A scanning electron microscopic study of the human ovarian surface epithelium: characterization of two cell types

The surface of the ovary has two types of epithelial cells. We have called these A and B cells and they are found in their own respective zones (A and B). To assess the scanning electron microscopic features of these cell types, 65 ovarian samples were collected from biopsies taken from 35 women with normal ovaries. Biopsies included developing follicles, corpora lutea and ovarian capsules. Type A cells were cuboidal and sometimes tall, with a mean diameter of 6.49 microns, and a mean density of microvilli of 6.48/microns 2. Type B cells, on the other hand, were flat squamous cells with broader and flat apices with mean diameters and microvillus density of 11.71 microns and 3.88/microns 2 respectively. The A and B zones were common to all surfaces including the distending follicle. Type A cells overlying the distended surface of a follicle had a mean diameter of 7.03 microns compared to a mean of 6.05 microns for the capsular surface. Type B cell diameters and the microvillus density of both types were more variable and did not differ significantly over any of the surfaces. We suggest that previous human studies which identified flattening of cells over the distending follicle were probably observing B cells. The relationship of the B zones to papillae and surface bridges on the ovarian surface, and the association of these with ovulation sites, suggests that B cells are probably metaplastic cells derived in response to chronic surface injury with ovulation.     

Isolation,growth and characteristics of human ovarian surface epithelium

The ovarian surface epithelium (OSE) is a key tissue in the pathogenesis of ovarian surface epithelial-stromal tumours and ovarian endometriosis, commonly encountered gynaecological diseases. Despite the high incidence of these diseases, experimental in vitro studies of OSE are few and so we used the scraping method with an enzymatic procedure to isolate human OSE and studied its characteristics in vitro. Nineteen normal ovaries were used. After incubation of the ovary for 40 min in collagenase type 1 solution (300 U/ml), the surface cells were removed by gentle scraping with a surgical blade. Cells obtained as a cluster after unit gravity sedimentation with 5% bovine serum albumin in medium 199 were cultured in medium 199 containing 15% fetal bovine serum. The viable cell number in a single ovary was 0.1–2.7×10⁶. The outgrowth of cells started from a homogeneous population of single cells, and the cell population doubling time was between 7 and 10 days. Confluent monolayers were formed after 13–20 days and subcultured from one to three times. The monolayers mostly had a cobblestone appearance, and fusiform or polygonal cells were also observed. By cytochemistry, immunocytochemistry and scanning and transmission electron microscopy, the cells were shown to have characteristics of mesothelial OSE cells in short-term culture. This experimental approach was efficient in providing cultured human OSE, which can be utilized to investigate pathobiology and carcinogenesis.     

Isolation, growth, and characteristics of human ovarian surface epithelium

To isolate human ovarian surface epithelium (OSE) and studied its characteristicsin vitro, we used the scraping method with an enzymatic procedure. After incubation of the ovary in collagenase type 1 solution, the surface cells were removed by gentle scraping with surgical blade. Cells obtained as a cluster after unit gravity sedimentation with 5% bovine serum albumin in medium 199 were cultured in medium 199 containing 15% fetal bovine serum. The viable cell number in a single ovary was 0.1 to 2.7×10⁶, and the cell population doubling time was between 7 to 10 days. Confluent monolayers were formed after 13–20 days and subcultured one to three times. The monolayers mostly had a cobblestone appearance, and fusiform or polygonal cells were also observed. By cytochemistry, immunocytochemistry and scanning and transmission electron microscopy, the cells in short-term culture were shown to have characteristics of mesothelial OSE cells.     

A light microscopical morphometric study of the luminal epithelium of human endometrium during the peri-implantation period

Different aspects of the histology of luminal epithelial cellswere examined quantitatively at the light microscopical level.For the first time, using a variety of morphometric indices,an attempt was made to quantify and compare the cellular eventsin a group of well defined fertile controls and infertile subjectsduring the peri-implantation period. The most striking observationwas that the majority of the features examined, including allnuclear parameters and most cell dimensions, were similar anddid not differ significantly between the fertile controls andthe infertile subjects over the period of study. The coefficientsof variation of most of the parameters investigated were similarand within a narrow range in both the fertile and infertilepopulation, indicating little variation either between individualsat a particular stage or between subjects on different days.This low inter-subject variability may suggest that the cellularevents which we have been able to quantify at the light microscopelevel in the uterine surface epithelium around the anticipatedtime of implantation are precisely regulated. Such precise regulationdoes not mean that changes do not occur in luminal epithelialcells during this time, but that those which do occur are wellcontrolled and co-ordinated. Possible changes in biochemistry,physiology and ultrastructure of these cells remain to be determined.     

Estimating human ovarian non-growing follicle number: the application of modern stereology techniques to an old problem

BACKGROUND Previous published reports on the number of non-growing follicles (NGFs) in the human ovary have employed model-based methods for number estimates. These methods are time-intensive, and require correction factors and assumptions that ultimately limit their accuracy. Here, we describe the modification, application and validation of a modern fractionator/optical disector technique for the estimation of human ovarian NGF number. METHODS Forty-eight pairs of normal human ovaries were collected from women (age 8-51 years) undergoing elective bilateral oophorectomy, organ donation, or from autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. The precision of individual NGF counts was determined by calculating the observed coefficient of error (OCE). Intra-observer variability and variation in NGF number between ovaries within a pair were also determined. RESULTS The mean OCE was 16.6% with larger variations observed at lower follicle counts. In recount experiments of the same ovary, NGF number estimates varied by 15-29%, except at very low follicle counts where variation was greater, but absolute differences were small. There was no significant difference in NGF number between ovaries within a pair (Wilcoxon signed rank test, P = 0.81). CONCLUSIONS Modern stereology methods provide an unbiased, efficient method for estimating NGF number in the human ovary. Both ovaries within a pair contain similar numbers of NGFs.     

Induction of proliferation in the primate ovarian surface epithelium in vivo

BACKGROUND: Epithelial ovarian cancer (EOC) is the primary ovarian malignancy affecting women. Proposed etiologies of EOC resist direct testing due to the absence of a suitable animal model, as EOC affects only primates, not other mammals. The role of proliferation in ovarian surface epithelium (OSE) transformation has been suggested but not demonstrated, nor has OSE proliferation been widely reported. We selected the rhesus macaque as a model to evaluate the unique primate OSE in vivo, and to determine whether it can undergo proliferative repair, which may relate to EOC etiology. METHODS: Macaque ovaries were collected at three stages of the cycle. Very late luteal phase ovaries were gently brushed during laparoscopy to remove a portion of the OSE, and ovaries (< or =3 per group) were collected 1-4 days later. Ovary samples were also collected from 10 women aged 33-74 years. Ovarian tissue sections were probed with OSE markers (keratin, beta-catenin, E- and N-cadherin), proliferation markers [proliferating cell nuclear antigen (PCNA), phosphorylated histone H3 (phospho-H3), and phosphorylated Retinoblastoma (pRb)], or labels of collagen and basement membrane. RESULTS: Brushing partially removed the OSE; did not cause tissue damage/adhesions; elevated the frequency of PCNA, phospho-H3 and pRb in the residual OSE, marking as many as 10-50% of cells in brushed regions (unbrushed areas contained <0.1% positive cells), and; did not induce proliferation in underlying stromal cells. CONCLUSIONS: The OSE can undergo proliferative repair, and thus its normal regulation could contribute to EOC etiology.     

A new model of reproductive aging: the decline in ovarian non-growing follicle number from birth to menopause

BACKGROUND: The primary determinant of reproductive age in women is the number of ovarian non-growing (primordial, intermediate and primary) follicles (NGFs). To better characterize the decline in NGF number associated with aging, we have employed modern stereology techniques to determine NGF number in women from birth to menopause. METHODS: Normal human ovaries were collected from 122 women (aged 0-51 years) undergoing elective oophorectomy, organ donation or autopsy. After gross pathologic examination, systematic random sampling was utilized to obtain tissue for analysis by the fractionator/optical disector method. Models to describe the resulting decay curve were constructed and evaluated. RESULTS: NGF decay was best described by a simple power function: log (y) = ax(b) + c, where a, b and c are constants and y = NGF count at age x (R(2) = 0.84, Sums of Squares Error = 28.18 on 119 degrees of freedom). This model implies that follicles decay faster with increasing age. CONCLUSIONS: Unlike previous models of ovarian follicle depletion, our model predicts no sudden change in decay rate, but rather a constantly increasing rate. The model not only agrees well with observed ages of menopause in women, but also is more biologically plausible than previous models. Although the model represents a significant improvement compared with earlier attempts, a considerable percentage of the variation in NGF number between women cannot be explained by age alone.     

Removal of the ovarian surface epithelium from the rabbit ovary--a cause of adhesions following a standard injury

To assess the effect of the removal of ovarian surface epitheliumon repair, a standard injury was induced in the ovaries of 10rabbits. In one ovary the surface cells were denuded, and inthe other they were left intact. The effect on adhesion formationwas assessed at 12 days. Adhesions were assessed by visual inspectionat laparotomy and histological examination of adhesion formation,including a stereological assessment of scar volume. On visualassessment the overall adhesion scores for the denuded ovarieswere greater than for the intact ovaries. Histology showed theadhesions were attached only to the site of injury. The Fallopiantube was adherent to 35 and 4% of the denuded and intact ovariansegments respectively (P = 0.003). The scar volumes for eachside were similar. After 12 days there was only partial re-epithelializationon the denuded ovaries. Electron microscopy confirmed the slowhealing, with much of the surface still covered by a fibrinous-likeexudate. The findings of this small study lend further weightto the importance of the surface epithelium in the control ofadhesion formation. Standard surgical procedures may generateadhesions by the inadvertent denuding of surface epitheliumfrom adjacent healthy tissues, possibly by the loss of plasminogenactivator activity that is found in the mesothelium of the peritoneum.This study highlights the importance of controlling for inadvertentcell loss whilst investigating methods for adhesion prevention.     

Cryopreservation of follicles in human ovarian cortical tissue. Comparison of serum and human serum albumin in the cryoprotectant solutions

BACKGROUND: Cryopreservation of follicles in ovarian cortical tissue has been suggested as a method for preserving fertility for young women who need to undergo cytotoxic therapy. Varying compositions of cryoprotectant solutions have been used to prevent tissue damage during cryopreservation and thawing. We compared human serum [20% (v/v)] and human serum albumin (HSA) (25 mg/ml) in cryoprotectant solutions containing propanediol and sucrose to evaluate whether serum-free medium could be used for this purpose. METHODS: Biopsies of ovarian cortical tissue were obtained from 23 subjects after informed consent. Fourteen underwent Caesarean section and nine underwent sterilization by laparoscopy. The cortical tissue was cut into pieces of 1-1.5 mm(3 )and cryopreserved in cryoprotectant solutions containing serum or HSA. After thawing, a total of 1318 follicles were analysed using light microscopy, transmission electron microscopy (TEM) or live/dead assay. RESULTS: Viability of the follicles was 99.3% in freshly dissected tissue. After thawing, 65% of the follicles and 75% of the oocytes were viable with serum, and 69 and 74%, respectively, with HSA. No significant differences were observed between results in solutions containing serum versus HSA. TEM showed similar results; however, poor survival of stromal tissue was evident in this analysis. The live/dead assay showed 82% viability after thawing for both groups. No benefits were seen from post-thawing culture for 4 h before histological preparation. CONCLUSIONS: A cryoprotectant solution containing HSA was equally effective as one containing serum. Good viability of follicles was confirmed when using propanediol and sucrose as cryoprotectants, with a large number of follicles.     

Asthenozoospermia and the human sperm mid-piece

This study provides a quantitative comparison between surfaceand ultrastructural features of motile spermatozoa in asthenozoospermicand fertile men. The study group consisted of 10 individualswith persistent asthenozoospermia and the controls were 10 fertiledonors to a sperm bank. Scanning electron microscopy and imageanalysis were used to objectively measure sperm mid-piece andtail dimensions. Sperm mid-piece length was significantly shorter(P < 0.01) in asthenozoospermic subjects compared with thecontrols, with mid-piece width and tail length being comparable.Mid-piece ultrastructure was then examined with the transmissionelectron microscope and the number of mitochondrial gyres andtheir configuration recorded. At the ultrastructural level theasthenozoospermic subjects demonstrated significantly fewermitochondrial gyres (P < 0.001) than their fertile counterparts.Energy for sperm movement is provided by mitochondria and adeficit in these organelles in the sub-fertile cohort providesan explanation for poor sperm function in these subjects.     

Brush cells in the human duodenojejunal junction: an ultrastructural study

Brush cells have been identified in the respiratory and gastrointestinal tract mucosa of many mammalian species. In humans they are found in the respiratory tract and the gastrointestinal apparatus, in both the stomach and the gallbladder. The function of brush cells is unknown, and most morphological data have been obtained in rodents. To extend our knowledge of human brush cells, we performed an ultrastructural investigation of human small intestine brush cells. Six brush cells identified in five out of more than 300 small intestine biopsies performed for gastrointestinal tract disorders were examined by transmission electron microscopy. Five brush cells were located on the surface epithelium and one in a crypt. The five surface brush cells were characterized by a narrow apical pole from which emerged microvilli that were longer and thicker than those of enterocytes. The filamentous core extended far into the cell body without forming the terminal web. Caveolae were abundant. Filaments were in the form of microfilaments and intermediate filaments. Cytoplasmic projections containing filaments were found on the basolateral surface of brush cells. In a single cell, axons containing vesicles and dense core granules were in close contact both with the basal and the lateral surface of the cell. The crypt brush cell appeared less mature. We concluded that human small intestine brush cells share a similar ultrastructural biology with those of other mammals. They are polarized and well-differentiated cells endowed with a distinctive cytoskeleton. The observation of nerve fibres closely associated with brush cells, never previously described in humans, lends support to the hypothesis of a receptor role for these cells.     

Proteomic analysis of human ovaries from normal and polycystic ovarian syndrome

Polycystic ovary syndrome (PCOS) is the most common cause of anovulatory infertility, affecting 5-10% of females of reproductive age. Currently, little is known about the changes in whole proteins between PCOS and normal ovaries. In the present study, a proteomic approach comprised two-dimensional gel electrophoresis (2DE) analysis and mass spectroscopy was used to identify proteins and examine expression patterns in three PCOS and normal ovaries. One hundred and ten protein spots were separated and showed different intensities between PCOS and normal ovaries. Sixty-nine proteins associated with cellular metabolism and physiological process were identified from 72 spots. Fifty-four proteins were up-regulated in PCOS ovaries and 15 other proteins were up-regulated in normal ovaries. These data demonstrate, for the first time, the complexity in the regulation of ovarian protein expression in human PCOS, and will provide important insight for a better understanding of the pathogenetic mechanisms underlying this clinical disorder.     

Characterization of 17beta-hydroxysteroid dehydrogenase type 4 in human ovarian surface epithelial cells

The human ovarian surface epithelium (hOSE) is a single layer of mesothelial-type primitive epithelial cells that are potential estrogen targets. It has been reported that hOSE cells can produce estrogen. However, the mechanisms that regulate estrogen level(s) in hOSE cells are not yet known. To elucidate the enzymes involved in these reactions, we examined gene expression of 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) in primary hOSE (POSE) and OSE2a cells using RT-PCR. We found that POSE cells and cells of the immortalized hOSE line, OSE2a, bidirectionally converted estrone (E1) and 17beta-estradiol (E2). Both cell types expressed mRNA for 17beta-HSD type 1 (17beta-HSD1), suggesting that the enzyme is involved in the E1 to E2 conversion. Interestingly, both cells expressed 17beta-HSD4 mRNA but not 17beta-HSD2 mRNA. We prepared an antibody against the carboxyl terminal of 17beta-HSD4 (anti-17beta-HSD4 antibody), which recognized the 80 and 48 kDa proteins in POSE and OSE2a cells based on immunoblot analysis. Furthermore, immunohistochemical study revealed the presence of 17beta-HSD4 in hOSE cells in the human ovary. These results suggest that 17beta-HSD4 is involved in estrogen inactivation and may protect against an excessive accumulation of E2 in hOSE cells.     

Primary ovarian angiosarcoma: A case report and literature review

Primary ovarian angiosarcoma is extremely rare. Only 16 cases have histologicaliy been reported to date In the Ilterature. A case of angiosarcoma arising In the right ovary of a 46-year-old female is presented. Grossly, the resected right ovary was completely replaced by a solid tumor mass, which revealed multiple necrotic and/or hemorrhagic foci. This case revealed the typical histological features of angiosarcoma with sinusoldal and solid patterns of anaplastic tumor cells. Immunohlstochemically, tumor cells were strongly and diffusely positive for CD31 and CD34, in particular, along the cytoplasmic membrane of the tumor cells. Ultrastructurally, tumor cells possessed the intermediate junctions between tumor cells, discontinuous basal laminae attached to the irregularly shaped blood vessels and occasional cytoplasmic pinocytotlc vesicles. These findings confirmed the case as being one of angiosarcoma of the ovary. The patient died 9 months after surgery as a result of developed multlfocal brain metastases. A total of 17 cases reported as primary ovarian anglosarcoma, including this presented case, are clinicopathologically reviewed.     

Selection of patients before and after anticancer treatment for ovarian cryopreservation

BACKGROUND: Although ovarian cryopreservation in patients with cancer should ideally be performed before the initiation of therapy, cryopreservation from such patients often becomes an option only later. The justification for the procedure needs to be elucidated. METHODS: Eighteen cancer patients before chemotherapy and 23 others after chemotherapy participated in the study. Freshly dissected ovarian samples were prepared for light microscopy to demonstrate follicular numbers and apoptosis, transmission electron microscopy to enhance intracellular changes, and staining with fluorescent markers (calcein AM, rhodamin 123 and ethidium homodimer) to test for viability. RESULTS: High numbers of preantral follicles were detected in ovaries of patients < or =20 years. No antral follicles were detected. All the follicles were viable and not apoptotic. Deterioration in follicular quality was observed after chemotherapy, manifested mainly as an increase in abnormal granulosa cell nuclei (P < 0.05-0.0001) and in oocyte vacuolization (P < 0.0001). CONCLUSIONS: Our study stresses the importance of prechemotherapy ovarian cryopreservation. However, the large number of viable, non-apoptotic follicles in ovaries of younger patients (age < or = 20 years) indicates that ovarian cryopreservation might be considered after treatment in this age group. Further studies of ovarian samples from women aged 20-30 years are needed to determine the exact age margin wherein postchemotherapy ovarian cryopreservation can be suggested.     

Effect of cooling rate and dehydration regimen on the histological appearance of human ovarian cortex following cryopreservation in 1, 2-propanediol.

Thin slices of human ovarian cortex were evaluated following cryopreservation in 1,2-propanediol (PROH)/sucrose under various conditions. Following rapid thawing, 1 microm sections were assessed by light microscopy and oocyte abnormalities were further examined by electron microscopy. Follicles (n = 503) were predominantly primordial (91%), with no follicles larger than the proliferating primary stage. Proportions of intact pre-granulosa cells and oocytes (expressed as percentages of the total numbers observed) were significantly reduced following cooling at three different rates with the highest levels of intactness (55 and 85% respectively) being achieved with slow cooling. The frequency of oocyte abnormalities [loss of organelles (mitochondria), organelle-free areas, and/or cytoplasmic vacuolation] was significantly increased at all cooling rates with slow cooling resulting in the highest proportion (56%) of normal oocytes. With slow cooling, increasing dehydration time increased the proportions of intact pre-granulosa cells and oocytes (maximum 74 and 91% respectively after 90 min dehydration). Under these conditions, the highest proportion of follicles with all pre-granulosa cells intact (44%) was observed, as was the highest proportion of 'normal' oocytes (85%). In this study, single step dehydration in PROH/sucrose for 90 min and slow cooling/rapid thawing results in the highest proportion of intact human primordial and primary follicles.     

Human ovarian tissue from cortex surrounding benign cysts: a model to study ovarian tissue cryopreservation

BACKGROUND: The scarcity of human ovarian tissue is a major problem in developing research on ovarian cryopreservation. We were interested in ovarian cortex surrounding benign ovarian cysts harvested during their requisite operations. METHODS: Ovarian tissue was collected from 25 women (mean age = 27.7 +/- 1.0 SEM) and frozen in serum-free cryoprotective medium. Histological and viability analysis were performed on fresh and frozen-thawed slices of tissue. RESULTS: Dermoid (n = 7), endometriosis (n = 13) and serous (n = 5) cysts were observed. Follicular densities (expressed per mm3) in ovarian cortex surrounding dermoid cysts were higher than in endometriosis and serous cysts for both histological (median of follicular densities: 13.04, 0.31 and 0.89 respectively) and viability analysis (2.93, 0.05 and 0.71 respectively). Freezing-thawing did not result in gross abnormality of follicle population either in number or morphology (80% of follicles preserved a normal pattern). However, a slight decrease of the density of living follicles (expressed per mm2) was reported. CONCLUSIONS: Ovarian cortex surrounding ovarian cysts, especially dermoid cysts, could be considered a source of ovarian tissue for future research. In our study, the cryopreservation procedure resulted in high follicular survival assessed by both histological and viability analysis. Nevertheless, further studies of in vivo and in vitro follicular maturation are needed to strengthen this model.     

A light and electron microscopic study of an ovarian and rectal carcinoid

An ovarian and rectal carcinoid within the same person is described. The ovarian carcinoid is non-argentaffinic with typical regular granules. The rectal tumour consists of two types of APUD cells, some argentaffinic and diazo-positive, whilst others are non-argyrophilic. These features are mirrored ultrastructurally, some cells containing the large pleomorphic argentaffinic granules while others have smaller regular granules. In addition, focal aggregates of microfilaments were found in both tumour types. These findings are discussed in terms of the APUD concept. The similarities of the APUD tumour cells to D1 APUD cells of the duodenum is highlighted, and an alternative explanation for their derivation is proposed.