To understand the possible treatment effects of interrupting Notch signaling with compound E or an anti-Dll4 BAb on gonadotropin-dependent folliculogenesis, one has to have a good understanding of where these molecules are expressed within the follicles. Complementary analysis of the expression of the Notch family proteins combined with preexisting data [2, 4, 5] has allowed us to obtain a better idea about which type of cell-to-cell Notch signalling occurs in growing follicles. We demonstrated that Notch3 is expressed exclusively in vascular smooth muscle cells (VSMCs), which are adjacent to theca layer endothelial cells . The presence of Notch3 together with Notch1  on VSMCs suggests a role in organizing the formation of a mature vasculature. It is very likely to involve interaction with the Notch ligand Jagged1 , which is expressed on ECs and VSMCs in the theca layer of growing follicles . It remains unclear as to why we were unable to detect Notch3 in GCs as described by Johnson et al. . Notch2 was consistently expressed in GCs of preantral and small antral follicles and sporadic Notch2 staining was also seen in preovulatory follicles. These findings suggest that Notch2 in GCs is activated by neighbouring GCs expressing Jagged2 , although we did not specifically stain for this protein. Our findings confirm the localization noted in in vitro models . Dll4 is exclusively expressed on ECs. Based on previous results [4, 5] and consistent with our data, this suggests that Dll4 expressed on ECs signals to a neighboring EC expressing Notch1 and possibly Notch4. As Jagged1 is present on ECs, it might not only signal to VSMCs Notch1/Notch3, but also compete with Dll4 regarding the interaction with the Notch1 receptor located on neighboring ECs, as suggested previously by Benedito .
Inhibition of Notch function with the γ-secretase inhibitor compound E significantly blocked gonadotropin-dependent follicle growth up to the preovulatory stage of development. Thus, the number of follicles evolving to the preovulatory stage was significantly decreased. Due to the blockage of gonadotropin-dependent follicle development, the following secondary effects were seen: 1) lesser degree of increase in ovarian weight due to the inability to develop tertiary follicles similar in number to control; 2) lesser degree of increase in uterine weight due to lower E2 secretion in the treatment group when compared to control. In contrast to the effects of VEGF receptor 2 (VEGFR-2) BAb on gonadotropin-dependent folliculogenesis , no reduction in follicular or interstitial area blood vessels is seen in ovaries subjected to compound E. Even though we did not quantify ECs or VSMCs, our visual inspection suggests that there might be a slight increase of these cell types in the treatment group. This supports the finding that vascular cell proliferation continued to occur at least at a level similar to control in the ovaries from compound E treated animals.
The salient feature of ovarian vasculature exposed to a γ-secretase inhibitor is its disorganized appearance. One has the impression that ECs and VSMCs have lost the ability to connect in an orderly fashion during angiogenesis. These observations may suggest that compound E induced perturbation of angiogenesis did not allow proper assembly of blood vessels.
It is of high interest that disruption of EC signaling through YW152F, an anti-Dll4 BAb  did not disrupt follicle growth to the preovulatory stage, nor did it affect ovarian or uterine weight or E2 production or secretion. The blocking of EC Notch1 activation seems to cause a mild level of disorganization of the interaction of ECs and VSMCs, but it is insufficient to block functional vascular growth and blood circulation to support follicle development to the preovulatory stage. In the retina, YW152F creates a phenotype of non-productive sprouting angiogenesis , which is very similar to the effects seen with γ-secretase inhbitors.
The weakness of our YW152F experiment is that one could argue that the absence of inhibiting effect on folliculogenesis in the treated animals might be due to ineffectiveness of the administered Dll4 BAb. Unlike with compound E, where the effect can be validated by observing goblet cell proliferation in the gut, there is no such readily available positive control for the YW152F treated animals. However, when administering YW152F during corpus luteum formation in the same animal model, there are profound differences in angiogenesis when Dll4 is blocked . This can indirectly serve as a proof of action and suggests that different types of angiogenic development and growth occur in follicular and luteal phase, indicating that circular elongation angiogenesis observed during follicular growth is quite different from sprouting angiogenesis in other tissues.
As Notch function is complex, several possibilities exist to explain our results at the molecular level.
Notch and angiogenesis
During inhibition of Notch function, through compound E or YW152F, PMSG driven VEGF production in GCs is maintained to stimulate vascular growth by activation of VEGFR-2 on endothelial cells [9, 15, 16]. Disruption of endothelial Notch1 signaling through blockage of Dll4 is not sufficient to disrupt coordination of vascular growth in a significant way. In contrast, interference with Notch1 signaling on endothelial cells, as well as Notch1 and Notch3 signaling on VSMCs in compound E treated animals disrupts critical coordination between these 2 cell types, which is necessary to form mature functional vasculature required for gonadotropin-dependent follicular growth. These observations suggest that Notch1 and Notch3 coordinate VEGF driven angiogenesis in the theca layer during gonadotropin-dependent folliculogenesis.
Effects of notch on non-angiogenic cells in the ovary
In-situ hybridization studies demonstrate that Notch2 and Notch3 are expressed on GCs . We did not detect Notch3 on GCs, but did see Notch2 expressed. Johnson et al. speculated that GCs Notch activity is necessary for proliferation and differentiation, while preventing follicular atresia due to apoptosis. In vitro models have shown that inhibition of Notch2 leads to reduction of c-Myc inhibiting granulosa proliferation. Therefore, we suggest that blockage of Notch2 through administration of compound E might have affected GCs proliferation and differentiation, which in turn could have contributed to the inhibition of follicle development. In this case, the absence of significant effects observed in YW152F treated animals would be plausible, since our immunohistochemistry stains did not demonstrate presence of Dll4 or Notch3 within the follicle and blocking this pathway may have no impact on notch signaling among granulosa cells. Thus, further experiments with specific inhibition of Notch2 and Jagged2 are needed.