Expression of Apelin and Apelin Receptor Protein in the Hypothalamo-Pituitary-Ovarian Axis during the Estrous Cycle of Mice

The reproductive function of mammals is driven by a neurohormonal system consisting hypothalamus, pituitary, and gonads, and this axis is known as the hypothalamic-pituitary-gonadal/ovarian axis (HPG or HPO) [1]. The physiological function of the HPO axis is to ensure the cyclic production of gonadotropin-releasing hormone (GnRH), gonadotropins, and steroid hormones from the respective place for reproductive activities [2]. The hypothalamus, a central brain region secretes GnRH, a decapeptide that controls the anterior pituitary to synthesize and secrets gonadotropin hormones, luteinizing hormone (LH), and follicle-stimulating hormone (FSH) that ultimately regulates the downstream production and secretion of ovarian steroids and oogenesis [3‒6]. However, ovarian steroids do not give direct feedback to GnRH neurons, whereas they rely on the kisspeptin system during the ovarian cycle. Kisspeptin neurons of the hypothalamus secrete kisspeptin, a decapeptide that regulates the reproductive axis by stimulating its receptor on the GnRH neurons [7]. The feedback signal from ovarian steroids for the GnRH surge and pulse is conveyed through the two populations of kisspeptin that exist in the anteroventral periventricular nucleus and arcuate (ARC) nucleus, respectively [8]. In mouse, the population of kisspeptin located in the anteroventral periventricular nucleus transduces the positive feedback to kisspeptin neurons that stimulate the occurrence of GnRH surge necessary for LH surge just before ovulation and the other population located in the ARC nucleus receive negative feedback and is more involved in tonic GnRH secretion [9].

The estrous cycle of mice has four repetitive sequential phases, namely, proestrus (Pro), estrus (Est), metestrus (Met), and diestrus (Di) and which last for 4–5 days [10]. During an estrous cycle, the functions of the hypothalamus, pituitary, and ovary are modulated in relation to their secretions of GnRH, gonadotropin, and ovarian steroids, respectively [2]. Although the GnRH-FSH/LH-E2/P4 pathways involved in HPG axis regulation are largely elaborated in mammals, the molecular and cellular events that interfere with this pathway are still unclear [11]. Previous studies suggested that several neuropeptides are involved in the modulation of this pathway through direct effect in synaptic transmission and/or act in a paracrine manner [12]. Apelin (APLN), an endogenous peptide isolated from bovine stomach tissue extracts, which has been identified in 1998 as the endogenous ligand of the human orphan Apelin receptor (APJ) [13]. Apelin receptor (APLNR, also known as APJ, APJR, AGTRL1, and HG11) is a class of G-protein coupled receptor consisting of 380 amino acids, which was discovered in 1993 [14]. Both APLN and its receptor APJ are expressed highly in the brain, heart, kidney, liver, placenta, gonads, and other reproductive organs in many species [15‒19]. The immunoreactivity of APLN has been identified in certain hypothalamic nuclei and pituitary, and it was suggested that this peptide may be a signaling molecule along the hypothalamic-hypophysial axis [20, 21].

Previous studies demonstrated the presence of APLN neurons in the supraoptic (SON) and paraventricular nuclei of the rat brain, which are involved in the central control of pituitary hormone release [22‒25]. The presence of APLN in the hypothalamic area like the supraoptic and the paraventricular nuclei suggested it as a neuropeptide factor as well [26]. The direct evidence of APLN-mediated LH and FSH release from the pituitary has been shown by Sandel et al [27] in male rats.

The APLN and APJ are also expressed in the granulose cells and oocyte and control the progesterone secretion from luteinizing granulosa cells [28]. APLN has been shown to stimulate the secretion of progesterone and estradiol in the bovine ovary [29]. Thus, the abovementioned literature established the presence of APLN and APJ in the HPO axis. There is evidence that suggests that various factors of the HPO axis change during the estrous cycle [30, 31]. The expression of the ovarian APLN system has been shown in the bovine estrous cycle [32]. To the best of our knowledge, no study has shown the expression of the APLN system in the HPO axis during the estrous cycle of mice and other mammals. Thus, the HPG axis in female mice was examined in terms of APLN and APJ expression and localization during estrous cycle phases.

In the present study, 32 virgin female Swiss Albino mice of age 3–4 months (weight 25 ± 5 g) were used from the reared colony from the laboratory animal house of Mizoram University, India. These mice were handled according to the protocol approved by the Mizoram University Institutional Animal Ethical Committee (Protocol Number, MZU/IAEC/2020/12), Mizoram University, Mizoram, India, and all animal experiments complied with the ARRIVE guidelines. All the mice were maintained under the standard laboratory conditions of 12 h light and dark, at a temperature of 25 ± 2°C, and provided water and food ad libitum.

The Apelin-13 (APLN) peptide (Gln-Arg-Pro-Leu-Ser-His-Lys-Gly-Pro-Met-Pro-Phe) used in this study was purchased from GMR Foundation, Tiruchirappalli, Tamil Nadu, India. The purity of the Apelin-13 (APLN) peptide was ≥95%.

The 4-oxo-6-((pyrimidin-2-ylthio)methyl)-4H-pyran-3-yl 4-nitrobenzoate (ML221), a potent APLN inhibitor or functional antagonist of APJ was purchased from Sigma-Aldrich Chemicals Pvt Ltd, St. Louis, MO, USA (cat# SML0919). The leuprolide acetate, a GnRH agonist used in this study was purchased from Sun.

The estrous cycle of the mice was observed according to the procedure described by Goldman et al. [33]. In brief, the vaginal smear of all the mice was taken with a dropper at around 8:00–9:30 a.m. or 5:00–6:00 p.m. and observed under the microscope. The estrous stages were identified and classified based on their vaginal cytology. The vaginal smear showed oval flat nuclear epithelial cells in the Pro phase, cornified cells in the Est phase, cornified and white blood cells in the Met, and mostly white blood cells in the Di.

The mice (n = 8/group) were divided into four groups Pro, Est, Met, and Di according to their vaginal smear observation. All the mice were sacrificed at the respective phase of the estrous cycle in the evening at around 5:00–6:00 p.m. and the hypothalamus, pituitary, and ovary samples were harvested immediately. The hypothalamus was excised following previous report by Daikoku and Shimizu [34]. Half of the pituitary samples (n = 4/group) were fixed in Bouin’s fluid for immunohistochemical study. Furthermore, other pituitary (n = 4/group) at Pro and Est are used for in vitro study. Half of the ovaries and hypothalamus (n = 4/group) were fixed in Bouin’s fluid for immunohistochemical study and the remaining were stored at −20°C for Western blotting analysis.

To explore the role of APLN in gonadotropin secretion, the pituitary (n = 4/group) at Pro and Est stages were cultured for 24 h. Briefly, pituitary were cultured in a medium of Dulbecco Modified Eagle’s and Ham’s F-12 (cat no-AL155 G, HiMedia, Mumbai, India) mixed with 100 U/mL penicillin, 100 μg/mL streptomycin, and 0.1% BSA (Sigma Aldrich, St Louis, MO, USA). The pituitary (1 pituitary per tube) was cultured in 500 μL of medium in a humidified atmosphere with 95% air and 5% CO₂ for 24 h at 37°C. The pituitary was divided into four groups: (i) control group (CON), cultured only in media, (ii) GnRH group, cultured in the presence of GnRH agonist at a dose of 100 ng/mL [35], (iii) GnRH+AP group, cultured in the presence of GnRH agonist and APLN-13 at a dose of 1 μg/mL [36], and (iv) GnRH+ML group, cultured in the presence of GnRH agonist and APLN inhibitor ML221 at a dose of 50 µm [37]. After 24 h, media was collected for ELISA.

Hypothalamus, ovaries, and pituitaries were fixed in Bouin’s fluid for 24 h and stored in 70% alcohol. Histological slides were prepared by the paraffin-embedded method described earlier by Tepekoy et al. [38]. Briefly, samples were dehydrated in alcohol grades (70%, 90%, and 100%), cleansed with xylene, and paraffin-embedded blocks were prepared. The blocks were sectioned (7 μm) with Leica rotary microtome (model RM2125 RTS). The hypothalamic sections were selected at the level of median eminence. The tissue sections were then spread in warm water placed in a poly-l-lysine coated slide and kept in a slide warming table at 37°C for overnight.

For immunohistochemistry, the slides were deparaffinized in xylene, followed by rehydrated with different alcohol grades (100%, 90%, and 70%) and then hydrated in distilled water. The endogenous peroxidase was blocked with 3% H₂O₂ and methanol solution and for blocking the nonspecific binding the slides were then treated with goat serum (1:100 with PBS) at room temperature and incubated in primary antibody (anti-APJ 1:50, cat# ABD43, Millipore and anti-apelin 1:50, cat#SAB4301741, Sigma Aldrich, USA) diluted with PBS at 4°C for overnight. PBS was used to wash off the unbound primary antibody and incubated with secondary antibody (1:400, goat anti-rabbit, cat# PI-1000, Vector Laboratories, Burlingame, CA, USA). The antibodies for apelin and apelin receptors (APJ) have been used for the localization of these proteins in mice [39]. The slides were then proceed in DAB solution containing 0.6 mg/mL solution of 3, 3-diaminobenzidine tetra hydrochloride Dihydrate (DAB) in Tris-HCl (pH 7.6) and 0.01% H₂O₂ until brown color developed and the reaction was stopped by distilled water. Counterstaining was done with hematoxylin. The stained slides were then dehydrated (70%, 90%, and 100%), and cleaned in xylene. Finally mounted with DPX and examined using a microscope (iScope series, Euromex, The Netherland). Negative control was also performed (shown in online suppl. File S1; for all online suppl. material, see https://doi.org/10.1159/000534838). The immunostaining was quantified by ImageJ using ImageJ’s threshold tool and e DAB-stained area was obtained, and the data were shown as a percentage of the staining area. Absorption control was also performed to confirm the specificity of the APLN antibody. In brief, the APLN antibody was pre-incubated with the APLN peptide (1:10 ratio) for 24 h at 4°C. This process allows the antibody to bind with the target antigen and becomes inactive. Then the pre-absorbed antibody was incubated with the tissue instead of the primary antibody following abovementioned method (shown in online suppl. File S2).

Immunoblotting was performed in the hypothalamus and ovary, as the method described earlier [40]. The hypothalamus was excised at depth to the optic chiasm and optic tract along the approximate margins from anterior to posterior (optic chiasm to infundibulum) and lateral to the optic tract [41, 42] (shown in online suppl. File S3). Protein concentration was estimated by the Bradford method [43]. An equal protein amount (50 μg) was loaded and resolved by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the resolved proteins were then transferred onto the polyvinylidene fluoride membrane using a wet apparatus. The membrane blocking was done with 5% nonfat skimmed milk (cat# GRM1254-500G; HiMedia Laboratory private limited, Mumbai) in PBST and incubated with the primary antibody, anti-APJ (1:1,000, lot# ABD43, Millipore), and anti-apelin (1:1,000, cat#SAB4301741, Sigma Aldrich, USA). PBST solution was used for washing unbound antibodies and then incubated with horseradish peroxidase-conjugated secondary antibodies (1:4,000, goat anti-rabbit, cat# PI-1000, Vector Laboratories, Burlingame, CA, USA). Again PBST was used to wash free secondary antibodies; the membranes were then incubated with electrochemiluminescence and developed onto X-ray film. To quantify the band intensities with respect to the loading control, ImageJ software (imagej.nih.gov/) was used. For loading control, the membrane was stripped and reprobed with β-Tubulin (1:1,500, cat#E7; DSHB, University of Iowa, Department of Biology, United States) and secondary antibody (1:4,000, Goat anti-mouse, cat#E-AB1001, Elabscience, Houston, TX, USA).

The media collected from the in vitro experiment were estimated for LH and FSH concentration by enzyme-linked immunosorbent assay. Commercially available mouse ELISA kit (Luteinizing Hormone, cat#E-EL-M3053; Follicle Stimulating Hormone, cat# E-EL-M0511, Elabscience, USA) was used following the instruction manual. The sensitivity of the LH and FSH are 0.94 ng/mL and 0.19 ng/mL, respectively. The coefficient of variation for both hormone kits is <10%.

Statistical analysis was performed with GraphPad Prism 9 (GraphPad Software, San Diego, CA, USA). The differences between the data were analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s test, and the data were expressed as mean ± standard error mean (SEM). p < 0.05 was considered as significant.

The expression of APLN in the hypothalamus during the estrous cycle was found to be significantly (p < 0.05) increased in the Met group as compared to other groups. There was no significant change in APLN expression in the Pro, Est, and Met groups (shown in Fig. 1A).

The expression of APJ in the hypothalamus during the estrous cycle showed a significant (p < 0.05) decrease in the Met groups as compared to other groups, whereas, in the Di group significantly (p < 0.05) increased as compared to other groups. There was no significant change in APJ expression between the Pro and Est groups (shown in Fig. 1B).

Immunostaining of APJ in the hypothalamus was observed in the median eminence during the estrous cycle (shown in Fig. 2A (a–h)). The APJ immunostaining in the median eminence showed strong staining in the Di (g, h), whereas, Met showed mild APJ immunostaining (e, f). The APJ showed moderate immunostaining in the Pro (a, b), and Est phases (c, d). The percentage (%) area of APJ staining in the hypothalamus was also measured (shown in Fig. 2B). The Met group showed significant (p < 0.05) decreased stained % area as compared to other groups. Whereas, there were no significant (p > 0.05) changes between the Pro, Est and Di groups.

Immunostaining of APLN in the pituitary gland during the estrous cycle was observed in various cell types of the anterior pituitary gland (shown in Fig. 3A (a–h)). APLN distribution was also observed in the intermediate lobe without any visible changes during the estrous cycle. The APLN immunostaining in the anterior lobe of the Est (c, d) and Di (g, h) showed intense immunostaining with abundant distribution of APLN-stained cells, whereas the Met (a, b) and Pro (e, f) group showed faint and considerably less distribution of APLN stained cells. The percentage (%) area of APLN staining in the pituitary was measured (shown in Fig. 3B). Est and Di groups showed significant (p < 0.05) increased stained % area as compared to Pro and Met. Whereas, no significant (p > 0.05) changes were observed between the Pro and Met groups. Furthermore, there was no significant (p > 0.05) change between the Est and Di groups.

Immunohistochemistry of APJ in the pituitary gland during the estrous cycle showed variation in the distribution and staining in various cell types of the anterior pituitary gland (shown in Fig. 4A (a–h)). Immunostaining of APJ showed intense staining in the anterior lobe of the Est (c, d) and Di (g, h) groups with abundant distribution of APJ stained cells, whereas Met (e, f) and Pro (a, b) group showed mild immunostaining and considerably less distribution of APJ stained cells. The percentage (%) area of APJ staining in the pituitary was measured (shown in Fig. 4B). Est and Di groups showed significant (p < 0.05) increased stained % area as compared to Pro and Met. However, the Pro group showed a significant (p > 0.05) increased % area as compared to the Met. There was no significant (p > 0.05) change observed between the Est and Di groups.

The LH concentration in the cultured media was detected as significantly (p < 0.05) higher in the only GnRH agonist-treated group (11.64 ± 0.14 ng/mL) and GnRH agonist plus APLN inhibitor (ML221)-treated groups (11.26 ± 0.65 ng/mL) compared to the control (1.07 ± 0.23 ng/mL) and GnRH plus APLN-treated groups (7.38 ± 0.78 ng/mL) whereas, GnRH plus APLN-treated group showed significant (p < 0.0001) increased than CON. However, there is no significant (p = 0.95) change between the only GnRH and GnRH plus ML221-treated groups (shown in Fig. 5A).

The FSH concentration in the cultured media was detected as significantly (p < 0.05) higher in the only GnRH treated (29.88 ± 0.34 ng/mL) and GnRH plus ML221-treated groups (36.64 ± 0.71 ng/mL) than the other groups, control (22.03 ± 2.11 ng/mL), and GnRH plus APLN-treated group (23.25 ± 2.01 ng/mL). Whereas, the GnRH plus Ml221-treated group showed a significant (p < 0.05) increase in FSH secretion than the only GnRH group. However, there are no significant (p = 0.94) changes observed between the control and GnRH plus APLN-treated groups (shown in Fig. 5B).

The LH concentration in the cultured media was detected as significantly (p < 0.05) higher in the only GnRH group (15.8 ± 1.66 ng/mL) compared to the other groups, CON (12.27 ± 0.45 ng/mL), GnRH plus APLN-treated group (13.45 ± 3.35 ng/mL), and GnRH plus ML221-treated group (15.01 ± 3.62 ng/mL). The GnRH plus APLN-treated group showed significantly (p < 0.05) decreased LH levels than other groups. However, GnRH plus ML221-treated groups showed no significant (p > 0.05) change between the control and GnRH plus APLN-treated groups (shown in Fig. 6A).

The FSH concentration in the cultured media was detected as significantly (p < 0.001) higher in the GnRH plus ML221-treated groups (29.37 ± 0.18 ng/mL) and GnRH plus APLN-treated group (26.82 ± 0.5) than the other groups, control (22.02 ± 0.54 ng/mL) and only GnRH (22.39 ± 0.27 ng/mL). Whereas, the GnRH plus APLN-treated group showed a significant (p = 0.002) decrease in FSH secretion than the GnRH plus ML221-treated group. However, there are no significant (p = 0.91) changes observed between the control and only GnRH groups (shown in Fig. 6B).

Immunostaining of APLN in the ovary during the estrous cycle was observed in all the follicles along with the oocytes, granulosa cells, and theca cells, and also in the corpus luteum (shown in Fig. 7A (a–t)). Immunohistochemistry of APLN showed intense staining in the corpus luteum and antral follicle of Pro (b, c) and Di groups (q, r), whereas moderate and mild staining in the corpus luteum and antral follicle of Met (l, m) and Est (g, h) groups, respectively. Granulosa cells, thecal cells, and oocytes of Pro (d, e), Met (n, o), and Di (s, t) showed moderate immunostaining whereas Est (i, j) showed very faint staining of APLN. The percentage (%) area of APLN staining in the ovary was measured (shown in Fig. 7B). Pro and Di groups showed significant (p < 0.05) increased stained % area as compared to Est and Met. However, there was no significant (p > 0.05) change observed between the Est and Met groups.

Immunohistochemistry of APJ in the ovary during the estrous cycle showed staining in follicles along with the oocytes, granulosa cells and theca cells, and also in the corpus luteum (shown in Fig. 8A (a–t)). Pro (b, c) and Di (q, r) showed intense immunostaining of APJ in the corpus luteum and antral follicle, whereas moderate and faint staining in the Est group (g, h) and Met (l, m) group, respectively. Pro and Di showed moderate immunostaining in the oocytes, granulosa cells, and thecal cells whereas Est showed very faint staining of APLN in these cells. The percentage (%) area of APLN staining in the ovary was measured (shown in Fig. 8B). Pro and Di groups showed significant (p < 0.05) increased stained % area as compared to Est and Met. However, there was no significant (p > 0.05) change observed between the Est and Met groups.

The expression of APJ in the ovary during the estrous cycle showed a significant (p < 0.05) decrease in the Est and Met groups as compared to other groups whereas, the Di group was found to be significantly (p < 0.05) increased as compared to other groups. Met and Est groups showed no significant (p > 0.05) change in APJ expression in the ovary (shown in Fig. 9).

The present study has investigated the expression and distribution of APLN and its receptor, APJ in the hypothalamus, pituitary, and ovary of mice during different phases of the estrous cycle. It is well known that the female reproductive process is controlled along the hypothalamo-pituitary-ovarian (HPO) axis by GnRH from the hypothalamus, FSH, and LH from pituitary and ovarian hormones [2]. APLN is an endogenous peptide, considered as neuropeptide [22] and adipokines [44] as well. It has been shown that APLN elicits biological action via binding to its receptor, APJ [45]. Our results showed that expression of APLN and APJ in the hypothalamus did not show a change from Pro to Est, however, expression of APJ was lower in Met along with elevated APLN. Expression of APJ in the hypothalamus was elevated in the Di phase along with decreased APLN expression. Our immunohistochemical study also showed mild staining of APJ in the Met, strong in the Di and moderate in the Pro and Est at the level of median eminence. The neuroendocrine system which includes the median eminence serves as a vital link between the hypothalamus and the pituitary gland [46]. A previous study has also shown that the APLN system is present in the median eminence [26]. Western blot analysis and quantification of immunohistochemistry of APJ did not show the same trend with respect to quantification in the hypothalamus. For Western blot entire hypothalamus was selected and immunohistochemistry represented only one area of the hypothalamus. This could be the reason for this discrepancy because APLN systems are also expressed in other hypothalamic areas [26]. Taken together, the expression of APLN and APJ during Met to Di could be considered as stimulated APLN signaling in these phases (postovulatory) of the estrous cycle in the hypothalamus. However, the presence of APLN and APJ during estrous also suggests its possible role in the hypothalamus. The exact role of the hypothalamic APJ system during the estrous cycle requires further investigation. Previous studies have shown that hypothalamic areas express the APJ system in the paraventricular, supraoptic, and ARC nuclei [24, 47]. It has also been shown that APLN fibers are present near GnRH neurons and also suggested that APLN could stimulate bursting and GnRH release in male rats [48]. Since we have not measured the physiological role of the hypothalamic APJ system in the female mice during the estrous cycle on GnRH secretion, thus, it would only be suggestive role of APJ on the GnRH secretion in female mice. Whether APLN stimulates or suppresses the GnRH in female mice remains unclear. This is also an important limitation of the study, which needs subsequent analysis. Moreover, the postovulatory elevated APLN system in the hypothalamus (Met and Di) might be slowing GnRH secretion. It has also been suggested that GnRH pulse frequency slows down during the postovulatory estrous/luteal phase [49].

Despite, the hypothalamus, the present study has also investigated the localization of APLN and APJ in the pituitary. Our results showed that APLN and APJ immunolocalization exhibited abundance in the anterior pituitary during the Est and Di phases of estrous. The intermediate lobe [50] also showed immunostaining of APLN and APJ during the estrous cycle, without any noticeable changes in the localization. Previous study has also shown the presence of APJ in the anterior pituitary of rats [51]. The presence of APLN and APJ was also noticed in the posterior pituitary; however, the APLN exhibited low immunostaining in the posterior pituitary. It has been shown that apelinergic nerve endings were found to be highest in the inner layer of the median eminence and the posterior pituitary. Moreover, arginine vasopressin (AVP) neurons also project toward the posterior pituitary [20, 52]. It has also been shown that intracerebroventricularly APLN infusion in rodents inhibits AVP neuron activity leading to low AVP and increased urination [26]. It has also been shown that water deprivation increases the APLN immunoreactivity in the hypothalamic nuclei [53] and inhibits its release. Our results showed a low immunoreactivity of APLN in the posterior pituitary. The exact reason for this low APLN immunoreactivity remains to be investigated in the posterior pituitary. Moreover, it could be suggested that water was given ad libitum in our experiment, thus, APLN expression could have been lower in the posterior pituitary due to decreased APLN synthesis at the hypothalamic level. A recent study has suggested that under physiological conditions, APLN, and AVP are released in balanced proportions from the magnocellular AVP neurons for water balance [54]; this could be a reason for low APLN immunoreactivity in the posterior pituitary during estrous cycle. It would have been interesting and worthy to measure the APLN release from the posterior pituitary during the estrous cycle to ascertain the reason for low APLN immunoreactivity in the posterior pituitary.

The presence of APLN and APJ in the anterior pituitary during the Est phase might be suggested to have some influence on gonadotropin secretion. Since we have not performed the co-localization of APLN systems in the gonadotrophs along with LH and FSH, thus, only a suggestive role of the APLN system in LH and FSH secretion might be proposed. The presence of an APLN system has been shown in corticotrophs and the possibility of an APLN system in the gonadotrophs has not been ruled out [21]. It is well known LH surge facilitates ovulation, thus, whether APLN could influence the LH and FSH secretion from the pituitary during Est, we have performed an in vitro study on the pituitary explants. The pituitary explants study was performed in two phases of the estrous cycle, Pro, and Est. Our results showed that the treatment of APLN 13 has suppressed that GnRH agonist stimulated FSH and LH secretion in Pro, however, treatment of APJ, antagonist, ML221 increased GnRH stimulated FSH secretion without affecting LH secretion. In the Est phase, GnRH showed a stimulatory effect on LH and no effect on FSH has been observed. It has also been shown that at the end of the follicular phase (Pro in rodents), the pituitary showed more responsiveness to GnRH [55] and LH surge is required for ovulation. Moreover, the treatment with APLN 13 again inhibited GnRH-stimulated LH secretion; surprisingly, ML221 also inhibited GnRH-stimulated LH secretion. The ML221-mediated LH inhibition needs further investigation. The secretion of FSH showed a mild increase after APLN 13 and ML221 treatment. To the best of our knowledge, our result is the first report on the direct role of APLN on LH and FSH secretion from mice pituitary. Our immunolocalization study also showed an increased abundance of APLN and APJ in the Di, thus, it may be suggested that the APLN system could be involved in the suppression of gonadotropin secretion during Di, as the next round of folliculogenesis is yet to start, however, increase abundance of APLN and APJ during Est remain unclear with respect to gonadotropin secretion. However, our in vitro results suggest that APLN might suppress the gonadotropin section from the pituitary. Previous studies have also shown that APLN inhibits LH secretion in male rats [27, 50]. The expression of the APLN system in the gonadotrophs remains unclear from the present study as well as from earlier study by Reaux-Le Goazigo et al. [21], however, the modulation of pituitary LH and FSH secretion by the APLN system during the estrous cycle of mice, prompted us to hypothesize that APLN system might be regulating gonadotropin secretion by modulating corticotrophs functions, although we do not have the supplementary results for this hypothesis. Furthermore, Reaux-Le Goazigo et al. [21] showed that APLN can stimulate basal ACTH secretion. Previous study also proposed that ACTH and cortisol might modulate the GnRH-stimulated LH secretion from pig pituitary in vitro, however, only cortisol, not ACTH showed an inhibitory effect on LH secretion [56]. Since the ACTH is derived from POMC, therefore, POMC-related or derived peptides (endogenous opiates) might be involved in APLN-mediated LH suppression. It has been shown that endogenous opiates exert an inhibitory action on LH secretion during the menstrual cycle [57]. It should be noted that there was a minor statistical change in GnRH-elicited LH secretion in the presence of APJ antagonist. Since this finding was based on our in vitro study and this could be due to a change in osmolarity, further study should be required to elicit its role in vivo conditions.

We have also examined the localization of APLN and APJ in the ovary during the estrous cycle. The intense immunostaining of APLN and APJ was observed in the corpus luteum as well as in the antral follicles of the Pro phase. Previous studies have also shown that the corpus luteum at Pro represents the regressing structure [58, 59] and the presence of APLN and APJ suggests its role in the regression of corpus luteum. The staining of APLN showed faint immunostaining in the corpus luteum and antral follicles of the Est phase, however, moderate immunostaining of APJ was observed in the corpus luteum and antral follicles of the Est phase. The presence of the APLN system in the corpus luteum and follicle suggests that APLN might have a modulatory effect on progesterone and estrogen secretion. Previous studies have also shown that APLN increases progesterone and estrogen secretion from human granulosa cells, buffalo ovarian follicles, and corpus luteum and bovine luteinizing granulosa cells [28, 60, 61]. It should also be noted that the abundance of APLN and APJ in the corpus luteum from Met to Di showed an increasing trend in the abundance of the APLN system. Based on these findings, it can be suggested that the APLN system might be involved in the luteinizing of granulosa cells and the maturation of the corpus luteum. Our result is in agreement with the previous study on the cow ovary where, involvement of the APLN-APJ system was shown in the maturation of corpus luteum [62]. The ovarian follicles also showed an increased abundance of APJ from Met to Est, which suggests that the APLN system could also be involved in folliculogenesis. Previous study has also suggested APLN system might regulate folliculogenesis and other aspect of ovarian function such as steroid hormone secretion, proliferation, or apoptosis [63]. It has also shown that the APLN system has a stimulatory role in bovine ovarian function in vivo and an inhibitory role in vitro study [28]. It should be noted that the present study has investigated only the expression of APLN and APJ at protein levels; however, it would have been worthy to analyze the expression of APLN and APJ at gene levels to strengthen the findings. This is a limitation of the present study, which requires further study.

In conclusion, this is the first report on the cyclic changes of the APLN system along the HPO axis during the estrous of mice. Our results showed that expression of the APLN system changes in the HPO axis during the estrous cycle of mice. The pattern of changes in the APLN system during the estrous cycle showed tissue-dependent variation along the HPO axis. Based on our findings and support from previous studies, it might be suggested that APLN might have an inhibitory role on the hypothalamus and pituitary and a stimulatory role on ovarian functions.

We acknowledged research facility provided to the Department of Zoology, MZU by DST-FIST, New Delhi.

The study protocol was approved by the Mizoram University Institutional Animal Ethical Committee (Protocol Number, MZU/IAEC/2020/12), Mizoram University, Mizoram, India, and all animal experiments complied with the ARRIVE guidelines.

All authors declare there is no conflict of interest.

This work has been funded by ICMR (File no: 5/10/FR/81/2020-RBMCH), New Delhi.

Borgohain Anima: conceptualization; methodology; investigation; writing – original draft; writing – review and editing; formal analysis; and data curation. Guruswami Gurusubramanian: conceptualization; methodology; investigation; writing – original draft; writing – review and editing; resources; and data curation. Vikas Kumar Roy: conceptualization; methodology; funding acquisition; supervision; writing – review and editing; writing – original draft; investigation; and data curation.

All data generated or analyzed during this study are included in this article. Further inquiries can be directed to the corresponding author.