Disruption of the tissue inhibitor of metalloproteinase-1 gene results in altered reproductive cyclicity and uterine morphology in reproductive-age female mice

Tissue inhibitor of metalloproteinase-1 (TIMP-1) is a multifunctional protein expressed in the uterus of essentially all species, yet the function of this protein is uncertain. To assess the role of TIMP-1 in the uterine events that occur during the murine estrous cycle, mature female TIMP-1 wild-type and null mice were monitored for reproductive cyclicity. Mice were sacrificed in each stage of the estrous cycle, and peripheral blood was collected and assayed for serum estradiol and progesterone content by RIA. Uterine morphology and TIMP-1, TIMP-2, TIMP-3, and TIMP-4 mRNA expression were also examined between genotypes in each stage of the estrous cycle. Disruption of the TIMP-1 gene product was associated with an altered reproductive cycle characterized by a significant decrease in the length of the estrus period in the null mice. Also during the period of estrus, null mice expressed significantly lower levels of uterine TIMP-3 mRNA expression, altered uterine morphology, significantly higher serum estradiol levels, and significantly lower serum progesterone levels compared to their wild-type counterparts. It is concluded from this study that TIMP-1 has a multifaceted role in regulating the murine reproductive cycle, and this control appears to be at the level of both the uterus and the ovary.     

Estrogen regulates the expression of the small proline-rich 2 gene family in the mouse uterus

Small proline-rich (SPRR) proteins are structural components of the cornified cell envelope (CE), a specialized structure beneath the plasma membrane of stratified squamous epithelia. They are divided into four families, of which SPRR2 is the most complex consisting of 11 members (2a-2k) in the mouse. To assess the possible influence of estrogen on expression of the SPRR2 family in the uterus, we examined the effect of 17b-estradiol (E2) on SPRR2 mRNA levels on ovariectomized (OVX) adult mice. We employed a combination of laser capture microdissection (LCM) and semiquantitative RT-PCR to examine expression in particular uterine cell types - luminal epithelia, and stromal and muscle cells. We also used quantitative real-time PCR to measure levels of the mRNA of several SPRR2 proteins in the mouse uterus over the estrous cycle and during early pregnancy. Expression of SPRR2a, 2b, 2c, 2d, 2e, 2f and 2g mRNA was increased by estrogen treatment. SPRR2a, 2b, 2d and 2e were highly expressed on day 1 and 2 of pregnancy, but decreased markedly by days 3-6. Interestingly, several members of the SPRR2 family were preferentially up-regulated at implantation sites compared to inter-implantation sites around day 4 of pregnancy. They were abundant during proestrus and estrus but declined rapidly during metestrus. These results indicate that estrogen is a key regulator of the expression of the SPRR2 family in the mouse uterus during the estrous cycle and early pregnancy. In addition, they suggest that some members of the family play an important role in uterine processes such as the estrous cycle, early pregnancy and implantation.     

Expression of insulin-like growth factor binding protein-4, insulin-like growth factor-I receptor, and insulin-like growth factor-I in the mouse uterus throughout the estrous cycle

Recent evidence suggests that a regulated insulin-like growth factor (IGF) system mediates the effects of estrogen, promoting the proliferation and differentiation of specific uterine cell types throughout the estrous cycle and during gestation in the rodent. Previous studies have shown that IGFs are differentially expressed in the mouse uterus during the periimplantation period. In the current study, we examined the expression of IGF binding protein-4 (IGFBP-4), IGF-I receptor (IGF-IR), and IGF-I in the mouse uterus throughout the estrous cycle. Ligand blot analysis was conducted on uterine homogenates using [125I]IGF-I. IGFBP-4 was detected in all uterine homogenates, varying in intensity throughout the estrous cycle. In situ hybridization studies at metestrus and diestrus demonstrated an intense IGFBP-4 mRNA signal in antimesometrial stromal cells between the luminal epithelium and the myometrium, but at proestrus and estrus, no IGFBP-4 signal was detected. No IGF-I mRNA was detected at any stage of the estrous cycle by in situ hybridization. However, by RT-PCR analysis, IGF-I mRNA was detected at all stages of the estrous cycle. RT-PCR analysis also showed IGF-IR mRNA throughout the estrous cycle. Using immunohistochemistry, IGF-IR immunostaining was detected throughout the estrous cycle and on days 2-7 of gestation, but was restricted to the glandular epithelium. These results suggest that uterine IGFBP-4 expression may not be dependent on uterine IGF-I expression. They also suggest that IGFBP-4 may play a role in uterine physiology independent of the inhibition of IGF-I action, and that IGF-IR is constitutively expressed in the mouse uterus.     

The mRNA expression of prostaglandin E receptors EP2 and EP4 and the changes in glycosaminoglycans in the sheep cervix during the estrous cycle

Transcervical artificial insemination in sheep is limited by the inability to completely penetrate the cervix with an inseminating pipette. Penetration is partially enhanced at estrus due to a degree of cervical relaxation, which is probably regulated by cervical prostaglandin synthesis and extracellular matrix remodeling. Prostaglandin E₂ acts via prostaglandin E receptors EP₁ to EP₄, and EP₂ and EP₄ stimulate smooth muscle relaxation and glycosaminoglycan synthesis. This study investigated the expression of EP₂ and EP₄ mRNA and glycosaminoglycans in the sheep cervix during the estrous cycle. Sheep cervices were collected prior to, during, and after the luteinizing hormone (LH) surge and during the luteal phase. The mRNA expression of EP₂ and EP₄ was determined by in situ hybridization, glycosaminoglycan composition was assessed by Alcian blue staining, and hyaluronan concentration was investigated by ELISA. The expression of EP₂ mRNA was greatest prior to the LH surge (P = 0.02), although EP₂ and EP₄ were expressed throughout the estrous cycle. Hyaluronan was the predominant glycosaminoglycan, and hyaluronan content increased prior to the LH surge (P < 0.05). Cervical EP₂ mRNA expression changed throughout the estrous cycle and was greatest prior to the LH surge. We propose that prostaglandin E₂ binds to EP₂ and EP₄ stimulating hyaluronan synthesis, which may cause remodeling of the cervical extracellular matrix, culminating in cervical relaxation.     

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.     

LacZ expression in Fut2-LacZ reporter mice reveals estrogen-regulated endocervical glandular expression during estrous cycle, hormone replacement, and pregnancy

The secretor gene (FUT2) encodes an alpha(1,2)fucosyltransferase (E.C. 2.4.1.69) that elaborates alpha(1,2)fucose residues on mucosal epithelium and secreted mucins. Though uterine alpha(1,2)fucosylated glycans have been proposed to be involved in embryo adhesion, mice with a homozygous null mutation of Fut2 displayed normal fertility. To help develop alternative hypotheses for function, the cell type and regulation of Fut2 expression during the estrous cycle, hormone replacement, and pregnancy was examined in Fut2-LacZ reporter mice containing targeted replacement of Fut2 with bacterial lacZ. LacZ expression in the reproductive tract of Fut2-LacZ mice is most prominent in the glandular epithelium of the endocervix during estrus and pregnancy. Nuclear LacZ expression identifies cell-specific expression of Fut2 in mucus-secreting cells of the endocervix, uterine glands, foveolar pit and chief cells of the stomach, and goblet cells of the colon. In ovariectomized Fut2-LacZ mice, estradiol treatment stimulates X-gal staining in endocervix and uterus but does not affect expression in stomach and colon. Northern blot analysis in wild-type mice shows 15-fold elevations of Fut2 steady-state mRNA with estradiol treatment, whereas Fut1 varies little. Fut2 levels in the glandular stomach and distal colon remain constant, and uterine Fut2 levels vary eightfold during the estrous cycle. These data represent the first demonstration of a glycosyltransferase gene under tissue-specific hormonal regulation in a LacZ reporter mouse model. Endocervical expression of Fut2 in estrus and pregnancy may modify cervical mucus barrier properties from microbial infection analogous to the potential role of mucosal glycans in humans.     

GRP78 expression and immunohistochemical localization in the female reproductive tract of mice

The 78-kDa glucose-regulated protein (GRP78) is an endoplasmic reticulum chaperone, with multiple functional roles in protein processing and provision of cellular protection. However, the physiological role of GRP78 in embryo development is not clear. Localization of GRP78 and expression of its mRNA in the reproductive organs throughout the estrous cycle in mice were investigated by immunohistochemistry and real-time polymerase chain reaction, respectively. Whereas there was intense staining for GRP78 in the oviduct at estrus, the ciliated cells of isthmus had better staining than those of infundibulum and ampulla at all phases of the cycle. Furthermore, GRP78 was located in the uterine luminal and glandular epithelial cells throughout the estrous cycle, particularly during the estrus phase. However, levels of GRP78 mRNA in the oviduct and uterus varied during the cycle, with peaks at estrus. In conclusion, GRP78 expression varied with the phase of the murine estrous cycle; this might be related to gamete transport, fertilization and early development of the zygote/embryo.     

Increased LH pulse frequency and estrogen secretion associated with termination of anestrus followed by enhancement of uterine estrogen receptor gene expression in the beagle bitch

The relationships among pulsatile LH secretion pattern, estrogen secretion, and expression of the uterine estrogen receptor gene were examined throughout the estrous cycle in beagle bitches. In Experiment 1, blood samples were collected from 30 bitches every 10 min for 8 h from a cephalic vein during different phases of the estrous cycle. An increase in the mean plasma levels of LH occurred from mid to late anestrus (P < 0.01). The LH pulse frequency increased (P < 0.01) from late anestrus to proestrus, and was strongly correlated (r = 0.96, P < 0.001) with the mean plasma level of estradiol-17 beta (E2). In Experiment 2, middle uterine samples, including the myometrium and endometrium, from 18 bitches were taken at 6 stages of the estrous cycle. The total number of estrogen receptors and nuclear estrogen receptor and its mRNA levels in the uterus also increased (P < 0.01) from late anestrus to proestrus. Mean plasma E2 level and the number of uterine estrogen receptor were positively correlated (r = 0.81, P < 0.05). In Experiment 3, nine bitches were ovariectomized in mid anestrus. Two weeks later they received a single injection of 10 or 50 micrograms/kg, i.m., estradiol benzoate. The number of uterine estrogen receptor and their mRNA levels for ovariectomized bitches were low, but increased (P < 0.05) after treatment with a low dose of estradiol benzoate. These results suggest that increases in LH pulse frequency and estrogen secretion are associated with termination of anestrus and that subsequent enhancement of uterine estrogen receptor expression may be up-regulated by estradiol.     

Concentrations of 13, 14-dihydro-15-keto-prostaglandin F(2)alpha, estradiol-17beta and progesterone during the peripubertal period in heifers

Twenty prepubertal Holstein heifers were utilized to assess plasma 13, 14-dihydro-15-keto-prostaglandin F(2)alpha (PGFM), serum progesterone (P(4)) and estradiol-17beta (E(2)) concentrations as well as the E(2):P(4) ratio during the onset of puberty in cattle. All animals were maintained as a group along with a sterile marker bull to assist in the detection of estrus. Upon detection of the first estrus (Day=O), daily blood samples were collected from a jugular vein until the heifers had completed 3 estrous cycles. The average body weight and age at first estrus were 247.6+/-4.8 kg and 304.0+/-7.5 days, respectively. Frequency of abnormal length estrous cycles was greater (P<0.02) during the first (40%) and second (35%) cycles than during the third estrous cycle (0%). All heifers had normal cycle lengths (18 to 24 days) by the third estrous cycle. Serum P(4) was greater during the third cycle (P<0.05) from Day 10 to Day 4 before the next estrus compared with the same period of the first estrous cycle. Serum E(2) did not peak until the day of estrus in the first cycle, whereas E(2) reached a maximal level 2 days before estrus in the third estrous cycle. Serum E(2) was higher (P<0.0001) 2 days before estrus in the third cycle than in the first estrous cycle. Plasma PGFM reached maximum concentrations 3 days before estrus in the third cycle compared with 1 day before estrus at the end of first estrous cycle. As estrus approached during the third cycle, PGFM rose 1 day before E(2) rose and P(4) declined, while the rise in PGFM and E(2) occurred simultaneously, with P(4) declining at the end of the first estrous cycle. During diestrus, the E(2):P(4) ratio was lower (P<0.07) in the third cycle than in the first, but it was higher (P<0.04) at estrus and 1 day before in the third estrous cycle. These data reveal a high incidence of abnormal length estrous cycles during the first two estrous cycles of the peripubertal period, and demonstrate anomalies in uterine and ovarian endocrine activity during the peripubertal period in cattle.     

基质金属蛋白酶-2、-9及其组织抑制因子(TIMPs)在动情周期大鼠子宫中的表达(英文)

基质金属蛋白酶（ＭＭＰｓ）家族的作用是降解所有细胞外基质,其活性受其特异性组织抑制因子（ＴＩＭＰｓ）的抑制。细胞外基质成分的降解与重组在动物生殖生长过程中起重要作用,其变化可以通过ＭＭＰｓ和ＴＩＭＰｓ两者表达水平的变化进行监测。大鼠虽然没有月经形成,但是在其子宫内膜也出现类似灵长类的生殖生物学变化。本文从ＭＭＰｓ和ＴＩＭＰｓ两者的表达水平,对大鼠子宫内膜的这些变化进行了研究。于大鼠动情周期的不同时期,将其处死、取子宫制备酶粗提液和组织切片,采用酶谱法（ｚｙｍｏｙｒａｎｈｎ）和原位杂交方法研究动情周期大鼠子宫中ＭＭＰ－２和－９的活性变化以及ＭＭＰ－２、－９和ＴＩＭＰ－１、－２、－３ｍＲＮＡ的表达。并通过光密度扫描方法对酶谱结果进行半定量分析。所用杂交探针见Ｔａｂｌｅ１。酶谱结果显示：在动情周期大鼠子宫中只检测到６７ｋＤａ的ＭＭＰ－２活性,而没有检测到ＭＭＰ－９的活性（Ｆｉｇ．１）。ＭＭＰ－２的活性在动情前期最高,动情期和动情后期次之,间情期最低（Ｆｉｇ．２）。原位杂交结果显示：ＭＭＰ－２、－９、ＴＩＭＰ－１、－２、－３ｍＲＮＡ主要在子宫内膜基底部的基质细胞中表达。ＭＭＰ－２和－９ｍＲＮＡ在动情前期、动情期和动     

Molecular mechanism of cell proliferation in rodent uterus during the estrous cycle

The rodent uterus is a widely studied target tissue for sexual steroid hormone action. The aim of the present study was to assess the molecular mechanism that participates in the initiation of cell proliferation of the rat uterine epithelial cells during the estrus (E)–metestrus (M) transition. Cell proliferation, ERα, c-fos, cyclin D1 and D3, cdk4, and cdk6 proteins were assessed in these animals by immunohistochemistry. Estradiol (E₂) and progesterone (P₄) plasma levels were assessed by RIA. The results indicate that the glandular epithelium starts to proliferate at 21:00 h on estrus day, and initiates at least 3 h before the luminal epithelium does. Fos expression was markedly increased during the afternoon of estrus day, and its increase was in parallel to ERα expression. Interestingly, both, cyclin D1 and D3 were abundantly expressed in the luminal and glandular epithelia, and nuclear immunolabelling of cyclin D1 and D3 precedes BrdU incorporation in the cell. cdk4 and cdk6 were localized in the nuclei in both epithelia throughout the studied time course. In addition, cdk4 was more abundant throughout estrus and metestrus days than cdk6. The overall results indicate that ERα, Fos and cyclins D1 and D3, cdk4 and cdk6 are expressed in both glandular and luminal epithelia of the rat uterus during the E–M transition. In conclusion, there is a good correlation between sequential expression of these proteins and cell cycle progression in the rat uterine epithelial cells during the estrous cycle. However, the differences observed in the cellular localization, time course of expression and the cellular types that express both cyclins between physiological and pharmacological conditions, demonstrated different mechanisms of regulation and should be due to the complex hormonal milieu during the estrous cycle.     

Messenger RNA levels of estrogen receptors alpha and beta and progesterone receptors in the cyclic and inseminated/early pregnant sow uterus

The aim of the present study was to investigate differences in the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus at different stages of the estrous cycle as well as in inseminated sows at estrus and during early pregnancy by use of solution hybridization and in relation to plasma levels of estradiol and progesterone. Uterine samples were collected at different stages of the estrous cycle and after insemination/early pregnancy. In the endometrium, the expression of ERalpha mRNA and PR mRNA was similar for cyclic and early pregnant groups. Both were highest at early diestrus/70 h after ovulation and ERalpha mRNA was lowest at late diestrus/d 19 while PR mRNA was lowest at diestrus and late diestrus/d 11 and d 19. The expression of endometrial ERbeta was constantly low during the estrous cycle but higher expression was found in inseminated/early pregnant sows at estrus and 70 h after ovulation. In the myometrium, high expression of ERalpha mRNA and PR mRNA was observed at proestrus and estrus in cyclic sows and at estrus in newly inseminated sows. Higher expression of myometrial ERbeta mRNA was found in inseminated/early pregnant sows compared with cyclic sows, although significant only at estrus. In conclusion, the expression of mRNAs for ERalpha, ERbeta and PR in the sow uterus differed between endometrium and myometrium as well as with stages of the estrous cycle and early pregnancy. In addition to plasma steroid levels, the differences between cyclic and inseminated/early pregnant sows suggest that other factors, e.g. insemination and/or the presence of embryos, influence the expression of these steroid receptor mRNAs in the sow uterus.