Expression of nitric oxide synthase and effect of substrate manipulation of the nitric oxide pathway in mouse ovarian follicles.
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.
Mitchell
Leila M
LM
Department of Biological Sciences, University of Warwick, Coventry CV4 7AL, UK.
Kennedy
C Richard
CR
Hartshorne
Geraldine M
GM
D000818:Animals; D001120:Arginine / Q000008:administration & dosage / Q000031:analogs & derivatives* / Q000494:pharmacology*; D002956:Citrulline / Q000008:administration & dosage / Q000494:pharmacology*; D003470:Culture Media / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D005260:Female; D066298:In Vitro Techniques; D051379:Mice; D008815:Mice, Inbred Strains; D009569:Nitric Oxide / Q000378:metabolism*; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D006080:Ovarian Follicle / Q000187:drug effects / Q000378:metabolism*; D010060:Ovulation / Q000187:drug effects; D012333:RNA, Messenger / Q000378:metabolism; D014022:Tissue Survival; D014168:Transferrin / Q000494:pharmacology
D003470:Culture Media; D012333:RNA, Messenger; D014168:Transferrin; D002956:Citrulline; D009569:Nitric Oxide; C068309:N(omega)-hydroxyarginine; D001120:Arginine; D019001:Nitric Oxide Synthase; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse