蛋白激酶H11基因在小鼠子宫中的表达及其性激素调节

为研究蛋白激酶H11基因在生殖系统中的作用,我们采用半定量RT-PCR和原位杂交方法,研究了蛋白激酶H11基因在小鼠中的组织特异性表达,在妊娠初始期胚胎植入位点、妊娠期子宫和胎盘以及正常动情周期子宫中的表达及其受性激素的调节。结果发现:蛋白激酶H11基因在小鼠多种组织中都有表达,在卵巢及子宫等一些生殖相关的组织中表达水平较高;妊娠初始期,蛋白激酶H11基因在小鼠子宫内膜植入位点处有明显的高表达,其mRNA定位于腔上皮细胞和基质细胞中。在动情周期中,蛋白激酶H11基因在动情前期子宫中表达水平较低;卵巢切除模型显示雌激素和孕激素均可显著上调蛋白激酶H11基因的表达。以上结果提示蛋白激酶H11可能参与了胚胎植入过程中腔上皮细胞凋亡和基质细胞增殖与蜕膜化以及动情周期小鼠子宫内膜细胞的功能调节[动物学报51(3):462-468,2005]。     

Dual roles for macrophages in ovarian cycle-associated development and remodelling of the mammary gland epithelium

Each ovarian cycle, the mammary gland epithelium rotates through a sequence of hormonally regulated cell proliferation, differentiation and apoptosis. These studies investigate the role of macrophages in this cellular turnover. Macrophage populations and their spatial distribution were found to fluctuate across the cycle. The number of macrophages was highest at diestrus, and the greatest number of macrophages in direct contact with epithelial cells occurred at proestrus. The physiological necessity of macrophages in mammary gland morphogenesis during the estrous cycle was demonstrated in Cd11b-Dtr transgenic mice. Ovariectomised mice were treated with estradiol and progesterone to stimulate alveolar development, and with the progesterone receptor antagonist mifepristone to induce regression of the newly formed alveolar buds. Macrophage depletion during alveolar development resulted in a reduction in both ductal epithelial cell proliferation and the number of alveolar buds. Macrophage depletion during alveolar regression resulted in an increased number of branch points and an accumulation of TUNEL-positive cells. These studies show that macrophages have two roles in the cellular turnover of epithelial cells in the cycling mammary gland; following ovulation, they promote the development of alveolar buds in preparation for possible pregnancy, and they remodel the tissue back to its basic architecture in preparation for a new estrous cycle.     

Effect of estradiol and progesterone on phosphatidylinositol metabolism in the uterine epithelium of the mouse

The effect of estradiol and progesterone on uterine phosphatidylinositol (PtdIns) metabolism was examined in whole uteri and separated uterine luminal epithelium of ovariectomized mice. Incorporation of [3H]myo-inositol in vitro, into inositol-containing phospholipids extracted from whole uteri, increased in mice injected with estradiol, with maximal incorporation at 9-12 h. The breakdown of PtdIns to inositol polyphosphates was also stimulated in whole uteri by estrogen, with an abrupt increase between 6 and 9 h. Comparable increases in both processes occurred in the uterine epithelium after estrogen stimulation and were inhibited by progesterone pretreatment which by itself had little or no effect. These results suggest that PtdIns metabolism is involved in the stimulation of uterine epithelial cell proliferation by estrogens, and its inhibition by progesterone.     

Sex hormone regulation of anti-bacterial activity in rat uterine secretions and apical release of anti-bacterial factor(s) by uterine epithelial cells in culture

In mature female rats, sex hormones regulate the reproductive (estrous) cycle to optimize mating and fertility. During the part of the estrous cycle when mating occurs, and when estrogen is the dominant sex hormone, the uterus is susceptible to infection with bacteria that can be deleterious for survival and fertility. The present study investigated whether sex hormones regulate innate immunity in the female reproductive tract by affecting the secretion of an anti-bacterial factor(s) in the rat uterus. Uterine fluids from intact rats at the proestrous stage of the estrous cycle significantly inhibited Staphylococcus aureus growth. When ovariectomized rats were treated with estradiol, anti-bacterial activity against both S. aureus and Escherichia coli increased in uterine secretions with hormone treatment. In contrast, rats injected with either progesterone and estradiol or progesterone alone displayed no bactericidal activity indicating that progesterone reversed the stimulatory effect of estradiol on anti-bacterial activity. In other studies, isolated uterine epithelial cells from intact animals were grown to confluence and high transepithelial resistance on cell inserts. Analysis of apical secretions indicated that a soluble factor(s) is released by polarized epithelial cells which inhibits bacterial growth. These results demonstrate that sex hormones influence the presence of a broad-spectrum bactericidal factor(s) in luminal secretions of the rat uterus. Further these studies suggest that epithelial cells which line the uterine lumen are a primary source of anti-bacterial activity.     

DNA replication in uterine cells of adult and prepubertal mice under normal and hormonally stimulated conditions detected by bromodeoxyuridine labeling method

To confirm the utility of the bromodeoxyuridine (BrdU) labeling method in the study of cell proliferation in mouse uterine tissues, changes in the labeling index in the luminal and glandular epithelia, the periluminal, periglandular and deep stromal regions and the myometrium were surveyed in normal adult mice during the estrous cycle and early pregnancy, in prepubertal mice and in ovariectomized adult and young animals treated with estrogen and/or progesterone. All results obtained were consistent with those obtained in previous histometric and autoradiographic studies and proved the effectiveness of the BrdU labeling method in the study of the uterus as well as many other organs. A marked rise in the labeling index was found in the luminal epithelium at metestrus, as well as on the proestrous morning, indicating the occurrence of extensive cell proliferation in the absence of estrogen stimulation. The change in the labeling index in adult mice was much more evident in the luminal epithelium than in the glandular epithelium in all conditions examined. On the other hand, the change in the stroma was more eminent in the periglandular region than in the periluminal and deep regions in most conditions. In immature mice, a great increase in labeling incidence occurred not only in luminal epithelium but also in muscle layers along with the process of puberty and at the time of estrogen stimulation. A moderate increase in the incidence also occurred in all other areas of the uterus including the perimetrium. Again, the increase was more prominent in the periglandular area than in other stromal regions.     

Light and electron microscopic radioautography of DNA synthesis in the endometria of pregnant-ovariectomized mice during activation of implantation window.

Pregnant mice were ovariectomized at pre-implantation stage and exogenous nidatory estradiol was administered to evaluate the DNA synthesis of the endometrial cells during activation of uterine receptivity for blastocyst implantation. After 0, 3, 6, 12 and 18 hrs. of estradiol treatment, the animals received 3H-thymidine injection, sacrificed 1 hr. later, and the uteri were prepared for light and electron microscopic radioautography. At time 0, no labelled stromal or epithelial cells was found in the endometrium. According to the time-lapse after estradiol induction, a gradual increase of labelled stromal and endothelial cells was seen in the endometrium. The highest labeling index was observed at the antimesometrial side of the implantation sites and the lowest value was found at the interimplantation site. The cells found at mesometrial side of the implantation site showed an intermediate labeling index. Eighteen hrs. after estradiol treatment, the labelled stromal cells found near the implantation chamber resembled the morphology of decidual cells while those labelled cells localized at the interimplantation sites were similar to the fibroblast. The uterine luminal epithelial cells showed low DNA synthesis after estradiol treatment resulting in only a few labelled cells at the interimplantation sites and no labelled cells at the implantation sites. A similar labeling pattern was seen in the glandular epithelium. The distribution of labelled cells seen among the regions of pregnant endometrium under estradiol effect suggest that DNA synthesis related to uterine activation for blastocyst implantation is a focal reaction, where the luminal epithelium does nt proliferate while the stromal and endothelial cells around the conceptus increase the DNA synthesis to prepare the endometrial decidualization.     

A collaboration of aquaporins handles water transport in relation to the estrous cycle in the bitch uterus

Fluid movement through uterine cell membranes is crucial, as it can modulate the tissue imbibition pattern in the different phases of the estrous cycle. To gain insight into the mechanisms underlying steroid-controlled water handling, the presence and distribution of aquaporins (AQPs), integral membrane channel proteins permitting rapid passive water movement, was explored in bitch uterine tissues. Immunohistochemistry and Western immunoblot analysis were used to study the presence of AQP1, AQP2, and AQP5 in the layers of the bitch uterine wall during the different estrous phases. Presence of endothelial nitric oxide-generating enzyme NO synthase (NOS3) was also investigated, as it is known that the vasodilator NOS3 might be involved in the development of uterine edema. The results demonstrated the following: (1) AQP1, AQP2, and AQP5 were present in the uterus of cycling bitches. (2) AQP1 was localized within uterine mesometrial, myometrial, and endometrial blood vessels and in the circular and longitudinal layers of myometrium. AQP1 localization and expression were unaffected by the estrous cycle. (3) The estrogenic milieu was probably at the basis of AQP2 expression in the glandular and luminal epithelium of the endometrium. (4) AQP5 water channels were present in the apical plasma membrane of uterine epithelial cells in coincidence with plasma progesterone increase. (5) NOS3 was localized in the myometrial and epithelial tissues as well as in blood vessels indicating a contribution of this vasoactive peptide to the uterine imbibition processes. Thus, we can hypothesize that a functional and distinctive collaboration exists among diverse AQPs in water handling during the different functional uterine phases.     

Hormonal regulation of apoptosis in the endometrium of common marmosets (Callithrix jacchus)

Phase-dependent apoptotic changes in the human endometrium during an ovarian cycle imply a potential role of steroids in the regulation of apoptosis. The present study was undertaken to determine the direct role of hormones in endometrial apoptosis in marmosets (Callithrix jacchus), a primate species which shows similarity to humans in terms of the cycle length and pattern. Endometrial apoptosis was detected by 3'-end labeling (TUNEL) in various phases of ovarian cycle in naturally cycling healthy marmosets (n=14) and also in ovariectomized marmosets (n=13) treated with either estradiol alone (E) or progesterone alone (P) or estradiol followed by progesterone (E+P). Expressions of apoptosis associated genes such as Bcl-2 family members (Bax and Bcl-2), proliferating cell nuclear antigen (PCNA)--a proliferation marker and steroid receptors, ERalpha and PR A were analysed by immunohistochemical methods. Apoptosis was intense in the glandular epithelial cells of endometrium during the mid-luteal phase as compared to other phases in naturally cycling animals; in the E+P group as compared to other groups of ovariectomized animals (P<0.05). Pronounced apoptosis in the mid-luteal phase was accompanied by the increased expression of Bax in glandular epithelial cells; while Bcl-2 immunoreactivity remained unchanged. PCNA expression was higher in the naturally cycling animals in the follicular phase and in the E group of the ovariectomized animals as compared those in the other groups. Immunoreactive ERalpha and PR A in glandular epithelial cells were most abundant during early follicular phase in naturally cycling animals and in both E and E+P groups among the ovariectomized animals. The present study highlights the importance of apoptosis in endometrial remodeling during the ovarian cycle and secondly, the role of both estradiol and progesterone in the regulation of apoptosis.     

The effects of sequential administration of 17 beta-estradiol on the synthesis and secretion of specific proteins in the immature rat uterus

We report here results of a study of the effect of sequential administration of 1 microgram 17 beta-estradiol in vivo on the incorporation of L-[35S]methionine into specific proteins in vitro in the immature rat uterus. One-dimensional SDS-PAGE analysis of labeled secreted uterine proteins and cellular proteins extracted from the luminal epithelial and from the stroma plus myometrial uterine fractions revealed that estradiol preferentially stimulated the synthesis of 110 K, 74 K and 66 K secreted proteins, 180 K and 110 K epithelial proteins and a 175 K stroma-myometrial protein among others, while it decreased the relative rate of synthesis of a 32.5 K secreted protein and a 70 K stroma-myometrial protein. The 110 K protein, a secreted luminal epithelial protein whose labeling in vitro dramatically increased greater than 60-fold per mg endometrial DNA after in vivo estrogen stimulation, may be a useful marker for studying estrogen action in the luminal epithelium of the immature rat uterus. Comparison of the secreted proteins labeled at 28 h (4 h after a second injection) and at 54 h (6 h after a third injection) revealed that estradiol effected a sequential change in the pattern of synthesis of secreted uterine proteins in vitro. Comparison of the number and magnitude of changes in the synthesis of specific proteins in the luminal epithelium and the stroma plus myometrium revealed that protein synthesis in the luminal epithelium is clearly more responsive to estradiol and readily distinguishable from the responsiveness of the stroma plus myometrium.     

REGENERATION OF UTERINE EPITHELIUM AFTER EXPERIMENTAL ABLATION IN THE RAT

Regeneration of the uterine luminal epithelium was studied after its mechanical removal in progesterone-primed rats, leaving one control horn intact. Pulse labelling with [³H]TdR during regeneration, showed a rapid peak of labelling index in remaining glands. A differentiated and highly labelled luminal epithelium reappeared at 34 hr, thereafter showing a rapidly declining LI. After initial depletion, the glandular cell population size was restored within 64 hr, whereas luminal epithelium cell numbers became stabilized at about half normal level. Grain counts after prelabelling showed more rapid dilution in gland cells of stripped uterine horns, indicating accelerated cycling of previously dividing cells. Thymidine labelling indices also showed that, after removal of the epithelium, almost all gland cells became rapidly committed to divide. On average, less than two cell cycles were necessary to restore stable glandular and epithelial population sizes. Numbers of labelled cells were also drastically increased in myometrium and serosa of treated horns. This suggests a non-specific mechanism for stimulation of mitotic activity after ablation of epithelium.     

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.     

G0101C03-3和L0254H10-3 基因在小鼠动情周期子宫中的差异表达

正常动情周期的维持是小鼠子宫多功能的必要条件 ,但对于其分子基础至今尚不十分明了。我们曾通过基因芯片技术分析了小鼠动情期与间情期子宫的基因表达谱 ,发现了许多差异表达的基因或表达序列标签(ESTs)。本实验选取了G0 1 0 1C0 3 3及L0 2 5 4H1 0 3两个差异表达的EST ,通过Northern印迹与原位杂交方法分析了它们在小鼠动情周期子宫中的时空表达模式。结果表明 :这两个基因的表达水平都发生周期性变化 ,在动情期表达量较少 ,而在间情期表达量较高 ;G0 1 0 1C0 3 3在动情期的表达量是间情期的 2 7% ,L0 2 5 4H1 0 3在动情期检测不到有表达 ,提示它们的表达受卵巢类固醇激素的调控 ;G0 1 0 1C0 3 3基因主要在子宫的腔上皮与腺上皮中表达 ,可能与子宫细胞的程序性死亡有关 ;而L0 2 5 4H1 0 3基因则主要位于基质细胞中     

Structural changes in the luminal epithelium of the porcine uterus between days 10 and 19 of the estrous cycle

Light and electron microscopy were used to study the morphology of uterine luminal epithelium from 4 or 5 gilts slaughtered on each of days 10, 13, 16, and 19 of the estrous cycle. Ultrastructural evidence indicated that metabolic activity and accumulation of glycogen by the uterine epithelium increased between days 10 and 16 of the estrous cycle. This phase of high synthetic activity had been terminated by day 19, as evidenced by the reduction or absence of glycogen deposits and decreased incidence of organelles associated with synthetic activity. Diffuse degeneration of epithelial cells occurred throughout the period of study but was maximal between days 16 and 19. Mitotic activity indicated that cell replacement also occurred between day 16 and day 19.     

Involvement of apoptosis during deciduomal regression in pseudopregnant hamsters effect of progesterone

We determined whether fragmentation of genomic DNA, apoptosis, occurs during deciduomal regression in pseudopregnant hamsters and the effect of progesterone on the apoptotic processes. Artificially induced deciduoma were obtained on different days of pseudopregnancy and separated into mesometrial and antimesometrial tissues. The deciduomal cell cycle progression and population profiles of both sides were compared by flow cytometry. The proportion of sub-G1 peak, which was correlated with the apoptotic cells, were about 10% on day 8 and reached to 40% in both tissues on day 10. Exogenous progesterone treatment by daily injection (2 mg; s.c.) on and after day 8 reduced the percentage of low molecular weight DNA in both tissues on day 10 and day 12 as compared to the nontreated control one, respectively. The appearance of DNA ladder was also delayed at least 24 h by progesterone administration. The intensity of DNA fragmentation was more pronounced in antimesometrial deciduoma. In situ 3'-end labeling of apoptotic cells further substantiated the apoptotic process. The apoptotic cells first appeared in the luminal region in antimesometrial deciduoma on day 8 and spreaded all over the entire deciduomal tissue on day 10. Progesterone treatment stimulated deciduomal proliferating cell nuclear antigen (PCNA) expression, maintained deciduoma until day 14 and retarded the differentiation and regeneration of the uterine epithelium.     

Apoptosis in endometrium of mouse during estrous cycle

The present study was carried out to evaluate apoptosis in endometrium and to correlate these changes with the circulating levels of estradiol and progesterone in the mouse. Apoptosis was observed in various compartments of mouse uterus i.e. stroma, glandular epithelium and luminal epithelium depending on the stage of cycle. Stromal cell apoptosis was observed during various stages of cyclicity except on estrus day. Luminal epithelial cells showed apoptotic changes during all stages of cyclicity except on diestrus day. During metestrus, apoptosis was observed in glandular and luminal epithelia as well as stromal cells. Steroid antagonists such as tamoxifen and onapristone altered the apoptotic changes in the uterus. The results suggest that epithelial cell apoptosis is regulated by estrogen while stromal cell apoptosis is under the control of progesterone.     

Magnum morphogenesis during the natural development of the quail oviduct: analysis of egg white proteins and progesterone receptor concentration

The histological development of the quail oviduct and the changes in concentrations of progesterone receptor, ovalbumin, conalbumin, ovomucoid and ovoglycocomponents are analyzed during the period spanning 7-35 days of age. The initiation of luminal epithelial cell proliferation is the first event of magnum growth. The epithelial cells begin to evaginate into subepithelial stroma and form tubular glands. Meanwhile, luminal epithelium starts cellular pleomorphism through ciliogenesis. No egg white proteins are detectable in the developing glands; at the same time, the concentration of the progesterone receptor increases from about 5500 sites/cell to 30,300 sites/cell. Tubular gland cells then begin to synthetize and accumulate egg white proteins, mucous cells differentiate in the luminal epithelium, and the cell proliferation decreases and finally stops. Compared with earlier studies dealing with the blood levels of estrogen and progesterone in developing quails during the same period, and the cellular changes induced in the oviducts of ovariectomized and ovariectomized-hypophysectomized quail by exogenous steroids, these results distinguish between the cellular responses that are physiologically controlled by estradiol and other responses that have multihormonal regulation.