Involvement of nitric oxide and the ovarian blood follicle barrier in murine follicular cyst development  Rashmi V Nemade, Ph.D., Odile Carrette, Ph.D., William J Larsen, Ph.D., Edith Markoff, Ph.D.  Fertility and Sterility  Volume 78, Issue 6, Pages 1301-1308 (December 2002) DOI: 10.1016/S0015-0282(02)04340-6

FIGURE 1 Effect of chronic hCG-only, hCG + L-NAME, and hCG + D-NAME treatment on ovarian morphology. Immature pregnant mare serum gonadotropin (PMSG)-primed mice were either sham-operated or implanted with osmotic pumps containing either hCG or two pumps containing hCG and L-NAME or hCG and D-NAME. (A), Section of an ovary from a sham-operated mouse at day 7 after surgery (original magnification, ×40) in which multiple layers of granulosa cells are present. (B), A section of an ovary from an hCG-treated mouse at day 7 after surgery (original magnification, ×40) in which significantly fewer granulosa cells (arrows) are present compared to ovary described in A. (C), Section of an ovary from an hCG + D-NAME mouse at day 7 after surgery (original magnification, ×40), which is similar to A. (D), Section of an ovary from an hCG + L-NAME mouse at day 7 after surgery (original magnification, ×40) in which more granulosa cells (arrows) are present compared to ovary described in B. F = normal preovulatory follicle; CF = cystic follicle. Nemade. NO in murine ovarian follicular cysts.Fertil Steril 2002. Fertility and Sterility 2002 78, 1301-1308DOI: (10.1016/S0015-0282(02)04340-6)

FIGURE 2 Ovulation patterns in hCG-only, hCG + L-NAME, and hCG + D-NAME-treated mice on indicated days. (A), Cumulus oocyte complexes were collected from sham-operated and hCG-treated superovulated mice from days 2 through 7. Sham-operated animals (n = 15 for each day) are represented by open bars and hCG-treated mice are represented by solid bars (n = 30 for each day). Data represent the mean ± confidence interval; P<.05. *Statistically significant difference from the previous day within the same treatment group. **Statistically significant difference between treatment groups on a given day. (B), Cumulus oocyte complexes were collected from superovulated mice on days 2, 3, and 6. Open bars indicate sham-operated control animals (n = 15). Solid bars represent animals treated with hCG-only (n = 30). Vertical bars show data for animals treated with hCG + L-NAME (n = 15). Horizontal bars show data for animals treated with hCG + D-NAME (n = 10). Data represents the mean ± confidence interval.*P<.01 statistically different from all other groups within a given time point. **P<.05 statistically different from sham-operated mice. ***P<.05 statistically different from sham-operated mice and hCG-only-treated mice. Nemade. NO in murine ovarian follicular cysts.Fertil Steril 2002. Fertility and Sterility 2002 78, 1301-1308DOI: (10.1016/S0015-0282(02)04340-6)

FIGURE 3 Immunolocalization of inter-α-inhibitor (IαI) in follicles of mice treated with hCG-only, hCG + L-NAME, and hCG + D-NAME. (A), Ovarian section from a sham-operated control mouse obtained 48 hours after pregnant mare serum gonadotropin (PMSG) injection. Mice show IαI present outside normal preovulatory follicles. (B), A positive control section from an ovary section obtained 48 hours after PMSG and 6 hours after hCG localizes IαI inside normal preovulatory follicles and around granulosa cells. Chronic hCG-treated mice were treated with an ovulatory dose of hCG on indicated days, euthanized 6 hours later, and ovaries were collected for IαI analysis (C–F). (C), Section from an ovary with 3 days of chronic hCG treatment shows IαI inside follicles and around granulosa cells. (D), Section from an ovary with 4 days of chronic hCG treatment shows no signal in a cystic follicle (arrow). (E), Section from an ovary with 6 days of chronic hCG treatment shows no signal in a cystic follicle (arrow), but shows IαI inside a normal follicle (arrow). (F), Section from an ovary with 7 days of chronic hCG treatment also shows no signal in a cystic follicle (arrow), but shows IαI inside a normal follicle (arrow). (G), Sodium nitroprusside was injected into 7-day hCG-treated mice and euthanized 6 hours later. Inter-α-inhibitor is present inside cystic follicles. (H), An ovary section from a mouse treated with hCG + L-NAME before an ovulatory surge shows mild to no IαI signal inside follicles. (I), An ovary section from a mouse treated with hCG + L-NAME after an ovulatory surge shows IαI inside follicles. F = normal follicle; CF = cystic follicle. Bar = 200 μm. Nemade. NO in murine ovarian follicular cysts.Fertil Steril 2002. Fertility and Sterility 2002 78, 1301-1308DOI: (10.1016/S0015-0282(02)04340-6)