S-100-immunoreactive nerves in the guinea-pig uterus with reference to ultrastructural correlations: Effects of chemical sympathectomy and pregnancy

Summary In the guinea-pig uterus, by use of an indirect immunofluorescence method, S-100 immunoreactivity was found to be restricted to nerves that corresponded in number, distribution and type to adrenergic axons and preterminals. With advancing pregnancy S-100 immunoreactivity completely disappeared in uterine tissue adjacent to a fetus, in parallel with an ultrastructural degeneration of the adrenergic innervation. In the cervix and the uterine horn devoid of a fetus, however, the number and distribution of S-100-immunoreactive nerves was seemingly unchanged and no ultrastructural changes were found in adrenergic nerves. In contrast, chemical sympathectomy produced by 6-hydroxydopamine did not change S-100 immunoreactivity of uterine nerves. These findings suggest that there are differences in the denervation effected by chemical and by pregnancy-induced sympathectomy. The latter probably represents a special type of adrenergic denervation by inducing a degeneration of Schwann cells in addition to destruction of neuronal structures. This may explain the differences in the speed of regeneration of uterine adrenergic nerves following the two types of denervation.     

Endometrial biopsy: a valuable clinical and research tool in bovine reproduction

Studies of postpartum endometrial physiologic and immune mechanisms in cows are compromised by the difficulty in acquiring tissue of suitable quality and in sufficient quantity (Bos taurus). Endometrial biopsy sampling has attracted concern regarding potential animal ill-health and perturbed subsequent fertility. Here, we describe a method of endometrial biopsy that obtains high-quality tissue samples and does not compromise fertility. Using a Hauptner instrument, endometrial biopsies were taken at 15, 30, and 60 d postpartum from 13 mixed-breed beef cows. The effects of repeat biopsy on health (heart rate, respiration rate, color of mucous membranes, rectal temperature), onset of estrous cyclicity, and first service conception rate were monitored. Extensive daily clinical examinations revealed no signs of ill-health. All cows had resumed estrous cyclicity at 60 d postpartum. A conception rate of 77% was achieved after estrus synchronization and artificial insemination. Each biopsy yielded intact endometrial tissue and nucleic acid suitable for extensive histologic and molecular analysis, respectively. We conclude that when carried out appropriately, bovine endometrial biopsy is a safe and reliable technique for assessing postpartum uterine function or health.     

Trophoblastic invasion and the development of uteroplacental arteries in the macaque: immunohistochemical localization of cytokeratins,desmin, type IV collagen,laminin, and fibronectin

The processes by which trophoblast cells invade and modify the walls of the uteroplacental arteries of macaques during the course of gestation were examined. Antibodies to cytokeratins were employed to identify trophoblast, anti-desmin antibody to identify smooth muscle, and antibodies to type IV collagen, laminin, and fibronectin to examine changes in extracellular matrix distribution in the arterial wall. During early gestation, endovascular trophoblast adhered to the arterial wall, often in an asymmetrical distribution. As trophoblast cells moved outwardly into the tunica media, the basement membrane underlying the endothelium was lost, as indicated by gaps in the layer when stained for type IV collagen and laminin. Trophoblast cells became sequestered in the vessel wall where they hypertrophied and became surrounded by a capsule containing type IV collagen and laminin. As the trophoblast cells became established in the vessel wall, the muscular layer of the artery became discontinuous. Throughout gestation it was common for trophoblast cells to invade the vessel intimal layer and share the lining of the artery with typical endothelial cells. This general disposition of endovascular and intramural trophoblast persisted into late gestation. In addition, and contrary to the results of earlier studies of macaques, we identified trophoblastic invasion and modification of myometrial segments of the uteroplacental arteries in later gestation. We also found evidence of interstitial trophoblast cells among the stromal cells of the endometrium, especially during early gestation.     

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]。     

Remodeling of uterine innervation

This minireview reports current hypotheses concerning the remodeling of sympathetic innervation in rodent and human uterus during the estrous cycle and gestation. Neural modulation in this organ is related to sexual hormone concentrations, and a reduction in nerve density is observed when estrogen levels are high during the estrous cycle. Estrogen receptor alpha is considered to be the major receptor mediating the action of estrogen. In the uterus, the expression of neurotrophins, such as nerve growth factor, which are involved in the survival and growth of nerve fibers, changes in response to steroid levels. Despite much research, further studies are necessary to clarify various aspects of nerve growth control under diverse physiological conditions. These studies could be of importance, since alterations of the biological mechanisms of uterus innervation may play significant roles in various pathologies, such as infertility and spontaneous abortion.     

不同固定液和染色方法显示山羊子宫内肥大细胞效果的比较

目的探讨用不同固定液和染色方法对显示处于间情期山羊子宫肥大细胞的影响。方法用四种不同的固定方法,应用改良甲苯胺蓝（MTB）染色法和阿尔辛蓝-番红花红（AB-S）染色法显示处于间情期山羊子宫肥大细胞。结果山羊子宫组织采用Carnoy氏液固定,MTB和AB-S染色对所有的肥大细胞均可获得良好的染色反应,但10％中性福尔马林,4％多聚甲醛,Bouin氏液固定的组织仅有少量肥大细胞着染。结论MTB和AB-S染色法均是山羊子宫肥大细胞良好的染色方法。     

谷胱甘肽-硫-转移酶M2基因在小鼠生殖器官中的表达

利用半定量RT-PCR和原位杂交的方法检测Gstm2基因在成年雄性和雌性小鼠生殖器官中的表达,并初步评价其在生殖过程中的作用。在雄性小鼠的睾丸、附睾、输精管和雌性小鼠的卵巢、输卵管、子宫、胎盘中,半定量RT-PCR的方法均检测到Gstm2的表达,在胎盘中表达水平较低,其余组织表达水平较高。利用原位杂交的方法在睾丸的间质细胞检测出较强的信号,在附睾中有微弱的信号,而输精管上皮细胞没有检测到信号;在输卵管上皮细胞和妊娠第3d的子宫上皮细胞中检出较强的信号。由于Gstm2在RNA水平在小鼠的生殖器官中广泛表达,因此我们推测Gstm2可能在小鼠精子发生、睾酮合成、精子的成熟和运输、卵子的发生和运输、胚胎着床等生殖过程中发挥作用,此结果为深入研究Gstm2在生殖生理中的功能打下基础。     

Estradiol receptor binding to the epithelium of uterine lumen and glands: region- and time-related changes during preimplantation and periimplantation periods studied by autoradiography

The presence and changes of estradiol nuclear binding and related functions in uterine luminal and glandular epithelium were studied before and after blastocyst implantation using receptor autoradiography with ³H-estradiol-17 in association with ³H-thymidine incorporation and immunocytochemical binding of antibody to estrogen receptor ER-. ³H-estradiol nuclear binding is present but variable during days 1.5–7.5 of pregnancy. Sites of strong nuclear binding of ³H-estradiol exhibit strong immunocytochemical staining with ER- antibody. Qualitative and quantitative evaluation of autoradiograms reveal that there is a general increase of nuclear ³H-estradiol binding during the first 3 days after fertilization in both luminal and glandular epithelium. The binding of estradiol is stronger in glandular epithelium from day 2.5 to day 7.5, paralleled by a rise in ³H-thymidine incorporation on day 2.5. By comparison, in the epithelium of the uterine lumen ³H-estradiol nuclear binding is low, but relatively high in epithelial cells at lateral branching of the lumen where the increase in ³H-estradiol binding corresponds to an increased labeling index with ³H-thymidine. A highly differentiated binding of ³H-estradiol to luminal and glandular epithelium was demonstrated with region- and time-specific changes of related effects on cell proliferation, differentiation, and secretion, probably involving involution and remodeling. The strong ³H-estradiol binding to glandular epithelium suggests that estradiol exerts pronounced effects on glandular activities in the periimplantation period.     

Unique sensitivity to alpha-galactosylceramide of NKT cells in the uterus

It was previously reported that NKT cells, which are mainly present in the liver of mice, are also present in the uterus and that these uterine NKT cells are associated with abortion under overactivated conditions. In this study, we further examined their phenotypic and functional properties. In parallel with the progression of pregnancy, the number of uterine lymphocytes increased. This increase accompanied an increase in the number of TCRalphabeta(+) T cells and NKT cells in the uterus, although the number of NKT cells decreased at late pregnancy. Approximately one-third of the TCRalphabeta(+) cells were NKT cells at the early pregnant stage, while this ratio decreased toward late pregnancy. These uterine NKT cells were found to respond to alpha-galactosylceramide (alpha-GalCer) differently in comparison with liver NKT cells. In contrast to the apoptotic response of liver NKT cells on day 1 after alpha-GalCer injection, uterine NKT cells expanded prominently without such apoptosis. The majority of liver NKT cells were CD4(+). In contrast, almost all of the uterine NKT cells were double negative CD4(-)8(-). However, similar to liver NKT cells, uterine NKT cells used an invariant chain of Valpha14Jalpha281 gene for TCRalpha. The resistance against apoptosis might be due to the high expression of bcl-2 on uterine NKT cells after alpha-GalCer activation. Other evidence was that macrophages which existed in the pregnant uterus carried an activation marker, CD69, and could potentially produce many cytokines by their activation. The present results suggest that uterine NKT cells and surrounding macrophages are quite different in function from those in the liver.     

Plasticity in developing rat uterine sensory nerves: the role of NGF and TrkA

In the present study we investigated the effects of infantile/prepubertal chronic oestrogen treatment, chemical sympathectomy with guanethidine and combined sympathectomy and chronic oestrogen treatment on developing sensory nerves of the rat uterus. Changes in sensory innervation were assessed quantitatively on uterine cryostat tissue sections stained for calcitonin gene-related peptide (CGRP). Uterine levels of NGF protein, using immunohistochemistry and ELISA, and mRNA, using Northern blots and in situ hybridization, were also measured. Finally, levels of TrkA NGF receptor in sensory neurons of T13 and L1 dorsal root ganglia (DRG), which supply the uterus, were assessed using densitometric immunohistochemistry. These studies showed that: (1) chronic oestrogen treatment led to an 83% reduction in the intercept density of CGRP-immunoreactive nerves; (2) sympathectomy had no effect on the density of uterine sensory nerves or on the pattern of oestrogen-induced changes; (3) NGF mRNA and protein increased following sympathectomy or chronic oestrogen treatment; and (4) oestrogen produced increased intensity of labelling (28%) for TrkA receptors in small-diameter sensory neurons, but decreased labelling (13%) in medium-sized neurons, which represent the large majority of the DRG neurons supplying the upper part of the uterine horn. Contrary to expectations, increased levels of NGF after sympathectomy and oestrogen treatment did not lead to increased sensory innervation of the uterus. The possibility that alterations in neuronal levels of TrkA contribute to the lack of response of uterine sensory nerves to the oestrogen-induced increase in NGF levels is discussed.This work was supported by The Wellcome Trust, UK (CRIG Grant 058122/Z/99/Z/JC/KO), and PEDECIBA, Universidad de la República, Montevideo, Uruguay