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D A Redmer

Publications and source records attributed to D A Redmer.

At least 55 records · Page 3Linked to original sources
PubMed1996

Gap junctional intercellular communication of bovine luteal cells from several stages of the estrous cycle: effects of cyclic adenosine 3',5'-monophosphate.

Cellular interactions mediated by both contact-dependent and contact-independent mechanisms are probably important to maintain luteal function. The objective of the present study was to evaluate the role of cAMP in regulation of contact-dependent gap junctional intercellular communication (GJIC) of bovine luteal cells from several stages of luteal development. In experiment 1, corpora lutea (n = 5) from the mid-luteal phase of the estrous cycle were dissociated with collagenase, and cells were preincubated in a medium with serum. Then the medium was changed to serum-free media containing a cAMP agonist (dbcAMP; 1 mM) or antagonist (Rp-cAMPS; 0, 3, 10, 30, or 100 microM). In experiment 2, corpora lutea from the early (n = 7), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were dissociated and preincubated as in experiment 1, and luteal cells were then incubated with no treatment, LH (100 ng/ml), dbcAMP (1 mM), forskolin (1 microM), Rp-cAMPS (100 microM), or LH+Rp-cAMPS. After incubation of luteal cells with treatments for 18-24 h, media were collected for determination of progesterone and cAMP concentrations. Then the rate of GJIC was evaluated for selected cells (small luteal cells in contact with small luteal cells, and large luteal cells in contact with small luteal cells) by using the fluorescence recovery after photo-bleaching technique and laser cytometry. In experiment 1, dbcAMP increased (p < 0.01) but Rp-cAMPS (p < 0.05) decreased GJIC between small luteal cells and between large and small luteal cells. In addition, dbcAMP stimulated (p < 0.01) but Rp-cAMPS did not affect progesterone secretion. In experiment 2, treatments affected (p < 0.05) GJIC and progesterone production of luteal cells from the mid- and late but not from the early luteal phase of the estrous cycle. GJIC between small luteal cells was increased (p < 0.01) by LH, dbcAMP, and forskolin. GJIC between large and small luteal cells was increased (p < 0.05) by dbcAMP and forskolin. Rp-cAMPS decreased (p < 0.01) GJIC between small luteal cells (mid-luteal phase) and between large and small luteal cells (mid- and late luteal phases). In addition, Rp-cAMPS inhibited (p < 0.05) the stimulatory effects of LH on GJIC between small luteal cells from the mid- and late luteal phases of the estrous cycle. For luteal cells from the mid- and late luteal phases, progesterone production was increased (p < 0.05) by LH, dbcAMP, forskolin, and LH+Rp-cAMPS, but was not affected by Rp-cAMPS. Across all stages of the estrous cycle, cyclic AMP accumulation in media was greater (p < 0.05) in LH- and forskolin-treated cultures than in control cultures and was greater (p < 0.01) in forskolin-treated than in LH-treated cultures. These data demonstrate that small and large luteal cells communicate with each other and that the rate of GJIC is modulated by LH and cAMP, as has been shown previously for other cell types. Thus, cAMP appears to be involved in the regulation of GJIC within the bovine corpus luteum, which probably is an important mechanism for coordinating luteal cell function.

Animals↗
PubMed1996

Growth and cellular proliferation of antral follicles throughout the follicular phase of the estrous cycle in Meishan gilts.

Meishan gilts were ovariectomized 2 h after an i.v. injection of 5'-bromo-2'-deoxyuridine (BrdU, a thymidine analogue; 5 mg/kg body weight) on Days 15-19 of the estrous cycle or 24-30 h after observed estrus (post LH, PLH). All antral follicles > or = 3 mm from one ovary were fixed in Carnoy's solution. Granulosa and thecal cell labeling indexes (LI; percentage of nuclei staining for BrdU) as well as LI of cells within the basal, middle, and antral thirds of the granulosa cell layer were estimated for each follicle. In addition, antral and granulosa cell layer volume, granulosa cell layer thickness, granulosa cell density, number of granulosa cells, and number of S-phase cells per hour were estimated for each follicle. Mean follicular diameter increased linearly (p < 0.01) from Day 15 to PLH, with a growth rate of 0.77 mm/day. Granulosa and thecal cell LI decreased (p < 0.01) from Day 15 to PLH; however, granulosa cell LI was greater (p < 0.01) than thecal cell LI on Days 15 and 16 but less (p < 0.05) than thecal cell LI on Day 19. Follicles collected from PLH gilts contained no labeled granulosa cells. Cells within the basal third of the granulosa cell layer contained fewer (p < 0.01) labeled nuclei than did cells within the middle or antral thirds. In addition, LI within the basal and middle thirds of the granulosa cell layer decreased (p < 0.01) from Days 15 to 18 and from Days 15 to 17, respectively, whereas LI within the antral third remained constant from Days 15 to 18. Granulosa cell layer thickness was greatest (p < 0.01) on Day 15, then decreased (p < 0.01) and was similar from Day 16 to PLH. Granulosa cell density was similar from Days 15 to 19, then decreased (p < 0.01) for PLH gilts. Antral and granulosa cell layer volumes increased linearly (p < 0.01) from Days 15 to 19 and Day 15 to PLH, respectively, resulting in 2.8 and 1.9 volume doublings and doubling times of 1.4 and 2.7 days, respectively. Number of granulosa cells per follicle increased linearly (p < 0.01) from Day 15 to PLH, resulting in 1.5 cell doublings and a doubling time of 3.3 days. Number of S-phase cells per follicle per hour was similar from Days 15 to 18 and then decreased (p > 0.01) from Day 18 to PLH. In summary, the percentages of proliferating granulosa and thecal cells decreased throughout the final stages of antral follicular development. Differentiation of granulosa cells occurred from the basal to the antral area as follicles matured. We proposed that, during the latter stages of follicular development, the rapid increase in follicular diameter resulted primarily from expansion of the antral cavity, whereas increases in the granulosa cell layer volume and number of granulosa cells per follicle maintained a constant granulosa cell layer thickness.

Animals↗
PubMed1996

Gap junctional protein connexin 43 in bovine corpora lutea throughout the estrous cycle.

The present study examined the pattern of expression of the gap junctional protein connexin 43 (Cx43) in bovine corpora lutea (CL) during growth, differentiation, and regression. CL from the early (n = 6), mid- (n = 6), and late (n = 6) luteal phases of the estrous cycle were weighed and divided into several portions. One portion of each CL was frozen in liquid nitrogen for evaluation of protein, DNA, progesterone, and presence of Cx43 by Western immunoblot analysis; another portion was frozen in liquid propane for immunofluorescent staining of Cx43. An additional portion of each CL was dispersed, and the luteal cells were cultured for 2 days, fixed, and used for immunofluorescent staining of Cx43. Weights and DNA, protein, and progesterone contents of CL increased (p < 0.05) from the early to mid-luteal phases and then decreased (p < 0.05) from the mid- to late luteal phases. The ratio of protein to DNA was similar in the early and mid-luteal phases and then decreased (p < 0.05) to the late luteal phase. Western immunoblot analysis revealed bands at 43 kDa that differed in volume (evaluated by densitometry); the early luteal phase volume was greater (p < 0.05) than that at the mid-luteal phase, which was greater (p < 0.05) than that at the late luteal phase. Immunofluorescent staining demonstrated that Cx43 was present in luteal tissues and cultured luteal cells throughout the estrous cycle, and the area of positive staining decreased (p < 0.05) as the estrous cycle progressed. Staining for Cx43 was punctate and localized to the cellular borders. Thus, levels of Cx43 in bovine CL are greatest early in the estrous cycle and are least late in the estrous cycle. These data demonstrate that gap junctions may be important for regulation of luteal growth, differentiation, and regression in the cow.

Animals↗
PubMed1996

Effects of gonadotropin treatment and withdrawal on follicular growth, cell proliferation, and atresia in ewes.

To determine the effects of FSH-P treatment and subsequent withdrawal on follicular growth, cell proliferation, and atresia, ewes (n = 4 ewes/treatment group) received twice daily injections of saline or FSH-P beginning on Day 13 of the estrous cycle (length of the estrous cycle = 16.5 days) and were slaughtered after 0, 48, or 72 h of treatment (i.e., on Days 13, 15, or 16). Some treatment groups received FSH-P from Day 13 until slaughter (FSH-P-treated), whereas some received FSH-P for 24-48 h followed by saline for 24-48 h (FSH-P withdrawal). All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analogue) 1 h before slaughter. For both ovaries from each ewe, the number and surface diameter of all visible antral follicles were determined, and antral follicles were classified as small (< or = 3 mm), medium (> 3 mm to < or = 6 mm), or large (> 6 mm). As an index of the rate of cell proliferation, BrdU was immunolocalized in paraffin-embedded tissue sections, and the labeling index (LI; BrdU-labeled nuclei as a percentage of total nuclei) was determined for granulosa and thecal cells of nonatretic and early atretic antral follicles of known diameter. Follicular status (atretic vs. nonatretic) was evaluated morphologically. Moreover, the presence of apoptosis was detected in situ by using the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling method. For untreated and saline-treated ewes, the number of small follicles per ewe increased (p < 0.01) from Day 13 to Day 15, then decreased again on Day 16, whereas numbers of medium and large follicles did not differ across days. Compared with saline-treated ewes, ewes receiving FSH-P from Day 13 until slaughter had fewer (p < 0.05) small but more (p < 0.05) medium and large follicles. Compared with FSH-P-treated ewes, FSH-P withdrawal resulted in fewer (p < 0.05) medium and large but more (p < 0.05) small follicles. Across all follicular size classes, granulosa and thecal cell LI of nonatretic follicles was decreased (p < 0.05) by FSH-P withdrawal compared with FSH-P treatment. Additionally, across all follicular size classes, FSH-P withdrawal increased (p < 0.01) the percentage of follicles that were atretic compared with saline or FSH-P treatment. Histochemical staining of early and advanced atretic follicles showed that granulosa cells are the predominant site of cell death (apoptosis) during follicular atresia. Thus, compared with continuous FSH-P treatment, withdrawal of FSH-P resulted in decreased numbers of medium and large follicles, decreased proliferation of follicular cells, and an increased incidence of atresia associated with granulosa cell death. This model should prove useful for studying the mechanisms regulating follicular growth and atresia in ewes.

Animals↗
PubMed1996

Estrogen and progesterone receptors, cell proliferation, and c-fos expression in the ovine uterus during early pregnancy.

To evaluate uterine growth during pregnancy and the potential roles of estrogen and progesterone in regulating uterine cell proliferation and c-fos expression, ewes were assigned randomly to slaughter on day 12 after estrus (nonpregnant, NP), and on days 12, 18, 24, or 30 after mating (pregnant, P) in Exp 1 (n = 7 ewes/day) and on days 12 or 14 after estrus and on days 12, 14, 16, 18, 21, or 24 after mating in Exp 2 (n = 3-6 ewes/day). In Exp 1, endometrial expression of c-fos mRNA was evaluated, and labeling index was determined both in vitro (incorporation of 3H-thymidine) and in vivo (iv injection of bromodeoyxuridine [BrdU], a thymidine analog). Endometrial expression of the c-fos proto-oncogene was increased by approximately 10-fold on days 18, 24, and 30 P compared with day 12 NP or P. Labeling index (proportion of cells incorporating 3H-thymidine or BrdU, which provides an index of the rate of cell proliferation) of endometrial caruncular and intercaruncular tissues was low for day 12 NP or P, increased on day 18 P, and remained elevated on days 24 P and 30 P. On day 18 P, labeling index also was greater for gravid than nongravid horns for both caruncular and intercaruncular tissues. In Exp 2, estrogen receptors (ER), progesterone receptors (PR), and proliferating (BrdU-positive) cells were immunolocalized. The percentage of cells exhibiting specific staining for ER, PR, and BrdU was quantified morphometrically for epithelial, stromal, and glandular tissues within luminal and deep regions, as well as for myometrial tissues. For luminal epithelium and glands, the rate of cell proliferation increased dramatically by day 18 P, even though ER and PR levels were low in these compartments. Conversely, the rate of cell proliferation remained low throughout early pregnancy in deep glands, deep stroma, and myometrium, in association with sustained or transient increases in ER and PR levels. For luminal stroma, the rate of cell proliferation increased by day 21 P even though ER levels were low and PR levels remained high. Thus, during early pregnancy, c-fos expression increased concomitantly with increased endometrial cell proliferation. In addition, during early pregnancy, ER and PR levels were inversely related to the rate of cell proliferation in most of the uterine tissue compartments except luminal stroma, which exhibited increased cell proliferation even though ER levels were low and PR levels remained high.

Animals↗
PubMed1996

Characterization and expression of vascular endothelial growth factor (VEGF) in the ovine corpus luteum.

The corpus luteum undergoes tremendous growth, development and regression each oestrous or menstrual cycle. These changes are reflected by equally impressive growth and regression of the luteal vasculature. We have previously shown that angiogenic factors from corpora lutea are primarily heparin binding and that one of these factors is similar to vascular endothelial growth factor (VEGF). In an effort to identify this factor, and to define its role in luteal vascular development, the cDNA for the coding region of ovine VEGF was sequenced and a sensitive RNase protection assay was developed to quantitate mRNA encoding VEGF in luteal tissues from ewes in the early (days 2-4), mid- (day 8) and late (days 14-15) stages of the oestrous cycle. In addition, an N-terminal peptide was synthesized from the translated ovine cDNA sequence for VEGF and an antiserum was raised against this peptide for use in western immunoblotting procedures. Nested reverse transcriptase (RT)-PCR of RNA from ovine corpora lutea resulted in three products that correspond in size to the alternatively spliced variants of VEGF (VEGF120, VEGF164, and VEGF188) predicted from other species. The RNase protection assay revealed that the proportion of mRNA encoding VEGF was 2- to 3-fold greater on days 2-4 than on day 8 or days 14-15. Densitometric analysis of gels from the RNase protection assay showed that VEGF120 represented approximately one third of the total mRNA encoding VEGF in the corpus luteum and that this proportion did not vary with stage of the oestrous cycle. SDS-PAGE and western immunoblot analysis of a homogenate from corpora lutea showed a single 18 kDa protein. These data demonstrate that VEGF is expressed in luteal tissue throughout the ovine oestrous cycle and that expression of mRNA encoding VEGF is upregulated during the period of rapid luteal development, when luteal vascular growth is at its maximum.

Amino Acid Sequence↗
PubMed1996

Angiogenesis in the ovary.

In adult tissues, capillary growth (angiogenesis) occurs normally during tissue repair, such as in the healing of wounds and fractures. Inappropriate capillary growth is associated with various pathological conditions, including tumour growth, retinopathies, haemangiomas, fibroses and rheumatoid arthritis in the case of rampant capillary growth, and nonhealing wounds and fractures in the case of inadequate capillary growth. The female reproductive organs exhibit marked, periodic growth and regression, accompanied by equally striking changes in their rates of blood flow. It is not surprising, therefore, that they are some of the few adult tissues in which angiogenesis occurs as a normal process. Ovarian follicles and corpora lutea have been shown to contain and produce angiogenic factors. These angiogenic factors appear to be heparin-binding and to belong to the fibroblast growth factor (FGF) and vascular endothelial growth factor (VEGF) families of proteins. In addition, factors regulating gap junctional communication may play a critical role in coordinating the interactions between luteal vascular and nonvascular tissues. Further elucidation of the specific physiological roles of these factors in follicular and luteal growth, development and function will ultimately lead to improved methods for regulating fertility in mammals.

Adult↗
PubMed1996

Production of heparin-binding endothelial mitogens by bovine uterine fibroblastic and epithelial cells.

This study was conducted to determine whether early-passage cultures of bovine endometrial fibroblastic (Fb, n = 7 uteri) and epithelial (Ep, n = 3 uteri) cells produce endothelial mitogens in vitro and to begin characterization of these mitogens. Confluent cultures of Fb and Ep were incubated for 72 h in serum-free media, and the resulting conditioned media (CM) were evaluated for effects on proliferation of bovine aortic endothelial cells. CM from these Fb cultures (n = 8) and Ep cultures (n = 4) stimulated (147 +/- 10% (mean +/- SEM), p < 0.01, and 124 +/- 8%, p < 0.10, respectively) proliferation of endothelial cells compared with control (unconditioned) media. Most of the mitogenic activity of a sample of Fb CM and a sample of Ep CM from one individual uterus bound to heparin-agarose, and each exhibited two major peaks of activity that eluted at 0.9-1.0 and 1.7-1.8 M NaCl; the Fb CM also exhibited an additional heparin-binding peak eluting at 0-0.1 M NaCl. Pooled Fb CM (n = 8 cultures from 7 animals) also contained mitogenic activity for endothelial cells that bound to heparin-agarose, but exhibited three major peaks, eluting at 0.6, 1.1, and 1.8 M NaCl. Pooled Ep CM (n = 4 cultures from 3 animals) showed only one peak of mitogenic activity, which eluted at 0.9 M NaCl. Further characterization indicated that heat treatment reduced the activity of all heparin-binding Fb CM and Ep CM peaks, except the Fb CM peak eluting at 1.7 M NaCl. Trypsin reduced the activity of all peaks except one. Protein-A-purified antibody against fibroblast growth factor 1 (FGF-1) had no or only a slight effect on the mitogenic activity of the peaks. Mitogenic activity of the Fb CM peak eluting at 0.6 M NaCl was reduced by antibody against FGF-2. Activity of the Fb CM and Ep CM peaks that eluted at 1.7-1.8 M NaCl also was immunoneutralized by antibody to FGF-2. These data demonstrate that early passage cultures of endometrial Fb and Ep cells produce heparin-binding endothelial mitogens that appear to be immunologically related to FGF-2. These heparin-binding endothelial mitogens may influence endometrial vascular growth.

Animals↗
PubMed1995

Immunohistochemical localization of 3 beta-hydroxysteroid dehydrogenase and P450 17 alpha-hydroxylase during follicular and luteal development in pigs, sheep, and cows.

Follicular and luteal morphology and steroidogenic function were investigated by immunohistochemistry for cytochrome P450 17 alpha-hydroxylase (P450c17) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) during the estrous cycle in pigs, sheep, and cows. The theca interna of all species expressed P450c17 during follicular development. In the pig, this constituted a continuous layer of cells around the follicle, but a sheath of cells lining the basement membrane appeared not to express P450c17. Neither was expression of P450c17 in ovine and bovine follicles uniform throughout the theca interna. In these two species, a beaded appearance was given by P450c17, since it was expressed in some regions but not in others. Therefore, staining for P450c17 defined functional sub-populations of cells within the theca interna of pigs, sheep, and cows. Ovulation was associated with a decrease in P450c17 in all species, but some expression persisted in theca-derived cells of developing and mature porcine CL. Expression of 3 beta-HSD in the preovulatory follicle was confined to the theca of the pig and sheep; in contrast, in the cow, it was highest in the granulosa. In general, 3 beta-HSD expression appeared to be greater in porcine than ovine or bovine follicles, the physiological relevance of which is discussed. Porcine and ovine theca continued to express 3 beta-HSD after ovulation, and granulosa-derived cells increased their 3 beta-HSD expression markedly as they luteinized in all three species. During early luteal development in pigs and sheep, theca-derived cells with high 3 beta-HSD encircled luteal lobules, but these cells appeared throughout the parenchyma of the mature CL. Luteal regression in sheep and cows was typified by the loss of many cells expressing 3 beta-HSD, whereas others, adjacent to them, appeared to be intact without loss of enzyme expression. These data further define differences in steroidogenesis during follicular and luteal development among the pig, sheep, and cow.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Proliferation and progesterone production of ovine luteal cells from several stages of the estrous cycle: effects of fibroblast growth factors and luteinizing hormone.

This study was conducted to evaluate the effects of fibroblast growth factor 1 (FGF-1), fibroblast growth factor 2 (FGF-2), or luteinizing hormone (LH) on proliferation and progesterone secretion of ovine luteal cells from days 5, 10, or 15 after estrus (estrus = day 0; n = 4 or 5 ewes/day). After enzymatic dispersion, luteal cells were incubated in the presence or absence of various doses of FGF-1, FGF-2, LH, or fetal bovine serum (FBS) (positive control) in serum-free media for 7 days in 24-well plates. Cells were counted on day 7 of culture and media analyzed for progesterone concentration. For all treatments, maximal effects (Emax) and dissociation constants (KD) were calculated. In addition, luteal cells were cultured in eight-chamber slides and treated as above, but on day 7 of culture cells were fixed and stained for the presence of 3beta-hydroxy-delta 5-steroid dehydrogenase (3beta HSD). The number of steroidogenic (3beta HSD positive) cells per unit area was counted for control cultures (no treatment) and cultures treated with the most effective doses of FGF-1, FGF-2, LH, OR FBS in proliferation and (or) progesterone assays. FGF-1, FGF-2, AND FBS stimulated (p < 0.05) proliferation of luteal cells from all stages of luteal development in a dose-dependent manner. In addition, LH increased (p < 0.01) the number of 3beta HSD-positive cells across all stages of luteal development. Moreover, LH and FBS increased (p < 0.05) progesterone secretion by luteal cells from all stages in a dose-responsive manner, but the effects of FGF-1 and FGF-2 were variable. For proliferation, the Emax of all factors was greatest (p < 0.01) on day 5, whereas the KD values were similar across days of the estrous cycle. For progesterone production, the Emax and KD of LH and FBS were similar and did not differ across the estrous cycle. These data demonstrate the luteal cells from the early luteal phase of the estrous cycle exhibit the greatest ability to proliferate and (or) increase their progesterone secretion in response to FGF-1, FGF-2, LH, or FBS. In addition, although LH does not affect the total number of luteal cells in culture, it does increase the number of steroidogenic cells. These data indicate that in addition to LH, fibroblast growth factors may be involved in regulation of luteal growth and differentiation in ewes.

Animals↗
PubMed1995

Utero-placental vascular development and placental function.

The rate of fetal growth and subsequent birth weight are major determinants of postnatal survival and growth. Because the placenta is the organ through which respiratory gases, nutrients, and wastes are transported between the maternal and fetal systems, its primary function is to supply the metabolic substrates necessary to support fetal growth. Placental growth and development, therefore, are critical for normal fetal growth and development. During the last half of gestation in mammals, growth of the fetus is exponential, whereas utero-placental growth slows or ceases. Nevertheless, unless placental transport capacity keeps pace with the continually increasing demands of the fetus, fetal growth will be compromised. Studies over the last two decades have shown that placental transport capacity does indeed keep pace with fetal growth. This increase in placental function can be accounted for primarily by continual increases in placental (uterine and umbilical) blood flows, associated with increased placental vascularity. Placental vascular growth and development, in turn, are probably regulated by angiogenic factors produced by the placental tissues themselves. These placental angiogenic factors are produced primarily by the maternal placental tissues, are heparin-binding, and seem to be related to the fibroblast growth factor family. Further elucidation of the factors responsible for placental growth and vascular development is critical for an improved understanding of uteroplacental-fetal interactions, which result in delivery of a healthy offspring.

Angiogenesis Inducing Agents↗
PubMed1995

Initial characterization of mitogenic activity of ovine corpora lutea from early pregnancy.

To characterize angiogenic factors produced by ovine corpora lutea (CL) during early pregnancy, two experiments were performed. In Experiment 1, luteal explants from days 12, 18, 24, and 30 (n=4 ewes/day) after mating were incubated in serum-free medium for 6 h. Luteal-conditioned media (LCM) were evaluated for their ability to stimulate proliferation of endothelial and 3T3 cells, as well as migration of endothelial cells. Pools of the LCM (one pool/day) then were characterized biochemically. In Experiment 2, two pools of LCM from days 24 of pregnancy were evaluated for their effects on endothelial cell, 3T3 cell, and ovine luteal cell proliferation. These pools of LCM then were concentrated by ultrafiltration and subjected to heparin-agarose affinity chromatography with salt gradient (0-4 M NaCl in buffer) elution, and fractions were evaluated for mitogenic activity for endothelial and 3T3 cells. The resulting five peaks of mitogenic activity from heparin-agarose chromatography were characterized biochemically. The five peaks of mitogenic activity were further purified by using chromatography, then were concentrated and subjected to SDS-PAGE and Western analysis for FGF-2. Ovine CL from each day of early pregnancy secreted mitogens (P<0.05) for endothelial (285 +/- 8% of unconditioned media controls) and 3T3 (142 +/- 7%) cells as well as factors which stimulated migration of endothelial cell (153 +/- 8% of controls). LCM pool from day 24 of pregnancy also stimulated (P<0.05) proliferation of ovine luteal cells in a dose-dependent manner. In Experiment 1, mitogenic activity for endothelial cells was greater than 100 kDa, heat-labile, trypsin-sensitive and bound to DEAE-Sephacel and heparin-agarose columns, but not to a CM-Sepharose column. Antibody against FGF-1 did not affect mitogenic activity of LCM for endothelial and 3T3 cells, whereas treatment with FGF-2 antibody decreased (P<0.05) mitogenic activity of LCM for both endothelial and 3T3 cells. In Experiment 2, heparin-agarose affinity chromatography resolved five peaks of mitogenic activity: a non-heparin-binding peak that was specific for 3T3 cells, three heparin-binding peaks that were specific for endothelial cells, and one heparin-binding peak that was specific for 3T3 cells. In Experiment 2, heparin-, heat-, or trypsin-treatment and immunoneutralization with FGF-1 or FGF-2 antibodies influenced mitogenic activity of all of the peaks. Whereas SDS-PAGE demonstrated several bands of protein within each peak, Western analysis was unable to detect the presence of FGF-2 in any of the heparin-binding peaks. These data demonstrate that ovine CL from early pregnancy produce mitogenic factors that can be resolved into 5 separate peaks of activity with differing affinities for heparin. These data also indicate that the endothelial mitogens produced by CL of early pregnancy are immunologically related to, but biochemically distinct from FGF-2. Mitogens for endothelial and other cells likely play a role in regulation of luteal function during early pregnancy in sheep.

3T3 Cells↗
PubMed1994

Studies of FSH-P induced follicular growth in cows.

Because cow ovaries do not contain a dominant follicle before Day 3 of the estrous cycle, we hypothesized that gonadotropin treatment early in the estrous cycle would induce growth of multiple follicles and could be used to induce superovulation. In Experiment 1, when 16 cows were treated with FSH-P beginning on Day 2 of the estrous cycle and were slaughtered on Day 5, all cows responded to gonadotropin treatment by exhibiting a large number ( approximately 19) of estrogenactive follicles >/= 6 mm. In Experiment 2, in response to FSH-P treatment from Day 2 to Day 7, and fenprostalene treatment on Day 6, 11 of 15 cows exhibited estrus and had a mean ovulation rate of 23.7 +/- 1.5. In Experiment 3, an FSH-P treatment regimen identical to that used in Experiment 2 was administered to cows beginning either on Day 2 (Day-2 cows; n=14) or Day 10 (Day-10 cows; n=11) of the estrous cycle. Twelve of 14 Day-2 cows and all Day-10 cows exhibited estrus after fenprostalene treatment. Day-2 cows exhibited 34.3 +/- 7.0 ovulations, which was less (P < 0.05) than that exhibited by Day-10 cows (48.3 +/- 4.4). However, the proportion of embryos recovered per corpus luteum was about 2-fold greater (P < 0.05) for Day-2 cows than for Day-10 cows (0.49 +/- 0.08 vs 0.27 +/- 0.06). These data indicate that beginning gonadotropin treatment early in the estrous cycle, when a dominant follicle is not present, provides an efficacious means to induce growth of multiple follicles and superovulation in cows. However, when FSH was administered for 6 d, beginning the treatment on Day 10 also resulted in a consistent and efficacious response.

Journal Article↗
PubMed1994

Mitogenic factors of corpora lutea.

The mammalian corpus luteum (CL), which plays a central role in the reproductive process because of its production of hormones such as progesterone, appears to be an exceptionally dynamic organ. Its rate of growth and development are extremely rapid and, even when the CL is functionally mature, its rate of cell turnover remains relatively high. Associated with this high rate of cell turnover, the mature CL receives the greatest blood supply per unit tissue of any organ, and also exhibits a relatively high metabolic rate. Although numerous growth factors have been identified in luteal tissue, their role in growth and differentiation of this dynamic organ remains unclear. Recently, while attempting to identify mitogenic factors of ovine and bovine CL, we have found that they produce several mitogens during the estrous cycle as well as pregnancy. The majority of these luteal-derived mitogenic factors are heparin-binding, and although some may represent previously identified factors, several appear to be novel heparin-binding growth factors. Isolation and purification of mitogenic factors produced by the CL will enable us to determine their roles in luteal growth, development and differentiated function, which will contribute to our understanding not only of the regulation of fertility but also of tissue growth and development in general.

Animals↗
PubMed1994

Size, number, cellular proliferation, and atresia of gonadotropin-induced follicles in ewes.

To determine the effect of exogenous gonadotropin on size, number, cellular proliferation, and atresia of follicles, ewes (n = 3-5/treatment/day) received an injection of vehicle or FSH-P (a pituitary extract) twice daily on Days 13, 14, and 15 (5, 4, and 3 mg FSH-P/injection, respectively; Day 0 = estrus) and were slaughtered on Days 14, 15, or 16 (i.e., after 24, 48, or 72 h of FSH-P treatment, respectively). An additional group of ewes (Day 13 control) received no treatment and were slaughtered on Day 13. All ewes received an i.v. injection of bromodeoxyuridine (BrdU, a thymidine analog; 5 mg/kg-1 BW) 1 h before slaughter. For both ovaries from each ewe, number and surface diameter of all visible follicles were recorded, and antral follicles were classified as small (< or = 3 mm), medium (> 3 mm to < or = 6 mm), or large (> 6 mm). To evaluate rate of proliferation of follicular cells, ovaries were fixed by perfusion with Carnoy's solution, and BrdU was immunolocalized in paraffin-embedded sections by use of a specific primary antibody and indirect immunoperoxidase detection. As an index of the rate of cellular proliferation, labeling index (LI: number of BrdU-labeled nuclei expressed as a percentage of total nuclei) of granulosa and thecal cells was determined by image analysis of antral follicles of known diameter. Follicular status (atretic vs. nonatretic) also was evaluated morphologically by using the histological sections. After 24 h of treatment (i.e., on Day 14), FSH-P-treated ewes had an increased (p < 0.01) number of medium follicles compared with vehicle-treated ewes.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Evaluation of growth, cell proliferation, and cell death in bovine corpora lutea throughout the estrous cycle.

To evaluate the kinetics of luteal growth, bovine CL were obtained from four stages (stage I, Days 1-4; stage II, Days 5-10; stage III, Days 11-17; stage IV, Days 18-21) of the estrous cycle, and luteal fresh weight as well as DNA, protein, and progesterone contents was determined. To evaluate the relative rate of cell proliferation, proliferating cell nuclear antigen (PCNA; a specific marker for cell proliferation) was immunolocalized in paraffin-embedded luteal tissue sections. To evaluate the relative rate of cell death, nucleosomal fragmentation of DNA (a specific marker for apoptosis) was detected by agarose gel electrophoresis and also by histochemical localization in paraffin-embedded luteal tissue sections. Luteal fresh weight and DNA, protein, and progesterone contents increased (p < 0.01) from stage I to stage II, were similar between stages II and III, and then decreased (p < 0.01) from stage III to stage IV. The ratio of protein to DNA (an index of average cell size) was similar for stages I, II, and III and then decreased (p < 0.01) at stage IV. For stage I (corpora hemorrhagica), most proliferating (PCNA-positive) cells were located in or around the core of the tissue infoldings (presumably thecal-derived areas), whereas relatively few proliferating cells were located at the periphery of the tissue infoldings (presumably granulosa-derived areas). For stages II, III, and IV, the majority of proliferating cells appeared to be small cells (i.e., small parenchymal cells, fibroblasts, and endothelial cells). The labeling index (LI; percentage of cells that were PCNA-positive) was greatest at stage I (20.3 +/- 1.1%); it then decreased (p < 0.01) by stage II and was similar at stages II, III, and IV (3.4 +/- 1.1%). Apoptosis, as determined by evaluation of nucleosomal DNA fragmentation by agarose gel electrophoresis and in situ localization, was detectable only at stage IV. These data demonstrate that luteal growth from stage I to stage II resulted from cell proliferation as shown by a high LI at stage I, accompanied by increased luteal DNA content but no change in average cell size, and by similar protein: DNA ratios. Luteal regression from stage III to stage IV was primarily associated with cell deletion and decreased cell size as shown by a decrease in luteal DNA content and the appearance of apoptosis along with a decrease in the luteal protein: DNA ratio.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Effects of dietary fiber on intestinal growth, cell proliferation, and morphology in growing pigs.

Growing pigs (initial BW 14.3 +/- 1.2 kg) were fed isocaloric (3.26 Mcal of ME/kg) and isonitrogenous (16% CP) diets containing either 0 (low fiber, LF; n = 4) or 10% (high fiber, HF; n = 4) wheat straw for ad libitum intake for 14 d. On d 14, each pig was injected i.v. with bromodeoxyuridine (BrdU, a thymidine analog; 5 mg/kg) and was slaughtered 1 h later. Visceral organs (liver, pancreas, and intestines) were weighed, and tissue samples were obtained. Feed consumption, daily gain, gain: feed, and final BW did not differ between treatments. Neither visceral weights nor visceral weights per unit of eviscerated BW were affected by diets. Tissue concentrations of DNA (milligrams/gram of tissue) were lower (P < .03) in HF than in LF only for jejunum, ileum, and liver. Contents of DNA and protein (milligrams) did not differ between LF and HF for intestinal segments or liver. Content of RNA (milligrams) was greater (P < .04) in HF than in LF only for colon. The number of crypt cell nuclei that were labeled with BrdU (indicating DNA synthesis and thus cell proliferation) was increased (P < .03) in HF relative to LF for jejunum and colon. The number of epithelial cells exhibiting DNA fragmentation (indicating programmed cell death) was greater (P < .07) in the HF than in the LF group for jejunum and ileum. Width of intestinal villi was increased (P < .10) in HF vs LF for jejunum and ileum. Depth of intestinal crypts was increased (P < .08) in HF vs LF for jejunum, ileum, and colon.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗