Rediscovering the rete ovarii, a secreting auxiliary structure to the ovary
- Department of Cell Biology, Duke University Medical Center, United States
- Section of Developmental Biology, Department of Pediatrics, University of Colorado Anschutz Medical Campus, United States
Figures
Figure 1 with 1 supplement
The rete ovarii (RO) undergoes dynamic changes during development alongside the ovary.
(a–d, top row) Diagrams depicting the development of the three regions (extraovarian rete [ER], magenta; connecting rete [CR], cyan; intraovarian rete [IOR], yellow) of the RO from E16.5 to P7 in the whole ovarian complex (ovary, light pink; oviduct, gray). (a–d, bottom rows) Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at E16.5 (a), E18.5 (b), P0 (c), and P7 (d) immunostained for PAX8 (yellow), GFRa1 (cyan), and KRT8 (magenta). (a–d, second row) are composite images, while the third row shows separate panels for KRT8 and the bottom row shows panels for GFRa1. *Note that GFRa1 and KRT8 do not co-localize and are specific to the CR and EOR, respectively. All figures are dorsal views of the ovary. Yellow asterisk indicates opening of the infundibulum for reference. Scale bar – 100 um.
Figure 1—figure supplement 1
Ventral view of ovary and extraovarian rete (EOR) during development.
Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at E16.5 (a), E18.5 (b), P0 (c), and P7 (d) immunostained for PAX8 (yellow), GFRa1 (cyan), and KRT8 (magenta). Top row are composite images, while the third row shows separate panels for KRT8 and the bottom row shows panels for GFRa1. *Note that GFRa1 and KRT8 do not co-localize and are specific to the connecting rete (CR) and EOR, respectively. All figures are dorsal views of the ovary. Yellow asterisk indicates opening of the infundibulum for reference. Scale bar – 100 um.
Figure 2
The extraovarian rete (EOR) is highly integrated with its extraovarian environment suggesting multifaceted communication.
(a) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for ENDOMUCIN (magenta) and PAX8 (cyan). Bottom panel is an optical section showing vasculature tightly surrounding the distal dilated tip (DDT) of the EOR (magenta). (b) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for aSMA (magenta) and KRT8 (cyan). Bottom panel is an optical section showing the EOR tightly ensheathed by smooth muscle (magenta). (c) Top panel is a whole ovarian complex maximum intensity projection of the confocal Z-stack at E18.5 immunostained for TUJ1 (magenta) and PAX8 (cyan). Bottom panel is an optical section showing direct contacts between the EOR and neuronal projections (magenta). (d) EOR at P0 immunostained for PAX8 (yellow), F4/80 (magenta), and LYVE-1 (cyan). Top and bottom panels are maximum intensity projection of the confocal Z-stack. Bottom panel shows the absence of LYVE1 macrophages (cyan) proximate to the EOR, in contrast to the closely associated F4/80 macrophages (magenta in top image). Arrowheads show regions devoid of LYVE1 macrophages where F4/80 macrophages are present. Scale bar – 100 um.
Figure 3
Fluid moves from the extraovarian rete (EOR) to the ovary.
(a, b) Schematic of dextran injections (syringe) for each group (a), EOR injections; (b,), ovary injections. Oviduct, gray; ovary, light pink; intraovarian rete (IOR), yellow; connecting rete (CR), cyan; EOR, magenta. (Second row) Maximum intensity projection of a confocal Z-stack of whole ovarian complexes at P7, where dextran was injected into the EOR. Presence of dextran in the ovary shows that when dextran is injected into the EOR, it diffuses throughout the ovary (dextran, cyan; E-CADHERIN, magenta). (Bottom row) Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at P7 where dextran was injected into the ovary. Absence of dextran signal in the EOR shows that dextran did not travel into the EOR when injected into the ovary near the IOR. Scale bar – 100 um.
Figure 4
The extraovarian rete (EOR) and connecting rete (CR) acquire the potential for fluid transfer.
(a–d) Cells of the CR acquire E-CADHERIN soon after birth. Maximum intensity projection from confocal Z-stacks of whole ovary/mesonephros complexes at E16.5-P7 immunostained for PAX8 (magenta) and E-CADHERIN (cyan). Arrowhead shows the region of the CR where E-CADHERIN expression is negligible at E16.5 (a), low at E18.5 (b), and fully expressed at P0 (c) and P7 (d). (e) Optical section from confocal Z-stacks of RO complexes at P0 immunostained for E-CADHERIN (yellow), CNN1 (magenta), and aSMA (cyan). Boxed regions show the absence of CNN1 in the aSMA+ sheath around the EOR at P0, and (f) its acquisition by P7, suggesting gain of contractility. (g) The RO expresses cilia marker ARL13b by P0. Maximum intensity projection from confocal Z-stacks of RO complexes at E18.5 immunostained for E-CADHERIN (cyan) and ARL13b (magenta). Outlined boxes show all regions of the EOR have ciliated cells. Scale bar – 100 um. (h) Airyscan optical section from confocal Z-stacks of RO complexes at E18.5 immunostained for KRT8 (cyan) and ARL13b (magenta). Scale bar – 25 um.
Figure 5
Presence of SNARE-complex members suggests a role for secretion.
(a) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) extraovarian rete (EOR) at E18.5 using hybridization chain reaction (HCR) for the detection of Clu expression (magenta). (b) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at E18.5 using HCR for Stxbp2 (magenta). (c) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for Clu (magenta). (d) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for Stxbp2 (magenta). (e) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-Cre; Rosa26mTmG (cyan) EOR at E18.5 immunostained for STX3 (magenta). Outlined higher resolution image acquired with Airyscan. (f) Optical section from confocal Z-stacks of PAX8-rtTa; Tre-Cre; Rosa26mTmG (cyan) EOR at E18.5 immunostained for RAB11 (magenta). Outlined higher resolution image acquired with Airyscan. Scale bar – 100 um.
Figure 6
Validation of Igfbp2 expression in the rete ovarii (RO).
(a) Maximum intensity projection of confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) extraovarian rete (EOR) at E18.5 using hybridization chain reaction (HCR) for the detection of IGFBP2 expression (magenta). (b) Maximum intensity projection of confocal Z-stacks of PAX8-rtTa; Tre-H2B-GFP (cyan) EOR at P7/8 using HCR for the detection of IGFBP2 expression (magenta). Bottom panels are outlined. Higher resolution images acquired with Airyscan. Scale bar top panel – 100 um. Scale bar bottom panel – 25 um.
Videos
Video 1
3D model of an XX ovarian complex at E18.5.
This video depicts the 3D rendering of native lightsheet images of an E18.5 ovary/mesonephros complex. It demonstrates how the rete wraps around the ovary. DNA (gray) ovarian surface epithelium (LGR5; green) and PAX8 (red).
Tables
Table 1
Cells of the extraovarian rete (EOR) secrete proteins essential for vesicle transport.
Candidate list of 15 proteins identified by mass spectrometry. Asterisked proteins were selected for validation due to role in vesicle and protein transport.
| Protein | Role |
|---|---|
| Agt | Pro-peptide for angiotensinogen |
| Bcam | Glycoprotein |
| C3 | Complement component |
| Cd200 | Glycoprotein |
| Cfi | Serine proteinase |
| Clu* | Secreted chaperone |
| Cp | Metalloprotein |
| Cpm | Membrane-bound arginine/lysine carboxypeptidase |
| Epcam | Antigen |
| Igfbp2 | Binds insulin-like growth factors I and II |
| Lama5 | Laminin |
| Napsa | Pro-peptide |
| Sema3c | Secreted glycoprotein |
| Slit3 | Secreted protein |
| Stxbp2* | Binds syntaxin |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | Crl:CD1(ICR) | Charles River | Strain code: 022; RRID:IMSR_CRL:022 | |
| Strain, strain background (M. musculus) | C57BL/6J | Jackson Laboratory | Stock #:000664; RRID:IMSR_JAX:000664 | |
| Genetic reagent (M. musculus) | Tre-H2B-Gfp (Tg(tetO-HIST1H2BJ/GFP)47Efu/J) | PMID:14671312 | MGI:J:90563; RRID:IMSR_JAX:005104 | |
| Genetic reagent (M. musculus) | Tre-Cre (B6.Cg-Tg(tetO-cre)1Jaw/J) | PMID:12145322 | MGI:J:78365; RRID:IMSR_JAX:006234 | |
| Genetic reagent (M. musculus) | mTmG Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo/J | PMID:17868096 | MGI:J:124702; RRID:IMSR_JAX:007576 | |
| Genetic reagent (M. musculus) | Pax8-rtTA (B6.Cg-Tg(Pax8-rtTA2S*M2)1Koes/J) | PMID:18724376 | MGI:J:140925; RRID:IMSR_JAX:007176 | |
| Genetic reagent (M. musculus) | Lgr5 (B6.129P2-Lgr5tm1(cre/ERT2)Cle/J) | PMID:17934449 | MGI:J:127123; RRID:IMSR_JAX:008875 | |
| Antibody | Smooth muscle alpha action (aSMA) (Cy3-conjugated mouse monoclonal) | Sigma-Aldrich | C6198; RRID:AB_476856 | (1:1000) |
| Antibody | E-Cadherin (rat monoclonal) | Zymed (Thermo Fisher Scientific) | 13-1900; RRID:AB_2533005 | (1:500) |
| Antibody | Endomucin (rat monoclonal) | Santa Cruz Biotechnology | sc-65495; RRID:AB_2100037 | (1:500) |
| Antibody | GFP (chicken polyclonal) | Abcam | ab13970; RRID:AB_300798 | (1:1000) |
| Antibody | GFRa1 (goat polyclonal) | R&D Systems | AF560; RRID:AB_2110307 | (1:150) |
| Antibody | KRT8 (rat monoclonal) | DSHB | TROMA-I; RRID:AB_531826 | (1:250) |
| Antibody | PAX8 (rabbit polyclonal) | Proteintech | A10336-1-AP; RRID:AB_2918972 | (1:500) |
| Antibody | CNN1 (rabbit polyclonal) | Proteintech | 13938-1-AP; RRID:AB_2082010 | (1:200) |
| Antibody | ARL13b (rabbit polyclonal) | Proteintech | 17711-1-AP; RRID:AB_2060867 | (1:1000) |
| Antibody | RAB11 (rabbit monoclonal) | Cell Signaling Technology | 5589; RRID:AB_10693925 | (1:500) |
| Antibody | LYVE-1 (goat polyclonal) | R&D Systems | AF2125; RRID:AB_2297188 | (1:500) |
| Antibody | F4/80 (rat monoclonal) | Bio-Rad | MCA497RT; RRID:AB_1102558 | (1:2000) |
| Antibody | STX3 (rabbit monoclonal) | Abcam | ab133750 | (1:200) |
| Antibody | TUJ1 (488-conjugated mouse monoclonal) | BioLegend | A488-435L; RRID:AB_10143904 | (1:1000) |
| Antibody | AF647 anti-Rabbit (donkey polyclonal) | Jackson ImmunoResearch | 711-605-152; RRID:AB_2492288 | (1:500) |
| Antibody | AF488 anti-Chicken (donkey polyclonal) | Jackson ImmunoResearch | 703-545-155; RRID:AB_2340375 | (1:500) |
| Antibody | AF488 anti-Rat (donkey polyclonal) | Life Technologies | A-21208; RRID:AB_2535794 | (1:500) |
| Antibody | Cy3 anti-Goat (donkey polyclonal) | Jackson ImmunoResearch | 705-165-147; RRID:AB_2307351 | (1:500) |
| Sequence-based reagent | Stxbp2 (B3 amplifier) | Molecular Instruments | Accession #: XR_001778418.1 | |
| Sequence-based reagent | Clu (B1 amplifier) | Molecular Instruments | Accession #: NM_013492.3 | |
| Sequence-based reagent | IGFBP2 (B3 amplifier) | Molecular Instruments | Accession #: NM_008342.3 | |
| Sequence-based reagent | F647 (B3 amplifier) | Molecular Instruments | ||
| Sequence-based reagent | F546 (B1 amplifier) | Molecular Instruments | ||
| Chemical compound, drug | Dextran, Alexa Fluor 568; 10,000 MW | Thermo Fisher | D22912 | |
| Chemical compound, drug | Dichloromethane | MilliporeSigma | 270997-1L | |
| Chemical compound, drug | Benzyl Ether | MilliporeSigma | 108014-1KG | |
| Chemical compound, drug | Quadrol=N,N,N′,N′-Tetrakis(2-Hydroxypropyl)ethylenediamine | MilliporeSigma | 122262 | |
| Software, algorithm | Zen Black Edition | Carl Zeiss | ||
| Software, algorithm | Imaris v9.6 | Bitplane | ||
| Software, algorithm | Adobe Creative Cloud | Adobe | Photoshop, Illustrator, Premier Pro |
Additional files
-
MDAR checklist
- https://cdn.elifesciences.org/articles/96662/elife-96662-mdarchecklist1-v1.docx
-
Supplementary file 1
PCR Genotyping Primer Sequences.
- https://cdn.elifesciences.org/articles/96662/elife-96662-supp1-v1.xlsx
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Rediscovering the rete ovarii, a secreting auxiliary structure to the ovary
eLife 13:RP96662.
https://doi.org/10.7554/eLife.96662.3
