Biomedical subjects

J E Gadsby

Publications and source records attributed to J E Gadsby.

At least 19 recordsLinked to original sources

PubMed2003

Expression and localization of vascular endothelial growth factor and its receptors in pig corpora lutea during the oestrous cycle.

Expression and localization of mRNAs for vascular endothelial growth factor (VEGF), VEGF receptor 1 (Flt) and VEGF receptor 2 (KDR) (VEGFR-1 and VEGFR-2, respectively) were investigated in pig corpora lutea. Northern blot analysis of total RNA indicated hybridization of pig VEGF, VEGFR-1 and VEGFR-2 cDNA probes to mRNA transcripts of approximately 3.9, 7.0 and 5.0 kb, respectively. The expression of mRNAs for VEGF and its receptors during the luteal phase (days 4, 7, 10, 13 and 15 after the onset of oestrus) were assessed by northern blot analysis, and hybridization signals were normalized to expression of pig 18S rRNA. Relative hybridization signals of expression of VEGF mRNA appeared to be constant; however, expression of VEGFR-1 mRNA was low on day 4, increased on day 7, and was higher on days 10, 13 and 15 (P<0.05, compared with day 4). In contrast, no changes in expression of mRNA for VEGFR-2 were evident on days 4-13, but a decrease was detected (P<0.05) at day 15. In situ hybridization revealed that VEGF mRNA was localized predominantly in large luteal cells, whereas both VEGFR-1 and VEGFR-2 were localized to small cells. These data indicate that the VEGF system may be involved in the regulation of luteal vasculature throughout the lifespan of the corpus luteum. Although the expression of VEGF mRNA was unchanged during the luteal phase, variations in the expression of VEGFR-1 and VEGFR-2 mRNAs indicate that differential regulation of expression of the VEGF receptors may play a role in the control of VEGF-mediated vascular growth at different phases of development and maturation of the pig corpus luteum.

Animals↗

PubMed2003

Steroidogenic responses of pig corpora lutea to insulin-like growth factor I (IGF-I) throughout the oestrous cycle.

This study was designed to investigate the roles of insulin-like growth factor I (IGF-I), IGF-type I receptor (IGF-IR) and IGF-binding proteins (IGFBPs) in regulating progesterone secretion by pig corpora lutea during the oestrous cycle, and the signal transduction pathways involved in mediating the steroidogenic actions of IGF-I. Corpora lutea were collected on days 4, 7, 10, 13 and 15 or 16 of the oestrous cycle, enzyme dissociated and the luteal cells were cultured for 24 h in Medium 199 with IGF-I (0-100 ng ml(-1)), long R(3)-IGF-I (0-100 ng ml(-1)), anti-IGF-I (Sm 1.2B; 0-10 microg ml(-1)), anti-IGF-IR (alphaIR3; 0-2 microg ml(-1)), or IGF-I signal transduction pathway inhibitors (phosphatidylinositol (PI)-3-kinase: 100 nmol Wortmannin l(-1) and 10 micromol LY 294002 l(-1); MAP kinase: 50 micromol PD 98059 l(-1)) to investigate their effects on IGF-I (100 ng ml(-1)) stimulated progesterone secretion. Pig luteal cells displayed dose-dependent responses to IGF-I and long R(3)-IGF-I on days 4 and 7 of the oestrous cycle, but not on days 10-16. There was no difference in the ED(50) or V(max) (maximal response) values between IGF-I and long R(3)-IGF-I. Neither anti-IGF-I nor anti-IGF-IR had significant effects on progesterone secretion, at any dose or day. Wortmannin and LY 294002 blocked IGF-I stimulated progesterone secretion, but PD 98059 was without effect. Finally, IGF-I (6 microg) infused into the ovary on day 7 in vivo significantly increased progesterone secretion within 45 min of infusion. The conclusions of this study are: (1) IGF-I has steroidogenic actions only on 'young' (days 4-7) pig corpora lutea; (2) endogenous IGF-I and IGFBP are insufficient to modulate progesterone secretion in vitro; and (3) the steroidogenic actions of IGF-I are mediated via PI-3-kinase.

Androstadienes↗

PubMed2003

Cloning of pig prostaglandin F2alphaFP receptor cDNA and expression of its mRNA in the corpora lutea.

Changes in the expression and localization of luteal mRNA for PGF(2alpha) (FP) receptors may be critical in determining the luteolytic action of PGF(2alpha) in pig corpora lutea. In this study, a full-length FP receptor (FPr) cDNA was isolated and cloned from pig corpora lutea. This isolate (GenBank accession no. U91520) contains an open reading frame of 1086 bases coding for a protein of 362 amino acids with seven potential transmembrane domains. The predicted amino acid sequence of this isolate was 83% identical to the FPr amino acid sequence of other species including sheep, cattle and humans. Northern blot analysis showed the presence of an FPr message of about 5 kb in mRNA from pig corpora lutea. Relatively weak FPr mRNA expression was detected on day 4 and day 7 of the oestrous cycle. The expression was greater (P < 0.05) on days 10, 13 and 15 than on days 4 and 7. In situ hybridization analysis revealed that mRNA for FPr was expressed predominantly in the steroidogenic large luteal subtype of cell, although there was some expression in small luteal cells, with histological appearance of steroidogenic small cells. Localization of hybridization signals of FPr was observed in luteal tissue at all stages examined. These data demonstrate that FPr is expressed in pig corpora lutea throughout the oestrous cycle and that upregulation of the FPr mRNA occurs when the corpora lutea becomes sensitive to PGF(2alpha). Direct luteal targets of PGF(2alpha) appear to be primarily large steroidogenic cells in this species.

Amino Acid Sequence↗

PubMed2000

Porcine ovarian cells express messenger ribonucleic acids for the acid-labile subunit and insulin-like growth factor binding protein-3 during follicular and luteal phases of the estrous cycle.

It has recently been shown that, in follicular fluid, as in the circulation, insulin-like growth factors (IGFs)-I and -II exist in a ternary complex with IGF binding protein-3 (IGFBP-3) and the acid-labile subunit (ALS). The current study was designed to determine whether ovarian follicular and luteal cells could synthesize IGFBP-3 and ALS. Ovaries were collected, during the follicular and early luteal phases, from mature pigs whose cycles were synchronized with PGF2alpha. We studied IGFBP-3 and ALS messenger RNA (mRNA) by in situ hybridization. These transcripts were colocalized with aromatase mRNA, a marker of healthy granulosa cells. IGFBP-3 mRNA was equally expressed in granulosa cells of all growing follicles. In contrast, granulosa cell ALS mRNA levels were higher (P < 0.05) in preantral and small antral follicles than in large antral follicles. In thecal cells, expression of mRNA for IGFBP-3, ALS and cyclin D1 (a marker of cell proliferation) was restricted to healthy (aromatase-expressing) follicles. In those follicles, thecal expression of IGFBP-3 mRNA was low or absent in preantral follicles but increased (P < 0.05) in antral follicles. Thecal cell ALS transcripts peaked in small antral follicles (P < 0.05) and then declined. In granulosa cells of atretic follicles, transcripts for aromatase were greatly reduced, whereas IGFBP-3 mRNA levels remained high. In contrast, ALS transcript levels were greatly reduced in both granulosa (P < 0.05) and thecal cells (P < 0.001) of atretic follicles. After ovulation, IGFBP-3 mRNA was moderately expressed in granulosa luteins but strongly detected in a few theca-derived cells and in vascular endothelial cells. This study demonstrates that follicular fluid IGFBP-3 and ALS, like the IGFs, originate (at least in part) from the ovary. The ability of follicular cells to synthesize, assemble, and store all components of the ternary complex may be critical in determining the bioavailability of follicular IGF-I and -II.

Animals↗

PubMed2000

Messenger ribonucleic acids for MAC25 and connective tissue growth factor (CTGF) are inversely regulated during folliculogenesis and early luteogenesis.

Cell proliferation, terminal differentiation, and angiogenesis occur during cycles of follicular and luteal development. In other paradigms, mac25, a potent tumor inhibitor is strongly induced in senescent epithelial cells, whereas CTGF stimulates angiogenesis and wound healing. Using in situ hybridization and immunohistochemistry, we have examined the possibilities that mac25 is inhibited, whereas CTGF is induced during active periods of follicular development and luteogenesis. Ovaries were collected during the follicular and early luteal phases from prostaglandin F2alpha-treated mature pigs and from slaughterhouse sows. CTGF transcripts were induced during the late preantral stage in granulosa and theca cells concomitantly with the appearance of endothelial cells in the theca. CTGF mRNA expression increased in granulosa cells to a maximum (P < 0.01) in mid-antral follicles but was down regulated (P < 0.01) in preovulatory follicles. In contrast, granulosa cell mac25 mRNA expression was undetectable between the preantral and mid-antral stage but was strongly induced in terminally differentiated granulosa cells of preovulatory follicles. CTGF mRNA and peptide were also detected in the theca externa/interstitium and in vascular endothelial cells of ovarian blood vessels, whereas mac25 transcripts, which were also abundant in ovarian blood vessels increased in the theca interna with follicular development. Transcripts of cyclin D 1, a marker of cell proliferation, appeared during the early antral stage and were moderate in granulosa cells but abundant in capillary endothelial cells in the theca interna, underneath the basement membrane. Following ovulation, CTGF and cyclin D1 mRNAs were associated with the migration of endothelial cells into the CL. Subsequently, there was a marked up-regulation of CTGF mRNA expression in granulosa luteins concomitantly with an increase in endothelial cell proliferation within the CL. We hypothesize that CTGF may promote ovarian cell growth and blood vessel formation during follicular and luteal development whereas mac25, a tumor inhibitor, may promote terminal differentiation of granulosa cells in preovulatory follicles.

Animals↗

PubMed2000

Insulin-like growth factor I receptor mRNA and protein expression in pig corpora lutea.

Insulin-like growth factor I (IGF-I) is believed to play a luteotrophic role in the pig corpus luteum during the oestrous cycle. Since the actions of IGF-I in target tissues are mediated by the type I IGF receptor, the concentrations of IGF-I receptor mRNA and protein were examined in pig corpora lutea at different stages of the oestrous cycle. Corpora lutea were collected from normally cyclic gilts on days 4, 7, 10, 13, 15 and 16 of the oestrous cycle (n = 4 animals per day). Corpora lutea on days 7, 10 and 13 were dissociated with collagenase, and large and small luteal cell sub-populations were separated by elutriation. Northern and slot blots were used to examine mRNA, and western blots were used to measure the concentrations of IGF-I receptor protein in the pig corpus luteum. On northern blots, luteal IGF-I receptor mRNA was present as a single 11 kb transcript. The slot blots showed that the steady state expression of IGF-I receptor mRNA increased significantly (P < 0.05) from its lowest value on day 4, to reach a maximum on days 13-16. IGF-I receptor mRNA was also expressed to a greater extent in large compared with small luteal cells (P < 0.05). On western blots, IGF-I receptor appeared as a 95 kDa protein band (beta-subunit) and IGF-I receptor protein concentrations were significantly higher (P < 0.05) on days 4-10 than on days 13-16. Finally, large luteal cells appeared to contain more IGF-I receptor protein than the small luteal cells. In conclusion, since IGF-I receptor was detected in the pig corpus luteum, it is a likely target tissue for IGF-I, especially during the early luteal phase. Furthermore, IGF-I receptor was localized primarily on large luteal cells, thus it is hypothesized that IGF-I may play a paracrine role in the pig corpus luteum.

Analysis of Variance↗

PubMed1999

Insulin-like growth factor (IGF)-I, IGF-I receptor, and IGF binding protein-3 messenger ribonucleic acids and protein in corpora lutea from prostaglandin F(2alpha)-treated gilts.

Insulin-like growth factor-I (IGF-I) is produced within the porcine corpus luteum (CL) and is thought to play an autocrine/paracrine role in CL development/function during the early luteal phase. This study examines the hypotheses that the luteolytic actions of prostaglandin F(2alpha) (PGF(2alpha)) during the early luteal phase may involve either a decrease in IGF-I or IGF receptor (IGF-IR), or an increase in IGF binding protein (IGFBP)-3, expression, any of which could interfere with the luteotropic actions of IGF-I in this tissue. Cycling gilts were treated twice daily with PGF(2alpha) (or saline) on Days 5-9 of the cycle to induce premature luteolysis. CL were collected on Days 6-9, and RNA, protein, or progesterone was extracted. By slot blot analysis, steady-state levels of IGF-I and IGFBP-3 mRNA were not different in PGF(2alpha)-treated vs. control animals; however, IGF-IR mRNA was increased in treated animals on Day 9. No changes in IGF-I content (ng/CL measured by RIA) were observed with respect to treatment. According to ligand blot analysis, the levels of IGFBP-3 increased on Day 6 and decreased on Days 8-9, while IGFBP-2 was higher on Days 6-7 and decreased on Day 9 in treated animals. IGF-IR levels, determined from Western blots, were higher on Day 7 (P < 0.05) and lower on Day 9 in PGF(2alpha)-treated animals vs. control animals (P < 0.05). In conclusion, PGF(2alpha)-induced premature luteolysis was associated with an increase in steady-state levels of IGF-IR mRNA, but it did not appear to be linked to changes in mRNA levels for IGF-I or IGFBP-3. However, since IGFBP-2 and -3 protein levels increased early in the treatment period (Days 6-7), it is possible that they may mediate the luteolytic actions of PGF(2alpha) by sequestering IGF-I and preventing its interaction with the IGF-IR.

Animals↗

PubMed1996

Expression of the messenger ribonucleic acids for insulin-like growth factor-I and insulin-like growth factor binding proteins in porcine corpora lutea.

In the pig, the corpus luteum (CL) can develop and function autonomous of pituitary gonadotropins for approximately 12 days. We hypothesized that the insulin-like growth factor (IGF) system may play an autocrine/paracrine luteotrophic role(s) during this period. In this study, we monitored the expression (i.e., steady-state levels of mRNAs) of IGF-I and IGF binding proteins (IGFBP)-2, -3, -4, -5, and -6 mRNAs in whole CL and in small and large luteal cells on Days 4-16 of the estrous cycle. CL were dissociated with collagenase, and large and small luteal cells were isolated by centrifugal elutriation. Whole CL and luteal cells were extracted to isolate total or poly(A)+ RNA, which was subjected to Northern and/or dot-blot analyses using [32P]-labeled cDNA probes for IGF-I and IGFBP-2, -3, -4, -5, and -6. Northern blots showed readily detectable transcripts for IGF-I (6.7 and 0.9 kb), IGFBP-2 (1.8 kb), IGFBP-3 (2.8 kb), IGFBP-4 (2.6 kb), and IGFBP-5 (6.0 kb), but not for IGFBP-6. IGFBP-3 and -5 transcripts were observed mainly in small luteal cells, while IGFBP-2 and -4 were seen in both cell types. Dot-blot analyses for IGF-I and IGFBP-3 mRNAs were performed on total RNA from small and large luteal cells; blots were counter-probed with 3-phosphoglyceraldehyde dehydrogenase (p-GAD) cDNA to assess RNA quantity and quality. IGF-I mRNA (ratio IGF-I:p-GAD mRNA) expression was approximately 2-fold greater in small than in large luteal cells on Days 4-10. However, steady-state levels of IGF-I mRNA in small, but not large, luteal cells decreased significantly on Days 12-16 (vs. Days 4-10). IGFBP-3 mRNA expression was significantly greater (approximately 3-fold) in small than in large luteal cells but did not vary significantly between Days 4-10 and 12-16 for either cell type. We conclude that porcine CL express mRNAs for IGF-I and IGFBP-2, -3, -4, and -5, and that while small luteal cells are the major sources of IGF-I and IGFBP-3 and -5, IGFBP-2 and -4 appear to be expressed to approximately the same extent in small and large luteal cells. These results further suggest that the IGF-I/IGF system may have autocrine/paracrine regulatory actions in CL development/function in the pig.

Animals↗

PubMed1995

Does increased PGF2 alpha receptor concentration mediate PGF2 alpha-induced luteolysis during early diestrus in the pig?

The mechanism by which multiple injections of PGF2 alpha result in premature luteolysis in pigs is unknown. In the present study we evaluated whether PGF2 alpha receptor concentrations on large luteal cells changed when gilts were injected IM with 12.5 mg of PGF2 alpha every 12 hours from the morning of day 5 of an estrous cycle (estrus = day 0) until ovariectomy on day 6, 7, 8, or 9. Luteal PGF2 alpha receptor concentrations remained constant from day 6 through 9 in the PGF2 alpha-treated group, but increased linearly (P > 0.05) in control gilts from day 6 to 9. Receptor affinity for PGF2 alpha did not change throughout the study in either PGF2 alpha-treated or control gilts. Luteal progesterone concentrations were significantly lower in PGF2 alpha-treated gilts than in control gilts only on day 9. Histological examination of luteal tissue obtained from PGF2 alpha-treated gilts revealed definite evidence of luteolysis by day 8. We conclude that PGF2 alpha-induced premature luteolysis is not mediated by an increase in luteal PGF2 alpha receptor concentrations and, based on luteal progesterone concentrations and histological features, that the PGF2 alpha-based protocol used to shorten the estrous cycle is accompanied by premature functional and structural luteal regression.

Animals↗

PubMed1994

Prostaglandin F2 alpha stimulates progesterone secretion by porcine luteal cells in vitro throughout the estrous cycle.

In this study we examined the stimulatory effects of PGF2 alpha on progesterone secretion by porcine luteal cells on different days of the estrous cycle, and the effects of PGF2 alpha, A23187 and PMA on progesterone secretion by isolated large and small luteal cells, in vitro. Corpora lutea were obtained from cycling pigs (days 6-16), collagenase dispersed and luteal cells incubated in medium 199 in the absence or presence of increasing doses of PGF2 alpha, A23187, and PMA. Progesterone concentrations in spent media were measured by RIA. PGF2 alpha stimulation of progesterone secretion by mixed luteal cells did not vary significantly throughout the estrous cycle. Progesterone secretion by large, but not small, luteal cells was increased (p < 0.05) in a dose-dependent fashion by PGF2 alpha. A23187 also caused a dose-dependent increase in progesterone secretion by large luteal cells but inhibited small luteal cells. Progesterone secretion by both large and small luteal cells was significantly increased by increasing doses of PMA. We conclude that the stimulatory response of luteal cells to PGF2 alpha in vitro did not correlate with PGF2 alpha receptor concentrations (not measured in this study), and we speculate that calcium/protein kinase C may be involved in mediating the stimulatory action of PGF2 alpha on luteal cell progesterone secretion.

3-Hydroxysteroid Dehydrogenases↗

PubMed1994

Differential effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells.

This study examined the effects of LH and PGE2 on progesterone secretion by small and large porcine luteal cells with or without low-density lipoproteins. Corpora lutea were isolated from gilts 13-14 days after administering gonadotrophins; enzymatically dissociated and small and large cells were isolated by elutriation. Culture plates, 24-well, were then seeded with 150,000 small or 30,000 large luteal cells suspended in 1 ml M199 medium supplemented with 5 micrograms insulin ml-1, 40 ng hydrocortisone ml-1 and with or without low-density lipoproteins (50 micrograms cholesterol ml-1) or PGE2. Cells were cultured for up to 24 h in a humidified incubator at 37 degrees C under 5% CO2 in air. The low-density lipoproteins stimulated (P < 0.05) progesterone secretion by large, but not small, luteal cells. Prostaglandin E2 stimulated (P < 0.05) progesterone production by large luteal cells in a dose-dependent manner, and the stimulatory effects of PGE2 were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. Progesterone secretion by small luteal cells was not significantly affected by PGE2. Progesterone production by small luteal cells was enhanced (P < 0.05) by LH, and the stimulatory effects of LH were greater (P < 0.05) in the presence than in the absence of low-density lipoproteins. In the absence of these lipoproteins, LH had no effect on progesterone secretion by large luteal cells; however, in the presence of low-density lipoproteins, LH increased (P < 0.05) progesterone secretion by large cells, though to a lesser (P < 0.05) extent than the effect of LH on small cells. These data demonstrate that progesterone secretion by porcine luteal cells is stimulated differentially by LH and PGE2 and that small luteal cells are more responsive to LH and PGE2 acts primarily on large luteal cells.

Animals↗

PubMed1993

Repeated administration of prostaglandin F2 alpha during the early luteal phase causes premature luteolysis in the pig.

Previous investigators considered pig corpora lutea refractory to the luteolytic effects of prostaglandin (PG) F2 alpha before Day 12 of the estrous cycle. This study was designed to determine whether multiple injections of PGF2 alpha would result in a sustained reduction of serum progesterone and luteolysis, leading to significant shortening of the estrous cycle and interestrous interval. On Days 5-10 of an estrous cycle, gilts (n = 4) received injections of 12.5 mg PGF2 alpha (dinoprost tromethamine) i.m. every 12 h, or vehicle (PBS; n = 4) according to the same schedule. Mean interestrous interval in PGF2 alpha-treated gilts was reduced (p < 0.001) to 13.3 +/- 0.5 days compared with 19.8 +/- 0.6 days for control gilts. Serum progesterone declined below 1 ng/ml by Day 10.5 in PGF2 alpha-treated gilts compared to Day 17.5 in control animals. Serum concentrations of estradiol-17 beta (E2) reached maximal levels in PGF2 alpha-treated gilts earlier (Day 12.5) in the cycle than in control gilts (Day 19.5). Peak E2 and LH concentrations coincided with the periestrous period, suggesting that PGF2 alpha-induced estrus is accompanied by normal follicular development and ovulation. These results demonstrate that the pig is susceptible to the luteolytic effects of PGF2 alpha before Day 12 if repeated injections are given from Day 5 through Day 10.

Animals↗

PubMed1993

Prostaglandin F2 alpha receptor concentrations in corpora lutea of cycling, pregnant, and pseudopregnant pigs.

The objective of this study was to measure and compare the concentrations of PGF 2 alpha receptors on luteal cells taken from cycling, pregnant, and pseudopregnant pigs. Corpora lutea were removed surgically from cycling, pregnant, and pseudopregnant (induced with 5 mg estradiol valerate/day i.m. beginning on Day 11) pigs on Days 12, 13, and 14 (postestrus) and were subjected to collagenase dissociation. Dissociated luteal cells (approximately 100,000 large viable cells per tube) were assayed for specific PGF 2 alpha binding by Scatchard analysis, using [3H]PGF 2 alpha and varying doses (0-5 microM) of unlabeled PGF 2 alpha. Luteal cells from all three types of pigs were shown to possess two specific PGF 2 alpha binding sites (high affinity, Kd = 9-47 nM; low affinity, Kd = 243-1359 nM). The concentrations of the high-affinity PGF 2 alpha binding site (PGF 2 alpha "receptor") on Days 12 and 13 were not significantly different (NS) between cycling (1.7 and 1.1 x 10(6) receptors per large luteal cell, respectively), pregnant (1.3 and 1.2 x 10(6)), and pseudopregnant (1.1 and 0.8 x 10(6)) pigs. However, on Day 14, luteal PGF 2 alpha receptor concentrations were significantly higher (p < 0.05) in cycling (4.2 x 10(6)) compared with pregnant (1.3 x 10(6)) and pseudopregnant (1.4 x 10(6)) pigs. We speculate that reduced luteal PGF 2 alpha receptor concentrations on Day 14 in pregnant and pseudopregnant compared with cycling pigs may lead to decreased luteal sensitivity to PGF 2 alpha in these animals, and that this mechanism may play a role in the maternal recognition of pregnancy in this species.

Animals↗

PubMed1992

The presence of glandular kallikrein in rabbit fetal placental conditioned medium.

Glandular (tissue) kallikreins are known to be involved in the posttranslational modification of protein hormones and growth factors in addition to their classical role as the bradykinin-releasing enzyme in tissues. They have been shown to be present in many tissues, such as the pancreas, salivary glands, pituitary, and testes. The objective of this study was to investigate the presence of kallikrein in the rabbit placenta. Day 21 pregnant rabbit fetal placentae were teased apart in medium 199, washed thoroughly, and incubated in fresh medium for 6 h at 37 C. The resulting placental conditioned medium (FPI) was found to have 31.35 +/- 4.3 U glandular kallikrein-like amidolytic activity/mg protein toward the chromogenic peptide substrate S-2266 (1 U is defined as the amount of p-nitroaniline liberated by 10 ng rat urinary kallikrein). Using an enzyme-linked immunosorbent assay with sheep serum raised against rat urinary kallikrein, the glandular kallikrein concentration of FPI was estimated to be 4.56 +/- 0.857 micrograms/mg protein. Upon Western blot analysis, FPI gave a positive band of 45,000 in the presence of beta-mercaptoethanol. In the absence of beta-mercaptoethanol, a band of 138,000 was observed, indicating that in FPI, kallikrein is present bound to a high mol wt binding protein. These results strongly indicate the presence of glandular kallikrein in rabbit fetal placentae.

Animals↗

PubMed1991

Acute stimulatory effects of prostaglandin F2 alpha on serum progesterone concentrations in pregnant and pseudopregnant pigs.

The objective of this study was to investigate whether PGF2 alpha, administered to pregnant and pseudopregnant gilts in vivo, would cause an acute increase in serum progesterone concentrations prior to luteolysis. Pregnant (n = 9) and pseudopregnant (n = 4) gilts were fitted with a jugular vein cannula on day 40, were treated with 3 ml vehicle (control) i.m. on day 42 and with 15 mg PGF2 alpha on day 45. Blood samples were collected at frequent (5 and 15 min) intervals from 1 h before until 1 h after control and PGF2 alpha injections, at 15 min intervals for 4 h, and then at 5, 6, 9, 21, 33, 45 and 57 h post injection. Progesterone was measured by radioimmunoassay (RIA) in all samples. Porcine LH was measured by RIA in samples collected frequently in the 1 h pre- and 1 h post-injection periods. Serum progesterone concentrations were unchanged in both pregnant and pseudopregnant animals in response to control injection on day 42. However, in both pregnant and pseudopregnant gilts, PGF2 alpha injection on day 45 resulted in an acute increase (approximately 75-80% above pre-treatment levels; p less than 0.05) in serum progesterone lasting approximately 1 h, followed by a return to pre-treatment levels by 2 h, and then a decline to 1 ng/ml or less by 45-57 h (pregnant) or 21-57 h (pseudopregnant), associated with luteolysis. Serum LH concentrations were unchanged between 1 h pre- and post-treatment periods in response to either control or PGF2 alpha-treatment, in both pregnant and pseuodpregnant gilts. These results indicate that PGF2 alpha-injection produces a rapid and transient increase in serum progesterone concentrations which may result from a rapid and direct stimulatory action of PGF2 alpha on porcine luteal cell progesterone synthesis/secretion in vivo.

Animals↗

PubMed1991

Luteal enzymes of the luteinizing hormone and beta-adrenergic signal transduction pathways in hypophysectomized rabbits do not require pituitary hormone support.

Experiments were conducted to determine whether continuous pituitary hormone support is required for expression of the LH and beta-adrenergic cAMP signal transduction pathways in rabbit CL during pseudopregnancy. Parameters of the LH and catecholamine cAMP signal transduction pathways in CL of estrogen-treated hypophysectomized rabbits were compared to those of pituitary-intact rabbits. Results showed that each of the parameters of the LH and beta-adrenergic cAMP signal transduction pathways was retained in CL taken from estrogen-treated pseudopregnant rabbits that had been hypophysectomized for as long as 13 days at levels not significantly different from those of estrogen-treated pituitary-intact rabbits. These included luteal basal, and LH-, epinephrine-, and fluoride-stimulated adenylyl cyclase activities; total luteal cAMP levels; the number and affinity of cAMP-dependent protein kinase regulatory subunit cAMP binding sites; binding activity of the type I and type II regulatory subunits; and the amount of catalytic subunit protein of cAMP-dependent protein kinase. We conclude that expression of the proteins of the cAMP signal pathway for LH and beta-adrenergic hormones in CL of estrogen-treated rabbits does not require pituitary hormone support.

Animals↗

PubMed1990

Prostaglandin F2 alpha receptors on enzyme-dissociated pig luteal cells throughout the estrous cycle.

A deficiency of luteal cell prostaglandin F2 alpha (PGF2 alpha) receptors might help explain the well documented refractoriness of pig corpora lutea to the luteolytic effects of PGF2 alpha administered in vivo before day 12 of the estrous cycle. Accordingly, experiments were conducted to measure the levels of [3H] PGF2 alpha-binding sites/receptors on collagenase-dispersed pig luteal cells taken at different stages of the estrous cycle. Pig corpora lutea were obtained surgically at various stages of the estrous cycle and dissociated with collagenase in medium 199. Dissociated mixed luteal cells (approximately 5-15 x 10(4) large luteal cells/tube) were assayed for [3H]PGF2 alpha-binding activity by saturation (Scatchard) analysis. In preliminary experiments it was determined that PGF2 alpha binding was maximal after incubation for 45 min at 30 C in assay buffer of pH 5.75. Additionally, it was determined that [3H]PGF2 alpha binding was displaceable by PGF2 alpha = PGD2 greater than PGE2 greater than 13,14-dihydro-15-keto-PGF2 alpha. Other eicosanoids did not inhibit [3H]PGF2 alpha binding. Two distinct classes of binding sites (high affinity Kd = 19-64 nM; low affinity Kd = 262-3103 nM) were observed at all stages of the estrous cycle. From studies using enriched (by elutriation) large (greater than 30 microns) and small (10-20 microns) luteal cells it appeared that the high affinity binding site was largely confined to large luteal cells, whereas the low affinity binding site was found on both large and small luteal cells. The concentrations (number per large luteal cell) of high affinity PGF2 alpha-binding sites of mixed (unelutriated) luteal cell preparations was low on days 6-8 (0.6 x 10(6) sites/cell) and increased gradually up to 1.4 x 10(6) sites/cell on day 12. The concentrations of binding sites were increased approximately 3-fold on day 13 (4.6 x 10(6) sites/cell; P less than 0.05 vs days 6-12) and remained elevated on days 14 and 16-17 (approximately 3 x 10(6) sites/cell). In summary, these results indicate the existence of a high affinity PGF2 alpha-binding site in pig (large) luteal cells, which is probably the luteal PGF2 alpha receptor. The numbers of these putative PGF2 alpha receptors are low during the early luteal phase (before day 12), but increase thereafter (days 13, 14, and 16-17). This may provide one explanation for the observed refractoriness in vivo of pig corpora lutea to PGF2 alpha before, and increased sensitivity to PGF2 alpha after, day 12 of the estrous cycle.

Animals↗

PubMed1989

Rabbit placental-conditioned medium stimulates progesterone accumulation by granulosa-lutein cells in culture: preliminary characterization of a placental luteotropic hormone.

The rabbit fetal placenta plays an important physiological role in luteal maintenance in pregnancy, probably via the secretion of an unidentified placental "luteotropin." The objective of these studies was to examine conditioned medium from fetal placental-tissue incubations (FPI) for the presence of placental luteotropic hormones/factors, using the stimulation of progesterone accumulation by rabbit granulosa-lutein cells in culture, as an in vitro luteotropic bioassay. Progesterone accumulation by rabbit granulosa-lutein cells (during 5 days of culture) was increased (compared with controls), 1.5-fold by 10(-8) M estradiol-17 beta (E2) and 11.5-fold by 100 ng/ml luteinizing hormone (oLH). FPI stimulated progesterone accumulation (approximately 3-fold) and this was further increased in the presence of E2 (FPI + E2; approximately 6-fold). Luteotropic bioactivity in FPI (+ E2) was retained after dialysis (6000-8000 MW cutoff; 7.8-fold) and heating (90-95 degrees C for 1 h; 7.5-fold), but was destroyed after incubation with trypsin (1 mg/ml, 1 h at 37 degrees C; 0.9-fold). Media conditioned with skeletal muscle (1.2-fold), heart (1.6-fold), liver (1.5-fold), and uterus (0.5-fold) and 5-10% serum (less than 1-fold), from pseudopregnant rabbits, had little or no luteotropic bioactivity. These data indicate that FPI contains a luteotropic hormone/factor that is probably a heat-stable, trypsin-sensitive, protein/peptide of greater than 6000-8000 MW that acts in synergy with E2 to promote granulosa-lutein cell steroidogenesis. This placental hormone/factor is a good candidate for the elusive rabbit placental luteotropin.

Animals↗