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. 2015 Sep;156(9):3358-69.
doi: 10.1210/en.2014-1874. Epub 2015 Jun 30.

Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

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Chemokine Ligand 20: A Signal for Leukocyte Recruitment During Human Ovulation?

Linah Al-Alem et al. Endocrinology. .
Free PMC article

Abstract

Ovulation is one of the cornerstones of female fertility. Disruption of the ovulatory process results in infertility, which affects approximately 10% of couples. Using a unique model in which the dominant follicle is collected across the periovulatory period in women, we have identified a leukocyte chemoattractant, chemokine ligand 20 (CCL20), in the human ovary. CCL20 mRNA is massively induced after an in vivo human chorionic gonadotropin (hCG) stimulus in granulosa (>10 000-fold) and theca (>4000-fold) cells collected during the early ovulatory (12-18 h) and late ovulatory (18-34 h) periods after hCG administration. Because the LH surge sets in motion an inflammatory reaction characterized by an influx of leukocytes and CCL20 is known to recruit leukocytes in other systems, the composition of ovarian leukocytes (CD45+) containing the CCL20 receptor CCR6 was determined immediately prior to ovulation. CD45+/CCR6+ cells were primarily natural killer cells (41%) along with B cells (12%), T cells (11%), neutrophils (10%), and monocytes (9%). Importantly, exogenous CCL20 stimulated ovarian leukocyte migration 59% within 90 minutes. Due to the difficulties in obtaining human follicles, an in vitro model was developed using granulosa-lutein cells to explore CCL20 regulation. CCL20 expression increased 40-fold within 6 hours after hCG, was regulated partially by the epithelial growth factor pathway, and was positively correlated with progesterone production. These results demonstrate that hCG dramatically increases CCL20 expression in the human ovary, that ovarian leukocytes contain the CCL20 receptor, and that CCL20 stimulates leukocyte migration. Our findings raise the prospect that CCL20 may aid in the final ovulatory events and contribute to fertility in women.

Figures

Figure 1.
Figure 1.
CCL20 expression across the ovulatory period. CCL20 mRNA increases in the early and late ovulatory phase in human ovary granulosa (A) and theca (B) cells. Results are the means ± SEM of n = 4–5. Bars that do not share a letter designation are significantly different (P < .05). Immunostaining of CCL20 protein expression and localization in the different stages of the human ovulatory cycle is shown. CCL20 staining is shown for PO (C), EO (D), LO (E), and negative control (F). Images are representative images from three patient samples. Lower magnification composite of a late ovulatory follicle illustrates CCL20 immunostaining (G). The boxed area depicts the approximate area shown in panel E. Scale bars (C–F), 100 μm; scale bar (G), 1000 μm.
Figure 2.
Figure 2.
CCR6-positive leukocytes are present in human IVF samples. A, Number of CD45+ cells, CCR6+ cells, and double-positive (CD45+CCR6+) cells. The approach to determine these percentages is illustrated in panels B–D. Representative figure of an unstained IVF sample and one in which the gates were set (B) are shown. An IVF sample that was stained for only CD45 (C), stained for only CCR6 (D), or a representative patient sample (E) is also shown.
Figure 3.
Figure 3.
Analysis of the leukocyte subtypes and the CCR6+ leukocyte population subtypes in human IVF samples. A, Distribution of various CD45+ leukocyte subtypes. Representative flow cytometry histograms for one patient are shown (B–H). B, Side scatter plot for cells stained for CD45+. Panels C–H depict an IVF sample that was stained for CD45 in conjunction with antibodies for one of the following: CCR6 (B), neutrophils (CD66b) (C), monocytes (CD14) (D), B cells (CD19) (E), NK cells (CD56) (F), or T cells (CD3) (G). The distribution of leukocyte subtypes that are CCR6+ is shown (H). Panels I–M are representative flow cytometry histograms for one patient. Panels depict an IVF sample that was stained for CD45 and CCR6 in conjunction with antibodies for one of the following: neutrophils (CD66b) (I), monocytes (CD14) (J), B cells (CD19) (K), NK cells (CD56) (L), or T cells (CD3) (M). Results are means ± SEM of n = 7 each. Bars that do not share a letter designation are significantly different (P < .05).
Figure 4.
Figure 4.
Human GLC expression of ovulatory genes in vitro. Human GLCs were cultured for 7 days and then serum starved for 1 hour in the presence or absence of hCG or vehicle control. Expression of mRNA for PTGS2 (A), AREG (C), and PGR (E) across days of culture and after temporal treatment of GLCs with hCG for PTGS2 (B), AREG (D), and PGR (F). Progesterone secretion increased after hCG treatment (G). Results are the means ± SEM of n ≥ 3. Coll, samples at the time of IVF collection. 0 hours represents samples after 6–7 days in culture at the initiation of hCG treatment. Bars that do not share a letter designation are significantly different (P < .05).
Figure 5.
Figure 5.
Human GLC expression of target genes in vitro. Human GLCs were cultured for 7 days and then serum starved for 1 hour in the presence or absence of hCG or vehicle control. Expression of mRNA for CCL20 (A), CCR6 (C), and CD45 (E) across days of culture and after temporal treatment of GLCs with hCG for CCL20 (B), CCR6 (D), and CD45 (F). Results are the means ± SEM of n = 3. Coll, samples at the time of IVF collection. 0 hours represents samples after 6–7 days in culture at the initiation of hCG treatment. Bars that do not share a letter designation are significantly different (P < .05).
Figure 6.
Figure 6.
The EGF pathway is involved in the induction of CCL20 expression in GLCs in vitro. Human GLCs were cultured for 7 days and then serum starved for 1 hour in the presence or absence of the following inhibitors: EGF receptor tyrosine kinase selective inhibitor, AG1478 (AG; 1 μM) (A), RU486 (RU; 1 μM) (B), PTGS-2 inhibitor, NS-398 (NS; 1 μM) (C), or the inhibitor of PTGS-1 and PTGS-2, indomethacin (200 nM) (D). Cells were then treated with or without hCG for 6 hours. Results are the means ± SEM. Bars that do not share a letter designation are significantly different (P < .05).
Figure 7.
Figure 7.
Proposed model for CCL20 action. The LH surge leads to an increased expression of CCL20 in the granulosa and theca cells of the dominant follicle. The presence of CCL20 causes leukocytes from the bloodstream that have CCR6 to migrate into the ovary to aid in the breakdown of the follicular wall leading to oocyte expulsion.

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