Human fallopian tube epithelium co-culture with murine ovarian follicles reveals crosstalk in the reproductive cycle

Abstract

Study question: Do interactions between human fallopian tube epithelium and murine follicles occur during an artificial reproductive cycle in a co-culture system in vitro?

Summary answer: In a co-culture system, human fallopian tissues responded to the menstrual cycle mimetic by changes in morphology and levels of secreted factors, and increasing murine corpus luteum progesterone secretion.

What is known already: The entire fallopian tube epithelium, including ciliated and secretory cells, can be regulated in the reproductive cycle. Currently, there are no in vitro culture models that can monitor fallopian tissues in real time in response to factors produced by the ovary. In addition, there are no reports on the impact of fallopian tissue on ovarian function during the menstrual cycle.

Study design, samples/materials, methods: Human fallopian tissue (n = 24) was obtained by routine hysterectomies from women (aged 26-50 years, mean age = 43.6) who had not undergone exogenous hormonal treatment for at least 3 months prior to surgery. CD1 female mice were used for ovarian follicle isolation. The human fallopian epithelium layers were either co-cultured with five murine multilayer secondary follicles (150-180 μm follicles, encapsulated in one alginate gel bead) for 15 days or received stepwise steroid hormone additions for 13 days. The fallopian tissue morphology and cilia beating rate, as measured by an Andor Spinning Disk Confocal, were investigated. Oviduct-specific glycoprotein 1 (OVGP1), human insulin-like growth factor 1 (hIGF1), vascular endothelial growth factor A (VEGF-A) and interleukin 8 (IL8) as biological functional markers were measured either by ELISA or western blot to indicate dynamic changes in the fallopian epithelium during the reproductive cycle generated by mouse follicles or by stepwise steroid hormone induction. Three or four patients in each experiment were recruited for replicates. Data were presented as mean ± SD and further analyzed using one-way ANOVA followed by Tukey's multiple comparisons test.

Main results and the role of chance: The cultured fallopian tube epithelium responded to exogenous steroid hormone stimulation, as demonstrated by enhanced cilia beating rate (~25% increase, P = 0.04) and an increase in OVGP1 secretion (P = 0.02) in response to 1 nM estradiol (E₂) treatment when compared with 0.1 nM E₂. Conversely, 10 nM progesterone plus 1 nM E₂ suppressed cilia beating rate by ~30% (P = 0.008), while OVGP1 secretion was suppressed by 0.1 nM E₂ plus 50 nM progesterone (P = 0.002 versus 1 nM E₂ alone). Human fallopian tube epithelium was co-cultured with murine secondary follicles to mimic the human menstrual cycle. OVGP1 and VEGF-A secretion from fallopian tissue was similar with stepwise hormone treatment and when cultured with murine follicles. However, the secretion patterns of hIGF1 and IL8 differed in the luteal phase when comparing steroid treatment with follicle co-culture. In co-culture, hIGF1 secretion was suppressed in the luteal versus follicular phase (P = 0.005) but stepwise hormone treatment had no effect on hIGF1. In co-culture, IL8 secretion was also suppressed on luteal phase day 15 (P = 0.013) versus follicular phase day 7, but IL8 secretion increased continuously under high E₂/progesterone treatment (P = 0.003 for D13 versus D3). In the co-culture system, the corpus luteum continuously produced progesterone in the presence of fallopian tube tissue until Day 18 while, without fallopian tissue, progesterone started to drop from Day 13.

Limitations, reasons for caution: One limitation of this study is that murine follicles were used to mimic the human menstrual cycle. However, although secretion patterns of peptide hormones such as inhibins and activins differ in mice and humans, the co-culture system used here did reveal interactions between the tissues that govern reproductive function.

Wider implications of the findings: In vitro co-culture models of fallopian reproductive tissues with ovarian follicles can provide an important tool for understanding fertility and for uncovering the mechanisms responsible for reduced fertility. In addition, the role of oviductal secretions and how they influence ovarian function, such as the production of progesterone during the menstrual cycle, can be uncovered using this model.

Large-scale data: None.

Study funding and competing interests: This work was funded by grants from the NIH (UH3TR001207), the American Cancer Society (RSG-12-230-01-TBG) and NIH (R01EB014806). The authors declare no competing financial interest.

Keywords: co-culture; fallopian tube; follicle; in vitro culture; luteal deficiency; menstrual cycle.

Figure 1

Human fallopian tube in vitro culture on the insert membrane. (A) The human fallopian tube was cut open and the epithelial layer was mechanically isolated. The epithelial layer was cut into 2 × 2 mm pieces and cultured on the insert membrane. Scale bar: 1 mm. (B) The epithelial layer was treated with low dose of estradiol (E₂) 0.1 nM for 14 days. Hematoxylin and eosin staining was used to evaluate the morphology of human fallopian tube epithelial layer after 14 days in culture compared with uncultured tissue. ERα, PR and oviduct-specific glycoprotein 1 (OVGP1) immunofluorescent staining was performed to characterize the human fallopian epithelial culture system. The scale bar indicated 100 μm. ER, estrogen receptor; PR, progesterone receptor.

Figure 2

Functional response of human fallopian tube epithelium cultured in the presence of E₂ and P₄ for 7 days. The human epithelial layers were treated with E₂ 0.1 nM, E₂ 1 nM, E₂ 1 nM + P₄ 10 nM or E₂ 0.1 nM + P₄ 50 nM for 7 days to mimic different stages of menstrual cycle. (A) After 7 days of culture in the indicated treatments, cilia beating frequency was measured using an Andor spin disk microscope with a 100 × objective and a 5-ms exposure time and 5-ms readout time. Quantification of cilia beating frequency for n = 3 fallopian endometrium cultures from 3 individual patients. (B) Immunoblot of OVGP1 in cultured fallopian epithelium after treatment described above for 7 days. Bar graph represents relative band density using α-tubulin as the loading control for n = 4 cultured fallopian endometrium lysates from four patients. ‘*’statistically significant differences between groups. The data are mean ± SD. A one-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, and P < 0.05 was considered statistically significant.

Figure 3

Human fallopian epithelium and murine follicle co-culture system mimic the menstrual cycle in vitro. (A) Schematic of follicle and fallopian epithelium co-culture model. Five secondary follicles (150 –180 μm) were encapsulated into a single 0.5% alginate bead, which was placed in the bottom of each well. Fallopian epithelium was cultured on a 0.4-μm insert membrane, which was placed into the 12-well plate containing encapsulated follicles. (B) Steroid secretion pattern in the follicle and fallopian co-culture system. E₂ and P₄ levels were measured in co-culture medium at different time points. The co-cultures were maintained in growth medium supplemented with 10 mIU/ml recombinant human FSH (rhFSH) for first 7 days. After 7 days, the follicles were treated with 1.5 IU/ml hCG for 16 h to induce IVM. The luteinized follicles were then cultured for another 7 days without rhFSH and hCG. ‘FO’ represented ‘follicle’ and ‘FA’ represented ‘fallopian’. Three patients’ tissue was included in this co-culture system. (C) Morphology of the fallopian epithelial tissue cultured alone or co-cultured with follicles for 7 days and 15 days. Scale bar: 100 μm in size.

Figure 4

Factors from the human fallopian epithelium undergo dynamic changes in the co-culture system. (A) Immunoblot of OVGP1 level in cultured fallopian epithelium in the co-culture system or fallopian culture only. The first 7 days for both co-culture and fallopian culture only has rhFSH present in the culture medium. On Day 7, the hCG was added in culture media for the co-culture system. On Day 8, the media for the co-culture was changed to the media without hCG and rhFSH. The fallopian tissue was harvested on Days 7 and 15. The tissue lysate was used for the OVGP1 western blot. The alpha-tubulin was used as loading control. The immunoblot represents the tissue status from one patient. Bar graph represents relative band density using α-tubulin as the loading control for n = 4 cultured fallopian epithelium lysates from four patients. ‘*’ corresponded to statistically significant differences between groups. The data are mean ± SD. A one-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, and P < 0.05 was considered statistically significant. (B) Human insulin-like growth factor 1 (hIGF1) levels were measured in the conditioned medium of the co-culture system at different time points by immunoblot analysis. Bar graph represents relative band density compared to culture day 5. Three individual experiments were performed using fallopian tissues from three patients. (C) Human vascular endothelial growth factor A (hVEGF-A) level and (D) human interleukin 8 (IL8) were measured in the conditioned medium of the co-culture system at different time points by human VEGF-A ELISA kit or human IL8 ELISA kit. Three patients were recruited for the experiments and two independent cultures for one experiment. * and ** are statistically significant differences between groups. The data are mean ± SD. One-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, P < 0.05 was considered statistically significant. ‘FO’ represented ‘follicle’ and ‘FA’ represented ‘fallopian’ in all of the panels.

Figure 5

Fallopian tube response to stepwise exogenous steroid hormone treatment. (A) Schematic of the stepwise exogenous steroid hormone treatment regimen. For the first 3 days, the fallopian tissue was treated with E₂ 0.1 nM followed by the increasing E₂ concentration 10 nM to match the E₂ level in the co-culture system for another 3 days. For the first 6 days, the growth medium was supplemented with 10 mIU/ml rhFSH to mimic the follicular phase. On Day 6, the tissues were treated with 1.5 IU/ml hCG to mimic the ovulation stage for 16 h, on Day 7 the culture medium changed into the growth medium without rhFSH and hCG, but with E₂ 10 nM and P₄ 10 nM for 3 days. On Day 10, the tissue was treated with E₂ 0.1 nM and P₄ 50 nM for another 3 days. (B) Immunoblot of OVGP1 in cultured fallopian epithelium in the stepwise steroid hormone treatment regimen. The fallopian tissue was harvested on Days 3, 6, 10 and 13. The tissue lysate was used for the OVGP1 western blot. The alpha-tubulin was used as loading control. The immunoblot represents one patient's tissue status. Bar graph represents relative band density using α-tubulin as the loading control for n = 3 cultured fallopian epithelium lysates from three women. The data are mean ± SD. ‘*’ Corresponded to statistically significant differences between groups. A one-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, and P < 0.05 was considered statistically significant. (C) hIGF1 levels were measured in the conditioned medium of the stepwise steroid hormone treatment at different time points by immunoblot analysis. Bar graph represents relative band density compared to culture day 3. Three individual experiments were performed using fallopian tissues from three patients. (D) hVEGF-A and (E) hIL8 were measured in the conditioned medium of stepwise steroid hormone treatment regimen at different time points using a human VEGF-A ELISA kit or human IL8 ELISA kit. Three individual experiments were performed using fallopian tissues from three patients, and two individual cultures for each experiment. The data are mean ± SD. ‘*’ corresponded to statistically significant differences between groups. One-way ANOVA followed by Tukey's multiple comparisons test was used for statistical analysis, P < 0.05 was considered statistically significant.

Figure 6

Human fallopian epithelium in the co-culture system affects murine follicle steroid hormone secretion. The E₂ (A) and P₄ (B) levels in the condition medium at the different time points from either the human fallopian epithelium and murine follicle co-culture system or from the murine follicle culture alone system. The corpora luteum either from co-culture or follicle-only culture after 18 days were fixed for the histological analysis (C, D). Three independent experiments were performed using tissue from three different patients. The data are mean ± SD.