Recent Aspects on the Paracrine/Autocrine Control of Luteal-Function in the Bitch
Bernd Hoffmann, DVM, Prof. Dr. med. vet.
Klinik für Geburtshilfe, Gynäkologie und Andrologie der Groß-und Kleintiere mit Tierärztlicher Ambulanz
The domestic dog is a mono-oestrous, in general a-seasonal breeder. The reproductive phase comprises the periods of pro-oestrus (13-16 days), oestrus (4-12 days), dioestrus (60-90 days) and anoestrus which--depending on the breed--may vary between 15-265 days. Different to other domestic animal species functional life span of the corpus luteum (Cl) is almost identical in non-pregnant and pregnant females, except that pregnant animals reach baseline concentrations of progesterone and estradiol earlier owing to the immediate prepartum decline of these hormones. Progesterone production of follicular origin commences at the end of pro-oestrus, reaching peripheral plasma levels of about 5 ng/ml at the time of ovulation. Formation of the Cl is indicated by the continuing increase of progesterone levels which in general reach maximum values during the first 20 days of dioestrus. This period is followed by a continuous decline of progesterone to anoestrous levels of < 1 ng/ml (Concannon, 1993, Hoffmann et al., 1996). Control of luteal function in the dog has been addressed in several studies. However, while a number of luteotropic factors has been characterised, control of luteolysis is still an enigma.
As was reviewed by Hoffmann et al. (1996) up to about 20-30 days the newly formed Cl seem to be independent of gonadotropic support; in the second half of dioestrus removal of the pituitary leads to luteolysis, as does a removal of prolactin and--somewhat later--of LH. Thus both hormones are luteotropic factors with prolactin, however, being the predominant one.
Prolactin levels increase in pregnant and non pregnant bitches during the second half of dioestrus commencing with the decline of progesterone. Also the availability of LH increases during this period in pregnant and non-pregnant dogs as was indicated by the area under the curve. However, regression occurs in spite of this increased gonadotropic support with apparently unchanged concentrations of LH-and prolactin receptor-sites in the Cl. Thus it was assumed that paracrine and/or autocrine mechanisms are major factors involved in the control of luteal function.
Aspects on the role of PGF2a
Other than in life stock luteolysis in the dog is independent of a uterine luteolysin. On the other side the immediate prepartal decline of progesterone coincides with an increase of PGF2a(Concannon et al., 1988). Such a prepartal PGF2a -release has been observed in all domestic animals. In general function relates to the induction of labor; however, it was also made responsible for prepartal luteolysis in the cow and goat. For the dog experiments with a competitive progesterone receptor-blocker have shown that this PGF2arelease and hence the preceding activation of cyclooxygenase (COX) is not triggered by the decrease of progesterone. In further experiments application of a COX-inhibitor prior to parturition lead to a block of peripheral PGF2a levels. However, only high dosages (> 5 mg indomethacin kg/bm/day), also leading to side effects, extended length of pregnancy. From these observations it was concluded that PGF2a might be a factor involved in luteal regression in the pregnant dog but that peripheral prostaglandin concentrations would not mimic activities at the level of the Cl (Hoffmann et al., 1999).
A possible role of PGF2a in luteolysis in the dog may also be deducted from observations that pregnancy can be terminated after about day 30 by use of PGF2a or its analogues.
However, again relatively high dosages or repeated treatments are necessary. In those dogs which aborted, progesterone declined to basal levels, in non aborting dogs the progesterone decrease was only temporary (Vickery, Mc Rae, 1980; Shille et al., 1984; Jackson et al., 1982).
To gain further information on the likely formation of prostaglandins in the Cl of the dog and its likely role as an autocrine / paracrine factor, expression of COX I and COX II, a key rate-limiting enzyme in prostaglandin biosynthesis, has been examined in non pregnant dogs during the course of dioestrus (Hoffmann et al., 2004a).
Cl were collected from non pregnant bitches on days 5, 15, 25, 35, 45 and 65 after ovulation. When tested by Real Time (Taq Man) RT-PCR, expression of mRNA for COX I and II could be detected during the whole course of dioestrus. Other than COX I, expression of COX II-mRNA was cycle dependent; it was highest on day 5 and decreased (p < 0.05) through day 15 to day 25 to show no further changes thereafter. Immunohistochemical detection of COX II revealed signals localised in the cytoplasm of luteal cells and few capillary pericytes. Staining was restricted to days 5 and 15 with signals being quantitatively and qualitatively stronger on day 5, confirming the results obtained by RT-PCR. These results indicate that expression of COX I more or less resembles the pattern of a housekeeping gene while expression of COX II seems to be dioestrus related, coinciding with the formation of the Cl rather than luteolysis. Hence a role of prostanoids in the formation of the Cl in the dog is suggested.
Likely role of luteal steroids as paracrine /autocrine factors
In granulosa and theca cell cultures progesterone had a stimulating effect on the activity of 3b-hydroxysteroid-dehydrogenease and the C20-side-chain-cleavage enzyme in a number of species; in bovine luteal cells cultures addition of synthetic progestagens stimulated progesterone production (reviewed by Papa 2001). These observations point towards a paracrine/autocrine activity of progesterone.
As in other species estradiol-17b is produced by luteal cells also in the dog (Nishiyama et al., 1999). In vitro studies with human cultured granulosa cells indicate a dose dependent effect of estradiol on progesterone production; in the pig estradiol stimulated luteal progesterone synthesis during the early and mid luteal phase. By potentiating the luteotropic effect of IGF I, estradiol also stimulated progesterone-synthesis in luteal cell cultures from the rabbit.
These observations and the resulting conclusions on a paracrine / autocrine activity of progesterone and estradiol in the Cl are supported by observations on the expression of luteal oestrogen (ER) and progesterone (PR) receptors in a number of species (reviewed by Papa 2001).
These similar observations in different species point to a common regulatory principle, allowing the assumption that corresponding mechanisms might also exist on the luteal level in the dog.
In order to test for this hypothesis we examined the expression of PR and ERa in the Cl of the bitch in relation to the stage of cycle; methods applied were immunohistochemistry and RT-PCR ( Hoffmann et al., 2004a).
The PR and ERa could clearly be detected in luteal and other cells (fibrocytes, capillary pericytes). The total number of PR receptor positive luteal cells was higher (p < 0.002) in the periphery compared to the inner part of the Cl, the effect of time was highly significant (p < 0.001); expression of the PR was highest on day 5, it had decreased by day 15 to constantly increase again until day 45. Similarly expression of the PR in the "other cells" was highest on day 5 with 75.3 ± 5.7% positive cells. Values had decreased to 48.8 ± 12.9% by day 15 and stayed between 50.1 and 42.5% until day 45, showing no further differences. Again the effect of time was highly significant (p < 0.001) but there was no effect of location.
Also expression of the ERa was higher (p < 0.004) in luteal cells located in the periphery of the CL compared to those in the inner part, but there was no effect of time. The number of other cells expressing the ERa varied between 61.7 ± 5.7% and 44.0 ± 8.5% with no effect of time and location.
These observations confirm our hypothesis of a paracrine and/or autocrine activity of progesterone and estadiol-17b also in the Cl of the dog. However, functional interpretations are difficult. The increased expression of PR with the formation of the Cl coincides with a significantly increased proliferation of luteal and non-luteal cells at the beginning of dioestrus (Klein et al., 2001) and still increasing progesterone concentrations, suggesting a stimulatory effect of progesterone in a paracrine /autocrine manner on the formation and the initial secretory activity of the Cl. Yet in spite of the re-increase of PR-positive luteal cells around day 35 to initial values, possibly accompanied by a slight increase of ERa-positive luteal cells, proliferative activity and progesterone concentrations continue to decrease. Apparently at this point of time active stimulatory mechanisms are either lost, downregulated or overcome by other mechanisms, possibly on the posttranscriptional level, governing luteal cell function.
Involvement of the immune-system
Observations in a number of species point towards an involvement of the immune system in regulation of luteal function (Pate & Keyes, 2001).
In view of this situation we hypothesised that also in the dog immune mediated events may play a role in controlling Cl-function and a study was designed aiming towards the detection of CD4-and CD8-positive lymphocytes and cells expressing MHC-II in the canine Cl during dioestrus (Hoffmann et al., 2004b). Consequently we have also tested for the expression of cytokines in the Cl by RT-PCR, based on the availability of known sequences (Engel et al., 2004).
When using specific monoclonal antibodies for immunohistochemistry, T-lymphocytes and macrophages stained positive for CD4. Invasion of these cells into the Cl could be detected at all stages of dioestrus (days 15, 30, 45, 60 and 75 after ovulation); similarly at any stage the presence of CD8-expressing T-lymphocytes and MHC-II positive staining cells could be demonstrated.
The number of positively stained cells (CD4-, CD8-antigen), respectively the area occupied by positively staining cells (MHCII-antigen), changed significantly during the course of dioestrus. There was a biphasic pattern for CD8-positive lymphocytes and the MHCII-complex with high values on days 15 and 60 (CD8) and days 15 and 75 (MHCII). Similarly, the number of CD4-positive lymphocytes was high on day 15, however, there were no further changes following the decrease to day 30.
In accordance with observations in other species (Bagvandoss et al, 1990) these data on one side demonstrate the presence of T-lymphocytes and macrophages in the juvenile canine corpus luteum. On the other side also luteal regression was matched by an increased number of CD8-positive lymphocytes and an increased expression of the MHCII-antigen.
For detection of cytokines Cl from groups of 4 to 5 dogs ovariohysterectomized on days 5, 12, 25, 35, 45 and 60-80 after ovulation were examined.
Unequivocal evidence for the expression of the mRNA for IL-8, IL-10, IL-12, TNF-a and TGF-b1 at any stage of the cycle was obtained. The mRNA for IL-4, IL-b and IL-2 could not be detected, and--based on the detection of mRNA--the expression of IL-6 and TFN-a was at the lower limit of the method with occasional positive results.
These data suggest that also in the dog cytokines have a modulatory function on differentiation, maintenance and regression of the Cl.
Morphological Aspects and Apoptosis
When examining Cl from days 5, 15, 30, 45, 60, 75 and 113 after ovulation by electron microscopy (Hoffmann et al., 2004a), until about day 30 luteal cells were characterised by round nuclei exhibiting homogenous chromatin, a distinct smooth endoplasmic reticulum (ER) and small lipid droplets almost evenly distributed in the cytoplasm; they were separated by well formed channels of extracellular matrix containing a high amount of capillaries. On day 45 intercellular distance of luteal cells had decreased and the smooth ER showed a reduced density. Marked signs of degeneration were first seen on day 60, when nuclei became polygonal and pyknotic, the chromatin was condensed. The smooth ER changed to whirl-like structures encircling large lipid droplets. From days 75 to 113, nuclear shape was pre-dominantly lobular and nuclei displayed increasing contents of heterochromatin.
These degenerative alterations correlated with the first signs of apoptosis detected on days 60-80 after ovulation by the caspase-3 method.
Cl of pregnant and non-pregnant bitches are independent of gonadotropic support until about days 20-30 after ovulation. Formation of the Cl is accompanied by an increased expression of COX II, of the PR and an increased invasion of CD-4, CD-8 and MHCII antigen positive staining immune cells. This points to a role of leucocyte derived cytokines and luteal cell derived prostanoids and steroids to act in a concentrated manner as paracrine/ autocrine factors in respect to the stimulation and formation of the early Cl. Different to that our observations seem to indicate that regression of luteal function in the dog is not an actively regulated process but rather a permissive one, possibly controlled on the post-transcriptional level.
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