Fate of the theca interna following ovulation in the ewe

Summary The fate of the theca interna after ovulation was studied in ewes, using light and electron microscopic histology and histochemistry. At the time of ovulation the theca interna was incorporated, apparently completely, into the margin of the developing corpus luteum and into the centres of many infoldings of the follicular wall. There was no evidence of degeneration of the more highly differentiated theca interna cells at or following the time of ovulation. Within 24 h of ovulation, cells derived from the theca interna began migrating from their original sites into the deeper, granulosa-derived areas of the luteal tissue. At later stages cells derived from the theca interna remained concentrated in septa derived from the follicular infoldings, but were also widely distributed throughout the luteal tissue. Structural evidence supported the view that the small luteal cells and fibroblasts of the corpus luteum were derived from the theca interna, and the large luteal cells from the membrana granulosa.The authors wish to thank Mrs. Linda Musk and Miss Anneke Veenstra for skilled technical assistanceDeceased on May 4, 1979     

In-vitro effects of angiotensin II on steroid production by hamster ovarian follicles and on ultrastructure of the theca interna

Summary Angiotensin II (AII) is present in the mammalian ovary and has been correlated with atresia in follicles. Since the theca interna may be one site at which atresia is intiated, we wished to determine whether AII exerts an effect on theca interna from explanted ovarian follicles of hamsters. Hamsters were sacrified on the morning of proestrus, and ovaries were removed. Preovulatory follicles were excised from the ovaries, and cultured with one of the following components: medium alone (control); medium plus AII (1x10^-6 M); the AII-receptor antagonist [Sar¹, Ile⁸] AII (1x10^-4 M); or AII plus antagonist. After 72 h, the follicles were processed for transmission electron microscopy (to determine quantities of theca interna organelles involved in the steroid synthetic pathway) or for protein determination (to normalize steroid production rates). The incubation medium was drawn off and analyzed by radioimmunoassay for progesterone, androstenedione, or estradiol-17. There was a significant positive correlation (r=0.92, P<0.01) between follicular androstenedione secretion and area comprising theca interna smooth endoplasmic reticulum. In the theca interna, AII induced a two-fold and 1.6-fold increase in lipid droplet number and area comprising smooth endoplasmic reticulum, respectively (P<0.05). Excess antagonist negated the increase in cell or-ganelles and also reduced androstenedione secretion compared with AII alone (P<0.05). Most importantly, AII significantly augmented the ratio of androstenedione: estradiol-17 secretion by 44% over that of control. The ultrastructural changes observed in this study and the increase in the andostenedione: estradiol-17 production ratio are consistent with atresia-like changes in ovarian follicles. We believe, therefore, that AII is involved, possibly at its membrane receptor, in an aspect of the overall process of follicular atresia, operating in part at the level of the theca interna.     

Structural changes occurring during atresia in sheep ovarian follicles

Summary The structural changes that characterize primary, secondary and tertiary atresia in sheep Graafian follicles have been studied by means of histological, histochemical and ultrastructural techniques.In primary atresia vacuoles representing swollen endoplasmic reticulum are prominent along the antral border together with disorganized granulosa cells containing pyknotic nuclei. Phagocytic cells, which increase in number as atresia progresses, were seen within the membrana granulosa and are considered to be transformed granulosa cells. Even in follicles classified as nonatretic, a few antral vacuoles and occasional pyknotic nuclei are present.During secondary atresia there is a large increase in the number of cells with pyknotic nuclei; many of these nuclei had been extruded and had fused to form the characteristic Feulgen-positive atretic bodies found along the edge of the antral cavity. These bodies usually have a diameter of up to 15 m but occasionally reached as much as 400 m. A second area of degeneration is frequently present in the membrana granulosa, two or three cell layers from the basal lamina, and it is at this level that exfoliation of granulosa cells occurs in tertiary atresia. In contrast to the membrana granulosa, there are during secondary atresia, only slight indications of degeneration in the cumulus.In tertiary atresia the membrana granulosa is highly disorganized; the atretic bodies are often fewer in number than at earlier stages. The basal lamina remains essentially intact. It is at this stage that the first clear signs of degeneration occur in the theca interna. Despite some disintegration of the cumulus, the integrity of the oocyte is maintained and its nucleus remains vesicular.Changes in the thecal microcirculation may play a key role in atresia: adjacent to the basal lamina of non-atretic follicles, there is a well-developed capillary network which is significantly reduced as atresia progresses.The authors are greatly indebted to Dr. H.M. Dott and Mr. G.C. Foster for carrying out the analysis with the Quantimet image analysing computer. The skilled technical assistance of Mrs. Linda Collins is also gratefully acknowledged     

The fine structure of the cumulus oophorus during follicular development in sheep

Summary The cumulus and membrana granulosa of non-atretic ovarian follicles from primordial up to a stage shortly before ovulation were studied by electron microscopy.The follicular cells of primordial follicles were undifferentiated and rested on a thick basal lamina. In secondary follicles the endoplasmic reticulum had proliferated forming an anastomosing network. In early antral and antral follicles (0.5–2.0 mm dia.) the ER was composed of short cisternae, the mitochondria had elongated and gap junctions were first observed. In late antral follicles (3.0–5.9 mm dia.) gap junctions were frequent. In the cumulus the glycogen was associated with electron lucent areas whereas in the granulosa it was invariably associated with membranes. In large antral follicles large membrane bound bodies were present in the basal cells of the cumulus. At early oestrus a distinctive mitochondrial morphology was noted in the granulosa but not elsewhere in the follicles. At mid oestrus numerous annular nexuses were present in the granulosa but not in the cumulus. At late oestrus numerous lipid droplets were formed in both cumulus and granulosa, the boundary with theca interna became indistinct and the basal lamina became incomplete.Deceased     

Ultrastructure of perfusion-fixed fetal capillaries in the human placenta

Summary The ultrastructure of human placental capillaries was investigated using perfusion fixation and the freeze-fracturing technique. The capillaries have a continuous endothelium especially rich in microfilaments, whereas micropinocytotic vesicles are exceedingly scarce. The endothelial cells are connected by three types of junctions: (1) zonulae occludentes characterized by 2 to 4 focal regions of membrane contact in thin-sectioned specimens and an equal number of ridges on the membrane E-face in freeze-fractured specimens; (2) small gap junctions associated with the zonula occludens. (3) attachment plaques resembling zonulae adhaerentes in their fine structure. Endothelial cells are provided with long, circularly oriented pseudopodial extensions, which may be responsible for intermittent constrictions of the vessel lumen. These findings indicate that diaplacental transport at the level of the fetal capillary is controlled by the cytoplasm of the endothelial cells and probably occurs only to a very limited extent by way of micropinocytotic vesicles.Dedicated to Prof. Dr. Drs. h.c. W. Bargmann on his 70. birthdayWith the support of the Deutsche ForschungsgemeinschaftThe authors acknowledge the technical help of Mrs. E. Benecci, and the criticism and discussion of Drs. D.W. Fawcett and S. Ito. We are also indebted to Mr. R. Partsch (Zeiss, Inc. New York) for helping us with the goniometric study     

Lysosomal breakdown of erythrocytes in the sheep placenta

Summary The breakdown of erythrocytes within the lysosomal apparatus of trophoblastic epithelial cells of the sheep placenta was studied at the ultrastructural level. Acid phosphatase activity could be demonstrated in the interspace between the erythrocyte membrane and the lysosomal membrane, but not inside ingested erythrocytes. The erythrocyte plasma membrane remained observable until the final stage of the breakdown process. Together with a peripheral layer of indigestible hemoglobin it might form a barrier for further penetration of lysosomal enzymes into the ingested erythrocyte. The hemoglobin of the erythrocyte is suggested to diffuse through the erythrocyte plasma membrane into the interspace between this membrane and the lysosomal membrane. Subsequently, the hemoglobin is digested in the interspace or in fragments pinched off from erythrocyte-containing lysosomes (=erythrolysosomes). The fragmentation of erythrolysosomes is considered to be the most efficient mechanism for the breakdown of red blood cells in the trophoblastic epithelium of the sheep placenta. The method of entry of hydrolytic enzymes into erythrocyte-containing phagosomes is discussed.     

Fusion of erythrolysosomes in epithelial cells of the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the breakdown of erythrocytes within complex erythrolysosomes was studied at the ultrastructural level.It was found that the formation of complex erythrolysosomes containing from two to several erythrocytes as a result of fusion of erythrolysosomes within the epithelial cells was a common occurrence when the epithelial cells engulfed a large number of erythrocytes. The erythrocytes enclosed in complex erythrolysosomes appear to be either in the same or in different stages of hemolysis.In the process of breakdown of erythrocytes within complex erythrolysosomes five successive stages of hemolysis could be distinguished. Acid phosphatase activity was demonstrated in the complex erythrolysosomes and appeared to be located in the angular interspaces between the erythrocytes and the lysosomal membrane. The fragmentation of complex erythrolysosomes with formation of small hemoglobin-containing lysosomes also occurred.The fusion of erythrolysosomes with formation of complex erythrolysosomes can be considered as an additional mechanism in the process of erythrocyte breakdown in the epithelial cells of the sheep placenta.     

Immunocytochemical localization of albumin in ovarian follicles of fertile rats

Summary The purpose of this study was to investigate whether albumin (Alb) can be detected in ovarian rat granulosa cells. Using immunocytochemistry and morphometrics, the percentages of Alb-positive follicles (follicle-index), of Alb-positive granulosa cells (granulosa-index), and of strongly reacting follicles (intensity-index) were evaluated in intact and regressing follicles of different diameter groups during different stages of the estrous cycle. In intact follicles, the follicle- and the granulosa-index increased from small-sized to large-sized follicles. Although the follicle-index did not change in any group during the stages of the estrous cycle, the granulosa-index was higher during proestrus than during the other stages. Intact follicles showed a stronger immunoreactivity than regressing follicles throughout the stages of the estrous cycle. Thus, Alb may be a requirement for the control of follicle growth in fertile rats. This Alb function may be attributable to Alb binding to specific cell-membrane components followed by the intracellular uptake of Alb-bound substances.Supported by a DFG grant no. Sp 232/2-2     

Final stages of erythrophagocytosis in the sheep placenta

Summary In trophoblastic epithelial cells of the sheep placenta the final stages of erythrocyte breakdown within the lysosomal apparatus were studied at the ultrastructural level.As a result of hemoglobin digestion lysosomes containing hemoglobin-derived pigments (HDP) were formed. The HDP-lysosomes were acid phosphatase-positive, highly electron-dense bodies of round to irregular shape containing whorled membranous formations. The accumulation of these lysosomes in epithelial cells led to fusion resulting in the formation of conglomerates. At the end of the gestation period the amount of HD Plysosomes and their conglomerates markedly increased.In addition to erythrocytes the trophoblastic epithelial cells in the erythrophagocytic regions phagocytosed maternal leukocytes and neighbouring epithelial cells and giant cells.By gradual accumulation of HDP-lysosomes and remnants of phagocytosed cells, highly electron-dense acid phosphatase-positive residual bodies of variable appearance were formed within the epithelial cells.At the end of pregnancy the spaces between juxtaposed villi of the trophoblastic epithelium in the erythrophagocytic zones were occluded by apposition of the epithelial cells. In these occluded regions an increase in highly electron-dense large-sized residual bodies (15–22 m of dimension) occurred as a result of multiple cell phagocytosis in combination with fusion. In these residual bodies the numerous incorporated HDP-lysosomes and the remnants of phagocytosed cells could still be recognized.     

The three-dimensional structure of the zona pellucida in growing and atretic ovarian follicles of the mouse

Summary The present study provides further details on the fine-structural three-dimensional architecture of the zona pellucida (ZP) in growing and atretic follicles of mice by use of ruthenium red in combination with the detergents Triton X100 and saponin. These detergents were used for extraction of the soluble fraction of the zonal proteins in an attempt to expose the structural zonal glycoproteins, which in turn can be viewed as minute three-dimensional networks upon transmission- and scanning electron-microscopic examination. By use of these methods, the ZP of growing follicles appeared to be formed by interconnected filaments which also bind to globular structures building up a three-dimensional lattice. In contrast, the ZP of stage I as well as other (II and III) stages of atretic follicles showed a structure characterized by the presence of closely packed granules connected with short filaments to form a close-mesh reticulum. This structural change of the ZP, which in the present study is also associated with the disappearance of gap junctions within the granulosa and cumulus cell population, might represent one of the early events involved in the onset of atresia. These changes, most probably depending on an altered secretory activity of both oocytes and follicle cells, might lead to a degradation of the ZP network structure and to its subsequent increased density (condensation). All these morphodynamic events eventually contribute to a sequestration of the oocyte in the early stage of atresia.     

Further studies of the surface epithelium covering preovulatory rabbit follicles with special reference to lysosomal alterations

Summary The fine structure of the surface epithelium over preovulatory rabbit follicles was examined parallel with visualization of acid phosphatase at the electron microscopical level. Small enzyme positive vesicles were pinched off from the Golgi cisternae and similar vesicles fused and got incorporated into larger lysosomes of dense body type. Some lysosomes appeared in direct continuity with tubular enzyme positive structures. Other possible ways of increase of the lysosomal pool are indicated and discussed.As in previous studies a maximal accumulation of lysosomes was found in the apical epithelium at 8 h after an ovulatory dose of human chorionic gonadotrophin (HCG); thereafter a gradual loss of lysosomes ensued. Before the lysosomes disappeared from the surface epithelium they changed in character. They became more electron-lucent and revealed a fine-fibrillar matrix. Dense bodies deep in the cell interior appeared to communicate with each other and with the extracellular space below the surface epithelium. Openings were never seen towards the peritoneal cavity. The loss of lysosomal content from the apical surface epithelium before follicle rupture appeared in many respects similar to the histamine release process in mast cells.The findings support our working hypothesis that the surface epithelium over Graafian follicles is an essential source of proteolytic enzymes and that these may be released extracellularly and actively contribute to the dissolution of the follicular apex before rupture.This investigation was supported by grants from the Swedish Medical Research Council (Projects No. B74-12X-78-09C and B75-12X-78-10A). The technical assistance of Miss Ingalis Fransson and Miss Kerstin Nilsson is greatly appreciated     

Relationships of changes in ultrasonographic image attributes to ovulatory and steroidogenic capacity of large antral follicles in sheep

Ovarian steroidogenesis and antral follicular development in ewes, following the treatment with medroxyprogesterone acetate (MAP) and equine chorionic gonadotrophin (eCG), are affected by the reproductive season. The objective of this study was to compare the ultrasonographic attributes of large antral follicles between cyclic (December) and seasonally anovular (June–July) ewes, after a 12-day treatment with MAP-soaked intravaginal sponges, with or without the administration of 500 IU of eCG at sponge removal, and to determine whether there is a correlation between the ultrasonographic attributes of the follicular wall and serum concentrations of oestradiol. Digital images of ovulatory follicles from cyclic ewes and eCG-treated anoestrous ewes (n = 34 follicles), and of anovulatory follicles attaining ≥5 mm in control anoestrous ewes (n = 8 follicles), were analysed using the spot and line techniques designed to determine the echotextural characteristics of the follicular antrum (central and peripheral), follicular wall and perifollicular ovarian stroma. The mean diameter of ovulatory follicles was greater (P < 0.001) in cyclic than anoestrous ewes, with or without the eCG treatment. The mean pixel heterogeneity (SD of numerical pixel values) of the follicular antrum (P < 0.05), as well as mean pixel intensity and heterogeneity of the peripheral antrum, follicular wall proper and perifollicular ovarian stroma (P < 0.05), were consistently greater in anoestrous than cyclic ewes at the time of sponge removal and 24 h after the treatment with MAP sponges or MAP/eCG. Mean oestradiol concentrations were greater (P < 0.05) in cyclic compared to anoestrous ewes in both MAP- and MAP/eCG-treated animals, from 1 to 2 days after sponge withdrawal. There was a moderate negative correlation (r² = 0.12, P < 0.05; Pearson's Product Moment and r² = 0.23, P < 0.05; ANCOVA) between mean pixel heterogeneity (standard deviation of mean pixel values) of the follicular wall proper (all follicles ≥5 mm in diameter) and serum concentrations of oestradiol after sponge withdrawal. Our results indicate that large antral follicles from cyclic and seasonally anovular ewes exhibit distinctive ultrasonographic characteristics. The differences in follicular echotexture appear to be related mainly to seasonal variations in ovarian follicular morphology and oestradiol production.     

Effect of holding medium, temperature and time on structural integrity of equine ovarian follicles during the non-breeding season

The objective was to evaluate the efficiency of phosphate-buffered saline (PBS) and Minimum Essential Medium (MEM) during the transport of equine preantral and antral follicles at various temperatures and incubation interval. Equine ovaries (n = 10) from an abattoir were cut into 19 fragments; one was immediately fixed in Bouin's solution (control) and the other fragments were placed in PBS or MEM solution at 4, 20, or 39 °C for 4, 12, or 24 h. After the respective incubation periods, all fragments were fixed in Bouin's solution for 24 h and then submitted to standard histologic analysis. In total, 2567 ovarian follicles were analyzed, including 1752 primordial, 764 primary, 34 secondary and seven antral follicles. Relative to the control group, the transport of equine ovarian fragments in both solutions significantly reduced the percentage of morphologically normal follicles with increasing time and temperature. At 4 °C for 4 h, considering primordial and developing follicles, PBS had a higher (P < 0.05) rate (98.9%) of morphologically normal follicles than MEM, 48.7%. At 39 °C for 12 h, all follicles in both solutions were degenerated. Regarding the stage of follicular development, primordial follicles were less (P < 0.05) affected by preservation than primary and secondary follicles in all media, times and temperatures tested, except at 4 °C for 12 h in PBS, in which the primary and secondary follicles were less (P < 0.05) affected. Overall, 43% of antral follicles were morphologically normal when maintained in MEM at 4 °C for 4 h. In conclusion, equine follicles were successfully preserved in ovarian fragments at 4 °C in phosphate-buffered saline for up to 4 h.     

Structural alterations of rabbit ovarian follicles after mating with special reference to the overlying surface epithelium

Summary Rabbit ovarian preovulatory follicles and in particular the overlying surface epithelium were studied by morphological and ultrahistochemical means at different times after mating.By light microscopy an increase of cytoplasmic granules was found in the surface epithelium at the follicle apex 4 h after mating. The granules increased in amount and showed maximal accumulation 8–9 h after mating. They then disappeared at the same time as the connective tissue elements in the underlying tunica albuginea and theca externa disintegrated.Transmission electron microscopy showed that the membrane-bounded granules or dense bodies fused with one another and by 8 h after mating they often changed character and appeared more electron lucent. Furthermore, open communications were found between altered granules and vacuoles and between vacuoles and the extracellular space below the epithelium. Acid phosphatase reaction product was localized to the granules and Golgi cisternae. Not all the dense bodies were enzyme positive. At later stages, close to the time of follicle rupture, the epithelial cells were attenuated and thin, with only a few granules.By scanning electron microscopy it was found that the epithelial cells at the follicle apex increased in size approaching the time of follicle rupture and that their microvilli decreased in number and in size. At 8 h and later, the contours of intracellular granules could be visualized.The results of this study were similar to those found when rabbits were induced to ovulate by HCG-stimulation. This further strengthens the hypothesis that the surface epithelium contributes proteolytic enzymes which help to disintegrate the follicle apex prior to rupture.     

Latent antithrombin does not affect physiological angiogenesis: an in vivo study on vascularization of grafted ovarian follicles

Latent antithrombin (L-AT), a heat-denatured form of native antithrombin (AT), is a potent inhibitor of pathological tumor angiogenesis. In the present study, we have investigated whether L-AT has comparable antiangiogenic effects on physiological angiogenesis of ovarian tissue. For this purpose, preovulatory follicles of Syrian golden hamsters were mechanically isolated and transplanted into dorsal skinfold chambers chronically implanted in L-AT- or AT-treated hamsters. Non-treated animals served as controls. Over 14 days after transplantation neovascularization of the follicular grafts was assessed in vivo by quantitative analysis of the newly developed microvascular network, its microvessel density, the diameter of the microvessels, their red blood cell velocity and volumetric blood flow as well as leukocyte-endothelial cell interaction using fluorescence microscopic techniques. In each group, all of the grafted follicles were able to induce angiogenesis. At day 3 after transplantation, sinusoidal sacculations and capillary sprouts could be observed, finally developing complete glomerulum-like microvascular networks within 5 to 7 days. Overall revascularization of grafted follicles did not differ between the groups studied. Interestingly, follicular grafts in L-AT- and AT-treated hamsters presented with higher values of microvessel diameters and volumetric blood flow, when compared to non-treated controls, which may be best interpreted as a reactive response to an increased release of vasoactive mediators. In conclusion, the present study demonstrates, that L-AT has no adverse effects on physiological angiogenesis of freely transplanted ovarian follicles. Thus, L-AT may be an effective drug in tumor therapy, which blocks tumor growth by selective suppression of tumor vascularization without affecting new vessel formation in the female reproductive system.     

Ultrastructure of the pars intermedia of the adult sheep hypophysis

Summary Light microscopy of coronal sections of the sheep pars intermedia revealed a compact, incompletely lobulated V-shaped region about 15–20 cells thick, situated between the pars distalis and the pars nervosa. A prominent hypophysial cleft and follicles containing a colloid-like substance were seen.Using electron microscopy, five cell types could be distinguished: pars intermedia glandular cells, pars distalis-like glandular cells, interstitial cells, follicular cells and cleft lining cells. The polyhedral to pear-shaped pars intermedia glandular cells predominated. They contained dense-cored, membrane-bound granules near the Golgi complex, and larger, irregular vesicles with finely granular contents of varying electron density throughout the remaining cytoplasm; exocytotic release of granules was occasionally observed. Smaller numbers of cells resembling those seen in the pars distalis were scattered throughout the pars intermedia. Interstitial cells usually possessed elongated cytoplasmic processes which extended between the glandular cells, and were characterized by deeply indented nuclei, elaborate junctional complexes and an absence of cytoplasmic granules. Cells lining the follicles resembled the interstitial cells. The major cells bordering the hypophysial cleft were triangular in section and bore irregular microvilli on their free surface. The pars intermedia appeared to be less vascular than the remainder of the hypophysis and only occasional fenestrated capillaries were seen. Nerve profiles were rare.     

Influence of vitrification techniques and solutions on the morphology and survival of preantral follicles after in vitro culture of caprine ovarian tissue

The objective was to compare the efficiency of various vitrification techniques and solutions for preserving morphology and viability of preantral caprine follicles enclosed in ovarian tissue. Fragments of ovarian cortex were cryopreserved by conventional vitrification (CV) in French straws, vitrification in macrotubes (MTV), or solid-surface vitrification (SSV). Six solutions containing 6 M ethylene glycol, with or without sucrose (SUC; 0.25 or 0.50 M) and/or 10% fetal calf serum (FCS) were tested (Experiment I). After 1 wk, samples were warmed and preantral follicles were examined histologically. To evaluate follicular viability (Experiment II), ovarian fragments were vitrified with the three techniques listed above, in a solution containing 0.25 M SUC and 10% FCS. After warming, follicles were assessed by the trypan blue dye exclusion test. In Experiment III, preantral follicles enclosed in ovarian tissue were vitrified using the protocol which yielded the highest percentage of viable preantral follicles (SSV with 0.25 M SUC and 10% SFB). After warming, the preantral follicles enclosed in ovarian tissue were cultured in vitro and then, were analyzed by histology and fluorescence microscopy (calcein-AM and ethidium homodimer-1). Every vitrification protocol significantly reduced the percentages of morphologically normal follicles relative to the control (88.0%); however, the addition of 0.25 M SUC and 10% FCS to the vitrification solution improved preservation of follicular morphology (67.4, 67.4, and 72.0% for CV, MTV, and SSV, respectively). Although follicular viability after SSV (80.7%) did not differ from that in fresh (non-vitrified) ovarian tissues (88.0%), after in vitro culture, percentages of viable follicles were significantly reduced (70.0%). Percentages of morphologically normal follicles after in vitro culture of vitrified ovarian tissue were similar (76.0%) to those in ovarian cortex fragments cultured without previous vitrification (83.2%). In conclusion, SSV using a solution containing 0.25 M SUC and 10% FCS, was the most efficient method for vitrifying caprine ovarian tissue.     

Ultrastructure of the vitelline follicles of Gorgoderina vitelliloba (Trematoda: Gorgoderidae)

An electron microscope study of the vitelline follicles of Gorgoderina vitelliloba indicates that they contain vitelline cells in various stages of development. Juvenile cells are small and characterised by a little cytoplasm. During differentiation a large amount of granular endoplasmic reticulum develops. In more mature cells, indistinct Golgi complexes give rise to globules of shell protein which migrate to form clusters at the periphery of the cell. Further maturation results in the appearance of large lipid bodies in the vitelline cell cytoplasm.Developing vitelline cells are ensheathed by nurse cell cytoplasm containing numerous small vacuoles which appear to be derived from smooth endoplasmic reticulum. It is suggested that nurse cells may have a role in selection and transport of nutrient material for vitelline cells and that they manufacture precursors of lipid which is subsequently stored as a food reserve in mature vitelline cells. Possible transport sites between parenchymal cells and nurse cells were identified.     

Ovulation and the mechanism of follicle rupture

Summary The rabbit Graafian follicles are encircled by a capillary network between the theca interna and the avascular membrana granulosa. After injection of an ovulatory dose of human chorionic gonadotrophin (HCG) the theca interna cells showed an increase in the amount of smooth endoplasmic reticulum, lipid droplets and mitochondria with tubular cristae. In addition, considerably more junctions, similar to the abutment nexuses of granulosa cells were found; annular nexuses also appeared. At 4 hours after injection of HCG a prominent oedema was evident in the theca interna layer, particularly in the apical region.Small fenestrations in the endothelium of the blood capillaries increased in amount after HCG injection, and close to the time of ovulation, large gaps or perforations, 1–3 in diameter, were found in the thin, distended part of the endothelial cells. The surrounding basement membrane became fragmented and partly lost, so that a seemingly free passage from the capillary lumen to the interstitium was eventually established. Leakage of fluid, causing interstitial oedema, presumably proceeds until the pressure in the pericapillary interstitium has risen to the pressure in the capillaries. Some hours before and up to ovulation the pericapillary interstitium has also broad communications with the cavity of the follicles. Therefore, both pressure and fluid can be passed from the capillaries-via the interstitium-to the follicle antrum. However, influx of fluid with subsequent follicle expansion and ovulation-at constant pressure-does not occur until the tensile strength of the follicle wall has decreased.This investigation was supported by grants from the Swedish Medical Besearch Council (Projects No. B72-12X-78-07A, B73-12X-78-08B and B74-12X-78-09C). The technical assistance of Miss Ingalis Fransson, Miss Kerstin Nilsson, and Mrs. Ulla-Britt Westman is greatly appreciated.     

Effect of methanol and Me2SO exposure on mitochondrial activity and distribution in stage III ovarian follicles of zebrafish (Danio rerio)

In this study the effect of cryoprotectants that have been shown to be the least toxic to zebrafish ovarian follicles (methanol and Me₂SO), on mitochondria of stage III ovarian follicles was evaluated. The mitochondrial distributional arrangement, mitochondrial membrane potential, mtDNA copy number, ATP levels and ADP/ATP ratios were assessed following exposure to cryoprotectants for 30 min at room temperature. Results obtained by confocal microscopy showed that 30 min exposure to 2 M methanol induced a loss of membrane potential, although viability tests showed no decrease in survival even after 5 h post-exposure incubation. Higher concentrations of methanol (3 and 4 M) induced not only a decrease in mitochondrial membrane potential but also the loss of mitochondrial distributional arrangement, which suggested a compromised mitochondrial function. Furthermore 3 and 4 M treatments resulted in a decrease in viability assessed by Fluorescein diacetate–Propidium iodide (FDA–PI) and in a decrease in mtDNA copy number and ADP/ATP ratio after 5 h incubation following methanol exposure, indicating a delayed effect. The use of Me₂SO, which is considered to be a more toxic CPA to zebrafish ovarian follicles than methanol, caused a decrease in viability and a sustained decrease in ATP levels accompanied by failure to maintain mtDNA copy number within 1 h post-exposure incubation. These results indicated that even CPAs that are considered to have no toxicity as determined by Trypan blue (TB) and FDA–PI tests can have a deleterious effect on mitochondrial activity, potentially compromising oocyte growth and embryo development.