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Background: Nitric oxide (NO) is involved in ovulation, but it is uncertain whether the mechanisms responsible are systemic or local. In vivo and in perfused ovaries, inhibition of nitric oxide synthase (NOS) reduces ovulation. This study used a well-characterized system of isolated follicle culture to assess which isoforms of NOS might be involved at the follicular level., Methods: NOS inhibitors, stimulators and NO donors were used to manipulate the environment of mouse cultured follicles, selectively targeting different isoforms of NOS. Follicle survival and ovulation in vitro were monitored using established markers., Results: Inhibition of endothelial NOS did not affect survival or ovulation of cultured follicles. Inhibition of inducible nitric oxide synthase (iNOS) had mild effects in this system, generally inhibitory of ovulation. NO donors had variable inhibitory effects, including toxic effects. Stimulators of iNOS appeared to show mixed, mostly inhibitory, effects that possibly involved different mechanisms., Conclusions: The results suggest that relatively minor effects on survival and ovulation in vitro are achieved by modulating the NO pathway, and the marked effects of NOS inhibitors in vivo do not occur in vitro. It is therefore probable that the effects of NOS inhibitors in vivo are mediated via effects outside the follicle., (Copyright 2004 European Society of Human Reproduction and Embryology)

Background: Nitric oxide (NO) is a cell messenger with multiple actions in different biological systems, implicated in the control of follicle and oocyte function. NO is formed from L-arginine by isoforms of nitric oxide synthase (NOS) via NG-hydroxy-L-arginine, with L-citrulline as a byproduct. This study aimed to show how modulation of NO by manipulating NOS substrates would affect mouse follicle growth and ovulation in vitro, where vascular effects of NO are attenuated., Methods: Immunohistochemistry [endothelial (eNOS) and inducible (iNOS)] and in situ hybridization (iNOS) were applied on mouse ovaries. Cultured follicles were also stained for iNOS by immunohistochemistry. For follicles cultured in the presence or absence of L-arginine, the ability of L-citrulline or NG-hydroxy-L-arginine to substitute for L-arginine was assessed in terms of follicle growth and ovulation., Results: iNOS and eNOS were localized in oocytes and theca, with some staining in granulosa. iNOS mRNA occurred predominantly in granulosa and oocyte. Omission of L-arginine significantly reduced follicle survival and ovulation. Partial compensation for L-arginine withdrawal was achieved with L-citrulline and NG-hydroxy-L- arginine. Specific abnormalities of follicle growth were noted., Conclusions: NOS is present in mouse follicles, and its action is necessary at a local level for normal follicle development in vitro. Reduced growth, persistent basement membranes and reduced ovulation were associated with in vitro disruption of NO.

Background: This study tested factors affecting mouse follicle growth in vitro, to determine end-points marking follicle function in vitro., Methods: Pre-antral follicles (mean 137 microm) from B6CBF1 mice were cultured in a substrate-adherent system for < or = 14 days. FSH (0-1000 mIU/ml) day of HCG (1.5 IU/ml days 9-14) protein supplement [fetal calf serum (FCS) (x2) mouse serum (x2) hypogonadal (hpg) mouse serum or human serum albumin (HSA)] were varied. Follicle survival timing of antrum formation incidence of ovulation within 16,24,40,48 h of HCG oocyte growth were assessed., Results: FSH (100 mIU/ml) produced the best antral development (P < 0.001 versus 10 and 1000 mIU/ml). Antra were observed from day 5. Transient antra formed occasionally in the absence of FSH. By 14 days significant senescence had occurred (P < 0.001) but the proportion of follicles ovulating within 16 h of HCG declined from day 9 onwards indicating this to be a more sensitive marker of follicle responsiveness. Optimal growth occurred in 5% FCS (x2) or hpg mouse serum although fewer follicles ovulated in hpg serum (P < 0.05). No normal growth occurred in normal mouse serum (x2) or HSA. Oocytes grew to full size within 9 days with 100 mIU/ml FSH FCS., Conclusions: These data provide sensitive end-points for assessing follicle growth in vitro.