Cdo deletion impairs embryo implantation by dys- 4 2 synthesize taurine, whereas Cdo deletion causes a severe regulating P and E signaling in mouse uterus. The 4 2 subfertility in female mice. CDO and endogenous taurine related molecular and genetic studies indicate that both synthesis are thus essential for embryo implantation and E and P play functions through their corresponding 2 4 the maintenance of animal reproduction. These are also receptor ERα and PR [48, 49] together with local tran- supported by our results here that taurine supplement scriptional and paracrine factors to govern the compli- to the Cdo KO mice can partly rescue the reproductive cated embryo-uterine crosstalk [11, 12, 43, 50]. It is much capacity. interested that our results here show that CDO involves Another important finding of this study is that Cdo in regulating embryo implantation by affecting P and E 4 2 KO causes defects in uterine epithelium receptivity and signaling. In support, the uterus PR mRNA and protein decline of pregnancy rate. It has been well documented levels in Cdo deleted mice are significantly decreased, that successful embryo implantation depends on the accompanied by decline of Ihh, Hoxa10 and Hand2 intimate connection between the blastocyst and mater- expressions, which are known PR targeting molecules nal endometrial surface [43, 44]. Healthy embryos at the [18, 43, 51]. These are also confirmed by PR and Hand2 blastocyst stage and receptive maternal endometrium are IHC results of this study. However, Cdo deletion does necessary for the implantation [45]. The results of this not affect ERα mRNA and protein levels, but the mRNA study show that Cdo deleted embryos do not have mor- levels of E -ERα responsive genes Lf and Muc1 [14, 15] phological defects, and behave normal implantation as are increased over 5 and 1.5 time in Cdo KO mouse uteri, that of the WT embryos in WT recipients. However, the especially the abnormal expression of Muc1 in Cdo KO implantation and birth rates are dramatically decreased LE and GE cells. In addition, CDO or/and taurine might after the WT embryos are transferred to the Cdo deleted participate in E -ERα signaling by affecting the ERα activ - recipients. These imply that Cdo deleted embryos have ity through ERα Ser 118 site phosphorylation [43, 52], normal capacity to get implantation. Another important although we did not detect it in this study. Collectively, character of uterine receptivity is the cessation in epithe- the results presented here demonstrate that CDO is a lial cell proliferation and robust proliferation in stroma crucial factor to affect uterine receptivity during embryo cells just prior to implantation [46, 47]. The results of implantation by involving in P -PR and E -ERα signaling 4 2 present study are in agreement with reports [44, 46] in mouse uterus, although the relating signaling pathways that the cell proliferation, marked by Ki67 IHC staining, and mechanisms need to be clarified in future study. is undetectable in the uterine epithelium, but the num- Furthermore, the regulating effects of P and E on 4 2 ber of Ki67 positive stroma cells significantly decreases uterine CDO are confirmed by using OVX mouse in Cdo KO mice compared with that of WT mice. These model. The in vivo results show that uterus CDO expres - collective data suggest that Cdo deletion leads to the sion level is decreased over 90% after 12  h E treatment abnormalities of uterine receptivity and embryo implan- in OVX mouse, which is in agreement with the reports tation, and the subfertility of Cdo KO mice is primarily of that E inhibits taurine synthesis through estrogen-ERs- uterine origin. CDO/CSAD signaling in liver and uterus [37, 38]. It is However, it has been reported that Cdo KO females are inversed that uterine CDO expression level is sharply fertile and carry their pregnancies to term [26], whereas elevated after P injection in OVX mouse. In addition, our results here demonstrate that 38.8% of plug-positive it is well known that E and P are key drivers of uter- 2 4 Cdo KO females are fertile, and the IS number and litter ine plasticity throughout the sexual cycle and early stage size of Cdo KO females are much less than that of WT of pregnancy, we thus propose that both E and P may 2 4 females. These discordant statements might be resulted play roles through their respective receptors to regulate from the limited assessment, and no description about uterine CDO expression and taurine synthesis, which the statistics analysis of the IS numbers and birth rates by subsequently affect the uterine receptivity and embryo (See figure on next page.) Fig. 5 The relations of P -PR and E -ERα signaling to uterine CDO expression. A and B, Relative Cdo mRNA levels in OVX mouse uteri after 0 4 2 (control), 2, 6 and 12 h treatments with 100 ng E (A) and 2 mg P (B). C, CDO IHC staining in OVX mouse uteri after 0 (control) and 12 h 100 ng E 2 4 2 or 2 mg P treatments. Scale bars: 50 μm. D, E and F, Relative Cdo mRNA (D) and protein levels (E and F) in OVX mouse uteri treated with E and E 4 2 2 inhibitor ICI182780 (ICI). G, H and I, Relative Cdo mRNA (G) and protein levels (H and I) in OVX mouse uteri treated with 2 mg P and PR inhibitor RU486. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n = 3) Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 11 of 14 Fig. 5 (See legend on previous page.) Zhang et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 12 of 14 Fig. 6 Taurine supplement increases the taurine levels in serum and uteri, and partly rescues the defects of embryo implantation in the CDO KO mice. A, Serum taurine concentration. B, Taurine concentration in uterus tissues. C, Representative images of implantation sites. Bars: 1 cm. WT: wide type female mice. KO: Cdo KO female mice. KO + taurine: Cdo KO female mice supplied with taurine. n = 3 for each genotype. D, Blastocysts flushed out from Cdo KO uterus. Bar, 50 μm. E, Numbers of implantation sites. Data are presented as Mean ± SEM. Different letters represent significant differences (P < 0.05, n ≥ 3) implantation. However, it remains to be elucidated about receptivity and embryo implantation by involving in the interactions or cross-talks among P -PR signaling, E -ERα and P -PR signaling pathways. These are crucial 4 2 4 E -ERα signaling, CDO and taurine in the duration of for our understanding the mechanism of embryo implan- embryo implantation. tation, and infer that taurine is a potential agent for Finally, the maintenance of taurine levels in uterus improving reproductive efficiency of livestock industry and serum relays on the endogenous taurine synthesis and/or for reproductive medicine. But it still remains to through the action of CSAD, CDO, and the active uptake be elucidated about the interactions among P -PR signal- from the diet [34, 35], while Cdo deletion impairs embryo ing, E -ERα signaling, CDO and taurine in the duration implantation and causes severe subfertility as it is showed of embryo implantation. in this study. Whereas taurine supplementation signifi cantly increases the litter size and parturition rate of Cdo Conclusions KO females (Table S2). These provide the in  vivo evi - The present study demonstrates that taurine and CDO dence that CDO and taurine are crucial factors for the play prominent roles for the uterine receptivity and maintenance and improvement of animal production, embryo implantation by involving in E -ERα and P -PR 2 4 and suggest that taurine may be potential agent for ani- signaling in early pregnancy stage. These elucidate a mal production. new mechanism for taurine regulating embryo implan- In conclusion, the present study demonstrates that tation. On the other side, abuse of steroid hormones in taurine and CDO play prominent roles for uterine stockbreeding industry usually gives rise to premature Zhang  et al. Journal of Animal Science and Biotechnology (2023) 14:6 Page 13 of 14 Consent for publication ovary failure and declines animal fertility. The balancing Not applicable. effect of CDO and taurine between P -PR signaling and E -ERα signaling shows that taurine is a potential agent Competing interests The authors declare no competing interests. for improving reproductive efficiency of livestock indus - try and reproductive medicine. Received: 19 July 2022 Accepted: 20 November 2022 Abbreviations CDO Cysteine dioxygenase E Estrogen Esr1 (ERα) Estr ogen receptor 1 References GE Glandular epithelium 1. Boivin J, Bunting L, Collins JA, Nygren KG. 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Journal

Journal of Animal Science and BiotechnologySpringer Journals

Published: Jan 5, 2023

Keywords: CDO; E2; Embryo implantation; P4; Taurine

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