Oxytocin stimulates progesterone release from microdialyzed bovine corpus luteum in vitro

A microdialysis system (MDS) was implanted in corpora lutea (CL) from cows (Days 5-7, 8-12, and 15-18 of the estrous cycle); the CL were maintained in organ culture chambers. With this system, active substances can be applied, and a collection of steroids released from luteal cells surrounding the microcapillary (cut-off point = 100 kDa) is possible, while luteal cells maintain cell-to-cell contact. Spontaneous pulses of progesterone release were observed in 90% of control (perfused with Ringer's solution only) at 60-80 min intervals. The infusion of bovine LH (bLH) for 20 min (0.1-10 micrograms/ml) stimulated dose-dependent release of progesterone. Both results indicate that the CL maintains the activity of progesterone release and the ability to respond to LH stimulation in this system. Oxytocin (1-100 microM) also stimulated progesterone release in a dose-dependent manner. Preexposure with oxytocin antagonist blocked the stimulatory effect of oxytocin (p less than 0.01) but not of LH (p less than 0.05), confirming the specificity of the effect. When CL were prestimulated with a low dose of oxytocin (1 microM, 20 min) twice before bLH application, the release of progesterone by bLH (1 micrograms/ml, 20 min) was more pronounced (p less than 0.05). A long-term infusion (3 h) with oxytocin and/or bLH stimulated the release of progesterone for the whole period of time. Oxytocin was most stimulative during the early luteal phase (Days 5-7) and decreased continuously from Days 8-12 to Days 15-18.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of several biogenic amines on in vitro progesterone secretion by the bovine corpus luteum

1. The effect of several biogenic amines on secretion of progesterone (P4) was examined using bovine corpora lutea (n = 6), removed on day 13 of the oestrus cycle, enzymatically dispersed and cultured in vitro. 2. Luteal cell cultures were co-incubated with 0, 10, 50 or 100 ng of luteinizing hormone (LH) and 50 ng of epinephrine (EPI), norepinephrine (NOR), dopamine (DOPA), melatonin (MEL), N-acetyl-50H-serotonin (N-acetyl-50H-tryptamine; NacS), serotonin (50H-tryptamine; 5HT) or saline control. 3. EPI, NOR and DOPA decreased baseline release of P4. 4. The magnitude of the response of P4 to LH was depressed when cells were co-incubated with DOPA, EPI and 5HT and stimulated when cells were co-incubated with MEL and NacS. 5. These data indicate that the biogenic amines might modulate ovarian steroidogenesis supplementary to hypothalamic-hypophyseal hormonal mechanisms.     

Regulation of prostaglandin synthesis by progesterone in the bovine corpus luteum

The objective of the present study was to investigate the influence of progesterone on prostaglandin synthesis by the corpus luteum (CL). Corpora lutea were obtained from dairy cows on days 4, 6, 10, and 18 of the estrous cycle, dissociated, and placed in serum-free culture. The addition of luteinizing hormone (LH) resulted in a slight, but non-significant (p greater than 0.05), increase in levels of 6-keto-PGF1 alpha, and had no effect on PGF2 alpha. Progesterone treatment caused a significant, dose-dependent decrease in both PGF2 alpha and 6-keto-PGF1 alpha in 6-day and 10-day corpora lutea, but not in 4-day or 18-day corpora lutea. In the 6- and 10-day corpora lutea, progesterone treatment resulted in a greater inhibition of PGF2 alpha than 6-keto-PGF1 alpha production. Therefore, progesterone treatment brought about an increase in the 6-keto-PGF1 alpha to PGF2 alpha ratio in these cells (12.9 vs. 21.3). It is concluded from these studies that progesterone can modulate luteal prostacyclin and PGF2 alpha synthesis, suggesting an interaction of progesterone and prostaglandin production within the corpus luteum.     

Two binding proteins for progesterone in the bovine corpus luteum

The cytosolic supernatant of bovine corpus luteum contains two proteins which bind progesterone specifically. Bovine luteal cytosol was fractionated on hydroxylapatite and the peaks of protein obtained subjected to equilibrium dialysis against progesterone. Progesterone-binding activities (Ka approx. 10(6) 1/mol) was eluted at 40 mM (Binding Protein 1) and 100 mM phosphate (Binding Protein 2). They sedimented differently (3.95 and 4.65, respectively) on sucrose gradients. In contrast to Binding Protein 1, Binding Protein 2 bound R5020 better than progesterone on sucrose gradients. Purification of the binding activity eluted by 40 mM phosphate from the hydroxylapatite column showed that it resided in a single protein (molecular weight 65,000 daltons). The function of these proteins is presently unknown, but they may participate in the biosynthesis and/or secretion of progesterone from bovine luteal cells.     

Electron transport and progesterone biosynthesis in the bovine corpus luteum homogenate

The role of electron transport in progesterone biosynthesis was investigated in bovine corpus luteum homogenate. Although amytal and malonate inhibited progesterone synthesis, rotenone under several different incubation conditions was without effect. On the basis of differences in the effects of amytal and rotenone it is suggested that for the side-chain cleavage of cholesterol, although formation of some high energy intermediate of oxidative phosphorylation is obligatory, the electron shuttle between NAD and flavin is not.     

The lipoxygenase pathways are involved in LH-stimulated progesterone production in bovine corpus luteum.

We have examined the effects of endogenous lipoxygenase products on basal progesterone (P4) production by cultured bovine mid-luteal cells. The involvement of lipoxygenase products in the stimulatory effect of LH on luteal cAMP accumulation and P4 production was also examined. Bovine luteal cells from mid-cycle corpora lutea (CL) were exposed for 16 h to a lipoxygenase inhibitor (nordihydroguaiaretic acid: NDGA; 0.33-33 microM). For the last 4 h of incubation, the cells were exposed to LH and/or three different lipoxygenase products, 5-, 12- and 15-hydroxyeicosatetraenoic acid (HETE). NDGA inhibited P4 production by the cells in a dose-dependent manner (P < 0.05). NDGA-reduced P4 production was reversed by the addition of 12-HETE, but not 5- or 15-HETE, whereas 5-, 12- and 15-HETE alone showed no significant effect on P4 production in the intact cells. Furthermore, NDGA (33 microM) blocked the stimulatory action of LH on P4 production (P < 0.05), without changing cAMP accumulation (P > 0.1). When the cells were exposed to 5-, 12- or 15-HETE with LH and NDGA, only 15-HETE maintained the stimulatory effect of LH on P4 production in the cells (P < 0.05). These results suggest that endogenous lipoxygenase products play important roles in P4 production by bovine CL, i.e. basal P4 production is supported by 12-HETE, and LH-stimulated P4 production is partially mediated via the activation of lipoxygenase and subsequent 15-HETE formation downstream of the LH-activated cAMP-PKA-phosphorylation pathway.     

Prostaglandin F and progesterone secretion by porcine endometrium and corpus luteum in vitro

Slices of porcine endometrium and corpus luteum tissue obtained from mature sows throughout the luteal phase of the oestrous cycle were incubated in culture medium which was analysed at regular intervals over a period of 8 hours for prostaglandin F and progesterone. Prostaglandin F secretion was greatest by endometrium obtained during the mid III to late I luteal stage of the cycle and the increased levels secreted by this tissue were paralleled by high levels of secretion from corpus luteum tissue. The addition of indomethacin (10 μg/ml) to the culture medium completely abolished prostaglandin F secretion by both endometrium and luteal tissue indicating that the high levels of the prostaglandin were due to synthesis. Progesterone secretion by the corpus luteum was maximal from early luteal tissue and had declined to considerably lower levels by late stage tissue when prostaglandin secretion was greatest. The possible physiological significance of luteal prostaglandin F secretion is discussed.     

Role of prostaglandin E2 in basal and noradrenaline-induced progesterone secretion by the bovine corpus luteum

The role of prostaglandin E2 (PGE2) in basal and noradrenaline (NA)-stimulated utilization of high density lipoprotein (HDL) as a source of cholesterol for progesterone synthesis was examined. In Experiment 1, a cannula was inserted into the aorta abdominalis through the coccygeal artery (cranial to the origin of the ovarian artery) in mature heifers, to facilitate infusion of NA (4 mg/30 min; n = 3) on day 10 of the estrous cycle. Three other heifers were similarly cannulated to serve as control. Before, during, and after NA or saline infusion, blood samples from the vena cava were collected every 5-15 min for analysis of PGE2, progesterone, and cholesterol. Each NA infusion stimulated (P < 0.01) secretion of both hormones in heifers. Short-duration increases (P < 0.05) in progesterone were observed due to the infusion of NA while cholesterol was not altered significantly. In addition, increases in PGE2 concentrations (P < 0.05) compared to controls were seen after NA infusion. Therefore, we used an in vitro model to verify the effect of PGE2 on HDL utilization by luteal cells from day 5 to 10 of the estrous cycle. In the preliminary experiment, 10(-6) M of PGE2 out of four different doses examined was selected for further studies, since it evoked the highest release of progesterone. In the next experiment, it was found that HDL increases progesterone secretion by luteal cells and both PGE2 and LH increased (P < 0.05) the response to HDL while NA did not. In the last in vitro experiment, progesterone stimulated PGE2 secretion by luteal cells. In conclusion, PGE2 may be directly involved in the utilization of cholesterol from HDL for progesterone synthesis. Furthermore, PGE2 may influence NA-stimulated progesterone secretion by the corpus luteum (CL). It is concluded that there is a positive feedback loop between progesterone and luteal PGE2 during days 5-10 of the estrous cycle.     

Role of frequency and amplitude of repetitive HCG stimulations for sustained progesterone secretion from the bovine corpus luteum in vitro.

This investigation aimed at evaluating a role for frequencies and amplitudes of repeated HCG stimulations for the optimal maintenance of progesterone (P4) secretion from the bovine corpus luteum in vitro. Slices (100-120 mg) of midluteal bovine corpora lutea were perifused with medium M199 (0.05% BSA, pH 7.2, 38.5 degrees C) and the perifusion effluent collected at 15 minute intervals for 20-29 hours. Unstimulated P4 release (n = 5) was distinctly pulsatile (by Pulsar pulse algorithm), with pulses occurring every 90 +/- 6 minutes (mean +/- SEM) and pulse amplitudes of 14.4 +/- 1.1 ng. Conversely, no pulses were detected in two control perifusions. Unstimulated P4 release increased during the first 5 perifusion hours (from 39.3 +/- 4.6 to 50.3 +/- 5.6 ng/15 min, p less than 0.01), but then appeared to decline (to 29.3 +/- 1.3 ng/15 min, p less than 0.05) towards the end of the perifusion periods. Hourly pulses of HCG (6.7 mM) did not change the P4 pulse amplitudes (16.6 +/- 2.0 ng), the pulse periodicities (105 +/- 15 min) and overall release rates (34.7 +/- 5.7 ng/15 min), nor did they prevent the decline in P4 secretion towards the end of perifusions (n = 5). In contrast, 2-hourly HCG stimulations maintained stable P4 release rates throughout the perifusion periods (34.7 +/- 6.8 ng/15 min), with P4 pulses of similar amplitudes (14.7 +/- 1.7 ng), but of lower periodicities (135 +/- 2 min, p less than 0.05) than during unstimulated conditions (n = 5).(ABSTRACT TRUNCATED AT 250 WORDS)     

Leptin in the bovine corpus luteum: receptor expression and effects on progesterone production

In cattle, leptin has been implicated in the control of ovarian function and has been shown to modulate steroid production by theca and granulosa cells in a number of species. However, a direct effect of leptin on bovine luteal function has not been demonstrated. This study was conducted to determine if the leptin receptor (OB-R) is expressed in the bovine corpus luteum (CL), and to examine the effects of leptin on progesterone production by dispersed luteal cells in vitro. RT-PCR was used to detect the presence of OB-R and, more specifically, the long, biologically active isoform (OB-Rb), in CL, collected on days 2-18 of the oestrous cycle (n=18). The effects of leptin on progesterone production were investigated in dispersed luteal cells prepared from CL collected on days 5 and 8 (n=14) of the cycle. The dispersed luteal cells were cultured for 24 hr with recombinant human leptin and/or LR3-IGF-1 and/or LH. OB-Rs, in particular, OB-Rb, were expressed in the CL at all stages of development. Progesterone production by luteal cells was increased (P<0.001) by treatment with LH (10 ng/ml) but treatment with leptin alone had no effect. However, in the presence of IGF-1 (100 ng/ml), leptin (10 ng/ml) caused a significant (P<0.005) increase in progesterone production. In conclusion, we have shown that the leptin receptor is expressed in the bovine CL and have demonstrated a modulatory effect of leptin on luteal progesterone production in vitro.     

FSH receptors in the bovine corpus luteum

Binding of follicle stimulating hormone (FSH) to a crude membrane fraction of bovine corpus luteum (CL) has been detected. This binding meets the usual criteria for a receptor based on specificity, time course of reaction and association constant (Ka = 8.5 x 10(10)M(-1)). Physiological studies with CL removed from heifers at specific times after estrus indicate that day-6 CL had the highest FSH binding. However, a correlation with physiological function was not obvious since some functional mid-cycle CL were high in progesterone and luteinizing hormone (LH) receptor but had nondetectable FSH receptor. Conversely, some late-cycle CL had low progesterone and LH receptor but significant quantities of FSH receptor.     

Conversion of cholesterol to progesterone by turtle corpus luteum

Homogenates of corpora lutea from the snapping turtle, $\text{Chelydra}$$\text{serpentina serpentina}$ were capable of converting small (< 1%) amounts of cholesterol-³H to progesterone-³H. There was about twice as much steroidogenic activity in corpora lutea taken from animals still carrying oviducal eggs as in those from animals that had laid their eggs. However, the latter showed up to an 80% increase in conversion of cholesterol to progesterone when turtle pituitary homogenate was incubated along with the luteal homogenate. Approximately 4 and 9 mg/g of free and esterified sterol, respectively, were found in the turtle corpus luteum. These results suggest that the corpus luteum of the oviparous turtle functions as a steroidogenic organ.     

Intercellular communication in the bovine corpus luteum

There is a growing body of evidence that intercellular communication is important in the regulation of luteal function. Although the nature of the interactions between small and large luteal cells are not yet clear, it seems likely that they do exist. Many of the substances to which luteal cells respond, such as prostaglandins, growth factors, oxytocin and progesterone, are produced locally. These substances may act as paracrine factors to modulate the response of luteal cells to hormonal signals. Endothelial cells also produce factors that can modify steroidogenesis, and luteal cell-stimulation of endothelial cell proliferation is necessary for the extensive angiogenesis that occurs during luteinization Finally, bidirectional intercellular communication likely occurs between luteal cells and resident immune cells. Immune cells produce cytokines that can modify progesterone and prostaglandin synthesis by luteal cells. Cytokines may also have direct cytotoxic effects on luteal cells, and dead cells are then phagocytized by resident macrophages. Also, factors secreted by luteal cells can serve as chemoattractants for immune cells, and can enhance or suppress immune cell functions. There is little doubt that intercellular communication within the corpus luteum is very complex. One must remember, however, that nearly all evidence collected thus far is based on in vitro studies. Eventually, technology will allow for study of these interactions in vivo, and may lead to new methods for control of luteal function.     

Changes in prostaglandin secretion by the regressing bovine corpus luteum

Secretion of prostaglandins (PGs) by the regressing corpus luteum (CL) was investigated in the cow. Six cows were implanted with microcapillary dialysis membranes of a microdialysis system (MDS) into the CL during Days 8-9 (Day 0 = estrus), and a prostaglandin (PG) F2alpha analogue (Estrumate) was injected intramuscularly (i.m.) to induce luteolysis. Acute increases in intraluteal release of PGF2alpha and PGE2 were observed during the first 4 h, followed by decreases over the next 8 h. Intraluteal release of both PGs gradually increased again during the period 48-72 h. Concentrations of PGF2alpha in ovarian venous plasma (OVP) were 4-13 times higher than those of jugular venous plasma (JVP) (P < 0.001) during the period of the experiment, and increased from 24 h after treatment with Estrumate (P < 0.05). Cyclooxygenase (COX)-2 mRNA expression increased (P < 0.05) at 2 and 24 h after treatment with Estrumate. The results indicated that local release of PGF2alpha and PGE2, and COX-2 mRNA expression were increased by Estrumate in the regressing CL at the later stages of luteolysis. Thus, luteal secretion of PGs may be involved in the local mechanism for structural rather than functional luteolysis.     

Biosynthesis of retinal in bovine corpus luteum

Bovine corpus luteum tissue was sliced and incubated with beta-[15,15'-(3)H]carotene. The conversion of radioactive beta-carotene into radioactive retinal was substantiated utilizing column chromatography, thin-layer chromatography, high-speed liquid chromatography, and a derivative formation. Lowering of the incubation temperature to 20 degrees C or boiling the tissue eliminated the conversion of beta-carotene to retinal. In addition, other carotenoids and possible oxidation products of beta-carotene in the corpus luteum were investigated. Our results indicate that the bovine corpus luteum possesses the ability to synthesize retinal in situ, which may play a role in reproductive functions.