MicroRNA-497 inhibition of ovarian cancer cell migration and invasion through targeting of SMAD specific E3 ubiquitin protein ligase 1

Ovarian cancer is the leading cause of death from gynecological malignancies worldwide. Understanding the molecular mechanism underlying ovarian cancer progression facilitates the development of promising strategy for ovarian cancer therapy. Previously, we observed frequent down-regulation of miR-497 expression in ovarian cancer tissues. In this study, we investigated the role of miR-497 in ovarian cancer metastasis. We found that endogenous miR-497 expression was down-regulated in the more aggressive ovarian cancer cell lines compared with the less aggressive cells. Exogenous expression of miR-497 suppressed ovarian cancer cell migration and invasion, whereas reduction of endogenous miR-497 expression induced tumor cell migration and invasion. Mechanistic investigations confirmed pro-metastatic factor SMURF1 as a direct target of miR-497 through which miR-497 ablated tumor cell migration and invasion. Further studies revealed that lower levels of miR-497 expression were associated with shorter overall survival as well as increased SMURF1 expression in ovarian cancer patients. Our results indicate that down-regulation of miR-497 in ovarian cancer may facilitate tumor metastasis. Restoration of miR-497 expression may be a promising strategy for ovarian cancer therapy.     

The effects of age and sex steroids on the macrophage population in the ovary of the chicken, Gallus domesticus

The role of macrophages in the function of the hen ovary has not yet been described, although these cells may be an important regulator of ovarian function in mammals. The aim of this study was to determine the changes in the frequency of macrophages during ageing and follicular atresia, and the effects of sex steroids on the macrophage population in the hen ovary. Cryostat sections of ovarian tissues of immature, young laying and old laying hens and those of immature hens treated with or without diethylstilboestrol (DES) or progesterone were immunostained for macrophage cells using mouse anti-chicken macrophage monoclonal antibody. Macrophages were observed under a light microscope and counted using a computer assisted image analyser. The frequency of macrophages in both the stroma and theca of primary follicles was significantly greater in young laying hens than in immature and old laying hens and these cells were more frequent in old laying hens than in immature hens (P < 0.01). Macrophages were more frequent in atretic follicles than in normal follicles (P < 0.01). The number of macrophages in both the stroma and theca of primary follicles of DES-treated birds was significantly greater than in those of progesterone-treated and control birds (P < 0.01). Progesterone had no significant effect on the population of macrophages. These results suggest that macrophages in the ovary increase in association with sexual maturation of birds and atresia of follicles and decrease during ageing. Oestrogen may be one of the factors that affect the population of macrophages in the hen ovary.     

Immunohistochemical profile of ovarian inclusion cysts in patients with and without ovarian carcinoma

Summary The expression of cytokeratin, epithelial membrane antigen, Leu-M1, B72.3, carcinoembryonic antigen, human placental lactogen, proliferating cell nuclear antigen, p53, and ovarian carcinoma-associated antigen OC-125 was evaluated in inclusion cysts in contralateral ovaries of patients with unilateral ovarian carcinoma. The findings were compared with the findings in inclusion cysts in ovaries of patients without ovarian carcinoma. Although there was more frequent expression of tumour markers B72.3 and CEA in patients with ovarian carcinoma, these differences did not reach statistical significance.     

Cell-specific localisation of apoptosis in the bovine ovary at different stages of the oestrous cycle

Apoptosis was localized in all ovarian cell types of 23 cows in various stages of the oestrous cycle, using the detection of active caspase-3, in situ end labelling (TUNEL) and DNA fluorescent staining (DAPI). Very few apoptotic cells were found in primordial, primary, secondary and vital tertiary follicles. In contrast, apoptosis in atretic tertiary follicles was much more frequent, and high apoptotic scores were recorded when using the TUNEL technique and lower scores with the caspase-3 assay. Cystic atretic follicles showed in general a higher apoptotic score than obliterative atretic follicles, with intermediate to high scores in granulosa cells and lower scores in theca cells. In corpora lutea, large and small lutein cells had intermediate to high scores using the caspase-3 assay, and intermediate to low scores using the TUNEL assay. Irrespective of the detection method, the scores were higher in lutein cells than in the capsular stroma cells. In all ovarian structures examined, variations in apoptotic scores were seen in the different cycle stages, suggesting a cycle-dependent influence on apoptosis, although correlations with plasma progesterone concentrations were low.     

Trophinin is a potent prognostic marker of ovarian cancer involved in platinum sensitivity

Ovarian cancer is the leading cause of death in women with gynecological malignancies, with prognosis of advanced stage tumors determined by chemotherapeutic response and the success of tumor resection. Since aberrant RAS pathway activation is frequent in ovarian cancer, study of in vitro RAS-induced transformation and accompanying genomic expression changes in ovarian surface epithelial cells is imperative for development of new therapeutic modalities and for understanding tumorigenesis. cDNA microarray analysis revealed TROPHONIN (TRO), a homophilic adhesion molecule involved in blastocyst implantation, was among the genes most downregulated by RAS induction. TRO expression is higher in cisplatin-sensitive cancer cell lines and positively correlates with prognoses in ovarian cancers. TRO knockdown by RNA interference conferred cisplatin resistance and led to increased invasiveness of cultured ovarian cancer cells. These findings underscore the importance of TRO in tumorigenesis, and suggest that TRO may be a useful biomarker for cisplatin sensitivity and invasive potential.     

Implication of BAG5 downregulation in metabolic reprogramming of cisplatin-resistant ovarian cancer cells via mTORC2 signaling pathway

Ovarian cancer is the most frequent cause of gynecologic malignancies associated death. Primary or acquired cisplatin resistance is frequently occurred during ovarian cancer therapy. Cancer stem cells (CSC) tend to form minimal residual disease after chemotherapy and are implicated in relapse. The ability of cancer cells to reprogram their metabolism has recently been related with maintenance of CSC and resistance to chemotherapies. The current study found that BAG5 expression was decreased in cisplatin-resistant ovarian cancer cells and clinical tissues. Our data demonstrated that BAG5 knockdown was implicated in metabolic reprogramming and maintenance of cancer stem cell (CSC)-like features of ovarian cancer cells via regulation of Rictor and subsequent mTORC2 signaling pathway. In addition, the current study demonstrated that Bcl6 upregulation was responsible for repression of BAG5 transactivation via recruitment on the BAG5 promoter in cisplatin-resistant ovarian cancer. The current study also demonstrated reverse correlations between BAG5 and Bcl6, BAG5 and Rictor in ovarian serous adenocarcinoma tissues. Collectively, the current study identified the implication of Bcl6/BAG5/Rictor-mTORC2 signaling pathway in metabolic reprograming and maintenance of CSC-like features in cisplatin-resistant ovarian cancer cells. Therefore, further studies on the mechanism underlying regulation of metabolic reprogramming and CSC-like characteristics of cisplatin-resistant ovarian cancer cells may contribute to the establishment of novel therapeutic strategy for cisplatin-resistance.     

Blood Cell Mitochondrial DNA Content and Premature Ovarian Aging

Primary ovarian insufficiency (POI) is a critical fertility defect characterized by an anticipated and silent impairment of the follicular reserve, but its pathogenesis is largely unexplained. The frequent maternal inheritance of POI together with a remarkable dependence of ovarian folliculogenesis upon mitochondrial biogenesis and bioenergetics suggested the possible involvement of a generalized mitochondrial defect. Here, we verified the existence of a significant correlation between blood and ovarian mitochondrial DNA (mtDNA) content in a group of women undergoing ovarian hyperstimulation (OH), and then aimed to verify whether mtDNA content was significantly altered in the blood cells of POI women. We recruited 101 women with an impaired ovarian reserve: 59 women with premature ovarian failure (POF) and 42 poor responders (PR) to OH. A Taqman copy number assay revealed a significant mtDNA depletion (P<0.001) in both POF and PR women in comparison with 43 women of similar age and intact ovarian reserve, or 53 very old women with a previous physiological menopause. No pathogenic variations in the mitochondrial DNA polymerase γ (POLG) gene were detected in 57 POF or PR women with low blood mtDNA content. In conclusion, blood cell mtDNA depletion is a frequent finding among women with premature ovarian aging, suggesting that a still undetermined but generalized mitochondrial defect may frequently predispose to POI which could then be considered a form of anticipated aging in which the ovarian defect may represent the first manifestation. The determination of mtDNA content in blood may become an useful tool for the POI risk prediction.     

Ovarian dysgenesis in individuals with chromosomal abnormalities

Summary To understand better the pathogenesis of ovarian dysgenesis in individuals with abnormalities such as 45,X Turner syndrome, trisomy 13, and trisomy 18, we have examined microscopically the ovaries of 36 infants with a number of chromosomal abnormalities confirmed by karyotype analysis. All infants with trisomy 13, trisomy 18, triploidy, and 45,X were found to have severe ovarian dysgenesis characterized by a virtual absence of primary oocytes. The ovaries of individuals with 21 trisomy and of those with partial deletion or duplication of an autosome demonstrated variable findings, which ranged from complete absence of oocytes to a mild diminution of oocyte numbers. The results of this study suggest that the attrition of germ cells in these infants is a result of faulty meiotic pairing and that ovarian dysgenesis is a more frequent finding in children with karyotypic abnormalities than has been realized previously.Presented, in part, at the 1989 David W. Smith Morphogenesis and Malformations Conference, Madrid, 1989, and at the 1990 Southern Society for Pediatric Research Meeting, New Orleans, 1990     

Inhibitory Effect of Tumor Suppressor p53 on Proinflammatory Chemokine Expression in Ovarian Cancer Cells by Reducing Proteasomal Degradation of IκB

Ovarian cancer, one of inflammation-associated cancers, is the fifth leading cause of cancer deaths among women. Inflammation in the tumor microenvironment is associated with peritoneal tumor dissemination and massive ascites, which contribute to high mortality in ovarian cancer. Tumor suppressor p53 is frequently deleted or mutated in aggressive and high-grade ovarian cancer, probably aggravating cancer progression and increasing mortality. We therefore investigated the influence of p53 on proinflammatory chemokines in ovarian cancer cells. A PCR array of the chemokine network revealed that ovarian cancer cells with low or mutated p53 expression expressed high levels of proinflammatory chemokines such as CXCL1, 2, 3 and 8. Transient transfection of p53 into p53-null ovarian cancer cells downregulated proinflammatory chemokines induced by tumor necrosis factor-α (TNF), a proinflammatory cytokine abundantly expressed in ovarian cancer. Furthermore, p53 restoration or stabilization blocked TNF-induced NF-κB promoter activity and reduced TNF-activated IκB. Restoration of p53 increased ubiquitination of IκB, resulting from concurrently reduced proteasome activity followed by stability of IκB. A ubiquitination PCR array on restoration of p53 did not reveal any significant change in expression except for Mdm2, indicating that the balance between p53 and Mdm2 is more important in regulating NF-κB signaling rather than the direct effect of p53 on ubiquitin-related genes or IκB kinases. In addition, nutlin-3, a specific inducer of p53 stabilization, inhibited proinflammatory chemokines by reducing TNF-activated IκB through p53 stabilization. Taken together, these results suggest that p53 inhibits proinflammatory chemokines in ovarian cancer cells by reducing proteasomal degradation of IκB. Thus, frequent loss or mutation of p53 may promote tumor progression by enhancing inflammation in the tumor microenvironment.     

Technologic advances in the imaging of ovarian morphology and their roles in ascertaining follicular growth and development in the rhesus monkey

Imaging follicular development has advanced notably from the invasive—laparotomy—to the relatively noninvasive—laparoscopy (making frequent observations over a single cycle possible)—to totally noninvasive approaches such as ultrasonography and magnetic resonance imaging (MRI). Now we can identify the dominant preovulatory follicle (DF) in macaques by day 6 of the menstrual cycle via laparoscopy and by day 6–8 via ultrasonography. MRI scans are remarkably correlated with histologic specimens. We obtained axial, coronal, and sagittal ovarian images with MRI software. Moreover, we are discerning structure/function relationships in ovarian estrogen-receptor systems using X-ray autoradiography and immunocytochemistry. We have localized the estrogen receptor in functional corpus luteum (CL) with ³H-estradiol and in germinal epithelium and granulosa cells with monoclonal antibody to the receptor. We believe that by using a combination of the aforementioned techniques, we will be able to investigate more fully the processes of recruitment and selection of ovarian follicles.     

Ovarian histology of the placentotrophic Mabuya mabouya (Squamata, Scincidae)

Ovarian structure and folliculogenesis of females at different reproductive stages are described for the viviparous placentotrophic lizard Mabuya mabouya. The small ovaries have a thin wall formed by the ovarian epithelium and a thin tunica albuginea. One to two germinal beds that contain numerous oogonia and developing primordial follicles are derived from the ovarian epithelium and are next to the ovarian hilum. The ovarian cortex contains follicles at different stages of development, corpora lutea, and atretic follicles. The yolk nucleus and Balbiani complex were not evident in the ooplasm of previtellogenic follicles. The follicular epithelium of these follicles is polymorphic, as in other species of Squamata, but the larger cells are spherical and monolayered rather than pyriform. The zona radiata of the preovulatory follicles is less developed than in lecithotrophic species. These features suggest a decrease in metabolic and absorptive processes during follicular growth. In preovulatory follicles (1.5-1.8 mm diameter), primordial yolk vacuoles and small cortical granules are deposited in the ooplasm instead of fatty yolk platelets, so that only one stage of vitellogenesis is observed. Polyovular atretic follicles occur in some females. Follicular atresia is minimal for preovulatory follicles, but is more frequent in follicles with polymorphic epithelia. In the corpus luteum, the luteal tissue is formed from granulosa cells and luteolysis occurs during the late gastrula -- late neurula embryonic stages. Thus, the maintenance of gestation from the pharyngula to preparturition stages seems to be related to secretion of extraluteal progesterone, possibly of placental origin. These observed ovarian features are related to the high degree of placental complexity of this species and show that the evolution of advanced placentotrophy in species of this lineage has been accompanied by concomitant changes in ovarian function.     

Steroid-mediated differentiation of neural/neuronal cells from epithelial ovarian precursors in vitro

We reported earlier that occasional neurons evolve in human cultures of pluripotent ovarian epithelial stem cells. In subsequent experiments, frequent transdifferentiation into neural stem cells (NSC) and differentiating neurons was observed in human ovarian epithelial stem cells and porcine granulosa cells after exposure to certain combinations of sex steroids. Testosterone (TS), progesterone (PG) or estradiol (E2) alone do not increase the emergence of neurons. However, a mixture of TS+PG after E2 pretreatment converted a majority of ovarian epithelial stem cells or porcine granulosa cells into NSC and differentiating neuronal cells within one to three hours. Cultured neurons manifested an interconnectivity resembling primitive neuronal pathways in culture. These converted cells expressed the cell markers SSEA-1, SSEA-4, NCAM, and Thy-1 glycoconjugates of NSC and neurons, and differentiating cells showed characteristic neuronal morphology. Emergence of NSC and neuronal cells was associated with significant cellular depletion of L-glutamic acid (glutamate), which serves as the major excitatory neurotransmitter in the vertebrate CNS and its fast removal is essential for preventing glutamate excitotoxicity. These observations suggest that certain sequential systemic treatment with common sex steroids and their mixture might be effective in the treatment or prevention of degenerative CNS disorders. The ovarian stem cell cultures readily obtainable from human ovaries regardless of the woman's age have the potential to produce NSC for autologous regenerative treatment of neurologic diseases in aging women. Finally, the proper combination of sex steroids could possibly be employed for transdifferentiation of adult bone marrow stem cells or mobilized peripheral blood cells into autologous NSC and stimulate their neuronal differentiation after homing in the CNS.     

Frequency of mortality due to cancer in the last 110 years (1870-1979) in the department of pathology of Cluj-Napoca

Records of 36,775 autopsies performed between 1870 and 1979 in the Department of Morphopathology, Cluj-Napoca, were re-studied. 10.82 per cent of deaths were due to cancer, whose incidence increased during the last 50 years. Cancer is more often seen in males and its highest incidence was noted between 50 and 70 years of age. Gastric and lung cancers are more frequent in males, while uterine, intestine, stomach and ovarian cancers are the most frequent localizations in females.     

Association of common WRAP 53 variant with ovarian cancer risk in the Polish population

Among many alterations within the TP53 gene the rs1042522 (C72G, p.Pro72Arg) has been associated with numerous cancers , however the results differ between populations for opposite Pro or Arg alleles. Similar thus inconclusive results are observed in ovarian cancer, which may suggest that the rs1042522 does not influence ovarian carcinogensis directly, but might be linked to another pathogenic alteration. WRAP53 which overlaps the TP53 is required to maintain normal levels of p53 upon DNA damage, but also when altered may independently increase the risk of cancer. To evaluate the association between three SNPs located in WRAP53–TP53 region: rs1042522, rs2287497, rs2287498 and ovarian cancer risk in Polish population we genotyped 626 cases and 1,045 healthy controls. Our results provide the evidence for an association between studied SNPs and a risk of invasive ovarian cancer in Poland. We found that CC homozygotes in rs1042522 were more frequent in cancers when compared to controls (OR = 1.46, p = 0.03). Similarly in WRAP53 both TT homozygotes in rs2287497 (OR = 1.95, p = 0.03) and AA homozygotes in rs2287498 (OR = 2.65, p = 0,01) were more frequent among cases than healthy individuals. There is also a suggestive evidence that specific homozygosity of studied SNPs in TP53–WRAP53 region is significantly overrepresented in ovarian cancer patients. In conclusion SNPs in WRAP53 (rs2287497 and rs2287498) have stronger association with an ovarian cancer risk than rs1042522 in TP53.     

Expression and function of HOXA genes in normal and neoplastic ovarian epithelial cells

We studied the roles of three HOXA genes in cultured normal ovarian surface epithelial (OSE) cells and ovarian cancer cells. They included HOXA4 and HOXA7 because, by cDNA microarray analysis, these were more highly expressed in invasive ovarian carcinomas than in benign or borderline (noninvasive) ovarian tumors, and HOXA9 because it characterizes normal oviductal epithelium, which resembles ovarian serous adenocarcinomas. The three HOXA genes were more highly expressed when OSE cells were dividing and motile than when they were confluent and stationary, and also when they dispersed in response to EGF treatment or to reduced calcium concentrations in culture media. The expression of the HOXA genes varied among ovarian cancer cell lines, but was highest in lines with compact epithelial morphologies. We focused on HOXA4 as the most highly expressed in the ovarian carcinoma array. HOXA4 expression did not parallel proliferative activities of either OSE or ovarian cancer lines. Moreover, modifying HOXA4 expression in ovarian cancer cell lines did not alter either E-cadherin expression or CA125 secretion. However, HOXA4 downregulation enhanced EGFR phosphorylation and migration in serum-starved OSE and ovarian cancer cells in response to EGF, and enhanced migration of all ovarian cancer lines in 5% serum even without EGF treatment. Thus, HOXA4 expression does not correlate with proliferation or with epithelial differentiation, but it increases in response to OSE cell dispersion and negatively regulates EGFR activation and the motility of OSE and of ovarian cancer cells. HOXA4 expression was highest in cancer lines with compact epithelial growth patterns, suggesting, again, an anti-dispersion function. In summary, increased HOXA4 expression in ovarian cancer appears to constitute a tumor-suppressive, homeostatic response to aberrant cell behavior, and, in particular, to cell dispersion and migration.     

Nanog 基因在卵巢癌和卵巢肿瘤干细胞中的表达及意义

目的:探讨胚胎干细胞关键因子Nanog基因mRNA及其蛋白在卵巢癌和卵巢癌肿瘤干细胞中的表达及意义。方法:选取10例正常卵巢上皮组织、10例卵巢良性肿瘤及60例卵巢癌组织,采用逆转录酶-聚合酶链反应(RT-PCR)方法和免疫组织化学PV-6000两步法检测Nanog mRNA和蛋白表达水平;采用无血清悬浮培养法从SKOV-3卵巢癌细胞株中分离培养肿瘤干细胞,流式细胞术鉴定肿瘤干细胞CD117表达,采用RT-PCR和Western Blot方法检测SKOV-3卵巢癌细胞及肿瘤干细胞中NanogmRNA及其蛋白的表达水平。结果:Nanog mRNA在卵巢癌组织中的表达水平均高于正常卵巢组织和卵巢良性肿瘤组织(P<0.05);Nanog mRNA在不同分化程度及临床分期的卵巢癌组织中表达水平不同,低分化组高于高分化组(P<0.05);III-IV期高于I-II期(P<0.05);免疫组化结果同RT-PCR。从SKOV-3卵巢癌细胞株中成功分离出肿瘤干细胞,SKOV-3卵巢癌细胞和肿瘤干细胞Nanog mRNA相对含量分别为0.6044±0.0368,0.8736±0.0537,差异具有统计学意义(P<0.05),两种细胞Nanog蛋白相对含量分别为0.6364±0.0169 1.2788±0.0314,差别具有统计学意义(P<0.05)。结论:Nanog基因在卵巢癌组织和SKOV-3细胞系中均高表达,其在组织中的表达强度与临床分期及病理分级关系密切,且在肿瘤干细胞中表达高于一般卵巢癌细胞,其与卵巢癌的发生发展关系密切,可能是卵巢癌干细胞的表面标志物,有望成为新的标志物。     

Overexpression of MEKK3 confers resistance to apoptosis through activation of NFkappaB

Many cancers have constitutively activated NFkappaB, the elevation of which contributes to cancer cell resistance to chemotherapeutic agent-induced apoptosis. Although mitogen-activated protein kinase/extracellular-regulated kinase kinase kinase-3 (MEKK3) has been shown to participate in the activation of NFkappaB, its relations to apoptosis and cancer are unclear. In this study, we established cell model systems to examine whether stable expression of MEKK3 could lead to increased NFkappaB activity and confer resistance to apoptosis. In addition, we investigated in breast and ovarian cancers whether MEKK3 expression may be altered and correlated with aberrant NFkappaB activity. We show that stable cell lines overexpressing MEKK3 not only had elevated levels of NFkappaB binding activity but also were more responsive to cytokine stimulation. These stable cells showed 2-4-fold higher basal expression of Bcl-2 and xIAP than the parental cells. Consistent with this increased expression of cell survival genes, MEKK3 stable cells showed reduced activation of caspases 3 and 8 and poly(ADP-ribose) polymerase cleavage and dramatically increased resistance to apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand, doxorubicin, daunorubicin, camptothecin, and paclitaxel. Intriguingly, analysis of human breast and ovarian cancers showed that a significant fraction of these samples have elevated MEKK3 protein levels with corresponding increases in NFkappaB binding activities. Thus, our results established that elevated expression of MEKK3 appears to be a frequent occurrence in breast and ovarian cancers and that overexpression of MEKK3 in cells leads to increased NFkappaB activity and increased expression of cell survival factors and ultimately contributes to their resistance to apoptosis. As such, MEKK3 may serve as a therapeutic target to control cancer cell resistance to cytokine- or drug-induced apoptosis.     

肿瘤干细胞与卵巢癌研究进展

近年来,肿瘤干细胞学说作为肿瘤发生发展的重要原因获得越来越多的认可。肿瘤干细胞是指肿瘤中存在的含量极少、具有无限增殖潜能的干细胞样肿瘤细胞,它们能自我更新、分化、迁徙,是导致肿瘤发生、发展、转移和耐药的重要原因。卵巢癌也可能是卵巢癌干细胞所致的疾病。卵巢癌干细胞的分离鉴定正处于起始阶段,针对卵巢癌干细胞的靶向治疗可能在卵巢癌治疗中具有重要作用,为临床彻底治愈卵巢癌带来希望。     

Histogenesis of the transmitting tract in Strelitzia reginae

Organisation and development of the stigmatic, stylar and ovarian parts of the transmitting tract in Strelitzia reginae were evaluated. They were characterised by 1) cell shape, 2) appearance of distal cell wall, 3) type of plastid, 4) and vacuolar system. The long stigmatic trichomes have a secondary irregular wall layer separated from the primary wall. Cell structures include pleomorphic plastids with vesiculated thylakoids and frequently a crystal or a lipidic globule. In early stages of bud development the extensive endoplasmic reticulum (ER) is smooth, whereas it is mainly rough in older buds. Coated vesicles are frequent, as are dictyosomes. Prominent invaginations along the plasma membrane contain floccular deposits in the older flower buds. These deposits are similar in structural appearance to material in the large vacuoles. The basal part of the stigma has wedge-shaped cells with wall ingrowths. Three stages of stigmatic secretion during the development were characterised. The stylar canal is initially narrow but widens subsequently. The cuticle is detached and the apical cell walls show a fringed surface; from this wall inbuddings develop shortly before anthesis. At this stage sheets of rough ER are evident in the cell cortex. The plastids have a few vesiculated thylakoids, proteinaceous crystals and starch grains. The epithelial cells of young buds have numerous vacuoles, the volume of which decreases in more mature cells. The stylar canal is filled with a secretion at all stages of bud development. The face of the ovarian transmitting tract, lining the placenta, is smooth in young buds but lobed in older ones due to the division pattern of the epithelial cells. These cells are large, elongated and culumnar as young but narrow and wedge-shaped when more mature. Cell wall inbuddings are formed late during bud development.