Pre-ovulatory graafian follicles are cooler than neighbouring stroma in pig ovaries

Using an infra-red camera, domestic pig ovaries were thermo-imaged almost instantaneously at laparotomy or within a closed abdomen by endoscopy. Rectal and jugular vein temperatures were recorded using thermo-probes. Graafian follicles (7-10 mm diameter) were cooler than ovarian stroma in all experimental models examined, and both compartments were cooler than rectal and jugular temperatures. The mean difference between follicles and stroma in 73 observations was 1.3 +/- 0.1 degrees C. When thermo-imaged under the fimbriated extremity of the Fallopian tube, follicles and stroma could still be distinguished. Follicles cooled slightly more rapidly than adjoining stroma during the first 10 s of a 60 s recording interval, after which curves for the two tissues remained parallel. Arresting ovarian blood supply for 5 min had a negligible influence on the temperature differentials. Endoscopy in three models recorded mean differentials between follicles and stroma of 0.6 +/- 0.1 degrees C to 1.1 +/- 0.1 degrees C. It is concluded that temperature gradients do exist in the ovarian tissues of mature animals, and that these are generated at least in part as a consequence of endothermic reactions within Graafian follicles.     

Mouse oocytes derived from in vitro grown primary ovarian follicles are fertile

A model culture system has been developed whereby individual,primary ovarian mouse follicles can be grown in vitro to theGraafian stage in the normal physiological time course, andthen ovulated in response to luteinizing hormone. We reporthere on the successful fertilization and subsequent embryo developmentof the oocytes from such follicles. This is the first time thatoocytes from in-vitro matured whole follicles have been fertilizedand shown to produce viable offspring in host animals. The studydemonstrates that the culture system mimics physiological conditionsfor normal follicle development.     

Hyaluronan and proteoglycans in ovarian follicles.

Proteoglycans are macromolecules formed by a protein backbone to which one or more glycosaminoglycan side chains are co-valently attached. They can be secreted by the cells, retained at the cell surface, or stored in intracellular vacuoles. Hyaluronan is an extremely long glycosaminoglycan which, at variance with other glycosaminoglycans, is released into the extracellular matrix as a free polysaccharide not co-valently linked to a core protein. Both proteoglycans and hyaluronan influence many aspects of cell behaviour by multiple interactions with other molecules. They are involved in matrix formation, cell-cell and cell-matrix adhesion, cell proliferation and migration, and show co-receptor activity for growth factors. Both proteoglycan and hyaluranon synthesis change significantly during ovarian follicle development and atresia. This review describes the structure of these molecules and their possible function in ovarian physiology.     

Oxidative phosphorylation and the tricarboxylic acid cycle are essential for normal development of mouse ovarian follicles

BACKGROUND: Mouse ovarian follicles are typically grown in upright drops of culture medium. Recently we found that culture of follicles at the medium-gas interface in inverted drops markedly improved follicular development, possibly due to improved access of oxygen to the follicle. In this study, we examined the importance of aerobic energy metabolism for follicle development by culturing mouse follicles (198 6 16.5 initial microm diameter, mean 6 SD) in the presence of phosphorylation and tricarboxylic acid (TCA) cycle inhibitors. METHODS: All inhibitors were tested in the inverted system using 100 microl medium drops in 96-well plates; certain inhibitors were also tested in upright drops with or without an oil overlay. RESULTS: The oxidative phosphorylation inhibitor rotenone (0.1, 0.5 and 1 micromol/l) totally abolished follicle growth in the inverted system; cyanide (1 mmol/l) totally abolished growth in the upright with oil system but not in the inverted system (possibly due to loss of cyanide gas due to the absence of an oil overlay). The mitochondrial uncoupler 2,4-dinitrophenol (0.5 and 1 mmol/l) also abolished growth in the inverted system. The TCA cycle inhibitor monofluoroacetate (10 mmol/l), significantly inhibited growth in all three culture systems (P < 0.01) but malonate (10 mmol/l) had no effect. CONCLUSIONS: Aerobic metabolism and an adequate oxygen supply are essential for normal follicular development.     

Histomorphometry of dominant and subordinate bovine ovarian follicles

The study was designed to quantitatively characterize the histomorphological attributes of dominant and subordinate follicles in relation to follicular wave dynamics. Heifers (n = 27) were examined daily using ultrasonography to record the growth of individual follicles from 2 days before ovulation until the day of ovariectomy to obtain growing (n = 7), early static (n = 6), late static (n = 6) and regressing (n = 5) phase anovulatory dominant follicles of Wave 1, as well as preovulatory (n = 6) and subordinate (n = 42) follicles. The wall thickness of Wave 1 dominant follicles decreased dramatically (P < 0.01) during the late-static (60.2 +/- 4. 3 microm) and regressing (41.8 +/- 4.3 microm) phases compared to earlier phases. Cells of the granulosa layer of the dominant follicle of Wave 1 became loose during the late-static phase, with an increase (P < 0.001) in number of degenerating cells. Dominant follicles of Wave 1 were lined by fibroblast-like flattened cells during the regressing phase. One day after wave emergence (i.e., before selection), the three largest follicles of the wave were histomorphologically indistinguishable. The wall of the preovulatory follicle close to the medulla of the ovary was thicker (P < 0.01) than the wall facing the ovarian surface. The wall of subordinate follicles was thinner (P < 0.01) and had a lower mitotic index (P < 0.01) than that of their dominant counterparts 3 days and 6 days after wave emergence. In summary, follicular status, ascribed by ultrasonography, was associated with quantitatively distinct histomorphological characteristics. Morphometric changes in the dominant follicle during immature, mature, and post-mature phases were similar to, but occurred later than, those of subordinate follicle. The dominant follicles of Wave 1 entered histological atresia at the time of emergence of Wave 2.     

Permeability of ovarian follicles and capillaries in mice

The permeability of ovarian capillaries and follicles in prepubertal and sexually mature (proestrus and metestrus) randomly bred Swiss Albino female mice (SCH:ARS HA ICR strain) was studied by intravenous injection of either ferritin or horseradish peroxidase (HRP), followed by examination with light and electron microscopes. The study revealed that capillaries in the interstitial and perifollicular regions were provided with a continuous endothelium that had constant permeability characteristics irrespective of sexual maturity or phase of the estrous cycle. Horseradish peroxidase left the capillaries primarily through interendothelial cell junctions and was present in all follicles within 30 seconds after administration of the tracer. Ferritin, on the other hand, was absent from endothelial cell junctions, and left the capillaries, at a slower rate than HRP, via cytoplasmic vesicular transport. Both tracers were found in the granulosa cells but rarely in the oocytes. The tracers reached the oocyte through the intercellular spaces between granulosa cells. These findings demonstrate that the follicular apparatus of the mouse is permeable to ferritin and HRP, and that follicular regions such as the basal lamina of the follicle and the zona pellucida do not stop or retard the passage of either tracer.     

Development-dependent responses of ovarian follicles to FSH and hCG.

Ovarian follicles removed from immature rats (preantral follicles) and immature rats treated in vivo with follicle stimulating hormone (FSH) (antral follicles) released progesterone in vitro in response to either human chorionic gonadotropin (hCG), hFSH, or DBcAMP in a time- and concentration-dependent fashion. Antral follicles produced approximately 20 times more progesterone than preantral follicles in response to both FSH and hCG at 10(-7) M and approximately 5 times more progesterone in response to 8 X 10(-3) M DBcAMP. After in vitro incubations, follicles were transplanted beneath the kidney capsules of recipient rats to assess their ability to luteinize after hormonal stimulation. Only antral follicles incubated with hCG, hFSH, and DBcAMP formed ectopic corpora lutea. Adenylate cyclase activity in preantral and antral follicle granulosa cells increased in response to both 10 mM KF and 10(-6) M hFSH with no major differences observed between membranes prepared from preantral or antral follicle granulosa cells. These results demonstrate that follicular maturation is associated with major changes in the ability of the granulosa cells to produce progesterone and luteinize in response to hormonal stimulation and that these changes may be, in part, independent of a functional hormone-responsive adenylate cyclase system.     

Stereo-electron microscopy of the ovarian follicles of cat and mouse

Three dimensional structure of developing ovarian follicles in the cat and the mouse were examined to clarify the cellular and the extra cellular components during follicular maturation by scanning electron microscopy. Epithelial cells of the membrana granulosa (MG) and the cumulus oophorus (CO) show variable morphology, which depends on the location of the cells in the follicle. In the small antral follicle, there is no morphological difference between the surface structure of the MG and the CO lining cells. In the large antral follicle, however, the lumenal lining cells of the MG change their spherical shape to a rather flattened one showing numerous pseudopodial processes creeping over the surrounding lumenal cells. The CO cells remain rounded with fine cytoplasmic protrusions on their outer surfaces which seem to make a network. The outer surface view of the zona appears as composed of labyrinthine canals with relatively large pores of various sizes, however, the inner surface is rather compact with a number of small pores.     

Morphological characteristics of histions of developing and atresic human ovarian follicles

The morphology of tissue elements of human ovarian follicles was studied in the course of normal folliculogenesis and in some forms of its disorders. Signs of abnormal development of follicles were detected that are typical of different stages of their development under the conditions studied. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 8, pp. 188–191, August, 1995     

Nuclear localization of E-cadherin but not beta-catenin in human ovarian granulosa cell tumours and normal ovarian follicles and ovarian stroma

Ohishi Y, Oda Y, Kurihara S, Kaku T, Kobayashi H, Wake N & Tsuneyoshi M
(2011) Histopathology 58 , 423–432
Nuclear localization of E‐cadherin but not beta‐catenin in human ovarian granulosa cell tumours and normal ovarian follicles and ovarian stroma Aims: The role of misregulated Wnt/beta‐catenin signalling in human ovarian granulosa cell tumour (GCT) has not been well characterized. The aim of this study was to confirm subcellular localization of key molecules of Wnt signalling (beta‐catenin and E‐cadherin) in human ovarian GCTs. Methods and results: Tissue samples taken from 32 human ovarian GCTs and 19 human normal ovaries containing 68 follicles were stained immunohistochemically using monoclonal anti‐beta‐catenin and anti‐E‐cadherin antibodies. None of the 32 GCTs and none of the 68 ovarian follicles showed beta‐catenin nuclear expression (0%). On the other hand, 28 of 32 GCTs (88%) and 53 of 68 normal ovarian follicles (78%) showed nuclear expression of E‐cadherin in granulosa cells. The ovarian stroma in all 19 normal ovaries showed nuclear expression of E‐cadherin but not beta‐catenin. Membranous and cytoplasmic expression was observed variously in ovarian GCT, follicles and stroma. Conclusions: We have confirmed frequent nuclear localization of E‐cadherin but not beta‐catenin in human ovarian GCT, ovarian follicles and stroma. There is no evidence of misregulated Wnt/beta‐catenin signalling (represented by nuclear expression of beta‐catenin) in human ovarian GCT. Nuclear translocation of E‐cadherin might contribute to ovarian folliculogenesis or granulosa/stromal cell differentiation.     

Observations on Graafian follicles and their oocytes during lactation and after the removal of pouch young in the marsupials Isoodon macrourus and Perameles nasuta

Ovaries from 63 bandicoots (Isoodon macrourus and Perameles nasuta) were collected in order to obtain Graafian follicles close to ovulation for light and electron microscopy. During the first 42 days of lactation (lactation c. 60 days), the follicles were less than 1.0 mm in diameter, whereas from 43 to 52 days, some animals had follicles up to 2.0 mm in diameter, or ovulation had occurred and new corpora lutea were present. This ovulation was associated with the lactation estrus that occurred in some animals. In general, the largest Graafian follicles of the bandicoots were morphologically similar and resembled those of many other mammals. These follicles protruded from the surface of the ovary and revealed a conspicuous theca interna. The granulosa cells exhibited an unusual feature in that they contained masses of glycogen, often associated with lipid droplets and filaments. The oocytes were similar in size (diameter c. 150 μm) to those of some other marsupials and were surrounded by a zona pellucida and cumulus cells attached to the granulosa layer. The cumulus cells did not form a corona radiata as in eutherian mammals. The oocyte nuclei were somewhat flattened, peripherally located and similar in size (c. 40 × 19 μm) to those in other marsupials. These nuclei, which stained lightly with Azure A and were electron-lucent and homogeneous, were unusually irregular in contour. The nuclei were unique in that nucleoli were always absent. Small cytoplasmic bodies which may have been extruded nucleoli were found in the oocytes of I. macrourus, but not in P. nasuta. The cytoplasm in the bandicoot oocytes resembled that of other marsupials and some eutherians in that it was highly vacuolated with most of the organelles concentrated peripherally. Within the central region of the bandicoot oocytes there were crystalloids which were similar to those in oocytes of primordial follicles and in unilaminar blastocysts of I. macrourus.     

The presence of midkine and its possible implication in human ovarian follicles

PROBLEM: Ovarian follicles undergo a dynamic change to provide a mature ovum, and the process involves angiogenesis, follicular cell proliferation and leukocyte recruitment. Midkine (MK) is a heparin-binding growth factor that has angiogenic, mitogenic, and chemotactic activities. In the present study, we investigated the presence of MK and its possible role in human ovarian follicles. METHOD OF STUDY: Follicular fluid (FF) and luteinized granulosa cells (LGC) were collected from women undergoing in vitro fertilization and embryo transfer. Expression of MK protein in FF was examined by Western blotting. Concentrations of MK, estradiol and oxygen in FF were measured. 5-bromo-2'-deoxyuridine (BrdU) incorporation assay was performed in LGC. Normal ovarian tissues were obtained surgically and used in in-situ hybridization of MK mRNA. RESULTS: The presence of MK protein was verified in FF. MK mRNA was expressed in both granulosa cells and theca cells of large follicles. There is a significant negative correlation between the concentrations of MK and oxygen in FF, and a significant positive correlation between the concentrations of MK and estradiol. MK promoted BrdU uptake in LGC. CONCLUSION: The present findings imply that hypoxic condition, a characteristic of growing follicles, associates with the production of MK. Given that MK is involved in granulosa cell proliferation and estradiol production in developing follicles, MK may play a role as a local regulator in the human ovary.     

Prolactin and lipopolysaccharide treatment increased apoptosis and atresia in rat ovarian follicles.

Follicular atresia is associated with the presence of increased macrophages within the follicle. What is not known is whether, in the adult rat, macrophages are instrumental in inducing apoptosis and/or atresia or whether they are simply secondary to a hormonally mediated event. As prolactin is an immunoreactive hormone and stimulates the expression of monocyte chemoattractant, the present experiments compared the effects of prolactin treatment with that of an immune challenge with lipopolysaccharide (LPS) on the invasion of macrophages into the follicular and luteal compartments of the ovary and the occurrence of apoptosis/atresia in relation to macrophage invasion. Rats were treated for 3 days with either prolactin or LPS and ovaries obtained at pro-oestrus or oestrus. Prolactin and LPS increased the number of atretic vs. healthy follicles (P < 0.008, chi2) in pro-oestrus ovaries and increased the mean number of apoptotic cells and macrophages (P < 0.05 for some groups). Macrophages were typically observed in the thecal layer, apoptotic cells in the granulosa cell layer, although 84% follicles which had macrophages within the granulosa cell layer also contained relatively high numbers of apoptotic nuclei. Prolactin and LPS treatment in vivo reduced the progesterone response to follicle stimulating hormone (FSH) (P < 0.001) in cultures of ovarian dispersates but did not inhibit the response to forskolin. In contrast, prolactin or LPS added in vitro to the cultures inhibited the progesterone response to forskolin. Results show that both prolactin and LPS increase follicular apoptosis and atresia and reduce the progesterone response to FSH.     

The influence of deep body temperatures and skin temperatures on respiratory frequency in the pig

1. The influences on respiratory frequency of ambient temperature, the temperature of the skin, the temperature and humidity of the inspired air, hypothalamic temperature, the temperature of the spinal cord, rectal temperature and some temperatures in the abdomen have been studied in the pig.2. At a constant ambient temperature the effect on respiratory frequency of heating a thermode in the hypothalamus was modified by the temperature of the skin of the trunk which was varied independently by means of a temperature-controlled coat. A cold skin inhibited panting; a warm skin enhanced panting. The effect of heating a thermode over the spinal cord was similarly modified by skin temperatures.3. Simultaneous heating of thermodes in the hypothalamus and spinal cord increased respiratory frequency more than heating either alone, and in a warm environment the rectal temperature influenced the extent to which respiratory frequency increased on heating the thermodes.4. Cooling the thermodes decreased respiratory frequency in a warm environment and the cooling of one thermode enhanced the effect of cooling the other.5. At a constant trunk skin temperature the effect on respiratory frequency of heating the thermode in the hypothalamus depended on ambient temperature.6. Changing the temperature of thermodes in the abdomen did not affect respiration nor was there any evidence that the temperature and humidity of the inspired air had a direct effect on respiration.     

Primordial and pre-antral follicles are not commonly observed in IVF aspirates

BACKGROUND: Follicular fluid recovered from IVF patients has been proposed to be a valuable source of pre-antral and primary follicles for patient therapy and research. We evaluated the recovery of immature follicles in follicular fluid from 54 patients undergoing IVF using several techniques. METHODS: Fluid from each patient underwent several methods of follicle recovery including: filtration through a cell strainer, Ficoll-Paque density gradient, isolate density gradient, histological slide preparation, and enzymatic digestion with collagenase and DNase. RESULTS: 34 primordial and primary follicles, mean 0.63 +/- 0.27/patient, and 14 pre-antral follicles, mean 0.26 +/- 0.14/patient, were found in this study. The serum estradiol level on the day of HCG injection was significantly lower (P < 0.05) in patients in which immature follicles were recovered, compared with those without immature follicles in the follicular fluid (1779.9 +/- 167.6 versus 2246.6 +/- 153.2 pg/ml). There were no women with advanced maternal age (>39 years) who had immature follicles in the follicular fluid. CONCLUSIONS: Follicular fluid cannot be considered an efficient or reliable source of immature follicles. The presence of any immature follicles appears to be associated with cause of infertility, the random placement of the aspirating needle and may be related to the age of patient.     

Course of structural changes in the microcirculation of growing and atretic ovarian follicles

Synchronized populations of growing and atretic vesicular follicles were obtained in the ovaries of prepubertal mice by injection of serum gonadotrophin. Light-optical and ultrastructural analysis by morphometric methods revealed definite correlation between the size of the follicles and the degree of their vascularization during growth and atresia. The results suggest that the microcirculation plays the leading role in the initiation of atresia.Department of Histology and Embryology of the Pediatric Faculty, and Laboratory of Electron Microscopy of the Research Institute, N. I. Pirogov Second Moscow Medical Institute, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 89, No. 2, pp. 229–232, February, 1980.     

Computerized detection and recognition of follicles in ovarian ultrasound images: a review

Observing changes in females?? ovaries is essential in obstetrics and gynaecological imaging, e.g., genetic engineering and human reproduction. It is particularly important to monitor the dynamics of ovarian follicles?? growth, as only fully mature and grown follicles, i.e., the dominant follicles have a potential to ovulate at the end of a follicular phase. Gynaecologists follow this process in two dimensions, but recently three-dimensional (3-D) ultrasound examinations are coming to the fore. This paper surveys the existing computer methods for detection, recognition, and analyses of follicles in two-dimensional (2-D) and 3-D ovarian ultrasound recordings. Our study focuses on the efficiency, validation, and assessment of proposed follicle processing algorithms. The most important processing steps were identified in order to compare their performances. Higher ranking solutions are suggested for the so-called best algorithm for 2-D and 3-D ultrasound recordings of ovarian follicles. Finally, some guidelines for future research in this field are discussed, in particular for 3-D ultrasound volumes.     

Immunohistochemical localization of gamma-enolase in normal human tissues other than nervous and neuroendocrine tissues

The neuron-specific enolase, gamma-enolase, is present at high concentrations in tissues of the nervous and neuroendocrine systems and at significant levels in other human tissues as detected by enzyme immunoassay. Its precise localization, however, has remained unclear. We report here the immunohistochemical localization of gamma-enolase in normal adult human tissues other than those of the nervous and neuroendocrine systems using direct and indirect enzyme-labeled antibody methods. The gamma-enolase was found in such smooth muscle cells as the media of aorta, fibromuscular tissue of the prostate, and the myometrium of the uterus, myoepithelial cells, the conducting system of heart, epithelial cells of loops of Henle, and macula densa cells of the kidney. It was also demonstrated in spermatogonia, lymphocytes, plasma cells, platelets, and megakaryocytes and in lesser amounts in bronchial epithelial cells and type II alveolar epithelial cells of the lung, and in secretory cells of the fallopian tube. The significance of its presence in these cells and the application of the gamma-enolase detection for diagnostic purposes in pathology are discussed.