Individual corpora lutea (CL) of Göttinger miniature pigs were implanted with an in vivo microdialysis system. This system functions like an artificial capillary, allowing diffusion of intraluteally secreted substances into the lumen of the dialysis system and administration of hormones into individual CL and simultaneous measurement of the response. After surgery the sows are fully awake and unrestrained. In the present study the in vivo release rates and secretion dynamics of progesterone (P) and oxytocin (OXT) were investigated. The dialysis system was implanted at day 2-4 of the estrous cycle, and dialysis experiments were performed throughout the next 3 days. Fractions were collected at 30 min intervals, and the concentrations of P and OXT were measured by RIA. Three major observations were made: Spontaneous intraluteal secretion of P and OXT occurred in a pulsatile manner. OXT secretion episodes in individual CL often coincide, indicating a simultaneous release from many CL of one ovary but also from the CL located in the contralateral ovary. OXT episodes also often coincide with P pulses; statistical evaluation revealed a significant correlation between P and OXT secretion. Intraluteal application of OXT stimulated luteal P and estradiol (E2) release in a dose-dependent manner. E2 added to the perfusates was also stimulatory to P release. The stimulation of P release by OXT could be antagonized by prior treatment of the CL with tamoxifen. We demonstrate for the first time in vivo the secretion of OXT from porcine CL. The microdialysis system enabled us to collect samples at the site of steroid and peptide release, i.e. within the intact luteal tissue. Our results suggest a stimulatory effect of OXT on P release from young and middle-aged CL and are in marked contrast to the previously demonstrated inhibitory effect of OXT on P release when luteal cells were cultured in vitro. A possible explanation for this apparent discrepancy is that OXT stimulates intraluteal release of E2, which is a powerful P releasing hormone, overcoming the direct inhibitory effect of OXT. This suggestion is substantiated by the observation that E2, when added to the perfusion medium, indeed stimulated P release.

In the porcine corpora lutea (CL), prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) but stimulate estradiol (E2) secretion from luteal cells kept under primary culture conditions. In vivo, both compounds are reported to have luteolytic properties when administered during the late luteal phase; in young CL, however, both substances stimulate P secretion, an effect which is E2-mediated. During the late luteal phase luteal cells appear to produce cytokines, and in addition, cytokine-producing macrophages invade the CL. We tested therefore whether cytokines, particularly tumor necrosis factor-alpha (TNF), have effects on basal or human CG-stimulated steroidogenesis. Furthermore, the interactions of cytokines with PGF2 alpha and/or OXT were investigated. TNF, and less potently interleukin (IL)-1 and IL-2 but not IL-6, inhibited basal as well as human CG-stimulated release of P and E2 in both small and large luteal cells. The inhibiting effect of PGF2 alpha and OXT on P secretion was augmented by these active cytokines. The stimulatory effect of PGF2 alpha and OXT on small and large luteal cell E2 production was completely inhibited. A profound stimulatory effect of E2 and small luteal cell P secretion was completely prevented by the cytokines, with TNF being more potent than IL-1 or -2. We conclude that the cytokines, particularly TNF, have luteolytic functions by their direct inhibiting effects on luteal cell P production. In addition, the cytokines inhibit synthesis and action of PGF2 alpha- and OXT-stimulated E2 secretion. Since E2 is a potent stimulator of luteal cell P production, this luteotropic signal is eliminated by cytokines, which add to the process of luteolysis.

There is ample evidence that prostaglandin F2alpha (PGF2alpha) is a luteolytic substance in sows, however, there is also some evidence that it may stimulate progesterone (P4) secretion in young corpora lutea (CL). In vitro studies also suggested that tumor necrosis factor alpha (TNF) is inhibitory to luteal cell P4 and estradiol-17beta (E2) release. Since E2 is a strong luteotropic substance in porcine CL, we studied the effects of intraluteal application of PGF2alpha and TNF alone and in combination on the secretion of P4 and E2 in freely moving sows. Furthermore, the effects of intraluteal infusion of E2 and its stereoisomer, estradiol-17alpha, on luteal function, were also determined. Microdialysis systems were implanted into CL at Day 10 of the estrous cycle. After a 24-h recovery period, PGF2alpha (10(-6) M) or E2 (10(-6) M) was applied daily for 6 h into the CL. PGF2alpha caused a stimulation of E2 and P4, and E2 also stimulated P4 secretion at Days 11 and 12, but the stimulatory effect of both substances diminished as the CL approached luteolysis. Intraluteal TNF application resulted in a transient increase of P4 secretion, which was followed by a dramatic reduction of P4 release. When TNF-pretreated CL were exposed to PGF2alpha at Day 11 of the estrous cycle, the prostaglandin was no longer able to stimulate but rather inhibited E2 and P4 secretion. Intraluteal application of estradiol-17alpha had no effect on P4 secretion. These results are suggestive that the PGF2alpha-induced E2 secretion in young and middle-aged CL is stimulatory to P4 secretion. Under the influence of macrophage-derived TNF production, E2 secretion is inhibited, and thereby PGF2alpha and TNF cause functional luteolysis.

Two days before ovulation ovarian follicles of sows were implanted with microdialysis systems (MDS) which function like artificial capillaries with exteriorized inlets and outlets. Steroid hormones and paracrine acting factors such as oxytocin (OXT) and angiotensin II (AII) diffuse from ovarian tissue into the fluid, which is pumped through the MDS and collected in fractions. This allows determination of dynamic changes of estradiol (E2), progesterone (P), OXT, and AII secretion during the pre-, peri-, and postovulatory periods in freely moving sows. More than 80% of such implanted follicles ovulate and form competent corpora lutea (CL) allowing continuation of experimentation during the early luteal phase. Follicular E2 release increases before ovulation and decreases with increasing blood LH concentrations. Twenty to 30 h after beginning of the preovulatory LH surge P secretion increases gradually. Both peptides OXT and AII are released episodically by the preovulatory follicle. During the time of decreased E2 and not yet increased P secretion, i.e. during the periovulatory period, mean AII secretion was highest in comparison to the late follicular and early luteal phase. E2 remains measurable during the early luteal phase. OXT and AII were also topically applied into the follicular wall and after ovulation into the CL. AII had no effect on steroidogenesis of both structures. Although OXT was ineffective in the follicle, in young CL it stimulated P secretion. These results indicate that the MDS can be used to study late follicular and early luteal steroid and peptide secretion. The function of OXT and AII in the follicle remains obscure, whereas OXT has a luteotropic effect in young porcine CL.

We hypothesize that spontaneous regression of corpora lutea (CL) involves short-lasting restructure of luteal tissue with an activation of matrix metalloproteinases (MMPs) and their respective inhibitors (tissue inhibitors of metalloproteinase, TIMPs). This was tested by determining the gene expression of MMP-1, MMP-2, and MMP-9 and respective TIMP-1 and TIMP-2 in luteal tissue from sows at the early, midluteal, and late luteal phase (Days 6-8, Days 9-11, and Days 13-15 of estrous cycle). Gene expression of the three MMPs was low in early, slightly higher in midluteal, and significantly elevated (P < 0.05) in regressing CL. An inverse pattern was found for gene expression of TIMP-1 and TIMP-2. Under culture conditions, the release of MMPs was determined from steroidogenic large luteal cells (LLC). LLC harvested from regressing CL released significantly (P < 0.05) more active MMPs than cells obtained from CL at the early luteal phase. As luteolysis can be induced by prostaglandin F(2alpha) (PGF(2alpha)) and tumor necrosis factor alpha (TNF), we studied their effects on LLC under culture conditions. Treatment of cells with PGF(2alpha) or TNF (10(-7) M or 3 x 10(-9) M, respectively) induced a significantly higher release of MMPs, and gene expression was also significantly stimulated in comparison to that in untreated LLC. The gene expression of TIMPs remained unaffected by either treatment. It is concluded that at the beginning of luteolysis, MMPs are expressed and released in high amounts and that this is essential for the structural regression of the CL.

No abstract

Previously, we have demonstrated an inhibitory effect of oxytocin (OXT) on progesterone (P) and androstenedione (A) release of porcine luteal cell cultures. The present study examines whether OXT modulates P, A, or estradiol (E2) release of so-called small luteal cells (SLC) or of granulosa-derived large luteal cells (LLC). To ensure clean Percoll-gradient separation of the 2 cell types, corpora lutea not older than 6 days were used. SLC, but not LLC, responded to human (h)CG (6 ng/ml) with increased P and A, but not E2, release. When OXT was added to the culture system, both basal as well as hCG-stimulated P release of SLC, but not of LLC, were dose dependently reduced. In contrast, E2 production of SLC and LLC was significantly stimulated by OXT whereas A release of SLC cultures, but not of LLC, was inhibited in response to OXT. In the presence of a specific OXT-antagonist, this inhibitory effect of OXT on P release was abolished, indicating a specific receptor-mediated effect of OXT on porcine luteal cells. When E2 was added to the culture medium, a dose-dependent stimulatory effect on P release of SLC was demonstrated. The presence of the E2 receptor antagonist monohydroxy-tamoxifen in the culture system prevented the E2-induced increase of P release of SLC. E2 was able to counteract dose dependently the OXT-induced inhibition of P release in SLC cultures. These results suggest that OXT may have a dual function in young corpora lutea. The reduction of P and A production can be interpreted as a luteolytic effect of OXT. The simultaneous increase of E2 production, however, may also point to an indirect luteotropic effect since E2 was shown to stimulate luteal P release and to counteract OXT-induced inhibition of P release excessively.

In the porcine corpora lutea (CL), prostaglandin F2 alpha (PGF2 alpha) and oxytocin (OXT) inhibit progesterone (P) but stimulate estradiol (E2) secretion from luteal cells kept under primary culture conditions. In vivo, both compounds are reported to have luteolytic properties when administered during the late luteal phase; in young CL, however, both substances stimulate P secretion, an effect which is E2-mediated. During the late luteal phase luteal cells appear to produce cytokines, and in addition, cytokine-producing macrophages invade the CL. We tested therefore whether cytokines, particularly tumor necrosis factor-alpha (TNF), have effects on basal or human CG-stimulated steroidogenesis. Furthermore, the interactions of cytokines with PGF2 alpha and/or OXT were investigated. TNF, and less potently interleukin (IL)-1 and IL-2 but not IL-6, inhibited basal as well as human CG-stimulated release of P and E2 in both small and large luteal cells. The inhibiting effect of PGF2 alpha and OXT on P secretion was augmented by these active cytokines. The stimulatory effect of PGF2 alpha and OXT on small and large luteal cell E2 production was completely inhibited. A profound stimulatory effect of E2 and small luteal cell P secretion was completely prevented by the cytokines, with TNF being more potent than IL-1 or -2. We conclude that the cytokines, particularly TNF, have luteolytic functions by their direct inhibiting effects on luteal cell P production. In addition, the cytokines inhibit synthesis and action of PGF2 alpha- and OXT-stimulated E2 secretion. Since E2 is a potent stimulator of luteal cell P production, this luteotropic signal is eliminated by cytokines, which add to the process of luteolysis.