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Importance of Uterine Cell Death, Renewal, and TheirHormonal Regulation in Hamsters that ShowProgesterone-Dependent Implantation

Qian Zhang and Bibhash C. Paria

Division of Reproductive and Developmental Biology, Department of Pediatrics, Vanderbilt University Medical Center,Nashville, Tennessee 37232

This study was initiated to investigate the significance of uter-ine cell death and proliferation during the estrous cycle andearly pregnancy and their correlation with sex steroids inhamsters where blastocyst implantation occurs in only pro-gesterone-primed uteri. The results obtained in hamsterswere also compared with mice where blastocyst implantationoccurs in progesterone-primed uteri if estrogen is provided.Apoptotic cells in the uterus were detected by using terminaldeoxynucleotide transferase-mediated deoxyuridine triphos-phate nick end labeling (TUNEL) technique. Uterine cell pro-liferation was determined by 5-bromo-2�-deoxyuridine label-ing followed by immunohistochemistry and methyl-tritiated[3H]thymidine labeling. Active caspase-3, an executor proteinof cell death, expression was assayed by immunohistochem-istry/immunofluorescence. Our results demonstrate that ep-ithelial proliferation on the second day after mating marksthe initiation of pregnancy-related uterine changes in both

species despite their differences in hormonal requirements.Hamsters and mice showed subtle differences in uterine pro-liferative and apoptotic patterns during early pregnancy andin response to steroids. There existed almost a direct corre-lation between apoptosis and caspase-3 expression, suggest-ing uterine cell death mostly involves the caspase pathway.Consistent with these findings, we showed, for the first time,that execution of uterine epithelial cell apoptosis by caspase-3is important for blastocyst implantation because a caspsase-3inhibitor N-acetyl-DEVD-CHO when instilled inside the uter-ine lumen on d 3 of pregnancy inhibits implantation in ham-sters and mice. The overall results indicate that uterine cellapoptosis and proliferation patterns are highly ordered cell-specific phenomena that play an important role in maintain-ing the sexual cycle and pregnancy-associated uterinechanges. (Endocrinology 147: 2215–2227, 2006)

THE UTERUS UNDERGOES cellular remodeling duringeach sexual cycle for a possible pregnancy. In the ab-

sence of pregnancy, uterine changes are reversible, permit-ting preparation for pregnancy in a subsequent cycle. In theevent of mating and successful fertilization, however, thechanges in the uterus take another route to support preg-nancy. The uterine cellular changes during the cycle andpregnancy are regulated by the circulating levels of ovariansex steroids progesterone (P4) and estrogen (E). One of thebasic events of uterine cellular change is the systemic cellturnover that consists of cell death by apoptosis and cellrenewal by proliferation. Cell apoptosis and cell proliferationhave been studied in the uterus of mice and rats (1–4). How-ever, there has been no detailed study of uterine cell apo-ptosis and proliferation during the estrous cycle and earlypregnancy and their possible relation to circulating sex ste-roid levels in hamsters. Unlike mice and rats that require bothovarian P4 and E for uterine receptivity and implantation(5–7), hamsters need only ovarian P4, but not ovarian E

(8–12). Furthermore, rabbits, pigs, monkeys, and perhapshumans show ovarian P4-dependent implantation like ham-sters (13–17).

Previous studies in mice and rats have reported an inversecorrelation between cell death and cell proliferation in theuterus (4). Although uterine epithelial proliferative indicesare at their highest level on the proestrous day, epithelialapoptotic indices are their lowest level on this day (18). Areverse scenario was demonstrated between uterine cell pro-liferation and apoptosis on the estrous day in rats (19). Theincreased uterine epithelial apoptosis on the estrous day wasattributed to a decline in serum E level (4). A number ofstudies in humans also showed that preovulatory ovarian Esecretion stimulates proliferation of uterine glandular epi-thelial cells during the proliferative phase (20). This alsoremains relatively high in the secretory phase when prede-cidual cells also show increased DNA synthesis. A rapidincrease in the apoptotic index was noted in both epitheliumand stroma during the last days of the cycle (late secretoryphase) with a maximum index on the second day of men-struation (21). However, the incidence of apoptotic stromalcells during late secretory phase is still controversial becauseothers have shown few apoptotic stromal cells at any stageof the cycle (22, 23).

In regard to the direct actions of P4 and E on uterus cellproliferation, animal studies have shown that whereas treat-ment of 17�-estradiol (E2) alone to ovariectomized mice stim-ulated the uterine epithelial cell proliferation, treatment with

First Published Online February 9, 2006Abbreviations: Ac-DEVD-CHO, N-Acetyl-Asp-Glu-Val-Asp-CHO;

AEC, aminoethylcarbazole; BrdU, 5-bromo-2�-deoxyuridine; dUTP, de-oxyuridine triphosphate; E, estrogen; E2, 17�-estradiol; P4, progesterone;PDZ, primary decidual zone; SDZ, secondary decidual zone; TdT, ter-minal deoxynucleotide transferase; TUNEL, terminal deoxynucleotidetransferase-mediated deoxyuridine triphosphate nick end labeling.Endocrinology is published monthly by The Endocrine Society (http://www.endo-society.org), the foremost professional society serving theendocrine community.

0013-7227/06/$15.00/0 Endocrinology 147(5):2215–2227Printed in U.S.A. Copyright © 2006 by The Endocrine Society

doi: 10.1210/en.2005-1555

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P4 alone induced stromal cell proliferation (1). However,treatment of P4-primed animals with E2 mainly maintainedthe proliferation in stromal cells but does not stimulate pro-liferation in epithelial cells (1, 24). In the mouse and hamsteruteri, apoptotic epithelial cell death mainly occurs because ofE withdrawal and can be blocked by E2 treatment (25, 26).

After a successful mating, preimplantation uteri of themouse first show luminal and glandular epithelial cell pro-liferation on the second day of pregnancy. On d 3 of preg-nancy, however, epithelial cell proliferation ceases and stro-mal cell proliferation begins because of increased P4

synthesis by corpora lutea. This stromal cell proliferation isfurther enhanced on d 4 in response to ovarian E secretionbefore initiation of implantation in mice (1, 27, 28). The com-bined effect of ovarian P4 and E in stromal cell proliferationis a prerequisite uterine change for blastocyst implantationin this rodent. The apoptotic patterns in uterine cells duringthe preimplantation period have not been studied in detail.

Initiation of blastocyst implantation induces proliferationof stromal cells that are situated only surrounding the im-planting blastocyst (1). These proliferating stromal cells failto undergo cytokinesis and become polyploid, forming theprimary decidual zone (PDZ) (29, 30). As the pregnancycontinues, cells of the PDZ on d 6 stop proliferating, butstromal cells next to the PDZ undergo proliferation andpolyploidy, forming the secondary decidual zone (SDZ) (1,3, 29). In regard to apoptosis, the general consensus is thatantimesometrial luminal epithelial cells surrounding the im-planting embryo die during implantation (31). As gestationprogresses, epithelial degeneration extends to the mesome-trial side. Because stromal decidualization is more at theantimesometrial area compared with the mesometrial area,cell death starts first at the PDZ and then at the SDZ of theantimesometrial area, causing a stepwise regression of de-cidual tissues and creating space for the growing embryo.Besides steroid hormones, various local factors such asgrowth factors and cytokines have been shown to be crucialmediators of apoptosis. However, our knowledge of the mo-lecular mechanisms underlying uterine apoptosis is stillpoor. There are two major pathways to activate proteases ofthe caspase family for apoptosis. The extrinsic pathway in-volves the death receptors and their ligands. Best studied arethe Fas ligands, TNF-�, TGF-�, and their receptors. The in-trinsic pathway induces oligomerization of the cytosolic ap-optotic protease-activating factor-1 (Apaf-1) and apopto-some formation by cytochrome c release from themitochondria. The caspase cascade is activated to execute theapoptotic cells in both pathways. Caspase-8 and caspase-9serve as initiators and are situated at the top of the cascade.Caspase-3 and caspase-6 are effector execution caspases thatdegrade cells (3).

In the present study, cell-type-specific apoptosis and pro-liferation was investigated in the uterus of hamsters duringthe estrous cycle and early pregnancy and after steroid treat-ment in ovariectomized animals. Uterine apoptotic patternswere correlated with expression of active caspase-3. Ourapoptotic and proliferation data in the hamster uterus werealso compared with findings in the mouse uterus. We alsoshow here the importance of caspace-3-mediated uterine cell

apoptosis in initiation of implantation both in the hamsterand mouse.

Materials and MethodsAnimals

Adult virgin male and female golden hamsters (Mesocricetus auratus)and CD1 mice were purchased from Charles River Laboratories (Wil-mington, MA). They were maintained in a 14-h light, 10-h dark cycle inthe Laboratory Animal Facility of the Vanderbilt University (Nashville,TN) with unlimited access to water and food according to the institu-tional and National Institutes of Health guidelines on the care and useof laboratory animals. All experimental protocols were approved by theVanderbilt University Committee of Use and Care of animals.

Uterine tissue collection during the estrous cycle ofthe hamster

The 4-d estrous cycle of hamsters was monitored by the presence ofcharacteristic vaginal discharge on the morning of the estrous day,which was also designated d 1 of the estrous cycle (32). Hamsters withat least three consecutive regular 4-d cycles were used in this study.Hamsters were killed at the estrus, metestrus, diestrus, and proestrus at0830–0900 h, 2 h after an injection (1 ml/100 g body weight) of 5-bromo-2�-deoxyuridine (BrdU) (catalog no. RPN201) purchased from Amer-sham Biosciences (Piscataway, NJ) to detect proliferative cells. Theiruteri were quickly removed, cut into small pieces, and rapidly frozen incold Super Friendly Freeze’it (Curtin Matheson Scientific, Houston, TX)and stored at �70 C for cell proliferation, cell apoptosis, and caspase-3expression studies. Unless otherwise mentioned, all materials were pur-chased from Sigma (St. Louis, MO) or Fisher Scientific (Pittsburgh, PA).

Preparation of pregnant hamsters and mice and uterinetissue collection

Female hamsters that showed three consecutive 4-d estrous cycleswere housed with fertile males overnight on the evening of proestrus.Finding of sperm in the vaginal smear the next morning (estrus) indi-cated the first day (d 1) of pregnancy (32). Three CD1 female mice wereplaced overnight with one fertile male for mating. Pregnancy was con-firmed the following morning by checking for the presence of a copu-latory plug in the vagina (d 1 � vaginal plug) (7). Animals were injectedip with either methyl-tritiated [3H]thymidine (25 �Ci/0.1 ml; catalog no.NET027Z; specific activity, 70–90 Ci/mmol; PerkinElmer Life Sciences,Boston, MA) (1) or BrdU (1 ml/100 g body weight) 2 h before they werekilled.

Hamsters and mice on d 1–3 of pregnancy were killed at 0830–0900h. Their whole uteri were removed and cut into small pieces afterconfirmation of pregnancy by flushing and recovering embryos fromoviducts (32). Although whole uteri were collected on the morning of d4 (0900 h), implantation sites were collected on the morning (0900 h) ofd 5 after an iv injection of Chicago Blue B dye solution [0.25 ml of 1%(wt/vol) dye in saline] in both species (7, 32). Implantation sites on thesedays were visualized by intermittent blue bands along the horns. On d6–8, implantation sites were distinct and were identified visually with-out blue dye injection. Uterine tissues were immediately frozen in coldSuper Friendly Freeze’it and stored at �70 C for cell proliferation, cellapoptosis, and caspase-3 expression studies.

To determine the effects of P4 and E on uterine cell proliferation andapoptosis, mice and hamsters were ovariectomized regardless of theirstage of estrous cycle and rested 15 d to eliminate circulating steroids.Control animals were injected with vehicle of steroids in sesame seed oil(0.1 ml/animal). Some animals were injected with a single injection ofeither P4 (2 mg/0.1 ml per mouse; 2 mg/0.1 ml per hamster) or E2 (100ng/0.1 ml per mouse; 1000 ng/0.1 ml per hamster) or a combination ofthe same doses of P4 plus E2 and killed 24 h later for uterine tissuecollection. Ovariectomized animals were also injected with P4 for 2 or 3 d(once each day) with or without an injection of E2 on the third day ofP4 treatment. The dose of P4 was selected on the basis of its ability tomaintain pregnancy in ovariectomized or hypophysectomized hamsters(8, 32). The dose of E2 was selected based on its ability to stimulateheparin-binding epidermal growth factor-like growth factor gene ex-

2216 Endocrinology, May 2006, 147(5):2215–2227 Zhang and Paria • Uterine Cell Turnover in Hamsters


pression in ovariectomized uteri of hamsters (32). All animals werekilled 24 h after the last injection. Animals were injected with either[3H]thymidine or BrdU 2 h before they were killed.

Apoptosis detection

DNA fragmentation during apoptosis was detected by the terminaldeoxynucleotide transferase (TdT)-mediated deoxyuridine triphosphate(dUTP) nick end labeling (TUNEL) technique using an apoptosis de-tection kit (Promega, Madison, WI; catalog no. G7130). TUNEL assaywas performed according to the manufacturer’s instruction with minormodification. Briefly, fresh-frozen sections (12 �m) were fixed in 10%(vol/vol) formalin for 30 min, washed in water, dehydrated in ascendingconcentrations [30, 50, 70, 85, 95, and 100% (vol/vol)] of ethyl alcohol,washed in xylene and acetone, rehydrated in descending concentrationsof ethyl alcohol, and finally washed in water and PBS before beingincubated with proteinase K (20 �g/ml) for 5 min. The sections werethen washed in PBS, refixed in 10% (vol/vol) formalin for 5 min, anddivided into three groups. At the beginning of the experiment, thepositive control sections were incubated with DNase (RQ1 RNase-freeDNase from Promega; catalog no. M6101) for 5 min and rinsed in water.Next, all three groups (positive, treatment, and negative) were incubatedwith equilibration buffer for 20 min. Subsequently, 250 �l TUNEL re-action mixture containing biotinylated nucleotide and TdT was addedto sections of the positive and treatment groups for 60 min at 37 C in thedark. For negative control, TdT was omitted from the reaction mixture.The reaction was terminated by transferring all the slides to the termi-nation buffer for 15 min. The sections were rinsed in PBS, incubated in0.3% (vol/vol) hydrogen peroxide (H2O2), washed in PBS, and incu-bated with streptavidin horseradish peroxidase solution for 60 min.After the sections were washed in PBS, aminoethylcarbazole (AEC)single solution (Zymed Laboratories Inc., San Francisco, CA; catalog no.00-1111) was added to the slide and developed color to detect biotin-ylated nucleotide. The sections were counterstained with hematoxylinand photographed in an ECLIPSE 80i Nikon microscope connected to acomputer with ACT-2U program for the DS-5M camera.

Caspase-3 immunocytochemistry

Freshly prepared cryosections were rapidly fixed (10 min) in acetoneat room temperature for 10 min. After rinsing in PBS, the blockingsolution of 10% (vol/vol) goat serum was added to the section for 10 minat room temperature followed by the primary antibody (antirabbit activecaspase-3 obtained from Promega) (1:40 dilution) in PBS at 4 C over-night. Subsequent immunostaining was performed using biotinylatedgoat antirabbit secondary antibody, streptavidin-horseradish peroxi-dase conjugate, and a substrate chromogen mixture (AEC single solu-tion) from Zymed. Sections were lightly counterstained with hematox-ylin. Brown deposits indicated the sites of immunoreactive proteins (33).Negative control studies were performed in which PBS was used insteadof primary antibody.

Double immunofluorescence (colocalization of caspase-3staining and TUNEL labeling)

A slide having cryosections from d-1 uterus of the hamster, and d-5implantation sites of both the hamster and mouse was first fixed inacetone for caspase-3 staining. The slide was first incubated with anti-rabbit active caspase-3 overnight at 4 C and visualized with a tetram-ethyl rhodamine isothiocyanate-labeled goat antirabbit secondary an-tibody (Zymed Laboratories Inc.). The same slide was then processed forTUNEL assay using the DeadEnd Fluorometric TUNEL system (Pro-mega), which measures the fragmented DNA of apoptotic cells by cat-alytically incorporating fluorescein-12-dUTP at 3�-OH DNA ends usingTdT. The double fluorescence was visualized directly by fluorescencemicroscopy using the Nikon Eclipse TS100 with an X-cite 120 fluores-cence illumination system.

Cell proliferation assays

BrdU incorporation. Proliferating uterine cells were assessed by BrdU-labeled cells using a kit from Calbiochem (catalog no. HCS30). Sectionswere soaked in 70% (vol/vol) ethyl alcohol for 1 h and then washed in

PBS for 5 min. Endogenous peroxidase was inactivated by covering thesection with 0.3% (vol/vol) fresh H2O2 in water for 10 min. Slides werethen washed and incubated with denaturing solution for 90 min. Afterwashing, sections were first treated with blocking solution for 30 minand then incubated at 4 C overnight with anti-BrdU mouse monoclonalantibody conjugated with biotin. After being rinsed in PBS, sections wereincubated for 90 min with streptavidin conjugated with peroxidase. Thesections were then rinsed in PBS, and peroxidase activity was revealedthrough the use of AEC single solution as chromogen. Control uterinesections obtained from the animal without BrdU injection were stainedin a similar manner. All sections were lightly counterstained with he-matoxylin and photographed.

[3H]Thymidine incorporation. To identify proliferating uterine cells, ham-sters and mice were treated ip with [3H]thymidine (25 mCi/0.1 ml insaline) 2 h before they were killed. Small pieces of uterine tissues werefrozen for cryosections. Cryosections mounted in poly-l-lysine-coatedslides were fixed in 4% (wt/vol) paraformaldehyde solution in PBS.Sections were rinsed in water; air dried, and dipped in NTB-2 emulsion.The slides were developed 5–6 d after dipping in NTB-2 emulsion(Eastman Kodak, Rochester, NY) to detect silver grains in the prolifer-ating nuclei (1). Sections were also counterstained with hematoxylin andphotographed (dark-field).

Blastocyst implantation after treatment of caspase-3inhibitor in pregnant hamsters

The ability of the caspase-3 inhibitor to inhibit or delay the initiationof implantation was determined in pregnant hamsters and mice. In thepresent study, an irreversible caspase-3 inhibitor N-acetyl-Asp-Glu-Val-Asp-CHO (Ac-DEVD-CHO) (EMD Biosciences, Inc., La Jolla, CA; cat-alog no. 235420) was chosen because caspase-3 appears to be one of thekey mediators of apoptosis (34). A single intraluminal injection of Ac-DEVD-CHO (10 �g/5 �l sterile saline in hamsters or 5 �g/2 �l sterilesaline in mice) was administered in one uterine horn at the ovarian endon the morning of d 3 (0900 h) when embryos were still in oviducts. Thecontralateral horn received an intraluminal injection of equal volume ofvehicle saline (5 and 2 �l sterile saline in hamsters and mice, respec-tively). Hamsters and mice were killed on the morning of d 5 (0900 h)15 min after blue dye injection. The number of implantation sites (bluebands) was visually recorded. The uterine horn not showing implan-tation sites was flushed with culture medium to recover blastocysts, andmorphological appearances of these blastocysts were checked by directvisualization under a stereo zoom microscope.

Embryo culture

Female hamsters were superovulated by ip injection of 20 IU preg-nant mare serum gonadotropin before 0900 h on the estrous day andwere housed with males overnight on the evening of proestrus (32). Tostudy the effects of Ac-DEVD-CHO on preimplantation embryo devel-opment, eight-cell embryos from superovulated hamsters and normalpregnant mice were flushed out from the uterus and oviduct, respec-tively, on d 3 (1300–1400 h). Hamster embryos were cultured in a groupof 10–15 embryos per drop (50 �l) in hamster embryo culture medium-6under silicon oil in an atmosphere of 10% CO2/90% air at 37 C for 24 h(35) in the presence or absence of Ac-DEVD-CHO (5 and 10 �g/ml).Mouse embryos were cultured in a group of eight to 10 embryos per drop(25 �l) in Whitten’s medium under silicon oil in an atmosphere of 5%CO2/95% air at 37 C for 48 h (36) in the presence or absence of Ac-DEVD-CHO (10 �g/ml). At the end of culture, the number of embryosthat developed to blastocysts was recorded.

Statistical analysis

All data were subjected to �2 test followed by Fisher’s exact test usingthe SAS 9.1 program (SAS Institute Inc., Cary, NC) to determine sta-tistical differences among groups. In all cases, P � 0.05 was consideredto be statistically significant.

Zhang and Paria • Uterine Cell Turnover in Hamsters Endocrinology, May 2006, 147(5):2215–2227 2217


ResultsApoptosis and proliferation in uterine cells during theestrous cycle in hamsters

Apoptosis and proliferation in uterine cells during theestrous cycle of hamsters is summarized in Fig. 1. Hamstersshow regular 4-d (estrus, metestrus, diestrus, and proestrus)estrous cycles in our colony. The apoptosis in luminal andglandular epithelial cells was first detected on the estrousday and continued to the metestrous day with lower fre-quency. Only a few apoptotic cells were noticed in stromaand muscle layers on both the estrous and metestrous days(Fig. 1). The number of apoptotic cells was much less in theepithelial, stromal, and muscular layers on the diestrous andproestrous days (Fig. 1). Caspase-3 expression was also no-ticed both in the luminal and glandular epithelial cells on theestrous day, which correlated to the pattern of TUNEL stain-ing. On the metestrous day, caspase-3 staining was observedin the luminal and glandular epithelial cells when epithelialcells were still undergoing apoptosis (Fig. 1). Immunoreac-tivity for caspase-3 was also noticed in a few subepithelialstromal cells. Caspase-3 staining appeared to be localized inthe nucleus. The number of caspase-positive cells were onlya few on the diestrous and proestrous days when there wasalmost no cellular apoptosis noticed (Fig 1). We next studiedthe cell proliferation pattern in the cyclic uterus of hamstersbecause apoptosis is followed by regeneration of new cells.We observed only a few proliferating cells in epithelial, stro-mal, and muscular compartments on the estrous day. Therewas a very small increase in the number of proliferating cells

in epithelial and stromal layers on the metestrous day (Fig.1). A noticeable increase in the number of stromal cell andglandular epithelial proliferations was noticed on thediestrous day, whereas no luminal epithelial cell prolifera-tion was found (Fig. 1). On the proestrous day, however, weobserved an increase in the number of proliferating luminalepithelial cells. Simultaneously, a reduction in the number ofstromal and glandular proliferating cells was noticed on thisday of the cycle. Cell proliferation in muscular layers re-mained unchanged on both the diestrous and proestrousdays (Fig. 1).

Separate effects of P4 and E2 in uterine cell apoptosisand proliferation

The effects of ovarian steroid hormones on uterine cellapoptosis and proliferation are shown in Figs. 2–4. TUNELresults revealed the presence of apoptotic cells more in theglandular epithelial cells and less in the luminal epithelialcells in the ovariectomized sesame oil-treated (vehicle fordissolving steroids) hamster (Fig. 2). However, there weremore apoptotic cells in the luminal epithelium than the glan-dular epithelium in the mouse (Fig. 3). There were only a fewapoptotic cells noticed both in the stroma and myometrium

FIG. 1. Sections of hamster uteri from each day of the estrous cyclewere immunostained to demonstrate cell-specific apoptosis and pro-liferation. Apoptosis were determined by TUNEL assay and activecaspase staining. Cell proliferation was determined by BrdU incor-poration. Arrow indicates positive immunostaining. Data representtwo to three animals from each day of the cycle. cm, Circular myo-metrium; ge, glandular epithelium; le, luminal epithelium; s, stroma.

FIG. 2. Sections of ovariectomized hamster uteri treated with vehi-cle, P4 and E2 were immunostained to demonstrate hormonal effectson cell-specific apoptosis and proliferation. Apoptosis were deter-mined by TUNEL assay. Cell proliferation was determined by BrdUincorporation. Arrow head indicates positive immunostaining. Datarepresent at least two to three animals in each treatment group. cm,Circular myometrium; ge, glandular epithelium; le, luminal epithe-lium; lm, longitudinal myometrium; s, stroma.



in both species. Although a single injection of P4 or E2 sig-nificantly attenuated the number of apoptotic cells in mice(Fig. 3), it has apparently no effect in hamsters (Fig. 2).

In regard to the uterine cell proliferation in ovariectomizedmice and hamsters, we noticed only a few scattered [3H]thy-midine-positive (Fig. 4) or BrdU-positive (Figs. 2 and 3) en-dometrial cells in the uterus of sesame oil vehicle-treatedanimals. Although a single injection of P4 did not cause anyproliferation of uterine cells in the ovariectomized hamsterat 24 h (Figs. 2 and 4), an injection of the same dose of P4 tothe ovariectomized mouse showed significant increase instromal cell proliferation at 24 h (Fig. 3). Treatment with P4for 48 h (once a day for 2 d), however, showed a moderateincrease in the number of proliferating uterine stromal cellsin hamsters (Fig. 2) compared with oil-treated control and P4treatment for 24 h. A similar P4 regimen in ovariectomizedmice reduced the proliferating stromal cell numbers at 48 hcompared with proliferating stromal cell numbers at 24 hafter P4 treatment (Fig. 3). A single injection of E2 to ovari-ectomized hamsters stimulated proliferation in both theglandular and luminal epithelia but not in the stroma, asdetected by the BrdU incorporation method (Fig. 2). A singleinjection of E2 to ovariectomized mice was also effective instimulating epithelial and glandular cell proliferation (Fig.

3). However, treatment of a single injection of E2 togetherwith P4 or to a P4-primed animal resulted in a significantincrease in the number of [3H]thymidine-labeled stromalnuclei by 24 h, suggesting that the presence of E2 enhancesP4 actions on stromal cell proliferation (Fig. 4). However,some epithelial DNA synthesis was also detected in theseuteri (Fig. 4). These results suggest that uterine cell apoptosisand proliferation processes are regulated by sex steroids ina species-specific manner.

Uterine cell apoptosis and proliferation during first 8 d ofpregnancy in relation to preparation of the receptive uterusfor implantation, initiation of implantation, and stromaldecidualization and progression of implantation processfor placentation

Apoptosis during d 1–4 of pregnancy in hamsters and mice.TUNEL and caspase-3 staining results during d 1–4 of earlypregnancy are presented in Figs. 5–7. During the days ofuterine preparation for implantation, TUNEL staining wasprimarily observed in uterine glandular and luminal epithe-lial cells on d 1 of pregnancy in hamsters (Fig. 5). AlthoughTUNEL staining was not found in the negative control with-out the TdT, it was present in all cells in the positive controltreated with DNase (data not shown). Caspase-3 stainingalmost parallels TUNEL assay on d 1 and showed its pres-ence in the luminal and glandular epithelial cells (Fig. 5). Thenegative control without the primary caspase-3 antibodyshowed no staining on d 1 (data not shown). We next triedto colocalize TUNEL staining with expression of caspase-3 ina d-1 uterine section to demonstrate caspase-mediated celldeath in the uterus. We observed that cells expressingcaspase-3 correspond to the cells showing TUNEL staining(Fig. 6). These findings suggest that most of the caspase-positive cells were undergoing apoptosis on d 1. Compared

FIG. 3. Sections of ovariectomized mice uteri treated with sesame oil(vehicle), P4, and E2 were immunostained to demonstrate hormonaleffects on cell-specific apoptosis and proliferation. Apoptosis was de-termined by TUNEL assay. Cell proliferation was determined byBrdU incorporation. Arrowhead indicates positive immunostaining.Data represent at least two to three animals in each treatment group.cm, Circular myometrium; ge, glandular epithelium; le, luminal ep-ithelium; lm, longitudinal myometrium; s, stroma.

FIG. 4. Dark-field pictures of uterine sections showing tritiated thy-midine incorporation in the nuclei of cells after treatment of sesameoil (vehicle), P4, and/or E2 to ovariectomized hamsters. Data representtwo animals in each treatment group. cm, Circular myometrium; le,luminal epithelium; lm, longitudinal myometrium; s, stroma.



with d 1 of pregnancy, the number of apoptotic cells inepithelial cells was reduced on d 2 of pregnancy but stillremained high compared with d 3 and 4 of pregnancy (Fig.5). Although the number of apoptotic cells was less in thestromal bed, apoptotic stromal cells were more on d 2 com-pared with d 1, 3, and 4. Caspase-3 staining was not observed

in the epithelial cells on d 2, but some cells in the stroma andmuscle layers showed caspase-3 staining. Only a fewTUNEL- and caspase-positive cells were present in the en-

FIG. 5. Sections of hamster uteri from d 1–4 of pregnancy were im-munostained to demonstrate cell-specific apoptosis and proliferation.Apoptosis was determined by TUNEL assay and active caspase stain-ing. Cell proliferation was determined by BrdU incorporation. Datarepresent at least two animals on each day of pregnancy. Arrowheadindicates positive immunostaining. cm, Circular myometrium; ge,glandular epithelium; le, luminal epithelium; lm, longitudinal myo-metrium; s, stroma.

FIG. 6. Colocalization of caspase-3 (red) and TUNEL(green) labeling as detected by double immunofluores-cence in sections from d-1 uterus of the hamster. Note thecolocalization of caspase-3 and TUNEL in the merger(arrowhead). Photographs were captured at �200 mag-nification. le, Luminal epithelium; s, stroma.

FIG. 7. Sections of mice uteri from d 1–4 of pregnancy were immu-nostained to demonstrate cell-specific apoptosis. Apoptosis was de-termined by TUNEL assay and active caspase staining. Arrowheadindicates positive immunostaining. Data represent at least two tothree animals on each day of pregnancy. cm, Circular myometrium;ge, glandular epithelium; le, luminal epithelium; lm, longitudinalmyometrium; s, stroma.



dometrial compartment of the uterus on d 3 and 4 (Fig. 5).In the mouse, however, apoptosis was primarily observed inboth the stromal and luminal epithelial layers on d 2 com-pared with other days (d 1, 3, and 4) of pregnancy (Fig. 7).The expression of caspase-3 was positively correlated withcell apoptosis showing maximum staining in luminal epi-thelial cells on d 2 of pregnancy (Fig. 7).

Apoptosis during d 5–8 of pregnancy in hamsters and mice. Uter-ine cell apoptosis during d 5–8 of pregnancy in hamsters andmice is shown in Figs. 8–10. After the initiation of implan-tation on d 5, we observed a few focal apoptotic cells in theuterine epithelium surrounding the implanting blastocyst inhamsters (Fig. 8) as well as in mice (Fig. 9). We also observedapoptotic cells in the trophoblast cell layers in both species(Figs. 8 and 9). There was no indication of apoptosis in stromaas well as luminal epithelial cells away from the implantationchamber in hamsters (Fig. 8). However, we noted the pres-ence of a few apoptotic cells in the subepithelial stromal layersurrounding the implanting embryo in the mouse (Fig. 9).Although a few caspase-3-positive luminal epithelial cellswere noticed surrounding the implanting blastocysts in mice(Fig. 9), only one or two caspase-3-staining cells were foundin this area in hamsters (Fig. 8). Double-immunofluorescenceanalyses were next performed to examine the colocalizationof caspase-3 and TUNEL labeling in d-5 implantation sites ofthe hamster and mouse (Fig. 10). The results showed thelocalization of TUNEL labeling in the luminal epithelial cellssurrounding the implanting blastocyst almost correspondedto that of caspase-3 protein staining in most of these cells.

On d 6, TUNEL staining was observed in luminal epithe-lium which starts to degenerate surrounding the implanting

embryo in both the species (Figs. 8 and 9). We also observedapoptotic cells in the PDZ. The apoptotic pattern was verysimilar on d 7 in both the species. Althoughapoptosis wasnoticed in decidual cells surrounding the embryo, luminalepithelial cells on the mesometrial side also showed apopto-sis. On d 8, apoptotic cells were more prominent in thehamsters compared with mice surrounding the growing em-bryo (Figs. 8 and 9). TUNEL-positive uterine cells weremainly observed just surrounding the embryo. Apoptoticcells were also seen in the growing embryo. Caspase-3 ex-pression in uterine cells was almost positively correlatedwith the apoptotic patterns on d 6–8 in mice. However, weobserved fewer of caspase-3-positive cells compared withapoptotic cells in hamsters from d 5–8 of pregnancy (Fig. 8).

Cell proliferation during d 1–4 of pregnancy in hamsters. Cellproliferation in the hamster uterus was studied by applyingtwo well known methods: BrdU and [3H]thymidine incor-porations. Only a few proliferating luminal epithelial cellswere noted in d-1 uterine section by the [3H]thymidine in-corporation method (Fig. 11), but not by BrdU incorporation(Fig. 5). Proliferating cells as detected by the BrdU incorpo-ration method were equally distributed in all compartmentsof the uterus as observed in the first day of the estrous cycle,the estrous day. Uterine cell proliferation was dramaticallyincreased on d 2 of pregnancy. Proliferation was seen mainlyin the luminal and glandular epithelial cells (Figs. 5 and 11).The negative control section obtained from the animal with-out the treatment of BrdU showed no staining on d 2 (datanot shown). We also saw some increase in the number ofproliferating cells in stromal and muscular compartments by[3H]thymidine incorporation (Fig. 11) but not by the BrdU

FIG. 8. Sections through implantation sites of hamstersfrom d 5–8 of pregnancy were immunostained to demon-strate cell-specific apoptosis and proliferation. Apoptosiswas determined by TUNEL assay and active caspase stain-ing. Cell proliferation was determined by BrdU incorpora-tion. Arrowhead indicates positive immunostaining. AM,Antimesometrial side; bl, blastocyst; cm, circular myome-trium; em, embryo; M, mesometrial side; PDZ, primarydecidual zone; SDZ, secondary decidual zone.



incorporation method (Fig. 5). Proliferation of epithelial cellcompletely ceased after d 2 of pregnancy. In contrast, wenoticed a significant increase in the number of proliferatingcells in the stromal compartment on d 3 and 4 of pregnancy(Figs. 5 and 11). Proliferating cells were also observed inmuscle layers from d 4.

Cell proliferation during d 5–8 of pregnancy in hamsters. With theinitiation of blastocyst implantation on d 5, an additionalincrease in stromal cell proliferation was observed at theimplantation site surrounding the implanting blastocyst. In-creased cell proliferation was also noticed in muscle layers.Implanting blastocysts also showed cellular proliferation(Figs. 8 and 11). However, with the gradual progression ofthe implantation process and transformation of stromal cellsto decidual cells from d 5 to d 8, gradual inhibition of stromalcell proliferation was noticed surrounding the implantingembryo (Figs. 8 and 11). However, stromal cells further awayfrom the implanting embryo still showed proliferation. Asurvey of serial sections showed the presence of some pro-liferating cells in the embryo as well in muscle layers (Figs.8 and 11).

A close similarity was observed in uterine cell proliferationpatterns during d 1–6 of pregnancy using two well estab-lished methods. Only a subtle difference in uterine cell pro-liferation pattern between these two methods was noticed ond 3 of pregnancy. Incorporation of [3H]thymidine was no-

ticed more in stromal cells just beneath the luminal epithe-lium, compared with equal distribution of BrdU incorpora-tion in the stromal area. We observed more proliferating cellsby the [3H]thymidine incorporation method compared withthe BrdU incorporation method, suggesting that the formermethod is more sensitive than the latter one (Figs. 5, 8, and11).

Caspase-3 inhibitor Ac-DEVD-CHO blocked blastocystimplantation in hamsters and mice

Because the activation of caspase-3 plays an important rolein executing apoptosis, we next determined whether its in-hibitor, Ac-DEVD-CHO, inhibits implantation by blockingluminal epithelial cell apoptosis. Compared with the vehicletreatment, uterine horns from five pregnant hamsters and sixpregnant mice that received administration of Ac-DEVD-CHO showed complete absence of implantation sites on d 5as determined by the blue-dye method (Table 1 and Fig. 12).A total of six and 26 blastocysts were recovered upon flush-ing drug-treated uterine horns of hamsters and mice, respec-tively (Table 1). Because hamster blastocysts do not undergodelay in implantation, these embryos mostly degenerate inthe event of interruption of the implantation process. Hence,blastocyst recovery from drug-treated hamster uterine hornswas low, and only six apparently normal looking zona-freeblastocysts were recovered from three horns. Twenty-sixblastocysts that were recovered from six drug-treated uterinehorns in mice had no sign of degeneration. Twenty of thoseblastocysts were zona free and the rest were zona encased.We observed a normal number of implantation sites in ve-hicle-treated uterine horns of both species. This drug is notreported to be toxic in mice (34). This drug is also not toxicto preimplantation embryos because normal blastocyst for-mation occurs when eight-cell embryos of hamsters and micewere cultured in vitro in the presence of this compound atdoses of 5 and 10 �g/ml (Table 2). Under our culture con-ditions, about 87% of hamster and 94% of mouse eight-cellembryos developed into blastocysts in the absence of Ac-DEVD-CHO (vehicle control). In the presence of Ac-DEVD-CHO, we observed no significant change in embryo devel-opment from eight-cell stage to the blastocyst stage (Table 2),suggesting no inhibitory or toxic effects of Ac-DEVD-CHOon embryo development. The morphological appearance ofthose blastocysts that formed in the presence of the drug wasindistinguishable from blastocysts that developed in the ab-sence of the drug. These results demonstrate that intralumi-nal administration of Ac-DEVD-CHO inhibits implantationin hamsters and mice by inhibiting uterine epithelial cellapoptosis at the implantation site.

Discussion

In this study, we aimed to establish a correlation betweenapoptosis and proliferation in uterine cell turnover duringthe estrous cycle and early pregnancy in hamsters that showP4-dependent implantation. The salient result of this study isthe evidence of significant epithelial cell proliferation on thesecond day (d 2) after mating, marking entry of the uterusinto the state of pregnancy in hamsters. This is also true inmice considering previously published results that showed

FIG. 9. Sections through implantation sites of mice from d 5–8 ofpregnancy were immunostained to demonstrate cell-specific apopto-sis. Apoptosis was determined by TUNEL assay and active caspasestaining. Arrowhead indicates positive immunostaining. AM, An-timesometrial side; bl, blastocyst; em, embryo; ge, glandular epithe-lium; M, mesometrial side, PDZ, primary decidual zone; SDZ, sec-ondary decidual zone.



significant increase in luminal epithelial cell proliferation ond 2 of pregnancy in this species (1). In general, an inverserelationship between apoptosis and cell proliferation pat-terns was noted except on d 2 of pregnancy in mice whereboth the proliferation (1) and apoptosis processes were hap-pening simultaneously in epithelial cell layers. The fre-quency of apoptosis in the stromal bed was not similar to thefrequency of proliferation during pregnancy, suggesting thata higher frequency of cell proliferation may well be relatedto rapid growth of the uterus during pregnancy. In respectto steroid hormonal regulations of the apoptosis and prolif-eration patterns in the uterus, our results clearly showed thatE2 was effective in stimulating epithelial cell proliferation inboth species. However, although P4 elicited stromal cell pro-liferation within 24 h in mice, it was not as effective inhamsters and took 48 h to obtain a moderate increase instromal cell proliferation. In addition, although both P4 and

E2 were ineffective in inhibiting apoptosis observed in ovari-ectomized hamsters, they were effective in inhibiting uterinecell apoptosis in ovariectomized mice. Our results also re-vealed the colocalization of caspase-3 in most of the apoptoticcells during early pregnancy. Thus, our observation thatcaspase-3 inhibitor Ac-DEVD-CHO adversely affects the im-plantation process shows, for the first time, that caspase-3-mediated uterine epithelial cell apoptosis may play an im-portant role in initiating the implantation process in both thehamster and mouse.

Previously reported data demonstrated that uterine epi-thelial cells undergo proliferation on the proestrous day ofthe estrous cycle of mice and rats (18). This pattern is partlysynchronous and corresponds to the period when serum Elevels are increasing (37). Consistent with these data, weobserved luminal epithelial cell proliferation on the morningof the proestrous day in hamsters. However, this could be

FIG. 10. Colocalization of caspase-3 (red) andTUNEL (green) labeling as detected by doubleimmunofluorescence in sections from d-5 implan-tation sites of hamsters and mice. Note the colo-calization of caspase-3 and TUNEL in the merger(arrowhead). Photographs were captured at �200magnification. bl, Blastocyst; le, luminal epithe-lium; s, stroma.



attributed to a gradual increase in E levels that peak at 1400 hand remain elevated for several hours on the diestrous day(38). Apparently, the second and large preovulatory E surgethat occurs on the estrous day between 0900 and 1500 h hasno effect on further uterine epithelial cell proliferation inhamsters because there was no significant epithelial cell pro-liferation at the estrous day. Proliferation of epithelial cellsin response to E2 was demonstrated in mice and rats (1, 39).We also observed proliferation of epithelial cells in responseto E2 in ovariectomized hamsters and mice, suggesting thatproliferation of epithelial cells in response to E2 is a commonphenomenon in the uterus. This was contradictory to the

previous belief that the hamster uterus is not quite as sen-sitive to E2 compared with rats (40). The rapid reduction inuterine epithelial cell proliferation was accompanied by thegradual increase in apoptosis on the estrous and metestrousdays. These epithelial changes were noted when serum Elevels were at their lowest limit after a peak on the proestrousday (39). This is consistent with the data published previ-ously that E withdrawal causes degenerative changes in theepithelial cells (25, 26). The hamster shows stromal cell pro-liferation on the diestrous day when the first peak of P4occurs (41, 42). These results are in agreement with previ-ously reported data that showed uterine stromal cell prolif-eration in response to P4 alone or when P4 and E2 were giventogether, P4 significantly suppressed the epithelial cell pro-liferation but increased the proliferation of stromal cells inmice (1, 24). We also observed a similar effect of E2 and P4in the ovariectomized hamster uterus. It was reported pre-viously that hamster uterus is more sensitive to P4 than E2(40). However, our results did not quite support this beliefbecause there was no noticeable increase in stromal cell pro-liferation by giving a single injection of P4 at the dose of 2 mgto ovariectomized hamsters. The increase in stromal cell pro-liferation in response to E2 plus P4 suggests that, whereas P4suppresses the E2 actions on epithelial cell proliferation, E2also potentiates P4 actions on the stroma. However, the rea-son for the diminished and delayed effectiveness of P4 in thehamster uterus in terms of stromal cell proliferation wasnot certain from our studies and will require furtherinvestigation.

FIG. 11. Dark-field pictures of uterine sections were showing[3H]thymidine incorporation in the nuclei of cells during d 1–6 ofpregnancy in hamsters. AM, Antimesometrial side; bl, blastocyst, cm,circular myometrium; em, embryo; ge, glandular epithelium; lm, lon-gitudinal muscle; M, mesometrial side; PDZ, primary decidual zone;SDZ, secondary decidual zone.

TABLE 1. Effect of caspase-3 inhibitor Ac-DEVD-CHO onblastocyst implantation in hamsters and mice

TreatmentNo. of

pregnantanimals

No. ofuterinehorns

Total no. ofimplantation sites

(mean � SEM)

Hamster 5Vehicle-treated horn 5 30 (6.00 � 0.63)Inhibitor-treated horn 5 1.0

Mouse 6Vehicle-treated horn 6 33 (5.45 � 0.10)Inhibitor-treated horn 6 00

Drug (hamster: 10 �g/5 �l saline per horn; mice: 10 �g/2 �l salineper horn) and vehicle (hamster: 5 �l saline per horn; mice: 2 �l salineper horn) were administered intraluminally in separate uterine hornof the same animal on d 3 of pregnancy at 0900 h. Implantation siteswere determined by the blue dye method on d 5 at 0900 h. A total ofsix blastocysts were recovered from uterine horns that did not showimplantation sites on d 5 of pregnancy in the presence of Ac-DEVD-CHO. Because hamsters do not exhibit delay in implantation, blas-tocysts that do not implant in normal time usually degenerate. A totalof 26 blastocysts were recovered from uterine horns that did not showimplantation sites on d 5 in the presence of Ac-DEVD-CHO in mice.

FIG. 12. An intraluminal application of caspase-3 inhibitor AC-DEVD-CHO inhibits implantation both in the hamster and mouse.Pregnant hamsters and mice received a single intraluminal injectionof AC-DEVD-CHO (10 �g/5 �l sterile saline in hamsters or 5 �g/2 �lsterile saline in mice) in one uterine horn and vehicle in the con-tralateral horn on d 3 of pregnancy when embryos were still inside theoviducts. Animals were examined for implantation sites at 0900 h ond 5 by the blue dye method. Arrowheads indicate the location ofimplantation sites in vehicle-treated uterine horns.



Uterine cell proliferation during early pregnancy in micehas been previously studied and showed epithelial, but notstromal, cell proliferation on the second day of pregnancyand stromal, but not epithelial, cell proliferation on the thirdand fourth days of pregnancy (1). We observed a similarpattern of uterine cell proliferation on d 2–4 of pregnancy inhamsters, suggesting that uterine cellular proliferating pat-terns in achieving uterine receptivity are similar in mice andhamsters. On the basis of these observations, it can be sug-gested that both in the mouse and hamster, mating on the dayof ovulation leads to a significant increase in luminal epi-thelial cell proliferation on the following day (d 2 of preg-nancy), marking the end of cycle-related and the beginningof pregnancy-related uterine changes. Another interestingfinding with respect to cell apoptosis between mice andhamsters was that although epithelial apoptosis andcaspase-3 activity were highest on d 1 of pregnancy in ham-sters, there was almost no apoptosis in d-1 uteri of mice. Ond 2 of pregnancy, however, the epithelial apoptosis andcaspase-3 activity was at the maximum level in mice whenepithelial proliferation is also reported to be occurring (1).This pattern is slightly different in hamsters. Fewer epithelialcells were undergoing apoptosis on d 2 in hamsters com-pared with mice. Maximum uterine cell apoptosis on d 1 ofpregnancy in hamsters is well correlated with the low levelof circulating E on this day (42, 43). Because circulating Elevels gradually start increasing from d 2 of pregnancy inhamsters (42, 44), epithelial cells clearly receive an E stimulusto divide on d 2 in hamsters. If the fall of proestrous E surgeis responsible for the induction of apoptosis in epithelial cellson d 2 of pregnancy, then the cause of epithelial cell prolif-eration on this day in mice remains a matter of debate be-cause there was no significant change in circulating E levelon this day (27). However, a previous study suggested thatd-2 uterine epithelial cell proliferation in mice could also bea result of proestrus E-stimulated epithelial c-myc expressionon d 1 of pregnancy (1). The occurrence of proliferation in thestroma and absence of apoptosis on d 3 and 4 of pregnancyin hamsters is probably the result of the gradual increase incirculating E and P4 levels during early pregnancy in thisspecies (42, 43). On the other hand, in mice, stromal cellproliferation on d 3 and 4 could be solely due to the increasein P4 levels during early pregnancy (44). However, it has beenpostulated that the brief increase in E levels on d 4 of preg-

nancy in mice (28) may accelerate the P4-induced stromalproliferation.

The immediate consequence of blastocyst implantation isthe stimulation of stromal cell proliferation and death ofluminal epithelium surrounding the implanting blastocyst(2). We observed that both of these events were occurring atthe implantation sites of both mice and hamsters. The firstepithelial cell death perhaps starts in a few cells at the an-timesometrial side and then extends to all epithelial cellssurrounding the embryo. From d 6–8, epithelial cell deathalso occurred in the mesometrial side of the luminal epithe-lium. Although the luminal epithelium starts apoptoticchanges on d 5, stromal cells surrounding the implantingblastocyst simultaneously undergo massive proliferation. Asgestation progresses, stromal cells immediately surroundingthe implanting blastocysts stopped proliferation and showedsign of apoptosis, whereas stromal cells next to it still showedproliferation. Electron microscopic studies showed that de-teriorating decidual cells were removed by autolytic activityand that their elimination was facilitated by trophoblasticphagocytosis (45). This elimination of decidual cells sur-rounding the implanting blastocyst is absolutely required toremodel the uterus to accommodate the growing embryo.Although we saw stromal cell death and proliferation indifferent regions of the decidua, we saw more proliferatingstromal cells than dying stromal cells, suggesting net growthof the uterus after implantation. Ovarian hormones are thekey regulatory elements for uterine apoptosis and prolifer-ation during the cycle and preimplantation period, but whatinduces uterine epithelial cell apoptosis and stromal cellproliferation at the implantation site is unknown. Becausethe blastocyst initiates uterine processes for implantation,embryo-derived molecules certainly play key roles. One ofthose embryonic molecules in hamsters is possibly E (32, 46).Although E-induced apoptosis has not been demonstrated inthe uterus, E-induced spatial and regional cell apoptosis hasbeen demonstrated in pituitary and prostate glands (47, 48).Furthermore, antiestrogen inhibits implantation in hamsters(49). Thus, depending on the circumstances, it is possible thatE produced by the embryo may induce epithelial cell apo-ptosis and stromal cell proliferation at the implantation site.However, these processes at implantation sites may alsorequire the involvement of cooperative actions of numerouslocal paracrine, autocrine, or juxtacrine factors to execute the

TABLE 2. Development of blastocysts after culture of eight-cell embryos in the absence or presence of caspase-3 inhibitor Ac-DEVD-CHO

Ac-DEVD-CHO(�g/ml)

No. of eight-cellembryos cultured

(no. of observations)

Duration ofculture

No. of embryosdeveloped toblastocysts

% Blastocystsdeveloped

Hamster 24 h0 63 (5) 55 875 37 (3) 31 8410 75 (6) 64 85

Mouse 48 h0 46 (5) 43 9410 48 (5) 44 92

Eight-cell embryos were cultured in a group of 10–15 embryos per drop for hamsters and 8–10 embryos for mice. Ac-DEVD-CHO was firstdissolved in water and later diluted in respective cultured medium for hamster and mouse embryos. No significant differences (P � 0.05) wereobserved between embryos cultured in the absence or presence of the caspase-3 inhibitor.



uterine remodeling process to accommodate the growingembryo.

Previous studies have proposed the involvement ofcaspase-3, a downstream executioner enzyme common tomany paradigms of programmed cell death in mediatingapoptosis of both germ and somatic cells (50). Becausecaspase-3 is an earlier event than DNA fragmentation and theapoptotic process lasts only a few hours (51), it is temptingto speculate that caspase-3 remains active in cells duringDNA fragmentation. In our studies, although we saw colo-calization of caspase-3 and TUNEL labeling in the same cellson d 1, we observed lower numbers of caspase-3-positivecells at the implantation sites as compared with TUNELassay in hamsters. This is not surprising, because both tech-niques might yield different results in certain cell types andeven in the same cell type on different times and days ofpregnancy, depending on the stimuli used or due to differentpathways in the apoptotic process in which caspase-3 may ormay not be involved. Using caspase-3 knockout mice, it hasbeen demonstrated that although caspase-3 is required forgranulosa cell apoptosis during follicular atresia, it is dis-pensable for germ cell apoptosis in females, suggesting co-existence of more than one cell death mechanism (52). Lu-minal epithelial cell loss at the implantation site via apoptosisis increasingly recognized as a key component of implanta-tion for trophoblast cell invasion and contact with the stro-mal/decidual cells. We studied the importance of uterineepithelial cell apoptosis in implantation by inhibiting epi-thelial cell apoptosis using a caspase-3 inhibitor. Our resultsshowed that Ac-DEVD-CHO completely blocked implanta-tion in the treated uterine horn as compared with the vehicle-treated contralateral uterine horn in both hamsters and mice.Thus, it appears that caspase-3-mediated uterine epithelialcell apoptosis plays an important role during the time ofestablishment of implantation in hamsters.

In summary, our comparative studies between hamstersand mice show that apoptosis is present in all cell typesduring the estrous cycle and periimplantation uterus, but theincidence of apoptosis was lower in the stroma and myo-metrium than the epithelium. There was a significant cor-relation of uterine cell apoptosis and proliferation with cir-culating steroid levels in hamsters. However, the reducedeffectiveness of progesterone in ovariectomized hamstersneeds further investigation. After initiation of implantation,epithelial cells only exhibited death, whereas stromal cellsshowed compartment-specific death and proliferation in re-sponse to growing embryo. Both in the mouse and hamster,stromal cells around the blastocyst first showed proliferationand then death a day later. This process starts at the subep-ithelial stroma and gradually moves toward the periphery.Substantial similarities have been noticed between the mouseand hamster in the pattern of uterine cell apoptosis andproliferation during early pregnancy. Further studies willconfirm whether a change in the ratio between proapoptoticand antiapoptotic regulators control uterine cell death andproliferative mechanisms. Our findings of almost parallelexpression of caspase-3 with TUNEL labeling and inhibitionof implantation by caspase-3 inhibitor indicate, for the firsttime, that caspase-3-mediated cell apoptosis may play an

important role in initiating the implantation process in ham-sters and mice.

Acknowledgments

We thank Drs. S. K. Dey and S. K. Das for unlimited laboratory accessand Monika Raposo and Xiaohong Wang for their excellent technicalassistance. The support of the National Cooperative program on Tro-phoblast-Maternal Tissue Interactions is gratefully acknowledged.

Received December 7, 2005. Accepted January 27, 2006.Address all correspondence and requests for reprints to: Bibhash C.

Paria, Division of Reproductive and Developmental Biology, Depart-ment of Pediatrics, Vanderbilt University Medical Center, D4124 Med-ical Center North, 21st Avenue South, Nashville, Tennessee 37232-2678.E-mail: [email protected].

This work was supported by National Institutes of Health GrantsHD044741 and UO1 HD042636(to B.C.P.).

Disclosure summary: both authors have nothing to declare.

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