Thyroid follicles with acid mucins in man: A second kind of follicles?

Summary Human thyroid follicles containing acid mucins have been regarded as a very rare finding and their significance has not yet been clarified. Therefore, a systematic anatomical, histochemical and immunohistochemical survey for the presence of such follicles in human thyroids was undertaken.Follicles with Alcian blue-positive acid mucins were practically confined to the 18% of sections that also contained ultimobranchial solid cell nests. Immunohistochemistry revealed that these follicles were mostly composed of and/or related to the presence of numerous calcitonin-immunoreactive cells, sometimes intermixed with occasional alcianophilic mucinous cells. These findings, with histometrical studies, demonstrate that there exists a relationship between mucinous C cell complexes and mucin/C cell-containing solid cell nests. The finding of calcitonin immunoreactivity in very occasional groups of cells with mucinous changes further suggests that at least some human follicular cells originate in ultimobranchial tissue.     

Müllerian inhibitory substance induces growth of rat preantral ovarian follicles

Müllerian inhibitory substance (MIS), also known as anti-Müllerian hormone, is best known as the hormone that regulates the regression of the Müllerian duct in males. In females, MIS is expressed in granulosa cells of preantral and early antral follicles. The specific MIS type II receptor is present in granulosa and theca cells of these small, growing follicles. Because the role of MIS in preantral follicle development is unknown, we have evaluated the effect of MIS on the growth, differentiation, and apoptosis of intact preantral follicles in a serum-free culture system. In this system, treatment with FSH induces an increase in both follicle diameter, cell number, and follicle cell differentiation based on increased inhibin-alpha synthesis. Of interest, treatment with MIS enhances the effect of FSH both on follicle diameter and cell number. Although treatment with activin A also enhances FSH effects on follicle growth, treatment with transforming growth factor (TGF)-ss inhibits the FSH effects on follicle growth. Based on in situ staining of fragmented DNA, MIS was found to have no effect on follicle cell apoptosis, unlike its proapoptotic action on Müllerian ducts. In contrast to MIS and activin, TGF-ss was a potent proapoptotic factor for preantral follicles in culture. Analysis of inhibin-alpha expression of cultured preantral follicles further indicated that in contrast to activin, treatment with MIS did not enhance FSH-stimulated follicle differentiation. Thus, MIS is a unique factor that promotes preantral follicle growth but not preantral follicle cell differentiation and apoptosis.     

Isolation of preantral follicles from nondomestic cats—viability and ultrastructural investigations

Large scale isolation of small preantral follicles (40–90 μm) from nondomestic cats (lion, puma, cheetah, jaguar, and three kinds of tigers) is described and compared with domestic cats. The viability of preantral follicles was estimated by trypan blue staining of granulosa cells and/or by 5-bromo-2′-deoxy-uridine (BrdU) incorporation into oocytes and granulosa cells during short term culture. Native and isolated preantral follicles were compared ultrastructurally. In nondomestic cats the mechanical dissection of ovaries provided 0–12 500 follicles per ovary with a viability of 20–50%, estimated by trypan blue staining. Even the follicles recovered from domestic cats, whose ovaries are considerable smaller than ovaries from all other felids, are characterised by only 28.7% viable follicles. The follicles from one Siberian tiger and three Indian lions were cultivated and their in vitro viability assessed by BrdU labelling. Lion follicles were comparable to domestic cat follicles with respect to BrdU incorporation. Tiger follicles were characterised by a decreased staining of granulosa cells.The ultrastructure of feline oocytes appears similar to that of most mammalian species and was only slightly affected during the isolation procedure. A central vesicular body was only observed in tiger oocytes.     

Cutaneous application of α-methylspermidine activates the growth of resting hair follicles in mice

Recent studies using transgenic animals have revealed a crucial role for polyamines in the development and the growth of skin and hair follicles. In mammals, the growth of hair is characterized by three main cyclic phases of transformation, including a rapid growth phase (anagen), an apoptosis-driven regression phase (catagen) and a relatively quiescent resting phase (telogen). The polyamine pool during the anagen phase is higher than in telogen and catagen phases. In this study, we used α-methylspermidine, a metabolically stable polyamine analog, to artificially elevate the polyamine pool during telogen. This manipulation was sufficient to induce hair growth in telogen phase mice after 2 weeks of daily topical application. The application site was characterized by typical features of anagen, such as pigmentation, growing hair follicles, proliferation of follicular keratinocytes and upregulation of β-catenin. The analog penetrated the protective epidermal layer of the skin and could be detected in dermis. The natural polyamines were partially replaced by the analog in the application site. However, the combined pool of natural spermidine and α-methylspermidine exceeded the physiological spermidine pool in telogen phase skin. These results highlight the role of polyamines in hair cycle regulation and show that it is possible to control the process of hair growth using physiologically stable polyamine analogs.     

Differential response of epithelial stem cell populations in hair follicles to TGF-β signaling

Epidermal stem cells residing in different locations in the skin continuously self-renew and differentiate into distinct cell lineages to maintain skin homeostasis during postnatal life. Murine epidermal stem cells located at the bulge region are responsible for replenishing the hair lineage, while the stem cells at the isthmus regenerate interfollicular epidermis and sebaceous glands. In vitro cell culture and in vivo animal studies have implicated TGF-β signaling in the maintenance of epidermal and hair cycle homeostasis. Here, we employed a triple transgenic animal model that utilizes a combined Cre/loxP and rtTA/TRE system to allow inducible and reversible inhibition of TGF-β signaling in hair follicle lineages and suprabasal layer of the epidermis. Using this animal model, we have analyzed the role of TGF-β signaling in distinct phases of the hair cycle. Transient abrogation of TGF-β signaling does not prevent catagen progression; however, it induces aberrant proliferation and differentiation of isthmus stem cells to epidermis and sebocyte lineages and a blockade in anagen re-entry as well as results in an incomplete hair shaft development. Moreover, ablation of TGF-β signaling during anagen leads to increased apoptosis in the secondary hair germ and bulb matrix cells. Blocking of TGF-β signaling in bulge stem cell cultures abolishes their colony-forming ability, suggesting that TGF-β signaling is required for the maintenance of bulge stem cells. At the molecular level, inhibition of TGF-β signaling results in a decrease in both Lrig1-expressing isthmus stem cells and Lrg5-expressing bulge stem cells, which may account for the phenotypes seen in our animal model. These data strongly suggest that TGF-β signaling plays an important role in regulating the proliferation, differentiation, and apoptosis of distinct epithelial stem cell populations in hair follicles.     

Amounts of an estrogen receptor β isoform increased in the theca of preovulatory follicles of sheep

Determination of the specific roles of the estrogen receptor (ER) forms in reproductive processes of different species remains incomplete. In the present experiment, cellular localization and changes in relative amounts of the ERα and ERβ in late developing ovarian follicles, oviduct, and uterus were determined during the follicular phase of the estrous cycle in sheep. Ewes in mid-luteal phase were treated with prostaglandin F(2α) (PG) to induce luteolysis and control the onset of the follicular phase. The oviducts, uterus, and the ovaries were collected at 0 (ewes not treated with PG), 4, 18, and 36 h after PG treatment (early, mid, and late follicular phase, respectively) and processed to evaluate the ERs using immunohistochemical (IHC) procedures. The ERα was localized to nuclei of granulosa cells of late developing follicles and most cells of the oviduct and uterus. The ERβ was detected only in ovarian follicles using two antibodies directed to different regions of the ERβ. Western immunoblotting demonstrated that the antibody directed against the N-terminal region of the ERβ detected one isoform (approximately 53 kDa) whereas the antibody directed against the C-terminus detected two ERβ isoforms (approximately 53 kDa and 59 kDa). Western and IHC results combined indicated presence of the 59 kDa ERβ in granulosa cells and the 53 kDa ERβ in both granulosa and theca cells. Relative amounts (immunostaining intensity) of the ERα increased (P<.05) in granulosa cells of preovulatory follicles and in the isthmian muscularis of the oviduct at the late follicular phase. Amounts of the ERα in the mucosal epithelium of the oviductal regions (isthmus, ampulla, and infundibulum), and in various uterine cell types (glandular and luminal epithelia, endometrial stromal cells, and myometrium) did not change (P>.05) throughout the follicular phase. A major increase (four-fold) in expression of the 53 kDa ERβ in the theca and a less pronounced increase in the granulosa occurred at the late follicular phase. The ERα is broadly expressed in reproductive organs of sheep and is upregulated only in few cell types during the late follicular phase. Immunoreactive ERβ was detected only in the ovary. Important estrogen actions in theca cells during preovulatory follicular development likely occur in association with a major increase in expression of an ERβ isoform.     

The growth of follicles in the rat ovary under the influence of Busulphan and Endoxan®

Summary Busulphan and Endoxan^® inhibit the normal course of ovarian follicular growth. Older secondary as well as all antral follicles perish within a very short time.Growing follicles, which at the beginning of the experiment exhibited only one layer, remained intact and single-layered during the entire duration of the experiment. The oocytes, however, continue growing, and the cytoplasmic structures, which are characteristic of older growing follicles, develop in them as well as in the follicle cells. Even a theca formation develops.In some of the growing follicles which have remained single-layered, after 10 days of Busulphan administration, some liquor folliculi is produced and accumulates in a fissure-shaped antrum between the zona pellucida and the follicular epithelium.     

Blood flow in the ovaries and ovarian follicles of Romanov and Préalpes-du-Sud ewes

Radioactive microspheres (15 microns diameter) were used to measure capillary blood flow rates in the ovaries and ovarian follicles (Qf) in high fecund Romanov and low fecund Préalpes-du-Sud ewes at the preovulatory stage of the oestrous cycle. Additionally, assessments of the percentage of arterial blood passing through ovarian arterio-venous anastomoses were obtained. The mean +/- s.e.m. Qf per unit volume of theca [ml/min) x 10(4)/mm3) for non-atretic follicles in Romanov ewes was significantly greater (P less than 0.05) than that in Préalpes ewes (365.8 +/- 42.4, n = 19, compared with 241.3 +/- 30.1, n = 14). For each breed, the mean Qf value for non-atretic follicles was 8-10 times greater than that for atretic follicles. In Romanov ewes, total Qf [ml/min) x 10(4) and Qf per unit volume of theca was greatest in small-sized follicles (3.1-5.0 mm) while in Préalpes ewes, maximum flow was attained in larger-sized follicles (5.1-7.0 mm). The elevated Qf in small-sized follicles in Romanov ewes may be conducive to more follicles achieving maturation at a smaller diameter in this breed than occurs in the Préalpes ewes. The absence of flow through ovarian arterio-venous anastomoses in the Romanov, but not in the Préalpes, ewes suggests different mechanisms for controlling the distribution of the total ovarian blood supply in the 2 breeds.     

Immunocytochemical study of anti-Müllerian hormone in sheep ovarian follicles during fetal and post-natal development

Anti-Müllerian hormone (AMH) was detected in perinatal and postnatal sheep ovaries, using avidin-biotin immunohistochemistry with a monoclonal antibody specific for ruminant AMH. Immunoreactivity was limited to granulosa cells, and was influenced both by the degree of follicular development, and by the age of the animal. In the fetus, only the most advanced follicles exhibited a faint immunoreactivity at 120 days gestation, and no reaction was observed in younger animals. Immediately before and after birth, primordial follicles were still negative, but a faint reaction was elicited in young growing follicles, increasing with follicle size. Strong immunoreactivity was visible in antral follicles, especially in the innermost granulosa cell layers, close to the oocyte and lining the antral cavity.     

Unilateral ablation of follicles ≥ 4 mm leads to compensatory follicle response from the contralateral ovary in heifers

In each of two experiments, heifers were assigned to a control group and a unilaterally ablated (UA) group (n = 6/group). In the UA group, follicles ≥ 4 mm in the left ovary were ablated by transvaginal ultrasound-guided technique at Hour 0 (8:00 AM) on the day of ovulation. Follicles in the CL-bearing right ovary remained intact. In Experiment 1, ablations continued until the next ovulation, and new follicles emerged in the right ovary in 9 of 14 (64%) waves. The number of follicles/wave (combined, 6.4 ± 0.4) did not differ between groups. In Experiment 2, follicles were counted at Hours 0, 4, 8, 12, and 24; the resistance index (RI) for blood flow in the ovarian pedicle was determined at Hours 0 and 12; and blood samples were collected every hour from Hours 0 to 12 and at Hour 24. An increase (P < 0.05) in the number of follicles in the follicle-intact ovary began at Hour 4 with complete compensation by Hour 24. Concentrations of FSH did not change between Hours 0 and 24 in the UA group but decreased (P < 0.05) in the controls by Hour 7. At Hour 12, RI to the right ovary approached being lower (P < 0.06) in the UA group than in the control group. Results indicated that unilateral ablation of follicles ≥ 4 mm led to compensatory follicle response in the follicle-intact ovary, and initially circulatory FSH concentrations were maintained and blood flow to the follicle-intact ovary increased.     

IFN-γ plays an essential role in the pathogenesis of gastric lymphoid follicles formation caused by Helicobacter suis infection

In this study, we aimed to assess the role of helper T cells in the development of gastric lymphoid follicles induced by Helicobacter suis infection. C57BL/6J mice were orally inoculated with H. suis. Six weeks after infection, gastric lymphoid follicles were observed in the gastric mucosa by hematoxylin and eosin staining, and the number of follicles was increased throughout the infection period. An immunohistological examination showed that the lymphoid follicles were composed of B cells, CD4-positive helper T cells, and dendritic cells (DC). It was also revealed that the mRNA expression level of interferon-γ (IFN-γ) in the gastric mucosa was significantly increased at 12 weeks after infection. No gastric lymphoid follicles were detected in IFN-γ-deficient mice that had been infected with H. suis at 12 weeks after infection, although the development of lymphoid follicles in IL-4-deficient mice infected with H. suis was similar to that seen in the wild-type mice. In conclusion, IFN-γ, a Th1 cytokine, is deeply involved in the pathogenesis of gastric lymphoid follicles induced by H. suis infection, and it is suggested that CD4-positive T cells and DC aid in the expansion of gastric lymphoid follicles.     

Correlated autoradiographic and ion-microscopic study of the role of iodine in the formation of “cold” follicles in young and old mice

Summary The role of iodine in the formation of cold follicles (not labeled on autoradiograms after radioiodine administration) was analysed in ICR female mice during aging and involution of thyroid hyperplasia, by use of light and electron microscopy and by comparing autoradiographic and analytical ion-microscopic images for the same follicle in serial sections. The proportion of cold and partly cold (displaying a patchy or ring labeling pattern on autoradiograms) follicles increased significantly during aging. This increase was more pronounced in old mice fed an iodine-rich diet as compared to mice fed a moderate iodine diet. Similarly, during goiter involution produced by refeeding iodine, the follicular heterogeneity of iodine metabolism was more accentuated with a high dose of iodine, regardless of the age of the mice. The follicular lumina of hot and cold follicles had the same concentration of stable iodine, as shown by analytical ion microscopy, and the cells of both types of follicles formed colloid droplets in response to TSH. Furthermore, when a goitrogenic treatment was induced in aged mice, some cold follicles persisted after 8 days, but all follicles resumed hot after 16 days. By analytical ion microscopy, ¹²⁷iodine was also found inside thyroid cells of old mice, but the cytoplasmic patches of ¹²⁷iodine were not labeled with ¹²⁵iodine. They corresponded to lipofuscin pigments and secondary lysosomes, as observed in serial sections at the electron-microscopic level. This intracellular stable iodine could constitute a slow turnover compartment not used for hormone synthesis.Portions of this work were presented at the 15th and 17th Annual Meetings of the European Thyroid Association (Stockholm 1986; Montpellier 1988). This work was supported in part by the Association de la Recherche sur le Cancer (ARC) and a cooperative programme Communauté Française de Belgique-France     

Correlation of plasma estradiol-17β and progesterone levels with ultrastructure and histochemistry of ovarian follicles in the white-spotted char,Salvelinus leucomaenis

Summary Plasma estradiol-17 and progesterone profiles were correlated with morphological changes in ovarian follicles during the preovulatory and postovulatory periods in the white-spotted char, Salvelinus leucomaenis. Plasma estradiol levels were highest in September, and were followed by a sharp drop in October; they remained very low throughout the postovulatory period. There was a good correlation between plasma estradiol levels and the gonadosomatic index, thus suggesting that estradiol is responsible for the synthesis of vitellogenic proteins. Plasma progesterone levels were very low in August, began to rise in September and reached a peak soon after ovulation; progesterone remained high for several days after ovulation. A preovulatory rise in plasma progesterone levels was recorded, and this is discussed in relation to the induction of oocyte maturation.In the preovulatory follicles, neither granulosa cells nor special thecal cells (ST cells) showed ⁵, 3-hydroxysteroid dehydrogenase (3-HSD) activity. In the young postovulatory follicles, in contrast, the ST cells showed intense 3-HSD activity with extensive agranular endoplasmic reticulum and numerous large mitochondria, while granulosa cells did not show 3-HSD activity. These results strongly suggest that the ST cells are the major sites of progesterone synthesis during the postovulatory period.Nanae Fish Culture Experimental Station Contribution No 14     

The Krüppel-like factor epiprofin is expressed by epithelium of developing teeth, hair follicles, and limb buds and promotes cell proliferation

We identified a cDNA clone for epiprofin, which is preferentially expressed in teeth, by differential hybridization using DNA microarrays from an embryonic day 19.5 mouse molar cDNA library. Sequence analysis revealed that this cDNA encodes a member of the Krüppel-like factor family containing three characteristic C2H2-type zinc finger motifs. The full-length cDNA was obtained by the 5' Cap capture method. Except for its 5'-terminal sequence, the epiprofin mRNA sequence is almost identical to the predicted sequence of Krüppel-like factor 14/Sp6 (specificity protein 6), which was previously identified in expressed sequence tag data bases and GenBank by an Sp1 zinc finger DNA-binding domain search (Scohy, S., Gabant, P., Van Reeth, T., Hertveldt, V., Dreze, P. L., Van Vooren, P., Riviere, M., Szpirer, J., and Szpirer, C. (2000) Genomics 70, 93-101). This sequence difference is due to differences in the assignment of the location of exon 1. In situ hybridization revealed that epiprofin mRNA is expressed by proliferating dental epithelium, differentiated odontoblast, and also hair follicle matrix epithelium. In addition, whole mount in situ hybridization showed transient expression of epiprofin mRNA in cells of the apical ectodermal ridge in developing limbs and the posterior neuropore. Transfection of an epiprofin expression vector revealed that this molecule is localized in the nucleus and promotes cell proliferation. Thus, epiprofin is a highly cell- and tissue-specific nuclear protein expressed primarily by proliferating epithelial cells of teeth, hair follicles, and limbs that may function in the development of these tissues by regulating cell growth.