Different phenotyes of cultured microvessel endothelial cells obtained from bovine corpus luteum

Summary Morphological heterogeneity has not been documented for cultured endothelial cells isolated from the microvascular bed of any organ. As the corpus luteum depends on a rich microvascularization, endothelial cells were dislodged from developing corpora lutea by mechanical dissection followed either by collagenase digestion or by no digestion. Cell separation was carried out by Percoll density centrifugation. Although the yield of intact cells was higher with collagenase treatment than without, successful endothelial cell cultures were only established when cells remained untreated. Viewed by light microscopy after an average lag phase of 10 days, five different phenotypes of endothelial cells were found under similar simple culture conditions: isomorphic epithelioid, polymorphic epithelioid, spindle-shaped, round, and phase-dense phenotypes. Monolayers appeared within 2–4 weeks. After an additional period of 2–4 weeks, tubular forms with a specific pattern were noted for types 1–3, the so-called pseudotubular forms for type 4, and none for type 5. Cell types differed in their cytochemical and immunocytochemical responses. Examined by SEM, type 1 displayed a more conspicuous surface anatomy than type 2. Types 3–5 demonstrated striking cell processes that were characteristic of each type. Tubular forms of types 1 and 2 showed cell borders and a marked increase in surface specializations, whereas tubular forms of type 3 lacked detectable cell borders in the absence of a striking surface anatomy. Pseudotubular forms of type 4 developed no particular spatial organization. Thus, for the first time, morphological evidence is provided that different endothelial cell types are obtained from diverse segments of the microvascular bed.     

Localization of thyrotropin receptor and thyroglobulin in the bovine corpus luteum

The receptor of the Thyroid Stimulating Hormone (TSHr) and thyroglobin (TGB), are two proteic factors necessary for the synthesis of hormones, in the thyrocite. In mammals, many immuno-histochemical reports indicate the presence of the TSHr in extra-thyroidal tissues, but not in the ovary. Triiodothyronine (T₃) and thyroxine (T₄) have been widely shown to affect ovarian functions and the synthesis of progesterone (P₄). The aim of this study was to determine if by immunohistochemistry techniques TSHr and TGB could be found in the bovine corpora haemorragica, lutea and albicantia.     

The ultrastructure of the corpus luteum of the guinea-pig

Summary An electron-microscopic investigation, based on the suggestion that differences seen in progesterone levels under differing hormonal conditions might be reflected in the ultrastructural organisation of the lutein cells of the guinea-pig was undertaken. Comparisons were made between corpora lutea taken from animals during the normal oestrous cycle, pregnancy and lactation, and after hysterectomy or hypophysectomy.The lutein cells from the oestrous cycle corpus luteum appeared to be of two types, light and dark. The former were more numerous. The main difference between them lay in the arrangement of the endoplasmic reticulum. Lutein cells from corpora lutea (with the exception of the old degenerating corpora lutea) all contained well-developed agranular endoplasmic reticulum, little granular endoplasmic reticulum, several electron-dense lipid granules, lysosomal bodies which ranged from small spherical bodies to large autophagic vesicles and mitochondria. The mitochondria were numerous, and in the corpus luteum of pregnancy, they were closely associated with the parallel arrays of granular endoplasmic reticulum.With minor exceptions, the lutein cells of the guinea-pig present a strikingly uniform picture despite their hormonal condition.The manner in which this uniformity of ultrastructure may be related to observed differences in progesterone levels in the corpus luteum of the guinea-pig is discussed.Meat and Livestock Commission (MLC) Scholar.The authors wish to thank Dr. J. S. Perry for doing the surgery involved in this work and for the specimens of corpora lutea of hysterectomy. They are also grateful to him for his helpful discussions and interest throughout.     

Ultrastructure of the reactivated corpus luteum after embryonic diapause in the marsupial,Macropus rufogriseus banksianus

Summary During embryonic diapause in the red-necked wallaby, M. r. banksianus, both the corpus luteum and uterine blastocyst remain dormant, and are reactivated following removal of the suckling pouch young (RPY). The morphology of dormant and reactivated corpora lutea has been studied throughout the 26.5 days of delayed gestation. Corpora lutea at 0, 2 1/2, 4, 9, 14, 21 and 25 days after RPY were fixed by perfusion. From day 4 to day 14 after RPY there was a progressive increase in the amount of smooth endoplasmic reticulum and the numbers of mitochondria. However there was a decrease in mitochondrial size from 1–2 m in diameter (0 days after RPY) to 0.5–1 m (14 days after RPY). Densely-staining granules (approximately 0.2 m in diameter) were first observed in the luteal cells at 4 days after RPY. The maximum density of granules was observed at 21 days after RPY. Shortly before birth (25 days after RPY) the number of secretory granules had significantly decreased and the features of cellular regression were evident. As with the eutherian mammals, the wallaby luteal cells have all the structural organelles associated with steroid hormone production. The numbers of densely-staining granules are greatest at 21 days after RPY and may reflect the luteal progesterone content since similar granules in the sheep and cow have been shown to be associated with elevated levels of progesterone.     

Immunocytochemical evidence for the presence of oxytocin and neurophysin in the large cells of the bovine corpus luteum

Summary The presence of neurophysin, oxytocin and vasopressin in the bovine corpus luteum was examined immunocytochemically. Tissue blocks of corpora lutea from pregnant and non-pregnant animals were fixed with glutaraldehyde/paraformaldehyde fixative and immunostained by the peroxidase-antiperoxidase (PAP) method. The simultaneous presence of immunoreactive oxytocin and immunoreactive oxytocin-neurophysin was demonstrated in large luteal cells of non-pregnant animals, while no staining for vasopressin or vasopressin-neurophysin was observed. None of the peptides were detected in the corpus luteum of pregnant animals. The small luteal cells were not found to be stainable at any time.     

The interphase microtubule cytoskeleton of five different phenotypes of microvessel endothelial cell cultures derived from bovine corpus luteum.

The interphase microtubule cytoskeleton of five different microvessel endothelial cell cultures, recently established from bovine corpus luteum, was analysed using anti-tubulin immunofluorescence. An antibody against acetylated microtubules detected four cell types each of which possessed a single cilia. The length of the cilia were up to 10 microns for cell types 1 and 2. Ciliary stubs had a length of up to 0.37 microns in cell types 4 and 5. Cilia were missing in cell type 3. Long and short cilia were located in the perinuclear region from where cytoplasmic microtubules radiated. Cell type 3 displayed straight microtubules rather than the wavy path seen in the other cell types. The amount of tyrosinated microtubules visualized by a specific antibody was consistently higher than that of posttranslationally acetylated microtubules. The latter were more apparent in cell types 4 and 5 than in the other cell types. We conclude: Differences in the cytoplasmic microtubule inventory of each microvessel endothelial cell type points at individual functions maintained in culture.     

Lipofuscin in the corpus luteum of macaque ovaries

The present study examined the histochemistry of pigments in the corpus luteum of the ovaries of cynomolgus monkeys (Macaca fascicularis), Japanese monkeys (Macaca fuscata), and rhesus monkeys (Macaca mulatta). Yellowish brown pigments were found in the regressing corpus luteum cells. Histochemical studies revealed that these pigments consisted of lipofuscin, the so-called age pigment. The findings obtained suggest that accumulation of lipofuscin might be related to cellular aging of the corpus luteum.     

Localization of relaxin in human gestational corpus luteum

Summary Corpora lutea from 12 pregnant women were prepared for immunohistochemical localization of relaxin using a highly specific antiserum. A positive response is given by luteal cells that are diffusely distributed throughout the corpus luteum. These cells do not form a distinctive group in any particular area. A negative response is seen in the adjacent ovarian tissue, and also in nongestational corpora lutea in an early luteal phase.     

Human corpus luteum: Immunocytochemical evidence for presence of prolactin

Summary Six human corpora lutea (day 17–25) of the menstrual cycle and 4 ovarian stromal tissues from 7 cycling women were examined for the presence of the hormone, prolactin, by immunohistochemistry using the indirect peroxidase-antiperoxidase method. After mounting tissue sections of 4 m, endogenous peroxidases were removed with hydrogen peroxide and the sections were incubated for l h at room temperature followed by 16 h at 4° C with a highly specific antisera for human prolactin, nonimmunized normal rabbit serum for a control reaction, or antiserum preadsorbed with excess human prolactin for specificity determination. Following the reaction with the second antibody (goat antirabbit IgG) for l h at room temperature, prolactin was localized using peroxidase anti-peroxidase and 3.3-diaminobenzidine as the chromogen. Prolactin was present and could be localized in the luteal cells of all 6 corpora lutea, but not in any of the ovarian stroma studied. Human adenohypophysis served as a positive tissue control for prolactin immunopositive staining. The localization of immunoreactive prolactin in the corpus luteum demonstrates directly the presence of this hormone in the human ovary, adding further evidence for its role in luteal function.     

Interrelationships among morphology, echotexture, and function of the bovine corpus luteum during the estrous cycle

It has been suggested that ultrasound image attributes are a potential indicator of physiological and functional status of the corpus luteum (CL) in several species, including cattle. The aims of this study were to evaluate CL morphological, functional and echotextural characteristics, and also to investigate the hypothesis that those attributes are correlated and change similarly throughout an estrous cycle. Ovaries of crossbred (Bos taurus taurus × Bos taurus indicus) heifers were evaluated using ultrasonography daily throughout an interestrus interval using a B-mode, real-time ultrasound machine equipped with a 5 MHz linear-array rectal transducer, during a natural estrous cycle (Experiment 1; n = 12) or during a shortened cycle, with luteolysis induction 10 d after estrus (Experiment 2; n = 6). Blood samples were collected for assay of plasma progesterone concentrations. Corpora lutea areas were measured and daily images of each CL were videotaped and digitized for computer-assisted analysis using custom-developed software. In Experiment 1, area of luteal tissue increased until a maximum value 10 d after estrus (P < 0.001), followed by a plateau phase, and then a decline beginning 14 d after estrus. Luteal tissue area was highly correlated to plasma progesterone concentrations (r = 0.86; P < 0.001). When luteolysis was induced in Experiment 2, loss of CL function (decrease in plasma progesterone concentrations to metestrous values) preceded tissue regression by 48 h (24 h compared with 72 h; P < 0.001). The mean pixel value of ultrasound images did not change in Experiment 1 (P > 0.70), but a day effect on this attribute was observed in Experiment 2 (P = 0.052). In contrast, mean pixel value was correlated to plasma progesterone concentrations in Experiment 1 (r = −0.63; P < 0.05), but not in Experiment 2 (r = −0.28; P > 0.10). In regard to CL heterogeneity, defined as the standard deviation of the mean pixel value of the luteal tissue, a time effect was observed following both natural (Experiment 1; P < 0.009) and luteolysis-induced (Experiment 2; P < 0.05) estrous cycles (P < 0.05). Moreover, this variable was correlated with plasma progesterone concentrations (r = −0.71 and −0.58 in Experiments 1 and 2, respectively; P < 0.01), indicating that CL images were more heterogeneous during metestrus and after luteolysis (functional regression). In summary, morphological and echotextural attributes were correlated with CL function and underwent similar changes during the estrous cycle. Luteal tissue heterogeneity, assessed by ultrasonography, is considered a potential indicator of CL functional status, because it is correlated to circulating progesterone concentrations.     

Fine structure of the corpus luteum of pregnancy in the gerbil (Meriones unguiculatus)

Summary Corpora lutea from gerbils on days 1, 4, 8, 12, 16, 20 and 24 of pregnancy were studied electron microscopically. Similarly, luteal tissue from animals on the day of parturition and one day postpartum was studied (gestation: 24 days ± 8–24h). Agranular endoplasmic reticulum increases in quantity through day 16 and thereafter is somewhat reduced. Granular endoplasmic reticulum and a population of small granules (type I) become abundant during late pregnancy and their possible role in the production and storage of relaxin is discussed. Luteal tissue undergoes a relatively rapid regression which begins on the day of parturition. Conspicuous in the regressing luteal tissue are large (type II) granules (possibly lysosomes), lipid droplets, leucocytic elements and macrophages. Functional correlates of these morphological findings are discussed.The author would like to thank Mrs. Yvis Tablada, Miss Mary Ann Anderson and Mr. Garbis Kerimian for their technical, secretarial and photographic assistance, respectively     

Expression and localization of progesterone receptor membrane component 1 and 2 and serpine mRNA binding protein 1 in the bovine corpus luteum during the estrous cycle and the first trimester of pregnancy

The aim of this study was to evaluate the mRNA and protein expression and the localization of progesterone receptor membrane component 1 (PGRMC1), PGRMC2, and the PGRMC1 partner serpine mRNA binding protein 1 (SERBP1) in the bovine CL on Days 2 to 5, 6 to 10, 11 to 16, and 17 to 20 of the estrous cycle as well as during Weeks 3 to 5, 6 to 8, and 9 to 12 of pregnancy (n = 5–6 per each period). The highest levels of PGRMC1 and PGRMC2 mRNA expression were found on Days 6 to 16 (P < 0.05) and 11 to 16, respectively, of the estrous cycle and during pregnancy (P < 0.001). The level of PGRMC1 protein was the highest (P < 0.05) on Days 11 to 16 of the estrous cycle compared with the other stages of the estrous cycle and pregnancy, whereas PGRMC2 protein expression (P < 0.001) was the highest on Days 17 to 20 and also during pregnancy. The mRNA expression of SERBP1 was increased (P < 0.05) on Days 11 to 16, whereas the level of its protein product was decreased (P < 0.05) on Days 6 to 10 of the estrous cycle and was at its lowest (P < 0.001) on Days 17 to 20. In pregnant cows, the patterns of SERBP1 mRNA and protein expression remained constant and were comparable with those observed during the estrous cycle. Progesterone receptor membrane component 1 and PGRMC2 localized to both large and small luteal cells, whereas SERBP1 was observed mainly in small luteal cells and much less frequently in large luteal cells. All proteins were also localized in the endothelial cells of blood vessels. The data obtained indicate the variable expression of PGRMC1, PGRMC2, and SERBP1 mRNA and protein in the bovine CL and suggest that progesterone may regulate CL function via its membrane receptors during both the estrous cycle and pregnancy.     

Ultrastructural localisation of oxytocin and neurophysin in the ovine corpus luteum

Summary Polyclonal rabbit antisera raised against oxytocin, bovine neurophysin I and vasopressin were used, together with an immunogold complex, to localise the peptides in ultrathin sections of ovine corpus luteum. The only organelle which consistently showed gold labelling was the secretory granule of the large luteal cell. In non-consecutive sections of the same large luteal cell all the granules showed a similar level of labelling after oxytocin or neurophysin I antisera: however no immunolabelling was detected for vasopressin. Oxytocin and neurophysin seem to be rapidly lost after secretion since exocytosed granule cores showed no labelling above background levels.     

Fine structure of the corpus luteum of the autografted ovary in the rat

Summary The rat ovary has been transplanted successfully to subcutaneous tissue areas by several investigators. Light microscopy has revealed that corpora lutea in ovarian autografts are formed by luteinization of intact follicles and contain entrapped ova. In the present study, corpora lutea from autografted ovaries in castrate rats were obtained at metestrus and examined electron microscopically to determine whether their cellular morphology correlated with the normal progesterone levels in these animals. Cellular features usually accepted as regressive were apparent. The findings suggest either structural luteolysis is occurring before functional luteolysis or that the adrenal has increased steroidogenic activity in the castrate with ovarian autografts to account for the normal progesterone levels.Supported in part by USPHS Grant T01-DE00241-04The authors wish to thank J. Canale and Y. Tablada for technical, G. Kerimian for photographic, and M.A. Anderson for secretarial assistance     

The evaluation of corpus luteum blood flow using color-flow Doppler ultrasound for early pregnancy diagnosis in bovine embryo recipients

The objective of this experiment was to evaluate corpus luteum blood flow (CLBF) as an early indicator of pregnancy status in bovine embryo recipients. Fifty crossbred beef cows were submitted to embryo transfer on Day 7 after estrus. On Days 7, 11, 13, 15, 17, 19, 21, 26, 33, and 40, a blood sample was taken, the CL examined using a color-flow Doppler ultrasound scanner, and video was recorded of each scanning session. Ultrasound data were grouped by the first day progesterone concentrations were <1 ng/mL (indicating early embryo loss, EEL) through Day 21 (EEL-17, n = 3; EEL-19, n = 9; EEL-21, n = 3), absence of an embryo on Days 26, 33, or 40 (late embryo loss; LEL; n = 12), or remained pregnant (P; n = 23). The first decrease in CLBF of EEL-17, EEL-19, and EEL-21 cows compared to P cows occurred on Days 17, 19, and 21, respectively (P < 0.05). There was no difference in CLBF between LEL and P cows on Days 17, 19, and 21. Six evaluators diagnosed pregnancy from randomized video clips on Days 17, 19, and 21. Evaluators made more (P < 0.004) correct diagnoses on Day 19 than Day 17. Sensitivity (82.9 ± 10.1%) was not affected by day. From Days 17 to 19, diagnostic specificity increased (P = 0.046) from 43.2 ± 3.0 to 54.3 ± 3.0% but remained unchanged thereafter. Due to low specificity and sensitivity, evaluation of CLBF alone was insufficient for early pregnancy diagnosis.     

Urokinase-type and tissue-type plasminogen activators have different distributions in cultured bovine capillary endothelial cells

The cell extracts and conditioned medium from cultured bovine capillary endothelial (BCE) cells were examined to determine the types of plasminogen activator (PA) present in each of these two fractions. The fractions were first analyzed by fibrin autography after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The cell extracts contained two species of PA of Mr 48,000 and 28,000. Multiple forms of PA were detected in the conditioned medium: variable amounts of the Mr 48,000 and 28,000 forms and a broad band of activity with Mr in the range of 67,000-93,000. The major fraction of the Mr 48,000 form was in the cell extract. Treatment of the cells with 12-0-tetradecanoyl phorbol-13-acetate or with a preparation containing angiogenic activity resulted in a proportionate increase in the levels of all forms. The Mr 48,000 form was demonstrated to be a urokinase-like PA, since it was immunoprecipitated with antibodies to urokinase. When conditioned medium or cell extracts from biosynthetically labelled BCE cells were incubated with antiserum to urokinase, the Mr 48,000 form was immunoprecipitated only from the cell extract. The Mr 67,000-93,000 forms were demonstrated to be tissue-type PAs, since they were immunoprecipitated with antibodies to tissue PA. When the same conditioned medium or cell extracts were incubated with antiserum to tissue-type PA, the Mr 67,000-93,000 forms were immunoprecipitated only from the conditioned medium. Therefore, BCE cells are able to produce both tissue-type PA, which is primarily secreted, and urokinase-type PA, which remains primarily cell associated.     

Nuclear volume and chromatin conformation of small and large bovine luteal cells: effect of gonadotropins and prostaglandins and dependence on luteal phase

Summary Change in nuclear volume and chromatin conformation are generally considered to reflect altered gene expression in eukaryotic cells. The present studies were undertaken to investigate whether these nuclear parameters of luteal cells can be altered by hormone treatment in vitro or change during the estrous cycle. The nuclear volume of small luteal cells was significantly lower than that of large luteal cells during the cycle and pregnancy. The nuclear volumes of small and large luteal cells from pregnancy did not change during incubation without any hormone or with 10 nM prostaglandin (PG)F₂. However, incubation with 1 nM human chorionic gonadotropin (hCG) or 10 nM PGE₁ resulted in a significant increase of nuclear volume of small luteal cells by 4 h and that of large luteal cells by 6 h. Small cells were more responsive to hCG than large luteal cells. The nuclear volumes of small and large luteal cells also significantly increased from early to mid luteal phase with no further change in late luteal phase. hCG and PGE₁, as well as PGF₂, treatment resulted in a change of chromatin conformation of small and large luteal cells. Dibutyryl cyclic AMP (10 mM) mimicked the hormones by increasing nuclear volumes and changing the chromatin conformation of small and large luteal cells. Chromatin conformation of small and large luteal cells also changed from early to mid luteal phase and mid to late luteal phase. In conclusion, in vitro, hCG and PGs can regulate nuclear volume and/or chromatin conformation of small as well as large bovine luteal cells. In vivo, these nuclear changes occur during the periods of luteal growth, development and regression in the estrous cycle.     

Systemic and intraovarian effects of corpus luteum on follicular dynamics during estrous cycle in hair breed sheep

The present study aimed to determine systemic and local effects of corpora lutea (CL), on follicular dynamics throughout the estrous cycle. All follicles >or=2 mm and CL were assessed by daily transrectal ultrasonography in 12 West African ewes. Blood samples were collected to determine plasma concentration of progesterone. Fifteen estrous cycles were evaluated with a mean interovulatory interval of 16.8+/-0.2 days. Two (13.3%), 10 (66.7%) and 3 (20%) of the estrous cycles had 2, 3 and 4 waves of follicular development, respectively. In sheep with three waves of follicular development, both the length of growing phase and the growth rate of dominant follicles from midluteal wave II were diminished (3.4+/-0.3 days, P<0.0001, and 0.4+/-0.1 mm/day, P<0.01, respectively) when compared to follicles from early luteal phase (wave I, 4.1+/-0.2 days, and 0.7+/-0.1 mm/day) or late luteal phase (wave III, 6.3+/-0.4 mm and 0.6+/-0.1 mm/day). The diameter of the dominant follicle was smaller during the midluteal phase (3.9+/-0.1 mm, P<0.0001) than in the early and late luteal phase (5.0+/-0.2 and 5.7+/-0.2 mm; respectively). The effect of the dominant follicle was less during midluteal phase, because number of accompanying smaller follicles was fewer (P<0.01) in waves I and III (6.3+/-0.9 compared with 3.4+/-0.8 and 2.3+/-0.7). The number of follicles was also different between ovaries that had CL and those that did not. The total number of large follicles during the luteal phase was less in ovaries with CL (0.9+/-0.5 compared with 2.7+/-0.3; P<0.01), as was the mean daily number of both large (0.1+/-0.02 compared with 0.2+/-0.02; P<0.001) and total number of follicles >or=2 mm (2.5+/-0.1 compared with 3.3+/-0.1; P<0.01). Current results indicate that the presence of a functional CL may exert both systemic and local effects on the population of follicles, affecting the dominance exerted by large follicles.     

Effect of cycloheximide on the fine structure of corpus luteum in intact and hypophysectomized rats

Summary In rats, one large intravenous dose of cycloheximide leads to extensive development of two types of membrane-formations in the cells of corpora lutea, within two hours. Both the laminated dense bodies (concentric layers of smooth membranes showing high electron density) and the tubular aggregates (tightly packed smooth tubules with diameter smaller than usual) exhibited obvious connections with endoplasmic reticulum membranes. The reorganization of tubular aggregates gave rise to crystalloids showing hexagonal symmetry. The crystalloids, being obviously unstable, were transformed into smooth fingerprints (concentric arrays of paired agranular membranes showing the same density as endoplasmic reticulum membranes). Hypophysectomy, performed 24 hours previously, moderated but did not totally abolish the development of membranous configurations. The described effect of cycloheximide is considered to represent cellular injury, probably due to membrane-denaturation.This work was supported in part by the Medical Research Council of Canada (Block Term Grant MT-1829), the Ministère des Affaires Sociales, Quebec, and Succession J.A. DeSève. The authors thank the Upjohn Company, Kalamazoo, Michigan, U.S.A., for the cycloheximide used in these experiments.Fellow of the Medical Research Council of Canada.     

Growth stimulation of ovarian and extraovarian mesothelial cells by corpus luteum extract

Summary Ovarian (OM) and extraovarian (EM) mesothelia represent a common source of gynecologic malignancies with yet unclear pathogenesis. Ovulation triggers a finite wave of DNA synthesis and morphogenesis only in native OM cells, probably through the activation of intraovarian growth factors. To evaluate their growth response to such factors, OM and EM cells were obtained from estrous New Zealand white rabbits by enzymatic dispersion and unit gravity sedimentation. Cell cultures were maintained in serumless, fibronectin-rich, HL-1 medium without or with rabbit corpora lutea tissue extracts (CLE). The growth effects of CLE were evaluated by measuring percent changes in cell number relative to controls (CCN), cell population doublings (CPD), cell population doubling time in hours (CPDT). After 7.5 days, CLE enhanced (P<0.001) the growth of both OM and EM cells, which exhibited, respectively, a CCN of 214 and 257%; a CPD of 2.89 and 2.87; and a CPDT of 54.39 and 59.49. CLE-treated OM and EM cells were smaller, formed more cohesive monolayers, and exhibited more frequent and diffuse microvilli than control cells. These data show a similar in vitro response of OM and EM cells to luteal growth factors, suggesting that the lack of postovulatory morphogenesis in native extraovarian mesothelia is due to the spatially restricted activity of intraovarian growth factors.