Keratinization of rat vaginal epithelium. III. Effect of estradiol on keratinization

Effect of estradiol-17 beta on rat vaginal epithelial cells (VEC) was studied by transmission electron microscopy. During the normal estrous cycle in adult rats keratinization and exfoliation of VEC was observed only in the estrus phase, though tonofilament bundles were seen distributed in the cytoplasm of the VEC during the proestrus and, to a lesser extent, in the diestrus phase. Electron microscope data on adult ovariectomised, immature and neonatally estradiol-primed rats demonstrated keratinization of the VEC within 24 h of injection. Proliferative activity was observed in the basal cells in primed animals. The basal cells remained cuboidal whereas cells of intermediate and luminal layers became flattened. These cells showed irregular profiles and, therefore, enhanced numbers of cellular junctions (desmosomes) were observed compared to control groups. Many tonofilament bundles were seen in the cytoplasm of cells belonging to intermediate and luminal layers in primed animals.     

Trypsin-like activity in the vaginal epithelial cells of the rat.

Rat vaginal epithelial cells have trypsin-like activity as shown by the formation of a colored product when the cells are incubated with alpha-N-methyl alpha-N-toxyl-L-lysine beta-naphthol ester and hexazotized pararosanilin. This enzyme activity in vaginal smears is maximal at proestrus, i.e., the day in the 5-day estrus cycle when plasma estrogen is maximal. Only the rounded nucleated epithelial cells present at late diestrus, proestrus and early estrus demonstrate the trypsin-like enzyme activity. These are the cells that stain blue in the Papanicolaou method. Preincubation of cell suspensions with the serine protease inhibitor, p-nitrophenyl p-guanidino benzoate, prevented the enzyme staining reaction, further demonstrating the trypsin-like nature of the cellular enzyme. The advantages of this enzyme staining technique over the fibrin plate method for the demonstration of trypsin-like enzymes in cells are increased resolution and ability to show trypsin inhibitor effects.     

Quantitative variations in the number and size of peritoneal mastocytes in the female rat during the normal sexual cycle, following ovariectomy and administration of estrogens

Female rat peritoneal mast cells were found more numerous and of more reduced sizes in metestrus and diestrus than in proestrus and especially estrus where a partial degranulation took place. Ovariectomy caused an important decrease in the number and size of mast cells. A regular treatment of spayed animals by 17 beta estradiol partly inhibited these effects.     

Differentiation of estradiol receptors in rat uterine cytosol by sensitivity to tamoxifen.

Analysis of rat uterine cytosol by means of immobilized antibody discloses the presence of two distinct high affinity, low capacity estradiol binding components. One of these is readily saturable by the antiestrogen tamoxifen (TS = tamoxifen sensitive), the other is tamoxifen insensitive (TI). Only TS-estradiol binding shows positive cooperativity at low ligand concentration. TS but not TI is lost when frozen tissue is thawed at 4°C then refrozen and stored for an additional 12 hours. Experiments with ovariectomized rats show that TI is formed only in the presence of estradiol. In the estrus cycle TS increases in the order: metestrus, diestrus, proestrus and estrus. The quantity of TI is the same in metestrus and diestrus and also the same but fractionally higher in proestrus and estrus.     

Hormone-sensitive lipase expression and IHC localization in the rat ovary, oviduct, and uterus

Hormone-sensitive lipase (HSL) is a key regulator of cholesterol esters metabolism. The aim of this study was to determine HSL localization in rat female reproductive organs during the ovarian cycle by IHC methods. HSL was located in the ovarian epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In mature follicles, HSL was found in oocytes and theca and granulosa cells. However, HSL expression in theca cells and oocytes decreased during follicular atresia. Luteal cells showed HSL staining in cytoplasm during proestrus and estrus, in the nucleus during metestrus, and in cytoplasm and the nucleus during diestrus. In the tubaric ampulla, HSL was located in the epithelial cells nuclei and in the cilia during proestrus and estrus but mainly in the nucleus during metestrus and diestrus. In the isthmus, cells showed HSL immunolabeling in the nucleus and cilia during proestrus, but only in the cilia during estrus, metestrus, and diestrus. In the uterus, HSL was found in the epithelial cells nuclei. HSL-immunoreactive bands at 84, 67, 54, and 43 kDa were found in rat female reproductive organs. HSL labeling in the nucleus of epithelial and germ cells suggests an as yet unknown function for this protein, probably related to oogenesis and cell proliferation.     

Prolactin receptors in the rat mammary gland. Change during the estrous cycle

Ovine prolactin (o-PRL) binding to mammary gland membranes was studied during the estrous cycle in the rat. Groups of rats were decapitated throughout the 4-day estrous cycle at 10 h00 on the days of diestrus I, diestrus II and estrus and at 10 h00, 12 h00, 16 h00 during the day of proestrus. Daily vaginal smears were taken to determine the stage of the estrous cycle which was also controlled by PRL and LH serum levels. Prolactin receptors were quantified in the 100 000 g pellet. For one Scatchard analysis, mammary gland membranes from 5 animals were pooled. Results given are the mean of 4 or 5 pools. Results obtained showed that the apparent affinity constant (KA) remained unchanged during the days of diestrus II and at all the times studied of proestrus and showed a slight but significant decrease on the days of estrus and diestrus I (or metestrus). The binding capacity did not vary from the day of diestrus II to the proestrus 16h00 (11.3 +/- 2.8 fmoles/mg protein) but sharply increased on the day of estrus (190.4 +/- 35.9 fmoles/mg protein). Binding capacity remained elevated on the day of diestrus I. This increase of PRL receptors on the day of estrous would appear to be an important step in preparing mammary gland for pregnancy and lactation.     

Relationship of Vaginal Cytology to Alterations of the Vaginal Microflora of Rats During the Estrous Cycle

The influence of cyclic changes occurring in the vaginal tract during the estrous cycle upon the indigenous microflora of the vagina has been investigated by semiquantitative techniques in virgin female rats. By plate counts performed on material lavaged daily from the vaginal tract of several rats, it is apparent that bacterial counts are elevated in the proestrus and estrus phases of the cycle to values several orders of magnitude greater than those observed during metestrus or diestrus phases. Increases in vaginal bacterial counts were associated with the presence of cornified epithelial cells in the vagina; these cells were predominantly nonviable. Decreases in the vaginal bacterial content were related to the influx of leukocytes into the vagina after estrus. When leukocytes were present in the vaginal tract, they were 90 to 100% viable. From these observations it has been concluded that the female vaginal tract and the bacteria which colonize it represent a dynamic ecosystem which is responsive to cyclic events occurring in the estrous cycle. The changing cellular content of the vaginal tract may have relevance to the observed cyclic changes in the bacterial content of the vagina.     

Localization of metallothionein in female reproductive organs of rat and guinea pig

We demonstrated the localization of metallothionein (MT) in rat uterus and ovaries and in guinea pig mammary glands. During the cyclic changes from one estrous period to the next, strong MT immunostaining was found in the glandular epithelium of the endometrium and weak immunostaining was observed in the simple columnar epithelium. Interestingly, during estrus, the intensity of MT immunostaining decreased in the cytoplasm, whereas during metestrus, diestrus, and proestrus the intensity of strong and similar immunostaining was observed in both the cytoplasm and nucleus. During proestrus and estrus, the number of vaginal epithelial cells containing MT increased on the luminal side of the epithelium and inside the lumen. In rat ovary, strong immunostaining was observed in the cytoplasm and nucleus of granulosa-lutein cells of the corpus luteum and in the cytoplasm of the ovum. In mammary gland of non-pregnant guinea pig, very strong but scattered MT immunostaining was demonstrated in both cytoplasm and nucleus of some epithelial cells of the lactiferous ducts. The mammary tissue of the pregnant guinea pig showed an increase in MT staining in alveolar cells that had proliferated due to pregnancy. The presence of MT in the female reproductive organs, the tissues of which actively grow under the control of female sex hormones, indicates some as yet unknown association of MT with cell proliferation and differentiation.     

Opposite regulation of XIAP and Smac/DIABLO in the rat endometrium in response to 17β-estradiol at estrus

During rat estrous cycle, the endometrium proliferates in response to sex steroids and specific endometrial epithelial cells undergo apoptosis in absence of embryonic factors. The central executioner of apoptosis is a family of aspartic acid-specific cysteine proteases known as caspases. Smac/DIABLO is released from the mitochondria during apoptosis and its stimulation promotes caspases activation by neutralizing members of the inhibitor of apoptosis proteins (IAPs) family, such as X-linked inhibitor of apoptosis protein (XIAP). The aim of this study was to investigate the involvement of Smac/DIABLO and XIAP in the control of caspases activation in endometrium of cycling rats. Polyoestrus female rats were sacrificed at each stage of estrous cycle (diestrus, proestrus, estrus, and metestrus). Endometrial protein extracts were collected to perform Western Blot analysis. Alternatively, uterine horns were sectioned for immunohistochemistry (IHC). We and others showed previously the presence of apoptosis at estrus in rat uterine epithelium. In the present study, cleaved caspase-3, -6, and -7 fragments were detected at estrus. IHC confirmed that caspase-3 was present only in luminal and glandular epithelium at estrus. XIAP was highly expressed at estrus in both epithelial and stromal cells. In contrast, expression of Smac/DIABLO was elevated at diestrus, proestrus and metestrus but was minimal at estrus. Treatment of ovariectomized rats with 17β-estradiol induced XIAP expression and inhibited Smac/DIABLO protein expression in the endometrium. Cleaved caspase-3, -6, and -7 fragments increased in endometrial protein extracts following 17β-estradiol treatment. Expression of NF-κB and IκB proteins, and IκB phosphorylation status were detected in the endometrium but were not influenced by the estrous cycle. These findings suggest that Smac/DIABLO and XIAP are regulated differently and may play important roles in the regulation of endometrial cell fate. Moreover, this study confirms a key role for executioner caspases in the control of apoptotic processes at estrus in the rat uterus.     

Effect of ethanol in the pre-ovulatory period on the ovarian cycle, progesterone and estradiol in the rat

The effect of high doses of ethanol (2 and 4 g/kg) administered to rats in pre-ovulatory periods (18th hour of diestrus) was studied. Plasma levels of estradiol and progesterone were measured at 10 hours of oestrus and changes in the ovarian cycle and the number of mature follicles were recorded. Compared with the control group, the receptive phase of estrus of the treated animals was longer lasting over 2 to 3 days. There was also an increase in the number of mature follicles as well as an increase in the plasma level of estradiol on the morning of estrus. Progesterone values showed no significant variations. Ethanol administered at the 18th hour of diestrus inhibits ovulation but not follicle development and allows the maintenance of high levels of estrogens on the morning of estrus. As a result the keratinization of the vaginal epithelium and the estrus phase are prolonged by 2 or 3 days.     

Actions of acetyl-L-carnitine on the hypothalamo-pituitary-gonadal system in female rats.

Acetyl-L-carnitine (ALC) is known to affect several aspects of neuronal activity. To evaluate the neuroendocrine actions of this compound, several endocrinological parameters were followed in ALC-treated and control animals during recovery from dark-induced anestrus. In treated animals, serum luteinizing hormone (LH) and prolactin levels were higher than those of controls during the proestrous and estrous phases of the cycle, and serum estradiol levels were higher during estrus. No significant changes were observed in serum levels of follicle-stimulating hormone and progesterone. Uterine weight was increased in ALC-treated rats during proestrus and estrus, but not in diestrus. The basal release of gonadotropin-releasing hormone (GnRH) from perifused hypothalamic slices of ALC-treated animals was elevated at proestrus and diestrus, and GnRH release elicited by high K+ was higher during all three phases of the cycle. The basal release of LH from perifused pituitaries of treated animals was elevated in diestrus, and the LH response to GnRH was higher in estrus and diestrus I. Depolarization with K+ caused increased LH secretion during proestrus and estrus in treated animals. In contrast to these effects of ALC treatment in vivo, no direct effects of ALC were observed during short- or long-term treatment of cultured pituitary cells. These results indicate that ALC treatment influences hypothalamo-pituitary function in a cycle stage-dependent manner, and increases the secretory activity of gonadotrophs and lactotrophs. Since no effects of ALC on basal and agonist-induced secretory responses of gonadotrophs were observed in vitro, it is probable that its effects on gonadotropin release are related to enhancement of GnRH neuronal function in the hypothalamus.     

Sexual dimorphism in the content of progesterone and estrogen receptors, and their cofactors in the lung of adult rats

Progesterone and estradiol play an important role in the regulation of lung functions such as ventilation and vasoconstriction. The genomic actions of progesterone and estradiol are mediated by their nuclear receptors: progesterone receptors (PR) and estrogen receptors (ER). These actions are linked to interactions between steroid receptors and some cofactors that function as coactivators or corepressors. In this work we determined the content of PR isoforms, ER-beta, one coactivator (SRC-1), and one corepressor (SMRT) in the lung of both female rats during the estrous cycle and intact males by Western blot. The rat lung presented a higher content of PR-A than that of PR-B during the estrous cycle. The highest content of both PR isoforms was observed on the day of proestrus whereas the lowest one was found on the day of estrus. In contrast, the content of ER-beta was the lowest on the day of proestrus and it increased at estrus. The content of SRC-1 and SMRT increased on the day of diestrus. SRC-1 content decreased at proestrus and estrus, while SMRT content decreased at proestrus and increased again at estrus. In the lung of adult male rats the content of PR isoforms, ER-beta and SMRT was lower than in that of females, whereas the content of SRC-1 was similar in both sexes. Our results suggest a sexual dimorphism in the content of PR, ER-beta, and SMRT in the rat lung as well as a relation of their content to the physiological levels of progesterone and estradiol.     

Expression of ghrelin in the porcine hypothalamo-pituitary-ovary axis during the estrous cycle

Ghrelin, a 28-amino acid acylated peptide produced mainly by the stomach, has various functions. Recent studies focus on its endocrine and/or paracrine effects in the regulation of the hypothalamo-pituitary-gonadal axis, that is, the role in reproduction. Previous data have shown that variation of ghrelin depended on the phases of estrous cycle in adult rat ovary. This study was to investigate the expression of ghrelin in the cyclic porcine hypothalamo-pituitary-ovary axis and stomach by semiquantitative RT-PCR and immunohistochemical method. Twenty virginal gilts were classified into four groups as the proestrus, estrus, diestrus1 and diestrus2. Results showed that expression of ghrelin mRNA in the hypothalamus changed with the estrous cycle, i.e., with the highest level in the proestrus and the lowest in the estrus. In the pituitary, the pattern of ghrelin mRNA expression during estrous cycle markedly decreased in the estrus and diestrus1. In the ovary, ghrelin mRNA exhibited with the highest level in the diestrus2 and the lowest in the proestrus, which was different from those in the hypothalamus and pituitary. In the stomach, the expression of ghrelin mRNA had the same tendency as that of the porcine ovary. In immunohistochemical experiment, ghrelin immunoreactive cells were predominantly located in the luteal compartment and growing follicles in the luteal phase of ovary. However, only few ghrelin immunoreactive cells were found in the proestrus ovary. In gastric mucosa, ghrelin immunoreactive cells were detected in the estrus, diestrus1 and diestrus2, but few ghrelin positive cells were seen in the proestrus. Results suggest that ghrelin may play a major role in the endocrine network that integrates energy balance and reproduction.     

酪氨酸对大鼠子宫胞浆雌,孕激素受体的影响

采用放射受体分析法,测定了动情周期不同阶段及去卵巢大鼠子宫胞浆雌二醇和孕酮受体含量,并观察了子宫腔内注射酪、丝、苏三种氨基酸对子宫胞浆雌。醇、孕酮受体含量的影响。结果表明：（１）Ｌ－酪氨酸对动情前期、动情期、间情期大鼠子宫胞浆雌二醇和孕酮受体都具有明显的降低作用。（２）Ｌ－酪氨酸也降低去卵巢大鼠子宫胞浆雌二醇和孕酮含量,即这一作用不是通过影响卵巢激素分泌实现的。（３）Ｌ－苏氨酸仅可降低动情期和间情期大鼠子宫胞浆孕酮受体含量,而对相应周期雌二醇受体没有明显作用。（４）Ｌ－丝氨酸和Ｌ－苏氨酸对去卵巢大鼠子宫胞浆雌二醇和孕酮受体均无影响。     

The effects of paradoxical sleep deprivation on estrous cycles of the female rats

The present purpose was to examine how sleep deprivation affects the estrous cycle of the female rat. Paradoxical sleep-deprived (PSD) adult female Wistar rats were compared to home-cage control (CTRL) on their estrous cyclicity. Forty-four PSD and forty-four CTRL female rats were distributed into 4 subgroups of 11 animals each according to the phase of estrous cycle and were subjected to sleep deprivation for 96 h by the multiple platform technique. After PSD period, vaginal estrous cycles were taken for an additional 9 days. Animals that were submitted to PSD in diestrus phase (PSD-diestrus) had their estrous cycles disrupted during the recovery period by showing a constant diestrus during the first week. As for hormone alterations, progesterone concentrations were statistically higher in PSD-diestrus compared to respective phase control and to PSD-proestrus and PSD-estrus rats while CTRL-metestrus had higher levels than CTRL-proestrus and estrus groups. Testosterone was significantly decreased in PSD-estrus in relation to PSD-proestrus and PSD-diestrus groups and was lower in CTRL-diestrus rats than in home-cage rats in proestrus. In addition, PSD-diestrus phase exhibited higher concentrations of corticosterone and lower estrogen than the respective control rats. These data indicate that PSD may modulate the ovarian hormone release through alterations in hormonal-neurochemical mechanisms.     

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     

N-terminal prolactin-derived fragments, vasoinhibins, are proapoptoptic and antiproliferative in the anterior pituitary

The anterior pituitary is under a constant cell turnover modulated by gonadal steroids. In the rat, an increase in the rate of apoptosis occurs at proestrus whereas a peak of proliferation takes place at estrus. At proestrus, concomitant with the maximum rate of apoptosis, a peak in circulating levels of prolactin is observed. Prolactin can be cleaved to different N-terminal fragments, vasoinhibins, which are proapoptotic and antiproliferative factors for endothelial cells. It was reported that a 16 kDa vasoinhibin is produced in the rat anterior pituitary by cathepsin D. In the present study we investigated the anterior pituitary production of N-terminal prolactin-derived fragments along the estrous cycle and the involvement of estrogens in this process. In addition, we studied the effects of a recombinant vasoinhibin, 16 kDa prolactin, on anterior pituitary apoptosis and proliferation. We observed by Western Blot that N-terminal prolactin-derived fragments production in the anterior pituitary was higher at proestrus with respect to diestrus and that the content and release of these prolactin forms from anterior pituitary cells in culture were increased by estradiol. A recombinant preparation of 16 kDa prolactin induced apoptosis (determined by TUNEL assay and flow cytometry) of cultured anterior pituitary cells and lactotropes from ovariectomized rats only in the presence of estradiol, as previously reported for other proapoptotic factors in the anterior pituitary. In addition, 16 kDa prolactin decreased forskolin-induced proliferation (evaluated by BrdU incorporation) of rat total anterior pituitary cells and lactotropes in culture and decreased the proportion of cells in S-phase of the cell cycle (determined by flow cytometry). In conclusion, our study indicates that the anterior pituitary production of 16 kDa prolactin is variable along the estrous cycle and increased by estrogens. The antiproliferative and estradiol-dependent proapoptotic actions of this vasoinhibin may be involved in the control of anterior pituitary cell renewal.     

Effects of thymulin and GnRH on the release of gonadotropins by in vitro pituitary cells obtained from rats in each day of estrous cycle

The effects of thymulin and GnRH on FSH and LH release were studied in suspension cultures of anterior pituitary cells from female adult rats sacrificed on each day of the estrous cycle. The spontaneous release of gonadotropins by pituitaries, as well as their response to GnRH or thymulin addition, fluctuated during the estrous cycle. Adding thymulin to pituitary cells from rats in diestrus 1 increased the concentration of FSH; while in cells from rats in estrus, FSH level decreased. Thymulin had a stimulatory effect on the basal concentration of LH during most days of the estrous cycle. Adding GnRH increased FSH release in cells from rats in diestrus 1, diestrus 2, or proestrus, and resulted in higher LH levels in cells obtained from rats in all days of the estrous cycle. Compared to the GnRH treatment, the simultaneous addition of thymulin and GnRH to cells from rats in diestrus 1, diestrus 2, or proestrus resulted in lower FSH concentrations. Similar results were observed in the LH release by cells from rats in diestrus 1, while in cells from rats in proestrus or estrus, LH concentrations increased. A directly proportional relation between progesterone serum levels and the effects of thymulin on FSH release was observed. These data suggest that thymulin plays a dual role in the release of gonadotropins, and that its effects depend on the hormonal status of the donor's pituitary.     

Regulation of the bacterial microflora of the vagina in cyclic female rats.

The bacterial microflora was examined in the vagina of cyclic female rats kept under normal laboratory conditions. Large variations occurred during the cycle with high numbers of bacteria (10(5)-10(8) per vagina) during proestrus and estrus and low numbers (10(1)-10(4) per vagina) during the diestrus period. Histological analysis of in situ vaginal tissue and transplanted vaginal tissue revealed an association of high bacterial numbers with the presence of large amounts of cellular debris in the vaginal lumen during the period of epithelial keratinization. Absence of phagocytosis in leucocytes at mestestrus suggested that leucocytes did not play an active role in reduction of bacterial numbers between estrus and metestrus. Accurate measurement of the pH in the vaginal lumen failed to reveal differences which could explain the reduction in bacterial numbers between estrus and metestrus. The cyclic changes in the bacterial population-consisting of species which are normally present in the intestinal flora-- seem to be controlled by cyclic changes in the amounts of cellular debris in the vaginal lumen.