In order to eventually elucidate the physiological roles of TIMP-1 and TIMP-2 in the CL, we first determined the temporal and spatial expression patterns of these two inhibitors during the estrous cycle.
In the present study, TIMP-1 mRNA was determined to be highly expressed in the early and mid cycle bovine CL, but decreased in the late stage. In the porcine CL, the TIMP-1 transcript was also highly expressed in the early stage, and is slightly reduced as the estrous cycle progresses before it decreases significantly in the regressing stages . In contrast, TIMP-1 mRNA expression in the ovine  and human  CL does not change throughout the estrous or menstrual cycle, respectively. In the pseudopregnant rat, a different pattern emerges whereby the strongest TIMP-1 mRNA expression is observed during CL formation and regression . Clearly, there are species differences with regard to the temporal expression of TIMP-1 transcript in the CL.
The expression of TIMP-1 protein in three ages of bovine CL was also demonstrated in the present study. Reverse zymography demonstrated that TIMP-1 was the predominant TIMP in the bovine CL, similar to the finding in sheep . In the present study, the patterns of TIMP-1 protein and mRNA paralleled each other, being high in the early and mid stages, but decreased in the late cycle CL. The high levels of TIMP-1 in the early and mid cycle CL may participate in regulating the extensive tissue remodeling events that occur during CL formation and development. The reduced TIMP-1 mRNA and protein expression in the late stage CL may portend the decline of this inhibitor observed during luteal regression in bovine , ovine [39, 40], porcine , and primate  CL. Collectively, these data implicate TIMP-1 as an important player in the physiology of the CL.
The TIMP-1 protein was localized in large luteal cells of early, mid, and late stage bovine CL. In the ovine CL, TIMP-1 was co-localized with oxytocin in secretory granules of large luteal cells . In addition, TIMP-1 expression was also detected in isolated ovine  and porcine  large luteal cells, and luteinized human granulosa cells . The presence of TIMP-1 in steroidogenic cells may be associated with its ability to enhance steroid production . This may be related in part to a 124 bp-nucleotide sequence similarity between the protein coding region of the bovine steroidogenic acute regulatory (StAR) gene and the 5' non-coding region of TIMP-1 . Additionally, although female mice lacking the TIMP-1 gene do not show detectable differences in serum estradiol-17β concentrations when compared to the wild type, the TIMP-1 deficient male mice have higher concentrations of total serum testosterone than the wild type [44, 45]. Furthermore, cell culture studies demonstrated that TIMP-1 is able to increase estradiol-17β production by granulosa cells from both TIMP-1 deficient and wild-type mice , and estradiol-17β and progesterone production from porcine thecal cells . Therefore, the strong expression of TIMP-1 in large luteal cells may be related to its collateral role in steroidogenesis.
Because of the predominant expression of TIMP-1 in large luteal cells, this inhibitor is used as a cellular marker for this cell type . However, other cell types in the CL are positive for TIMP-1 expression as well. For example, the present study showed that TIMP-1 was also localized in the vascular smooth muscle cells (VSMC) of the bovine CL. Although the cellular source was not specified, the capillary compartments in ovine  and rat  CL stain positively for TIMP-1. The localization of TIMP-1 in the vascular smooth muscle compartment supports its potential role during angiogenesis and vascular maintenance. Indeed, overexpression of TIMP-1 in VSMC by exogenous gene transfer reduced VSMC cell proliferation and migration [49, 50]. In addition, stimulation of TIMP-1 expression impaired angiogenesis in a variety of tumor types [51–53]. These data collectively suggest that TIMP-1 may act as a negative regulator of blood vessel formation [32, 54].
Extensive angiogenesis occurs during early CL development when theca-derived luteal cells and fibroblasts invade through the breached basement membrane into the cavity of the ruptured follicle. These early events in the angiogenic process require MMP activity. However, in the last step of angiogenesis, recruitment and maintenance of pericytes (VSMC) are critical for vascular maturation and survival [55, 56]. Thus, the high level of TIMP-1 expression in VSMC of 4-day old bovine CL may provide an environment where MMP action was curbed as the vasculature matures. As the CL ages, its structure remains relatively stable. Although angiogenesis is ongoing, it is slowed during CL maintenance [57–59]. This may be the reason for the absence of TIMP-1 in the VSMC compartment at this stage of the bovine estrous cycle. In the 16-day old CL, TIMP-1 was detected again in the VSMC compartment. This localized expression of TIMP-1 is consistent with the hypothesis of Redmer et al. , who proposed that maintenance of the vasculature may be necessary for the transport of degraded products during luteal regression, which ultimately results in a massive decrease in CL size and weight as regression ensues.
Although TIMP-1 was the most abundant TIMP in luteal tissues, the other three TIMPs were also detected in the bovine CL by reverse zymography. Among them, we chose to focus our attention on TIMP-2. It has been reported that TIMP-2 may be involved in the pro-MMP-2 activation process by binding an MT1-MMP to form a "co-receptor" for pro-MMP-2 on the cell surface . This bound pro-MMP-2 would then be presented to an adjacent MT1-MMP for activation [23, 25]. Although a transmembrane-deleted MT1-MMP is capable of activating pro-MMP-2 without the participation of TIMP-2 , TIMP-2 deficient mice have a dramatically reduced ability to activate pro-MMP-2 . In the present study, both TIMP-2 mRNA and protein levels were low in the early CL, but significantly increased in the mid and late stages. This expression pattern was similar to that for active MT1-MMP and MMP-2 , suggesting that the MT1-MMP/TIMP-2/pro-MMP-2 tri-molecular system may be available for MMP-2 activation in vivo in the bovine CL. The coordinate expression of these three molecules is also observed during embryonic development , which supports the presence of this activation system in a variety of tissue types.
In addition to the temporal correlation of TIMP-2 with active MT1-MMP and MMP-2 expression in the CL, TIMP-2 was also co-localized with these two molecules in endothelial and large luteal cells. TIMP-2 has a variety of functions in endothelial cells. On the one hand, the stoichiometrically correlated expression with MT1-MMP and pro-MMP-2 may facilitate the activation process of pro-MMP-2, which is critical for the angiogenesis process. On the other hand, inhibition of angiogenesis by TIMP-2 cannot be excluded since TIMP-2 has now been demonstrated to be a potent inhibitor of both physiological and stimulated angiogenesis in vivo and over-expression of TIMP-2 blocks vascular smooth muscle cell invasiveness  and reduces angiogenic ability [54, 66]. The latter observation is due, in part, to down-regulation of vascular endothelial cell growth factor (VEGF) .
The localization of TIMP-2 in large luteal cells may also contribute to the activation of pro-MMP-2 in this cell type by assembling the trimeric complex on the cell membrane. The in situ activated MMP-2 is then able to bind integrin αvβ3 , where localized pericellular proteolysis ensues. This resulting degradation of the ECM is needed to accommodate the enlargement of large luteal cells from the early to mid and late stages. Although there is no direct evidence demonstrating involvement of TIMP-2 in steroidogenesis, the dynamic interactions between large luteal cells and their local ECM may induce biochemical changes related to the steroid biosynthetic process in this cell type . For example, disruption of the links between ECM, integrins, and the cytoskeleton  may perturb the intracellular transport of substrates, such as cholesterol, for steroidogenesis . Additional in vitro and in vivo studies are needed to elucidate the physiological roles of these TIMPs in this ovarian endocrine gland.