Neurotensin drives theca cell migration during ovulation in primates

Abstract

Theca cells are a critical steroidogenic cell type of the ovarian follicle and corpus luteum. The ovulatory luteinizing hormone (LH) surge (or human chorionic gonadotropin (hCG)) stimulates theca cell relocation from the stroma surrounding the dominant follicle to full integration into the developing corpus luteum. Luteinizing hormone/human chorionic gonadotropin also stimulates granulosa cells to produce local mediators of ovulation, including the peptide neurotensin (NTS). To determine if hCG-stimulated NTS regulates theca cell relocation within the ovulatory follicle, vehicle or an NTS receptor antagonist was injected into a macaque dominant follicle, and ovaries were removed 48 h after hCG administration. Additional ovaries with dominant follicles were collected without administration of hCG (pre-hCG). Theca cells were sparse in the ovarian stroma surrounding pre-hCG follicles, while theca cells were abundant in the stroma and granulosa cell layer of recently-ovulated, hCG-treated follicles. Intrafollicular injection of a general NTS receptor antagonist or antagonist selective for a specific NTS receptor (NTSR1 or SORT1) reduced theca migration into the granulosa cell layer after hCG. In vitro, NTS stimulated macaque theca cell migration in conventional and 3-dimensional migration assays, and NTS receptor antagonists blocked NTS-stimulated migration. Neurotensin-stimulated theca cell migration in vitro was influenced by ovarian extracellular matrix components, with laminin reducing theca cell migration. NTS also increased theca cell number in vivo and stimulated theca cell proliferation in vitro. In summary, hCG-stimulated NTS acts directly at theca cells via NTSR1 and SORT1 to stimulate theca cell migration during ovulation and transformation of the ovulatory follicle into the corpus luteum.

Theca cells actively migrate in response to the paracrine mediator neurotensin during ovulation and luteinization.

Graphical Abstract

Graphical Abstract.

Introduction

Steroidogenic theca cells, often referred to as theca interna cells, are first noted in the ovarian stroma when ovarian follicles reach the secondary follicle stage [1]. Theca cells proliferate as follicles grow to the antral stage and become large, preovulatory follicles. After the ovulatory luteinizing hormone (LH) surge, theca cells integrate into the luteinizing granulosa cell layer. Now called theca-lutein cells, these cells form an essential part of the mature corpus luteum [2].

The major role of theca cells in the follicle and corpus luteum is steroid hormone production. Luteinizing hormone is the primary endocrine stimulus for theca cells [3]. Luteinizing hormone and human chorionic gonadotropin (hCG) bind the LH/CG receptor (LHCGR) to stimulate theca steroidogenesis [1, 4]. Androgens, most notably androstenedione, are produced in large amounts by theca cells [1]. In the follicle, most thecal androgens are rapidly converted to estrogens by neighboring granulosa cells, with some theca-produced androgens also entering the systemic circulation [3]. Theca-lutein cells of the corpus luteum continue to produce androgens for conversion to estrogens by granulosa-lutein cells [5]. Close proximity between theca and granulosa cells is likely necessary for optimal steroidogenesis in both the follicle and corpus luteum.

The LH surge (or hCG in experimental models) is the endocrine trigger of ovulatory changes in the follicle [3]. While some ovulatory actions of LH are direct via LHCGR-coupled signal transduction, the LH surge also acts at follicle cells to initiate production of paracrine mediators of ovulation. Neurotensin (NTS) was recently identified as a critical paracrine mediator of primate ovulation as blocking NTS action resulted in 75% of follicles failing to ovulate [6]. Neurotensin is produced primarily by granulosa cells, and granulosa cell NTS mRNA increases rapidly and dramatically after the ovulatory LH surge [6–8]. Neurotensin has three known receptors: NTSR1, NTSR2, and SORT1 [9]. Expression of each NTS receptor has been reported in the ovulatory follicle [6–8], and NTSR1 has been implicated in NTS-stimulated oocyte maturation and ovulation in mice [10, 11]. However, little is known regarding expression of NTS receptors by theca cells, ovulatory actions of NTS directly at theca cells have not been reported.

The present study was conducted to determine if the paracrine mediator NTS acts directly at theca cells to stimulate critical ovulatory changes in theca cells. In vivo and in vitro studies were performed using macaques, primates with menstrual cycles very much like those of women. Overall, these studies demonstrate that NTS promotes theca cell migration as the ovarian follicle ovulates and undergoes a major structural transformation to become the corpus luteum.

Methods

Monkeys

Whole ovaries were collected from adult female cynomolgus macaques (Macaca fascicularis, aged 4–8 years) at Eastern Virginia Medical School (EVMS) as previously described [12]. All animal protocols were performed in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals [13] and with the approval of the Institutional Animal Care and Use Committee of EVMS. Briefly, animals were monitored daily for menstruation, with the first day of menstruation was designated as day 1 of the menstrual cycle. Blood samples were collected under ketamine chemical restraint (10 mg/kg body weight) via femoral or saphenous venipuncture. Serum was stored at −20°C. Serum estradiol and progesterone levels were quantified using the ADVIA Centaur CP Immunoassay System (Siemens). Surgeries were performed aseptically under isoflurane anesthesia, followed by postoperative analgesia (buprenorphine, with either ketoprofen or meloxicam as needed [12]). For each experiment using monkey ovaries or ovarian cells, n reflects the total number of tissues or distinct primary cell lines, each collected from a different animal. Additional tissues (e.g., colon, kidney, pancreas) were collected at necropsy, flash frozen in liquid N₂, and stored at −80°C.

Controlled ovulation with follicle injection

Delivery of test materials directly to the follicular fluid of a naturally selected ovulatory follicle was previously described [14]. Serum concentrations of estradiol and progesterone were monitored daily beginning 5–7 days after menstruation. Once serum estradiol levels increased above 150 pg/mL, recombinant human FSH (60IU, Follistim, Organon & Co., Jersey City, NJ) and recombinant human LH (60IU, Serono Reproductive Biology Institute, Rockland, MA) were administered once a day for 2 days to maintain the healthy growth of the ovulatory follicle. The gonadotropin-releasing hormone (GnRH) antagonist Acyline (60 μg/kg per day; Eunice Kennedy Shriver National Institute of Child Health and Human Development) was also administered daily to prevent an endogenous LH surge. On the following day, surgery was performed to inject the ovulatory follicle. Control injections were performed with either sterile water (n = 2) or a non-targeting control IgG (n = 4) and are collectively referred to as Control. Additional follicles were injected with either the general NTS receptor antagonist SR142948 (Tocris Bioscience 2309, Bristol, UK; 100 μM, n = 4), the NTSR1 antagonist SR48692 (Tocris Bioscience 3721; 50 μM, n = 3), or the SORT1 antagonist AF38469 (Cayman Chemical 35,530, Ann Arbor, Michigan; 0.25 μM, n = 3). Sterile water was used as the vehicle for the SR142948 and AF38469 injection solution. Sterile water with a final concentration of dimethyl sulfoxide <0.1% was used as the vehicle for the SR48692 injection solution. Injection of dimethyl sulfoxide to achieve a final concentration of 0.1% [14, 15] or non-targeting IgG [6, 16, 17] have previously been demonstrated to have no adverse effect on ovulation. Immediately after surgery, 1000 IU hCG (Ovidrel, EMD Serono, Rockland, MA) was administered intramuscularly to initiate ovulation. Ovariectomy was performed 48 h after hCG to remove the injected ovary, with ovulation expected at around 40 h [18].

To obtain large preovulatory follicles consistent with the time of follicle injection and hCG administration (pre-hCG), two approaches were used. One ovary with a large, preovulatory follicle was obtained after 2 days of FSH, LH and Acyline administration as described above; this ovary was removed in the absence of hCG administration. Two additional ovaries with large preovulatory follicles were obtained from monkeys experiencing natural menstrual cycles [18]. Briefly, once-daily serum sampling was performed to monitor rising estradiol. Ovariectomy was performed on the day consistent with peak serum estradiol in a prior menstrual cycle. Subsequent serum analysis confirmed that estradiol was high and both progesterone and LH were low at the time of ovariectomy, consistent with a healthy preovulatory follicle without exposure to an ovulatory gonadotropin stimulus.

Whole ovaries were fixed in 10% formalin for 24 h and embedded in paraffin, oriented such that sections included the follicle apex and follicle wall opposite the apex at the maximal follicle diameter [6]. Ovaries were serially sectioned at 5 μm, with each section retained in order. Histologic structure, luteinization, angiogenesis, and ovulation have been previously reported for water- and IgG-injected controls as well as NTS receptor antagonist-injected follicles [6, 16, 17, 19, 20].

Theca migration and proliferation in vivo

Monkey ovary tissue sections were deparaffinized, hydrated, and blocked with Image-iT™ FX signal enhancer (Thermo Fisher Scientific, Waltham, MA) for 30 min at room temperature. Then blocked in PBS + 5% goat serum for an additional 30 min. Slides were also blocked for endogenous biotin, streptavidin receptors, and protein via the Streptavidin/Biotin Blocking Kit (Vector Laboratories, Burlingame, CA) per manufactures instructions. Slides were incubated overnight at 4°C with CYP17A1 rabbit polyclonal antibody (Proteintech Cat#14447-1-AP (RRID:AB_2292527); 350 μg/mL) in PBS + 5% goat serum. Post primary incubation slides were incubated with biotinylated goat anti-rabbit IgG (Vector Laboratories BA-1000; 7.5 μg/mL) in PBS + 5% goat serum for 2 h at room temperature. CYP17 positive cells were then fluorescently tagged with AF488-conjugated secondary antibody (Thermofisher CAT#A11034 (RRID:AB_2576217); 4 μg/mL) for 30 min. Tissue sections were treated with 1% Sudan black in 70% methanol and mounted with Prolong Gold antifade mountant with DAPI (Thermo Fisher Scientific, Waltham, MA). Omission of primary antibody served as a negative control.

Theca cell invasion into the granulosa cell layer (migration) and theca cell number (proliferation) were assessed quantitatively, similar to methods previously described for assessment of angiogenesis [16]. Tissue sections containing the approximate maximal diameter of the ovulatory follicle were selected, and images were obtained of the follicle wall directly across from the follicle apex (maximal rupture or thinnest apical tissue in unruptured follicles). Images were obtained using a Zeiss Observer Z1 microscope fitted with an AxioCam MRm camera and Zen software v3.8 for image acquisition (Zeiss, Oberkochen, Germany). For each image, the distance between a stromal theca cell and theca cell closest to the follicle antrum was determined using ImageJ (NIH), with the line of measurement perpendicular to the granulosa cell basal lamina. In addition, cells that contained a clearly visible nucleus surrounded by CYP17 detection were counted for each image. For both invasion and cell number, four to eight replicate measurements were made per ovary to obtain an average.

Extracellular matrix proteins in monkey ovaries in vivo

Two approaches were used to localize extracellular matrix proteins to monkey ovarian tissues with preovulatory follicles or after controlled ovulation and follicle injection. Immunocytochemistry was used to detect fibronectin and laminin, essentially as previously described [16]. Briefly, deparaffinized tissues sections were rehydrated in PBS, heated in citrate antigen retrieval buffer, and incubated in 2% hydrogen peroxide in MeOH to inactivate endogenous peroxidase. Tissues were then blocked in PBS containing 3% goat serum and incubated with primary antibodies (either fibronectin (Thermofisher Cat#PA5-29578 (RRID:AB_2576217), 0.34 μg/mL) or laminin (Thermofisher Cat#PA116730 (RRID:AB_2133633), 5 μg/mL)) in PBS + 3% goat serum overnight at 4°C. Omission of primary antibody served as a negative control. Colorimetric detection proceeded using a rabbit Vectastain ABC kit (Vector Labs Cat#PK-4001 (RRID:AB_2336810)) and DAB kit (Vector Labs) according to manufacturer’s instructions. Tissues were counterstained with hematoxylin, dehydrated, and permanently coverslipped. Collagen staining was achieved using Masson trichrome and was performed by the Histology Service Core located in the EVMS Biorepository. Images were obtained using an Olympus microscope with a DP70 digital camera system and associated software (Olympus, Melville NY).

Monkey theca cells

Theca cells were obtained from monkeys experiencing a modified ovarian stimulation protocol as previously described [21]. Briefly, monkeys received recombinant human follicle stimulating hormone (90 IU FSH, Organon) for 6 days. A GnRH antagonist (0.03 mg/kg Ganirelix; Organon) was also administered daily to prevent an endogenous LH surge. Ultrasonography and serum estradiol were used to confirm the presence of multiple small antral follicles 2–3 mm in diameter on day 5–6. On day 7, aseptic surgery was performed to remove whole ovaries.

Monkey theca tissue was isolated from ovarian follicles as previously described [22]. Briefly, small antral follicles (2–4 mm) were dissected free of ovarian stroma and bisected. Granulosa cells were scraped free, and a thin layer of theca interna tissue was pulled free from the interior of the follicle. Theca tissue was minced and incubated in base medium (1:1 mixture of low glucose DMEM (Sigma), and Ham F-12 medium (Sigma) supplemented with 5.96 g/L HEPES (Sigma), 2.4 g/L NaHCO₃ (Sigma), 3.33 mU/mL insulin (Humulin R U-100, Lilly), 20 nM selenium (Sigma), 1 μM vitamin E (Sigma), and antibiotic/antimycotic mixture (Sigma); pH 7.4; [23]) containing collagenase I and DNAse as previously described [22]. Theca cells were plated on fibronectin-coated culture ware and maintained in growth media (base media plus 5% fetal bovine serum (R&D Systems), 5% horse serum (Gibco), and 2% UltroSer G (PALL Life Sciences) [23]) in a humidified cell culture incubator at 37°C in an atmosphere of 5% O₂, 5% CO₂, 90% N₂. Identity of primary cells as >95% theca cells was verified by immunodetection of CYP17A1 but not CYP19A1 (aromatase) as previously described [22]. Theca cells maintain phenotype (CYP17A1+, CYP19A1-) for at least eight passages. Theca cells produced androstenedione (34.66 ± 2.95 pg/mL, n = 3) and testosterone (187.46 ± 9.81 pg/mL, n = 5) after 24 h in serum-free media, as determined by ELISA (DRG International, Inc., Springfield, NJ). Experiments were performed on theca cells from passages 3–6. Theca cells were cultured in serum-free media (base media plus 1.0 mg/mL bovine serum albumin (Sigma) and 100 μg/mL apo-transferrin (Sigma); [23]) for 1 h or overnight before use in experiments. All experiments were conducted using serum-free medium with treatments and vehicles as indicated. Viability was determined after incubation in serum free medium containing indicated concentrations of SR142948, SR48692, or AF38469 for 24 h treatment using trypan blue (Sigma) exclusion according to the manufacturer’s protocol. Theca cells were >90% viable at the concentrations of SR142948, SR48692, and AF38469 used in these experiments (Supplemental Figure S1).

RNA isolation and quantitative PCR

Theca cells were grown to ≥90% confluence, then cultured overnight serum-free medium. Theca cells were treated with NTS (0–5 μM) in serum-free media for 24 h, then extracted for RNA using the RNeasy® Mini Kit (Qiagen, Hilden Germany) per manufacturer’s instructions. Quality and quantity of RNA was assessed via NanoDrop 1000 (NanoDrop Technologies, Wilmington, DE). RNA (500 ng) was converted to cDNA using the Qiagen RT2 First Strand Kit per manufacturer’s instructions. Quantitative PCR (qPCR) was performed using primers designed against M. fascicularis sequences (Table 1) and the FastStart SYBR® Green Master kit (Roche Diagnostics GmbH; Mannheim Germany) per manufacturer’s instructions with the CFX96 Real-Time System (Bio-Rad Laboratories). Cycling conditions were as follows: 10 min at 95°C; then 45 cycles of 15 s at 95°C, 30 s at 57°C (NTS), 59°C (NTSR1 and NTSR2), 60°C (SORT1), or 61.7°C (GAPDH), and 45 s at 72°C. A melt curve was performed ranging from 65°C to 95°C in 0.5°C increments. Expression of target mRNA was calculated via 2^-ΔΔCT method and normalized to expression of GAPDH. Any sample showing lack of amplification after 40 cycles was determined to have non-detectable level of the targeted mRNA.

Table 1.

Primers for qPCR.

Target	Forward & reverse sequence	Annealing temperature	Accession #
NTS	F-CAGAGCACCTCTCATAGTTCA R-GGAACTGAAAGCCAGGAGTA	57°C	XM_005571668.2
NTSR1	F-CGCCTCATGTTCTGCTACAT R-TTGATGGTGGAGCTGACGTA	59°C	XM_005569538.2
NTSR2	F-ATCCAGGTGAATGTGCTGGT R-GAAGTGGACGGCACTTGG	59°C	NM_001283319.1
SORT1	F-GGGGGACGTTTCCTTTTTGC R-TCCGCCTGTGGTAGTGTAGA	60°C	XM_005542475.2
GAPDH	F-TTCAACAGCGACACCCACTC R-GCCAATTCGTTGTCATACCAGG	61.7°C	XM_045364618.2

Western blotting

Theca cells were grown till confluence in a fibronectin-coated 6-well plate, then cultured in serum-free medium+1%FBS overnight. The following day, theca were exposed to serum-free medium for an additional 24 h. Theca cell lysate was collected as previously described and protein quantity was determined via the bicinchoninic acid assay (Sigma) [24]. Lysate was also obtained as above from M. fascicularis kidney, pancreas, and intestine. Sample and control protein lysates were normalized to 12 μg and mixed with 2X Laemmli buffer then heated to 95°C for 5 min. Samples were loaded onto a 4%–12% polyacrylamide gradient gel (Invitrogen) and transferred to a polyvinylidene fluoride membrane (Immobilon; Millipore, Billerica, MA). Membranes were blocked in 5% nonfat dry milk in Tris-buffered saline (TBS, Santa Cruz Biotechnology SC-24951) overnight for SORT1 and 2 h for NTSR1. To detect NTS, the membrane was blocked overnight in 5% bovine serum albumin with 0.25% gelatin in 0.05% Triton and TBS. Membranes were then probed with antibodies against SORT1 (Sigma-Aldrich Cat#HPA006889 (RRID:AB_1080056), 0.4 μg/mL) in blocking buffer with 0.1% Tween 20; NTSR1 (Thermofisher Cat#PA3–214 (RRID:AB_10979876), 1:500, protein concentration not provided by manufacturer) in blocking buffer with 0.05% Tween 20 and 0.05% Igepal CA-630 (Sigma); or NTS (1:1000; ImmunoStar, Cat#20072 (RRID:AB_572254); protein concentration not provided by manufacturer) in blocking buffer with 0.1% Tween 20 and TBS for two days. Membranes were then incubated with an anti-rabbit HRP-conjugated secondary antibody (Vector Labs Cat#PI-1000 (RRID:AB_2336198), 0.1 μg/mL) in their appropriate buffer for 1 h at room temperature then at 4°C overnight. Protein bands were visualized with Amersham ECL Western Blotting Detection Reagents (Cytiva, Marlborough, MA) using the Odyssey CLx (LI-COR, Lincoln, NE) and Image Studio Version 5.2. software (LI-COR, Lincoln, NE).

Immunocytochemistry

Theca cells were grown to 95% confluence on 8-well chamber slides. Theca cells were cultured in serum-free medium overnight, then exposed for 24 h to either base media or base media containing 5 μM of NTS. Theca cells were then fixed in 4% paraformaldehyde for 20 min. Slides were blocked with 5% normal goat serum in PBS plus 0.1% Triton X-100 and then incubated overnight at 4°C with a primary antibody targeted against NTS (ImmunoStar, 20,072, 1:100 dilution, protein concentration not provided by manufacturer), NTSR1 (Thermofisher PA3–214, 1:400, protein concentration not provided by manufacturer), or SORT1 (Sigma, HPA006889, 4 μg/mL). Immunodetection and imaging proceeded as described for extracellular matrix proteins (above). Monkey pancreas and colon served as positive controls.

Migration in vitro

Migration in vitro was assessed as previously described for vascular endothelial cells [15]. Migration membranes with 8 μm pores (BD Biosciences, San Jose, CA) were pre-coated with fibronectin (10 μg/mL; Gibco #33014–015), collagen III (10 μg/mL; Millipore #CC054), collagen IV (10 μg/mL; Sigma #C5533), laminin (0.0001–3 μg/mL; Sigma #L4544), or no matrix in PBS and rinsed with PBS before plating cells. Theca cells were seeded directly onto cell culture inserts containing membranes in serum-free medium. Recombinant human NTS (0–50 μM; Bachem, Torrance, CA) or hCG (20 IU/mL; Sigma) was added to the culture well below the membrane. NTS receptor antagonists, when used, included SR142948 (25 μM), SR48692 (25 μM), or AF38469 (0.1 μM) and were added to media 1 h before addition of NTS. After 24 h of NTS treatment, membranes were fixed in 100% EtOH and stained with hematoxylin and eosin. Images (n = 5 per membrane) were taken; cells were counted and averaged for each membrane.

Migration was also quantified using a 3-dimensional (3D) assay as previously described for vascular endothelial cell sprout formation [15, 25]. Briefly, theca cells were incubated with inert Cytodex polymer beads (GE Healthcare). Cell-coated beads were embedded in a fibrin matrix (about 25 beads per 0.5 mL matrix prepared in serum-free medium) and overlayed with serum-free medium containing NTS (0–50 μM). Individual cell-covered beads (five per treatment) were imaged at the start of culture (day 0) and 24 h later (day 1) using an Olympus microscope, Infinity Lite camera, and associated software (Lumenera). For each bead imaged, each theca cell moving away from the surface of the bead was counted, and the distance between the cell and the surface of the bead was determined (ImageJ, NIH). For beads with many migrating cells, the five longest distance measurements were used. In the absence of migrating cells, a score of zero (0) was recorded for both cell number and distance for that individual bead.

After 48 h (day 2), matrix-embedded beads were fixed with 4% paraformaldehyde for 20 min and permeabilized in PBS + 0.05% Triton X-100 for 15 min. Beads were blocked with Image-iT™ FX signal enhancer for 30 min at room temperature and then in PBS + 1% goat serum for an additional 30 min. Beads were incubated at room temperature for 30 min with Alexa Fluor 546 phalloidin (Molecular Probes A22283, 66 nM) in PBS + 1% goat serum. Beads were then mounted with Prolong Gold antifade mountant with DAPI and visualized using the Keyence BZ-X810 All-in-One Fluorescence Microscope (Keyence, Itasca, IL). Cells on beads were imaged using the Plan Fluorite 20X LD PH lens (Keyence, BZ-PF20LP) and automated haze reduction was performed using the Advanced Analysis Software (Keyence, BZ-H4A).

Proliferation in vitro

For assessment of proliferation, theca cells were grown on glass chamber slides (Nunc, Thermo Fisher) precoated with fibronectin (μg/mL) until 50% confluent. After overnight incubation with serum-free medium+1%FBS, serum free medium containing hCG (20 IU/mL) or NTS (0–50 μM) was added. If treatments included NTS receptor antagonists, then serum-free medium containing SR142948 (25 μM), SR48692 (25 μM), or AF38469 (0.1 μM) was added for 1 h before the addition of NTS (0–50 μM). Slides were fixed in 10% formalin after 24 h of NTS treatment and used for detection of KI67 by immunohistochemistry as previously described [26], using a mouse monoclonal antibody against KI67 (Agilent Cat#M7240 (RRID:AB_2142367), 1:100, protein concentration not provided by manufacturer) and procedure similar to that described for NTS and NTS receptors above. Images (five images per chamber, at least 50 cells/image) were collected, each cell was scored (Ki67+/Ki67-), and the average was expressed as percent positive for each treatment group.

Data analysis

Data were assessed for heterogeneity of variance by Bartlett tests. Bartlett test for data in Figures. 1F, 1K, and  6E yielded P < 0.05, so these data were log10 transformed before further analysis. To test differences between treatments, analysis was performed using 2-tailed paired student t-tests or one-way analysis of variance (ANOVA) without or with one repeated measure, followed by Duncan post hoc test as indicated in each Figure legend. P < 0.05 was considered significant. Statistical analysis was performed using StatPak version 4.12 software (Northwest Analytical, Portland, OR). Data are presented as mean ± SEM.

Figure 1.

Theca cells express NTS and NTS receptors. Monkey theca cells expressed mRNA for NTS (A), NTSR1 (F), and SORT1 (K) after culture with 0 or 5 μM NTS. For all panels, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by paired, two-tailed t-test; no groups were found to be different (P > 0.05). Western blot detection of NTS (B; arrowhead at 15 MW), NTSR1 (G; arrowhead identify theca NTSR1 bands at 40 MW (short, non-glycosylated form) and 60–70 MW (full length, glycosylated forms)), and SORT1 (L; arrowhead at 110 MW) in monkey theca cell (Th) lysate; lysate of monkey colon (C (panel B)), pancreas (P (panel G)), and kidney (Ki (panel L)) served as positive controls. Immunocytochemical detection of NTS (C, D), NTSR1 (H, I), and SORT1 (M, N) in monkey theca cells cultured with 0 μM (C, H, M) or 5 μM NTS (D, I, N). In images of theca cells, arrows indicate nuclear/perinuclear localization, and arrowheads indicate cytoplasmic localization. Detection of NTS in monkey colon (E), NTSR1 in monkey pancreatic islets (J), and SORT1 in monkey colon (O) served as positive controls. In positive control tissues, arrowheads indicate specific staining at approximately same location in low magnification image and high magnification inset. For images C–D, H–I, and M–N, scale bar = 40 μm. For images E, J, and O, scale bar = 200 μm for low magnification image and 100 μm for high magnification inset For all immunohistochemistry, brown represents specific detection of target protein, and nuclei are counterstained with hematoxylin (blue).

Figure 6.

Theca cell migration is modulated by extracellular matrix. Theca cells (n = 3 lines) were plated on migration membranes pre-coated with fibronectin (10 μg/mL; A), collagen III (10 μg/mL; B), collagen IV (10 μg/mL; C), laminin (3 μg/mL; D), no matrix (E), or laminin (0.0001 μg/mL; F); theca cells were seeded and treated with either basal medium (0 μM NTS) or 5 μM NTS for 24 h before assessing for migration. For each panel, numbers of migrated cells are shown, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by paired t-test; groups with no common letters are different, P < 0.05.

Results

Theca cells express neurotensin and neurotensin receptors

Our prior reports detail expression of NTS and NTS receptors in ovulatory follicles of a number of mammalian species [6–8], including initial demonstration of NTS and NTS receptor expression in theca cells [6, 7]. To more completely characterize NTS and NTS receptor expression in theca cells of monkey ovulatory follicles, primary monkey theca cells were assessed for NTS, NTSR1, NTSR2, and SORT1 mRNA and protein.

NTS mRNA was detected in monkey theca cells (Figure 1A). Neurotensin western blot detected a single band at 15 MW in monkey theca cell lysate (Figure 1B). Neurotensin was also detected by immunostaining in theca cells (Figure 1C and D). Treatment with NTS did not alter NTS mRNA levels (Figure 1A) or protein localization within theca cells (Figure 1D; compare with untreated in Figure 1C). Neurotensin was detected in monkey colon lysate by western blot (Figure 1B) and was localized to monkey colon by immunocytochemistry (Figure 1E), which served as a positive control [27].

NTSR1 mRNA was present in monkey theca cells (Figure 1F). NTSR1 protein was detected as multiple bands in monkey theca cells (Figure 1G). Detection of multiple size bands for NTSR1 is consistent with a previous report of glycosylation and proteolytic cleavage of NTSR1 [28]. NTSR1 protein was localized to theca cells by immunocytochemistry (Figure 1H and I). Treatment with NTS did not alter NTSR1 mRNA levels (Figure 1F) or protein localization within theca cells (Figure 1I; compare with untreated in Figure 1H). NTSR1 was detected in monkey pancreas lysate by western blot (Figure 1G) and localized to monkey pancreatic islets by immunocytochemistry (Figure 1J), which served as a positive control [6].

SORT1 mRNA was present in monkey theca cells (Figure 1K). SORT1 protein was detected as a single band of 110 MW in monkey theca cells (Figure 1L). SORT1 protein was localized to theca cells by immunocytochemistry (Figure 1M and N). Treatment with NTS did not alter SORT1 mRNA levels (Figure 1K) or protein localization within theca cells (Figure 1N: compare with untreated in 1M). SORT1 was detected in monkey kidney lysate by western blot (Figure 1L) and localized to monkey colon by immunocytochemistry (Figure 1O), which served as positive controls [29, 30].

NTSR2 mRNA in theca cells was below the threshold of detection, so NTSR2 protein detection was not pursued. NTSR2 mRNA was detected in monkey pancreas and served as a positive control [6].

Neurotensin stimulates theca cell migration and proliferation in vivo

The LH surge or hCG stimulates NTS production by the cells of the ovulatory follicle [6–8]. For this reason, an antagonist approach was utilized to block the action of endogenously produced NTS at NTS receptors on follicle cells. Using our macaque model of controlled ovulation and follicle injection, preovulatory follicles were injected with vehicle (Control) or an NTS receptor antagonist, followed by administration of an ovulatory dose of hCG. Ovaries were removed 48 h later, with ovulation expected 40 h after hCG [18]. Additional ovaries with large, preovulatory follicles were collected in the absence of an endogenous LH surge or follicle injection and hCG (pre-hCG follicles). The impact of NTS receptor antagonist follicle injection on ovulation and luteinization has been previously reported [20].

In pre-hCG follicles, theca cells formed a discontinuous cell layer on the stromal side of the granulosa cell basal lamina (Figure 2A). Control follicles showed structural changes consistent with exposure to an ovulatory gonadotropin stimulus, including an expanded granulosa cell layer, stromal invaginations into the granulosa cell layer, and enlarged stromal vessels (Figure 2B; quantified in [20]). Importantly, Control follicles contained numerous theca cells appearing to invade the luteinizing granulosa cell layer (Figure 2B).

Figure 2.

Neurotensin regulates theca cell location and number during ovulation and luteinization. Monkey ovaries were collected before (pre-hCG; A) and 48 h (B–E) after intrafollicular injection and administration of an ovulatory dose of hCG. Follicle injections included vehicle (Control IgG injected follicle shown in panel B), SR142948 (C), SR48692 (D), and AF38469 (E). CYP17 detection (green) identifies theca cells; nuclei are blue (DAPI). Images in panels A–E are oriented as in panel A, with follicle antrum (an) at top, granulosa cells (gc) central, and stroma (st) in the lower portion of each image. A stromal vessel (ve) in panel B is indicated. All images are at the same magnification; bar in panel A = 100 μm. Theca cell invasion into the luteinizing granulosa cell layer (μm; panel F) and theca cell number (cell number per 123 μm²; panel G) were determined for monkey ovulatory follicles before and after intrafollicular injection and administration of an ovulatory dose of hCG (n = 3–4 ovaries/treatment group). For panels F and G, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by ANOVA and Duncans post hoc test; groups with no common letters are different, P < 0.05. Panel H. Representative measurements of theca cell invasion (yellow lines) in a Control follicle; scale bar = 100 μm. Panel I. Representative theca cells (white arrows) include a nucleus (blue) surrounded by CYP17 immunodetection (green) in a Control follicle. A total of 23 theca cells were identified in Panel I. For Panel I, scale bar = 50 μm.

Follicle injection of NTS receptor antagonists altered the location and apparent number of theca cells. Injection of SR142948, which blocks NTS action at all NTS receptors, resulted in a few theca cells along the edge of stromal invaginations (Figure 2C). Injection of the NTSR1 selective antagonist SR48692 resulted in few theca cells located along the stromal edge of the granulosa cell basal lamina (Figure 2D), similar to pre-hCG follicles (Figure 2A). Injection of the SORT1 selective antagonist AF38469 resulted in many theca cells and modest invasion into the luteinizing granulosa cell layer (Figure 2E).

To determine the impact of NTS acting via each NTS receptor on theca cell movement during ovulatory changes in vivo, invasion into the granulosa cell layer was quantified (Figure 2F and H). In pre-hCG follicles, the theca cell layer had nominal thickness, which is reflected in measurement of invasion. Theca cell invasion was also quantified after follicle injection and 48 h of exposure to hCG. Theca cells of Control follicles were present both in stroma and within the expanding granulosa cell layer; the most invasive theca cells were detected 79 ± 20 μm from theca cells remaining in the ovarian stroma. Injection of the pan-NTS receptor antagonist SR142948 or SORT1-selective antagonist AF38469 reduced theca invasion into the granulosa cell layer when compared to Control follicles (Figure 2F). Injection of the NTSR1-selective antagonist SR48692 resulted in no significant theca cell movement, with invasion distance lower than Control follicles and not different from pre-hCG follicles (Figure 2F).

To quantify the impact of NTS acting via each NTS receptor on theca cell number in vivo, theca cells were identified by CYP17 immunodetection and counted (Figure 2G and I). Pre-hCG follicles had few theca cells. In contrast, hCG treatment of Control follicles resulted in more than twice the number of theca cells per unit area (Figure 2G). Follicle injection of either SR142948 or SR48692 resulted in fewer theca cell than Control follicles, similar to pre-hCG follicles (Figure 2G). Follicle injection of AF38469 yielded theca cell numbers similar to Control follicles (Figure 2G).

Theca cell migration is modulated via neurotensin in vitro

Our in vivo studies suggest that NTS stimulates theca cell invasion into the expanding granulosa cell layer during ovulation. To determine if NTS acts directly at theca cells to stimulate migration, an in vitro migration assay was used. Theca cells migrated through a porous membrane in response to NTS in a concentration-dependent manner, with both 5 and 50 μM NTS increasing theca cell migration over migration with no (0 μM) NTS (Figure 3A). In a 3D model of migration, polymer beads coated with theca cells were treated without or with NTS (Figure 4). After 1 day in vitro, 50 μM NTS increased the number of migrating theca cells leaving the bead (Figure 4E). Neurotensin at concentrations of both 5 and 50 μM increased the distance that migrating theca cells traveled away from the bead (Figure 4F). Both doses of NTS also appeared to increase the length and number of filopodia extending toward the direction of migration after 2 days in vitro (Figure 4G–I).

Figure 3.

Neurotensin stimulates theca cell migration in vitro. (A) Theca cells (n = 4 lines) were cultured on porous membranes and treated with basal medium (0 μM NTS) or NTS (0.5, 5, or 50 μM), then assessed for migration after 24 h. (B) Theca cells (n = 3 lines) were pretreated with the general NTS receptor antagonist SR142948 (25 μM), the NTSR1 selective antagonist SR48692 (25 μM), or the SORT1 selective antagonist AF38469 (0.1 μM) for 1 h before addition of NTS (5 μM), then assessed for migration 24 h after addition of NTS. For all migration assays, NTS-stimulated migration in each theca line was expressed relative to migration with no treatment, which was set equal to 100%. For all panels, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by ANOVA with one repeated measure and Duncans post hoc test; groups with no common letters are different, P < 0.05. Panels C–F show representative migration membranes after treatment with 0 μM NTS (C), 0.5 μM NTS (D), 5 μM NTS (E), and 50 μM NTS (F). In panel C, the arrow indicates a migrated cell, and arrowhead indicates a migration pore.

Figure 4.

Neurotensin stimulates theca cell migration in 3D. Panels A–D show representative theca-coated beads at the time of plating (day 0; A) and after 1 day of treatment with either 0 μM NTS (B), 5 μM NTS (C), and 50 μM NTS (D). In panels B–D, arrowheads indicate migrating cells. Beads are approximately 100 μm in diameter but vary slightly in size. All panels A–D are shown at the same magnification. Theca cells on polymer beads were assessed for number of migrating cells (E) and migration distance (F) after 1 day of culture. For cell number and distance, NTS-stimulated changes in each theca line (n = 3) were expressed relative to no treatment (0 μM NTS), which was set equal to 100%. For all panels, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by ANOVA with one repeated measure and Duncans post hoc test; groups with no common letters are different, P < 0.05. Panels G–I show representative theca cells after 2 days of treatment with either 0 μM NTS (G), 5 μM NTS (H), and 50 μM NTS (I) and staining for actin (red) and DNA (blue). In panels G–I, arrows indicate leading edges of migrating cells. Beads (not included in image) are to the left of the cells shown, and cells are migrating away from the bead (see arrow at bottom of image). All panels G–I are shown at the same magnification, with bar = 50 μm.

To identify the specific NTS receptors mediating NTS-stimulated theca migration, additional theca cells were plated on migration membranes with no NTS, NTS alone, or NTS + an NTS receptor antagonist (either SR142948, SR48692, or AF38692). Neurotensin alone stimulated migration above levels seen with no NTS (Figure 3B). Furthermore, each NTS receptor antagonist blocked NTS-stimulated migration to levels comparable with no NTS (Figure 3B).

Extracellular matrix proteins of the monkey ovulatory follicle

Composition of the extracellular matrix may influence structural changes in the follicle during ovulation and luteal formation [3]. The granulosa cell basal lamina and stroma of the theca interna layer have been reported to contain collagen III, collagen IV, laminin, and fibronectin, with other extracellular matrix proteins also present in small amounts [31–36]. To identify the extracellular matrix proteins present in monkey follicles around the time of ovulation, pre-hCG follicles as well as follicles injected with vehicle (Control) and exposed to hCG in vivo were stained for fibronectin, collagen, and laminin (Figure 5). Fibronectin staining was strong in the granulosa cell layer of pre-hCG follicles (Figure 5A) as well as the ovarian stroma surrounding pre-hCG follicles (Figure 5A) and throughout the ovary (Figure 5D, inset). Fibronectin was also detected surrounding the granulosa cells of Control follicles, with variable detection in the stroma surrounding luteinizing follicles (Figure 5B). Histological stain for collagens shows moderate to intense staining in the ovarian stroma of pre-hCG and Control follicles (Figure 5F and G). Follicles experiencing hCG-induced structural luteinization showed that collagen was present in stromal invaginations into the luteinizing granulosa cell layer (Figure 5G). Laminin detection was intense in the granulosa cell layer of pre-hCG follicles (Figure 5K), with weak laminin staining in the ovarian stroma surrounding pre-hCG follicles (Figure 5K) and throughout the ovary (Figure 5N, inset). Control follicles showed apparently reduced laminin staining in the granulosa cell layer, with weak laminin staining in the surrounding stroma (Figure 5L). Structural luteinization of these pre-hCG and Control injected/hCG treated follicles was quantified in a prior report [20].

Figure 5.

ECM proteins in monkey ovulatory follicles. Monkey ovaries were collected before (pre-hCG; A, F, K) and 48 h after intrafollicular injection and administration of an ovulatory dose of hCG. Follicle injections included vehicle (Control IgG injected follicle shown in panels B, G, L), SR142948 (C, H, M), SR48692 (D, I, N), and AF38469 (E, J, O). Immunocytochemical localization of fibronectin (panels A–E) is brown; nuclei are counterstained blue. Collagens (F–J; blue) were detected by Masson trichrome. Arrowheads indicate collagen in the area of the granulosa cell basal lamina. Arrows indicate collagen invaginations into the luteinizing granulosa cell layer. Immunocytochemical localization of laminin (panels K–O) is brown; nuclei are counterstained blue. Inset in panel K shows lack of immunodetection when primary antibodies were omitted. Insets show zona pellucida of a primary follicle oocyte staining negative for fibronectin (inset, D) and positive for laminin (inset, N); these insets also show ovarian stroma surrounding primary follicles is positive for fibronectin (inset, D) and negative for laminin (inset, N) All images are in the same orientation as indicated in panel C, with antrum (an) at top, granulosa cells (gc) central, and stroma (st) at bottom of image. All images are at the same magnification; scale bar in panel E = 50 μm.

Fibronectin, collagen, and laminin were also evaluated in follicles injected with the NTS receptor antagonists SR142948, SR48692, or AF38469. Fibronectin detection was similar in the granulosa cell layer of Control follicles and all follicles injected with an NTS receptor antagonist, with the intensity of staining correlating with the degree of follicle luteinization (Figure 5B–E). Fibronectin was also present in the stroma surrounding Control and NTS receptor antagonist-injected follicles (Figure 5B–E). Well-luteinized follicles (Control, SR142948, and AF38469 treatment groups) showed evidence of collagens in the luteinizing granulosa cell layer (Figure 5G, H, J; arrows). Collagen staining was most intense in stroma surrounding follicles injected with SR48692, which showed very limited luteinization (Figure 5I), similar to pre-hCG follicles (Figure 5F). Laminin was detected weakly, if at all, between granulosa cells and in the stroma immediately adjacent to Control and NTS receptor antagonist-injected follicles (Figure 5L–O). Structural luteinization of these follicles was quantified in a prior report [20]. Overall, the presence and distribution of fibronectin, collagen, and laminin correlated most closely with the degree of luteinization present in the follicle did not appear to be strongly influenced by NTS receptor activity. Importantly, these extracellular matrix proteins are present in the monkey ovulatory follicle as theca cells migration from the surrounding stroma into the granulosa cell layer during luteinization.

Theca cell migration is influenced by extracellular matrix

To assess the impact of individual extracellular matrix components on theca migration, porous membranes were pre-coated with an individual extracellular matrix protein (3–10 μg/mL) prior to adding theca cells. Migration was robust when membranes were coated with fibronectin, the matrix used routinely for these studies. Similar to data shown in Figure 3A, NTS increased theca cell migration through the fibronectin coated membrane (Figure 6A). Theca cells migrated through collagen III, but NTS did not increase migration above levels seen without NTS (Figure 6B). Collagen IV permitted theca cell migration, similar to fibronectin and collagen III; NTS increased migration through collagen IV-coated membranes when compared with no NTS (Figure 6C). Interestingly, laminin severely limited migration in the absence and presence of NTS (Figure 6D).

To further explore the impact of laminin on theca migration, matrices were coated with a range of laminin concentrations (0–1 μg/mL). Limited theca cell migration was observed with laminin at concentrations of 0.01–1 μg/mL, while laminin at concentrations of 0.001 and 0.0001 μg/mL permitted limited theca cell migration in this preliminary, dose-ranging experiment (Supplemental Figure S2). In fully-powered experiments, theca cells migrated when plated on no matrix, and treatment with 5 μM NTS increased migration over migration when treated with no NTS (0 μM NTS) (Figure 6E). Theca cells showed some migration when membranes were pre-coated with laminin at a concentration of 0.0001 μg/mL, but NTS did not stimulate migration above levels seen with no NTS (Figure 6F).

Theca cell proliferation is modulated via multiple neurotensin receptors

In vivo studies (Figure 2) indicate that hCG-stimulated NTS increases the number of theca cells present in the follicle 48 h after hCG. To determine if NTS acts directly at theca cells to stimulate proliferation, theca cells were treated with a range of NTS concentrations in vitro. Theca cell proliferation increased in response to NTS in a concentration-dependent manner, with both 5 and 50 μM NTS increasing theca cell number over levels present with no NTS (Figure 7A). Additional theca cells were cultured with no NTS, NTS alone, or NTS + an NTS receptor antagonist (either SR142948, SR48692, or AF38692) (Figure 7B). Neurotensin alone stimulated proliferation above levels seen with no NTS, and each NTS receptor antagonist blocked NTS-stimulated proliferation to levels seen with no NTS.

Figure 7.

Neurotensin promotes theca cell proliferation in vitro. (A) Theca cells (n = 3 lines) were treated in vitro with basal medium (0 μM NTS) or NTS (0.5, 5, or 50 μM) for 24 h and assessed for expression of the proliferation antigen KI67. (B) Theca cells (n = 3 lines) were pretreated with no antagonist, the general NTS receptor antagonist SR142948 (25 μM), the NTSR1 selective antagonist SR48692 (25 μM), or the SORT1 selective antagonist AF38469 (0.1 μM) for 1 h before addition of NTS (5 μM). Cells were assessed for proliferation 24 h after addition of NTS. For all proliferation assays, NTS-stimulated proliferation in each theca line was expressed relative to proliferation in basal medium (no NTS, no antagonist), which was set equal to 100%. For all panels, bars represent mean + SEM, and white circles represent actual data points. Within each panel, data were assessed by ANOVA with one repeated measure and Duncans post hoc test; groups with no common letters are different, P < 0.05. Panels C–F show representative KI67 detection (brown) after treatment with 0 μM NTS (C), 0.5 μM NTS (D), 5 μM NTS (E), and 50 μM NTS (F). In panel C, arrow indicates a KI67+ cell, and arrowheads indicate KI67− cells.

Discussion

In response to the ovulatory gonadotropin surge, theca cells relocate from the periphery of the follicle to the interior of the luteinizing follicle and ultimately become fully integrated into the forming corpus luteum. The initial location of theca cells in the stroma surrounding the dominant ovarian follicle, as well as the final location of theca-lutein cells in mature corpus luteum, have been described by histologists [2]. In the dominant follicle and the corpus luteum, theca cells are located near both the vasculature and granulosa (or granulosa-lutein) cells, an optimal position to access cholesterol-containing lipoproteins and supply androgens as precursors for estrogen synthesis. Cells move within tissues by a variety of methods [37–40]. The mechanism by which theca cells relocate has not been extensively investigated. We have previously demonstrated that the small peptide NTS is produced by granulosa cells of the follicle in response to the ovulatory gonadotropin surge [6–8], functions as a paracrine mediator of ovulation [6], and is required for structural reorganization associated with luteinization of the primate ovulatory follicle [20]. Here we provide support for the concept that theca cells actively migrate in response to NTS as the dominant ovarian follicle ovulates and transforms into the corpus luteum (Figure 8).

Figure 8.

Concept diagram of NTS-stimulated theca migration. 1. The ovulatory gonadotropin stimulus (either the LH surge or hCG) acts at granulosa cells to stimulate the production of NTS. NTS is present at a high concentration in the granulosa cell layer and diffuses into the theca layer 2. Neurotensin binds to and activates NTSR1 and SORT1 located on the theca cell plasma membrane. 3. A gradient of NTS stimulates theca cells to migrate towards the granulosa cell layer. Luteinizing hormone/human chorionic gonadotropin-stimulated luteinization includes alterations in extracellular matrix proteins, such as reduction in laminin, that likely facilitate theca cell migration as the follicle luteinizes and progresses towards ovulation. Created in BioRender. Miller, J. (2025) https://BioRender.com/j1jcc1p

Migration involves cell movement in response to a specific stimulus. In the present report, we confirm that monkey theca cells express NTS mRNA and protein, consistent with initial reports from our group [6, 7]. Importantly, the LH/hCG surge significantly stimulates NTS expression by granulosa cells (reviewed in [9]), rapidly increasing the NTS concentration in the vicinity of the dominant follicle. Evidence that theca cell invasion into the granulosa cell layer is NTS-dependent is provided by the ability of NTS receptor antagonists to reduce theca invasion into the luteinizing follicle in vivo. It is well established that the LH surge or hCG stimulates theca cell movement from the ovarian stroma towards the granulosa cell layer (present study and reviewed in [3]). For this reason, we utilized in vitro migration models to confirm that NTS acts directly at theca cells to promote cell movement through a porous membrane (2D) and in a 3D model of migration. Macaque theca cells do express LHCGR [41]. However, antagonists against one or multiple NTS receptors blocked NTS-stimulated migration in vivo and in vitro, suggesting that NTS effectively promotes theca cell migration. Overall, NTS increases the number of and distance traveled by migrating theca cells. Neurotensin-stimulated migration has also been reported for several types of cancer cells [42–44] as well as macrophages [45] and vascular endothelial cells [20, 46–48]. Taken together, these findings support the concept that theca cells migrate in response to NTS.

Cells migrating must express receptors for the migratory stimulus. Monkey theca cells expressed mRNA and protein for the NTS receptors NTSR1 and SORT1, with NTSR2 not detected. Given the presence of multiple NTS receptors, an antagonist approach was used to query the role of all NTS receptors (SR142948), NTSR1 (SR48692), and SORT1 (AF38692) in theca cell migration in vivo and in vitro. When the NTS receptor antagonist SR142948, SR48692, or AF38469 was injected into a macaque follicle prior to administration of an ovulatory dose of hCG, theca cell migration from the stroma into the dominant follicle was reduced. Similarly, treatment with any of these NTS receptor antagonists prevented NTS-stimulated migration through a porous membrane in vitro. In our prior report of NTS-stimulated migration in monkey ovarian vascular endothelial cells [20], we showed that receptor-selective antagonists or siRNA knockdown of either NTSR1 or SORT1 prevented NTS-stimulated migration, suggesting that both receptors are required to mediate NTS actions. NTSR1 and SORT1 are both high affinity receptors for NTS [49]. NTSR1 is a seven transmembrane domain receptor shown to couple to multiple G proteins [50], while SORT1 has a single transmembrane domain and is best known for its role in intracellular protein trafficking [51]. Neurotensin action at NTSR1 and SORT1 may impact different aspects of the migratory pathway, such that each receptor is independently required for migration. In addition, NTSR1 and SORT1 have been shown to form a complex to generate intracellular signals in response to NTS stimulation [52, 53]. Further studies will be needed to determine if NTSR1 and SORT1 cooperate or operate independently to modulate theca migration into an ovulatory follicle.

Migration is an active process whereby a cell moves in a specific direction. While stiffness of the surrounding extracellular matrix and electrical fields can provide direction, migration is often stimulated by a chemical, with cells moving towards the source of the chemical (reviewed in [54]). Theca cells express NTS mRNA and protein (present report and [6, 7]). However, granulosa cells are likely the major source of NTS in the ovulatory follicle, since NTS mRNA is one of the most highly upregulated mRNAs in granulosa cells after the LH surge (reviewed in [9]). Theca cells migrate towards NTS-producing granulosa cells in vivo, and theca cells migrate towards NTS in a concentration-dependent manner in vitro. Many migrating cells express chemoattractant receptors preferentially on one side of the cell during migration; this asymmetrical sensing of the migratory stimulus defines direction of movement towards the source of the chemoattractant [54]. Additional studies will be needed to determine if NTS receptors are located on the leading edge of migrating theca cells, directing theca movement towards the NTS gradient produced by granulosa cells. Protrusions (typically filopodia or lamellipodia) characterize the leading edge of the migrating cell [55], and actin fibers aligned in the direction of movement interact with myosin to contract and propel the cell forward [56]. Theca cells migrate in 2D as typical migrating cells, with cell protrusions at the leading edge of the cell and actin filaments aligned in the direction of migration. When theca cells migrate in 3D, surrounded by matrix and lacking contact with tissue culture ware, they assume a more elongated phenotype with more pronounced and extended filopodia and lamellipodia. Accordingly, NTS treatment augmented the migratory features of this phenotype in 3D matrix by increasing the length and number of filopodia-like structures extending out and away from the cell body and nucleus. Future studies will be needed to further investigate the structural features of theca cells as they migrate in 2D and 3D. However, our findings do support the concept that NTS forms a chemical gradient to attract theca cells towards granulosa cells during ovulatory and luteal formation.

In addition to promoting theca cell migration, NTS also increases proliferation of theca cells. In vivo, hCG increased the number of theca cells detected in the luteinizing follicle, and this increase in theca cell number was reduced when an NTS receptor antagonist was injected into the follicle. It is possible that NTS receptor antagonists reduced detection of theca cells in vivo by causing theca cell death. However, these ovarian follicles did not show TUNEL staining in a prior study [20], suggesting that theca cell apoptosis was not induced by administration of hCG or intrafollicular injections of NTS receptor antagonists. Additional studies confirmed that NTS stimulates theca cell proliferation in a concentration-dependent manner in vitro, and NTS-stimulated proliferation was blocked by the pan-NTS receptor antagonist SR142948, the NTSR1 selective antagonist SR48692, or the SORT1 antagonist AF38692. Neurotensin action at NTSR1 and SORT1 has been reported to stimulate both migration and proliferation in a variety of cancer cell models [57]. Proliferation can be viewed as a confounding variable in some migration assays. For example, an increase in the number of theca cells could ultimately be detected as migration in the membrane migration assay and could result in an increase in the number of migrating cells in the 3D migration assay. However, theca invasion in vivo and migration distance in the 3D migration assay in vitro are unlikely to be influenced by an increase in theca cell number. In a prior report, we observed that NTS stimulated migration, but not proliferation, in monkey ovarian microvascular endothelial cells [6]. The observation that NTS stimulates both migration and proliferation of theca cells is unusual in that activation of a receptor by a stimulus will typically promote either migration or proliferation due to regulation of small GTPases Rac and Rho. Active Rac supports migration and inhibits Rho, while Rho supports proliferation [55]. VEGFA can stimulate both migration and proliferation in monkey and human ovarian microvascular endothelial cells [58, 59]. However, the ability of VEGFA to promote proliferation is mediated via different receptors, with KDR stimulating migration, while proliferation is mediated via FLT1 [58]. Our findings suggest that both NTSR1 and SORT1 mediate NTS-stimulated migration and proliferation in vivo and in vitro. The intracellular signals generated by NTS-receptor binding and how these signals regulate small GTPases and other cell functions associated with migration and proliferation will require further study.

Cell migration is influenced by extracellular matrix components. Prior reports of human and bovine follicles confirm dynamic regulation of extracellular matrix proteins in the granulosa cell layer and surrounding stroma of the dominant follicle at the time of ovulation, including fibronectin [60, 61], collagens [31, 32, 35, 62], and laminin [32, 35, 63, 64] . Consistent with this literature, our results show that the monkey dominant follicle includes significant amounts of fibronectin, collagens, and laminin. In our studies in vitro, theca cells migrated through membranes coated with fibronectin, collagen III, and collagen IV. The very limited ability of theca cells to migrate through laminin-coated membranes in our 2D migration assay indicates that laminin forms a barrier to block theca migration. Our studies are consistent with the concept that the coordinated breakdown of laminin, located in the basal lamina and granulosa cell layer of the dominant follicle, is required for theca cells to migrate into the follicle during ovulation. Migrating cells express integrins at the leading edge of the cell, which anchor the front of the cell to extracellular matrix; actin/myosin contraction moves the rear of the cell towards the integrin attachment points [56]. RNAseq studies indicate that theca cells express a variety of integrins [65–67]. Of particular importance may be ITGA6/ITGB1, which has been previously shown to limit cell movement [68]. Our findings suggest that reduction in or removal of laminin is necessary to permit theca migration in vivo. Luteinizing hormone/human chorionic gonadotropin-stimulated changes in the location of ECM proteins were noted in monkey follicles, most notably decreased detection of laminin in the basal lamina and granulosa cell layer of the luteinizing follicle. Our qualitative histological findings do not support a key role for NTS in the removal of laminin during the transformation of the dominant follicle into a young corpus luteum. A migrating theca cell would begin its journey in the ovarian stroma, move through the granulosa basal lamina, and enter the follicle along with vessels as stromal tissue integrates into the granulosa cell layer. Once laminin has been removed, NTS can stimulate theca cell migration into the luteinizing granulosa cell layer, along with vessels and stromal matrix components, as a key step in the formation of the corpus luteum.

Author contributions

J.S.M.: developed concept, generated and analyzed data, drafted and approved manuscript. M.A.G.S.: developed concept, generated and analyzed data, drafted and approved manuscript. T.E.C.: obtained funding, developed concept, approved manuscript. D.M.D.: obtained funding, developed concept, generated and analyzed data, drafted and approved manuscript.

Conflict of Interest: The authors have no conflicts of interest to declare.

Data availability

All data are included in the manuscript.