Biomedical subjects

W Hansel

Publications and source records attributed to W Hansel.

At least 19 recordsLinked to original sources

PubMed1995

Contact-associated interactions between large and small bovine luteal cells during the estrous cycle.

This experiment was designed to study the effects of cell-to-cell contact, arachidonic acid (10 microM; AA), oxytocin (10 microM), and luteinizing hormone (5 ng; LH) on bovine luteal cell function. Corpora lutea collected from Holstein cows between Days 10 and 12 (n = 4; midluteal stage) or 17 and 18 (n = 4; late-luteal stage) of the estrous cycle (Day 0 = estrus) were dispersed, and small and large cells were separated by unit gravity sedimentation and flow cytometry. Large and small luteal cells were either incubated together, allowing intercellular contact, or separately, without intercellular contact, with culture well inserts. Cells were incubated in a modified Ham's F-12-N-hydroxyethylpiperazine-N'-2-ethanesulfonic acid medium. After an 18-hr preincubation period, treatments were introduced and cells were incubated for 240 hr. Media samples were collected and treatments were replaced at 48-hr intervals. Incubations were maintained at 37 degrees C in 5% CO2 in humidified air. Overall, progesterone secretion decreased with increased incubation time (P < 0.0001), regardless of treatment, stage of the cycle, or cell arrangement. During the 18-hr pretreatment period, large and small luteal cells with contact secreted more progesterone than did luteal cells without contact during both the mid- (P < 0.0001) and late-luteal stages (P < 0.06) of the estrous cycle. After treatments were initiated, both mid- and late-stage luteal cells treated with LH secreted more (P < 0.0001) progesterone than occurred with any other treatment; oxytocin, AA, and control treatments were similar.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Interactions between large and small luteal cells collected during the mid- or late-luteal stages of the bovine oestrous cycle.

The effects of contact between large and small bovine luteal cells together with those of luteinizing hormone (LH) or arachidonic acid (AA) on progesterone production during the oestrous cycle were investigated. Corpora lutea were collected during the mid-luteal stage (Days 10-12; n = 4) and late-luteal stage (Days 17-18; n = 4) of the oestrous cycle. Large and small luteal cells were dispersed and separated and then incubated together or separately. Mid-luteal stage cells were treated with LH (0 or 5 ng) whereas late-luteal stage cells were treated with LH (0 or 5 ng) or AA (0 or 10 microM). Culture medium was collected and replaced 1, 3 and 6 h after starting treatments. Progesterone production decreased (P < 0.0001) with increased incubation time irrespective of cell arrangement, the stage of the oestrous cycle or treatment. During the 18 h before treatment, cells in the contact arrangement produced more progesterone (P < 0.003) than cells without contact in both mid- and late-luteal stages of the oestrous cycle; progesterone production within cell arrangements between prospective treatment groups was similar. After initiating treatments, mid-luteal stage cells in the control group without contact produced more progesterone (P < 0.01) than cells with contact. Mid-luteal stage cells treated with LH produced more (P < 0.0001) than control cells; progesterone production between cell arrangements within the LH treatment group was similar. In the late-luteal stage cells, both LH and AA increased (P < 0.01) progesterone production by comparison with control cells; LH and AA treatment groups produced similar results.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Stimulation of progesterone production in bovine luteal cells by co-incubation with bovine blastocyst-stage embryos or trophoblastic vesicles.

A study was conducted to determine whether bovine blastocyst-stage embryos and trophoblastic vesicles stimulate the production of progesterone in bovine luteal cells during incubation in vitro. The effects of co-incubation of these embryos and vesicles with uterine endometrial tissue on progesterone production was also investigated. Bovine small and large luteal cells were obtained on day 12 of the oestrous cycle, dispersed by unit gravity sedimentation and recombined to provide preparations free of accessory cells. Blastocyst-stage embryos were obtained on day 7 and trophoblastic vesicles were obtained from bovine embryos on day 12. A uterine endometrial tissue sample was obtained from the same cow from which the corpus luteum was taken. Treatment groups were arranged in 24-well plates as follows: luteal cells alone; luteal cells and one trophoblastic vesicle; luteal cells and one blastocyst embryo; luteal cells and a 10 mg uterine endometrial sample; luteal cells, one trophoblastic vesicle and a uterine endometrial sample; and luteal cells, one blastocyst embryo and a uterine endometrial sample. All treatment groups were incubated (at 37 degrees C under 5% CO2) in Ham's F-12 medium supplemented with antibiotics (100 micrograms penicillin ml-1 and 100 U streptomycin ml-1, L-glutamine (0.29 mg ml-1), insulin (5 micrograms ml-1), transferrin (5 micrograms ml-1) and selenium (5 ng ml-1) for 12 h. Samples of the medium were harvested 10 min (basal concentration) and 2, 6 and 12 h after incubation to determine the concentrations of progesterone and prostaglandin.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Interactions between large and small bovine luteal cells in a sequential perifusion co-culture system.

The objectives of this experiment were to study large and small luteal cell interactions and examine the effect of arachidonic acid (AA) on progesterone production by separated bovine luteal cells. Corpora lutea collected from Holstein cows (n = 5) on d 12 of the estrous cycle were dispersed, and small (SLC) and large (LLC) luteal cells were separated by unit gravity sedimentation and flow cytometry. Cells were incubated at 37 degrees C in separate perifusion chambers with a modified Ham's F-12-HEPES medium and aerated with 95% O2:5% CO2. The flow rate of medium was 100 microL/min, and fractions were collected at 30-min intervals for 4 h. Luteal cells were arranged in tandem so that medium from the first cell type would pass through the chamber containing the second cell type. Luteal cells were arranged so that medium flowed from 1) SLC to SLC, 2) LLC to LLC, 3) SLC to LLC, 4) LLC to SLC, 5) SLC to LLC, 6) LLC to SLC; medium for arrangements 5 and 6 contained 10 microM AA. Cells in arrangements 5 and 6 were perifused for 30 min before AA was added. Progesterone was measured with an enzymeimmunoassay. The LLC to LLC arrangement had a greater (P < .05) average progesterone secretion rate than all other cell arrangements, and the SLC to SLC arrangement had the least progesterone secretion rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Role of platelet-derived growth factor in development of in vitro matured and in vitro fertilized bovine embryos.

This experiment was designed to determine whether the stimulatory effects of bovine oviductal epithelial cells (BOEC) on development of early bovine embryos are due to platelet-derived growth factor (PDGF). Four hundred and twenty five 8-cell bovine embryos derived from in vitro maturation and in vitro fertilization procedures were equally and randomly allotted to one of the following culture treatment groups: control medium alone (Menezo's B2 medium; MB2), MB2 with 1 ng PDGF ml-1 (PDGF), 1 ng PDGF ml-1 and 10 micrograms anti-PDGF antibody ml-1 (PDGF + Ab), BOEC or BOEC and 10 micrograms anti-PDGF antibody ml-1 (BOEC + Ab). All embryos were cultured in 100 microliters of serum-free MB2 medium supplemented with 2 mg fatty-acid-free bovine serum albumin ml-1. Embryos for all treatment groups were incubated at 39 degrees C and 5% CO2 in humidified air in groups of five embryos per well in 96-well culture plates until 7 days after in vitro insemination. A higher proportion of embryos developed to > 8-cell and to the morula stage following culture with PDGF, BOEC or BOEC+Ab than with MB2 alone. Incubation of PDGF and BOEC-treated embryos with anti-PDGF reduced development to the morula and blastocyst stages. However, anti-PDGF did not completely inhibit blastocyst development when added to BOEC. In addition, embryos incubated with BOEC and anti-PDGF contained a reduced number of inner cell mass cells compared with embryos incubated with BOEC alone. These results indicate that PDGF provides a developmental stimulus similar to BOEC for bovine embryos at the fourth cell cycle.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Stimulation of development of in vitro-matured and in vitro-fertilized bovine embryos by platelets.

In vitro-fertilized bovine embryos were incubated in Menezo's B2 medium (MB2) supplemented with 2 mg/mL of BSA. In Exp. 1, eight-cell stage embryos were allotted to one of the following groups: control medium (MB2), MB2 with 20 ng/mL of platelet-activating factor (PAF), 1 x 10(7) bovine blood platelets (Platelets), oviductal cells (BOEC), BOEC and 20 ng/mL of PAF (BOEC+PAF), or BOEC and 1 x 10(7) platelets (BOEC+Platelets). In Exp. 2, eight-cell embryos were allotted to one of the following groups: control medium (MB2), MB2 with 1 x 10(7) platelets (Platelets), 1 x 10(7) platelets and 10 micrograms/mL of platelet-derived growth factor antibody (Platelets+anti-PDGF), 1 x 10(7) platelets and 1 microgram/mL of indomethacin (Platelets+Indomethacin), or 1 x 10(7) platelets and 3 micrograms/mL of mianserin (Platelets+Mianserin). Embryos were incubated at 39 degrees C in 5% CO2 in groups of five until 8 d after in vitro fertilization (IVF). In Exp. 1, Platelets stimulated embryo development to the morula, blastocyst, and expanded blastocyst stages. Embryo development was greatest in the BOEC+Platelets group on d 7 and 8 after IVF. Only embryos incubated in the BOEC+Platelets treatment group reached the hatched blastocyst stage on d 8. In Exp. 2, embryos incubated in the Platelets treatment group had the greatest (P < .05) proportion develop beyond the eight-cell stage. Embryos incubated in the Platelets + anti-PDGF group had less (P < .05) development beyond the eight-cell stage and to the morula stage. These results indicate that the stimulatory effects of PDGF on bovine embryo development may be derived from both the oviductal epithelium and platelets.

Animals↗

PubMed1992

Coculture of in vitro fertilized bovine embryos with oviductal epithelial cells originating from different stages of the estrous cycle.

Bovine embryos derived from in vitro fertilization procedures were cocultured in vitro with oviductal cells obtained from heifers between d 4 and 6 or d 14 and 16 of the estrous cycle. In addition, proteins secreted by oviductal cells isolated between d 4 and 6 or d 14 and 16 of the cycle were monitored. Embryos (2- to 4-cell) were incubated in Tissue Culture Medium-199 with 10% fetal bovine serum with or without oviductal cells at 39 degrees C for 10 d following in vitro insemination. There were more morulae, blastocysts, and hatched blastocysts following coculture with oviductal cells than with culture in medium alone. However, no differences were noted in embryo development following coculture with oviductal cells obtained between d 4 and 6 or d 14 and 16 of the estrous cycle. Also, no differences were detected in the amount of [35S]methionine-labeled proteins secreted by oviductal cells isolated from different days of the estrous cycle. These results indicate that oviductal epithelial cells isolated from early and late luteal phases of the estrous cycle will effectively support early embryonic development following prolonged in vitro culture.

Animals↗

PubMed1992

Role of arachidonic acid and its metabolites in the regulation of progesterone and oxytocin release from the bovine corpus luteum.

We have examined the effects of arachidonic acid (AA) and some of its metabolites on progesterone (P4) and oxytocin (OT) release by corpora lutea obtained from Holstein heifers at day 8 of the estrous cycle (Day 0 = estrus). The luteal cells were dispersed with collagenase and small and large cells were separated by unit gravity sedimentation and flow cytometry. After an 18-hr preincubation period, the cells were incubated in the presence of various treatments for 1 hr, followed by a 23-hr incubation period with no treatment. OT was secreted by the large, but not by the small, luteal cells into the incubation medium. AA elicited a significant (P less than 0.05) release of OT from the large cells and P4 from both the large and small cells within 1 hr of incubation, having a specific effect at a concentration of 10 microM. Larger doses (25 and 100 microM) of AA adversely affected the cell viability. Phospholipases A2 (0.5 unit/ml) and C (0.05 unit/ml) and calcium ionophore A23187 (0.1 microM) stimulated OT release from the large cells to the same extent as AA (10 microM). Inhibition of the AA cyclooxygenase metabolic pathway by indomethacin did not affect AA-induced release of OT and P4, although exogenous prostaglandins F2 alpha and I2 (5-25 ng/ml) stimulated the release of OT. Lipoxygenase products of AA (hydroxyeicosatetraenoic acid and leukotrienes; 25 ng/ml) also stimulated OT release. Inhibition of the lipoxygenase metabolic pathway by nordihydroguaiaretic acid abolished AA-induced release of both OT and P4. These results suggest that intracellular accumulation of free AA may modulate secretory functions in the bovine corpora lutea, including OT and P4 release.

Analysis of Variance↗

PubMed1991

Bovine corpus luteum function after removal of granulosa cells from the preovulatory follicle.

Experiments were conducted to determine the effects of removing granulosa cells from bovine preovulatory follicles on subsequent corpus luteum (CL) function. Holstein heifers were assigned to three groups: untreated controls (n = 6), removal of granulosa cells (n = 9) and removal and return of granulosa cells (n = 7). Surgery was performed 18-24 hr after the onset of estrus and in all cases after the preovulatory luteinizing hormone (LH) surge. Jugular venous blood was collected and estrous activity monitored twice daily. Corpora lutea were formed in six heifers in each group. Concentrations of plasma progesterone were reduced (P less than 0.05) on Days 7-17 in heifers from which granulosa cells were removed when compared to the other two groups. There were no differences in the lengths of the estrous cycles nor concentrations of LH in the three groups. In summary, these experiments support the concept that granulosa cells make a substantial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. These experiments also establish the fact that granulosa cells develop into functional luteal cells after their removal and return to the preovulatory follicle.

Animals↗

PubMed1991

Cellular distribution and cycle phase dependency of gonadotropin and eicosanoid binding sites in bovine corpora lutea.

Bovine luteal functions are regulated by gonadotropins and eicosanoids. The specific binding sites that presumably mediate the actions of these regulatory agents have previously been characterized in bovine luteal tissue. However, the cellular distribution and/or the cycle phase dependency of these binding sites have never been investigated. In the present study, we investigated these parameters by using quantitative light microscope autoradiography. The results showed that both small and large luteal cells contained binding sites for LH/hCG, prostaglandin (PG)E2, PGF2 alpha, PGI2, and leukotriene (LT)C4. In addition, luteal blood vessels contained LH/hCG and LTC4 binding sites and luteal fibroblasts contained PGE2 binding sites. On a per cell basis, there were more binding sites for all ligands in large luteal cells as compared to small or nonluteal cells. After correction for the cellular area differences, small luteal cells contained more LH/hCG, PGE2, PGI2, and LTC4 binding sites, while large luteal cells contained more PGF2 alpha binding sites. The small and large luteal cell binding of hCG, PGE2, PGI2, and LTC4 increased from early to mid luteal phase, followed by a decline in the late luteal phase. PGF2 alpha binding, on the other hand, increased from early to late luteal phase. In contrast to luteal cells, binding of hCG and LTC4 to luteal blood vessels and binding of PGE2 to luteal fibroblasts did not change during the cycle. These results suggest that LH/hCG and eicosanoid regulation of luteal function is more complex than previously envisioned and it involves both small and large luteal cells and, in some cases, also nonluteal cells.

Animals↗

PubMed1991

Differential origin and control mechanisms in small and large bovine luteal cells.

Studies of the calcium requirement and the relationship of intracellular calcium to progesterone synthesis in highly purified preparations of bovine luteal cells reveal a remarkably close relationship between intracellular calcium levels and steroidogenesis. The differential responses of the two cell types, summarized in Table 2, are beginning to reveal how the two cell types may co-operate to produce both luteotrophic and luteolytic responses at different stages of the oestrous cycle and early pregnancy. The luteotrophic mechanisms in the small cells are fairly clear; in addition to the luteotrophic effects of LH and cAMP, activation of protein kinase C leads to increased progesterone synthesis. Accordingly, PGF-2 alpha and several other prostanoids are luteotrophic in these cells. PGF-2 alpha stimulates phospholipase C activity in the small cells but does not reduce LH-stimulated cAMP or progesterone accumulation (Davis et al., 1989). This acute stimulus of protein kinase C activation to progesterone production in bovine small luteal cells is rapidly desensitized, although its stimulus to prostanoid production continues for at least 24 h. Large cells respond to LH, but only at relatively high levels. In addition, we have no good evidence for a role for protein kinase C in the control of progesterone synthesis in the large bovine luteal cells from mid-cycle corpora lutea. Phorbol esters have no effect on steroidogenesis and it is not yet established that protein kinase C provides the same high affinity receptor for phorbol esters that is found in the small cells. Experiments with inhibitors of protein kinase C, such as staurosporine, in large cells have been inconclusive. Evidence for several species suggests that both cell types co-operate, in ways not yet fully understood, to bring about maximal progesterone production at mid-cycle. Some evidence suggests that they may also co-operate to bring about luteolysis. The concept that PGF-2 alpha initiates luteolysis by inhibiting LH stimulated progesterone production in the large cells must be revised in light of the relative insensitivity of these cells to LH and the fact that they probably constitutively express the cholesterol side-chain cleavage enzymes (P-450scc) that represent the rate-limiting step in progesterone production. Oonk et al. (1989) have reported that, once P-450scc mRNA is induced in rat granulosa cells by the LH surge, it is constitutively maintained by the luteinized cells in the absence of gonadotrophins and is no longer regulated by cAMP.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1991

Prolonged gestation in a Holstein cow: adenohypophyseal aplasia and skeletal pathology in the offspring.

A Holstein fetus was delivered by Caesarean section at a gestational age of 441 days. The pituitary pars distalis was aplastic and the adrenal and thyroid glands were severely hypoplastic. Arrested or retarded cartilage cell maturation resulted in absence or minimal development of epiphyseal ossification centers, delayed ossification of carpal bones, and arrest of longitudinal growth of bones. The pathophysiology of prolonged gestation and of skeletal pathology is discussed.

Adrenal Glands↗

PubMed1990

Differential effects of calcium on progesterone production in small and large bovine luteal cells.

We studied the effects of calcium (Ca2+) ions in progesterone (P) production by separated small and large luteal cells. Corpora lutea were collected from 31 heifers between days 10 and 12 of the estrous cycle. Purified small and large cells were obtained by unit gravity sedimentation and flow cytometry. P accumulation in cells plus media was determined after incubating 1 x 10(5) small and 5 x 10(3) large cells for 2 and 4 h respectively. Removal of Ca2+ from the medium did not influence basal P production in the small cells (P greater than 0.05). However, stimulation of P by luteinizing hormone (LH), prostaglandin E2 (PGE2), 8-bromo-cyclic 3',5' adenosine monophosphate (8-Br-cAMP) and prostaglandin F2 alpha (PGF2 alpha) was impaired (P less than 0.05) by low Ca2+ concentrations. LH and PGE2-stimulated cAMP production was not altered by low extracellular Ca2+ concentrations, and PGF2 alpha had no effect on cAMP. In contrast, basal as well as LH and forskolin-stimulated P production were attenuated (P less than 0.05) in Ca2(+)-deficient medium in the large cells. However, P production stimulated by 8-Br-cAMP was not altered in Ca2(+)-deficient medium. Steroidogenesis in large cells was also dependent on intracellular Ca2+, since 8-N, N-diethylamineocytyl-3,4,5-trimethoxybenzoate (TMB-8), an inhibitor of intracellular Ca2+ release and/or action, suppressed (P less than 0.05) basal, LH and 8-Br-cAMP stimulated P. In contrast, basal P in small cells was not altered by TMB-8; whereas LH-stimulated P was reduced 2-fold (P less than 0.05). The calcium ionophore, A23187, inhibited LH-stimulated P in small cells and both basal and agonist-stimulated P in large cells. These studies show that basal P production in small cells does not require Ca2+ ions, while hormone-stimulated P production in small cells and both basal and hormone-stimulated P in large cells do require Ca2+. The inhibitory effect of Ca2+ ion removal was exerted prior to the generation of cAMP in the large cells, but distal to cAMP generation in hormone-stimulated small cells. The calmodulin/protein kinase C antagonist, W-7, also inhibited both basal and hormone-stimulated P production in both small and large luteal cells, indicating that P production in luteal cells also involves Ca2(+)-calmodulin/protein kinase C-dependent mechanisms.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1990

Phorbol ester receptors in bovine luteal cells: relationship to protein kinase C.

We investigated the binding kinetics of the tumor-promoting phorbol ester, phorbol-12,13-dibutyrate (PBt2) to dispersed total bovine luteal cells, purified small luteal cells, and purified luteal protein kinase C (PKC). Saturation analysis and competitive displacement techniques were used. Binding of [3H]PBt2 to total luteal cell preparations resulted in two distinct affinities. The high affinity component was characterized by a Kd of 4.5 +/- 1.5 nM. Analysis of [3H]PBt2 binding to total cells using competitive displacement demonstrated that the low affinity binding was specific and displaceable but dependent on concentrations of [3H]PBt2 far above the Kd for the high affinity binding. In contrast to the total cell preparations, only high affinity binding was observed in intact purified small luteal cells (Kd = 0.96 +/- 0.04 nM). Partial purification of luteal cytosolic PKC by DEAE-Sephadex chromatography resulted in co-elution of PKC enzyme activity and the [3H]PBt2 binding activity. Under conditions of saturating calcium (0.1 mM) and phosphatidylserine (PS) (100 micrograms/tube) concentrations, binding to the partially purified PKC preparation was found to be of a single high affinity and exhibited a Kd (1.3 +/- 0.2 nM) similar to the high affinity binding observed in intact cells. These results suggest that the primary phorbol ester receptor in luteal cells is PKC. However, a low affinity, high capacity [3H]PBt2 binding site also exists within the corpus luteum, either in the large cells or in the accessory cell fraction which consists mainly of endothelial cells.

Animals↗

PubMed1990

Arachidonic acid and its metabolites increase cytosolic free calcium in bovine luteal cells.

We studied the effects of arachidonic acid and its metabolites on intracellular free calcium concentrations ([Ca2+]i) in highly purified bovine luteal cell preparations. Corpora lutea were collected from Holstein heifers between days 10 and 12 of the estrous cycle. The cells were dispersed and small and large cells were separated by unit gravity sedimentation and flow cytometry. The [Ca2+]i was determined by spectrofluorometry in luteal cells loaded with the fluorescent Ca2+ probe, Fura-2. Arachidonic acid elicited a dose-dependent increase in [Ca2+]i in both small and large luteal cells, having an effect at concentrations as low as 5 microM; and was maximally effective at 50 microM. Several other fatty acids failed to exert a similar response. Addition of nordihydroguaiaretic acid (NDGA) or indomethacin failed to suppress the effects of arachidonic acid. In fact, the presence of both inhibitors resulted in increases of [Ca2+]i, with NDGA exerting a greater stimulation of [Ca2+]i than indomethacin. Prostaglandin F2 alpha (PGF2 alpha) as well as prostaglandin E2 (PGE2) increased [Ca2+]i in the small luteal cells. These results support the idea that arachidonic acid exerts a direct action in mobilizing [Ca2+]i, in the luteal cells. Furthermore, they demonstrate that the cyclooxygenase (PGF2 alpha and PGE2) and lipoxygenase products of arachidonic acid metabolism also play a role in increasing [Ca2+]i in bovine luteal cells. Since the bovine corpus luteum contains large quantities of arachidonic acid, these findings suggest that this compound may regulate calcium-dependent functions of the corpus luteum, including steroid and peptide hormone production and secretion.

Animals↗

PubMed1990

Oxytocin-specific RNA, oxytocin and progesterone concentrations in corpora lutea of heifers treated with oxytocin.

Sixteen virgin Holstein heifers were used to study the role of oxytocin in luteal function. Oxytocin (100 i.u.) was administered on Days 2-5 (Exp. 1) or Days 2-6 (Exp. 2), and the corpora lutea were collected and weighed on Day 6 or Day 8, respectively. Plasma progesterone values and tissue concentrations of progesterone and oxytocin were determined by radioimmunoassay. In addition, the tissue level of the oxytocin-specific transcript was measured by dot-blot hybridization. Expression of the oxytocin-specific transcript was not affected by oxytocin treatment in either experiment. In contrast, plasma progesterone values and tissue progesterone and oxytocin concentrations were all decreased after 5 days of oxytocin treatment (Exp. 2), but not after 4 days (Exp. 1). Since oxytocin concentrations were reduced while the level of its transcript remained unaffected, oxytocin injections might be influencing either peptide processing or release.

Animals↗

PubMed1989

Second messenger systems and progesterone secretion in the small cells of the bovine corpus luteum: effects of gonadotropins and prostaglandin F2a.

The present studies were conducted to determine the effects of gonadotropins (LH and hCG) and prostaglandin F2a (PGF2a) on the production of "second messengers" and progesterone synthesis in purified preparations of bovine small luteal cells. Corpora lutea were removed from heifers during the luteal phase of the normal estrous cycle. Small luteal cells were isolated by unit-gravity sedimentation and were 95-99% pure. LH provoked rapid and sustained increases in the levels of [3H]inositol mono-, bis-, and trisphosphates (IP, IP2, IP3, respectively), cAMP and progesterone in small luteal cells. LiCl (10 mM) enhanced inositol phosphate accumulation in response to LH but had no effect on LH-stimulated cAMP or progesterone accumulation. Time course studies revealed that LH-induced increases in IP3 and cAMP occurred simultaneously and preceded the increases in progesterone secretion. Similar dose-response relationships were observed for inositol phosphate and cAMP accumulation with maximal increases observed with 1-10 micrograms/ml of LH. Progesterone accumulation was maximal at 1-10 ng/ml of LH. LH (1 microgram/ml) and hCG (20 IU/ml) provoked similar increases in inositol phosphate, cAMP and progesterone accumulation in small luteal cells. 8-Bromo-cAMP (2.5 mM) and forskolin (1 microM) increased progesterone synthesis but did not increase inositol phosphate accumulation in 30 min incubations. PGF2a (1 microM) was more effective than LH (1 microgram/ml) at stimulating increases in inositol phosphate accumulation (4.4-fold vs 2.2-fold increase for PGF2a and LH, respectively). The combined effects of LH and PGF2a on accumulation of inositol phosphates were slightly greater than the effects of PGF2a alone. In 30 min incubations, PGF2a had no effect on cAMP accumulation and provoked small increases in progesterone secretion. Additionally, PGF2a treatment had no significant effect on LH-induced cAMP or progesterone accumulation in 30 min incubations of small luteal cells. These findings provide the first evidence that gonadotropins stimulate the cAMP and IP3-diacylglycerol transmembrane signalling systems in bovine small luteal cells. PGF2a stimulated phospholipase C activity in small cells but did not reduce LH-stimulated cAMP or progesterone accumulation. These results also demonstrate that induction of functional luteolysis in vitro requires more than the activation of the phospholipase C-IP3/calcium and -diacylglycerol/protein kinase C transmembrane signalling system.

Animals↗

PubMed1989

The effects of platelet-activating factor and platelet-derived compounds on bovine luteal cell progesterone production.

This study was conducted to characterize bovine platelets with respect to serotonin (5-HT) concentration and platelet-activating factor (PAF)-activation and to examine the in vitro effects of PAF and platelet-derived compounds on bovine luteal progesterone (P4) production. The concentration of 5-HT in platelets, as determined by high-performance liquid chromatography, was 538.8 +/- 40.83 ng/1 x 10(8) platelets. Based on a circulating platelet concentration range of 2.3 x 10(8) 5.8 x 10(8) platelets/ml, the circulating concentration of 5-HT would be approximately 1239-3125 ng/ml of blood. Bovine platelets were found to aggregate in response to PAF (1-40 ng/0.5 ml), with maximal aggregation occurring at 20-40 ng/0.5 ml. Coincubation of luteal cells with platelets (1 x 10(7)-4 x 10(8] enhanced luteal P4 production (p less than 0.05). Addition of the 5-HT receptor antagonist mianserin blocked the platelet-induced increases in P4 (p less than 0.05). Preincubation of platelets with indomethacin did not alter the production of P4 (p greater than 0.05), nor did the addition of propranolol (p greater than 0.05). Platelet-derived growth factor at 8 and 16 ng/ml enhanced basal P4 production (p less than 0.05) but had no effect on the responsiveness of luteal cells to luteinizing hormone (LH) (p greater than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗