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REPRODUCTION-DEVELOPMENT |
Cardiovascular Research Center and Reproductive Endocrine Unit (S.L.), Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02129
Address all correspondence and requests for reprints to: Paul L. Huang, M.D., Ph.D., Cardiovascular Research Center, Massachusetts General Hospital, 149 Thirteenth Street, Charlestown, Massachusetts 02129-2060. E-mail: . huangp{at}helix.mgh.harvard.edu
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Abstract |
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-nNOS splice variants. We generated a new line of nNOS knockout mice (KN2) lacking exon 6, which codes for the heme-binding domain of nNOS. KN2 mice are viable, but mated homozygotes do not produce litters, indicating that either one or both sexes are infertile. Male KN2 mice show decreased gonad weights, but sperm counts are normal. KN2 males do not display mating behavior, and consequently do not leave vaginal plugs when housed with wild-type (WT) females. KN2 females show decreased ovary weight, and histology reveals decreased corpus luteum counts. RIAs show that KN2 males have decreased plasma FSH, whereas KN2 females have increased levels of plasma LH and increased hypothalamic GnRH content. Experimental ovarian transplantation suggests that central, rather than ovarian, processes are influenced by nNOS, as KN2 ovaries ovulate at near-normal rates under WT hormonal control, whereas WT ovaries transplanted into KN2 mice have decreased ovulation rates. We observed pyloric stenosis in KN2 mice, but plasma leptin levels are normal, and no ketones are found, indicating that hypogonadism is not a result of malnutrition. We conclude that nNOS is required for normal central hormonal regulation of reproductive function. |
Introduction |
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NOS mutant mice have been useful tools in dissecting the roles of individual NOS isoforms (19). Mice deficient in endothelial NOS (eNOS) show a prolonged estrous cycle, a reduced ovulation rate, and elevated estradiol levels (20, 21). eNOS mutant mice also produce smaller litters (22, 23). In contrast, inducible NOS mutant mice display no deficiency in estrous cycle length or ovulation rate (20). Mutant mice lacking exon 2 of the nNOS gene (KN1) show no overt deficiency in reproductive function (24). However, expression of alternatively spliced RNA forms, nNOSß and nNOS, is still detected in KN1 mice, resulting in residual NOS activity (24, 25). To produce mice fully deficient in nNOS and to circumvent the problem of alternative splice variants, we deleted exon 6 of the nNOS gene in embryonic stem (ES) cells. Exon 6 codes for the catalytic heme-binding domain of nNOS (26). Thus, even if truncated nNOS mRNAs and proteins were expressed, they would be functionally inactive. We report here that complete ablation of nNOS function in mice results in hypogonadism and infertility, which are due to sexually dimorphic alterations in hormone secretion in the hypothalamo-pituitary axis.
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Materials and Methods |
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nNOS RT-PCR
RNA was extracted from wild-type (WT) and KN2 brains using Ultraspec (Biotecx Laboratories, Houston, TX). RT was performed using random hexamers and SuperScript II reverse transcriptase (Life Technologies, Inc., Grand Island, NY). Intron-spanning primers were targeted to nNOS exon 2 (AGC TGT CGA TCT GTC TCG CC), exon 3 (GTG CAG TTT GCC GTC GAG GT), exon 5 (ATC ATG CTG CCA TCC CAT CA), and exon 6 (GGA CCA CTG GAT CCT GCC CA). Amplification of nNOS cDNA with exon 2 and exon 3 results in a 340-bp PCR product, whereas amplification with exon 5 and exon 6 primers results in a 264-bp PCR product.
[3H]Citrulline assay
NOS catalytic activity was assayed by measuring the conversion of [3H]arginine to [3H]citrulline (28). After centrifuging brain homogenates at 20,000 x g for 20 min, tissue supernatant (25 µl) is added to 100 µl assay buffer [50 mM Tris-HCl (pH 7.4), 1 mM NADPH, 1 mM EDTA, 1 mM EGTA, 2.25 mM CaCl2, and 0.1 µCi [3H]arginine] and incubated for 15 min at room temperature. The reaction is terminated by adding 1 ml stop buffer [20 mM HEPES (pH 5.5), 1 mM EDTA, and 1 mM EGTA] and applied to 1-ml Dowex AG50WX-8 columns. [3H]Citrulline is quantitated in the eluate by scintillation counting.
Hormone RIA
LH and FSH levels in plasma samples were determined by RIA using assay materials provided by the NIDDK. The standard used in the LH assay was LH RP-3. The sensitivity of the LH RIA was 20 pg/tube, and the intraassay coefficient of variation was less than 6%. The interassay coefficient of variation at 0.18 ng/tube was 9%. For FSH RIA, the FSH RP-2 standard preparation was used, and the intra- and interassay coefficients of variation were 5.3% and 4.1%, respectively.
Histological analysis and nNOS immunohistochemistry
For hematoxylin-eosin staining, 10-µm-thick fresh-frozen sections were fixed in 80% ethanol for 1 min, rinsed in H2O, submerged in Harris’ hematoxylin (Fisher Scientific, Fairlawn, NJ) for 3 min, rinsed in 1% acid alcohol, and stained with eosin (VWR Scientific, West Chester, PA) for 2 min. For nNOS immunohistochemistry, sections were fixed in 4% paraformaldehyde and washed in PBS (pH 7.4). Sections were blocked with 1.5% horse serum and incubated with a rabbit polyclonal anti-nNOS antibody (Zymed Laboratories, Inc., San Francisco, CA; 1:400 dilution) for 16 h at 4 C. This polyclonal antibody was raised against the N-terminal 195 amino acids, which includes, but is not limited to, the heme-binding domain. The antibody shows cross-reactivity with mouse nNOS, and as a polyclonal antibody, it would be expected to react with intact nNOS as well as nNOS lacking exon 6. Incubation with an antirabbit secondary antibody and avidin-biotin complex was performed using the Vectastain Elite ABC kit (Vector Laboratories, Inc., Burlingame, CA). nNOS immunoreactivity was visualized using 3,3-diaminobenzidine and H2O2 (Sigma).
Ovarian transplantation
Under deep isoflurane anesthesia, ovaries were removed from newborn (5- to 7-d-old) female mice and placed in sterile L15 medium (Life Technologies, Inc.) containing 1% penicillin/streptomycin at room temperature. Recipient mice (2–4 months old) were anesthetized with Avertin (2%, 22 ml/kg), and from a dorsal midline incision one kidney was exposed. Using sterile microtweezers a small hole was torn on the kidney capsule, and one newborn ovary was placed between the kidney tissue and its capsule. Simultaneously, an ovariectomy was performed on the recipient mouse. This procedure was repeated for the other kidney through the same incision. The muscle and skin were sutured, and mice were observed regularly throughout convalescence for signs of pain and discomfort. Analgesics (butorphanol, 1.5 mg/kg) were administered as required. Eight weeks later, recipient mice were killed by pentobarbital overdose (0.1 g/kg), and kidneys with attached ovarian tissue were removed for histological analysis. All procedures were performed using sterile instruments and aseptic techniques.
Morphometric analysis of follicular development
Primordial follicles were defined as having a small oocyte with a single layer of squamous granulosa cells. Preantral follicles had multiple layers of GCs, but no antrum; antral follicles were distinguished by the presence of an antrum within the granulosa cell layers enclosing the oocyte. Follicles were determined to be atretic by evidence of disintegration of the granulosa cell layer.
Statistical analysis
Hormone levels and body and organ weights were compared using ANOVA and Fisher’s protected least significant difference test. Follicle and corpus luteum (CL) counts were compared with Mann-Whitney U test. All data are expressed in the text and figures as the mean ± SD.
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Results |
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.) We used RT-PCR to assess residual nNOS mRNA expression after deletion of exon 6 and to compare mRNA expression between exon 2 (KN1) (24) and exon 6 (KN2) mutant mice. Intron-spanning primers were used to detect the presence of exon 2 (primers located on exons 2 and 3) and exon 6 (primers on exons 5 and 6; Fig. 2A) on the nNOS mRNA. RT-PCR performed on brain extracts revealed that both KN1 and KN2 mice express residual nNOS mRNAs, with exon 2 missing in KN1 and exon 6 missing in KN2 (Fig. 2B). Analysis of NOS enzymatic activity by [3H]arginine to [3H]citrulline conversion showed that NOS activity in KN2 brain extracts is only 0.3% that in WT brain extracts (WT, 9.1 x 105 cpm/mg protein; KN2, 2.9 x 103 cpm/mg protein), indicating that even in the presence of residual truncated mRNA, NOS activity is nearly completely disrupted when the heme-binding domain is absent. To further demonstrate the absence of nNOS, immunostaining was performed using a polyclonal nNOS antibody raised against the N-terminal 195 amino acids. Intense nNOS staining was found in cells of the anterior hypothalamus of WT mice, with characteristic presence of immunoreactivity in the cytoplasm, neurites, and varicosities of these neurites (Fig. 3). This staining pattern was absent in KN2 mice. Similarly, no nNOS immunostaining was found in the cortex and striatum of KN2 mice (data not shown).
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Homozygous KN2 mice were infertile when mated to each other. To determine whether infertility was due to male or female abnormality, adult KN2 males were housed with WT females, and adult KN2 females were housed with WT females. During the 3-wk observation period, each morning females were examined for the presence of vaginal plug indicative of successful copulation. In addition, mating pairs were visually observed for 15 min immediately after setting up mating pairs. KN2 males (n = 6) did not mount WT females within this first 15-min observation period. Moreover, no vaginal plugs were found in WT females mated with KN2 males during the 3-wk observation period. Consequently, no pregnancies occurred among the KN2 male-WT female mating pairs. When KN2 females were mated with WT males, two pregnancies were observed of six mating pairs (33%) within the 3-wk mating period, compared with WT x WT mating pairs, where five pregnancies occurred in six mating pairs (83%).
Gonad weighs of adult mice (>10 wk for males, >6 wk for females) were markedly lower in KN2 males and females compared with WT mice [WT testis, 88 ± 10 mg; KN2 testis, 60 ± 20 mg (P < 0.0001); WT ovary, 4.1 ± 0.9 mg; KN2 ovary, 1.7 ± 0.6 mg (P < 0.0001); Fig. 4]. Examination of histological sections of WT and KN2 ovaries revealed a marked decrease in the number of CL (WT, 16.1 ± 13.4 CL/ovary; KN2, 6.3 ± 5.2 CL/ovary; P < 0.05). The number of follicles at different developmental stages is shown in Table 1. Mean values for developing, preantral, and antral follicles were all lower in KN2 ovaries compared with WT ovaries, although these differences did not reach statistical significance. In male mice, on the other hand, sperm counts obtained by squeezing the caudal epididymis indicated normal spermatogenesis in KN2 mice (WT, 2.9 x 106 spermatozoa/epididymis; KN2, 3.5 x 106 spermatozoa/epididymis).
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). There were no differences in plasma LH (WT, 0.10 ± 0.01 ng/ml; KN2, 0.12 ± 0.01 ng/ml) and testosterone levels between male WT and KN2 mice. Conversely, in female KN2 mice, the hypothalamic GnRH content was higher (WT, 4.82 ± 3.74 ng/ml; KN2, 11.13 ± 2.71 ng/ml; P < 0.05), and plasma LH was also higher in KN2s compared with WT mice (WT, 0.45 ± 0.21 ng/ml; KN2, 1.69 ± 0.94 ng/ml; P < 0.05; Fig. 6).
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As both brain and ovaries express nNOS, and gonadotroph levels are higher, rather than lower, in KN2 females, the question arises as to whether the failure to ovulate in KN2 females is due primarily to an ovarian defect or to a central hormonal effect. To address this question, ovaries of newborn KN2 mice were transplanted under the kidney capsule of ovariectomized adult WT female mice, and newborn WT ovaries were transplanted under the kidney capsule of ovariectomized KN2 mice. In each group, 6 recipient animals received a total of 12 transplanted ovaries. Eight weeks later, the recipient mice were killed, and the transplanted tissue underwent histological analysis. Five WT recipient mice and 3 KO recipient mice had viable ovarian transplants, as determined by the presence of multiple developing follicles in the transplanted ovaries. The ovulation rate was estimated by counting CLs through the whole ovary. Transplantation resulted in the reversal of ovarian phenotypes; KN2 ovaries started ovulating at near-normal rates under WT hormone control (7.75 ± 2.39 CL/ovary), whereas WT ovaries turned near quiescent in KN2 mice (0.67 ± 0.67 CL/ovary; P < 0.05), demonstrating the importance of nNOS in the central hormonal regulation of ovulation (Fig. 7).
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Discussion |
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Exon 6 codes for a critical part of the oxidative domain of nNOS, which is responsible for the catalysis of L-arginine to NO and L-citrulline (31). The first line of nNOS knockout mice (KN1) lacks exon 2 and shows 5% residual NOS activity in the brain due to the presence of nNOSß and nNOS RNA splice variants (25). We hypothesized that this residual activity might mediate additional functions for nNOS, which cannot be discovered using KN1 mice. Our gene expression studies in KN1 mice using RT-PCR show the presence of nNOS transcript(s) lacking exon 2, confirming reports of the presence of nNOSß and nNOS splice variants in KN1 mice. KN2 mice also appear to express a truncated form of nNOS mRNA, but without exon 6, as demonstrated by RT-PCR. Even if a truncated protein is translated from this mRNA, it would not be expected to be catalytically active. Indeed, [3H]citrulline measurements show that although NOS activity in KN1 mice is 5% of that in WT mice, this value in KN2 is only 0.3%. It also must be considered that if these truncated mRNAs are indeed translated into a truncated protein, they might interfere with the functions of other proteins, such as eNOS, in a dominant negative fashion. However, the observation that heterozygote KN2s show a normal phenotype suggests that either this truncated mRNA is not translated, or its expression does not alter other proteins’ function. Furthermore, immunohistochemical studies with a polyclonal antibody fail to detect nNOS epitopes in KN2 mice.
KN2 mice show a sexually dimorphic phenotype. Both male and female KN2 mice have lower gonad weight; however, spermatozoa development appears normal in the testis, whereas in the ovary, a severe decrease in the number of CLs is observed, indicating a defect in ovulation. The hormonal status is also markedly different between male and female KN2 mice. Plasma FSH is decreased in KN2 males. FSH is a main regulator of testicular development; thus, the low FSH level is a likely cause of the decreased testicular weight. In female KN2, however, plasma FSH levels are higher than normal, and plasma LH is more than 3 times higher than that in WT animals. As LH is responsible for the induction of ovulation, abnormally high levels might cause impaired ovulation in KN2 females. One possibility is that abnormally high circulating LH levels down-regulate LH receptors in the ovary. Alternatively, abnormally elevated LH levels might be already at a ceiling that cannot be significantly elevated by rising estrogen levels, which normally trigger the preovulatory spike of LH. Finally, it is possible that the normal pulsatile pattern of LH secretion is lost in KN2 mice. NO might directly modulate the function of gonadotrophs of the anterior pituitary, as nNOS protein and mRNA can be detected in gonadotroph and folliculostellate cells of the rat anterior pituitary (7). Indeed, experimental evidence suggests an inhibitory role for NO on gonadotrophs in the male rat, as inhibiting NOS in dispersed pituitary cells increases GnRH-stimulated LH release (7). Similarly, nitroglycerine taken sublingually by healthy men attenuates GnRH-induced LH secretion (8). On the other hand, sodium nitroprusside was found not to alter basal or GnRH-induced LH release from freshly isolated hemipituitaries (13). In males, our LH measurements in WT and KN2 mice do not support a significant role for NO in LH secretion. In female KN2 mice, however, LH secretion is greatly increased, suggesting an inhibitory role for NO in LH secretion. Whether this effect is the result of a direct interaction in the pituitary or is secondary to altered hypothalamic function is not known. In support of the pituitary effect is the observation that a sharp increase in nNOS immunoreactivity in gonadotrophs can be seen in proestrus in response to the GnRH surge in female rats (32). In vitro studies, on the other hand, lend support to NO’s influence on the hypothalamus. In the hypothalamic cell line GT1-7, both the glutamate analog, N-methyl-D-aspartate, and the NO donor, sodium nitroprusside, repress GnRH mRNA, as measured with Northern blot, and the inhibitory effect of N-methyl-D-aspartate is eliminated when it is coincubated with the NOS inhibitor N
-nitro-L-arginine, indicating that the inhibitory effect of glutamate on GnRH gene expression is mediated by NO (33, 34). Our finding that GnRH levels are significantly higher in the hypothalamus of female mice in the absence of nNOS supports this theory. If NO indeed inhibits GnRH synthesis, it is quite plausible that increased GnRH levels are responsible for the elevated LH levels, which, in turn, cause the impaired ovulation in KN2 mice.
As nNOS is also expressed in the gonads, we considered the possibility that a local gonadal defect is responsible for the infertility, and the increased hormone levels are a result of central hormonal compensation. However, the ovarian transplant experiment indicates that KN2 ovaries are capable of normal rates of ovulation, and conversely, ovulation by WT ovaries is suppressed in the KN2 hormonal environment. A plausible explanation for this is that a central nNOS mediated regulatory mechanism for ovulation exists. In support of this theory is the presence of a dense nNOS-immunoreactive neuronal network in the anterior hypothalamus, the altered GnRH and LH levels in the female KN2 mice, and the increase in ovulation rate after transplantation into WT mice. An alternative explanation must also be considered: that alterations not directly related to reproductive functions might prevent ovaries from thriving in KN2 mice. Lower body weight, pyloric stenosis, and higher mortality are all indicative of impaired general health, and malnutrition can cause ovulation to pause. However, normal leptin levels in KN2 mice suggest that malnutrition is unlikely to be the cause of the ovulation defect. In addition, GnRH and LH levels are not low, but high, in KN2 mice, indicating a regulatory failure rather than a general illness. We cannot, however, exclude the possibility that nonneuroendocrine alterations due to the absence of nNOS in peripheral tissues are the cause of the ovulatory defect. Further studies using tissue-specific nNOS KO mice might help elucidate this question.
Our observations of the lack of mating behavior in male KN2 mice point out the importance of brain nNOS in sexual behavior. It has been shown that intracerebroventricular injection of the NOS inhibitor N-monomethyl-L-arginine prevents progesterone-facilitated sexual receptive behavior (lordosis) in ovariectomized, estrogen-primed female rats, whereas injection of the NO donor sodium nitroprusside induces lordosis even in the absence of progesterone stimulation (18). We found that in the male mouse, nNOS-derived NO is required for normal sexual behavior. The hormonal findings in male KN2 mice support a stimulatory role for NO in regulating FSH secretion and consequent stimulation of testicular development. The low FSH secretion corresponds well with earlier observations showing that NO is important in regulating the release of GnRH at the median eminence (11, 12). Our attempts at measuring GnRH release from isolated mouse hypothalamus were not successful.
KN2 mice suffer from pyloric stenosis, similar to KN1 mice. To improve the nutritional status of KN2 mice, all experimental animals were kept on a liquid rodent diet ad libitum. To exclude the possibility that hypogonadism is a result of malnutrition, plasma ketones and plasma leptin levels were monitored. No evidence of ketones was found in KN2 plasma, indicating the absence of lipid catabolism. Leptin is released by adipocytes, and it is thought to be a permissive signal for puberty in the hypothalamus (35). Leptin levels were similar in WT and KN2 mice, indicating that KN2 mice accumulate sufficient amounts of fat tissue to produce normal levels of leptin.
In conclusion, these data show that NO generated by nNOS is necessary for normal reproductive function in the mouse. In the male, NO is required for normal mating behavior, FSH secretion, and testicular development. In the female, NO facilitates ovulation by a mechanism that involves regulation of LH secretion. Whether NO regulates FSH and LH release directly or by modulating GnRH production is the subject of ongoing investigations.
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Acknowledgments |
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Footnotes |
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Abbreviations: CL, Corpus luteum; eNOS, endothelial nitric oxide synthase; ES, embryonic stem; hsv-tk, herpes simplex virus thymidine kinase; neor, bacterial neomycin resistance; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; WT, wild-type.
Received November 26, 2001.
Accepted for publication April 2, 2002.
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