Effect of neuronal nitric oxide synthase serine-1412 phosphorylation on hypothalamic–pituitary–ovarian function and leptin response

Abstract

Hypothalamic neuronal nitric oxide synthase (nNOS) potentiates adult female fertility in rodents by stimulating gonadotropin releasing hormone (GnRH) secretion, which in turn promotes luteinizing hormone (LH) release and ovulation. The mechanism of hypothalamic nNOS activation is not clear but could be via nNOS serine1412 (S1412) phosphorylation, which increases nNOS activity and physiologic NO effects in other organ systems. In female rodents, hypothalamic nNOS S1412 phosphorylation reportedly increases during proestrus or upon acute leptin exposure during diestrus. To determine if nNOS S1412 regulates female reproduction in mice, we compared the reproductive anatomy, estrous cycle duration and phase proportion, and fecundity of wild-type and nNOS serine1412➔alanine (nNOS^S1412A) knock-in female mice. We also measured hypothalamic GnRH and serum LH, follicle stimulating hormone (FSH), estradiol, and progesterone in diestrus mice after intraperitoneal leptin injection. Organ weights and histology were not different by genotype. Ovarian primordial follicles, antral follicles, and corpora lutea were similar for wild-type and nNOS^S1412A mice. Likewise, estrous cycle duration and phase length were not different, and fecundity was unremarkable. There were no differences among genotypes for LH, FSH, estradiol, or progesterone. In contrast to prior studies, our work suggests that nNOS S1412 phosphorylation is dispensable for normal hypothalamic–pituitary–ovarian function and regular estrous cycling. These findings have important implications for current models of fertility regulation by nNOS phosphorylation.

nNOS serine-1412-alanine knock-in mice demonstrate normal estrous cycles and hypothalamic–pituitary–ovarian (H–P–O) function.

Introduction

Female fertility reportedly requires nitric oxide (NO) stimulation of the hypothalamic–pituitary–ovarian (H–P–O) axis [1–4]. Pharmacologic neuronal NO synthase (nNOS) inhibition suppresses preovulatory luteinizing hormone (LH) release in estradiol (E₂)-primed ovariectomized mice [3], and partial (nNOSα KO) or complete (nNOS^exon6Δ) genetic ablation of nNOS in mice causes extended diestrus and anovulatory infertility [3, 4]. NO enhances gonadotropin releasing hormone (GnRH) secretion via the soluble guanylate cyclase (sGC)-cGMP-protein kinase G (PKG) pathway [1, 5–7]. PKG promotes presynaptic vesicle release [8], and both PKG1 and PKG2 are widely expressed in the mammalian hypothalamus [9].

Despite a clear role for NO, the mechanism underlying preovulatory nNOS activation is controversial. Stimuli for nNOS activity include Ca²⁺/calmodulin during N-methyl-D-aspartate (NMDA) receptor opening [10] and nNOS serine-1412 (S1412) phosphorylation [11, 12]. S1412 phosphorylation by Akt or PKA enhances nNOS Ca²⁺/calmodulin affinity to increase nNOS enzymatic activity. Phosphorylation of nNOS S847 by CamKII, by contrast, decreases nNOS Ca²⁺/calmodulin affinity to lower nNOS activity [11–14]. During rat proestrus, nNOS association with the NMDA receptor and nNOS S1412 phosphorylation both increase [2, 15], and knockdown of PSD-95 (mediating nNOS-NMDA receptor association) disrupts estrous cycles [2]. These correlations do not conclusively prove that nNOS S1412 phosphorylation alters hypothalamic regulation of female reproduction. In this study, our first objective was to evaluate whether nNOS S1412 phosphorylation modulates female mouse fertility.

The adipokine leptin stimulates hypothalamic NO production [16, 17] and pituitary LH release [1, 18, 19] in rodents. In leptin-deficient mice (Lep^ob/ob), pharmacologic nNOS inhibition or nNOSα knockout blocks the ability of intraperitoneal leptin injection to restore LH release [1]. These findings suggest that hypothalamic nNOS mediates leptin effects on the H–P–O axis [17]. Leptin promotes hypothalamic Stat3 phosphorylation and alters hypothalamic gene transcription in rodents 30 min after intraperitoneal administration [20, 21]. In addition to the JAK-Stat pathway, leptin receptor agonists promote PI3K-Akt activation [22]. Akt activation promotes nNOS S1412 phosphorylation [3, 23], and acute (15-min) injection of intraperitoneal leptin reportedly stimulates hypothalamic nNOS S1412 phosphorylation in adult diestrus female mice [1]. Therefore, leptin could stimulate Akt phosphorylation of hypothalamic nNOS S1412 to enhance LH release. However, it is not clear that hypothalamic GnRH secretion, LH release, or ovulation definitively requires nNOS S1412 phosphorylation in regularly cycling adult females. Leptin receptor deletion in hypothalamic GnRH or nitrergic neurons minimally affects fertility in female mice [18, 24]. Additionally, cohort studies detected weak correlation between serum leptin and LH levels in premenopausal women [25]. Therefore, our second objective was to evaluate whether nNOS S1412 phosphorylation is required for leptin regulation of the adult female hypothalamus.

We recently created a mutant knock-in mouse that cannot be phosphorylated at nNOS S1412 (nNOS^S1412A). Male nNOS^S1412A mice have no gross anatomic abnormalities, but they exhibit impaired NO-dependent relaxation of the ileum mediated by the enteric nervous system [26, 27]. However, the nNOS^S1412A mutation does not affect enteric NO signaling as severely as the nNOSα KO mutation [26]. We hypothesized that if nNOS S1412 phosphorylation is critical for hypothalamic control of fertility, nNOS^S1412A female mice will have altered ovulation and irregular estrous cycles. Likewise, we hypothesized that nNOS^S1412A mice will exhibit a decreased LH surge in response to intraperitoneal leptin. Unexpectedly, we found that nNOS^S1412A mice exhibited normal estrous cycles and follicle development, and their acute response to intraperitoneal leptin injection was indistinguishable from WT animals. These results contrast with the conclusions of prior reports on the role of hypothalamic nNOS S1412 phosphorylation.

Materials and methods

Animals

The University of Colorado School of Medicine Institutional Animal Care and Use Committee approved all experiments involving animals (Protocol #90). nNOS^S1412A mice were generated in our laboratory with assistance from the University of Colorado Anschutz Bioengineering Core. The male phenotype was reported previously and demonstrated to be deficient in NO-dependent ileal relaxation [26, 27]. For these studies, we used 3–4 month-old sibling-matched wild-type (WT) or nNOS^S1412A knock-in virgin females. Vendor-purchased C57/BL6 WT mice (Charles River Laboratories #027) were indistinguishable from nNOS^S1412A sibling-matched WT mice for all measured parameters and were used when age-matched WT siblings were unavailable. To promote regular estrous cycling, female mice were single-housed for 2 weeks in a 12-h light/dark cycle vivarium maintained at 20 °C with ad libitum food and water. For breeding trials, we single housed WT and nNOS^S1412A virgin females overnight, after which we added WT male bedding to synchronize cycles and enhance receptivity. A total of 12 h later, we individually paired WT males with females. Time to first litter was determined relative to the introduction of male mice to female cages. Offspring sex was determined 21 days postpartum at weaning.

Acute leptin administration

Methods for leptin injection/analysis are those described by Bellefontaine et al. [1] and confirmed with those authors. To determine estrous cycle length and phase proportions, we tracked estrus phases at 12 h intervals for 14 days by vaginal lavage and crystal violet cytology [28]. We assigned estrus phase from the dominant cell type in the vaginal exfoliate—i.e., leukocytes (L) for metestrus and diestrus, nucleated epithelium (N) for proestrus, and cornified epithelium (C) for estrus [28]. After 14 days, we collected vaginal swabs for immediate cytology once daily at 7:15 am to select animals in diestrus. We administered recombinant murine leptin (3 mg/kg; #450–31, PeproTech; Rocky Hill, NJ) or saline vehicle to diestrus mice between 11:00 am to 12:00 pm by intraperitoneal injection. Precisely 15 min after leptin treatment, we euthanized mice by CO₂ asphyxiation and cervical dislocation and transferred to ice. Serum was obtained via left ventricular cardiac puncture and transferred to a Serum Separator Tube (#365967, BD Biosciences; La Jolla, CA). Hypothalami were dissected immediately, flash-frozen in liquid N₂, and stored at −80 °C. As a control to assess the bioactivity of intraperitoneal leptin, we sacrificed mice 30 or 90 min after leptin or saline injection and measured hepatic Stat3 phosphorylation, as has been demonstrated for hypothalamus and adipose [29, 30].

Serum and hypothalamic hormone analysis

The University of Virginia Ligand Assay and Analysis Core determined serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), 17β-estradiol (E₂), and progesterone (P₄). LH and FSH levels were determined by radioimmunoassay; E₂ and P₄ levels were determined by ELISA. To quantify hypothalamic GnRH levels, we adapted Koch and Baram’s acidified methanol extraction procedure [31] as follows. We homogenized hypothalami at 4 °C in methanol +10% (v/v) acetic acid +5x protease inhibitors (P8340; Millipore-Sigma; St. Louis, MO) with <2.0 mm steel beads in a Bullet Blender mill (Next Advance; Troy, NY) and then centrifuged lysates at 16 000 × g for 5 min at 4 °C. Pellets were retained for immunoblotting. To evaluate GnRH, we heated supernatants to 95 °C for 5 min, dried by speed-vac, re-suspended in 4x PBS (P3813; Millipore-Sigma), and quantified GnRH (pg/ml extract) using a competitive ELISA (#LS-F13002–1, LifeSpan Biosciences; Seattle, WA) according to the manufacturer’s instructions. GnRH content was expressed relative to immunoblot level of α-tubulin for the same specimen, similar to previous reports [32].

Immunoblots

To assess hypothalamic protein expression, after acidified methanol extraction, we re-suspended the pellets in 6x Laemmli buffer [375 mM Tris-HCl, 9% (w/v) SDS, 50% (v/v) glycerol, 9% (v/v) β-mercaptoethanol, 0.03% (w/v) bromophenol blue], heated to 95 °C for 10 min, and resolved the lysates by SDS-PAGE. Proteins were transferred to Immobilon FL PVDF membranes (Millipore-Sigma) and visualized with antibodies against nNOS (1:500; Immunostar #24431), soluble guanylate cyclase β (sGCβ; 1:1000; Cayman Chemical #160897; Ann Arbor, MI), protein kinase G 1 (PKG1; 1:1000; Cell Signaling #3248S; Beverly, MA), and α-tubulin (1:5000; Abcam #ab4074; Burlingame, CA) using previously validated antibodies [26]. Quantification of nNOS, sGCβ, and PKG1 is relative to α-tubulin abundance. To validate the biological activity of intraperitoneal leptin, we measured hepatic Stat3 phosphorylation, which is induced by leptin [30]. We lysed flash-frozen liver in 100 mM Tris-HCl (pH 7.5 at 25 °C) + 150 mM KCl + 2 mM EGTA+ 2 mM EDTA+ 0.4% (v/v) Triton X-100 + 3.3% (v/v) glycerol + 1 mM dithiothreitol + 50 mM sodium fluoride + 5 mM sodium pyrophosphate + 30 mM β-glycerophosphate + 1 mM sodium orthovanadate + 2x protease inhibitors and performed immunoblots for Stat3 (1:1000; Cell Signaling #4904S) and Stat3 phosphorylated at tyrosine 705 (pStat3) (1:1000; Cell Signaling #9131S). Quantification of Stat3 phosphorylation is relative to total Stat3 abundance.

Histology

All histology was examined by a qualified mouse histopathologist (DJO) [33–36]. We dissected and cleaned the ovary, oviduct, uterus, adrenal, kidney, stomach, liver, gastrocnemius muscle, heart, and brain. Organs were weighed, fixed overnight in Bouin’s solution (#HT10132, Millipore-Sigma), and stored in 70% (v/v) ethanol. Fixed tissues were paraffin-embedded and sectioned onto glass slides for hematoxylin and eosin (H&E) staining and examination using a BH-2 microscope (Olympus; Waltham, MA) or an Olympus BX51 microscope equipped with a 4MP Macrofire digital camera (Optronics; Goshen, IN) using the PictureFrame Application 2.3 (Optronics). To assess oviducts and uteri, we qualitatively compared interstitial glands and vessels. We examined surface columnar epithelia and glands for hypertrophy, apoptosis, or dysplasia. We also examined the lamina propria for vessel defects and inflammatory infiltrate, the musculature for thickness, quantity and quality of myocytes, large vessels between the muscularis layers for quantity and quality, and the serosa for inflammatory cells. To assess ovaries, we verified that all follicle stages were present in multiple animals of each genotype. Follicle counts were conducted by a qualified ovarian biologist (JJ) [37, 38]. Briefly, paraffin-embedded ovaries were cut into 8 μm serial sections and stained with hematoxylin and picric methyl blue by AML Laboratories. We determined the number of non-atretic primordial and antral follicles and corpora lutea in every fifth section using established methods [37, 38]. Ovarian biologists (ELC, CN, JJ) were blinded to group/strain assignment. Data are reported as number of follicles per ovary per mouse.

Statistics

We analyzed data with Kruskal–Wallis and Dunn’s post tests or by Mann–Whitney tests as appropriate, using Prism (GraphPad; San Diego, CA). We selected nonparametric statistical tests due to small sample size and/or nonnormally distributed data [39]. Ovarian follicle box and whisker plots depict interquartile ranges and extrema. All other data are reported as mean ± standard error of the mean (SEM). Significance was set at P < 0.05. N refers to the number of animals per group.

Results

General characterization of female nNOS^S1412A mice

In mice, hypothalamic nNOS phosphorylation reportedly increases during proestrus or with pharmacologic stimulation of LH release by leptin [1, 15]. To evaluate the role of nNOS S1412 phosphorylation in H–P–O function, we used a knock-in mouse created by our laboratory in which nNOS phosphorylation is eliminated by mutating serine-1412 to nonphosphorylatable alanine (nNOS^S1412A) [26]. Similar to males [26], female WT and nNOS^S1412A heterozygous and homozygous mice appear healthy overall and demonstrate indistinguishable adrenal, kidney, stomach, liver, gastrocnemius, heart, and brain tissues (Supplemental Figure S1). In contrast to males [26], female nNOS^S1412A homozygote brains were 16% larger than WT, and adrenals were 26% smaller (Supplemental Table S1). However, when we controlled for body mass, normalized organ weights were not different (Table 1).

Table 1 .

Organ weights for WT siblings and nNOS^S1412A homozygotes and heterozygotes.

Genotype	Age (days)	Adrenal	Kidney	Stomach	Liver	Heart	Brain	Pituitary	Ovary and oviduct	Uterus
WT (N = 13)	145 ± 7.8	0.62 ± 0.05	14.2 ± 2.01	16.2 ± 1.55	41.7 ± 1.90	5.5 ± 0.50	0.46 ± 0.50	0.46 ± 0.05	0.54 ± 0.03	4.2 ± 0.09
nNOSS1412A Het (N = 10)	156 ± 10.7	0.48 ± 0.04	12.6 ± 0.71	18.2 ± 1.76	49.2 ± 1.37	5.5 ± 0.49	18.6 ± 0.74	0.40 ± 0.03	0.67 ± 0.06	4.1 ± 0.51
nNOSS1412A Hom. (N = 7)	167 ± 12.1	0.46 ± 0.06	11.3 ± 0.76	18.9 ± 2.80	45.1 ± 2.36	4.7 ± 0.24	18.8 ± 1.07	0.51 ± 0.09	0.51 ± 0.05	5.1 ± 1.01
P-value	0.284	0.036	0.285	0.396	*0.024	0.697	0.057	0.520	0.154	0.432
Post test P-value (WT vs Hom)	N.A.	0.066	N.A.	N.A.	0.780	N.A.	N.A.	N.A.	N.A.	N.A.

Data are expressed as mg organ per g whole animal weight. ^*: P < 0.05 by Kruskal–Wallis or Dunn’s post tests.

Female nNOS^S1412A mice exhibit normal reproductive organ histology

If nNOS S1412 phosphorylation critically regulates ovulation, nNOS^S1412A mutant mice might exhibit reproductive organ changes. However, we found no difference in ovary/oviduct weight for WT and nNOS^S1412A heterozygote or homozygote siblings (Table 1). There was also no difference between WT C57Bl/6 females recently obtained from our vendor and sibling-matched WT mice from heterozygous knock-in crosses (data not shown). Uterine weights were also indistinguishable among genotypes (Table 1). Likewise, histology of the ovary, oviduct, and uterus did not differ among WT and nNOS^S1412A heterozygous or homozygous females (Figure 1A). These findings indicate that nNOS S1412 does not influence female genital tract development. To determine whether nNOS S1412 phosphorylation influences ovarian follicle number or recruitment, we quantified primordial follicles, antral follicles, and corpora lutea in ovaries from WT and nNOS^S1412A heterozygous and homozygous mice. There was no difference among the three genotypes, with similar numbers of primordial follicles, antral follicles, and corpora lutea (Figure 1B–D). Thus, nNOS S1412 phosphorylation is not critical for normal post-pubertal follicle development and luteinization.

Figure 1.

Female nNOS^S1412A mice have anatomically normal reproductive organ histology. (A) H&E staining of ovaries, oviducts, and uteri from wildtype (WT) mice and nNOS^S1412A heterozygotes (Het) and homozygotes (Hom). All scale bars: 200 μm. Ovary. AF: antral follicle. CL: corpus luteum. PF: primary follicle. Oviduct. M: muscularis. MC: mucosa. S: serosa. Uterus. EN: endometrium. MY: myometrium. (B–D) Quantification of primordial follicles (B), antral follicles (C), and corpora lutea (D) from ovaries of WT (black), Het (green), and Hom (blue) mice. Follicles were quantified from 8 μm serial sections of hematoxylin and picric blue-stained ovaries. Box plots denote median and interquartile range. Error bars: maxima and minima. N: mice. n.s.: not significant by Kruskal–Wallis test.

nNOS^S1412A mutation does not affect murine female reproduction

nNOSα KO mice, which retain ~ 10% WT nNOS activity, exhibit irregular estrous cycles with extended diestrus [3]. Since S1412 phosphorylation promotes nNOS activity [12, 15, 26], we hypothesized that nNOS^S1412A mice would have longer estrous cycles and protracted diestrus compared with WT animals. To assess this, we tracked estrous cycles for 14 or more days from eight or more mice per genotype (Figure 2 and Supplemental Figure S2) and found similar total estrous cycle duration in sibling-matched WT and nNOS^S1412A heterozygotes or homozygotes (Figure 2). Moreover, we found no difference in the proportion of any estrous cycle stage among genotypes (Figure 2). These data indicate that estrous cycle and phase duration do not require nNOS S1412 phosphorylation.

Figure 2.

The nNOS^S1412A mutation does not influence estrous cycling. We determined estrous cycle length and stage in WT and nNOS^S1412A heterozygous (Het) and homozygous (Hom) mice by vaginal lavage and crystal violet staining. Staging was based on dominant cell types in vaginal exfoliate. Lk: leukocytes (met/diestrus). Cn: cornified epithelium (estrus). Nc: nucleated epithelium (proestrus). n.s.: not significant by Kruskal–Wallis test.

We also evaluated fecundity of nNOS^S1412A dams. When paired with WT males, nNOS^S1412A homozygous females produced litters indistinguishable from sibling-matched WT controls in time to first litter, offspring per litter, and offspring sex (Table 2). These data suggest that female fertility and pregnancy are independent of nNOS S1412 phosphorylation.

Table 2 .

Homozygous nNOS^S1412A mice exhibit normal fertility.

Female breeding measurements
Parameter	WT (n = 5)	nNOSS1412A HOM (n = 4)	P-Value
Maternal age at pairing (wk)	10.4 ± 0.36	9.6 ± 0.12	0.127
Time to first litter (d)	23.0 ± 2.03	24.5 ± 2.63	0.452
Pups/first litter	4.0 ± 0.32	5.5 ± 0.96	0.206
Overall pups/litter	5.4 ± 0.43	5.1 ± 0.86	0.756
Offspring sex (% male)	57.8 ± 11.76	58.9 ± 4.85	0.583

Female WT or nNOS^S1412A homozygous mice (9–10 weeks) were paired with WT males. Values are mean ± SEM. Non-parametric Mann–Whitney tests did not reveal significant differences.

Diestrus nNOS^S1412A mice exhibit normal circulating pituitary and ovarian hormones

Exogenous leptin reportedly stimulates hypothalamic nNOS S1412 phosphorylation and increases LH secretion in rodents acutely [1, 40]. To test if nNOS S1412 phosphorylation facilitates leptin stimulation of LH release, we replicated a previously published protocol by administering leptin to diestrus WT and nNOS^S1412A heterozygous and homozygous female mice (Figure 3A). In contrast to prior reports [1, 41, 42], leptin did not increase serum LH in WT animals relative to vehicle after 15 min (Figure 3B). Moreover, LH levels were similar among WT and nNOS^S1412A heterozygote or homozygote siblings after leptin injection (Figure 3B). Serum concentrations of FSH, P₄, and E₂ were comparable to previously reported values for diestrus rodents [43, 44] and indistinguishable among genotypes (Figure 3C–E). Intraperitoneal leptin stimulated hepatic Stat3 phosphorylation (Figure 3F–G), confirming leptin bioactivity and the efficacy of intraperitoneal delivery [20, 21]. Thus, leptin administration did not acutely influence serum levels of pituitary or ovarian hormones in diestrus mice.

Figure 3.

Acute leptin administration does not influence serum pituitary or ovarian hormone levels in WT or nNOS^S1412A heterozygous (Het) or homozygous (Hom) diestrus mice. (A) Method to measure effects of intraperitoneal leptin on hypothalamic–pituitary–ovarian (H–P–O) function in diestrus mice. D: diestrus. P: proestrus. E: estrus. M: metestrus. We tracked estrous cycle phases at 12 h intervals by crystal violet staining of vaginal exfoliate for 2 weeks. After confirmation of diestrus, we administered leptin or saline vehicle by intraperitoneal (i.p.) injection. We euthanized mice 15 min after i.p. injection and assessed H–P–O hormones. (B–E) Serum concentrations of luteinizing hormone (LH), follicle stimulating hormone (FSH), estrogen (17β-estradiol; E₂), and progesterone (P₄). (F–G) Intraperitoneal leptin induces hepatic Stat3 tyrosine 705 phosphorylation (pStat3) in WT mice. Mice were fasted overnight, injected with leptin or vehicle intraperitoneally, and euthanized after the indicated times. GAPDH: glyceraldehyde-3-phosphate dehydrogenase (loading control). N = 3 mice for each treatment. (F) Representative immunoblots. (G) Summary graph. Error bars: SEM. ^*: P < 0.05 by Mann–Whitney test. n.s.: not significant.

nNOS^S1412A mutation does not affect hypothalamic GnRH levels or NO signaling proteins

Pharmacologic NO donors induce hypothalamic release of GnRH [5]. Therefore, we hypothesized that diminished basal NO production in nNOS^S1412A mice could influence hypothalamic GnRH content. However, we found no significant difference in hypothalamic GnRH among leptin-treated diestrus WT and nNOS^S1412A heterozygous or homozygous females. We also saw no change in WT hypothalamic GnRH after leptin injection compared with vehicle controls (Figure 4A). Because NO-induced GnRH release requires classical sGC-cGMP-PKG signaling [5, 45], we wondered if the nNOS^S1412A mutation altered hypothalamic protein expression of these pathway transducers. We found no differences in hypothalamic nNOS, sGC β-subunit (sGCβ), or PKG1 protein among the three genotypes, and leptin did not alter WT NO signaling pathway protein expression either (Figure 4B and C). This suggests that hypothalamic GnRH and the sGC-cGMP-PKG pathway are intact in the knock-in mouse hypothalamus and that nNOS S1412 phosphorylation does not significantly regulate GnRH release or the NO signaling pathway. Consistent with the lack of effect on pituitary and ovarian hormones, acute leptin did not alter hypothalamic GnRH synthesis in post-pubertal WT female mice. In summary, nNOS S1412 phosphorylation is not critical for female genitourinary development, ovarian follicle development or recruitment, luteinization, estrous cycling, or female fertility.

Figure 4.

Hypothalamic GnRH and classical NO signaling protein levels do not differ among wildtype (WT) or nNOS^S1412A heterozygous (Het), or homozygous (Hom) mice. We obtained gonadotropin releasing hormone (GnRH) by methanol extraction of hypothalami. We extracted residual hypothalamic protein with Laemmli buffer. (A) GnRH is expressed as pg/ml extract per alpha tubulin (αTub) immunoblot fluorescence intensity (arbitrary units). (B–E) Acute leptin treatment and the nNOS^S1412A knock-in mutation do not affect hypothalamic nNOS, PKG1, or sGCβ protein expression, and there is no difference in expression among the three strains. (B) Immunoblots. (C–E) Summary graphs. N: mice. Error bars: SEM. n.s.: not significant by Kruskal–Wallis test. All quantitation is normalized to αTub and relative to WT + Veh.

Discussion

The major finding of this study is that nNOS S1412 phosphorylation is not required for normal murine female reproduction. Using mice with a missense mutation in nNOS S1412 (nNOS^S1412A), we found histologically normal ovaries, unremarkable estrous cycles, normal fertility, normal litter sizes, and normal hypothalamic and serum sex hormones. Thus, hypothalamic nNOS S1412 phosphorylation may be functionally coincidental with (but not required for) ovulation. This contrasts with prior reports that normal H–P–O axis function involves hypothalamic nNOS S1412 phosphorylation. The physiologic importance of nNOS S1412 phosphorylation has been demonstrated in other models, such as ileal relaxation and penile erection [12, 26, 27]. Although pharmacologic or genetic nNOS inhibition reduces ovary size and decreases corpora lutea counts in mice [1, 3, 4, 46], previously there were only correlative data on nNOS S1412 phosphorylation and estrous cycles or serum LH changes. Normal fertility and H–P–O signaling decline with drastic reduction in nNOS activity as in nNOS KO mice or upon pharmacologic NOS inhibition [1, 4, 47]. However, blockade of nNOS phosphorylation via nNOS^S1412A mutation does not attenuate nNOS activity as much as nNOSα KO mutation [26]. In contrast to knockout models, nNOS^S1412A mice exhibit apparently intact facilitation of GnRH-E₂ positive feedback prior to the LH surge and ovulation.

Our second major finding is that there was no immediate functional role for leptin stimulation of hypothalamic nNOS S1412 phosphorylation in adult female mice. Acute (30–60 min) intraperitoneal leptin exposure alters hypothalamic protein expression [20, 21]. However, we did not see LH release in WT diestrus mice after leptin injection. Additionally, we found that serum E₂, P₄, FSH, LH, and hypothalamic GnRH were similar between WT and nNOS^S1412A mice. This contrasts with a prior report that acute intraperitoneal leptin promotes hypothalamic nNOS S1412 phosphorylation and stimulates LH release in diestrus mice [1, 6]. Our study had larger N, and we used a genetic model that directly assesses the importance of nNOS S1412 phosphorylation. Variation in the quality of leptin across manufacturing lots is possible, although our observation that leptin enhanced hepatic Stat3 phosphorylation appears to confirm leptin bioactivity and delivery efficacy. Together, our data suggest that leptin does not augment H–P–O signaling acutely and that hypothalamic nNOS S1412 phosphorylation (even if leptin-induced) does not influence H–P–O function.

Our findings on adult mouse estrous cycling do not preclude developmental roles for leptin-stimulated nNOS S1412 phosphorylation in puberty onset. Leptin is an essential factor for puberty initiation in humans and rodents. Leptin increases with adiposity in juveniles and permits hypothalamic GnRH synthesis and pubertal H–P–O maturation only with sufficient growth and adequate energy stores [17, 19, 48]. We did not examine pubertal parameters in nNOS^S1412A mice. We also did not examine nNOS S1412 phosphorylation regulating hypothalamic GnRH expression during mini-puberty (the transient activation of the H–P–O axis in male and female neonates) [49]. The proposed switch of hypothalamic GnRH control by NO, from opposition following mini puberty to facilitation during mature cycling, may reflect differential phosphorylation of S1412 or the concurrent influence of other nNOS regulatory sites such as nNOS S847 [14, 50]. In addition, leptin/nNOS S1412 signals could be relevant in adults during chronic negative energy balance or low adiposity states (e.g., starvation, anorexia nervosa, or female athlete triad) [51–53]. It is also possible that the nNOS^S1412A mutation induces changes in nNOS activation and H–P–O signaling that compensate for the lack of S1412 phosphorylation. NO is a “volume transmitter” whose potency sometimes depends on the total regional activity of nNOS rather than individual synaptic connectivity between nitrergic neurons and target cells [6, 54]. Thus, upregulation of nNOS activating pathways such as leptin-mediated NMDA receptor stimulation [55] and nNOS S847 dephosphorylation [14] may still occur in nNOS^S1412A homozygotes.

In summary, blocking nNOS S1412 phosphorylation does not appear to alter estrous cycles, primordial ovarian follicle or corpora lutea counts, levels of GnRH, FSH, LH, E₂, or P₄, or female fertility. Exogenous leptin did not change serum LH levels acutely in our hands. Using nNOS^S1412A mice provides rigorous evidence that contrasts with prior pharmacologic or observational studies, and we found that nNOS S1412 phosphorylation is dispensable for neuroendocrine control of ovulation. Nitrergic modulation of the H–P–O axis may depend on unidentified factors such as NMDA receptor activation or S847 phosphorylation. Our findings do not exclude roles for nNOS S1412 in mini puberty, leptin activation of the H–P–O axis at puberty, oocyte quality, or pregnancy maintenance. Hypothalamic NO may mediate GnRH-E₂ synergy by kisspeptin and leptin in rodents [1, 3, 56], but nNOS S1412 phosphorylation is not required for H–P–O regulation of female fertility and reproduction.

Grant Support: This work was supported by a perinatology–neonatology T32 training grant (5T32HD007186-37, to DDG) and a Society for Maternal Fetal Medicine/American Association of Obstetricians and Gynecologists Foundation Scholar Award (to KJH).

Conflict of interest

The authors have no conflicts of interest.