Morphometric and ultrastructural characterization of Bos indicus preantral follicles

The aim of the present study was to characterize the ultrastructure of zebu cow preantral follicles (PAFs). Ovarian cortex samples were processed for light and transmission electron microscopy. Primordial follicles consisted of an oocyte surrounded by one layer of flattened or flattened-cuboidal granulosa cells. The oocyte contained a large and usually eccentric nucleus. Most organelles were located at the perinuclear ooplasm. Round shaped mitochondria, which contained electron-dense granules, smooth and rough endoplasma reticulum and a Golgi apparatus were also observed. Vesicles and coated pits were often observed in the cortical ooplasm. In primary follicles, the oocyte was surrounded by one layer of cuboidal granulosa cells. Short microvilli were observed on the oolema. Secondary follicles consisted of an oocyte surrounded by a variable number of layers of cuboidal granulosa cells. Small secondary follicles had an ultrastructure very similar to that observed in primary follicles. At this follicular stage, the zona pellucida was beginning to form around the oocyte. In large secondary follicles, the zona pellucida was totally developed around the oocyte. Several granulosa cell projections could be detected that were encroaching into the zona pellucida and protruding towards the oocyte, where gap junctions were observed between oocyte and granulosa cell membranes. Organelles within the oocyte were located at the periphery of the ooplasm, and clusters of cortical granules were observed. Round mitochondria were abundant in all developmental stages. In conclusion, this study described the ultrastructure of zebu cow PAFs, and some unique characteristics could be observed as compared with what has been reported for follicles of Bos taurus cattle.     

Anatomical and scanning electron microscopic characteristics of the oropharyngeal cavity (tongue,palate and laryngeal entrance) in the southern lapwing (Charadriidae: Vanellus chilensis,Molina 1782)

This investigation aimed to determine the morphological characteristics of the tongue, palate and laryngeal entrance of southern lapwing by gross anatomy and scanning electron microscopy. For this purpose, the organs of three birds were used as material. Numerous densely distributed acicular projections were found on the lingual apex. Papillary crest consisting of sharp conical papillae were observed between the body and root of the tongue. Conical papillae of the lateral border of the papillary crest were triangular in form, and other conical papillae of the papillary crest were shorter in form. There were no papillary projections or papillae on the smooth surfaces of the lingual body and radix. On the median part of the palate, larger conical papillae, which were directed caudally, also surrounded entrance of the choanal cleft. The transversal papillary rows of conical papillae were observed between the rostral and caudal parts of the choanal cleft and on the caudal border of the infundibular cleft. The laryngeal entrance was surrounded by smooth mucosa without conical papillae. However, in the caudal border of the glottic fissure, there was a conical papillary row formed by numerous conical papillae. There were no anatomical differences between female and male birds.     

Papillary projections at the lingual margin in the striped dolphin,Stenella coeruleoalba,with Special Reference to Their Development and Regression

Papillary projections along the anterolateral margin of the tongue were observed in fetal and young stages of the striped dolphin, Stenella coeruleoalba. Papillary projections appear in the prenatal period and attain maximum development in the early postnatal period. They almost disappear by weaning. Vestigial eminences remain at the corresponding region, but they completely disappear in the adult. The papillary projections observed differ markedly from the lingual papillae of the general mammalian tongue as they are temporary, localized at the anterolateral margin and large in size. The projections were also present in young individuals of some other dolphins. No taste buds could be seen on the projections in any of the stages of all specimens observed. Such projections may have important mechanical functions during suckling in these mammals.     

Cutaneous and pulmonary aspergillosis in rabbits.

Cutaneous and pulmonary aspergillosis was diagnosed in rabbits. Dichotomous branching, septate hyphae typical of Aspergillus spp were observed in cystic hair follicles. Atypical, short, knobby mycelia with radiating projections were seen in nodular lesions in the lung.     

A diet rich in n-3 polyunsaturated fatty acids reduced prostaglandin biosynthesis, ovulation rate, and litter size in mice

Successful cryopreservation is usually measured in terms of cell survival. However, there may also be more subtle effects within cells that survive. Previous studies on zebrafish have produced evidence of mitochondrial DNA (mtDNA) damage in cryopreserved embryonic blastomeres and, after exposure to cryoprotectants, alterations in mtDNA replication in embryos and decreased mitochondrial membrane potential, mtDNA and ATP production in ovarian follicles. This study shows that the decreased ATP levels previously observed in stage III zebrafish ovarian follicles exposed to ≥3 M methanol persisted in those follicles that subsequently developed to stage IV. However, the decreased mtDNA levels were restored in those follicles. In order to determine whether mitochondrial distribution and/or their transport network was affected by the methanol exposure, immunocytochemistry analysis of tubulin and mitochondrial cytochrome c oxidase I (COX-I) was performed, along with phalloidin staining of polymerized actin. Neat arrangements of all proteins were observed in control follicles, with COX-I and tubulin being colocalized near granulosa cell nuclei, while actin formed hexagonal and/or polygonal structures nearer granulosa cell membranes and projected into the oocyte surface. Exposure to methanol (2 to 4 M) disrupted the COX-I and tubulin arrangements and the hexagonal and/or polygonal actin distribution and actin projections into the oocyte. These effects were still observed in those follicles that developed to stage IV, although the severity was reduced. In summary, the disruption to function and distribution of mitochondria in ovarian follicles exposed to > 2 M methanol may be mediated via disruption of the mitochondrial transport system. Some recovery of this disruption may take place after methanol removal and subsequent follicle maturation.     

Ultrastructure of the ovarian follicles in the placentotrophic Andean lizard of the genus Mabuya (Squamata: Scincidae)

We studied the ultrastructural organization of the ovarian follicles in a placentotrophic Andean lizard of the genus Mabuya. The oocyte of the primary follicle is surrounded by a single layer of follicle cells. During the previtellogenic stages, these cells become stratified and differentiated in three cell types: small, intermediate, and large globoid, non pyriform cells. Fluid‐filled spaces arise among follicular cells in late previtellogenic follicles and provide evidence of cell lysis. In vitellogenic follicles, the follicular cells constitute a monolayered granulosa with large lacunar spaces; the content of their cytoplasm is released to the perivitelline space where the zona pellucida is formed. The oolemma of younger oocytes presents incipient short projections; as the oocyte grows, these projections become organized in a microvillar surface. During vitellogenesis, cannaliculi develop from the base of the microvilli and internalize materials by endocytosis. In the juxtanuclear ooplasm of early previtellogenic follicles, the Balbiani's vitelline body is found as an aggregate of organelles and lipid droplets; this complex of organelles disperses in the ooplasm during oocyte growth. In late previtellogenesis, membranous organelles are especially abundant in the peripheral ooplasm, whereas abundant vesicles and granular material occur in the medullar ooplasm. The ooplasm of vitellogenic follicles shows a peripheral band constituted by abundant membranous organelles and numerous vesicular bodies, some of them with a small lipoprotein core. No organized yolk platelets, like in lecithotrophic reptiles, were observed. Toward the medullary ooplasm, electron‐lucent vesicles become larger in size containing remains of cytoplasmic material in dissolution. The results of this study demonstrate structural similarities between the follicles of this species and other Squamata; however, the ooplasm of the mature oocyte of Mabuya is morphologically similar to the ooplasm of mature oocytes of marsupials, suggesting an interesting evolutionary convergence related to the evolution of placentotrophy and of microlecithal eggs. J. Morphol., 2010. © 2010 Wiley‐Liss, Inc.     

Melanin and dopa-positive cells in the skin of tropical cattle.

Various histochemical and histological techniques were used to study the melanin and dopa-positive cell distribution in the skin of some tropical and temperate breeds of cattle in Nigeria. Melanin pigments were concentrated in the basal and lower spinous layers of the epidermis and in the hair cortex, follicle sheaths and papillae of the various breeds. In the White Fulani and N'Dama breeds, melanin pigments were however found in all layers of the epidermis. Dopa-positive cells (melanocytes) were observed in the epidermis, dermis and hair follicles; the distribution pattern varied among breeds, being copiously disposed in the basal epidermis and papillary dermis in the White Fulani and Muturu and, except in areas of thick epidermal ridges, scanty in the epidermis and dermis of the Friesian and N'Dama. Mast cell distribution pattern in the various breeds was similar to that of the dopa-positive cells. Peroxidase-positive cells were present in the basal epidermis and upper dermis of the Muturu, widespread in the subepidermal layer of the N'Dama and very scanty in the dermis of the White Fulani and Friesian. Acid phosphatase activity was intense in the granular layer of the Muturu and N'Dama breeds and also in the papillary dermis and hair follicles, whereas alkaline phosphatase-positive dendritic cells, and 'clear' cells were also observed in the basal and upper epidermis.     

Vitrification of caprine secondary and early antral follicles as a perspective to preserve fertility function

The present study aimed to evaluate the structure, survival and development of isolated caprine (secondary-SEC and early antral-EANT) follicles, after vitrification in the presence of synthetic polymers and in vitro culture. Additionally, transzonal projections (TZPs) and p450 aromatase enzyme were evaluated. After isolation, SEC and EANT follicles were in vitro cultured for six days or vitrified. After one week, SEC and EANT follicles were warmed and also in vitro cultured for six days. Data revealed that the percentage of morphologically normal follicles was similar between fresh and vitrified follicles in both follicular categories and antrum formation rate was similar between fresh and vitrified SEC follicles. Fluorescence by calcein-AM did not show difference between fresh and vitrified (SEC and EANT) follicles, however, the trypan blue test showed low viability for vitrified follicles. The integrity of TZPs was not affected between fresh and vitrified SEC follicles, however, in vitrified EANT follicles, there were signs of TZPs loss. Regarding steroidogenic function, it was observed a positive staining for p450 aromatase enzyme in fresh and vitrified SEC and EANT follicles. It was concluded that SEC follicles seem to be more resistant to vitrification than EANT follicles, as shown by the trypan blue test and TZPs assay. Future studies may confirm this hypothesis, in order to consolidate the use of SEC and EANT follicles as an alternative to ovary cryopreservation.     

Cx37 and Cx43 Localize to Zona Pellucida in Mouse Ovarian Follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Cx37 and Cx43 localize to zona pellucida in mouse ovarian follicles

In the ovarian follicle, granulosa cells adjacent to the oocyte extend processes through the zona pellucida matrix, and these projections establish gap junctions both with the oocyte and with neighboring transzonal projections. The identity of connexins contributing to gap junctions between transzonal projections has not been extensively studied. Here, we examined the expression pattern of Cx37 and Cx43 in mouse zona pellucida using multiple connexin-specific antibodies. Immunofluorescence staining revealed abundant Cx37 and Cx43 puncta within the zona pellucida of both preantral and antral follicles. Cx37 persisted in the zona pellucida of mature follicles up to 5 h after an ovulatory stimulus whereas Cx43 was reduced in the zona pellucida by 3 h after an ovulatory stimulus. We suggest that in addition to its role in oocyte-granulosa cell communication, Cx37 could enable a distinct communication pathway between those granulosa cells that are in direct contact with the oocyte.     

Experimental study of follicle formation in suppressed parathyroid glands of Mongolian gerbils.

Parathyroid follicle formation was studied in Mongolian gerbils subjected to different concentrations of calcium in vivo and in vitro, using light and electron microscopic methods, including the potassium pyroantimonate technique and x-ray microanalysis for identification of cations. Follicles were frequent at high calcium concentration, but sparse at intermediate and low levels of calcium. Two main types of follicle were differentiated: "degenerative follicles" containing cellular debris and lined by smooth-surfaced epithelium which occasionally showed degenerative changes; and "secretory follicles" characterized by amorphous and granular contents, and an epithelium possessing microvilli and cytoplasmic projections. Amorphous masses were also seen in dilated intercellular spaces and in dilated cisterns of rough endoplasmic reticulum in the follicle epithelium. Calcium-containing precipitates were found in degenerating chief cells, and between degenerating cells and follicles. Parathyroid follicles are believed to be formed by degeneration of suppressed chief cells (degenerative follicles), and by secretion of hormonal and/or other substances into dilated intercellular spaces which progressively increase in size to form follicular cavities (secretory follicles), thereby possibly reducing the level of metabolically active parathyroid hormone. Functional suppression is believed to underlie the development of parathyroid follicles.     

Studies on the morphology of the isolated perfused rabbit ovary

Summary Ovulation was induced in rabbits by intravenous administration of human chorionic gonadotrophin (HCG), and 4–5 h later the ovaries were isolated and introduced into an in-vitro perfusion system containing synthetic medium with albumin. Rupture of follicles occurred in vitro within the physiological time range (mean 11.3 h after injection of HCG), although with a reduced frequency. Preovulatory and ruptured follicles were studied in detail by light and electron microscopy.In the granulosa layer of ruptured or preovulatory follicles cytoplasmic blebbing activity, disappearance of CallExner bodies and differentiation toward luteinized cells were found. Perhaps the most important sign of normal preovulatory development in vitro was that the basement membrane surrounding the granulosa layer was penetrated by projections of granulosa cells. In the absence of this penetration phenomenon the granulosa layer prolapsed out of the follicle. Immediately before rupture, follicles showed marked degeneration, restricted to the outer layers of the apical wall, which is compatible with the hypothesis that degradative enzymes are released close to the surface of preovulatory follicles.Although the majority of follicles that ovulated under in-vitro conditions showed the same kind of morphological alterations as can be seen in vivo, occasional atypical ruptures occurred without any overt signs during perfusion. Also technical manipulations of the perfusion system, e.g., nonphysiological increase of perfusion pressure, could force follicles to rupture. This illustrates the importance of careful morphological study of all ovaries perfused in vitro before conclusions are drawn.     

Distinction between colloid nodules and follicular neoplasms of the thyroid. Further observations on cell blocks

The differentiation between colloid nodules and follicular neoplasms may be difficult in lesions yielding only microfollicles by fine needle aspiration (FNA). In a retrospective study of 35 follicular lesions, the FNA smears and cell blocks and the excised specimens were reviewed for possible distinguishing features. Columnar cells lining follicles and prominent sinusoidal stroma were seen only in follicular neoplasms; these were more easily appreciated in the cell blocks. Nuclear features and fibrosis were not helpful because they were found in both groups of lesions. Attention to the nuclear morphology was important, however, for the diagnosis of the follicular variant of papillary carcinoma. A repeat FNA might be helpful in obtaining hyperplastic papillae and fragments of dilated follicles for a diagnosis of colloid nodule in half of the cases. A few lesions with predominant microfollicular patterns were even problematic to diagnose on the excised specimens.     

Development and cell phenotypes in primary follicles of foetal sheep lymph nodes

Lymph nodes from sheep foetuses and postnatal lambs were examined to determine the participation of different leucocyte populations in primary follicle formation, with special emphasis on the emergence and subsequent development of follicular dendritic cells during late gestation and early postnatal life. A series of immune and enzyme histochemical markers was used. The first 5′-nucleotidase-positive primary follicles were found at 80 days gestational age (gestation in sheep is 150 days) in superficial cervical lymph nodes. In the last month of gestation the primary follicles possessed follicular dendritic cells, macrophages, dendritic cells, and CD5-positive lymphocytes, in addition to IgM-positive cells. Follicular dendritic cells in primary follicles were found to be ultrastructurally immature. These follicular dendritic cells were characterised by a few, coarse surface projections and many ribosomes attached to the endoplasmic reticulum. A final differentiation to mature follicular dendritic cells was coincident with the postnatal germinal centre reaction. Computer-assisted morphometric analysis demonstrated that the size of 5′-nucleotidase-positive primary follicles in the distal jejunal lymph node, but not in the superficial cervical lymph node, increased significantly during late gestation. It was concluded that stromal cells in primary follicles of foetal sheep lymph nodes were a continuously developing population but that ultrastructural maturity was only achieved in the germinal centres of postnatal lambs.     

The fine structure of the follicular cells in growing and atretic ovarian follicles of the domestic goose

Summary The structure of follicular layer of growing and atretic follicles in the ovary of the domestic goose, was studied by electron microscopy. In small follicles, the wall is lined with a narrow layer of tightly packed small, cuboidal cells separated from the thecal tissue by the basal lamina. During growth, they transform into tall, columnar cells arranged in a single row. The cells display several peculiar ultrastructural features. First, annulate lamellae are commonly observed. Second, cytoplasmic dense-cored granules accumulate in close association with fenestrated cisternae and networks of tubuli derived from the RER. They consist of spheres and strands of amorphous substance of unknown origin. Third, the cells contain many transosomes, a unique organelle of the avian follicle cell consisting of a dense plaque associated with ribosome-like particles. The mature forms of transosomes are located at the tips of lateral and apical cell projections, while bodies thought to be their precursors, are found in the apical cytoplasm. In follicles larger than 8 mm in diameter, most of the transosomes and their precursors have disappeared. Follicular atresia occurs in all of the size-classes of follicles investigated. A loss of transosomes (in follicles up to 8 mm in diameter) and an accumulation of lipid droplets are the first atretic events detectable by electron microscopy. Morphologic features, including deep nuclear indentations, accumulation of lipid droplets frequently encireled by membrane whorls, dilation and disintegration of RER cisterns, swelling of mitochondria and accumulation of dense irregular masses of unknown origin in the cytoplasm, are taken as evidence for advanced degradation. We conclude that necrosis is the dominant type of cell death of the follicular cells during atresia. However, a small fraction of cells, characterized by dark condensed cytoplasm, seems to die by apoptosis.     

The structure of the spiny mouse (Acomys cahirinus) ovary during development

The study presents the structure of the ovaries of the spiny mouse (Acomys cahirinus) during the first months of life. The ovaries in neonate females exhibit a large number of primordial and primary follicles, sometimes clustered in nests. The growing follicles were also observed within the ovary at that period. The first, early antral follicles appeared in the ovary during the second week of life. In the group of 60-day old females, the structure of the ovaries was characterized by a significant increase in the connective tissue elements. Moreover, ovarian follicles at various stages of development were observed, except for the antral ones with cumulus oophorus. The first mature follicles were identified in 3-month old females. In the ovarian follicles, apoptosis occurs at all stages of follicle development, especially in the early antral follicles. In the atretic follicles, apoptotic cells were identified in the layer of granulosa cells.     

Follicular variant of papillary thyroid carcinoma – a cytological study

The cytological diagnosis of classical papillary carcinoma is easily established based on the characteristic architectural and nuclear features. However, the follicular variant of papillary thyroid carcinoma(FVPTC) poses a diagnostic challenge. In this study we analysed the cytological features of 14 histopathologically proven cases of FVPTC. We inferred that a combination of architectural features such as follicles and syncytial clusters and nuclear features, viz grooves, pseudoinclusions and enlarged nuclei with fine chromatin, were helpful in establishing the diagnosis. It is hence suggested that based on the combination of the aforesaid features a diagnosis of FVPTC be offered whenever it is possible. This helps in patient management, obviating the need for a second surgical intervention.     

Cytologic findings in the follicular variant of papillary carcinoma of the thyroid.

Fine needle aspirates from 8 thyroid papillary carcinomas, follicular variant, were studied. Histologically the tumors were composed exclusively of follicles (four cases) or an admixture of follicular and trabecular structures (four cases). Follicles were identified in smears from seven cases (87.5%) and sheets reminiscent of a trabecular pattern in one (12.5%). Colloid material presented as intraluminal (one case) or extraluminal, dense, round masses (six cases). Nuclear cytoplasmic inclusions occurred in seven (87.5%) of the cases. All these features have been described for follicular lesions or neoplasia together with occasional nuclear grooves, as occurred in two (25%) of the studied cases. In the other six (75%) cases, a moderate to high proportion of neoplastic cells with nuclear grooves facilitated the diagnosis of papillary carcinoma. Our findings suggest that a careful microscopic search for nuclear grooves should be attempted in aspirates yielding a diagnosis of follicular neoplasia that could otherwise be indistinguishable from the follicular variant of papillary carcinoma of the thyroid.     

In vitro effect of leptin on steroids' secretion by FSH- and LH-treated porcine small, medium and large preovulatory follicles

The role of exogenous leptin in the follicular steroidogenesis in pigs has not been fully elucidated and available data are controversial. In the current study porcine follicles were recovered from ovaries during early, middle, and preovulatory stages of the follicular phase of the estrous cycle. Follicles were cultured in the presence of the recombinant ovine leptin (oLEP) with or without LH (100 ng/ml) or FSH (100 ng/ml). Medium estradiol (E(2)), testosterone (T) and progesterone (P(4)) concentrations were determined after 48h of culture. Leptin at a dose of 2 ng/ml had no effect on basal E(2) and T secretion by small and medium follicles but decreased E(2) secretion by large follicles. Significant synergistic action of FSH and leptin resulting in a 2 - 5 fold stimulation of E(2) secretion by small and medium follicles was observed. The aromatase inhibitor, CGS 16949A augmented T secretion and inhibited E(2) secretion by control and FSH-treated medium follicles. In FSH and leptin-treated follicles, the inhibitory action of CGS 16949A on E(2) secretion was observed. However, there was no augmentation of T secretion. In leptin-treated follicles the stimulatory action on P(4) secretion was observed only during the preovulatory stage. In these follicles, significant synergistic action of leptin with LH on P(4) secretion was also noted. These results indicate that there is a maturation-dependent action of leptin on both E(2) and P(4) secretion. They also suggest a synergistic action of leptin and FSH on E(2) secretion by small and medium follicles as well as leptin and LH on P(4) secretion by large follicles in pigs.     

Age-related alterations in follicular development and hormonal profiles in rats with 4-day estrous cycles

Two experiments were conducted to examine the hypothesis that an alteration in follicular development is associated with advancing maternal age in the absence of prolonged estrous cycles. In Experiment 1, serum and four follicles (from one ovary per rat) were collected from young and middle-aged, 4-day cycling rats on estrus or metestrus. Number and diameter of nonatretic antral follicles greater than 200 microns in diameter were determined from serial sections of the other ovary from each rat. In Experiment 2, serum and follicles (12 +/- 2) from both ovaries were collected from young and middle-aged rats on each day of a 4-day estrous cycle. All microdissected follicles were assayed for estradiol-17 beta (E2) and all sera were assayed for E2, follicle-stimulating hormone, and luteinizing hormone by radioimmunoassay. Numbers of follicles greater than 400 microns in diameter did not differ, while numbers of follicles 200-400 microns in diameter were reduced in middle-aged rats compared to young rats (Experiment 1). The mean diameter of follicles greater than 400 microns in diameter and the follicular content of E2 was greater in middle-aged than in young rats. In Experiment 2, a greater proportion of large follicles were observed in middle-aged rats than in young rats on all days, and a greater proportion of follicles with high concentrations of E2 were observed on diestrus. We interpreted these data as indicative of an early age-related change in the control of follicular recruitment, growth, and maturation.