Inhibition of delta-like ligand 4 induces luteal hypervascularization followed by functional and structural luteolysis in the primate ovary

Abstract

Using specific inhibitors established that angiogenesis in the ovarian follicle and corpus luteum is driven by vascular endothelial growth factor. Recently, it has been demonstrated that the Notch ligand, delta-like ligand 4 (Dll4) negatively regulates vascular endothelial growth factor-mediated vessel sprouting and branching. To investigate the role of Dll4 in regulation of the ovarian vasculature, we administered a neutralizing antibody to Dll4 to marmosets at the periovulatory period. The vasculature was examined on luteal d 3 or d 10: angiogenesis was determined by incorporation of bromodeoxyuridine, staining for CD31 and cell death by staining for activated caspase-3. Ovulatory progesterone rises were monitored to determine effects of treatment on luteal function and time to recover normal cycles in a separate group of animals. Additionally, animals were treated in the follicular or midluteal phase to determine effects of Dll4 inhibition on follicular development and luteal function. Controls were treated with human IgG (Fc). Corpora lutea from marmosets treated during the periovulatory period exhibited increased angiogenesis and increased vascular density on luteal d 3, but plasma progesterone was significantly suppressed. By luteal d 10, corpora lutea in treated ovaries were significantly reduced in size, with involution of luteal cells, increased cell death, and suppressed plasma progesterone concentrations. In contrast, initiation of anti-Dll4 treatment during the midluteal phase produced only a slight suppression of progesterone for the remainder of the cycle. Moreover, Dll4 inhibition had no appreciable effect on follicular development. These results show that Dll4 has a specific and critical role in the development of the normal luteal vasculature.

Fig. 1.

Marmoset ovaries stained with H&E after control Fc treatment (panel A) or inhibition of Dll4 during the early luteal phase (panel B). Each ovary exhibits a recently formed corpus luteum and measurement of luteal area (C) showed no significant effect of treatment. Dual staining for BrdU and CD31 (black nuclei, red cytoplasm, respectively) in corpora lutea from, control (panel D) and Dll4 antibody-treated animal (panel E) shows the high proportion of proliferating cells that are endothelial (green arrows) in both groups. Note the increased incidence of these cells in the corpora lutea from the treated marmoset. Histogram (F) shows a significant increase (P < 0.05) in area of BrdU staining after Dll4 treatment. Immunostaining for CD31 in control ovary (panel G), and Dll4 antibody-treated animal (panel H). Both show a well-developed microvasculature, but that in the corpora lutea of the treated marmoset is exceptionally dense, a feature that is depicted in a higher power magnification (J and K). I, The quantification of CD31 immunocytochemistry expressed as percentage area of corpus luteum stained in control (open bar) and treated (closed bar). Dll4 treatment caused a significant (P < 0.05) increase in area of CD31 staining. L, Plasma progesterone concentrations in Fc control (○) and Dll4 antibody-treated marmosets (●). Treatment started on the day of expected ovulation (d 0) and was repeated on d 2. Dll4 antibody treatment was associated with a significant suppression of progesterone by d 3 (n = 4 per group). Scale bar, 500 μm (A, B, G, and H) and 50 μm (D, E, J, and K). CL, Corpus luteum.

Fig. 2.

Marmoset ovaries on d 10 of the luteal phase stained with H&E after control Fc treatment (panel A) or inhibition of Dll4 during the early luteal phase (panel B). A normal corpus luteum is central to the control ovary whereas in the ovary of the treated animal small corpora lutea are shown (*). C, Quantification of total corpus luteum area was significantly lower in the treated group. Immunohistochemistry for BrdU in corpora lutea from control (panel D) and Dll4 antibody-treated animal (panel E). Note the moderate incidence of proliferating cells (black nuclei) that are endothelial in the control and the healthy appearance of the hormone-producing cells whereas the corpus luteum from the treated marmoset shows increased cell density as a result of involution of hormone-producing cell nuclei and cytoplasm. BrdU stained cells are evident. F, Areas of BrdU staining in both groups were not significantly different. Immunostaining for activated caspase-3 (brown) is demonstrated in control corpus luteum (panel G) and Dll4 antibody-treated animal (panel H). The control shows an isolated positive cell surrounded by healthy cells. In contrast the corpus luteum of the treated marmoset shows numerous positive cells that when quantified (panel I) show a significant increase compared with controls. CD31 staining of whole ovaries is illustrated in ovaries from control (panel J) and after inhibition of Dll4 (panel K). Note the extensive mocrovascular tree in the two corpora lutea in the control ovary whereas staining for CD31 is dense in the treated ovary but the corpora lutea (*) are regressed. N, Plasma progesterone concentrations in Fc control (○) and Dll4 antibody-treated marmosets (●). Treatment started on the day of expected ovulation (d 0) and was repeated on d 2. Dll4 antibody treatment was associated with a significant suppression of progesterone by d 3 that continued until d 10 when ovaries were collected (n = 4 and 5) for Fc and Dll4 treated, respectively. Scale bar, 500 μm (A, B, J, and K) and 50 μm (D, E, G, H, L, and M). CL, Corpus luteum; bv, blood vessel.

Fig. 3.

A, Effect of treatment with Fc control (○) or Dll4 antibody (●) on the day of expected ovulation (d 0) and d 2 on plasma progesterone concentrations. Dll4 antibody treatment was associated with a significant suppression of progesterone by d 3 after which time progesterone secretion declined to follicular phase concentrations before a rise around d 16. B, A representative progesterone profile from a marmoset treated with Dll4 antibody, illustrating failure of normal luteal progesterone rise followed by an apparent ovulation around d 15, which resulted in a short period of progesterone secretion. This was followed by a normal posttreatment luteal phase beginning on d 28. Fc controls (n = 4) and treated with Dll4 antibody (n = 3). Numbers are means ± sem.

Fig. 4.

A. Effect of treatment with Fc control (○) or Dll4 antibody (●) on d 8 and d 10 of the luteal phase on plasma progesterone concentrations. Dll4 antibody treatment was associated with a significant suppression of progesterone on d 4 and d 5 after treatment. B, A representative progesterone profile from a marmoset treated with Dll4 antibody to show luteal regression was followed by a follicular phase of normal duration and progesterone secretion associated with normal ovulation (n = 4 per group). Numbers are means ± sem.

Fig. 5.

A–C, H&E-stained marmoset ovaries showing that large antral follicles (LAF) can develop despite inhibition of Dll4. A, Ovary from a marmoset on d 10 of the luteal phase after treatment with an antibody to Dll4 on luteal d 0 and d 2. Note the presence of two large antral follicles (LAF), in marked contrast to the restricted follicular growth expected at this stage of the cycle (compare with Fig. 2A). B, Ovary from a control marmoset treated with Fc for the duration of the follicular phase. Note the emergence of three large antral follicles and a recently ovulated follicle (CL). C, Ovary after treatment with Dll4 antibody throughout the follicular phase showing emergence of large antral follicles. D–-F, High-power magnifications of the wall of a large antral follicle in respective ovaries shown in panels A–C dual stained with BrdU (black nuclei) and CD31 (red cytoplasm). Note that the development of the vasculature within the thecal wall, shown by CD31 and the proliferating endothelial cells (shown by green arrows) appears to be similar to the control despite treatment with Dll4 antibody. Proliferating cells (black nuclei) in the granulosa cell layer (g) are also evident in each LAF. Scale bar, 500 μm (A–C) and 50 μm (D–F). CL, Corpus luteum.