ARTICLE |
Correspondence to: Maria V.T. Lobo, Servicio de Neurobiología, Departamento de Investigación, Hospital Ramón y Cajal, Ctra. de Colmenar Km 9, 28034 Madrid, Spain. E-mail: rafael.martin@hrc.es
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Summary |
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The distribution of the amino acid taurine in the female reproductive organs has not been previously analyzed in detail. The aim of this study was to determine taurine localization in the rat ovary, oviduct, and uterus by immunohistochemical methods. Taurine was localized in the ovarian surface epithelium. The granulosa cells and oocytes of primordial follicles were immunonegative. In primary and antral follicles, taurine was found mainly in theca cells and oocytes, whereas the zona pellucida, antrum, and most granulosa cells were unstained. However, taurine immunoreactivity in theca cells and oocytes decreased during follicular atresia. During corpora lutea development, the number of immunopositive theca lutein cells increased as these cells invaded the granulosa-derived region. Therefore, most luteal cells from the mature corpora lutea were stained. In the regressing corpora lutea, however, taurine staining in luteal cells decreased. In the fimbriae, infundibulum, and uterotubal junction, taurine was localized in most epithelial cells. In the ampullar and isthmic segments, taurine was found in the cilia of most ciliated cells and in the apical cytoplasm of some non-ciliated cells. In the uterus, most epithelial cells were immunopositive during diestrus and metestrus, whereas most of them were immunonegative during estrus and proestrus. Moreover, taurine immunoreactivity in the oviduct and uterus decreased with pregnancy. (J Histochem Cytochem 49:11331142, 2001)
Key Words: taurine, ovary, oviduct, uterus, immunohistochemistry
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Introduction |
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Taurine (2-aminoethanesulfonic acid) is a free -amino acid. Because of its zwitterionic nature at physiological pH, it has an extremely low capacity to diffuse across cell membranes (
The roles of taurine in the reproductive system are multiple and complex. Sperm cells contain high amounts of taurine (
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Materials and Methods |
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Adult female SpragueDawley rats (250 g) were used for this study. Rats were maintained on a 14L:10D cycle (lights-on at 0500 hr) with food and water provided ad libitum. Vaginal smears were taken to determine estrous cycle phase. A group of virgin female rats were mated and the first day of gestation was determined by the detection of a vaginal plug. Animals in all phases of the estrous cycle and on Days 9 and 18 of pregnancy were anesthetized with a mixture of ketamine, lidocaine, and atropine and perfused through the ascending aorta with 100 ml sodium phosphate buffer (0.12 M, pH 7.4), followed by 450 ml of one of four fixatives: 4% paraformaldehyde with 0.5%, 1%, or 1.5% glutaraldehyde in 0.12 M phosphate buffer, and 4% paraformaldehyde in the same buffer (control specimens). Tissue samples were removed from the rats and stored for 624 hr in the same fixative used during perfusion, washed in buffer, dehydrated, and embedded in paraffin. Streptavidinperoxidase immunostaining was performed as previously described (
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Results |
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The specific localization of taurine in the ovary (Fig 1 and Fig 2), oviduct (Fig 3), and uterus (Fig 4) of the rat was studied. Similar results were obtained with the use of the different glutaraldehyde-containing fixatives (not shown). No immunostaining was observed in the negative controls (Fig 1k and Fig 3a). In the cerebellar sections (positive controls), many Purkinje cells were stained, whereas glial cells were unstained (Fig 2i).
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Most cells of the ovarian surface epithelium (germinal epithelium) were intensely immunolabeled (Fig 1a and Fig 1g). In this cell type, taurine was localized in the cytoplasm, whereas the nucleus was weakly stained (Fig 1j). However, the collagen fibers and most cells of the tunica albuginea were unstained (Fig 1j). In follicle cells, the pattern of taurine staining varied with the different stages of follicle development. The single layer of granulosa cells and the immature oocytes of primordial follicles were immunonegative (Fig 1b). In contrast, when follicle development has begun and primary follicles contain two or three layers of granulosa cells, the oocytes became immunostained, whereas the granulosa cells remained unstained (Fig 1c). In preantral follicles showing a morphologically distinct theca layer, taurine was found in theca cells and in the oocytes, whereas the zona pellucida and the granulosa cells were poorly stained or unstained (Fig 1d). In antral follicles (Fig 1e and Fig 1f) taurine was detected in the endothelial cells of the capillaries of the theca layer and in most cells of the theca interna. Moreover, the oocyte and some granulosa cells adjacent to the oocyte and the follicular antrum were also stained. However, granulosa cells were less intensely immunolabeled than theca cells and the oocyte. In these antral follicles, the zona pellucida, the follicular antrum, and most of the granulosa cells were not stained (Fig 1e and Fig 1f). When the oocytes were immunolabeled, taurine appeared mainly distributed in the cytoplasm whereas the nucleus was usually weakly stained (Fig 1c and Fig 1e). However, taurine was localized in the nucleus and cytoplasm of theca cells. In contrast to maturing follicles (Fig 1b1f), taurine immunoreactivity progressively disappeared from the oocyte and theca cells during follicular atresia (Fig 1g1i). Atretic follicles were identified by the presence of pyknotic cells in the granulosa layer, cell debris in the follicular antrum, the shrinking oocyte, accumulation of lipid droplets in theca and granulosa cells, and the final appearance of the collapsed follicle with a hypertrophied theca layer. In early atretic follicles (Fig 1g), identified by the presence in serial sections of a few pyknotic granulosa cells, taurine was detected in some cytoplasmic areas of the oocyte and in some theca cells. However, in the late stages of atresia taurine almost disappeared from most follicle cells, including the oocyte (Fig 1h and Fig 1i).
Taurine immunoreactivity varied in relation to the different stages of corpora lutea formation and regression (Fig 1a and Fig 2). In the post-ovulatory period, taurine was detected in most theca lutein cells and in some cells scattered in the granulosa-derived region, whereas most granulosa lutein cells were unstained (Fig 2a). Moreover, the endothelial cells of the luteal vessels (mainly large-diameter capillaries) were also stained. A similar labeling pattern was observed in the early luteal phase (Fig 2b and Fig 2c). During the luteinization process, when theca-derived cells invade the granulosa-derived region, an increase in the number of immunopositive luteal cells was observed. Finally, most cells from the mature corpora lutea were stained (Fig 2d2f). However, in the regressing corpora lutea, taurine immunoreactivity decreased in luteal cells (Fig 2g and Fig 2h). Positive immunostaining for taurine was also found in some polyhedral interstitial endocrine cells of the cortical stroma. In the cortex and medulla, some vascular endothelial cells and fibroblasts were immunolabeled, whereas collagen and elastic fibers were unstained.
The oviduct comprises four morphologically distinct segments: preampulla (which includes both the fimbriae and infundibulum), ampulla, isthmus, and uterotubal junction. The distribution of taurine in these regions was studied in the oviduct of nonpregnant (Fig 3a3n) and pregnant rats (Fig 3o and Fig 3p). In all oviducts examined, taurine was mainly found in the luminal epithelium which contained two main cell types, ciliated and nonciliated cells. The ciliated cells were identified by the presence of kinocilia on the apical cell surface (Fig 3f and Fig 3g). The nonciliated cells presented microvilli extending into the lumen, and the apical surface of these cells was either at the same level as the ciliated surface or protruded into the oviduct lumen (Fig 3g and Fig 3j). In nonpregnant rats, taurine immunoreactivity was higher in the uterotubal junction (Fig 3b) and preampulla (Fig 3k3n) than in the isthmus (Fig 3c3g) and ampulla (Fig 3h3j). In the ampullar and isthmic segments, taurine was found in the apical surface of the epithelium (Fig 3c3j). In ciliated cells, it was mainly found in the cilia, whereas the rest of the cell was usually unstained (Fig 3d3f). Some scattered nonciliated cells were stained in these segments, taurine being found mainly in the apical cell part (Fig 3g and Fig 3j). In the fimbriae (Fig 3k3m), infundibulum (Fig 3k and Fig 3n), and uterotubal junction (Fig 3b), both main cell types of the oviductal epithelium were stained. In nonciliated cells taurine was mainly found in the apical cytoplasm and in the nucleus (Fig 3b and Fig 3l). Ciliated cells showed a variable degree of taurine immunostaining in these segments. In some ciliated cells taurine was mainly restricted to the ciliated surface (Fig 3l), whereas in other cells it was distributed all along the cell, including the ciliated surface (Fig 3m). In the oviduct of pregnant rats, taurine was found only in the fimbrial epithelium, whereas all other segments were weakly stained or unstained (Fig 3o and Fig 3p). In all oviducts examined, only a few scattered cells of the oviductal stroma contained taurine, and the smooth muscle cells showed a variable degree of immunostaining, being weak in some cells and strong in other smooth muscle cells.
No cyclic changes of taurine immunoreactivity were found in the ovary and oviduct. However, dramatic cyclic variations were observed in the epithelial cells of the uterus. Most cells of the luminal and glandular epithelia were immunopositive for taurine during diestrus (Fig 4a4c) and metestrus (Fig 4h), whereas most epithelial cells were immunonegative during proestrus (Fig 4d) and estrus (Fig 4e4g). Moreover, many stromal cells (fibroblasts, vascular endothelial cells, lymphocytes, and macrophages) were stained. No cyclic changes in the stromal staining were observed (Fig 4a4h). The smooth muscle cells of the uterus were usually weakly stained or unstained (Fig 4a). However, moderate staining was found in some muscle cells during estrus (Fig 4i). In the perimetrium, some vascular endothelial cells and fibroblasts were stained (Fig 4i). In addition, most cells in the epithelium, stroma, and myometrium of the gravid uterus were unstained (Fig 3p and Fig 4j).
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Discussion |
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The distribution of taurine in several cell types is highly dynamic and involves variations in the cellular taurine concentration under modifications of the cellular environment or functional state (
It has been demonstrated by RT-PCR that the rat ovary contains the mRNA of a taurine transporter, which has been cloned from human thyroid cells (
In the ovary, theca cells are intensely stained for taurine, whereas granulosa cells are weakly stained or unstained. The primary role of theca cells is to synthesize and to provide an aromatizable androgen substrate (i.e., testosterone, androstenedione) for granulosa cell estrogen biosynthesis. In the testis, taurine is mainly localized in Leydig cells (
The oviduct epithelium consists of two kinds of cell, ciliated and nonciliated (secretory) cells. Ciliated cells play important roles in the transport of gametes, whereas nonciliated cells synthesize and release specific secretory products to the oviduct fluid (reviewed in -amino acid that shows a tight relationship with taurine) is present in the rat oviduct at high concentrations, 5 µmol GABA/g tissue (
Increasing research interest has recently addressed the identification and characterization of the secretory products released by the oviduct epithelium. They have special relevance in relation to the physiological events occurring in the oviduct, such as gamete transport, capacitation, fertilization, and early embryo development (reviewed in
No cyclic changes in the proportions of ciliated and nonciliated cells have been found in the oviduct of the golden hamster and rat. However, in other species, such as the primates and the cow, cyclic variations in the relative number of both cell types have been reported ( and ß) in the female reproductive tract demonstrated that estrogen receptor-
is the predominant subtype in uterus and that its expression changes during the estrous cycle (
This study is the first to demonstrate the distribution of taurine in the female reproductive organs. Although the physiological significance of these results remains to be determined, a growing body of evidence indicates that taurine may have important roles on the reproductive system. Future studies on the taurinergic mechanisms in these organs may lead to a better understanding of the physiology of the reproductive system.
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Acknowledgments |
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We thank Dr Carlos Correa of the Experimental Surgery Service at the Hospital Ramón y Cajal, who carried out examinations of the animals and vaginal smears.
Received for publication November 22, 2000; accepted March 28, 2001.
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