L-arginine is a versatile amino acid, serving as a precursor for many molecules including NO and polyamines . The plasma concentration of L-arginine has been reported to be around 200 μmol/L in humans during the fed state [4, 41]. Therefore, we sought to determine the effect of L-arginine on endometrial RL95-2 cells at physiological (200 μmol/L) and supraphysiological (800 μmol/L) concentrations. The presence of NOS and/or arginase enzymes in the endometrium of many species indicates the ability of the endometrium to catabolize L-arginine [20, 23–28]. In females, NO is produced in the endometrium  and is involved in embryo implantation and development [43–45]. Polyamines are also produced by the endometrium [46, 47] and have been shown to be important for embryo implantation, as inhibition of polyamine synthesis reduced pregnancy rates in mice .
L-arginine has been reported to be present in the uterine flushes of sheep , cows , rats , and humans , with concentrations in human uterine flushes ranging from 220 μmol/L to 330 μmol/L depending upon the phase of the menstrual cycle . Additional work has revealed that mRNA of the L-arginine transporters SLC7A1, SLC7A2, and SLC7A3 are present in ovine uterine luminal epithelial . Furthermore, the positive influence that L-argnine has on cell signaling, proliferation, hypertrophy, hyperplasia, and migration of ovine trophectoderm cells [9, 17] suggests that L-arginine is transported into the uterine lumen to support growth and development of the peri-implantation embryo.
In addition to supporting the peri-implantation embryo, L-arginine may also have a direct effect on the uterine luminal epithelium. Proliferation of the endometrium has been implicated as a vital process which provides an optimal environment for embryo adhesion and implantation , and this argument is further supported by the observation that increasing endometrial thickness is associated with improved implantation rates in humans [53–55]. Interestingly, the uterine lumen concentration of L-arginine is greatest during the proliferative phase of the menstrual cycle , suggesting that L-arginine may have a role in the proliferation of the endometrial epithelium which must regenerate following menstruation. L-arginine and its metabolites, NO and polyamines, have a dual role in cell proliferation and apoptosis. In some cell types, L-arginine, NO, and polyamines stimulate cell proliferation and reduce apoptosis [8, 9, 56, 57], yet they inhibit cell proliferation and promote apoptosis in others [58–61]. Results from the current study indicate that L-argrinine enhances endometrial RL95-2 cell proliferation at physiological and supraphysiological concentrations. Moreover, Nor-NOHA, an arginase inhibitor, and 7-NI, an NOS inhibitor, reduced the positive effect that L-arginine had on endometrial RL95-2 cell proliferation. Conversion of L-arginine to ornithine, via arginase, is the first enzymatic process involved in polyamine synthesis . Likewise, NOS is responsible for converting L-arginine to NO . Together, the inhibitory effect that Nor-NOHA and 7-NI exhibited in the presence of L-arginine indicates that L-arginine enhances endometrial RL95-2 cell proliferation through polyamine and NO mediated pathways, which both have a positive influence on cell proliferation [5–11].
Cell proliferation is often inversely related to apoptosis [39, 62–64], and a reduction in apoptosis is a contributing factor in the enhancement of cell proliferation . Therefore, we hypothesized that the enhancement of cell proliferation in the presence of L-arginine would be associated with decreased endometrial RL95-2 cell apoptosis. Apoptosis in the endometrium is a key feature of the human menstrual cycle and aids in maintaining endometrial homeostasis by eliminating cells from the functionalis layer during the late secretory phase . In the functionalis layer of the endometrium, apoptosis exhibits a cyclic pattern with the least amount being observed during the proliferative phase followed by an increase during the secretory phase and the maximum being observed during menstruation [67, 68]. The exposure of endometrial RL95-2 cells to physiological and supraphysiological concentrations of L-arginine reduced the proportion of cells that exhibited DNA fragmentation as assessed by TUNEL assay. Activation of endonucleases  and the subsequent DNA fragmentation  are considered to be hallmark characteristics of cells undergoing apoptosis. To this end, the current results demonstrate that the presence of L-arginine reduces the proportion of endometrial RL95-2 cells experiencing apoptosis. Apoptosis can occur through either receptor-ligand mediated pathways or mitochondrial mediated pathways, with both resulting in DNA fragmentation . Receptor-ligand mediated apoptosis requires an external signal, while mitochondrial mediated apoptosis occurs through the disruption of the mitochondrial membrane . As the presence or absence of L-arginine would represent an intracellular event rather than receptor mediated extracellular signaling, we hypothesized that L-arginine’s prevention of apoptosis in endometrial RL95-2 cells is mediated through the mitochondria. The presence of L-arginine in the culture media increased the ratio of cells with a healthy mitochondrial membrane compared to cells with an altered mitochondrial membrane potential. Thus, the current study indicates that L-arginine reduces the incidence of endometrial RL95-2 cell apoptosis by preventing the disruption of mitochondrial membrane potential, suggesting a role for L-arginine in the regulation of endometrial epithelial apoptosis.
Mitochondrial membrane potential is highly influenced by proteins that belong to the BCL2 family . The pro-apoptotic protein BAX and the anti-apoptotic protein BCL2 are often studied together as indicators of apoptosis. In healthy cells, a balance exists in which BCL2 is normally found imbedded in the mitochondrial membrane . Under apoptotic conditions, activated BAX will embed in the mitochondrial membrane with BCL2 and disrupt the mitochondrial membrane potential . Accordingly, we examined if L-arginine’s prevention of apoptosis is through a BCL2 and BAX mediated event. Interestingly, the presence of L-arginine did not increase the ratio of BCL2 to BAX in endometrial RL95-2 cells. In fact, the BCL2 to BAX mRNA and protein ratios were higher in endometrial RL95-2 cells not exposed to L-arginine which were undergoing apoptosis through a mitochondrial mediated pathway. Despite the anti-apoptotic properties of BCL2, upregulation of BCL2 mRNA and protein has been reported in cells undergoing apoptosis . Moreover, increased expression of BCL2 protein can lead to disruption of mitochondrial membrane potential , as caspases can cleave BCL2 into a BAX-like molecule which can serve as a latent pro-apoptotic stimuli in apoptotic cells .
Because exposure to L-arginine did not increase the ratio of BCL2 to BAX, we hypothesized that L-arginine might decrease endometrial RL95-2 cell apoptosis through an alternative mechanism. In addition to BCL2 and BAX, BAD is another member of the BCL2 family of proteins that affects mitochondrial membrane potential. The presence of L-arginine in the culture media did not affect the levels of total BAD. However, L-arginine increased p-BAD (Ser-136) levels in endometrial RL95-2 cells and increased the ratio of p-BAD to BAD, indicating that L-arginine enhances the phosphorylation of BAD protein at serine residue 136 in endometrial RL95-2 cells. When BAD is phosphorylated at either serine residue 112 (Ser-112) or 136 (Ser-136), it is bound by 14-3-3 and sequestered in the cytosol . In contrast, non-phosphorylated BAD interacts with BCL2 and BCL-XL embedded in the mitochondrial membrane and inhibits their anti-apoptotic properties [77, 78] and causes release of cytochrome C . In this regard, L-arginine reduces mitochondrial membrane disruption and, thus, apoptosis through phosphorylation of BAD in endometrial RL95-2 cells. BAD protein is phosphorylated at serine residue 136 through the kinase activity of PI3K-dependent Akt-1 . L-arginine increases phosphorylation, and therefore the activity, of Akt-1 in ovine trophectoderm cells . Moreover, NO can stimulate phosphorylation of Akt-1 [14–16], and Akt-1 phosphorylation is also enhanced in cells with elevated expression of ornithine decarboxylase , the enzyme responsible for converting ornithine to first polyamine putrescine. Thus, it is likely that the presence of L-arginine in the culture media increased p-BAD levels in endometrial RL95-2 cells by influencing Akt-1 phosphorylation through the action of polyamines and/or NO.