Multiple mechanisms are involved in apoptotic cell death in the mouse uterus and vagina after ovariectomy

doi:10.1016/S0890-6238(03)00011-X

Reproductive Toxicology

Volume 17, Issue 3, May–June 2003, Pages 289-297

https://doi.org/10.1016/S0890-6238(03)00011-X Get rights and content

Abstract

Withdrawal of sex hormones by gonadectomy results in rapid involution of mouse reproductive organs. To study the regression mechanism in the uterus and vagina after ovariectomy, histologic and biochemical changes were examined. Apoptotic cells were detected by in situ 3′-DNA nick end labeling method and electron microscopy, while the number of cells showing incorporation of bromo-deoxyuridine (BrdU) decreased in the uterus and vagina after ovariectomy. DNA fragmentation in the uterus was observed even at estrus and the degree of fragmentation increased after ovariectomy. DNA fragmentation in the vagina occurred 1–5 days after ovariectomy. Semi-quantitative RT-PCR revealed that expression of Fas-ligand and tumor necrosis factor-α (TNF-α) mRNA in the uterus and vagina was increased by ovariectomy. These results suggest that apoptotic cell death is induced by ovariectomy through the mediation of both Fas and TNF-α in the mouse uterus and vagina; however, uterine and vaginal cells in CBA lpr^cg/lpr^cg mice lacking functional Fas showed apoptosis, indicating that Fas is not the sole regulator of apoptosis in female reproductive organs in mice.

Introduction

Cell proliferation, differentiation, and regression of the uterus during the estrous cycle are regulated by ovarian estrogen (17β-estradiol; E₂) and progesterone in rats [1]. In the endometrium of the human uterus, apoptosis is an integral component of the menstrual process [2]. Apoptosis can be induced by sex hormone withdrawal in the uterus and prostate in rats and mice [3], [4], [5], [6]. We have demonstrated that regression of the uterus and vagina after ovariectomy was accompanied by induction of new proteins [7], indicating that some specific factors could be involved in the regression mechanism in male and female reproductive organs. Transforming growth factor-β (TGF-β) was increased in the prostate on the first day after androgen removal in the rat [8] and addition of TGF-β1 to primary culture of rabbit uterine epithelial cells caused apoptosis in vitro [9]. Fas, a member of the NGF/TNF receptor family, transduces an apoptotic signal in immunocytes [10], [11] and is thought to be involved in cell death in the human endometrium [12]. The expression of tumor necrosis factor-α (TNF-α) mRNA and TNF-α protein in the human uterus was correlated with the menstrual cycle [13], [14] and mouse estrous cycle [15]. On the other hand, bcl-2 and related proteins also regulate the survival and death of cells [16], [17], [18], [19]. In human endometrium, expression of the bcl-2 gene family changes during the menstrual cycle [20], [21]. Menstruation is correlated with changes in bcl-2, bax, and TNF-α expression level [22], [23]. These results raise the question of whether these factors could be participating in apoptosis in the uterus and vagina after ovariectomy.

The present study was designed to further examine the regression mechanism of the mouse uterus and vagina induced by hormone withdrawal. C57BL/Tw female mice were ovariectomized at estrus, and histologic and biochemical changes were examined by the in situ 3′-end labeling method, electron microscopy, and RT-PCR. In order to study the involvement of Fas, histologic changes in the uterus and vagina were examined in lpr^cg female mice that were ovariectomized at estrus. The lpr^cg mice have a point mutation of the gene encoding Fas, resulting in an autoimmune disorder [24]. The lpr^cg mice are capable of reproduction, suggesting that their reproductive organs might be the under control of ovarian hormones. Our results indicate that apoptotic cell death is induced by ovariectomy through the mediation of both Fas- and TNF-α-systems; however, uterine and vaginal cells in CBA lpr^cg/lpr^cg mice lacking functional Fas showed apoptosis indicating that the Fas system may not be essential.

Section snippets

Animals

Female mice of the C57BL/Tw strain were kept under 12 h light/12 h dark at 23–25 °C and fed laboratory chow (CE-2, CLEA, Tokyo, Japan) and tap water ad libitum. Mice were killed at estrus, and 1, 2, 3, and 5 days after ovariectomy, which was carried out at estrus. The estrous cycle was determined by vaginal smears. The uteri and vaginae from five mice in each group were weighed and fixed in 4% buffered formaldehyde for histologic preparation. Portions of the organs were frozen in liquid nitrogen

BrdU labeling index and apoptotic index

Weights of uteri and vaginae decreased following ovariectomy (Fig. 1). BrdU labeling index in uterine luminal and glandular epithelia was low at estrus, but it increased significantly 5 days after ovariectomy (Fig. 2A). In vaginal epithelium, the labeling index was high at estrus but it decreased significantly 2 days after ovariectomy. BrdU labeling index was not altered in the stromal cells of either the uterus or vagina by ovariectomy (Fig. 2B).

Apoptotic index in uterine luminal epithelial

Discussion

Apoptotic cell death plays a critical role in tissue homeostasis. Morphologic changes of apoptosis are electron-microscopically characterized by compaction and segregation of chromatin in sharply circumscribed masses that abut the inner surface of the nuclear envelope [30]. The nuclear fragments and condensation of the cytoplasm are associated with extensive cell surface protrusion, followed by separation of the surface protuberances to produce membrane-bounded apoptotic bodies of varying sizes

Acknowledgments

We thank Emeritus Professor Noboru Takasugi at Yokohama City University, for his valuable advice and critical readings of this manuscript. This work was supported by a Grant-in-Aid for Scientific Research on Priority Areas (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Research Grant from Kihara Memorial Yokohama Foundation for the Advancement of Life Sciences (to T.I. and T.S.), a Grant for Support of the Promotion of Research at Yokohama City

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Estrogen levels decline in both sexes with age, but more dramatically in females. Activation of the Wnt/β-catenin signaling pathway is central to the regulation of bone mass accrual and maintenance and in response to mechanical loading. Using the ovariectomized mouse model we examined the effect of estrogen loss on the osteocyte's ability to activate the Wnt/β-catenin pathway following mechanical loading. Female TOPGAL mice underwent ovariectomy (OVX) (n = 10) or sham surgery (n = 10) at 16 weeks of age. Four weeks post-surgery, a single loading session (global strain of 2200 με for 100 cycles at 2 Hz) was performed on the right forearm with the left as a non-loaded control. Mice (n = 5) were sacrificed at 1 or 24 hr post-load. Ulnae were stained for β-catenin activation, femurs were used for μCT and 3-pt bending/biomechanical testing, and tibiae were used for histology analysis and to determine osteocyte lacunar size using SEM and high resolution micro-XCT. A 2.2-fold increase in β-catenin signaling activation was observed 24 hr post-load in the Sham group but did not occur in the OVX group. The OVX group versus control had significant losses (p < 0.05) in trabecular BMD (−8%), BV/TV (−35%) and thickness (−23%), along with cortical thickness (−6%) and periosteal perimeter (−4%). The OVX group had significantly higher trabecular bone osteoclast numbers (63%), OCS/BS (77%) and N.OC/BPm (94%) and a significant decrease in osteoblast number (53%), OBS/BS (37%) and N.OB/BPm (40%) compared to the sham group (p < 0.05). Cortical bone lacunar number/lacunar volume and bone biomechanical properties did not change between groups. Given that the ulna is a cortical bone loading model and the lack of changes in osteocyte lacunar number/volume in cortical bone, which would alter strains experienced by osteocytes, these data suggest the absence of estrogen resulted in intrinsic changes in the ability of the osteocyte to respond to mechanical load, rather than changes in the biomechanical and architectural properties of bone.
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Overexpression of ERBBs is associated with carcinogenesis in the reproductive organs (Dickson and Lippman, 1995) and ERBB2 is frequently amplified and overexpressed in cancer cells (Hynes and Stern, 1994). Apoptotic cell death occurs in vaginal epithelial cells and there is decreased DNA synthesis during the estrous cycle and after ovariectomy in intact rats and mice (Sato et al., 1997, 2003). In neonatally DES-exposed adult mice, however, ovariectomy did not induce reduction of DNA synthesis and only a slight increase in apoptotic cells in vaginal epithelium (Sato et al., 2004).
In the early 1960's, at Professor Bern's laboratory, University of California, Berkeley) in the US, Takasugi discovered ovary-independent, persistent vaginal changes in mice exposed neonatally to estrogen, which resulted in vaginal cancer later in life. Reproductive abnormalities in rodents were reported as a result of perinatal exposure to various estrogenic chemicals. Ten years later, vaginal cancers were reported in young women exposed in utero to the synthetic estrogen diethylstilbestrol (DES) and this has been called the “DES syndrome”. The developing organism is particularly sensitive to developmental exposure to estrogens inducing long-term changes in various organs including the reproductive organs. The molecular mechanism underlying the persistent vaginal changes induced by perinatal estrogen exposure was partly demonstrated. Persistent phosphorylation and sustained expression of EGF-like growth factors, lead to estrogen receptor α (ESR1) activation, and then persistent vaginal epithelial cell proliferation. Agents which are weakly estrogenic by postnatal criteria may have major developmental effects, especially during a critical perinatal period. The present review outlines various studies conducted by four generations of investigators all under the influence of Prof. Bern. The studies include reports of persistent changes induced by neonatal androgen exposure, analyses of estrogen responsive genes, factors determining epithelial differentiation in the Müllerian duct, ESR and growth factor signaling, and polyovular follicles in mammals. This review is then expanded to the studies on the effects of environmental estrogens on wildlife and endocrine disruption in Daphnids.
The Bax/Bcl-2 apoptotic pathway is not responsible for the increase in apoptosis in the RU486-treated rat uterus during early pregnancy
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The blocking of the glucocorticoid receptors by RU486 binding may account for the shift in the Bax/Bcl-2 ratio toward a more anti-apoptotic environment, however, this does not account for the increase in observed apoptotic activity within the RU486-treated rat endometrium. Sato et al. [25] demonstrated increased levels of TNF-α, and Fas and Fas-ligand (FasL) 24 h after ovariectomy within the endometrium. Both TNF-α and Fas and FasL have been reported to be involved in the up-regulation of apoptosis [28,29].
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Polymorphism and parent-of-origin effects on gene expression of CAST, leptin and DGAT1 in cattle
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Fetus age varied from 70 to 130 days, determined by crown-rump length (Evans & Sack, 1973). Tissues (uterus and fetal skin) were submitted to DNA extraction with organic solvents according to Sato, Fukazawa, Kojima, Ohta, and Iguchi (2003) and used for genotyping of cows and fetuses. Fetal tissues (muscle, liver and skin) were submitted to RNA extraction with Trizol (Invitrogen) and reverse transcription with the SuperScript III First-Strand Synthesis SuperMix kit (Invitrogen) and 2.5 μM of oligo (dT), according to the manufacturer's protocol, and used in gene expression studies.
This study aimed to investigate differential allele expression (DAE) and polymorphism and parent-of-origin effects on expression of genes related to beef traits. CAST, related to meat tenderness, and DGAT1 and leptin, related to fat deposition, were evaluated. In bovine fetal tissues CAST was expressed twice as much (P < 0.05) in muscle of homozygous GG than in heterozygous AG. Leptin was expressed about one-tenth as much (P < 0.05) in heterozygous TpCm (allele T of paternal origin and allele C of maternal origin) than in homozygous CC. No DAE was observed. The evidence of polymorphism effect on expression of CAST and parent-of-origin effect on leptin contributes to a better understanding of events controlling the expression of genes of economic interest in cattle. Furthermore, if the parent-of-origin effects observed in fetal tissues are confirmed in adult tissues and associated to phenotypic variation, this parental origin criterion may be considered in marker-assisted selection of beef traits.
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Ovariectomy-induced reduction in the active form of AKT, which is also an anti-apoptotic factor [37], may rather indicate reduced anti-apoptotic capability of the vagina after estrogen withdrawal. Indeed, a considerable level of apoptosis has been observed in the mouse vagina as early as 1 day after ovariectomy [38]. At present, the exact mechanism of estrogen-mediated eNOS (Ser-1177) phosphorylation in the rat vagina is unknown.
Estrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown.
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We localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme's activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina.
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¹: Present address: Division of Cerebral Structure, National Institute for Physiological Sciences, 38 Nishigonaka, Myodaiji, Okazaki 444-8585, Japan.

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Multiple mechanisms are involved in apoptotic cell death in the mouse uterus and vagina after ovariectomy

Abstract

Introduction

Section snippets

Animals

BrdU labeling index and apoptotic index

Discussion

Acknowledgments

Cell

J. Biol. Chem.

Blood

Cell

Cancer Lett.

Exp. Cell. Res.

Reprod. Toxicol.

Ultrastructure as a basis for dating of rat endometrium

Anat. Rec.

Atrophy and apoptosis in the cyclical human endometrium

J. Pathol.

Characterization of uterine epithelium apoptotic cell death kinetics and regulation by progesterone and RU 486

Am. J. Pathol.

Relationship between DNA fragmentation and apoptosis in the programmed cell death in the rat prostate following castration

Prostate

The role of androgen in the regulation of programmed cell death/apoptosis in normal and malignant prostatic tissue

Semin. Cancer Biol.

Androgen suppressed apoptosis is modified in p53 deficient mice

Oncogene

Effect of ovariectomy on histological change and protein expression in female mouse reproductive tracts

In Vivo

Expression of transforming growth factor-β in the rat ventral prostate during castration-induced programmed cell death

Mol. Endocrinol.

Coordinated regulation of apoptosis and cell proliferation by transforming growth factor β1 in cultured uterine epithelial cells

Proc. Natl. Acad. Sci. U.S.A.

Fas ligand, Fas antigen and Bcl-2 expression in human endometrium during the menstrual cycle

Mol. Hum. Reprod.

Distinct regional and menstrual cycle dependent distribution of apoptosis in human endometrium. Potential regulatory role of T cells and TNF-α

Endocr. J.

Tumor necrosis factor-α messenger ribonucleic acid and protein in human endometrium

Biol. Reprod.

Mouse endometrial tumor necrosis factor-α mRNA and protein: localization and regulation by estradiol and progesterone

Endocrinology

Bcl-2 and the regulation of programmed cell death

J. Cell. Biol.

Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death

Cell

Cyclic bcl-2 gene expression in human uterine endometrium during menstrual cycle

Lancet

Differential expression of members of the bcl-2 gene family in proliferative and secretory human endometrium: glandular epithelial cell apoptosis is associated with increased expression of bax

J. Clin. Endocrinol. Metab.