Ovary and ovulation: Cell-specific localization of nitric oxide synthases (NOS) in the rat ovary during follicular development, ovulation and luteal formation

Abstract

Nitric oxide (NO) has emerged as one of several important intraovarian regulatory factors. In particular, NO has been implicated in the processes of ovulation and atresia-related apoptosis. The aim of the present study was to investigate the presence and distribution of the NO-generating nitric oxide synthase (NOS) enzymes in the ovary during follicular development, ovulation and luteal formation of the equine chorionic gonadotrophin (ECG)/human chorionic gonadotrophin (HCG)-primed rat. NADPH diaphorase activity was used as a histochemical marker for NOS within the ovary. Diaphorase reactivity was most abundant in the stroma (S) of the ovary and in the theca (T) layer of the follicle. In luteinized ovaries, weaker diaphorase reactivity was present within the corpora lutea (CL). Two different isoforms of NOS, the constitutively expressed endothelial NOS (eNOS) and the inducible isoform of NOS (iNOS), were immunolocalized in ovaries of immature rats and in ECG/HCG-primed rats during the periovulatory period from HCG injection until 2 days after ovulation. In addition, ovarian concentrations of eNOS and iNOS were quantified by immunoblotting. Immunoblotting with a monoclonal anti-eNOS antibody demonstrated the presence of eNOS mainly in the residual ovary (ROV) during the periovulatory period. In luteinized ovaries, higher concentrations of eNOS were seen in CL, while those in the ROV at this stage were lower than in the periovulatory ovary. Immature ovaries contained diminutive amounts of eNOS, detectable mostly in the ROV compartment. In contrast, iNOS was barely detectable during follicular development to the preovulatory stage. A slight elevation of iNOS was observed in the granulosa cells at 6 h after the HCG injection. The levels of iNOS during the luteal phase were also low. Immunohistochemical analysis using polyclonal eNOS and iNOS antibodies revealed the localization of these two isoforms primarily in the S and the T of the periovulatory ovary. In luteinized ovaries, positive immunoreactivity was also seen within the CL. With a monoclonal antibody against eNOS, intense immunoreactivity was observed in the S, T and within CL. There was a particularly strong staining in blood vessels. These data demonstrate the presence of an intraovarian NO-generating system. The localization of this system to the S, T and CL suggests a role for NO in the ovulatory process and in the regulation of CL function.

Expression and localization of nitric oxide synthase isoforms during porcine oocyte growth and acquisition of meiotic competence

“…The increased amount of protein in the nucleus and cytoplasm in partially meiotically competent oocytes may be related to the acquisition of meiotic competence and the requirement of nitric oxide synthesis. Our results confirmed that eNOS is a frequently expressed isoform in mammalian oocytes (Van Voorhis et al, 1994;Jablonka-Shariff and Olson, 1997;Nishikimi et al, 2001;Mitchell et al, 2004) and is also abundant in the somatic compartments of the ovary (Zackrisson et al, 1996;Jablonka-Shariff and Olson, 1997;Jablonka-Shariff et al, 1999;Nakamura et al, 1999;Yamagata et al, 2002;Mitchell et al, 2004). The expression and distribution of eNOS in porcine oocytes do not deviate from this pattern Takesue et al, 2003).…”

“…The expression of all three NOS proteins in one cell type is not surprising; it is known that the expression of several NOS isoforms occurs both in somatic cells (Mehta et al, 1995) and in mammalian oocytes (Zackrisson et al, 1996;Jablonka-Shariff and Olson, 1997;Tao et al, 1997;Jablonka-Shariff et al, 1999). The expression of NOS mRNA was also observed in the cumulus and granulosa cells which enclose the oocyte during its growth period.…”

“…However, works that concentrate on NOS isoform expression and localization contribute to its clarification. eNOS and iNOS isoforms were detected in fullygrown rat oocytes (Van Voorhis et al, 1994Zackrisson et al, 1996;Jablonka-Shariff and Olson, 1997;Jablonka-Shariff et al, 1999;Nakamura et al, 1999;Yamagata et al, 2002;Tao et al, 2004) and in mouse oocytes (Nishikimi et al, 2001;Mitchell et al, 2004). The nNOS isoform was not detected in oocytes.…”

Nitric oxide in follicle development and oocyte competence

“…In the rat, Zhang et al (2011) demonstrated cellular expression and immunolocalization of three different NOS isoforms in the ovary before puberty. In adult rats, several authors assessed the expression of eNOS in granulosa cells, thecal layer, and ovarian stroma (Zackrisson et al 1996, Jablonka-Shariff & Olson 1997, Jablonka-Shariff et al 1999, Nakamura et al 1999, while iNOS was localized only in somatic cells of follicle and luteal cells (JablonkaShariff & Olson 1997, Tao et al 1997. Even though eNOS has been considered a constitutively expressed enzyme isoform, many experimental works document that the pattern of protein and mRNA expression within the ovary is subjected to changes during follicular and luteal phases of the estrous cycle.…”

“…This transition may be due to local transformation of mesenchymal cells into endothelial cells or active migration of endothelial cell precursors from preexisting blood vessels. Apart from the effect of NO on follicular blood flow demonstrated in the rat (Griffith 1994, Zackrisson et al 2000, Mitsube et al 2002 and human (Zackrisson et al 1996), suppressive effects of NO on angiogenesis in the bovine follicle have been postulated by observing a negative relationship between VEGF and NO levels in porcine granulosa cells (Grasselli et al 2002).…”