Figure 1.
Genotyping, ovarian morphology and weight.
Genotyping was performed as above using two reactions to distinguish between heterozygotes and Edn2KO mice. Lanes 1, 2, and 3 show Edn2+/?, Edn2KO, and WT mice that were genotyped from a single litter, respectively. Ovaries were collected from 23 to 31 day-old Edn2KO (n = 4) and age-matched Edn2+/? mice (n = 3) at the time of euthanasia 24 hours after superovulation. Note that Edn2KO ovaries and uteri were smaller than heterozygote siblings, and Edn2KO ovaries were also lighter when ovarian masses from mice aged 23–31 days were averaged (wet weight, p = 0.030). When average ovarian weight was normalized to average body weight, no significant difference remained between groups (p = 0.318).
Table 1.
Table 1. Ovarian function of heterozygous Edn2+/? mice and WT mice.
Figure 2.
Ovarian histology of Edn2KO mice and heterozygous littermates.
Ovaries collected from 5 to 7 week old Edn2KO mice and Edn2+/? littermates (n = 5 for each genotype) were serially sectioned and stained with hematoxylin and eosin. All ovaries contain follicles of various stages, but no CL were present in the Edn2KO ovaries. Representative images are displayed; the two Edn2KO ovaries at right display prominent congested blood vessels. CL: corpus luteum; AF: antral follicle; GC: granulosa cells; IT: interstitium; PF: primary follicles; TH: theca layer; RBC: red blood cells.
Figure 3.
Vascularization and distribution of atretic follicles in the Edn2KO ovary.
25 day-old Edn2KO mice and heterozygous littermates were induced for superovulation. Ovaries were collected 16 hours after hCG injection and serially sectioned and stained with hematoxylin and eosin, or underwent immunohistochemical staining for CD31 (PECAM-1; an endothelial membrane marker) or Caspase 3 (Casp-3; an apoptotic cell marker). Dark brown membranal immunolabeling by anti-CD31 antibody presented multifocal staining in the medullary interstitium and the theca layer in both genotypes. Additional staining was present within the CL of Edn2+/? ovaries. Rare and multifocal anti-Casp-3 antibody immunolabeling was equally present in the cytoplasm of secondary and antral follicles of each genotype. There was no difference in endothelial cell or atretic staining observed between genotypes. Arrows indicate CD31 and Casp-3 staining; CL: corpus luteum; GC: granulosa cells.
Table 2.
Numbers of oocytes retrieved at hCG 16 hours.
Table 3.
Numbers of oocytes retrieved at hCG 24 hours.
Figure 4.
Lack of CL formation in the Edn2KO ovary following superovulation induction.
Adjacent ovarian sections of the Edn2KO mice and heterozygous littermates collected at hCG24 were stained with αSMA antibody and P450scc antibody. Edn2+/? ovaries display αSMA and P450scc staining within CL, as well as in the interstitium around clearly demarcated follicles. In the Edn2KO ovaries, CL are absent and multiple large Graafian follicles are instead present in the periphery of the ovaries. Some, but not all, contain an oocyte with a partial or complete cumulus layer. Histology was similar between all Edn2KO ovaries regardless of number of oocytes ovulated. CL: corpus lutea.
Figure 5.
Serum progesterone concentrations.
Blood samples were collected by cardiac puncture at 16h and 24h after hCG injection, and serum progesterone concentrations were measured by RIA. The numbers of mice (n) used for each group and the time points of assays are indicated. The progesterone concentrations in the Edn2KO mice that ovulated multiple oocytes at hCG 24 h were significantly lower than heterozygous littermates (p = 0.046), but similar to the Edn2KO mice that did not ovulate normally (p = 0.355). The mean serum progesterone concentrations were lower in Edn2KO mice than littermates when the concentrations were compared at hCG 16 h. Error bars (SEM) extend the range of recorded values for columns in which n = 2. For * and **, p = 0.011 and 0.046, respectively; for hCG 16h, p = 0.064.
Figure 6.
Ovulation and CL formation in Edn2KO mice after EDN2 peptide injection.
Oocytes and ovaries were collected at hCG 16h from Edn2KO mice and control littermates that were induced for superovulation and were given intraovarian injections of EDN2 peptide, PBS, or no injection at hCG12. Ovarian sections were stained with H&E, or against αSMA or P450scc. Note that P450scc staining is present in the CL of Edn2+/? ovaries and in the granulosa cells of larger follicles of Edn2KO ovaries that had received EDN2 treatment, but not in vehicle-treated Edn2KO ovaries. The number of oocytes ovulated by each ovary is displayed in the corresponding oocyte image in the lower right corner. Histology images are taken at 4X; inset images of P450staining are at 40X.
Table 4.
Numbers of oocytes retrieved after EDN2 injection.
Figure 7.
CD31 expression in Edn2KO and Edn2+/? ovaries after EDN2, vehicle, or no injection.
Frozen sections were made from Edn2+/? or Edn2KO mouse ovaries collected at hCG16. Mice received injection with EDN2 peptide, PBS, or no injection at hCG12. Ovaries were stained with CD31 (PECAM-1) to visualize the vascular network. Note that positive staining was seen inside the CL of heterozygote ovaries, but was not present inside any demarcated structures of Edn2KO ovaries treated with EDN2 or PBS. Arrows indicate vascularization within representative CL of ovaries treated with PBS or no injection.
Figure 8.
Inhibition of endothelin receptors prevents CL formation and progesterone synthesis.
WT mice were induced for superovulation and received vehicle or tezosentan injections every 2 hours from hCG12 to hCG22. Mice (n = 4) were sacrificed at hCG24 hours. Tezosentan-treated mice ovulated significantly fewer oocytes (p = 0.038) and had lower serum progesterone levels (p = 0.043). Frozen ovarian sections were stained for CD31 and αSMA. Note that CL from PBS-injected mice showed clear CD31 staining in the CL, but CD31 staining was limited to the theca layers of tezosentan-injected ovaries. Little αSMA staining was seen in the CL of PBS treated mice or within tezosentan treated mouse ovaries. Arrows indicate CL in PBS-treated ovaries.