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Remodeling of bovine endometrium throughout the estrous cycle
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Remodeling of bovine endometrium throughout the estrous cycle Miki Arai, Shin Yoshioka, Yukari Tasaki, Kiyoshi Okuda ∗ Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
a r t i c l e
i n f o
Article history: Received 1 May 2013 Received in revised form 2 August 2013 Accepted 6 August 2013 Available online xxx Keywords: Cattle Endometrium Remodeling Proliferation and apoptosis
a b s t r a c t The mammalian endometrium changes morphologically and functionally throughout the estrous cycle. In some species, endometrial cells also undergo periodic proliferation and degeneration. However, the remodeling of bovine endometrium throughout the estrous cycle remains unclear. In the present study, we examined how the remodeling of bovine endometrium varied through the estrous cycle by measuring the relative rates of cell proliferation and apoptosis. Cells positive for both KI-67 (a proliferation marker) and cleaved caspase-3 (CCP3: an apoptotic cell marker) were immunohistochemically evaluated throughout the estrous cycle in the luminal and glandular epithelia, and the stroma of bovine endometrium. Percentages of KI-67-positive cells tended to be higher at the early luteal and follicular stages than at the mid and late luteal stages in all cell types. Similarly, percentages of CCP3-positive cells were higher at the early luteal stage than at the mid and late luteal stages in the luminal epithelium and stroma. Furthermore, CCP3 expression levels by Western blot analysis agreed with these immunohistological observations. On the other hand, DNA fragmentation was detected in the bovine endometrium without significant differences during the estrous cycle by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Together, these results show that cell proliferation and apoptosis undergo cyclic patterns in the bovine endometrium, and suggest that the bovine endometrium is remodeled in each estrous cycle. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In mammals, the uterus is the organ of pregnancy (Senger, 2011). To establish pregnancy, endometrial cells change morphologically and functionally throughout the estrous cycle (Weitlauf, 1994). Ovarian steroids such as estradiol-17 and progesterone play important roles in regulating the function of the endometrium (Jabbour et al., 2006). In humans, estradiol-17 promotes endometrial
∗ Corresponding author at: Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan. Tel.: +81 86 251 8333; fax: +81 86 251 8333. E-mail address: [email protected] (K. Okuda).
cell proliferation, and then progesterone differentiates the function and morphology of endometrial glandular and stromal cells (Jabbour et al., 2006). However, if pregnancy does not occur, the functional layer of the endometrium is shed by menstruation in primates (Mihm et al., 2011). Apoptosis is induced in the human endometrium during the late secretory and menstrual phase (Hopwood and Levison, 1976), and plays important roles for the functional maintenance in various tissues including the primate endometrium (Kerr et al., 1972; Harada et al., 2004; Jin and El-Deiry, 2005). Caspases are central factors in the execution of apoptosis and are synthesized as inactive zymogens and activated by proteolytic cleavage at two sites following the induction of apoptosis (Jin and El-Deiry, 2005). In mammals, at
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Please cite this article in press as: Arai, M., et al., Remodeling of bovine endometrium throughout the estrous cycle. Anim. Reprod. Sci. (2013), http://dx.doi.org/10.1016/j.anireprosci.2013.08.003
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least 14 caspases, caspase-1 to -14, have been identified (Jin and El-Deiry, 2005). Caspase-3 (CASP3) is recognized as a crucial executioner caspase, and cleaved caspase-3 (CCP3) is used widely for identifying apoptosis cells (Duan et al., 2003; Jin and El-Deiry, 2005). The functional layer of the endometrium that is lost by menstruation is remodeled by cell proliferation at the proliferative phase, and subsequently the glandular and stromal cells differentiate functionally at the secretory phase to support pregnancy in woman (Gargett et al., 2008; Mihm et al., 2011). Therefore, the proliferation and cell death of endometrial cells throughout the menstrual cycle is important for the maintenance of endometrial function in human (Cavagna and Mantese, 2003; Harada et al., 2004; Gargett et al., 2008). On the other hand, since menstruation does not occur in non-primate species, the morphological change of the endometrium seems to be less than that in primates. However, cyclic cell proliferation and cell death by apoptosis have been observed in the uterus of murine (Wood et al., 2007), rat (Sato et al., 1997), canine (Van Cruchten et al., 2004), equine (Gerstenberg et al., 1999; Roberto da Costa et al., 2007) and sow (Sukjumlong et al., 2003; Okano et al., 2007). In addition, mitosis occurs in endometrial glandular and stromal cells in nonpregnant cow (Ohtani et al., 1993; Ohtani and Okuda, 1995). Based on those results, we hypothesized that bovine endometrial cells are remodeled during the estrous cycle to maintain normal endometrial function. To test the above hypothesis, we investigated (1) the relative rate of cell proliferation by immunohistochemical localization of a proliferation marker (KI-67), (2) the relative rate of apoptosis by TUNEL assay and immunohistochemical localization of cleaved caspase-3 (CCP3), and (3) CASP3 mRNA and CCP3 protein levels by quantitative RTPCR and Western blot analysis in the bovine endometrium throughout the estrous cycle.
2.2. Immunohistochemistry Four-m sections were deparaffined, rehydrated in a graded series of ethanol and washed in tap-water. Antigen retrieval was performed by microwaving in 0.01 M citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) using microwave for 15 min at 600 W. Endogenous peroxidase activity was quenched in methanol with 0.3% H2 O2 for 10 min. Nonspecific binding was blocked at room temperature for 20 min in PBS-5% skim milk then incubated in 10% (v/v) normal horse serum (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 10 min. The sections were incubated with either (1) mouse monoclonal anti KI-67 antibody, clone MIB-1 (M7240; Dako-Cytomation, Glostrup, Denmark; dilution 1:150) as the proliferation marker (Scholzen and Gerdes, 2000) for 2 h at room temperature (RT), (2) rabbit polyclonal anti cleaved caspase 3 (Asp175) antibody (#9661; Cell Signaling Technology, Boston, USA; dilution 1:800) as the apoptotic cell marker (Duan et al., 2003) overnight at 4 ◦ C or (3) normal mouse or rabbit serum instead of the primary antibody (for negative control). After incubation, the sections were washed with PBS 3 times and incubated with ImmPRESS UNIVERSAL reagent, anti-mouse/rabbit Ig (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 30 min according to the manufacturer’s instruction. The sections were visualized with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.01 M PBS (pH 7.4) containing 0.01% H2 O2 and counterstained with Mayer’s hematoxylin, washed in DW, dehydrated in a graded series of ethanol, cleared in xylene and covered by coverslips. 2.3. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method DNA fragmentation was detected with on In Situ Cell Death Detection Kit, POD (1684817; Roche, Mannheim, Germany) according to the manufacturer’s instructions.
2. Materials and methods 2.4. Quantification methods 2.1. Collection of endometrial tissues Apparently healthy uteri of Holstein cows without a visible conceptus were obtained within 10–20 min after exsanguinations and were immediately transported to the laboratory. Stages of the estrous cycle were confirmed by macroscopic observation of the ovary and uterus as described previously (Okuda et al., 1988a; Miyamoto et al., 2000). For mRNA and protein determination, endometrial tissues (n = 6 cows/stages) were collected from cows at four different stages of the estrous cycle (estrus = Day 0; early luteal: Days 2–3; mid luteal: Days 8–12; late luteal: Days 15–17 and follicular stage: Days 19–21). Intercaruncular endometrial tissues from the uterine horn, ipsilateral to the CL or the dominant follicle, were used for experiments. Endometrial tissues were immediately frozen in liquid nitrogen, and stored at −80 ◦ C until processed for mRNA extraction and protein isolation. Endometrial tissues for immunohistochemistry (n = 4 cows/stages) were fixed in phosphate buffered saline (PBS) with 4% (v/v) paraformaldehyde for 24 h.
To calculate the percentage of KI-67- and CCP3positive cells in the bovine endometrium, sections of the endometrium including luminal and glandular epithelia were selected randomly and examined at ×400 magnification. A test grid covered each area (approximately 0.12 mm2 ) on the sections. Each cell number was counted by the “point count method” as described previously (Okuda et al., 1988a; Hojo et al., 2009) with some modifications. Briefly, each crosspoint on the grid that coincided with positive immunostaining (DAB and hematoxylin) or the negative immunostaining (only hematoxylin) was counted. In each endometrial structure (luminal epithelium, glandular epithelium and stroma), the percentages of the KI-67and CCP3-positive cells were calculated as X/Y × 100, where X was the total number of positive cells and Y was the total number of cells under crosspoints. Three sections were randomly chosen from each animal, and counting was performed for three randomly chosen sections from each animal. Three randomly chosen areas (microscopic fields)
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Fig. 1. Immunohistochemical localization of cells showing positive nuclear staining for KI-67 antigen in the bovine endometrium (LE: luminal epithelium, STR: stroma and GE: glandular epithelium) throughout the estrous cycle (A: the early luteal, B: mid luteal, C: late luteal and D: follicular stages). Cells showing positively stained nuclei indicate proliferating cells. Scale bar represents 100 m.
on each section were counted by three persons who were blind to the stage of the estrous cycle. Average counts of three persons were used for statistical analysis.
the 2-CT method was used (Livak and Schmittgen, 2001). 2.6. Western blot analysis
2.5. Total RNA extraction and quantitative RT-PCR Total RNA was extracted from endometrial tissue using TRIsure (#BIO-38033; Bioline, London, UK) according to the manufacturer’s directions. The total RNA (1 g) was reverse transcribed using iScript RT Supermix for RT-qPCR (1708840; Bio-Rad, Hercules, CA, USA). Quantifications of mRNA expression were determined by Quantitative RT-PCR using MyiQ (Bio-Rad) and SooAdvanced SYBR Green Supermix (Bio-Rad) starting with 2 ng of reverse-transcribed total RNA as described previously (Sakumoto et al., 2006). GAPDH expression was used as an internal control. Specific primers for CASP3 and GAPDH were used in the previous study (Kamemori et al., 2011). Briefly, the primers for CASP3 were 5 -AAGCCATGGTGAAGAAGGAA-3 (5 primer, 20 mer) and 5 -GGCAGGCCTGAATAATGAAA-3 (3 primer, 20 mer), for GAPDH were 5 -CACCCTCAAGATTGTCAGCA-3 (5 primer, 20 mer) and 5 -GGTCATAAGTCCCTCCACGA-3 (3 primer, 20 mer). Protocol conditions were consisted of denaturation at 95 ◦ C for 3 min, followed by 45 cycles at 94 ◦ C for 15 sec, 60 ◦ C for 20 sec and 72 ◦ C for 15 sec with a final dissociation (melting) curve analysis. Use of the iQ SYBR Green supermix at elevated temperatures resulted in reliable and sensitive quantification of the RT-PCR products with high linearity (Pearson correlation coefficient r > 0.99). To analyze the relative level of expression of each mRNA,
CCP3 protein levels in endometrial tissues were assessed by Western blot analysis. Endometrial tissues were homogenized on ice in the homogenization buffer by a tissue homogenizer (NS-50; Physcotron; Niti-on, Chiba, Japan), followed by filtration with a metal wire mesh (150 m). For CCP3 protein analysis, tissue homogenates were centrifuged at 600 × g for 10 min and supernatants were collected as cytoplasmic fractionations. Protein concentrations were determined by the BCA method (Osnes et al., 1993). The proteins were heated with SDS gel-loading buffer containing 1% (v/v) -mercaptoethanol (137-06862; Wako Pure Chemical Industries) at 95 ◦ C for 10 min. Samples (25 g protein) were subjected to SDS-PAGE (15%) for 1.5 h at 200 V. Separated proteins were transferred to a polyvinylidene difluoride membrane electrophoretically (RPN303F; GE Healthcare, Milwaukee, WI, USA) for 30 min at 200 mA by Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was washed in TBS with 0.1% Tween 20 (TBST), incubated in PVDF Blocking Reagent for Can Get Signal (NYPBR01; TOYOBO, Osaka, Japan) for 60 min at room temperature, incubated separately with specific primary antibodies to CCP3 (19 and 17 kDa: #9661; Cell Signaling Technology, Boston, USA; 1:1000 dilution) and ACTB antibody (42 kDa: A2228; Sigma–Aldrich; 1:4000 dilution) in Can Get Signal
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Fig. 2. Percentage of KI-67-positive cells in the bovine endometrium during the estrous cycle (mean ± SEM, n = 4 experiments). Percentages in each endometrial structure (A: luminal epithelium, B: stroma and C: glandular epithelium) were derived from dividing the number of positively stained cells by total number of cells under crosspoint in each histological section at ×400 magnification. Cells were counted in three randomly chosen areas. Different superscript letters indicate significant differences (P < 0.05).
Immunoreaction Enhancer Solution 1 (NKB-201; TOYOBO) overnight at 4 ◦ C, incubated with secondary antibodies to CCP3 (anti-rabbit Ig, HRP-linked whole antibody produced in donkey; NA934; GE Healthcare; 1:4000 dilution) and ACTB (NA931; anti-mouse Ig, HRP-linked whole antibody produced in sheep; GE Healthcare; 1:40,000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 2 (NKB301; TOYOBO) for 60 min at room temperature, incubated in Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck) and captured using a ChemiDocTM XRS+ System (Bio-Rad). The intensity of the immunological reaction was estimated by measuring the optical density in the defined area by computerized densitometry using an Image LabTM software (170-8265J1PC; Bio-Rad)
2.7. Statistical analysis All experimental data are shown as the mean ± SEM. The statistical significance of differences was assessed by analysis of variance (ANOVA) followed by Tukey’s multiple
comparisons test using GraphPad Prism ver. 6.0 for Mac (GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Localization and quantification of KI-67-positive cells throughout the estrous cycle In the bovine endometrium, KI-67 expressions were localized in luminal and glandular epithelia and the stroma throughout the estrous cycle (Fig. 1), and the percentage of KI-67-positive cells was calculated based on these chromatic fields (Fig. 2). The rate of KI-67-positive cells was higher at the follicular stage than at the mid and late luteal stages in the luminal epithelium (Fig. 2A; P < 0.05), and was higher at the follicular stage than at the late luteal stage in the stroma (Fig. 2A; P < 0.05). Furthermore, the percentage of KI-67-positive cells was higher at the early luteal and follicular stages than at the mid and late luteal stages in the glandular epithelium (Fig. 2 C; P < 0.05).
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Fig. 3. TUNEL-positive cells (arrows) in sections of bovine endometrium prepared at the early luteal (A), mid luteal (B), late luteal (C) and follicular (D) stages of the estrous cycle. Scale bar represents 100 m.
Fig. 4. CCP3-positive cells (arrows) in sections of bovine endometrium prepared at the early luteal (A), mid luteal (B), late luteal (C) and follicular (D) stages of the estrous cycle. Scale bar represents 100 m.
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Fig. 5. Percentage of CCP3-positive cells in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 4 experiments). Percentages of CCP3positive cells in each endometrial structure (A: luminal epithelium, B: stroma and C: glandular epithelium) were derived by dividing the number of CCP3-positive cells by total number of cells under crosspoint in the histological section at a magnification of ×400. Cells were counted in three randomly chosen areas. Different superscript letters indicate significant differences (P < 0.05).
3.2. Localization of TUNEL-positive cells throughout the estrous cycle
3.4. CASP3 mRNA and CCP3 protein expression throughout the estrous cycle
TUNEL-positive cells were found in the luminal and glandular epithelia and in the stroma during the estrous cycle. However, the number of stained cells was very few and did not significantly change during estrous cycle (Fig. 3).
CCP3 mRNA expression did not change significantly during the estrous cycle (Fig. 6), whereas 19 kDa and 17 kDa
3.3. Localization and quantification of CCP3-positive cells throughout the estrous cycle CCP3 was expressed throughout the estrous cycle, but predominantly in the luminal epithelium (Fig. 4). The percentage of CCP3-positive cells was higher at the early luteal stage than at the mid and late luteal stages in the luminal epithelium (Fig. 5A; P < 0.05), and was higher at the early luteal stage than at the other stages in the stroma (Fig. 5B; P < 0.05). In contrast, the number of CCP3-positive cells did not significantly change during the estrous cycle in the glandular epithelium (Fig. 5 C).
Fig. 6. Changes in the relative amounts of CASP3 mRNA expression in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 5 experiments).
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Fig. 7. Changes in the relative amounts of CCP3 protein (A: 19 kDa and B: 17 kDa) expressions in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 5 experiments). Different superscript letters indicate significant differences (P < 0.05).
CCP3 protein levels were higher at the early luteal and follicular stages than at the mid and late luteal stages (Fig. 7A and B; P < 0.05). 4. Discussion In the present study, the percentages of KI-67-positive cells were higher from the follicular stage to the early luteal stage in all types of endometrial cells. These results are consistent with previous studies that showed cyclic cell proliferation in rat (Sato et al., 1997), sow (Sukjumlong et al., 2003), equine (Gerstenberg et al., 1999), ewe (Johnson et al., 1997b) and human (Gargett et al., 2008). Estradiol17 secreted by the ovary induces the proliferation of endometrial cells in human (Jabbour et al., 2006), rodents (Tong and Pollard, 1999; Zhang and Paria, 2006) and sheep (Johnson et al., 1997a). Additionally, estradiol-17 also induces the expressions of growth factors throughout the estrous cycle in the bovine endometrium (Robinson et al., 2000; Tasaki et al., 2010) and promotes the proliferation of bovine endometrial epithelial cells by stimulating the production of a variety of growth factors in stromal cells (Xiao and Goff, 1998). Therefore, the cell proliferation observed in the present study at the early luteal and follicular stages may result from growth factors stimulated by estradiol17. The removal of unwanted cells by apoptosis plays an important role in the maintenance of homeostasis in various tissues including the endometrium (Harada et al., 2004; Jin and El-Deiry, 2005). In our study, CCP3-positive cells were observed throughout the estrous cycle. In addition, the percentage of CCP3-positive cells increased at the follicular and early luteal stages in the bovine endometrium. These findings are coincident with the previous finding that apoptotic cells were barely detected at the mid and late luteal stages (Days 12–18) in the bovine endometrium (Groebner et al., 2010). Endometrial apoptosis is mainly promoted at the periods of luteolysis or periovulatory in
several species (Hopwood and Levison, 1976; Sato et al., 1997; Van Cruchten et al., 2003; Okano et al., 2007; Roberto da Costa et al., 2007; Wood et al., 2007). When pregnancy does not occur, the endometrium may be renewed to prepare for the next chance to be pregnant. Furthermore, endometrial apoptosis is induced by withdrawal of progesterone in rabbit (Rotello et al., 1992), by withdrawal of estradiol-17 in hamster (Sandow et al., 1979) and mouse (Jo et al., 1993) or by the withdrawal of both in human (Song et al., 2002). In the present study, CCP3-positive cells were relatively higher at both the follicular (low progesterone level) and early luteal stages (low estradiol-17 level). The above findings suggest that the withdrawal of progesterone of estradiol-17 or both contributes to induce apoptosis in the bovine endometrium. CCP3-positive cells were fewer than KI-67-positive cells in the bovine endometrium throughout the estrous cycle in the present study. KI-67 protein is thought to be expressed in all active phases of the cell cycle, for approximately 24 h in certain human cells in culture (Cooper, 2000), whereas apoptotic cells are quickly engulfed by phagocytes (Jin and El-Deiry, 2005; Erwig and Henson, 2007). Therefore, the reason why CCP3-positive cells were fewer than KI-67positive cells, may be that apoptotic cells are removed rapidly by phagocytosis in the bovine endometrium. Furthermore, apoptotic mammary epithelial cells are extruded from the epithelial layer, preserving its structure in the rat mammary gland (Monks et al., 2008). In fact, CCP3-positive cells were found to be extruded from the luminal epithelium to the side of the uterine cavity in the present study. TUNEL-positive cells appeared to be fewer than CCP3positive cells, although the difference was not significant. Similar results were reported for dogs (Van Cruchten et al., 2003), horses (Roberto da Costa et al., 2007) and cats (Liman et al., 2013). The reason for the fewer TUNEL-positive cells than CCP3-positive cells may be that CASP3 are cleaved before apoptotic DNA fragmentation (Nagata, 2000; Duan et al., 2003).
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Apoptosis is regulated by continuous cleavages and activations of caspases, especially CASP3 (Jin and El-Deiry, 2005). In the present study, CASP3 mRNA expression in bovine endometrium during the estrous cycle disagreed with CCP3 protein expression. CASP3 mRNA expression is readily detected in a variety of tissues (Earnshaw et al., 1999), whereas the cleavage and activation of CASP3 needs initiator caspases (e.g., CASP-8 and CASP-9) activated by diverse apoptotic stimuli (Jin and El-Deiry, 2005). Therefore, the inconsistency of expressions of CASP3 mRNA and CCP3 protein may be due to the differences between the transcriptional and activation mechanisms of CASP3. The overall results show that cell proliferation and apoptosis undergo cyclic patterns in the bovine endometrium, and suggest that the endometrium in cow is also remodeled in each estrous cycle. Acknowledgement This research was supported by Grant-in-Aid for Scientific Research (No. 24380155) of the Japan Society for the Promotion of Science (JSPS). References Cavagna, M., Mantese, J.C., 2003. Biomarkers of endometrial receptivity – a review. Placenta 24 (Suppl. B), S39–S47. Cooper, G., 2000. The eukaryotic cell cycle. In: The Cell: A Molecular Approach, 2nd edition. Sinauer Associates Inc, Sunderland, Massachusetts. Duan, W.R., Garner, D.S., Williams, S.D., Funckes-Shippy, C.L., Spath, I.S., Blomme, E.A., 2003. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J. Pathol. 199, 221–228. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424. Erwig, L.P., Henson, P.M., 2007. Immunological consequences of apoptotic cell phagocytosis. Am. J. Pathol. 171, 2–8. Gargett, C.E., Chan, R.W., Schwab, K.E., 2008. Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells. Mol. Cell. Endocrinol. 288, 22–29. Gerstenberg, C., Allen, W.R., Stewart, F., 1999. Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle. J. Reprod. Fertil. 116, 167–175. Groebner, A.E., Schulke, K., Unterseer, S., Reichenbach, H.D., Reichenbach, M., Buttner, M., Wolf, E., Meyer, H.H., Ulbrich, S.E., 2010. Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis. Placenta 31, 168–177. Harada, T., Kaponis, A., Iwabe, T., Taniguchi, F., Makrydimas, G., Sofikitis, N., Paschopoulos, M., Paraskevaidis, E., Terakawa, N., 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update 10, 29–38. Hojo, T., Al-Zi’abi, M.O., Skarzynski, D.J., Acosta, T.J., Okuda, K., 2009. Changes in the vasculature of bovine corpus luteum during the estrous cycle and prostaglandin F2␣-induced luteolysis. J. Reprod. Dev. 55, 512–517. Hopwood, D., Levison, D.A., 1976. Atrophy and apoptosis in the cyclical human endometrium. J. Pathol. 119, 159–166. Jabbour, H.N., Kelly, R.W., Fraser, H.M., Critchley, H.O., 2006. Endocrine regulation of menstruation. Endocr. Rev. 27, 17–46. Jin, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139–163. Jo, T., Terada, N., Saji, F., Tanizawa, O., 1993. Inhibitory effects of estrogen, progesterone, androgen and glucocorticoid on death of neonatal mouse uterine epithelial cells induced to proliferate by estrogen. J. Steroid Biochem. Mol. Biol. 46, 25–32. Johnson, M.L., Redmer, D.A., Reynolds, L.P., 1997a. Effects of ovarian steroids on uterine growth, morphology, and cell proliferation in ovariectomized, steroid-treated ewes. Biol. Reprod. 57, 588–596.
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Remodeling of bovine endometrium throughout the estrous cycle Miki Arai, Shin Yoshioka, Yukari Tasaki, Kiyoshi Okuda ∗ Laboratory of Reproductive Endocrinology, Graduate School of Natural Science and Technology, Okayama University, Okayama 700-8530, Japan
a r t i c l e
i n f o
Article history: Received 1 May 2013 Received in revised form 2 August 2013 Accepted 6 August 2013 Available online xxx Keywords: Cattle Endometrium Remodeling Proliferation and apoptosis
a b s t r a c t The mammalian endometrium changes morphologically and functionally throughout the estrous cycle. In some species, endometrial cells also undergo periodic proliferation and degeneration. However, the remodeling of bovine endometrium throughout the estrous cycle remains unclear. In the present study, we examined how the remodeling of bovine endometrium varied through the estrous cycle by measuring the relative rates of cell proliferation and apoptosis. Cells positive for both KI-67 (a proliferation marker) and cleaved caspase-3 (CCP3: an apoptotic cell marker) were immunohistochemically evaluated throughout the estrous cycle in the luminal and glandular epithelia, and the stroma of bovine endometrium. Percentages of KI-67-positive cells tended to be higher at the early luteal and follicular stages than at the mid and late luteal stages in all cell types. Similarly, percentages of CCP3-positive cells were higher at the early luteal stage than at the mid and late luteal stages in the luminal epithelium and stroma. Furthermore, CCP3 expression levels by Western blot analysis agreed with these immunohistological observations. On the other hand, DNA fragmentation was detected in the bovine endometrium without significant differences during the estrous cycle by the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method. Together, these results show that cell proliferation and apoptosis undergo cyclic patterns in the bovine endometrium, and suggest that the bovine endometrium is remodeled in each estrous cycle. © 2013 Elsevier B.V. All rights reserved.
1. Introduction In mammals, the uterus is the organ of pregnancy (Senger, 2011). To establish pregnancy, endometrial cells change morphologically and functionally throughout the estrous cycle (Weitlauf, 1994). Ovarian steroids such as estradiol-17 and progesterone play important roles in regulating the function of the endometrium (Jabbour et al., 2006). In humans, estradiol-17 promotes endometrial
∗ Corresponding author at: Laboratory of Reproductive Physiology, Graduate School of Environmental and Life Science, Okayama University, Okayama 700-8530, Japan. Tel.: +81 86 251 8333; fax: +81 86 251 8333. E-mail address: [email protected] (K. Okuda).
cell proliferation, and then progesterone differentiates the function and morphology of endometrial glandular and stromal cells (Jabbour et al., 2006). However, if pregnancy does not occur, the functional layer of the endometrium is shed by menstruation in primates (Mihm et al., 2011). Apoptosis is induced in the human endometrium during the late secretory and menstrual phase (Hopwood and Levison, 1976), and plays important roles for the functional maintenance in various tissues including the primate endometrium (Kerr et al., 1972; Harada et al., 2004; Jin and El-Deiry, 2005). Caspases are central factors in the execution of apoptosis and are synthesized as inactive zymogens and activated by proteolytic cleavage at two sites following the induction of apoptosis (Jin and El-Deiry, 2005). In mammals, at
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least 14 caspases, caspase-1 to -14, have been identified (Jin and El-Deiry, 2005). Caspase-3 (CASP3) is recognized as a crucial executioner caspase, and cleaved caspase-3 (CCP3) is used widely for identifying apoptosis cells (Duan et al., 2003; Jin and El-Deiry, 2005). The functional layer of the endometrium that is lost by menstruation is remodeled by cell proliferation at the proliferative phase, and subsequently the glandular and stromal cells differentiate functionally at the secretory phase to support pregnancy in woman (Gargett et al., 2008; Mihm et al., 2011). Therefore, the proliferation and cell death of endometrial cells throughout the menstrual cycle is important for the maintenance of endometrial function in human (Cavagna and Mantese, 2003; Harada et al., 2004; Gargett et al., 2008). On the other hand, since menstruation does not occur in non-primate species, the morphological change of the endometrium seems to be less than that in primates. However, cyclic cell proliferation and cell death by apoptosis have been observed in the uterus of murine (Wood et al., 2007), rat (Sato et al., 1997), canine (Van Cruchten et al., 2004), equine (Gerstenberg et al., 1999; Roberto da Costa et al., 2007) and sow (Sukjumlong et al., 2003; Okano et al., 2007). In addition, mitosis occurs in endometrial glandular and stromal cells in nonpregnant cow (Ohtani et al., 1993; Ohtani and Okuda, 1995). Based on those results, we hypothesized that bovine endometrial cells are remodeled during the estrous cycle to maintain normal endometrial function. To test the above hypothesis, we investigated (1) the relative rate of cell proliferation by immunohistochemical localization of a proliferation marker (KI-67), (2) the relative rate of apoptosis by TUNEL assay and immunohistochemical localization of cleaved caspase-3 (CCP3), and (3) CASP3 mRNA and CCP3 protein levels by quantitative RTPCR and Western blot analysis in the bovine endometrium throughout the estrous cycle.
2.2. Immunohistochemistry Four-m sections were deparaffined, rehydrated in a graded series of ethanol and washed in tap-water. Antigen retrieval was performed by microwaving in 0.01 M citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) using microwave for 15 min at 600 W. Endogenous peroxidase activity was quenched in methanol with 0.3% H2 O2 for 10 min. Nonspecific binding was blocked at room temperature for 20 min in PBS-5% skim milk then incubated in 10% (v/v) normal horse serum (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 10 min. The sections were incubated with either (1) mouse monoclonal anti KI-67 antibody, clone MIB-1 (M7240; Dako-Cytomation, Glostrup, Denmark; dilution 1:150) as the proliferation marker (Scholzen and Gerdes, 2000) for 2 h at room temperature (RT), (2) rabbit polyclonal anti cleaved caspase 3 (Asp175) antibody (#9661; Cell Signaling Technology, Boston, USA; dilution 1:800) as the apoptotic cell marker (Duan et al., 2003) overnight at 4 ◦ C or (3) normal mouse or rabbit serum instead of the primary antibody (for negative control). After incubation, the sections were washed with PBS 3 times and incubated with ImmPRESS UNIVERSAL reagent, anti-mouse/rabbit Ig (MP-7500; Vector Laboratories Inc, Burlingame, CA, USA) for 30 min according to the manufacturer’s instruction. The sections were visualized with 0.05% 3,3-diaminobenzidine tetrahydrochloride (DAB) in 0.01 M PBS (pH 7.4) containing 0.01% H2 O2 and counterstained with Mayer’s hematoxylin, washed in DW, dehydrated in a graded series of ethanol, cleared in xylene and covered by coverslips. 2.3. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) method DNA fragmentation was detected with on In Situ Cell Death Detection Kit, POD (1684817; Roche, Mannheim, Germany) according to the manufacturer’s instructions.
2. Materials and methods 2.4. Quantification methods 2.1. Collection of endometrial tissues Apparently healthy uteri of Holstein cows without a visible conceptus were obtained within 10–20 min after exsanguinations and were immediately transported to the laboratory. Stages of the estrous cycle were confirmed by macroscopic observation of the ovary and uterus as described previously (Okuda et al., 1988a; Miyamoto et al., 2000). For mRNA and protein determination, endometrial tissues (n = 6 cows/stages) were collected from cows at four different stages of the estrous cycle (estrus = Day 0; early luteal: Days 2–3; mid luteal: Days 8–12; late luteal: Days 15–17 and follicular stage: Days 19–21). Intercaruncular endometrial tissues from the uterine horn, ipsilateral to the CL or the dominant follicle, were used for experiments. Endometrial tissues were immediately frozen in liquid nitrogen, and stored at −80 ◦ C until processed for mRNA extraction and protein isolation. Endometrial tissues for immunohistochemistry (n = 4 cows/stages) were fixed in phosphate buffered saline (PBS) with 4% (v/v) paraformaldehyde for 24 h.
To calculate the percentage of KI-67- and CCP3positive cells in the bovine endometrium, sections of the endometrium including luminal and glandular epithelia were selected randomly and examined at ×400 magnification. A test grid covered each area (approximately 0.12 mm2 ) on the sections. Each cell number was counted by the “point count method” as described previously (Okuda et al., 1988a; Hojo et al., 2009) with some modifications. Briefly, each crosspoint on the grid that coincided with positive immunostaining (DAB and hematoxylin) or the negative immunostaining (only hematoxylin) was counted. In each endometrial structure (luminal epithelium, glandular epithelium and stroma), the percentages of the KI-67and CCP3-positive cells were calculated as X/Y × 100, where X was the total number of positive cells and Y was the total number of cells under crosspoints. Three sections were randomly chosen from each animal, and counting was performed for three randomly chosen sections from each animal. Three randomly chosen areas (microscopic fields)
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Fig. 1. Immunohistochemical localization of cells showing positive nuclear staining for KI-67 antigen in the bovine endometrium (LE: luminal epithelium, STR: stroma and GE: glandular epithelium) throughout the estrous cycle (A: the early luteal, B: mid luteal, C: late luteal and D: follicular stages). Cells showing positively stained nuclei indicate proliferating cells. Scale bar represents 100 m.
on each section were counted by three persons who were blind to the stage of the estrous cycle. Average counts of three persons were used for statistical analysis.
the 2-CT method was used (Livak and Schmittgen, 2001). 2.6. Western blot analysis
2.5. Total RNA extraction and quantitative RT-PCR Total RNA was extracted from endometrial tissue using TRIsure (#BIO-38033; Bioline, London, UK) according to the manufacturer’s directions. The total RNA (1 g) was reverse transcribed using iScript RT Supermix for RT-qPCR (1708840; Bio-Rad, Hercules, CA, USA). Quantifications of mRNA expression were determined by Quantitative RT-PCR using MyiQ (Bio-Rad) and SooAdvanced SYBR Green Supermix (Bio-Rad) starting with 2 ng of reverse-transcribed total RNA as described previously (Sakumoto et al., 2006). GAPDH expression was used as an internal control. Specific primers for CASP3 and GAPDH were used in the previous study (Kamemori et al., 2011). Briefly, the primers for CASP3 were 5 -AAGCCATGGTGAAGAAGGAA-3 (5 primer, 20 mer) and 5 -GGCAGGCCTGAATAATGAAA-3 (3 primer, 20 mer), for GAPDH were 5 -CACCCTCAAGATTGTCAGCA-3 (5 primer, 20 mer) and 5 -GGTCATAAGTCCCTCCACGA-3 (3 primer, 20 mer). Protocol conditions were consisted of denaturation at 95 ◦ C for 3 min, followed by 45 cycles at 94 ◦ C for 15 sec, 60 ◦ C for 20 sec and 72 ◦ C for 15 sec with a final dissociation (melting) curve analysis. Use of the iQ SYBR Green supermix at elevated temperatures resulted in reliable and sensitive quantification of the RT-PCR products with high linearity (Pearson correlation coefficient r > 0.99). To analyze the relative level of expression of each mRNA,
CCP3 protein levels in endometrial tissues were assessed by Western blot analysis. Endometrial tissues were homogenized on ice in the homogenization buffer by a tissue homogenizer (NS-50; Physcotron; Niti-on, Chiba, Japan), followed by filtration with a metal wire mesh (150 m). For CCP3 protein analysis, tissue homogenates were centrifuged at 600 × g for 10 min and supernatants were collected as cytoplasmic fractionations. Protein concentrations were determined by the BCA method (Osnes et al., 1993). The proteins were heated with SDS gel-loading buffer containing 1% (v/v) -mercaptoethanol (137-06862; Wako Pure Chemical Industries) at 95 ◦ C for 10 min. Samples (25 g protein) were subjected to SDS-PAGE (15%) for 1.5 h at 200 V. Separated proteins were transferred to a polyvinylidene difluoride membrane electrophoretically (RPN303F; GE Healthcare, Milwaukee, WI, USA) for 30 min at 200 mA by Trans-Blot® SD Semi-Dry Transfer Cell (Bio-Rad). The membrane was washed in TBS with 0.1% Tween 20 (TBST), incubated in PVDF Blocking Reagent for Can Get Signal (NYPBR01; TOYOBO, Osaka, Japan) for 60 min at room temperature, incubated separately with specific primary antibodies to CCP3 (19 and 17 kDa: #9661; Cell Signaling Technology, Boston, USA; 1:1000 dilution) and ACTB antibody (42 kDa: A2228; Sigma–Aldrich; 1:4000 dilution) in Can Get Signal
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Fig. 2. Percentage of KI-67-positive cells in the bovine endometrium during the estrous cycle (mean ± SEM, n = 4 experiments). Percentages in each endometrial structure (A: luminal epithelium, B: stroma and C: glandular epithelium) were derived from dividing the number of positively stained cells by total number of cells under crosspoint in each histological section at ×400 magnification. Cells were counted in three randomly chosen areas. Different superscript letters indicate significant differences (P < 0.05).
Immunoreaction Enhancer Solution 1 (NKB-201; TOYOBO) overnight at 4 ◦ C, incubated with secondary antibodies to CCP3 (anti-rabbit Ig, HRP-linked whole antibody produced in donkey; NA934; GE Healthcare; 1:4000 dilution) and ACTB (NA931; anti-mouse Ig, HRP-linked whole antibody produced in sheep; GE Healthcare; 1:40,000 dilution) in Can Get Signal Immunoreaction Enhancer Solution 2 (NKB301; TOYOBO) for 60 min at room temperature, incubated in Immobilon Western Chemiluminescent HRP Substrate (WBKLS0500; Merck) and captured using a ChemiDocTM XRS+ System (Bio-Rad). The intensity of the immunological reaction was estimated by measuring the optical density in the defined area by computerized densitometry using an Image LabTM software (170-8265J1PC; Bio-Rad)
2.7. Statistical analysis All experimental data are shown as the mean ± SEM. The statistical significance of differences was assessed by analysis of variance (ANOVA) followed by Tukey’s multiple
comparisons test using GraphPad Prism ver. 6.0 for Mac (GraphPad Software, San Diego, CA, USA).
3. Results 3.1. Localization and quantification of KI-67-positive cells throughout the estrous cycle In the bovine endometrium, KI-67 expressions were localized in luminal and glandular epithelia and the stroma throughout the estrous cycle (Fig. 1), and the percentage of KI-67-positive cells was calculated based on these chromatic fields (Fig. 2). The rate of KI-67-positive cells was higher at the follicular stage than at the mid and late luteal stages in the luminal epithelium (Fig. 2A; P < 0.05), and was higher at the follicular stage than at the late luteal stage in the stroma (Fig. 2A; P < 0.05). Furthermore, the percentage of KI-67-positive cells was higher at the early luteal and follicular stages than at the mid and late luteal stages in the glandular epithelium (Fig. 2 C; P < 0.05).
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Fig. 3. TUNEL-positive cells (arrows) in sections of bovine endometrium prepared at the early luteal (A), mid luteal (B), late luteal (C) and follicular (D) stages of the estrous cycle. Scale bar represents 100 m.
Fig. 4. CCP3-positive cells (arrows) in sections of bovine endometrium prepared at the early luteal (A), mid luteal (B), late luteal (C) and follicular (D) stages of the estrous cycle. Scale bar represents 100 m.
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Fig. 5. Percentage of CCP3-positive cells in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 4 experiments). Percentages of CCP3positive cells in each endometrial structure (A: luminal epithelium, B: stroma and C: glandular epithelium) were derived by dividing the number of CCP3-positive cells by total number of cells under crosspoint in the histological section at a magnification of ×400. Cells were counted in three randomly chosen areas. Different superscript letters indicate significant differences (P < 0.05).
3.2. Localization of TUNEL-positive cells throughout the estrous cycle
3.4. CASP3 mRNA and CCP3 protein expression throughout the estrous cycle
TUNEL-positive cells were found in the luminal and glandular epithelia and in the stroma during the estrous cycle. However, the number of stained cells was very few and did not significantly change during estrous cycle (Fig. 3).
CCP3 mRNA expression did not change significantly during the estrous cycle (Fig. 6), whereas 19 kDa and 17 kDa
3.3. Localization and quantification of CCP3-positive cells throughout the estrous cycle CCP3 was expressed throughout the estrous cycle, but predominantly in the luminal epithelium (Fig. 4). The percentage of CCP3-positive cells was higher at the early luteal stage than at the mid and late luteal stages in the luminal epithelium (Fig. 5A; P < 0.05), and was higher at the early luteal stage than at the other stages in the stroma (Fig. 5B; P < 0.05). In contrast, the number of CCP3-positive cells did not significantly change during the estrous cycle in the glandular epithelium (Fig. 5 C).
Fig. 6. Changes in the relative amounts of CASP3 mRNA expression in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 5 experiments).
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Fig. 7. Changes in the relative amounts of CCP3 protein (A: 19 kDa and B: 17 kDa) expressions in the bovine endometrium throughout the estrous cycle (mean ± SEM, n = 5 experiments). Different superscript letters indicate significant differences (P < 0.05).
CCP3 protein levels were higher at the early luteal and follicular stages than at the mid and late luteal stages (Fig. 7A and B; P < 0.05). 4. Discussion In the present study, the percentages of KI-67-positive cells were higher from the follicular stage to the early luteal stage in all types of endometrial cells. These results are consistent with previous studies that showed cyclic cell proliferation in rat (Sato et al., 1997), sow (Sukjumlong et al., 2003), equine (Gerstenberg et al., 1999), ewe (Johnson et al., 1997b) and human (Gargett et al., 2008). Estradiol17 secreted by the ovary induces the proliferation of endometrial cells in human (Jabbour et al., 2006), rodents (Tong and Pollard, 1999; Zhang and Paria, 2006) and sheep (Johnson et al., 1997a). Additionally, estradiol-17 also induces the expressions of growth factors throughout the estrous cycle in the bovine endometrium (Robinson et al., 2000; Tasaki et al., 2010) and promotes the proliferation of bovine endometrial epithelial cells by stimulating the production of a variety of growth factors in stromal cells (Xiao and Goff, 1998). Therefore, the cell proliferation observed in the present study at the early luteal and follicular stages may result from growth factors stimulated by estradiol17. The removal of unwanted cells by apoptosis plays an important role in the maintenance of homeostasis in various tissues including the endometrium (Harada et al., 2004; Jin and El-Deiry, 2005). In our study, CCP3-positive cells were observed throughout the estrous cycle. In addition, the percentage of CCP3-positive cells increased at the follicular and early luteal stages in the bovine endometrium. These findings are coincident with the previous finding that apoptotic cells were barely detected at the mid and late luteal stages (Days 12–18) in the bovine endometrium (Groebner et al., 2010). Endometrial apoptosis is mainly promoted at the periods of luteolysis or periovulatory in
several species (Hopwood and Levison, 1976; Sato et al., 1997; Van Cruchten et al., 2003; Okano et al., 2007; Roberto da Costa et al., 2007; Wood et al., 2007). When pregnancy does not occur, the endometrium may be renewed to prepare for the next chance to be pregnant. Furthermore, endometrial apoptosis is induced by withdrawal of progesterone in rabbit (Rotello et al., 1992), by withdrawal of estradiol-17 in hamster (Sandow et al., 1979) and mouse (Jo et al., 1993) or by the withdrawal of both in human (Song et al., 2002). In the present study, CCP3-positive cells were relatively higher at both the follicular (low progesterone level) and early luteal stages (low estradiol-17 level). The above findings suggest that the withdrawal of progesterone of estradiol-17 or both contributes to induce apoptosis in the bovine endometrium. CCP3-positive cells were fewer than KI-67-positive cells in the bovine endometrium throughout the estrous cycle in the present study. KI-67 protein is thought to be expressed in all active phases of the cell cycle, for approximately 24 h in certain human cells in culture (Cooper, 2000), whereas apoptotic cells are quickly engulfed by phagocytes (Jin and El-Deiry, 2005; Erwig and Henson, 2007). Therefore, the reason why CCP3-positive cells were fewer than KI-67positive cells, may be that apoptotic cells are removed rapidly by phagocytosis in the bovine endometrium. Furthermore, apoptotic mammary epithelial cells are extruded from the epithelial layer, preserving its structure in the rat mammary gland (Monks et al., 2008). In fact, CCP3-positive cells were found to be extruded from the luminal epithelium to the side of the uterine cavity in the present study. TUNEL-positive cells appeared to be fewer than CCP3positive cells, although the difference was not significant. Similar results were reported for dogs (Van Cruchten et al., 2003), horses (Roberto da Costa et al., 2007) and cats (Liman et al., 2013). The reason for the fewer TUNEL-positive cells than CCP3-positive cells may be that CASP3 are cleaved before apoptotic DNA fragmentation (Nagata, 2000; Duan et al., 2003).
Please cite this article in press as: Arai, M., et al., Remodeling of bovine endometrium throughout the estrous cycle. Anim. Reprod. Sci. (2013), http://dx.doi.org/10.1016/j.anireprosci.2013.08.003
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Apoptosis is regulated by continuous cleavages and activations of caspases, especially CASP3 (Jin and El-Deiry, 2005). In the present study, CASP3 mRNA expression in bovine endometrium during the estrous cycle disagreed with CCP3 protein expression. CASP3 mRNA expression is readily detected in a variety of tissues (Earnshaw et al., 1999), whereas the cleavage and activation of CASP3 needs initiator caspases (e.g., CASP-8 and CASP-9) activated by diverse apoptotic stimuli (Jin and El-Deiry, 2005). Therefore, the inconsistency of expressions of CASP3 mRNA and CCP3 protein may be due to the differences between the transcriptional and activation mechanisms of CASP3. The overall results show that cell proliferation and apoptosis undergo cyclic patterns in the bovine endometrium, and suggest that the endometrium in cow is also remodeled in each estrous cycle. Acknowledgement This research was supported by Grant-in-Aid for Scientific Research (No. 24380155) of the Japan Society for the Promotion of Science (JSPS). References Cavagna, M., Mantese, J.C., 2003. Biomarkers of endometrial receptivity – a review. Placenta 24 (Suppl. B), S39–S47. Cooper, G., 2000. The eukaryotic cell cycle. In: The Cell: A Molecular Approach, 2nd edition. Sinauer Associates Inc, Sunderland, Massachusetts. Duan, W.R., Garner, D.S., Williams, S.D., Funckes-Shippy, C.L., Spath, I.S., Blomme, E.A., 2003. Comparison of immunohistochemistry for activated caspase-3 and cleaved cytokeratin 18 with the TUNEL method for quantification of apoptosis in histological sections of PC-3 subcutaneous xenografts. J. Pathol. 199, 221–228. Earnshaw, W.C., Martins, L.M., Kaufmann, S.H., 1999. Mammalian caspases: structure, activation, substrates, and functions during apoptosis. Annu. Rev. Biochem. 68, 383–424. Erwig, L.P., Henson, P.M., 2007. Immunological consequences of apoptotic cell phagocytosis. Am. J. Pathol. 171, 2–8. Gargett, C.E., Chan, R.W., Schwab, K.E., 2008. Hormone and growth factor signaling in endometrial renewal: role of stem/progenitor cells. Mol. Cell. Endocrinol. 288, 22–29. Gerstenberg, C., Allen, W.R., Stewart, F., 1999. Cell proliferation patterns in the equine endometrium throughout the non-pregnant reproductive cycle. J. Reprod. Fertil. 116, 167–175. Groebner, A.E., Schulke, K., Unterseer, S., Reichenbach, H.D., Reichenbach, M., Buttner, M., Wolf, E., Meyer, H.H., Ulbrich, S.E., 2010. Enhanced proapoptotic gene expression of XAF1, CASP8 and TNFSF10 in the bovine endometrium during early pregnancy is not correlated with augmented apoptosis. Placenta 31, 168–177. Harada, T., Kaponis, A., Iwabe, T., Taniguchi, F., Makrydimas, G., Sofikitis, N., Paschopoulos, M., Paraskevaidis, E., Terakawa, N., 2004. Apoptosis in human endometrium and endometriosis. Hum. Reprod. Update 10, 29–38. Hojo, T., Al-Zi’abi, M.O., Skarzynski, D.J., Acosta, T.J., Okuda, K., 2009. Changes in the vasculature of bovine corpus luteum during the estrous cycle and prostaglandin F2␣-induced luteolysis. J. Reprod. Dev. 55, 512–517. Hopwood, D., Levison, D.A., 1976. Atrophy and apoptosis in the cyclical human endometrium. J. Pathol. 119, 159–166. Jabbour, H.N., Kelly, R.W., Fraser, H.M., Critchley, H.O., 2006. Endocrine regulation of menstruation. Endocr. Rev. 27, 17–46. Jin, Z., El-Deiry, W.S., 2005. Overview of cell death signaling pathways. Cancer Biol. Ther. 4, 139–163. Jo, T., Terada, N., Saji, F., Tanizawa, O., 1993. Inhibitory effects of estrogen, progesterone, androgen and glucocorticoid on death of neonatal mouse uterine epithelial cells induced to proliferate by estrogen. J. Steroid Biochem. Mol. Biol. 46, 25–32. Johnson, M.L., Redmer, D.A., Reynolds, L.P., 1997a. Effects of ovarian steroids on uterine growth, morphology, and cell proliferation in ovariectomized, steroid-treated ewes. Biol. Reprod. 57, 588–596.
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Please cite this article in press as: Arai, M., et al., Remodeling of bovine endometrium throughout the estrous cycle. Anim. Reprod. Sci. (2013), http://dx.doi.org/10.1016/j.anireprosci.2013.08.003