The isolated perfused rabbit ovary — A model for studies of ovarian function

Summary In the present investigation the ultrastructure of isolated rabbit ovaries, perfused with different media for various time periods, was studied. The steroid hormone production by the perfused ovary was also determined. Perfusion with Medium 199 results in prominent interstitial ovarian oedema which increases with perfusion time. Even after the addition of 6–10 % Dextran T40, oedema appears in the interstitial tissue of the ovary. Perfusion solutions with osmotically active colloid particles of large molecular size (Dextran T70; average molecular weight 70,000 and bovine serum albumin), cause less distortion in the ovarian structure, and ultrastructurally the ovarian tissues appear essentially the same as in the control ovaries.The results indicate that the perfused rabbit ovary, under strictly controlled conditions, can be used as an experimental model for studies of various aspects of ovarian function, including follicular rupture.     

Ovulation and the mechanism of follicle rupture

Summary The effects of penicillin and chlortetracyline HCl on the fine structure of the intracellular symbiotes of the pea aphid were studied in an attempt to remove the symbiote population. High penicillin concentrations, 1% and 0.1%, caused symbiote breakdown but were toxic and/or repellent to the aphids; at 0.1% specific effects were observed on the symbiotes' cell walls. After the use of 0.01% penicillin in the aphid diet, the symbiotes had abnormal cell walls and were abnormally dilated; however, symbiote division and transmission from one aphid generation to the next seemed unaffected and the aphids appeared normal. Aphids fed 0.1% chlortetracycline failed to reproduce. After 7 days, their symbiotes were found to break down at a high rate but aphid mitochondria were also adversely affected at this stage. Following 0.002% chlortetracycline, the aphids produced aposymbiotic progeny with apparently normal mitochondrial populations; these larvae failed to develop.Research supported by the College of Agricultural and Life Sciences, University of Wisconsin, and by a research grant (GB 31904 X) from the National Science Foundation.     

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     

Studies on the morphology of the isolated perfused rabbit ovary

Summary Ovulation was induced in rabbits by intravenous administration of human chorionic gonadotrophin (HCG), and 4–5 h later the ovaries were isolated and introduced into an in-vitro perfusion system containing synthetic medium with albumin. Rupture of follicles occurred in vitro within the physiological time range (mean 11.3 h after injection of HCG), although with a reduced frequency. Preovulatory and ruptured follicles were studied in detail by light and electron microscopy.In the granulosa layer of ruptured or preovulatory follicles cytoplasmic blebbing activity, disappearance of CallExner bodies and differentiation toward luteinized cells were found. Perhaps the most important sign of normal preovulatory development in vitro was that the basement membrane surrounding the granulosa layer was penetrated by projections of granulosa cells. In the absence of this penetration phenomenon the granulosa layer prolapsed out of the follicle. Immediately before rupture, follicles showed marked degeneration, restricted to the outer layers of the apical wall, which is compatible with the hypothesis that degradative enzymes are released close to the surface of preovulatory follicles.Although the majority of follicles that ovulated under in-vitro conditions showed the same kind of morphological alterations as can be seen in vivo, occasional atypical ruptures occurred without any overt signs during perfusion. Also technical manipulations of the perfusion system, e.g., nonphysiological increase of perfusion pressure, could force follicles to rupture. This illustrates the importance of careful morphological study of all ovaries perfused in vitro before conclusions are drawn.     

Studies on the morphology of the isolated perfused rabbit ovary

Summary Isolated ovaries from untreated, sexually mature rabbits were introduced into an in vitro perfusion system and perfused with a chemically defined medium containing albumin. The ovaries were perfused for up to 15 h (mean 11.5 h) and then processed for morphological investigation. Both at the light- and electron-microscopical levels, most of the ovaries exhibited a normal structure comparable with ovaries in situ. In two cases, however, marked accumulations of bacteria were found, although not inside the follicles.Since ovulation in the rabbit normally occurs between 9.5–13 h after mating or human chorionic gonadotrophin treatment, this model seems adequate for studies of ovulation in vitro. It is, however, important to study the ovaries microscopically after the perfusion to detect artifacts, e.g., bacterial infection, that may have influence on the process of ovulation.     

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     

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.     

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     

促排卵周期中未破裂黄素化卵泡综合征与性激素的关系探讨

目的:对未破裂黄素化卵泡综合征(LUFS)周期及正常排卵周期患者在不同时间性激素进行测定与比较,探讨LUFS的发生与性激素的关系。方法:对自然周期有排卵障碍的患者使用克罗米芬+HCG促排,分别在月经周期的第3天(C3)、第10天(C10)、卵泡成熟日(CM)(卵泡直径≥18mm)、尿LH阳性或注射HCG后48小时(CL)进行性激素测定,根据患者是否有排卵分为:正常排卵组(A组);未排卵出现LUFS组(B组)。选择同期自然周期正常排卵者为对照组(C组),同上法进行性激素测定。对A、B、C组患者不同时间的血清性激素进行比较。结果:1、FSH:在CM日A、B两组的促卵泡成熟激素(FSH)均低于C组(P<0.01)。2、LH:在C3、C10、CL日测定的A、B组LH值均高于C组(P<0.01),在CM日测定的A、B组LH值低于C组(P<0.01)。3、P:在CM日A、B两组的孕酮值低于C组(P<0.01);4、E2:在C3日测定的B组E2值低于C组(P<0.01),在CL日测定的B组E2值高于C组(P<0.01);5、PRL:在不同时间各组的PRL值均无显著性差异(P>0.05)。6、T:在不同时间各组的T值均无显著性差异(P>0.05)。结论:性激素在LUFS的发生中起重要作用,但以C3日E2值与LUFS的发生的关系最大,E2值低者易发生LUFS。     

Ultrastructural characterization of porcine oocytes and adjacent follicular cells during follicle development: lipid component evolution

The objective of this study was to characterize the morphometry and ultrastructure of porcine preantral and antral follicles, especially the lipid component evolution. Ovarian tissue was processed for light microscopy. Ovarian tissue and dissected antral follicles (< 2, 2-4, and 4-6 mm) were also processed for transmission electron microscopy using routine methods and using an osmium-imidazole method for lipid detection. Primordial follicles (34 ± 5 μm in diameter, mean ± SD) had one layer of flattened-cuboidal granulosa cells around the oocyte, primary follicles (40 ± 7 μm) had a single layer of cuboidal granulosa cells around the oocyte, and secondary follicles (102 ± 58 μm) had two or more layers of cuboidal granulosa cells around the oocyte. Preantral follicle oocytes had many round mitochondria and both rough and smooth endoplasmic reticulum. In oocytes of primordial and primary follicles, lipid droplets were abundant and were mostly located at the cell poles. In secondary and antral follicles, the zona pellucida completely surrounded the oocyte, whereas some microvilli and granulosa cells projected through it. Numerous electron-lucent vesicles and vacuoles were present in the oolemma of secondary and antral follicles. Based on osmium-imidazole staining, most of these structures were shown to be lipid droplets. As the follicle developed, the appearance of the lipid droplets changed from small and black to large and gray, dark or dark with light streaks, suggesting that their nature may change over time. In summary, although porcine follicles and oocytes had many similarities to those of other mammalian species, they were rich in lipids, with lipid droplets with varying morphological patterns as the follicle developed.     

Ultrastructure of the theca interna of ovarian follicles in sheep

Summary The theca interna of non-atretic ovarian follicles from 2.0 mm in diameter up to the stage shortly following ovulation was studied by light and electron microscopy.In follicles <3.0mm in diameter, the theca interna consisted of about 8–12 layers of flattened cells, together with many capillaries and small bundles of collagen. Two main forms of cellular differentiation were seen. These were towards either fibroblast-like cells or presumed steroidogenic cells whose cytoplasm contained large amounts of predominantly smooth tubular endoplasmic reticulum, to which some ribosomes were attached. The majority of cells were of relatively undifferentiated or intermediate structure.In larger follicles up to the early stages of oestrus the theca interna cells became larger and less flattened, and cells rich in tubular endoplasmic reticulum became proportionately more numerous. By 18 h after the onset of oestrus the theca interna was oedematous, and many cells possessed pseudopodia. Many cells also contained numerous lipid droplets, but there were no signs of thecal cell degeneration or death. Shortly after ovulation the basal lamina of the membrana granulosa was incomplete, and it became more difficult to distinguish between theca and granulosa layers. Structural heterogeneity, with two major cell types and cells of intermediate structure, was present at all stages.It was concluded that: (1) the theca interna of 2.0–2.9 mm follicles contained many cells whose structure was compatible with a steroidogenic capacity; (2) changes in the differentiated thecal cells up to the early stages of oestrus were quantitative rather than qualitative, and suggestive of an increased steroidogenic capacity; (3) the accumulation of lipid in many cells of the theca interna by 18 h after the onset of oestrus probably reflected a reduction in steroidogenic activity; and (4) there was no evidence of any structural specialization to facilitate the transport of steroids from the theca interna to the membrana granulosa.     

Effect of melengestrol acetate on the bovine ovary

Summary To investigate the relation of ovarian morphology to increased body weight gain during administration of melengestrol acetate (17-acetoxy-6-methyl-16-methylenepregna-4,6-diene-3,20-dione, or MGA, a progestagen), ovaries of 100 Holstein dairy heifers were examined for characteristics of follicular maturation, signs of lipid secretion, and the presence of steroid-producing-type cell organelles. The heifers were fed either a normal or a high-protein level ration with or without 0.30–0.45 mg daily of MGA from 2.5 months of age to the age of first estrus or to breeding age (120 cm withers height). The high-level ration effect was not consistently mediated by the special development of ovarian structures. MGA effect, like the weight-gain effect, was consistently evident after the time of first estrus. MGA suppressed estrus and ovulation, and resulted in the presence in the ovary, after regression of corpora lutea, of an increased number of large follicles. The largest follicles corresponding in structure to developing proestrous, mature estrous, or early atretic follicles contained hypertrophied theca interna cells showing lipid secretion signs and ultrastructural characteristics of steroid synthesis. The majority of the largest follicles, however, were atretic or late atretic and did not contain steroid-type cells. The development of replacement follicles was evident in ovaries with late atretic largest follicles. The weight-gain effect of MGA may therefore be associated with a steroidogenic, probably estrogenic, effect in the cow.Dedicated to Prof. W. Bargmann of Kiel.This investigation was supported by scholarships of the Ministère Français des Affaires Étrangères and the Alexander von Humboldt-Stiftung to Dr. J. Priedkalns.Drs. R. G. Zimbelman and L.S. Goyings of the Upjohn Company, Kalamazoo, Michigan, and Drs. D.E. Pritchard, R.W. Purchas, H.A. Tucker, H.D. Hafs, and L.J. Boyd of the Department of Dairy Science, Michigan State University, East Lansing, Michigan, designed and conducted a study on the effects of melengestrol acetate on Holstein heifers and provided ovaries for this investigation. The author wishes to thank Dr. J. W. Lauderdale of the Upjohn Company for aid in statistical analyses; the Department of Veterinary Anatomy, University of Minnesota, St. Paul, Minnesota, and the Laboratoire d'Histologie de l'École Nationale Vétérinaire de Lyon, for assistance in the processing of the ovarian tissues for electron microscopic examination; and the Laboratoire d'Histophysiologie du Collège de France, Paris, for providing facilities for a part of the histochemical study of the ovaries.A brief report of this investigation has been presented to the Société des Sciences Vétérinaires de Lyon (Priedkalns, 1971).     

Post-coital depletion of dense-core vesicles from the external layer of the rabbit median eminence

Summary The effects of mating on the depletion of osmiophilic material from dense-core vesicles (DCV) within the external layer of the rabbit median eminence (ME) were investigated. Two different populations of DCV were demonstrated within the ME external layer of the non-mated control animals. A plot of the diameters of these vesicles showed two peaks, one at 90–100 nm, and the other at 120–130 nm. These two populations of DCV were found in separate axons and axon terminals in contact with, or near the perivascular spaces of, portal capillaries. Within these axons and terminals an occasional membrane profile or vesicle ghost was also observed.The same two populations of DCV were evident in the ME external layer of animals that were sacrificed at 10 minutes post-coitus. In these experimental animals, the number and size of the smaller population of DCV were the same as those of the none-mated controls. However, there was an obvious decrease in the number of the large (120–130nm) DCV, together with a simultaneous marked increase in the number of vesicle ghosts. These ghosts had a mean diameter of 137±14 nm. On the basis of their size, it is suggested that the vesicle ghosts represent large DCV that have been depleted of their content following mating. Ultrastructural evidence for the depletion of osmiophilic material from the large DCV of mated animals is provided.According to previous writers the smaller DCV within the ME are aminergic, whereas the larger DCV may contain releasing factors (RF). Our findings provide further morphological evidence to support this hypothesis. Furthermore, RF and biogenic amines appear to be contained within separate nerve terminals in the rabbit ME.This investigation was supported by grants to Drs. D. G. Montemurro and R. P. Singh from the Medical Research Council of Canada. The authors gratefully acknowledge the technical assistance of Miss A. Overweel and Mrs. S. Griffin.     

Ovulation rate and its relationship with follicle diameter and gene expression of the LH receptor (LHR) in Nelore cows

The objective was to determine the relationship among the diameter of ovarian follicles, ovulation rate, and gene expression of the LH receptor (LHR) in Nelore cattle. In Experiment 1, ovulation was synchronized in 53 Nelore cows. Three days after ovulation, ovaries were assessed with ultrasonography, all cows were given 6.25 mg LH im, and they were allocated into three groups, according to diameter of their largest ovarian follicle: G1 (7.0-8.0 mm); G2 (8.1-9.0 mm); and G3 (9.1-10.0 mm). For these three groups, ovulation rates were 9, 36, and 90%, respectively, (P <0.03; each rate differed significantly from the other two). In Experiment 2, granulosa and theca cells were subjected to total RNA extraction, and gene expression of the LHR was determined by RT-PCR. Follicles were allocated in three groups based on their diameter (similar to the Experiment 1), which were denoted Groups A, B, and C. Expression of the LHR gene in granulosa cells was lower in Group A than Group C (P < 0.05). However, there were no significant differences among groups in expression of the LHR gene in theca cells. We concluded that ovulatory capacity in Nelore cattle was related to increased follicular diameter and expression of the LHR gene in granulosa cells.     

Primordial follicle activation and follicular development in the juvenile rabbit ovary

Of all the stages of mammalian folliculogenesis, the primordial to primary follicle transition is the least understood. In order to gain new insights into this process, we have conducted a comprehensive morphological, morphometric and molecular study of ovarian organisation and early follicle development in the rabbit. The structure of ovaries collected from rabbits aged from 2–12 weeks (a period encompassing primordial follicle formation, activation and the first wave of folliculogenesis in this species) has been analysed by light microscopy and the follicles present have been measured and scored for their developmental stage. To establish useful molecular markers of activation, we have further classified follicles according to their expression of the proliferative marker, proliferating cell nuclear antigen, and the zona pellucida protein, ZPB. The activation of primordial follicles is initiated immediately following their formation in the rabbit ovary and is characterised by oocyte growth, granulosa cell morphogenesis and increased granulosa cell mitosis. Enhanced ZPB protein expression at the oolemma is also associated with follicle activation and development. Few primordial follicles in the juvenile rabbit ovary are lost by atresia, as assessed by the TUNEL assay. The appearance of apoptotic granulosa cells is however coincident with the development of antral follicles. This study thus describes the temporal and spatial regulation of early follicular development in the post-natal rabbit ovary and, for the first time, shows that the primordial to primary transition in the juvenile rabbit is a highly ordered process occurring within quantifiable parameters.K.J.H. was supported by the Pest Animal Control CRC and Post Graduate scholarships from the Australian National University.     

Ultrastructure and function of follicle cell in the ovary of Branchiostoma belcheri

The ultrastructure of follicle cells in the ovary at different developmental stages ofBranchiostoma has been observed in detail with a transmission electron microscope. The results indicate that only one kind of follicle cell exists with structural features related to steroid hormone biosynthesis: (i) oval or round mitochondria with tubules; (ii) smooth surfaced endoplasmic reticulum; (iii) several large lipid droplets in the cytoplasm; (iv) a well developed Golgi complex and tubular rough surfaced endoplasmic reticulum, as can be found in mammalian theca interna cells. In addition, as steroid hormone synthesizing cells, they obviously play an important role in the phagocytosis of relict gametes and cellular debris and may have a nutritive function for the oocytes. They can produce abundant secretory granules in stages III-IV ovaries. In mature ovaries they transform into flat epithelial cells with numerous microfilaments which may play a role in ovulation.     

Changes in the surface morphology of the rabbit endometrium related to the estrous and progestational stages of the reproductive cycle

Summary Changes occurring on the surface of the uterine luminal epithelium of the rabbit during the estrous and progestational stages of the reproductive cycle were examined by scanning and transmission electron microscopy. The findings demonstrate that the uterine epithelium, or endometrium, contains two cell types: ciliated cells and nonciliated, microvillous cells. In estrous animals, ciliated cells, although not very numerous, were usually observed in small groups. However, at increasing intervals of time following mating, ciliated cells progressively disappeared from the endometrium until approximately eight to ten days post coitum, when they became scare. From several hours to four to five days following mating, extensive changes occurred on the surfaces of microvillous cells. When observed by TEM, these elements contained organelles typical of cells involved in the synthesis and secretion of glycoproteins. Furthermore, microvillous cells during this period displayed numerous apical protrusions of different sizes and shapes and containing material of varying electron density. Parallel SEM examinations of the same material confirmed the presence of these protrusions. Some of the protrusions appeared as spheroidal masses attached to the cytoplasm by means of a cytoplasmic strand. Other surface masses were clearly unattached to the cell surface and were distributed (1) on the surface of microvillous cells, (2) on the cilia of adjacent ciliated cells, and (3) on the surface of spermatozoa.Changes occurring on the luminal surface during the early postcoital period are interpreted as an expression of morphodynamic processes likely involving coupled secretion (exocytosis) and resorption (endocytosis) of luminal material. The observations presented here also demonstrate that between six and ten days post coitum, the rabbit endometrium contained increasing numbers of enlarged, nonciliated cells that probably arose by the fusion of smaller, microvillous elements.The work reported here was supported by C.N.R. contracts No. CT 760128809 and CT 77014239 (to P.M.) and NIH. Grant HD-04274 (to J.V.B.)