Vascular endothelial growth factor A (VEGFA), a member of the VEGF family, occupies a central place among the regulators of angiogenesis in the ovary. Also known as vascular permeability factor (VPF), VEGFA is the major specific stimulator of endothelial cell proliferation and migration, acting through two tyrosine kinase receptors, VEGFR-1 (flt-1) and VEGFR-2 (KDR; Ferrara and Davis-Smyth 1997).
In the CL, VEGFA expression (mRNA and protein) is upregulated soon after the LH surge and remains elevated at least until the mid-late luteal phase (Berisha et al. 2000; Jiang et al. 2011; Tesone et al. 2005). In most species, VEGFA mRNA is detected in the new CL, in granulosa-derived lutein cells (Wulff et al. 2001; Berisha et al. 2000). Additionally, several studies had verified that VEGFA is essential for optimal CL function (Fraser et al. 2010; Yamashita et al. 2008). Daily intraluteal injections with VEGFA antibody (from Day 1 to Day 8) markedly decreased the bovine CL volume, plasma P4 concentration and steroidogenesis (Yamashita et al. 2008). The in vivo observations on the VEGF inhibitory effect on plasma P4 are most probably due to the disruption of luteal blood vessels. However, in the mare, VEGFA could directly stimulate P4 and prostaglandin E2 (PGE2) production by in vitro cultured luteal cells (Galvão et al. 2012a). Also, critical is the up-regulation of VEGFA transcription by hypoxia-inducible factor (HIF)-1α protein (Hewitson et al. 2007). The hypoxia-induced up-regulation of VEGFA occurs at the transcriptional level by HIF1 proteins, which bind to the hypoxia response element region in the VEGF promoter (Hewitson et al. 2007; Kim et al. 2008). The biological activity of secreted VEGFA is further influenced by hypoxia-inducible expression of the Flt-1 and KDR receptors (Kim et al. 2008). At the early stage of luteal development, due to insufficient blood supply, hypoxic conditions exist in the CL (Tesone et al. 2005) and act as a potent stimulus for VEGF expression by granulosa/luteal cells (Kim et al. 2009; Klipper et al. 2010). Remarkably, in the ovary, unlike in other tissues, LH/hCG alongside with hypoxia can augment VEGF expression (Christenson and Stouffer 1997; Klipper et al. 2010). Indeed, chronic or acute exposure to hCG directly stimulates VEGF production and secretion by granulosa cells of several mammalian species, including farm animals (Christenson and Stouffer 1997; Klipper et al. 2010).
Fibroblast growth factor 2 (FGF2) is another potent luteal pro-angiogenic factor. It stimulates the proliferation of endothelial cells derived from bovine corpora lutea (Woad et al. 2012; Zalman et al. 2012) and is expressed at particularly high levels during the bovine follicular–luteal transition (Robinson et al. 2007). The FGF family encompasses 23 structurally related heparin-binding angiogenic peptides (Ornitz and Itoh 2001), which are pleiotropic factors that act on various cells, including endothelial cells. Intracellularly, FGFs interact with heparin sulphate proteoglycans (HSPGs) and FGF receptors with tyrosine kinase activity. The CL, especially at early stages of its development, is a rich source FGF2 (Robinson et al. 2007). In fact, FGF2 was first extracted and identified in the bovine CL (Gospodarowicz et al. 1985; ). In association with VEGF, FGF2 provides the essential pro-angiogenic support to the developing CL necessary to establish its complex vascular system (Schams and Berisha 2004; Robinson et al. 2007; Yamashita et al. 2008; Woad et al. 2009). As a matter of fact, it was also shown that FGF2 was a potent inducer of angiogenic characteristics in luteal derived endothelial cells, enhancing cell migration and proliferation (Zalman et al. 2012). However, FGF2 is known to be more potent at inducing endothelial cell proliferation than VEGF. Treatment with a FGF receptor signalling inhibitor almost completely blocked luteal endothelial network formation in vitro (Woad et al. 2009, 2012). This occurred even in the presence of exogenous angiogenic growth factor VEGFA, indicating that FGF2 is critical for the formation of luteal endothelial networks (Woad et al. 2009). Furthermore, mentioned findings also suggest that these factors could have complementary rather than redundant actions (Woad et al. 2009). The significance of FGF2 to luteal development was also demonstrated when neutralization of FGF2, achieved by injecting FGF2 antibody into the developing bovine CL, altered luteal growth and function, probably by inhibiting the establishment of a new vascular network (Yamashita et al. 2008).
Fibroblast growth factor 2 levels, like those of VEGFA, are elevated during luteinization. However, FGF2 peaks at a very early luteal phase (Robinson et al. 2007) strikingly overlapping the hypoxic conditions in the CL and the profile of HIF-1α. Nevertheless, the relationship between FGF2 and hypoxia in the CL has not been thoroughly studied yet. Also, the cellular distribution of these two pro-angiogenic factors is different. While VEGFA is more abundant in the steroidogenic luteal cells, FGF2 is highly expressed in both steroidogenic and endothelial cells types with a clear preference towards the endothelial cells. A limited number of studies have shown that FGF2 may also affect granulosa and luteal cell functions and illustrate pleiotropic nature of this growth factor.
Immune cells present in the CL throughout the oestrous cycle are considered to be determinant for ovarian function regulation (Penny et al. 1999; Townson and Liptak 2003). Generally, immune cells action in the CL is carried out through the production and secretion of cytokines tumour necrosis factor-α (TNF), interferon-γ (IFNγ) or interleukins (ILs), and other factors as: PGs, granulocyte–macrophage colony–stimulating factor (GM-CSF), macrophage colony–stimulating factor (M-CSF) and angiogenic factors (Pate and Keyes 2001; Skarzynski and Okuda 2010). Cytokines role on luteal function regulation in cattle has been thoroughly reviewed before (Pate and Keyes 2001; Okuda and Sakumoto 2003; Skarzynski and Okuda 2010). The mRNA of TNF, IFNγ and IL-1 is transcribed not only in immune cells but also in steroidogenic and endothelial cells (Sakumoto et al. 2000, 2011; Okuda and Sakumoto 2003; Nishimura et al. 2004). This finding suggests that, besides being determinant during luteal regression, these cytokines may also regulate luteal steroidogenesis as well as vascular function (Petroff et al. 1999; Skarzynski et al. 2003a, 2007; Korzekwa et al. 2008a). Tumour necrosis factor-α was shown to control several ovarian processes like follicular development, ovulation and luteal regression, by acting specifically on different receptors: the TNF-RI (death receptors), and the TNF-RII (survival receptors; Terranova 1997). Furthermore, numerous in vitro studies have evidenced that TNF induces luteal cell death exclusively in association with INFγ or other intraluteal factors, that is, endothelin (EDN)-1 (reviewed by Pate and Keyes 2001; Meidan et al. 2005; Skarzynski and Okuda 2010).
As a pleitropic factor, TNF can exert opposite biological functions. Previous in vivo studies confirmed TNF luteolytic action and its influence on bovine CL lifespan (Skarzynski et al. 2003a). In fact, lower doses of TNF increased prostaglandin F2α (PGF2α) and nitrite/nitrate (stable NO metabolites) levels, decreased P4 level and, consequently, shortened the oestrous cycle. Interestingly, TNF administration in high doses stimulated the synthesis of P4 and PGE2, prolonging the lifespan of bovine CL (Skarzynski et al. 2003a, 2007; Korzekwa et al. 2008a). These in vivo results were recently in vitro supported (Szostek et al. 2011). Still regarding TNF luteotropic action, the TNF at higher concentrations increased P4 output by microdialysed bovine CL (Fig. 1). Thus, TNF may act in the bovine CL in a dose-dependent manner, supporting steroidogenic luteal cells function including PGE2 and P4 stimulation during the luteal phase and early pregnancy (Sakumoto et al. 2000; Skarzynski et al. 2003a, 2007; Korzekwa et al. 2008a).
Figure 1. Effect of tumour necrosis factor (TNF) α on pulsatile progesterone (P4) output by bovine corpora lutea (CL) in vitro. The bovine CLs (Day 8–10 of the oestrous cycle) were cut vertically, through the face of the apex, in four pieces and perfuzed in a microdialysis system (MDS) with TNF at luteotropic dose 1 μg/ml for 4 h (b). Saline was run as a control (a). The pulses of P4 were determined when a minimum of two or more adjacent measurements presented a concentration value 25% higher than the mean of all the 12 measurements. The asterisks indicate the significant statistical differences in the pulses of P4 (p < 0.05). Adapted and modified from Szostek et al. (2011)
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Not only steroidogenic cells can be a target of cytokine actions within the bovine CL. Specific binding sites for TNF are also present in luteal endothelial cells derived from bovine CL (Petroff et al. 1999; Okuda et al. 1999; Friedman et al. 2000; Korzekwa et al. 2011). Cytokines may stimulate cell proliferation or induce apoptosis of bovine luteal endothelial cells (Friedman et al. 2000; Davis et al. 2003; Pate and Keyes 2001). Also, TNF contributes to the production of PGE2, PGI2 and PGF2α in in vitro cultured bovine luteal endothelial cells (Okuda et al. 1999; Korzekwa et al. 2011). Therefore, several cytokines (including TNF) could also promote the formation of bovine CL, as it was recently reported for equine CL (Galvão et al. 2012a,b).
A peptide implicated in ovulation and luteal formation is the endothelin (EDN)-2. It represents a member of a 21-amino acid peptides family that includes also EDN1 and EDN3 (Inoue et al. 1989; Meidan and Levy 2007). Both EDN1 and EDN2 are the forms expressed in the ovary, presenting the closest amino acidic structure and differing only by two amino acids (aa). Considering EDN3, it differs from other EDNs by six aa (Inoue et al. 1989; Meidan and Levy 2007). Similarly to EDN1, EDN2 is known for its strong vasoconstricting activity. Nevertheless, it acts in a different physiological context and is produced not only by the epithelial cells but also in granulosa cells (Ko et al. 2006; Kim et al. 2009; Klipper et al. 2010).
The multiple effects of EDNs are mediated by G protein-coupled receptors, termed ETA and ETB. The coupling of activated EDN receptors to multiple G-proteins may explain the involvement of EDNs in diverse cellular processes (Inoue et al. 1989; Meidan and Levy 2002). Ko et al. (2006) firstly reported that EDN2, localized in the granulosa cells, abruptly increased after hCG injection into PMSG-primed rats. Treatment with mixed ETA/ETB EDN receptor antagonist before ovulation caused a substantial delay in ovulation and decreased the number of released oocytes in mice and rats (Ko et al. 2006; Palanisamy et al. 2006). Which receptor type mediates EDN2 action in bovine granulosa cells is still unclear and awaits further research. In cows, EDN2 mRNA displayed a transient pattern of expression during the luteal phase. The highest EDN2 mRNA levels were observed in the early CL, 60 h post-GnRH-induced ovulation and also in cyclic CL collected before the Day 5 of the cycle. The EDN2 mRNA then declined to basal levels in mid-luteal phase (Klipper et al. 2010). Notably, unlike EDN2, EDN1 mRNA was not elevated at any time point during GnRH-induced folliculo-luteal transition (Klipper et al. 2010). In the early CL, EDN2 mRNA was identified mainly in bovine luteal steroidogenic cells, but not in endothelial cells that expressed the EDN1 gene (Klipper et al. 2010). Similarly, in pre-ovulatory follicles, EDN2 was expressed in the granulosa cells (Klipper et al. 2010) and not in the vascular theca interna, similarly to what was observed in mice (Ko et al. 2006; Palanisamy et al. 2006). Besides LH, hypoxia was found to be a strong inducer of EDN2 transcription in all species examined thus far (Kim et al. 2009; Klipper et al. 2010). It is noteworthy that these same factors (LH and hypoxia) have been long known to induce VEGF in the CL (Christenson and Stouffer 1997). Interestingly, not only LH/hCG but also EDN2 itself could also directly induce VEGF in granulosa cells (Klipper et al. 2010). Like LH/hCG, EDN2 induced in these cells proliferation as well as up-regulation of VEGF and cyclooxygenase-2 (mRNA and protein levels). Together, these data suggest that elevated EDN2 in the early bovine CL, triggered by the LH surge and hypoxia, may facilitate CL formation by promoting angiogenesis, cell proliferation and differentiation.
Phospholipids metabolites: prostaglandins, leukotrienes and lisophosphatidic acid
Prostaglandins and leukotriens (LTs) are produced from AA, which is released from the phospholipid membrane (Hansel 1996). A rate-limiting enzyme of PG synthesis is the PG-endoperoxide synthase 2 (PTGS2), responsible for AA conversion into PGG2. During the oestrous cycle in the cow, mRNA expression of PTGS2 is higher in the early CL than in other phases (Tsai et al. 1996; Kobayashi et al. 2002). Cell-specific synthases and isomerases, such as PGES and PGFS, are responsible for PGE2 and PGF2α production, respectively. Both PGs are secreted in the bovine CL throughout the luteal phase. However, the highest PGs output level and expression of their synthetic enzymes were found in the early luteal phase (Milvae and Hansel 1983; Arosh et al. 2004).
Prostaglandin E2 is known as a luteotropic factor, as it stimulates the in vitro P4 production by bovine luteal steroidogenic cells (Alila et al. 1988; Kotwica et al. 2003; Bowolaksono et al. 2008). Moreover, PGE2 also promotes the secretion of luteotropic LTB4 from bovine steroidogenic and endothelial luetal cells, as well as inhibits cytokine-induced apoptosis in both early and mid-luteal stages (Korzekwa et al. 2010b; Bowolaksono et al. 2008).
The early bovine CL also produces the highest amount of PGF2α, comparing with other luteal phases (Milvae and Hansel 1983; Kobayashi et al. 2001). The PGF2α binds to the luteal cell through its specific plasma membrane receptor named PTGFR (Anderson et al. 2001). In vivo studies demonstrated that PGF2α acutely decreased P4 secretion by inhibiting 3βHSD and StAR mRNA expression and other rate-limiting steroidogenic enzymes (Tsai and Wiltbank 1998; Atli et al. 2012). Simultaneously, PGF2α was shown to promote the production of luteolytic mediators, like EDN1 (reviewed by Meidan et al. 1999; Meidan and Levy 2002, 2007), LTC4 (Korzekwa et al. 2010a,b) and nitric oxide (NO; Skarzynski et al. 2003b; Shirasuna et al. 2008; Lee et al. 2010). Other in vitro reports demonstrated that PGF2α stimulates P4 secretion by cultured bovine luteal cells (Miyamoto et al. 1993; Mamluk et al. 1999; Bah et al. 2006; Korzekwa et al. 2008b; Pate et al. 2012). Likewise, PGF2α and PGE2 can suppress apoptosis of bovine steroidogenic and endothelial cells (Bowolaksono et al. 2008). Both PGs at the concentration of 1 μm increased cell viability and suppressed cell death, decreased caspase (CASP) 3 and CASP8 mRNA expression, and inhibited TNF and IFNγ induced CASP3 activity (Bowolaksono et al. 2008). Nonetheless, Pate et al. (2012) have recently proposed that the observed PGF2α luteotropic effects are mainly related with its utilization at high concentrations, which may represent supraphysiologic doses considerably higher than those seen during spontaneous luteolysis in vivo. At such levels (1 μm), PGF2α could bind PGE2 receptor rather than PGF receptor (Rao 1974; Anderson et al. 1999) and, once activated, PGE2 receptor could stimulate P4 production. Still, it should be noticed that the referred PGF2α concentration decreased P4 secretion by CL slices (Girsh et al. 1995; Doerr et al. 2008). This emphasizes the importance of other cell types, besides steroidogenic cells (endothelial and immune cells), for the modulation of PGF2α actions in the bovine CL.
Leukotrienes are synthesized by 5-lipoxygenase (5-LO), which catalyses the conversion of AA into LTA4. The intermediary product LTA4 is further hydrolysed to LTB4 or conjugated with glutathione to form LTC4 and its metabolites: LTD4 and LTE4 (Zipser and Laffi 1985). Messenger RNA for LT receptors (both for LTB4 and C4) and 5-LO is expressed in the bovine steroidogenic and endothelial cells of the CL, throughout the luteal phase (Korzekwa et al. 2010a,b). Leukotriene B4 was shown to promote bovine CL growth by stimulating PGE2 secretion in different in vitro conditions, where just luteal endothelial cells from early and mid CL stages were cultured (Korzekwa et al. 2010b), or all luteal cell populations were used (Blair et al. 1997). In mid-luteal stage, LTB4 also presents a luteotropic action, stimulating P4 output from the bovine CL (Korzekwa et al. 2010b). Contrarily, LTC4 increases PGF2α production by bovine luteal endothelial cells (Korzekwa et al. 2010b) and by microdialysed CL (Blair et al. 1997), suggesting its putative luteolytic action. Regarding NOEDN-1 production, LTC4 was shown to stimulate its secretion by endothelial cells from Day 2–4 CL (Korzekwa et al. 2010b). In conclusion, on one hand, endogenous LTC4 inhibits P4 secretion in the oestrous cycle, as its antagonist – Azelastine elevated P4 output, and on the other hand, LTB4 action enhances P4 secretion, as its antagonist – Dapsone inhibited P4 output in the oestrous cycle in vivo (Korzekwa et al. 2010a). Further studies are needed to better understand LTs role in CL angiogenesis and/or angioregression.
Lysophosphatidic acid (LPA), the biologically active phospholipid, is critical for several physiological and pathological processes, including inflammation, cell proliferation and differentiation, cytoskeletal rearrangement, angiogenesis, wound healing and cancer invasion. The LPA has been shown to affect female reproductive functions in several mammalian species, including the cow (Woclawek-Potocka et al. 2009, 2010). In vivo, LPA administered into the abdominal aorta stimulated P4 and PGE2 secretion during the luteal phase and prolonged bovine CL lifespan (Woclawek-Potocka et al. 2009, 2010). Recently, it has been confirmed that bovine CL presents a dual function regarding LPA action, representing both the site of LPA synthesis and the target for LPA action (Kowalczyk-Zieba et al. 2012a). Lysophosphatidic acid concentrations in the CL tissue increased towards the end of the cycle and were stable during early pregnancy. No changes in the expression of LPA receptors (LPARs) occurred during the oestrous cycle. However, expressions of LPAR2 and LPAR4 on Days 17–19 of pregnancy were higher than those on the respective days of the oestrous cycle, and even higher than those on Days 8–10 of pregnancy. It was also shown that LPA stimulates P4 production and 3βHSD, at mRNA and protein levels, in the luteal cells from Days 8–10 of the oestrous cycle (Fig. 2; Kowalczyk-Zieba et al. 2012a).
Figure 2. Effect of lysophosphatic acid (LPA) on progesterone (P4) production by the cultured bovine luteal cells at mid-luteal phase. Lysophosphatic acid (10–6 m), interferone τ and both reagents together were added 12-h before the end of culture. (a) P4 concentrations in the culture medium; (b) mRNA expression of 3β-hydroxysteroid dehydrogenase (3β-HSD) in the cells (real-time PCR); (c) Densitometric quantification of 3β-HSD protein content in the luteal cells (Western blot). All values are expressed as means ± SEM. Different letters indicated significant differences (p < 0.05). Adapted and modified from Kowalczyk-Zieba et al. (2012a)
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In humans, LPA in preovulatory follicles may be important for luteal angiogenesis via increasing IL-8 and IL-6 levels (Chen et al. 2008). Thus, LPA could be a potential modulator of endothelial cell growth and function. Panetti et al. (1997) demonstrated that LPA at the dose of 3 μm is a potent stimulator of bovine aortic endothelial cells (BAEC) growth. Also, endothelial NOS (eNOS) was forcefully activated by LPA, in BAEC (Kou et al. 2002). Conversely, thrombospondin-1 (TSP1), a known inhibitor of angiogenesis and endothelial cell proliferation, inhibited LPA-stimulated mitogenesis of BAEC (Panetti et al. 1997). Moreover, in vitro studies reported that LPA inhibited NO and TNF/IFNγ cytokine-induced apoptosis via abrogation of the stimulatory effect of these factors on mRNA and protein levels of BAX/Bcl2 ratio, Fas–FasL system and pro-inflammatory cytokine receptors (Kowalczyk-Zieba et al. 2012b). Evidently, LPA supported bovine luteal cells survival. Therefore, it could be suggested that LPA is an additional auxiliary luteosupportive factor in the bovine CL (Kowalczyk-Zieba 2012a,b).
Rekawiecki and co-workers recently reviewed the regulation of P4 synthesis and its auto/paracine actions in the bovine CL (Rekawiecki et al. 2008). Progesterone receptor (PR) is expressed in both small and large luteal cells, and also in vascular endothelial cells (Sakumoto et al. 2010). Bovine CL exhibited specific nuclear staining for PR, which was more evident on Days 5–10 after ovulation in cycle animals, comparing with other stages of the oestrous cycle or pregnancy (Kotwica et al. 2004). Messenger RNA level of PR significantly changes during the oestrous cycle, presenting the highest transcription level in the early CL of the cow (Sakumoto et al. 2010). It is well known that P4 considerably influences the vascular function of the female reproductive system, throughout the oestrous cycle (Reynolds and Ford 1984). Progesterone has also been shown to regulate cell survival in the bovine CL (Friedman et al. 2000; Rueda et al. 2000; Okuda et al. 2004; Rueda et al. 2000). Use of P4 antagonists promoted DNA fragmentation (Rueda et al.2000) and P4 could abolish TNF-induced apoptosis of luteal endothelial cells (Friedman et al. 2000). Furthermore, P4 inhibited cytokine-induced apoptosis in bovine luteal cells through the inhibition of Fas and CASP3 mRNA expression (Okuda et al. 2004).
Considering P4 production, it has been shown that this steroid is able to regulate its own synthesis in the bovine CL (Kotwica et al. 2004). After 6 h of incubation with P4, its concentration in CL slices, as well as 3β-HSD activity, increased over 3-times when compared with the control. These effects were seen only in CL from Days 5–10 of the cycle. To avoid the putative influence of secreted P4 present in the culture medium after in vitro culture, cells were treated with a specific P4 antagonist (onapristone). As a consequence, in early luteal cells, secretion of P4, OT, PGF2α and PGE2 was reduced by onapristone (Skarzynski and Okuda 1999; Okuda et al. 2004). Undoubtedly, these data suggest that P4 promotes its own synthesis and secretion from bovine CL, with special significance during CL growth and development.
Another important finding related with P4 action in CL regards the NO generation system. The inducible NO synthase (iNOS) isoform was also inhibited by auto/paracrine P4 actions in bovine luteal endothelial cells (Yoshioka et al. 2012). Moreover, an antagonist of P4 blocked the suppressive effect of P4 on NO generation. Interestingly, eNOS expression and its activity were unaffected by P4, in the bovine CL (Yoshioka et al. 2012). One may conclude that P4 plays important roles in regulating CL function by controlling NO generation and action.
Prostaglandin F2α-induced corpus luteum regression
In the absence of an embryonic signal, the CL will regress. Luteal regression is necessary for the initiation of a new reproductive cycle. Nonetheless, extension of luteal life span and P4 secretion are absolutely required for pregnancy maintenance.
In a non-fertile cycle, PGF2α is secreted from the uterus and then initiates a series of events culminating in its demise (McCracken et al. 1999). Initially, luteal function is impaired and plasma P4 declines (Niswender et al. 2000). This is followed by apoptosis and structural elimination of the CL (Meidan et al. 1999; Yadav et al. 2002; Korzekwa et al. 2006). Exogenously administered PGF2α can initiate luteolysis only in the mature CL (McCracken et al. 1999). For instance, the bovine CL is resistant (or refractory) to the luteolytic actions of PGF2α before Day 5 of the oestrous cycle (Pursley et al. 1995; Tsai and Wiltbank 1998; Levy et al. 2006). The refractory period exists even though early bovine CL contains the receptors for PGF2α and can respond to its injection, by changing hormone secretion and gene expression patterns (Pursley et al. 1995; Levy et al. 2006). The reader is also referred to previous reviews on the luteolytic actions of PGF2α (McCracken et al. 1999; Niswender et al. 2000; Pate 2003; Schams and Berisha 2004; Meidan et al. 2005; Skarzynski and Okuda 2010; Shirasuna et al. 2012a, b; Pate et al. 2012).
Luteal stage-specific responses: Role of immune cells and angiogenic-related factors
Important novel findings regarding the luteolytic cascade and especially luteal stage-specific responses to PGF2α were recently made (Zalman et al. 2012; Atli et al. 2012). These studies attempted to identify PGF2α-induced changes in the transcriptome of bovine CL that are specific to mature CL (Mondal et al. 2011; Zalman et al. 2012). The microarray analysis was performed on PGF2α-responsive (Day 11) CL vs. refractory (Day 4) CL with GeneChip Bovine Genome Arrays. Accentuated gene expression response in Day 11 CL was accompanied by specific enrichment of PGF2α-regulated genes in distinctive gene ontology categories (Mondal et al. 2011). This study revealed that a considerable proportion of transcripts (25%) regulated at 4 h after PGF2α administration in Day 11 CL was similarly regulated in Day 4 CL that fails to regress (Mondal et al. 2011). The significant but transient gene expression response on day 4 suggested that, although initial response had occurred (at 4 h post-PGF2α administration), it was subsequently blocked or failed to amplify (at 24 h; Mondal et al. 2011). The mechanisms responsible for persistent enrichment of PGF2α-regulated genes in immune-related gene ontology categories in the Day 11 (PGF2α responsive) but not in the Day 4 (PGF2α refractory) CL may therefore be attributed to the enhanced crosstalk between immune cells and other luteal cell populations in the mature responsive CL. The induction of a battery of endothelial adhesion molecules such as chemokine (C-C motif) ligand 2 (CCL2), selectin (SEL) E and SELP would facilitate leucocyte recruitment and endothelial transmigration. Immune cells present in the mature CL (Townson et al. 2002; Bauer et al. 2001) may account for the more vigorous and persistent response to PG in the immune-related genes and the luteolytic cascade in Day 11 but not in Day 4 CL.
Interestingly, among the novel PGF2α-regulated genes recently described, quite few are involved in angiogenesis (being pro or anti-angiogenic; Fig. 3). Moreover, PG regulated the expression of angiogenesis-modulating factors in a luteal stage–dependent manner (Mondal et al. 2011; Zalman et al. 2012; Atli et al. 2012). A robust increase in FGF2 expression (mRNA and protein) occurred in the PGF2α-refractory Day 4 CL (Fig. 4). Elevation in FGF2 by PG at an early stage (particularly when no FGF2 inhibitors exist below) would act as a survival signal for both endothelial (Zalman et al. 2012; Woad et al. 2009) and steroidogenic cells of the CL (Grazul-Bilska et al. 1995). Support of blood vessel growth and its stabilization are expected to enhance the supply of nutrients and hormones to the gland, promoting its survival and contributing to its ability to become resistant to luteolysis. Activity of FGF2 in the extracellular milieu is controlled by its interaction with various extracellular matrix proteins and binding factors. Pentraxin-3 (PTX3) and TSP1 and TSP2 are prominent examples of such factors (Zalman et al. 2012; Fig. 3). It was found that inhibitors of FGF2 action, TSPs and their receptor (CD36), and (PTX3) were upregulated by PG specifically in Day 11 CL undergoing luteolysis (Zalman et al. 2012; Mondal et al. 2011), but not Day 4 CL. Furthermore, pronounced inhibition of FGF2-induced proliferation and/or migration of luteal endothelial cells was observed in vitro in response to TSP1 and PTX3 treatment. Dispersed luteal steroidogenic and endothelial cells alike expressed these genes, but TSP1 and FGF2 were more abundant in luteal endothelial cells. Reduced angiogenic support due to lower levels of FGF2 and VEGF, along with increased anti-angiogenic factors expression in Day11 PGF2α-responsive CL (Zalman et al. 2012; Atli et al. 2012), is expected to destabilize luteal vasculature and reduce its hormonal output characteristic of luteal regression. The increased angiopoietin-1 mRNA and protein, soon after PGF2α administration (Tanaka et al. 2004; Berisha et al. 2010), would also contribute to vessels destabilization. Only in mature gland these events along with increased blood vessel constriction by PGF2α-elevated EDN-1 would further reduce P4 secretion and promote regression of the CL (Meidan et al. 1999; Shirasuna et al. 2004; Doerr et al. 2008).
Figure 3. Anti-angiogenic genes expression in the bovine corpus luteum (CL) after prostaglandin (PG)F2α administration. Effects of i.m. administration of PGF2α on the abundance of mRNA for Pentraxin 3 (PTX3), thrombospondin (TSP)1 and TSP2, and their receptors (CD36) in PGF2α-refractory (Day 4) and PGF2α-responsive (Day 11) bovine CL collected before (0 h) and 4 h after PGF2α administration (n = 5 each). Data were normalized relative to the abundance of GPDH mRNA in the same samples. Letters denote significance for Day 4 (lowercase) and Day 11 (uppercase) CL (p < 0.05). Adapted and modified from Zalman et al. (2012)
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Figure 4. Effects of prostaglandin (PG)F2α administration on fibroblastic growth factor (FGF)2 in PGF2α-refractory (Day 4) and PGF2α-responsive (Day 11) bovine corpus luteum (CL) collected before (0 h) and 4 h after PGF2α administration (n = 5 each). (a) The abundance of mRNA. Data were normalized relative to the abundance of GPDH mRNA in the same samples. Letters denote significance for Day 4 (lowercase) and Day 11 (uppercase) CL (p < 0.05). (b) Densitometric quantification of FGF2 protein content. Data were normalized relative to the abundance of β-Actin in the same samples. Significant (p < 0.05) difference from time 0. Inset: a representative Western blot for FGF2 with a major band at 18 kDa and actin (42 kDa) as the loading control is shown. Adapted and modified from Zalman et al. (2012)
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These recent studies, based on transcriptome analyses, provide an insight into stage-specific responses to PGF2α and potential mediators of luteolysis (Zalman et al. 2012; Mondal et al. 2011). They reinforce the significant role of infiltration and activation of immune cell types suggested before (Shirasuna et al. 2012a; Pate 1995; Pate and Keyes 2001). Particularly, these reports introduce the concept that by tilting the balance between pro- and anti-angiogenic factors, PGF2α can potentially control the ability of the CL to resist or evolve towards luteolysis (Fig. 5).
Figure 5. Prostaglandin F2α (PGF2α) – induced luteolysis is related to its ability to promote or inhibit angiogenesis. A significant increase in pro-angiogenic fibroblast growth factor-2 (FGF2) occurred in the PG-refractory Day 4 corpus luteum (CL). Conversely, inhibitors of FGF2 action, thrombospondin (TSP)1 and 2, their receptor (CD36), and Pentraxin 3 (PTX3) were upregulated by PGF2α specifically in mature CL undergoing luteolysis. Vascular endothelial growth factor (VEGF) mRNA decreased 4 h post-PGF2α in both Day 4 and Day 11 CL. See text for detailed description
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Death-ligands and their receptors in corpus luteum regression
Structural involution of the CL mainly involves apoptosis, or programmed cell death (Davis and Rueda 2002; Korzekwa et al. 2006). Apoptosis is defined as the fragmentation of DNA into oligonucleosomal units (Tilly 1996). During apoptosis, three major pathways have been identified: death receptor pathway, mitochondrial and nucleus pathways (Tilly 1996). Many cytokine receptors (Sakumoto et al. 2000; Petroff et al. 2001), second messengers including calcium ions [Ca2+] (Orrenius et al. 1992) and regulatory proteins (Antonsson 2001) are involved in apoptotic process. Fas ligand, a member of TNF super family, primarily engages its receptor (Fas) to induce apoptosis (Okuda and Sakumoto 2003). Members of the Bcl-2 protein family regulate the mitochondrial pathway of apoptosis (Antonsson 2001). Among other proteins, BAX – another member of Bcl-2 family – is also increased during CL regression (Sugino et al. 2005) and can ultimately lead to disruption of steroidogenesis and cell death (Davis and Rueda 2002). Ratio of pro-apoptotic BAX to anti-apoptotic Bcl-2 protein determines the induction of apoptosis (Anderson et al. 2001). Proteins of Bcl-2 family are caspase activators, resulting on the mitochondrial release of apoptogenic factors (Gross et al. 1999; Davis and Rueda 2002). The mechanism of apoptosis is known to occur as a cascade of sequential activation of initiator and effector caspases (Carambula et al. 2002). Caspases, a family of aspartic acid-specific cysteine proteases, are pivotal mediators of apoptosis during CL regression (Carambula et al. 2002). Of the 14 identified caspase family members, caspase-3 is the best-characterized enzyme, which participates in apoptotic signal transmission from the cytoplasm to the nucleus (Carambula et al. 2002; Davis and Rueda 2002). During luteal regression, besides apoptotic DNA degradation, cleavage of the putative caspase-3 substrate poly (ADP) ribose polymerase (PARP) also occurs (Davis and Rueda 2002).
Apoptosis of luteal cells and CL vascular regression are regulated by many different factors (see Fig. 6). A large number of factors have been implicated in PGF2α-induced structural luteolysis in cattle, such as pro-inflammatory cytokines (TNF, IFNγ), FasL (Davis and Rueda 2002; Okuda et al. 2002), EDN1 (Watanabe et al. 2006) and NO (Korzekwa et al. 2006). Messenger RNA for TNF, TNF death receptors (TNF-RI), Fas and IFNγ significantly increase during luteolysis in bovine CL (Sakumoto et al. 2000; Taniguchi et al. 2002; Neuvians et al. 2004; Korzekwa et al. 2008a). Contrary to the evidences that mRNA for TNF is present in the bovine CL throughout its lifespan (Petroff et al. 1999; Sakumoto et al. 2011), secreted TNF protein or its bioactivity could be detected only after the initial decrease in plasma P4 in bovine CL (Shaw and Britt 1995). This evidences the role of TNF on luteolysis promotion. As TNF and IFNγ receptors have been described in the microvasculature, their activated pathways can also modulate endothelial cell function (Okuda et al. 1999; Davis et al. 2003). Once endothelial cells are the first cellular content to undergo apoptosis in the regressing CL, these cytokines were considered to regulate bovine luteolysis, by inducing endothelial cells apoptosis (Sawyer et al. 1990; Okuda et al. 1999; Hojo et al. 2010). The above-mentioned cytokines,TNF and IFNγ), can also be integrated in the regulation of luteal endothelial cells (Korzekwa et al. 2006, 2011) and interact with EDN1 and PGF2α, inhibiting luteal steroidogenesis and stimulating luteal cells death (Ohtani et al. 2004; Okuda et al. 2002). In addition to TNFR, other cytokine receptors, second messengers including [Ca2+] and regulatory proteins are involved in apoptosis of steroidogenic and endothelial cells of the CL (Friedman et al. 2000; Petroff et al. 2001; Taniguchi et al. 2002). The FasL, a member of the TNF super family, primarily engages its receptor (Fas) to induce apoptosis (Taniguchi et al. 2002; Okuda et al. 2004). The expression of Fas mRNA was increased by IFNγ, and TNF augmented the stimulatory action of IFNγ on Fas expression (Taniguchi et al. 2002). Moreover, apoptotic bodies were observed in luteal cells treated with FasL, in the presence of IFNγ and/or TNF. Clearly, leucocyte-derived TNF and IFNγ play an important role on FasL-Fas-mediated luteal cell death in the bovine CL.
Figure 6. Possible luteolytic actions of pro-inflammatory cytokines (tumour necrosis factor α – TNFα, interferone γ – IFNγ), Fas Ligand (Fas-L) and nitric oxide in the bovine luteal cells. Pro-inflammatory cytokines, Fas-L and NO in concert, play a crucial role in both functional (inhibition of the basal and luteinizing hormone-stimulated P4 synthesis and regulation of corpus luteum blood flow) and structural luteolysis (induction of apoptosis of the luteal cells). They stimulate luteolytic arachidonic acid (AA) metabolites: prostaglandin (PG)F2α and leukotriene (LT)C4 and inhibit P4 secretion. Moreover, pro-inflammatory cytokines, Fas-L and NO are involved in induction of apoptosis stimulating caspase-3 expression and activity, stimulating BAX mRNA expression, and consequently, the ratio of pro-apoptotic BAX to anti-apoptotic BCL2 is increased. See text for detailed description. Modified and adapted from Sakumoto and Okuda (2003); Skarzynski et al. (2005), Skarzynski and Okuda (2010)
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Another factor involved in apoptosis in the CL is the NO. Inhibition of NOS avoided the decrease in P4 and prolonged the luteal phase in cow (Jaroszewski and Hansel 2000). In fact, NO mediates PGF2α action during the first steps of the luteolytic cascade in cattle (Skarzynski et al. 2003a,b; Shirasuna et al. 2008; Acosta et al. 2009). Additionally, the molecular mechanisms of NO action during structural luteolysis were also demonstrated (Korzekwa et al. 2006). The NO strongly decreased luteal cell viability in vitro. Treatment of bovine luteal cells with NO donor more significantly increased DNA fragmentation than the one with PGF2α. Moreover, NO increased the mobilization of [Ca2+] in steroidogenic luteal cells. Although NO donor did not affect Fas-L and bcl-2 gene expression, it stimulated Fas and BAX mRNA and CASP3 expression. Also, the ratio of bcl-2 to BAX mRNA level decreased in cells treated with NO donor (Korzekwa et al. 2006). The overall results suggest that NO plays a crucial role for regulating the oestrous cycle, not only during the first steps of functional luteolysis (Skarzynski et al. 2003b; Shirasuna et al. 1998; Acosta et al. 2009), but also as a potent mediator of PGF2α in structural luteolysis by inducing apoptosis of steroidogenic CL cells in cattle (Korzekwa et al. 2006; Fig. 6). Similar effects of NO in steroidogenic luteal cells apoptosis induction have been reported in another species (Jee et al. 2003; Preutthipan et al. 2004; Al-Gubory et al. 2005).