Neurotensin: a neuropeptide induced by hCG in the human and rat ovary during the periovulatory period

Abstract

Neurotensin (NTS) is a tridecapeptide that was first characterized as a neurotransmitter in neuronal cells. The present study examined ovarian NTS expression across the periovulatory period in the human and the rat. Women were recruited into this study and monitored by transvaginal ultrasound. The dominant follicle was surgically excised prior to the luteinizing hormone (LH) surge (preovulatory phase) or women were given 250 μg human chorionic gonadotropin (hCG) and dominant follicles collected 12–18 h after hCG (early ovulatory), 18–34 h (late ovulatory), and 44–70 h (postovulatory). NTS mRNA was massively induced during the early and late ovulatory stage in granulosa cells (GCs) (15 000 fold) and theca cells (700 fold). In the rat, hCG also induced Nts mRNA expression in intact ovaries and isolated GCs. In cultured granulosa-luteal cells (GLCs) from IVF patients, NTS expression was induced 6 h after hCG treatment, whereas in cultured rat GCs, NTS increased 4 h after hCG treatment. Cells treated with hCG signaling pathway inhibitors revealed that NTS expression is partially regulated in the human and rat GC by the epidermal-like growth factor pathway. Human GLC, and rat GCs also showed that Nts was regulated by the protein kinase A (PKA) pathway along with input from the phosphotidylinositol 3- kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways. The predominat NTS receptor present in human and rat GCs was SORT1, whereas NTSR1 and NTSR2 expression was very low. Based on NTS actions in other systems, we speculate that NTS may regulate crucial aspects of ovulation such as vascular permeability, inflammation, and cell migration.

Introduction

Neurotensin (NTS) is a small, 13 amino acid neuropeptide that was originally identified in the bovine hypothalamus in the mid-1970s during the purification of substance P [1]. NTS has been extensively studied in the central nervous system, mainly in the dopaminergic and cholinergic systems, where it has been shown to be involved in the regulation of luteinizing hormone (LH) and prolactin release [2–4]. In addition, NTS has been reported in the brain to be involved in appetite control, endocrine functions, pain modulation, and pathogenesis of mental disorders [5]. However, since its discovery in the central nervous system, NTS has been found throughout the body including the cardiovascular system, the gastrointestinal tract, the endocrine system, and the reproductive system [6–8]. For the endocrine system, NTS regulates hormone secretion leading to an increase in adrenocorticotropic hormone (ACTH), LH, follicle stimulating hormone (FSH), and prolactin secretion [1]. Thus, NTS has a variety of functions in numerous tissues.

NTS conveys its function through three well-characterized receptors: NTSR1, NTSR2, and SORT1 (also known as sortillin or NTSR3). NTSR1 and NTSR2 are classical G protein-coupled, seven transmembrane spanning domain receptors that differ in their affinity for NTS. NTS has a high affinity for NTSR1 and a lower affinity for NTSR2 [9]. Unlike NTSR1 and NTSR2, SORT1 is a single transmembrane receptor [10, 11]. The action of NTS is dependent on the specific receptor to which it binds, the abundance of each receptor, the specific tissue location of the NTS receptor, and the interaction between the NTS receptors [12–15].

There are a limited number of studies investigating the role of NTS in the female reproductive system. In the neuroendocrine axis, NTS levels are increased in the preoptic region of the brain in the presence of estrogen in the rat [16]. In addition, when NTS antiserum is injected into rats, the magnitude of the LH surge decreases, whereas the timing of the LH surge is unchanged, suggesting that NTS in the brain may play a role in the LH surge [17]. Similarly, Ferris et al. [18] showed that NTS has a major role in the regulation of LH release in the rat. However, Lemko et al. [2] have reported that NTS does not have a role in the release of LH from the brain in the mouse suggesting that species differences may exist in the actions of NTS in modulating LH release.

In contrast to the extensive neuroendocrine examination of NTS, there are limited reports of neurotensin in the ovary. A peptidomic analysis of the ascidian, Ciona intestinalis, revealed a neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was able to downregulate the growth of vitellogenic oocytes [19]. These findings suggest that neurotensin-like peptides in the ascidian act not only as neuropeptides in neural tissue but also as hormones in nonneuronal tissues [19]. In concordance with these studies regulating oocyte growth in the ascidian, NTS mRNA was observed in mouse cumulus cells [20]. During their studies exploring neurotensin action on sperm capacitation and the acrosome reaction in mice, Hiradate et al. [20] observed NTS mRNA levels in cumulus cells were markedly higher in hCG-induced ovulated COCs compared with immature COCs. In the human, Wissing et al. [21] noted that NTS mRNA expression increased 15.7 fold in granulosa cells (GCs) after the administration of hCG. Thus, because of the scarcity of data regarding the expression and role that NTS plays in the ovary, we aimed to investigate the expression and regulation of NTS in the ovary throughout the periovulatory period in the human and rat.

Materials and methods

Cells, media, and reagents

Unless otherwise specified, all chemicals and reagents were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Culture media, Superscript Reverse transcriptase, Oligo-dT, dNTPs, and RNaseOUT were purchased from Invitrogen Life Technologies (Carlsbad, CA).

Human tissue collection: in vivo ovulatory follicles

Human ovarian granulosa and theca cells were collected from the dominant follicle of patients across the periovulatory period as previously described [22–24]. Collections were performed at the Division of Gynaecology and Reproductive Medicine with the approval of the Human Ethics Committee of the Sahlgrenska Academy at the University of Gothenburg. Healthy 30–38-year old women with previous proven fertility and regular menstrual cycles who were undergoing laparoscopic tubal sterilization were recruited and all women provided informed written consent before enrollment. The women had not been on any form of hormonal contraceptives for at least 3 months prior to their enrollment. All women were monitored by repeated transvaginal ultrasound during one to three menstrual cycles before surgery to ascertain cycle regularity and normal increase in the size of the dominant follicle during the follicular phase. During the actual cycle of the laparoscopy, transvaginal ultrasound was performed every 1–2 days from cycle day 9 until surgery to ascertain that the dominant follicle followed the normal increase in follicle diameter of about 2 mm/day [23, 24]. By these examinations, the surgery was scheduled at the precise stage of the menstrual cycle when the dominant follicle had a diameter of more than 14 mm, which is considered as a size of full LH responsiveness. Detailed description of patient characteristics can be found in Al-Alem et al. [25]. Surgery was performed for retrieval of preovulatory follicles at four predetermined periovulatory phases to cover the period from before the LH surge until after follicular rupture. In the preovulatory group, follicles were collected prior to the endogenous LH surge (patients did not receive hCG). The three remaining groups received hCG (250 ug recombinant hCG, Ovitrelle; Merck Serono), and the follicles were dissected between 12 and ≤ 18 h post hCG (early ovulatory group or EO), between 18 and ≤ 34 h (late ovulatory group or LO), and between > 44 and ≤ 70 h (postovulatory group or post). The dominant follicle was excised using laparoscopic scissors and diathermy was not used. Once excised, the follicles were either fixed and then processed for immunostaining or were bisected and the inside of the follicle was scraped to collect the GCs and the theca layer was then separated. Granulosa and theca cell samples were snap frozen at −80°C for subsequent mRNA expression analysis described below.

Human tissue collection: IVF granulosa-luteal cells

Because of the fact that collecting timed in vivo human samples is extremely difficult and time consuming, we aimed to utilize a human granulosa-luteal cell culture model to examine NTS expression and regulation as previously described [25]. Granulosa-luteal cells (GLCs) were collected from women undergoing IVF at the Bluegrass Fertility Center, Lexington, KY as approved by the Institutional Review Board of the University of Kentucky Office of Research Integrity. These women were administered recombinant hFSH to induce ovarian hyperstimulation. After 9–11 days, patients were given 10 000 IU of hCG and the follicles were aspirated 36 h post hCG. Oocytes were removed and the cumulus cells and GLCs were collected. Cumulus cells were snap frozen. GLCs were subjected to a percoll gradient to remove the red blood cells and the remaining GLCs were cultured for 6–7 days as described and validated previously [25]. Samples were collected throughout the 7 days and NTS expression was determined. After 6–7 days, GLCs were serum starved for 1 h and then treated with or without 1 IU of hCG for 6, 12 or 24 h. Alternatively, GLCs were treated with AG1478 (1 μM), for 1 h and then with or without 1 IU hCG.

Rat tissue collection

All animal procedures for these experiments were approved by the University of Kentucky IACUC. Immature female rats (Harlan Sprague–Dawley Inc., Indianapolis, IN) were injected with pregnant mare serum gonadotropin (PMSG, 10 IU s.c.) between 0900 and 1000 h on the morning of day 24–25 of age to stimulate follicle growth. Forty-eight hours after PMSG injection, animals received 5 IU of hCG to induce ovulation. Rats were killed at varying time points before and after hCG treatment (0, 4, 8, 12, and 24 h) for tissue collection as follows. Whole intact ovaries were isolated and either: used intact, or used to isolate GC for culture experiments. Intact ovaries were used for quantification of mRNA as described previously [26]. To collect COCs and GC, ovaries were collected at 12 h after hCG and punctured with a 26G needle to release the GC and COCs. COCs were visually identified and isolated. The remaining GC were partially purified by filtration through a 40-micron pore size nylon filter to remove tissue debris, pelleted by centrifugation at 300xg, snap-frozen, and stored at −80°C for later analysis.

For the in vitro studies, GCs were isolated from intact ovaries collected from PMSG 48 h primed rats. Briefly, GCs were isolated by follicular puncture, pooled, filtered, pelleted by centrifugation, and resuspended in Opti-MEM medium supplemented with 0.05 mg/ml of gentamycin and 1x ITS (insulin, transferin, and selenium). Cells were plated and then treated with or without 1 IU hCG, culture at 37 °C in a humidified atmosphere of 5% CO₂ and collected at the time of culture and after 4, 8, 12, or 24 h. To determine the pathways regulating NTS expression, GC were pretreated with one of the following signaling pathway inhibitors for an hour: the epidermal-like growth factor (EGF) receptor inhibitor (AG1478;1 μM), progesterone receptor antagonist (RU486; 1 μM), prostaglandin (PG) synthase 2 inhibitor (NS398; 1 μM) or the PG synthase 1 and 2 inhibitor (Indomethacin 1 μM (IND)) [27]. Alternatively, rat GC were incubated for 1 h with the following signaling inhibitors: H89 (PKA inhibitor, 10 μM), GF (GF109203x; protein kinase C (PKC) inhibitor 1 μM), LY (LY294002; PI3K inhibitor 25 μM), U0 (U0126; MEK1/2 inhibitor, 10 μM), PD (PD90859, MEK1/2 inhibitor, 10 μM), or SB (SB203580; p38 MAPK inhibitor 20 μM). GCs were then treated with or without I IU of hCG for 4 h (rat) or 6 h (human) after which the cells were collected and mRNA was isolated as described below. This approach of using signal pathway inhibitors to begin to unravel LH/hCG downstream signaling mechanisms has been widely used to understand key regulatory events in the ovulatory process [28–33]. However, this approach also has limitations as some of the signaling inhibitors cross-react with other pathways thereby limiting definitive identification of all of the specific LH/hCG downstream regulators. For example, it should be noted that RU486 also acts as a glucocorticoid receptor antagonist, thus it also possesses antiglucocorticoid activities [34]. We have employed this approach as a first step to begin to understand NTS regulation.

RNA isolation and mRNA analysis

To examine the mRNA expression of NTS, total RNA was isolated from in vivo or in vitro human or rat samples using an RNeasy kit from Qiagen (Valencia, CA) as per the manufacturer’s protocol. The mRNA was reverse transcribed, and the expression was subsequently analyzed by real-time PCR using 20XTaqMan Gene Expression Assay primers from Applied Biosystems (Foster City, CA) for hNTS (Hs00175048_m1), rNTS (Rn01503265_m1), hSORT1 (Hs00361760_m1), rSORT1 (Rn01521847_m1), hNTSR1 (Hs00901551_m1), rNTSR1 (Rn01415846_m1), hNTSR2 (Hs00892563_m1), and rNTSR2 (Rn00575514_m1). The thermal cycling steps: 2 min at 50°C to permit AmpErase uracil-N-glycosylase optimal activity, a denaturation step for 10 min at 95°C, 15 s at 95°C and 1 min at 60°C for 50 cycles, followed by 1 min at 95°C, 30 sec at 58°C and 30 s at 95°C for ramp dissociation. The relative amount of mRNA in each sample was calculated following the 2-ΔΔCT method and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in human samples and to Rpl32 in rat samples.

Immunostaining

Human follicles were fixed in 4% neutral buffered formalin, embedded in paraffin, sectioned (7 μm), and processed for immunostaining as previously described [35]. The sections were incubated in a humidified chamber at 4°C overnight with NTS primary antibody (Immunostar 20072, Hudson, WI) at a concentration of 1:2000 dilution. Biotinylated Trekkie Rabbit Link (Biocare, Concord, CA) was used. The immunostaining was visualized using a rabbit Vectastain ABC kit (Vector laboratories, Burlingame, CA). Sections were counterstained with hematoxylin. Negative control slides were prepared in an identical manner and processed without a primary antibody. Staining was completely eliminated by pretreatment with 10 μg of neurotensin per 1 mL of diluted antibody (per the manufacturer, Immunostar 20072).

Statistical analysis

Data are presented as means ± standard error of mean. The Students t-test, one or two-way analysis of variance (ANOVA) was used to test differences among treatments as appropriate. Sample values that were acquired for the human experiments were log transformed because of the variability between patients, and ANOVA was used post transformation. The Tukey post hoc test was performed in order to identify significant differences among treatments. Means were compared with p ≤ 0.05 considered significant. Statistical analysis was performed using GraphPad Prizm (GraphPad 5.00, La Jolla, CA).

Results

Expression of NTS mRNA increases in human granulosa and theca cells collected in vivo across the periovulatory period

Expression of NTS mRNA was examined in follicles collected across the periovulatory period. Administration of hCG resulted in a 10 000 fold increase in the GC expression of NTS mRNA between the preovulatory and early ovulatory periods and NTS mRNA levels remained elevated (up to a 15 000-fold increase) during the late ovulatory period (Figure 1A). In theca cells, hCG-induced NTS mRNA expression during the early ovulatory period reaching a maximum of approximately 800 fold at the late ovulatory period (Figure 1B). Immunohistochemistry was performed to determine if the NTS protein expression mimicked the mRNA expression profile. The localization of NTS in sections of the dominant follicle at timed intervals during the periovulatory period showed that NTS protein expression was extremely low prior to hCG and increased in the granulosa and theca layer by the late ovulatory period (Figure 1C, D, and E).

Figure 1.

Neurotensin (NTS) mRNA expression across the ovulatory period in the human. Data are presented as the fold change relative to the preovulatory (Pre, no hCG) time point. NTS mRNA increases in the early and late ovulatory phase in the human granulosa (A) and theca (B) cells. Results are the means ± standard error of mean of n = 4–5. A one-way analysis of variance was used to test differences across the time points for the granulosa and theca cells. The Tukey post hoc test was performed and bars that do not share a letter designation are significantly different (p < 0.05). Immunostaining of NTS protein expression across the different stages of the human ovulatory cycle demonstrates low expression of NTS prior to hCG (C). Expression is present in the granulosa cells (gc) during the early (D) and late ovulatory periods (E). an = antrum, st = stroma.

NTS mRNA expression is elevated in human cumulus cells

Interestingly, when levels of NTS mRNA were determined in granulosa-luteal cells at the time of IVF collection and compared with cumulus cells from the same patient collected at the same time, NTS mRNA expression was approximately 200 fold higher in cumulus cells compared with their respective GLC (Figure 2A and B).

Figure 2.

Neurotensin (NTS) mRNA expression in cumulus cells of matched GLC patients. NTS expression is higher in cumulus cells when normalized to GLC from matched patients collected at the time of oocyte retrieval as analyzed by the Student t-test (A). When taken collectively, there is a > 200 fold increased NTS expression compared with GLC at the time of collection (B).

NTS mRNA expression increases in human granulosa-luteal cells in vitro

In order to explore the expression of NTS mRNA in vitro, we used a model where human GLCs are collected after IVF and cultured for 6–7 days during which period they regain their responsiveness to hCG. This model has been previously used to examine the hCG regulation of known ovulatory genes such as AREG, PTGS2, PGR, and others [25, 36–38]. The expression of NTS mRNA at the time of IVF collection (36 h after hCG) was arbitrarily set at 1. NTS expression decreased across the days in culture reaching the lowest levels after 4 days of culture (Figure 3A). At 6 or 7 days after culture, cells were treated with or without hCG for 0, 6, 12, or 24 h, and NTS expression was determined temporally. NTS expression increased approximately 250 fold after 6 h of hCG treatment, reached a maximum of approximately 450-fold change, and remained elevated through 24 h of treatment (Figure 3B).

Figure 3.

Neurotensin (NTS) mRNA expression in human GLC in vitro. Human GLCs were collected at the day of IVF retrieval (day 0) and NTS expression was examined across the 7 days of culture (A). After 6–7 days of culture, GLCs were serum starved for 1 h and then treated with vehicle control (−) or hCG (+) for 24 h (B). “Coll” represents samples at the time of IVF collection. A total of 0 h represents samples after 6–7 days in culture at the initiation of hCG treatment. Results are the means ± standard error of mean of n = 3. A two-way analysis of variance was used to test differences with or without hCG treatment across the time points. The Tukey post hoc test was performed and bars that do not share a letter designation are significantly different (p < 0.05).

Administration of hCG stimulates Nts mRNA expression in the rat ovary

In order to determine whether induction of Nts mRNA expression occurred in the rodent, we examined the expression of Nts mRNA in both intact ovaries across the periovulatory period and in cultured GCs. For the intact ovary studies, the expression of Nts was measured in whole ovaries after hCG administration. The mRNA expression of Nts increased approximately 6 fold at 12 h after hCG and remained elevated after ovulation at the 24 h time point (Figure 4A).

Figure 4.

Nts mRNA expression across the ovulatory period in the rat. Nts expression in intact ovaries increases during the late ovulatory period prior to follicular rupture (12 h) and remains elevated (A). Nts mRNA expression in isolated rat granulosa cells in vitro is stimulated by 4 h after hCG and declines to control levels by 24 h (A). Results are the means ± standard error of mean of n = 4. A one-way analysis of variance (ANOVA) was used to test differences across the time points in intact ovaries. A two-way ANOVA was used to test differences across the time points with or without hCG treatment in isolated granulosa cells. The Tukey post hoc test was performed and bars that do not share a letter designation are significantly different (p < 0.05).

In cultured rat GCs, GCs were treated with or without 1 IU hCG for 4, 8, 12, or 24 h. The expression of Nts increased after 4 h of hCG treatment by approximately 7 fold and remained elevated at 8 h after which Nts expression declined to basal levels by 24 h (Figure 4B).

In the COC, levels of Nts mRNA were different in the rat compared with the human. In contrast to the human, levels of Nts mRNA in the rat COC were extremely low (Ct values approaching 50 cycles, data not shown) suggesting species differences in NTS expression in the cumulus cells.

Regulation of NTS mRNA expression in vitro

To begin to understand the LH/hCG-induced expression of NTS mRNA in vitro, rat GC or human GLCs were cultured with or without inhibitors of major LH/hCG signaling pathways such as the progesterone receptor, PG synthase, and the EGF pathway. In the rat, treatment of GC with specific pathway inhibitors followed by hCG for 4 h, revealed that hCG induction of Nts mRNA expression was partially blocked by AG1478 suggesting that Nts is regulated in part via the EGF pathway (Figure 5A). Treatment with inhibitors of PG synthase or the progesterone receptor had no effect on hCG-induced Nts expression (Figure 5A).

Figure 5.

Regulation of Neurotensin (NTS) mRNA expression in human GLC and rat granulosa cells in vitro. Inhibitors for hCG signaling pathways were used to determine which signaling pathway is involved in the expression of NTS. Rat granulosa cells (A) and human granulosa-luteal cells (B) were incubated for 1 h with the following hCG pathway inhibitors: AG (AG1478; EGF receptor inhibitor), RU (RU486; progesterone receptor antagonist), NS (NS398; prostaglandin synthase 2 inhibitor) or IND (Indomethacin; a prostaglandin synthase 1 and 2 inhibitor). The hCG induction of NTS mRNA expression was partially by the EGF receptor inhibitor. Rat granulosa cells (C) and human GLC (D) were incubated for 1 h with the following signaling inhibitors: GF (GF109203x; PKC inhibitor), LY (LY294002; PI3K inhibitor), H89 (PKA inhibitor), SB (SB203580; p38 MAPK inhibitor) or U126 (MEK1/2 inhibitor). The hCG induction of NTS mRNA expression was regulated by the PKA pathway in both species. However differences existed in other signaling pathways between the species. Results are the means ± standard error of mean of n = 6. A two-way analysis of variance was used to test differences with or without hCG treatment across the different signaling pathway inhibitors. The Tukey post hoc test was performed and bars that do not share a letter designation are significantly different (p < 0.05).

In human GLCs the expression of NTS mRNA increased approximately 50 fold within 6 h after hCG induction. Similar to the findings in the rat, the AG compound partially reduced the expression of NTS by approximately 60% (Figure 5B). Importantly, the hCG-induced expression of NTS was not altered by treatment with inhibitors of PG synthase or the progesterone receptor (data not shown).

To further explore the regulation of NTS, human GLC, and rat GCs were pretreated with signaling inhibitors including the PKC inhibitor GF109203x (GF), the PI3K inhibitor LY294002 (LY), the PKA inhibitor (H89), the p38 MAPK inhibitor SB203580 (SB), or the MEK1/2 inhibitor U0126 (U0) or PD325901 (PD). In the rat, the expression of Nts mRNA was decreased by LY, H89, SB and U0 (Figure 5C). These results indicate that the PKA pathway, PI3 kinase, MAPK and MEK1/2 ERK1/2 are involved in the regulation of Nts via hCG in rat GC.

In the human GLC, GF and H89 decreased the hCG-induced expression of NTS mRNA, whereas LY and SB partially inhibited NTS expression (Figure 5D). These results indicate that the PKA pathway and PKC pathway along with PI3 kinase and MAPK are involved in the regulation of NTS via hCG in human GLC.

SORT1 is the most abundant receptor for NTS in GCs

There are three different known NTS receptors designated as NTSR1, NTSR2, and NTSR3 or Sortlin 1 (SORT1). In order to better understand how NTS conveys its function in the ovary, we determined the expression of the three NTS receptors in GC. In human GCs collected in vivo across the periovulatory period and in cultured human granulosa-luteal cells, the expression of NTSR1 was extremely low (cycle threshold values above 40 cycles) and displayed no change with hCG treatment (data not shown). Likewise, NTSR2 mRNA expression was also low but decreased after hCG in human GCs collected in vivo (Figure 6A) yet was unchanged after hCG administration in cultured human GLC (Figure 6C). In human GCs collected in vivo, SORT1 mRNA expression decreased after hCG administration (Figure 6B). In cultured human GLCs, SORT1 expression increased across the time in culture and this increase was diminished by the administration of hCG (Figure 6D).

Figure 6.

Expression of Neurotensin (NTS) receptors in human and rat granulosa cells in vitro. The expression of mRNA after treatment with hCG is shown for NTSR2 (A) and SORT1 (B) in human granulosa collected across the periovulatory period. NTSR2 (C) and SORT1 (D) mRNA expression is also shown in cultured human GLCs. Human GLCs were cultured for 7 days and then treated with or without hCG. The expression of NTS receptors was also determined in isolated rat GC treated with or without hCG. The expression of mRNA for Ntsr2 (E) and Sort1 (F) after temporal treatment with hCG is shown. Results are the means ± standard error of mean of n = 3–5. A two-way analysis of variance was used to test differences with or without hCG treatment across the time points. The Tukey post hoc test was performed and bars that do not share a letter designation are significantly different (p < 0.05).

In rat GCs, the expression of Ntsr1 had cycle threshold values above 40 cycles indicating low levels of expression and expression did not change with hCG treatment (data not shown). Similarly, the expression of Ntsr2 had high cycle threshold values (40 cycles), which also did not change with hCG treatment (Figure 6E). Sort1 was present in rat GCs but expression did not change over time or with hCG treatment (Figure 6F). These findings suggest that Sort1 was the most abundant receptor present in rat GCs, although its expression was not regulated by hCG.

Discussion

The present findings demonstrate for the first time the induction of NTS mRNA by hCG in both the granulosa and theca cell compartments of dominant follicle across the periovulatory period in the human ovary. Similarly, hCG acts to induce Nts mRNA expression in the rat ovary across the periovulatory period. In addition, this induction appears to be regulated through classical hCG signaling pathways acting, in part, through the EGF pathway.

There are limited studies describing NTS in the ovary. In the invertebrate tunicates, a neurotensin-like peptide, C. intestinalis neurotensin-like peptide 6, was able to downregulate the growth of vitellogenic oocytes [19]. The only study that has reported the induction of NTS in the mammalian ovary is a provocative study by Wissing et al. [21] where NTS mRNA expression increased 15.7 fold in GCs collected from the same woman undergoing IVF prior to hCG administration and at the time of oocyte retrieval (36 h after hCG). In the present study, NTS mRNA increased almost 15 000 fold between human GCs collected prior to hCG and GCs collected at the late ovulatory period immediately prior to ovulation. Both the current study and the previous work [21] demonstrate a significant induction of this neuropeptide by hCG; however, the magnitude of the difference in the relative induction by hCG may reflect differences in the models. The present study utilized GCs collected from a single dominant follicle during a natural cycle, whereas Wissing et al. collected GCs from women undergoing IVF who had at least eight periovulatory follicles (with a size of at least 14 mm diameter). Another alternative in the relative change in expression may be related to the method of detection. The present study utilized TaqMan Real Time PCR where the former study analyzed gene expression patterns via Affymetrix Microarrays. In the current study, the striking induction may reflect extremely low levels of NTS mRNA prior to the preovulatory LH stimulus. Irrespective of the relative change, it is readily apparent that NTS is induced in the human preovulatory follicle.

Localization of NTS by immunohistochemistry revealed a pattern of protein expression where NTS was extremely low prior to hCG and increased in the granulosa and theca layer by the late ovulatory period similar to the changes in mRNA. Although this pattern of localization mimicked the changes in NTS mRNA, there was a substantial difference in the magnitude of expression of NTS protein compared with the mRNA induction. This apparent difference could be due to discrepancies between mRNA and protein levels, or it could be due to the rapid synthesis and release of NTS [39–41].

In the rat, hCG also induces an increase in NTS expression prior to ovulation. The in vitro studies suggest that this increase seen in vivo is due in part to hCG stimulation of Nts mRNA expression in GCs. Utilizing in vitro models for both the human and the rat allowed investigation of the potential hCG-induced mediators of NTS expression. This was accomplished through the pharmacological approach of inhibiting various hCG-induced downstream pathways, which revealed that hCG acts through the classical signaling pathways such as the PKA, PKC, and MAPK pathways to induce NTS mRNA. Slight differences in the hCG-induced signaling pathways were observed between the two model systems. This may reflect species differences between the human and the rat, small differences in the time collection after hCG (4 h in the rat, 6 h in the human), or variability in the different models as the rat GCs are primary cells, whereas the human GLC have been in culture for 6 days. Irrespective, the cAMP/PKA pathway appears to be a major transducer of hCG induction of NTS expression in GCs.

These findings in GCs are in agreement with previous studies in other systems that have demonstrated induction of NTS mRNA by the cAMP/PKA signaling pathway. For example, in a neuroblastoma cell line and hypothalamic neurons, estrogen increases cAMP and the phosphorylation of the cAMP response element-binding protein in a time frame that precedes induction of NTS gene transcription [42]. Furthermore, interference with the cAMP/protein kinase A signal transduction cascade blocks the ability of estrogen to elicit increases in NTS transcription in neuroblastoma cells and hypothalamic neurons [42]. Similarly, in a human endocrine cell line, NTS expression is regulated by a cAMP-responsive element (CRE)/AP-1-like element that binds both the AP-1 and CRE-binding protein/ATF proteins [43]. These observations support the current findings of a cAMP dependence in the regulation of NTS.

Further evidence for cAMP regulation of NTS is found in mouse cumulus cells. Hiradate et al. [20] observed that Nts mRNA levels in cumulus cells were markedly higher in hCG-induced ovulated COCs compared with immature COCs. Further examination of this observation revealed that Nts expression in the cumulus cells was stimulated by FSH and EGF. Using a MAPK kinase inhibitor, U0126, the FSH and EGF induction of Nts was abolished, similar to the current findings in rat GCs [20]. The authors proposed that NTS secreted from cumulus cells might be one of the enhancers of acrosome reaction. The elevated expression in human cumulus may also play a role in enhancing the acrosome reaction and facilitating fertilization. However, the levels of Nts in rat cumulus cells in the present study were very low suggesting that NTS may have different levels and functions in different species.

NTS signals through three predominate receptors characterized as NTSR1, NTSR2, and SORT1 [13–15, 44]. In the present study, NTSR1 and NTSR2 mRNA were extremely low in both human and rat GCs. However, SORT1 mRNA was highly expressed in both species. These observations in the ovary are similar to findings in human and murine microglia cells which express SORT1 but not NTSR1 and NTSR2 [14, 45]. The actions of NTS in these microglia cells, as well as other cells, set in motion events similar to those associated with ovulation. For example, in these human and murine microglia cells, NTS stimulation induces an increase in proinflammatory cytokines and chemokines as well as cell migration [46, 47]. Likewise, in brain mast cells NTS can stimulate the release of inflammatory mediators that disrupt the blood–brain barrier, increase vascular permeability, stimulate microglia, and cause focal inflammation [48]. Although the actions of NTS in the ovary are currently unknown, it is possible that NTS acts as in other systems to regulate cell proliferation, cell migration, vascular permeability, and inflammation, all key aspects of the ovulatory process.

NTS expression in the ovary has been described in ovarian cancer (OvCa) cells. In women, NTS has been identified in OvCa and is increased with high-grade serous ovarian carcinoma without identifiable serous tubal intraepithelial carcinoma [12, 49, 50]. Follow-up studies with OvCa cell lines demonstrated increased expression of NTS and NSTR1, whereas the NTSR3 receptor was lower in all OvCa cells compared with fallopian tube epithelial cells (FTE 237, FTE 240, and FTE 246) and immortalized human ovarian surface epithelium cells [12]. Treatment with the NTSR1 inhibitor (SR48692) decreased cell proliferation but increased cell migration in OvCa cells [12]. Evidence that the effects of SR48692 were due to NTSR1 was confirmed by transient RNAi knockdown of NTSR1, which mimicked the increased migratory effects on OvCa cells. In contrast, the knockdown of NTSR3 mimicked the antiproliferative effects on OvCa cells, and the knockdown of NTSR1 or NTSR3 was associated with acquisition of distinct morphological phenotypes, epithelial, or mesenchymal, respectively [12]. These findings suggest that neurotensin signaling plays a key role in cell proliferation and epithelial-mesenchymal transition in high-grade serous ovarian carcinoma [12]. These findings may have an impact on the ovulatory actions of NTS in that the type of receptor may dictate NTS actions toward cell proliferation, migration, or differentiation.

In summary, these findings demonstrate the hCG induction of NTS expression prior to ovulation in both the human and rat ovary with elevated levels of expression in the GC compartment. Although the actions of NTS in the ovulatory process remain to be elucidated, the extrapolation of findings in other systems would predict that NTS regulates many of the crucial aspects of the ovulatory process such as vascular permeability, inflammation, and cell migration.

Author contributions

LAA: designed study, performed research, analyzed data, and wrote the paper.

MP: performed research, analyzed data, and wrote the paper.

KR: performed research, analyzed data, and wrote the paper.

MJ: performed research, analyzed data, and wrote the paper.

DMD: performed research, analyzed data, and wrote the paper.

MB: collected the human ovarian samples and wrote the paper.

JA: collected the human IVF samples and wrote the paper.

YC: performed research, analyzed data, and wrote the paper.

TEC: designed study, performed research, analyzed data, and wrote the paper.

KS: performed research, analyzed data, and wrote the paper.

Conflict of interest

The authors have declared that no conflict of interest exists.