Lisa M. Olson

Hormonal Regulation of Nitric Oxide Synthases and Their Cell-Specific Expression during Follicular Development in the Rat Ovary

Nitric oxide (NO) has emerged as a novel regulator of several ovarian events, such as ovulation, steroidogenesis, and apoptotic cell death. The NO synthases (NOS) are a family of enzymes that catalyze the oxidation of l-arginine to NO and l-citrulline. The purpose of the present study was to localize NOS isoforms in the rat ovary and to examine their hormonal regulation. We conducted immunohistochemistry and Western blot analysis using isoform-specific antibodies against brain NOS, endothelial NOS (eNOS), and inducible NOS (iNOS). Immature rats were superovulated by injecting PMSG (10 IU sc) followed by an injection of human CG (hCG; 10 IU sc) 48 h later. Ovaries were obtained from control rats (no PMSG), 24 h and 48 h after PMSG treatment and 2 h, 8 h, 12 h, 20 h or 6 days and 10 days after hCG injection (n = 3–5 rats/group). Rat ovaries were clearly devoid of brain NOS staining at any of the time points studied. In control ovaries, eNOS was detected in the theca cell layer, ovarian stroma, and on the su...

Inhibitors of Nitric Oxide Synthase Influence Oocyte Maturation in Rats

Objective: To examine the effect of inhibiting nitric oxide synthase (NOS) on the number of ovulated oocytes and on occyte meiotic maturation. Methods: Female Sprague-Dawley rats (25 days old) were superovulated with a subcutaneous injection of 10 U pregnant mares serum gonadotropin, followed 52 hours later by a subcutaneous injection of 10 U human chorionic gonadotropin (hCG). Three hours before and 3 hours after hCG injection, the rats were treated orally with the vehicle (0.5% methylcellulose) as the control or with either of two NOS inhibitors, Nω-nitro-L-arginine methyl ester (L-NAME) and L-N6-(1-iminoethyl)-lysine (L-NIL). The rats were killed 20 hours after hCG injection, and oocytes present in the oviduct were flushed, counted, and classified for stages of meiosis. In addition, ovarian occytes (12 hours post-hCG) and ovulated occytes were treated with an immunofluoresect stain for the presence of endothelial NOS (eNOS). Results: Strong positive staining for eNOS was observed in the cytoplasm of ovarian and ovulated oocytes. Control rats ovulated an average 43.0 ± 4.1 oocytes each, which was lowered with either L-NAME or L-NIL (23.8 ± 4.3 and 23.5 ± 4.0 oocytes per rat, respectively; P < .002). We observed that significantly fewer ovulated oocytes obtained from rats treated with NOS inhibitors were at metaphase II (P < .006), the normal stage of meiosis for unfertilized oocytes, and a significantly greater percentage of oocytes displayed atypical morphology as compared with control oocytes (P < .0001). Conclusion: Ovarian nitric oxide synthesis is required for maximal ovulation, and a lack of nitric oxide during the periovulatory period results in severe defects in oocyte maturation.

Effective diminution of amniotic prostaglandin production by selective inhibitors of cyclooxygenase type 2

OBJECTIVE Cyclooxygenase inhibitors are effective tocolytic agents, but significant adverse effects limit their use. We hypothesized that selective inhibitors of the isozyme cyclooxygenase 2 would effectively diminish labor-associated prostaglandin production. STUDY DESIGN We analyzed cyclooxygenase type 1 and 2 expression in amnion, chorion, decidua, and myometrium from laboring or nonlaboring women and tested the efficacy of selective cyclooxygenase 2 inhibition in diminishing prostaglandin production. RESULTS The expression of cyclooxygenase 2 in amnion from women in labor, either preterm or at term, was significantly higher than in amnion before labor. In contrast, cyclooxygenase 1 expression was unchanged by labor. The enhanced expression of amniotic cyclooxygenase 2 was associated with increased prostaglandin E(2) levels in laboring women. Amniotic prostaglandin E(2) production was effectively diminished by the selective cyclooxygenase 2 inhibitors SC-236 and NS-398 but not by the cyclooxygenase 1 inhibitor SC-560. CONCLUSION Selective inhibitors of cyclooxygenase 2 are effective in diminishing prostaglandin production in vitro and may be useful in prevention of preterm deliveries.

Gene expression of nitric oxide synthase in cultured human term placental trophoblast during in vitro differentiation

The human placental syncytiotrophoblast is derived from differentiating cytotrophoblasts and is in contact with maternal blood. This endothelial function positions the trophoblast to regulate maternal-fetal exchange and to influence circulatory dynamics through paracrine interactions in the placenta. Two isoforms of nitric oxide synthase (NOS) are expressed in placenta, and northern analysis, reverse transcription-polymerase chain reaction (RT-PCR), and immunocytochemistry were used to correlate expression of the type II, inducible NOS (iNOS) and the type III, endothelial NOS (eNOS) with state of differentiation in cultured trophoblast from term placentae. It was also tested whether cytokines known to induce NOS in other cell systems would induce iNOS in human trophoblast. The mRNA for eNOS was detected by RT-PCR, but not by Northern analysis, in cultures grown for 24 h when cytotrophoblasts were dominant. In contrast, eNOS mRNA was abundant in cultures grown for 72 h when syncytiotrophoblast was present. Immunocytochemical staining for eNOS protein showed specific fluorescence in a few cells in cultures at 24 h, but the vast majority of cells expressed eNOS at 72 h. The iNOS isoform was expressed neither basally in any trophoblast culture nor was this isoform induced in cultures exposed to interleukin-1, tumour necrosis factor-alpha, interferon-gamma and lipopolysaccharide. The in vitro pattern of trophoblast eNOS expression models the in vivo pattern of eNOS expression described for villous trophoblast. The results suggest that eNOS plays a role in human trophoblast differentiation and function.

Ovarian Nitric Oxide: A Modulator of Ovulation and Oocyte Maturation

Nitric oxide (NO) is a highly diffusable and lipophilic gas that has been identified as a major secretory product of mammalian cells (1–3). It is synthesized from arginine by nitric oxide synthase (NOS), yielding NO and citrulline (1–3). Three isoforms of NOS have been identified, each of which is encoded by a separate gene (4, 5). Two constituitive isoforms, first identified in the endothelium and brain, require calcium and calmodulin for activity and respond to stimuli by producing small quantities of NO for short periods (1, 2, 5, 6). A third inducible NOS (iNOS) is transcriptionally regulated by a number of cytokines and hormones, and results in a sustained synthesis of NO over long periods (1, 2, 4).

The murine tub (rd5) mutation is not associated with a primary axonemal defect.

Abstract Some genetic syndromes causing loss of hearing and vision, such as some forms of Usher’s syndrome, also cause reduced sperm cell motility, bronchiectasis, and other pathologies involving cilia- and flagella-bearing cells. In some Usher’s patients, ultrastructural defects of axonemes within photoreceptor ciliary bridges, nasal cilia, and sperm cell flagella have been found, indicating a primary defect of axonemal conformation. Mice homozygous for the tub (rd5) mutation exhibit progressive retinal degeneration, sensorineural hearing loss, reduced fertility, and obesity, and presently represent the only animal model with neuroepithelial degeneration of both cochlea and retina without other neurological abnormalities. They provide a good phenotypic match to human genetic sensory syndromes, particularly human sensory/obesity syndromes, such as Alstrom’s and Bardet/Biedl, although no human candidate genes have been identified. Because of their unique phenotype, tubby mice are an appropriate model in which to look for a primary axonemal defect. We studied the axonemal ultrastructure of photoreceptors and sperm cells and performed functional testing of sperm in tub/tub mice before and after the onset of obesity. Approximately 15% of photoreceptor axonemes appeared abnormal in tub/tub animals, compared to 0% in controls. Both tub homozygotes and controls exhibited approximately 10% abnormal sperm cell axonemes, and no differences in sperm cell motile function were found at any age. The modest occurrence of axonemal defects in photoreceptors of tub/tub animals is likely to be a secondary effect of retinal degeneration. We conclude that the tubby phenotype is not associated with a generalized defect of cilia- and flagella-bearing cells and that the tub mutation does not primarily affect axonemal structure.