Impact of pituitary FSH purification on <i>in vitro</i> early folliculogenesis in goats

Porcine pituitary follicle stimulating hormone (pFSH) is known to regulate the production of growth factors that have an essential role in early foliculogenesis. However, the effects of different preparations of pFSH on the survival and development of caprine follicles are not yet known. The aim of this study was to evaluate the effects of different pFSH (Stimufol^® and Folltropin^®) on the in vitro survival and growth of caprine preantral follicles. Pieces of caprine ovarian tissues were cultured for either one or seven days in a supplemented Minimum Essential Medium, alone or containing either Stimufol^® (50 ng/mL) or Folltropin^® (10, 50, 100 and 1000 ng/mL). Fresh control ovarian tissues as well as cultured tissued were processed for histological and ultrastructural studies. The results showed that after seven days, only Stimufol^® maintained follicular morphology similar to control. Moreover, follicular degeneration was higher in medium alone or with Folltropin^® at 50, 100 and 1000 ng/mL. However, at day seven, the percentage of growing follicles was higher in 100 ng/mL of Folltropin^® than Stimufol^®. In conclusion, FSH preparations affect differently the performance of in vitro culture of caprine preantral follicles. Stimufol^® was better to preserve follicular morphology while Folltropin^® was more efficient to promote follicular growth.     

Scanning electron microscopic changes in granulosa cells during follicular atresia in Caprine ovary

During this study, topographic changes in healthy and atretic granulosa cells have been investigated during follicular atresia in goat ovary. Under scanning electron microscopy atresia was marked by asymmetrical shrinkage and vacuolization of cytoplasm. The specific topographical alterations observed in atretic cells were loss of micro extensions, disruption of cell-cell interaction, and smooth-textured membrane with a number of uneven depressions and ruffles. Some portions of the cell membrane were marked by extensive shrinkage due to condensation of cytosol. Irregular membrane at occasions was studded with blunt microextensions. The findings of present investigation will help in understanding the cellular changes in granulosa cells during follicular atresia and will find applications in screening of follicles for in vitro culture, in vitro fertilization and Embryo transfer technology.     

Ultrastructural characterization of apoptotic granulosa cells in caprine ovary

The unique phenomenon of cell proliferation and apoptosis is encountered in the ovarian follicles undergoing early stages of atresia. The aim of this study was to verify the morphological variations in these two physiologically distinct processes operating in antral follicles of caprine ovaries using histological and ultrastructural techniques. Histologically the degenerating granulosa cells were characterized by condensed cytoplasm, and nucleus fragmentation in hazy cytosol. The pyknotic nuclei of degenerating cells stained darkly with haematoxylin and giemsa while the cytoplasm was eosinophilic. Under electron microscopy, apoptosis was marked by asymmetrical shrinkage, vacuolization of cytoplasm, swollen and vacuolated mitochondria, increased irregularity and/or fragmentation of nucleus, chromatin condensation and finally, production of membrane enclosed nuclear fragments containing intracellular material, the apoptotic bodies. The parallel use of these two methods on caprine ovaries has enabled us to analyse the decline in the frequency of granulosa cells during follicular atresia due to apoptosis.     

Immune system involvement in the regulation of ovarian function and augmentation of cancer

Increasing evidence indicates a role for the immune system and mesenchymal-epithelial interactions in the regulation of ovarian function. Cytokines produced by mesenchymal cells can stimulate development and regression of ovarian structures. We report here that mesenchymal cells releasing surface molecules among epithelial cells--namely vascular pericytes and monocyte-derived cells (MDC)--and intraepithelial T lymphocytes are associated with oogenesis and formation of new primary follicles in both fetal and adult human ovaries. These activated mesenchymal cells interact with the ovarian surface epithelium, which appears to be a source of secondary germ cells and granulosa cells. Activated pericytes and MDC are also associated with stimulation of thecal development during selection of growing secondary follicles from the cohort of primary follicles. However, survival of the dominant follicle during mid-follicular phase selection is associated with a lack of activity of mesenchymal cells and retardation of thecal development, since immature granulosa cells lacking aromatase are unable to resist high levels of thecal androgens. Once the selected follicle matures (late follicular phase), it shows enhanced activity of thecal mesenchymal cells and advanced thecal development. Corpus luteum (CL) development is accompanied by a high activity of vascular pericytes and MDC. In mature CL and CL of pregnancy, luteal MDC and pericytes show a stable (inactive) state. Regression of the CL is associated with regression of pericytes, transformation of MDC into dendritic cells, infiltration by T lymphocytes, and binding of immunoglobulin G to the luteal cells. The immunoglobulin M (IgM) binds to young but not mature luteal cells. In the CL of pregnancy, IgM binds to luteal vessels, but not to luteal cells. Regressing CL shows IgM binding to both luteal cells and vessels. In ovarian cancers, highly activated MDC and sometimes activated pericytes (poorly differentiated carcinomas) are present. IgM binding is similar to that seen in the CL of pregnancy. These data indicate that vascular pericytes, MDC, T cells, and immunoglobulins may play an important role in the regulation of ovarian physiology and contribute to the augmentation of ovarian cancer growth.     

Lectin-binding pattern of the Senegalese sole Solea senegalensis oogenesis

The glycoconjugate pattern of developing ovarian follicles in wild and cultured Senegalese sole Solea senegalensis was investigated by means of lectin histochemistry. Ovaries from cultured fish contained oocytes up to the late vitellogenic stage, whereas they reached the hydration stage in wild specimens. The follicular cells bound MAL II, SBA, HPA, DBA, Con A, KOH-sialidase (K-s)-WGA, GSA I-B(4) in the late vitellogenic stage, and in wild fish also SNA and K-s-PNA, whereas in the hydration stage SBA, HPA, DBA, and GSA I-B(4) only. The zona radiata reacted with SBA, HPA, DBA, Con A, and GSA I B(4) in the late vitellogenic stage and in cultured fish also with UEA I, whereas in the hydration stage it stained with SBA only. The cortical alveoli bound SBA, HPA, RCA(120) during the late vitellogenic stage, also SNA, PNA, K-s-PNA, GSA I-B(4) in cultured fish, DBA, and K-s-WGA in wild ones which stained with SBA, HPA, and GSA I-B(4) in the hydrated stage. The yolk reacted with Con A in the late vitellogenic oocytes, and also with MAL II, SNA, K-s-PNA, SBA, HPA, K-s-WGA, GSA I-B(4), UEA I in the hydrated ones. From perinucleolus to late vitellogenic stages, the oocyte nucleoplasm bound Con A, GSA I-B(4), GSA II, UEA I, and in wild fish also MAL II, SNA, LTA but only GSA I-B(4) reactivity in the early maturation stage. These findings demonstrate that the glycan pattern of fish ovarian follicles changes during the maturative stages and that it is affected by culture-rearing conditions.     

Germinal vesicle chromatin configurations of bovine oocytes

A common feature in the configuration of germinal vesicle (GV) chromatin in most species is that diffuse chromatin condenses into a perinucleolar ring during follicular growth; however, no such ring was observed in goat oocytes. Reports on whether bovine GV chromatin condenses into a perinucleolar ring are controversial. Besides, it is not known whether the perinucleolar ring in an oocyte represents a step toward final maturation or atresia. Changes in GV chromatin configurations during growth and maturation of bovine oocytes were studied using a new method that allows a clearer visualization of both the nucleolus and the chromatin after Hoechst and chromomycin A(3) staining. On the basis of the degree of condensation and distribution, the GV chromatin of bovine oocytes were classified into five configurations: NSN with diffuse chromatin in the whole nuclear area, N with condensed netlike chromatin, C with clumped chromatin, SN with clumped chromatin surrounding the nucleoli, and F with floccular chromatin near the nucleoli and near the nuclear envelope. Most of the oocytes were at the NSN stage in the <1.4-mm follicles, but the NSN pattern disappeared completely in follicles larger than 1.5mm. The SN pattern began to emerge in 1.5-mm follicles, and the number of SN oocytes increased while the number of oocytes with N and C configurations decreased with follicular growth. During maturation in vivo, while the number of N, C, and SN oocytes decreased, that of the F oocytes increased and reached maximum at 51h post prostaglandin injection. After that, the number of F oocytes decreased significantly because of germinal vesicle breakdown (GVBD). During maturation in vitro, GV chromatin configurations changed in a similar manner as during maturation in vivo. Fewer oocytes were at N, C, and SN stages, but more were at F and GVBD stages in the atretic than in the healthy follicles. Serum starvation slowed the F-GVBD transition of the in vitro maturing oocytes. More oocytes were of the SN or C configuration when ovaries were transported at 45-40 degrees C than at 35-30 degrees C. Most of the heated oocytes were blocked at the SN stage during in vitro maturation. It is concluded that (i) bovine GV chromatin condenses into a perinucleolar ring during follicular growth; (ii) bovine oocytes were synchronized at the F stage before GVBD; (iii) oocyte GV chromatin configurations were affected by serum starvation, high temperature, and follicular atresia.     

Ovarian acetylcholine and muscarinic receptors: hints of a novel intrinsic ovarian regulatory system

More than two decades ago, the degrading enzyme of the neurotransmitter acetylcholine (ACH) was reported in nerve fibers of the rat ovary. Subsequently, it was assumed that ACH is a neurotransmitter of ovarian nerves, although the sole presence of the degrading enzyme, ACH-esterase, does not allow such a conclusion. That ACH may be involved in the complex regulation of ovarian functions, including hormone production, was indicated by studies using, for example, granulosa cells (GCs). The lack of detailed information about both source(s) and functions of ACH in the ovary prompted us to examine sites of ovarian ACH-synthesis and ACH-receptor-bearing target cells. We also started to identify functions of ACH in cultured human GCs. While ovarian innervation and recently described neuron-like cells of the ovary were not immunoreactive for the ACH-synthesizing enzyme, choline-acetyl transferase (CHAT), we found immunoreactivity in GCs of rodents and primates. Isolated human and rat GCs produced ACH and contained the vesicular ACH transporter (VACHT). These results indicate that endocrine GCs are an unexpected non-neuronal source of ACH in the ovary. Moreover, these cells and GCs in vivo contain ACH-receptors of the muscarinic subtype (MR), namely M1R and M5R. In contrast, oocytes express M3R. MR of human GCs are functional and cholinergic stimulation is linked to rapid increases in intracellular Ca(++) levels. M1/5R activation also led to increased cell proliferation of human GCs in vitro and this stimulatory effect was found to be associated with rapid disruption of gap junction communication. Ongoing studies begin to identify regulation of ion channels and altered gene expression as consequences of MR stimulation. Thus, our results outline first details of an unexpected intraovarian, non-neuronal cholinergic system, and suggest that it may be involved in the regulation of cell proliferation in the ovary.     

Cell surface labelling with gold colloid particulates: the use of avidin and staphylococcal protein A-coated gold in conjunction with biotin and fc-bearing ligands

Procedures for preparing gold colloid particles stabilized with either avidin or protein A are described. Methods of using these general utility tracers for localizing biotinylated and fc bearing immunoglobulins are outlined and, as examples of the way in which these methods can be applied, procedures for identifying epidermal growth factor receptors and surface fibronectin on ovarian granulosa cells are described.     

A new method for obtaining scanning electron microscopic images of the reorganization process of human dermal microvascular endothelium in vitro

A new method for obtaining scanning electron microscopic images of the reorganization process of endothelial cells has been developed. When covered with a collagen-coated disk, all the cultured endothelial cells reorganized on the collagen of the disk, which was easily taken out from the dish to process for SEM. The reorganization process could be divided into four stages: endothelial cell growth (Stage 1), reticular network formation (Stage 2), tubular structure formation (Stage 3), and cytolysis of the tube (Stage 4). Between Stages 1 and 2 the endothelial cells transformed from a cobblestone to a spindle-shaped pattern and fused each other, forming a board-like structure. Between Stages 2 and 3 break up of parts of the board-like structure and outflow of a necrotic mass from the centre of the structure occur. At Stage 3 a tubular structure is formed following enwrapping of the cleared centre by the surrounding endothelial cells. This method produces a means to study the angiogenesis in a variety of disorders including tumours and wound-healing process using SEM.     

Ultrastructural correlates of meiotic maturation in mammalian oocytes

Immature mammalian oocytes reside in ovarian follicles with junctionally coupled granulosa cells. When released from a currently undefined meiotic arresting influence, these oocytes resume meiosis to progress from late diplotene (germinal vesicle stage) through the first meiotic division to metaphase II. Oocytes remain at metaphase II until fertilization activates them to complete meiosis. This review summarizes ultrastructural events that occur during meiotic maturation in mammals. Developmental correlates that promise a clearer understanding of regulatory mechanisms operating to control maturation are emphasized. By use of TEM of thin sections, freeze-fracture analysis, and replicated oocyte cortical patches, we demonstrate stage-specific changes in the oocyte nucleus, reorganization of cytoplasmic organelles, correlations between oocyte maturational commitment and the junctional integrity of associated granulosa cells, and definition of the components comprising the oocyte cortical cytoplasm.     

Morphodynamics of ovarian follicles during oogenesis in mice

In the mouse, oogonia enter the prophase of the first meiotic division and differentiate into oocyte while developing in the fetal ovary. Shortly after birth, all oocytes are arrested in the dictyate stage of late prophase in the developing follicles; a small number of follicles reach the ovulatory stage; the rest are lost by apoptosis. The resumption of meiotic division and nuclear progression to metaphase II (oocyte maturation) occur in the ovulatory follicles. In this article we review recent morphological data that have clarified how cytokines and glycosaminoglycans (GAGs) are involved in mouse follicular development, atresia, and maturation during oogenesis, as exogenous/endogenous factors. (1) Microvascular networks and angiogenic factors (epidermal growth factor; GAGs) are deeply involved in selective mouse oocyte growth beyond approximately 20-30 microm in diameter. (2) Gonadotropin-inducible neuronal apoptosis inhibitory protein may indirectly affect oocyte survival as a result of the inhibition of apoptotic granulosa-cell death during folliculogenesis. (3) The pattern of oocyte degeneration depends on follicle and oocyte developmental stages, and follicle stimulating hormone accelerates the process of degeneration of oocytes. (4) The process of degeneration of mouse oocytes/eggs is modulated by tumor necrosis factor-alpha that is accumulated in the expanded cumulus during oocyte maturation. (5) A colloidal iron-positive substance was detected in the intercellular spaces of follicular tissue, especially in the cumulus mass. Cells located where the cumulus mass and granulosa cell layer interwound became enlarged during the resumption of oocyte meiosis. Colloidal iron-positive substances accumulated extensively within the intercellular spaces of the enlarged cells.     

Organ culture of the bovine subcommissural organ: evidence for synthesis and release of the secretory material

The subcommissural organ (SCO) is a brain circumventricular organ formed by ependymal and hypendymal secretory cells. It secretes glycoproteins into the cerebrospinal fluid of the third ventricle where they condense into a thread-like structure known as Reissner's fiber (RF). The present study was designed to investigate whether or not the bovine SCO continues to synthesize and release glycoproteins after a long-term culture. Cultured explants of SCO survive for several months. The content of the secretory granules present in the cultured ependymocytes displayed immunoreactive and lectin-binding properties similar to those of the core glycosylated glycoproteins found in the bovine SCO. The explants actively incorporated (35)S-cysteine. In the cultured ependymocytes, the pattern of distribution of the radioactive label and that of the immunoreactive secretory material was similar, thus indicating that this material has been synthesized during culture. At the ultrastructural level, the cultured tissue exhibited a high degree of differentiation comparable to that of the bovine SCO in situ. A striking finding was the observation of similar results when cerebrospinal fluid was used as a culture medium. The addition of antibodies against RF-glycoproteins into the culture medium allowed visualization, by means of different immunocytochemistry protocols, deposits of extracellular immunoreactive secretory material on the free surface of the cultured ependymocytes, indicating that release of secretory glycoproteins into the culture medium does occur. Primary culture of dispersed SCO ependymocytes, obtained either from fresh or organ cultured bovine SCO, showed that these cells release RF-glycoproteins that aggregate in the vicinity of each cell. The present investigation has shown that: (1) two types of secretory ependymocytes become evident in the cultured SCO; (2) under culture conditions, the SCO cells increase their secretory activity; (3) explants of bovine SCO synthesize RF-glycoproteins and release them to the culture medium; (4) after release these proteins aggregate but do not form a RF; (5) a pulse of anti-RF antibodies into the culture medium blocks the secretion of RF-glycoproteins for several days.     

Neurotrophic control of ovarian development

Substantial evidence now exists indicating that the neurotrophins, a family of growth factors required for the survival, development, and differentiation of various neuronal populations of the nervous system, are also important for the development of nonneuronal tissues. Such a function was first suggested by studies showing the presence of high-affinity neurotrophin receptors in a variety of nonneuronal tissues including those of the cardiovascular, endocrine, immune, and reproductive systems. Within the latter, the gonads appear to be a preferential site of neurotrophin action as suggested by the presence in the mammalian ovary of at least four of the five known neurotrophins and all of the neurotrophin receptors thus far identified. While the various functions that the neurotrophins may have in the ovary are still being elucidated, it is now clear that in addition to recruiting the ovarian innervation, they play a direct role in the regulation of two different maturational periods that are critical for the acquisition of female reproductive function: early follicular development and ovulation. Neurotrophins facilitate the development of newly formed follicles by promoting the initial differentiation and the subsequent growth of primordial follicles. These actions appear to be related to the ability of neurotrophins to sustain the proliferation of both mesenchymal and granulosa cells, and to induce the synthesis of follicle stimulating hormone (FSH) receptors. At the time of the first ovulation, neurotrophins contribute to the ovulatory cascade by increasing prostaglandin E(2) release, reducing gap junction communication, and inducing cell proliferation within the thecal compartment of preovulatory follicles.     

Preovulatory rise of NGF in ovine follicular fluid: possible involvement in the control of oocyte maturation

Since nerve growth factor (NGF) is produced in vitro by granulosa cells after gonadotropin stimulation, the present research has been designed to investigate whether this neurotropin is involved in the events triggered by the gonadotropin surge that lead the follicle to ovulate a mature oocyte. To this aim, NGF levels in follicular fluid, collected before or 20 hours after the gonadotropin surge, was measured by ELISA. To evaluate whether NGF may have a non-neurotropic effect on follicle cells, the presence of NGF receptors was investigated by immunohistochemistry and further evaluated by analysing the tyrosine-phosphorylation pattern after NGF stimulation in vitro. The effect of NGF on the degree of cumulus expansion, cumulus-oocyte metabolic coupling, and meiotic maturation was finally studied by using the culture of follicle-enclosed oocyte. The results demonstrate that GnRH causes a dramatic rise of NGF in large follicles. Immunohistochemistry revealed a discrete positivity for trkA receptors localised in cumulus cells. Tyrosine phosphorylation pattern confirms that somatic cells are capable to transduce NGF signal. By contrast, all the oocytes examined were negative for trkA and did not change the phosphorylation pattern after NGF. In vitro NGF (100 ng/ml) induced a marked cumulus expansion and a progressive cumulus-oocyte uncoupling similar to that produced by gonadotropins. The addition of NGF also caused the resumption of meiosis in more than 70% of the oocytes analysed with an effect that is only slightly less pronounced than that of gonadotropins (80%). The increase in NGF secretion following gonadotropin surge suggests that this neurotropin may be involved in the control of oocyte maturation.     

Structural changes and cell properties of human ovarian surface epithelium in ovarian pathophysiology

The surface epithelial cells of the ovary, which are modified peritoneal cells, form a single, focally pseudostratified layer. The Müllerian ducts differentiate after invagination of the coelomic mesothelium over the gonadal ridges during the 6th week of embryonic life. On the basis of the embryologically putative Müllerian potential of this epithelium, endometriosis can be explained by coelomic metaplasia from the peritoneum, including ovarian surface epithelium. Some pelvic endometriosis specimens have shown that epithelial cells on the ovary or pelvis are serially changed to endometriotic gland cells. Immunohistochemistry as well as scanning electron microscopy also reinforce the light-microscopical findings. A three-dimensional culture system demonstrated that human ovarian surface epithelial cells exhibited a glandular-stromal structure when they were cocultured with endometrial stromal cells in an estrogen-rich environment. Ovarian carcinomas in the epithelial-stromal category are thought to arise from the surface epithelium and its inclusions. The ovarian surface epithelium is physiologically involved in follicular rupture, oocyte release, and the subsequent repair of follicle wall during reproductive age. Simultaneously, ovulation may cause a loss of integrity of the surface epithelium, followed by accumulation of multiple mutations. The cortical invagination, surface stromal proliferation, and Müllerian differentiation of these cells are likely not to be an early step in the cancer development. However, the inclusion cysts are closely related with carcinogenesis because they are significantly more common in ovaries contralateral to those containing epithelial cancers than in control ovaries. As an in vitro study, ovarian carcinoma cell lines were established from simian virus 40 large T antigen-transformed human surface epithelial cells of the ovary. Further investigations of these cell lines may lead to insights into the preneoplastic and early stages of carcinomas. To clarify the pathogenesis of endometriosis and epithelial ovarian cancer, specifically designed studies of ovarian surface epithelium are required.     

Use of microwave fixation in the preparation of cell cultures for observation with the scanning electron microscope

This paper describes a method for primary fixation of cultured cells for scanning (SEM) and transmission (TEM) electron microscopy using microwaves alone. This method of fixation takes 8 seconds and is therefore quicker and less expensive than conventional fixation techniques. The preservation of cell morphology is excellent and cultures of mammalian immune system cells and peripheral nervous tissue have been examined using this fixation.     

Optimization of culture conditions for an efficient xeno‐feeder free limbal cell culture system towards ocular surface regeneration

Ex vivo expansion of limbal stem cells from a small biopsy and its subsequent transplantation is the golden choice of treatment for limbal stem cell deficiency. Use of murine 3T3 feeder layer is a prerequisite for this ex vivo expansion. There is an ever‐increasing demand for feeder free cultures to avoid xenotoxicity and transmission of xeno‐diseases to human system. This study was aimed to establish an efficient xeno‐feeder free limbal culture system towards ocular surface regeneration. To study the effect of initial dispase treatment and culture system used, migratory distance of cells from explants was analyzed from phase contrast images using “interactive measurements” of Qwin software (Leica). Expression of p63 in different culture systems was studied by immunofluorescent staining, followed by quantitative confocal microscopy (Carl Zeiss). Results showed dispase treatment was not necessary for establishing limbal explant culture. A combination of Iscove's modified Dulbecco's medium and Panserin 801 resulted in formation of autofeeder layer with maintenance of progenitor characteristics, thus mimicking natural tissue architecture. Further analysis of this culture system showed that cells could be cultured till confluency. Immunofluorescent staining of ABCG2 revealed presence of stem cell marker in the confluent cell layer. Scanning Electron Micrographs demonstrated homogenous population of tightly packed cells in this culture system. Replacement of bovine serum with autologous serum did not affect morphology or growth of cells in this culture system. This study will be a major step in the development of xeno‐feeder free epithelial equivalents towards ocular surface reconstruction. Microsc. Res. Tech. 73:1045–1052, 2010. © 2010 Wiley‐Liss, Inc.     

The structure and formation of the egg membranes in Nasonia vitripennis (Walker) (Hymenoptera, Pteromalidae)

A histochemical and fine structural study of the vitelline membrane and chorion was carried out. Following the uptake of protein at the oöcyte plasma membrane during yolk synthesis, the egg membranes are laid down in sequence in the intercellular space between the oöcyte and the follicle cells. The follicular epithelial cells alone contribute towards their formation. The structure of the chorion is much simpler than that of Bombus, the only other hymenopteran previously investigated.     

Follicular development and morphological changes in the vaginal epithelium during the estrous cycle of Galea spixii

The current study aimed to determine if characteristics observed in vaginal cytology during the estrous cycle of female SYT cavies corresponded with proliferation of the vaginal epithelium, characterized by proliferating cell nuclear antigen (PCNA) immunolocalization, and with follicular development at different phases of the estrous cycle. After determining estrous cycle phases by vaginal cytology, females were euthanized at metestrus, diestrus, proestrus, and estrus. Histological study of the vaginal epithelium and ovary were then performed. Immunohistochemistry for PCNA in vaginal tissue at each cycle phase was also performed. Superficial cornified cells and early post‐ovulatory follicles were found at estrus. Few nuclei below the enucleate superficial cells were immunoreactive to PCNA. At metestrus, the vaginal epithelium underwent desquamation and lost the superficial cornified cells; basal and intermediate cells appeared, and the post‐ovulatory follicle formed an early corpus luteum. No PCNA immunoreactivity was observed. At diestrus, the corpus luteum was developed, and the vaginal epithelium contained basal and intermediate cells. There was PCNA immunoreactivity in the cellular nucleus in the germinative stratum of the epithelium. Because of the growth and maturation of ovarian follicles, the vaginal epithelium suffered intense proliferation at proestrus. Vaginal cytology revealed large intermediate cells and nucleated and enucleated superficial cornified cells. In the ovary, mature follicles were present. More apparent immunoreactivity of PCNA in the germinative layer was found. In summary, we inferred that vaginal exfoliative findings matched the proliferation process of the vaginal epithelium. PCNA immunolocalization occurred as well as corresponding follicular development in the ovaries.     

Morphological abstraction of thyroid tumor cell nuclei using morphometry with factor analysis

Various morphonuclear studies by digital image analysis have successfully been applied to quantify the nuclear morphology, including chromatin distribution pattern, in cytology of various organs; however, the majority of past reports have not shown correlation between the quantitative data by digital image analysis and cytological findings in practical diagnosis. In this report, we present the usefulness of morphological abstraction to combine the objective data and subjective observation in cytological diagnosis. Randomly selected, 100 cells in each Papanicolaou-stained ABC smear samples of 39 benign and malignant thyroid tumor cases were studied. Gray-level image data provided seven parameters for nuclear size, four parameters for nuclear shape, and 16 parameters showing the nuclear chromatin patterns from high-dimensional texture analysis of using co-occurrence and run-length matrices. To statistically abstract nuclear morphology, factor analysis was used. Factor analysis classified morphological nuclear characters as abstraction parameter into five abstract parameters composed of nuclear size, shape, heterogeneity, and contrast and homogeneity of chromatin pattern. The nuclei of papillary carcinoma showed larger size, more irregular shape, and higher contrast of chromatin pattern than those of the benign group. The follicular carcinomas have larger nucleus in each cell and more monotonous chromatin pattern among cells in each case than those of the benign group. Morphological abstraction by morphometry with factor analysis may provide a practical approach to the detection of the underlying characteristics of nuclear morphology in aspiration biopsy cytology.