Surface Ultrastructure of Ultimobranchial Remnants in the Thyroid Gland of Buffalo (Bos bubalis)

The remnant of ultimobranchial (UB) body in the thyroid gland of adult buffalo was studied. This remnant appeared in the form of irregular and elongated follicles of various size and shape with folded lumen. These structures occupied a peripheral location beneath the capsule, as well as being embedded mostly in the vascular connective tissue of the thyroid. Calcitonin cells were predominantly localized in the UB follicles. Scanning electron microscopy revealed that surface of most UB follicular cells was almost flat and polyhedral or hexagonal in shape. Other follicles possessed cells with dome shaped apical surface demarcated by a shallow intercellular depression. The luminal surface of the follicular cell lining was studded with microvilli that appeared with various density and length and were numerous at the cellular borders. Interestingly, most of UB follicular cells were provided with single cilium, projecting over the cell surface. Notably some UB follicles presented various stages of apocrine activities.     

Perineuronal Nets in the Central Nervous System

The remnant of ultimobranchial (UB) body in the thyroid gland of adult buffalo was studied. This remnant appeared in the form of irregular and elongated follicles of various size and shape with folded lumen. These structures occupied a peripheral location beneath the capsule, as well as being embedded mostly in the vascular connective tissue of the thyroid. Calcitonin cells were predominantly localized in the UB follicles. Scanning electron microscopy revealed that surface of most UB follicular cells was almost flat and polyhedral or hexagonal in shape. Other follicles possessed cells with dome shaped apical surface demarcated by a shallow intercellular depression. The luminal surface of the follicular cell lining was studded with microvilli that appeared with various density and length and were numerous at the cellular borders. Interestingly, most of UB follicular cells were provided with single cilium, projecting over the cell surface. Notably some UB follicles presented various stages of apocrine activities.     

Ultrastructure of the thyroid gland of the one-humped camel (Camelus dromedarius)

Follicular epithelium of the thyroid gland of the one-humped camel was examined by light and electron microscopy. It consisted of a single type of epithelial cell which varied from flattened to columnar in shape. Follicular epithelial cells were characterized by the presence of markedly dilated cisternae of rough-surfaced endoplasmic reticulum, well developed Golgi apparatus, abundant small vesicles (150 nm to 200 nm in diameter) in the apical cytoplasm, and electron-dense colloid droplets measuring from 250 nm to 1600 nm. Follicular epithelial cells frequently showed apocrine secretion into colloidal lumens. Apocrine protrusions with smooth surface were dome-like or balloon-like structures and contained a fine granular matrix. These findings indicate that the morphological features of the follicular epithelial cells of the thyroid gland of the camel are essentially similar to those of mammals except for the presence of apocrine secretion, which is unique to the camel.     

Microscopic changes in intercaruncular endometrium and ovarian follicles in cows 35 days after parturition]

Surface epithelium of intercaruncular endometrium in pluriparous cows, crossbreds of Bohemian Pied cattle with Holstein-Friesian cattle, was not coherent until day 15 after parturition, with a variable height of cells (16-64 microns). Oedematous nature of lamina propria was subsiding gradually. From day 15 to day 20 after parturition the cell height of coherent surface epithelium and uterine glands became equable and stabilized (16-32 microns), and the lamina propria assumed its cellular nature with marked infiltration of polymorphonuclears and lymphocytes. Noticeable symptoms of atresia were demonstrated to occur more frequently in ovaries in the follicular population until day 15 after parturition. From day 10 to day 25 after parturition an increasing occurrence of luteinized follicles with diameters of maximally 3 mm was demonstrated.     

Ultrastructural features of the follicular wall in developing follicles of the sexually immature ostrich (Struthio camelus)

The ultrastructure of the follicular wall in primordial, previtellogenic and vitellogenic follicles of the sexually immature ostrich is described in the present study. The follicular wall consists of a zona radiata, granulosa cell layer, basal lamina and thecal layer. Cytoplasmic processes from the plasma membranes of the granulosa cell layer and the ovocyte form the zona radiata in previtellogenic and vitellogenic follicles. The granulosa cell layer transforms from simple cuboidal epithelium in primordial follicles to simple columnar or pseudostratified columnar epithelium in previtellogenic and vitellogenic follicles. Transosomes were observed along the apical and lateral plasma membranes of granulosa cells. The thecal layer in previtellogenic and vitellogenic follicles consists of interna and externa components. The fibroblasts in the theca externa contain microfilaments, which are thought to be actin filaments. The study revealed ultrastructural features, which are associated with the transportation of yolk precursors and nutrients into the ovoplasm. In addition, the study indicates that, although the cells in the theca externa contain microfilaments, they do not possess the ultrastructural characteristics of smooth muscle cells.     

A case of canine cutaneous clear cell adnexal carcinoma with prominent expression of smooth muscle actin

Cutaneous clear cell adnexal carcinoma was found in the right lip of a 14-year-old male castrated Shih Tzu. Histologically, the tumor mostly consisted of neoplastic cells with clear or vacuolated cytoplasms and contained frequent tubular structures. Neoplastic cells showed coexpression of pan-cytokeratin (CK) and vimentin by double-labeled immunofluorescence staining. In addition, immunohistochemistry revealed that the tumor cells were positive for pan-CK (AE1/AE3, KL1, CAM 5.2), CK-7, CK-8, CK-14, CK-15, CK-18, vimentin and alpha-smooth muscle actin (SMA) with varied intensity and positivity. Among these marker proteins, SMA was positive in 75% of the tumor cells. On the other hand, CK-15, which is a specific marker of follicular stem cells, was expressed in less than 1% of the tumor cells. Based on these findings, the tumor showed diverse differentiation in apocrine sweat glands and the inner and outer root sheaths of hair follicles, indicating the follicular stem cell to be the origin of this tumor.     

Quantitative Histochemical Study of Perineural Nets in the Retrosplenial Cortex of Albino Rats

Thyrocalcitonin cells (C cells) were predominantly demonstrated in the ultimobranchial (UB) structures of the thyroid gland of one-humped camels. The UB population was observed as clear cells, either singly distributed or as groups of cells between the component cellular elements lining the UB structures. They displayed various shapes: oval, pear-like, rounded, cubical, columnar and or ellipsoid. Those populating the cell nests or solid cellular masses were almost polygonal or ovoid in shape. The cytoplasm contained well-developed golgi complexes, several mitochondria and characteristically presented rounded or oval membrane-bound electron dense granules of different sizes. The latter granules were irregularly distributed within the cytoplasm especially at the vascular pole. The nucleus of C cells was relatively large, indented, occupying a more or less central position in the cytoplasm. It presented various chromatin contents; the heterochromatin variety was predominantly demonstrated at the marginal zone. However, the euchromatin occupied a more central position within the nucleus.     

A morphological and immunohistochemical study of healthy and atretic follicles in the ovary of the sexually immature ostrich (Struthio camelus)

The morphology of healthy and atretic follicles in the ovary of the sexually immature ostrich was described in the present study. In addition, the distribution of the intermediate filaments desmin, vimentin and smooth muscle actin, in these ovarian follicles, was demonstrated. Healthy and atretic primordial, pre-vitellogenic and vitellogenic follicles were present in the ovaries of the sexually immature ostrich. Atresia occurred during all stages of follicular development. Atretic primordial and pre-vitellogenic follicles were characterized by the presence of a shrunken oocyte surrounded by a multilayered granulosa cell layer. Two forms of atresia (types 1 and 2) were identified in vitellogenic follicles. In the advanced stages of type 1 atresia the follicle was dominated by a hyalinized mass. In contrast, in type 2 atresia the granulosa and theca interna cells differentiated into interstitial gland cells. Positive immunostaining for desmin was observed in the granulosa cells of only healthy primordial and pre-vitellogenic follicles. Atretic primordial and pre-vitellogenic follicles were immunonegative for desmin. Vimentin immunoreactivity was demonstrated in the granulosa cells of all follicles except the vitellogenic atretic follicles. The results of the present study indicate that ovarian follicles in the sexually immature ostrich undergo a cycle of growth and regression, which is similar to that reported in other avian species. Furthermore, based on the results of the immunohistochemical study, it would appear that the distribution and immunostaining of intermediate filaments changes during follicular development and atresia.     

Comparison of surface diameters and dissected diameters of bovine ovarian follicles

Comparisons were made between diameters of 54 bovine follicles greater than 5.9 mm from 32 pairs of ovaries measured on the ovarian surface and diameters of the same follicles subsequently dissected from the ovaries. Seventy-eight percent of follicles measured on the ovarian surface were within 1.9 mm of the size measured after dissection. The remaining 22% of follicles measured on the surface had diameters recorded that were 2.0 to 3.9 mm different than their diameter after dissection. Surface diameter tended to underestimate dissected diameter for small follicles (less than 8.0 mm) and to overestimate dissected diameter for large (greater than or equal to 12.0 mm) follicles. The correlation coefficient between surface and dissected follicular diameters was .83. We conclude that measuring the diameter of the largest follicles on the ovarian surface and after dissection yield approximately equivalent results.     

Morphology of thyroid follicular cells of methimazole-treated rats

Thyroids of rats treated with methimazole (MMI) were analysed histologically, immunohistochemically and electron microscopically. Thyroids of MMI-treated rats displayed greater hypertrophy and were heavier than those of control rats. Thyroids of MMI-treated rats were composed of variant or small follicles and contained more columnar cells than those of control rats. Strong staining of thyroid granules by azocarmine G was evident in the apical regions of follicular cells from MMI-treated rats. Anti-thyroglobulin positivity was detected in the apical regions of follicular epithelial cells, especially those of MMI-treated rats. After application of a post-embedding method to detect thyroglobulin, colloidal gold particles were often observed in apical vesicles of MMI-treated rats. Our study suggests that apical vesicles may serve as markers of hyperthyroidism. When the functional state of the thyroid gland is known from histological analysis, use of the Azan stain could provide useful additional information.     

Relationship between apoptosis and proliferation in granulosa and theca cells of cystic follicles in sows

To investigate the causes of the occurrence and persistence of porcine cystic follicles, we evaluated the apoptosis and proliferation of follicular cells in these cysts. Apoptotic frequencies were examined by TUNEL assay and the expression of apoptosis regulators (XIAP, bax, bc1-2 and caspase-3) by immunohistochemistry, Western blotting and real-time quantitative PCR; cell proliferation activity was evaluated by PCNA immunohistochemistry and proliferation of in vitro cultured granulosa and theca cells. The low apoptotic frequency and weak proliferative activity were found in cystic follicles. Low frequency of apoptosis might be associated with decreased amounts of apoptotic-related factors (bax and caspase-3) and increased amounts of anti-apoptotic factors (XIAP and bcl-2) in cystic follicles. Significantly lower proliferation activity was detected in granulosa and theca cells from cystic follicles, and lesser PCNA-positive cells were found in cystic follicles. Our results indicate that the programmed cell death and cell proliferation system were altered in cystic follicles. The disorder between apoptosis and proliferation was responsible for maintaining a static condition without degeneration, which leads to the long-term persistence of follicles. These findings provide important novel insights into the pathogenesis of follicular cysts in sows.     

Expression of nitric oxide synthase-3 in porcine oocytes obtained at different follicular development

The present study was designed to determine the localization of nitric oxide synthase-3 (NOS-3) in porcine follicles during follicular development. A 130-kDa NOS-3 protein was found with greater frequency much in the oocytes than in the cumulus cells, as revealed by Western blotting analysis. The content of NOS-3 in the oocyte was higher in large follicles (> 7-mm diameter) than in small follicles (< 2-mm). The data by Western blotting showed the same pattern as the observations obtained from the immunohistochemical studies, in which the periphery of the oocyte stained strong positive. The inner surface cell layer of granulosa cells and cumulus cells were positive staining, especially in large antral follicles. In the primordial follicles, NOS-3 was restricted to the cytoplasm of oocytes, and no stained product was observed in the nucleus of oocytes or granulosa cells. A significant synthesis of NO by oocytes was observed in the presence of ionomycin, but not in the absence of ionomycin, indicating that oocyte NOS-3 functions in response to transient elevations in the intracellular calcium level. We concluded that NOS-3 is expressed in the oocyte from the primordial follicular stage to antral follicular stage, and that it is functional at least in the antral follicles.     

Scanning electron microscopic features of the ovine interdigital sinus

This article describes the scanning electron microscopic (SEM) features of the ovine interdigital sinus. The lumen was filled with a dense secretory material and quite a number of hairs embedded in the luminal content. For SEM purposes, the sinus was divided into three parts: base, body and neck. At the cut surface, the wall exhibited significant folds which were almost absent in the base, the very short blind end of the sinus. The wall had three layers: epidermis, dermis and fibrous capsule. Stratified epithelium with a prominent keratin layer faced the lumen. The inner surface was similar to the skin surface; however, it was coarser due to folds. The fibrous capsule was composed mainly of dense connective tissue, constituting the outermost layer of the wall. The dermis contained common skin structures including sebaceous glands, hair follicles, arrector pili muscles and apocrine glands. Sebaceous glands appeared as groups of bubbles if they were not collapsed. Apocrine glands generally appeared as a group of coiled tubules. They frequently exhibited apocrine blebs, which is a feature of apocrine secretion. SEM was able to locate some secretory vesicles in the lumen of apocrine tubules which is frequently filled by secretory content. Thus, the apocrine tubules exhibited classical features of apocrine secretion.     

Ultramicroscopical Study on Thyrocalcitonin Cells in the Camel (Camelus dromedarius)

Thyrocalcitonin cells (C cells) were predominantly demonstrated in the ultimobranchial (UB) structures of the thyroid gland of one‐humped camels. The UB population was observed as clear cells, either singly distributed or as groups of cells between the component cellular elements lining the UB structures. They displayed various shapes: oval, pear‐like, rounded, cubical, columnar and or ellipsoid. Those populating the cell nests or solid cellular masses were almost polygonal or ovoid in shape. The cytoplasm contained well‐developed golgi complexes, several mitochondria and characteristically presented rounded or oval membrane‐bound electron dense granules of different sizes. The latter granules were irregularly distributed within the cytoplasm especially at the vascular pole. The nucleus of C cells was relatively large, indented, occupying a more or less central position in the cytoplasm. It presented various chromatin contents; the heterochromatin variety was predominantly demonstrated at the marginal zone. However, the euchromatin occupied a more central position within the nucleus.     

Distribution of Cytochrome P450-side Chain Cleavage in the Theca Interna Layers of Bovine Small Antral and Cystic Follicles

Cystic follicle is anovulatory follicular structure that is caused by an endocrine imbalance. The activity of cytochrome P450‐side chain cleavage (P450scc) is essential for the initiation of steroidogenesis in the follicle. The present study was designed to compare the frequency of cells containing P450scc between healthy and atretic small antral follicles, and among several types (I, II and III, classified based on the presence of granulosa layer) of cystic follicles. Paraffin sections of healthy (2–5 mm in diameter), atretic (2–5 mm) and cystic follicles (>25 mm) were immunohistochemically stained with rabbit polyclonal antibody to bovine P450scc. The P450scc‐positive cells were counted in four different regions of the follicles from the apical to the basal side. In small antral follicles and cystic follicles, P450scc‐positive cells were localized in the theca interna layers but not granulosa layers. The P450scc‐positive cell populations decreased in the late atretic follicles compared with the early and advanced atretic follicles at all the regions of follicle. Type III cystic follicles showed significantly lower frequencies of P450scc‐positive cells than those in the types I and II cystic follicles. These results suggest that in both small and cystic follicles in cows, total loss of granulosa cells may be associated with the reduction of frequency of P450scc‐positive cells in theca interna layer.     

Oestrogen receptor evaluation in Pomeranian dogs with hair cycle arrest (alopecia X) on melatonin supplementation

The role of oestrogen receptors in dogs with hair cycle arrest (alopecia X) was investigated by immunohistochemistry. The purpose of this study was to determine if hair regrowth in dogs with hair cycle arrest treated with melatonin was associated with a decrease in follicular oestrogen receptors. Fifteen Pomeranians (excluding intact females) with hair cycle arrest were enrolled. Two biopsies were obtained from alopecic areas of the trunk before and after 3 months on melatonin. Haematoxylin and eosin-stained tissues were examined and oestrogen receptor-alpha was demonstrated immunohistochemically. Common histopathological findings included hyperkeratosis, follicular keratosis, excessive tricholemmal keratinization (flame follicles), thin epidermis, few small anagen bulbs, epidermal pigmentation and melanin aggregates within follicular keratin. Melanin aggregates within basal cells and hair were an occasional finding. After 3 months, 40% (six) dogs had mild to moderate hair regrowth. Biopsies from six dogs showed histological evidence of an increase in anagen hairs and eight dogs had a decrease in epidermal pigmentation. Moderate to marked staining intensity of oestrogen receptor-alpha was noted in all sebaceous gland basal cells, all small hair bulbs and follicular epithelium of telogen hairs. There was no oestrogen receptor-alpha staining of nuclei within the epidermis, apocrine glands or dermal fibroblasts. Large anagen hair bulbs had minimal to no oestrogen receptor staining. Hair regrowth was not associated with a change in oestrogen receptor-alpha staining.     

Gene expression and immunolocalization of fibroblast growth factor 2 in the ovary and its effect on the in vitro culture of caprine preantral ovarian follicles

This study quantified Fibroblast growth factor 2 (FGF-2) mRNA and localized FGF-2 protein in different categories of follicles isolated from goat ovaries. In addition, we verified the effects of this factor on the in vitro culture of preantral follicles isolated from goats. For mRNA quantification, we performed real-time PCR using primordial, primary and secondary follicles, as well as cumulus-oocyte complexes (COCs) and mural granulosa and theca cells of small and large antral follicles. For FGF-2 protein localization, the ovaries were subjected to conventional immunohistochemical procedures. Preantral follicles were isolated and cultured in vitro for 12 days in either control (basic) or supplemented with FGF-2 medium. The expression of FGF-2 mRNA was detected in all categories of follicles and there was no difference in preantral follicles and COCs or granulosa/theca cells from small and large antral follicles. However, in large antral follicles, COCs showed expression levels significantly lower than in granulosa/theca cells (p < 0.05). We observed moderate expression of FGF-2 protein in preantral follicles but not in granulosa cells of primordial follicles and theca cells of secondary follicles. In both small and large antral follicles, strong, moderate and weak staining was observed in oocytes, granulosa and theca cells, respectively. The addition of FGF-2 caused a significant increase in the daily follicular growth rate compared to the control group. We conclude that FGF-2 mRNA is expressed throughout follicular development and that its protein can be found in different patterns in preantral and antral follicles. Furthermore, FGF-2 increases the follicular growth rate in vitro.     

Distribution of immunoreactive von Willebrand factor in the microvascular network of bovine cystic follicles

The present study was undertaken to examine whether the von Willebrand factor (vWF) expression in the follicular microvasculature in the cystic follicles differs from that in the atretic follicles. Paraffin sections of healthy, atretic and cystic follicles were immunostained with rabbit monoclonal antibody to vWF. The vWF-positive cells were counted in four different regions of a follicle from the apical to the basal side. In all types of follicles, immunoreactions for vWF were observed in the endothelial cells of capillaries as well as veins and arteries in the theca interna and externa. In the theca interna, vWF-positive areas were significantly lower in the Type A and B cystic follicles compared to advanced and late atretic follicles. In the theca externa, the vWF-positive blood vessels and vWF-positive area were significantly smaller in all types of cystic follicles than in the healthy or atretic follicles. From these results, it is suggested that in the cystic follicles the induction of vWF in the follicular microvasculature system is reduced, which may suppress the degeneration of vascular system. Continuation of stability in vasculature may be one of the factors that delays the tissue regression in the cystic follicles, and also contributes to the accumulation of follicular fluid that originates from the serum.     

A morphological study of the thyroid gland in Risso's Dolphin,Grampus griseus

To accumulate histological information of cetaceans and basic information about metabolic systems of marine mammals, the thyroid gland of Risso's dolphins was examined by gross anatomical and light and electron microscopic observations. Gross anatomically, right and left lobes of the thyroid were not clearly discriminated, and no isthmus was observed. By light microscopy, irregular or oval follicular lumens were seen, and surrounded by follicular epithelial cells. By electron microscopy, the rough endoplasmic reticulum (rER) was seen adjacently to mitochondria at the basal and lateral regions of the follicular epithelial cells. RERs at the basal side of the cells sometimes contained flocculent material with the same electron density as the follicular lumen component. Microvilli were poorly developed at the apical surface of the cells. In the apical regions of the cells, there were typical Golgi complexes, multivesicular bodies, and granules with various size and electron density. The parafollicular cells were recognized among the follicular epithelial cells and in the interstitial regions but never protruded into the follicular lumen. These cells were present singly and/or formed clusters among the follicular epithelial cells, and often located adjacent to capillaries. They were obviously discriminated from follicular epithelial cells by higher electron density of their granules. In their cytoplasm, well-developed rERs, primary lysosomes, secondary lysosomes, multivesicular bodies, and phagosomes were recognized.