2023.11.08.566085v2.full-html.html

Abstract

The

rete ovarii

(RO) is an appendage of the ovary that has been given little attention. Although the RO appears

in drawings of the ovary in early versions of Gray’s Anatomy, it disappeared from recent textbooks, and is

often dismissed as a functionless vestige in the adult ovary. Using PAX8 immunostaining and confocal

microscopy, we characterized the fetal development of the RO in the context of the ovary. The RO consists

of three distinct regions that persist in adult life, the intraovarian rete (IOR), the extraovarian rete (EOR), and

the connecting rete (CR). While the cells of the IOR appear to form solid cords within the ovary, the EOR

rapidly develops into a convoluted tubular epithelium ending in a distal dilated tip. Cells of the EOR are ciliated

and exhibit cellular trafficking capabilities. The CR, connecting the EOR to the IOR, gradually acquires tubular

epithelial characteristics by birth. Using microinjections into the distal dilated tip of the EOR, we found that

luminal contents flow towards the ovary. Mass spectrometry revealed that the EOR lumen contains secreted

proteins potentially important for ovarian function. We show that the cells of the EOR are closely associated

with vasculature and macrophages, and are contacted by neuronal projections, consistent with a role as a

sensory appendage of the ovary. The direct proximity of the RO to the ovary and its integration with the

extraovarian landscape suggest that it plays an important role in ovary development and homeostasis.

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Introduction

The

Rete Ovarii

(RO) is an epithelial structure that is directly connected to the ovary, first described over a

century ago (Waldeyer, 1870) as a multi-region structure of mesonephric origin. Despite high conservation

among mammalian species including guinea pigs, cows, cats, sheep, swine, prairie deer mice, camels, dogs,

monkeys (Cassali et al., 2000; Kim et al., 2012), cows (Santos et al., 2012), and humans (Khan et al., 1999),

previous work on the RO did not arrive at a consensus on the function of this structure. Thus, it has remained

a mysterious orphan structure.

Although the RO has been proposed to be the female homologue of the rete testis (Wenzel and Odend'hal,

1985), our recent work revealed that it is more complex (McKey et al., 2022). The RO is composed of three

distinct regions: (1) the extraovarian rete (EOR) which consists of columnar epithelial cells that create a single

convoluted tubule structure ending in a blind distal dilated tip (DDT), (2) the connecting rete (CR) which

consists of pseudo-columnar cells; and (3) the intraovarian rete (IOR) which consists of squamous epithelial

cells that branch and form a fine network of thin solid cell cords approximately 1-3 cells thick (Byskov and

Lintern-Moore, 1973; McKey et al., 2022). It was previously shown that the RO consists of ciliated and non-

ciliated cells rich in apical microvilli and mitochondria (Czernobilsky et al., 1985). Fine granular Periodic acid-

Schiff (PAS) positive material was found in the cytoplasm of the EOR and CR cells, indicating the presence

of polysaccharides (e.g. glycogen and mucins) (Stein and Anderson, 1979). Because the proportion of PAS-

positive material in the RO was found to be influenced by the estrous cycle in cows (Wenzel et al., 1987),

researchers concluded that the luminal contents of the RO were under endocrine control (Archbald et al.,

1971). Although these results strongly suggested a secretory role for the RO, this function was not

experimentally confirmed, and no proteomic or metabolomic investigations were pursued. Previous

characterization of the RO relied heavily on imaging serial sections to study the structure. However, these

studies lacked contextual information, and the use of sections made it challenging to determine whether

tubules were connected to one another or were isolated structures (Wenzel and Odend'hal, 1985; Woolnough

et al., 2000). Furthermore, some investigators reported that the whole RO contracted postnatally and that the

DDT of the EOR separated from the RO and degenerated (Byskov and Lintern-Moore, 1973). Perhaps for

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these reasons, the RO was deemed a functionless vestige, and has been omitted from recent textbook

representations of the female reproductive tract (Girsh, 2021).

The RO was rediscovered and highlighted in our recent study (McKey et al., 2022), where we used confocal

and lightsheet imaging of whole ovaries to study the integration of ovary morphogenesis with the development

of surrounding tissues, including the RO. Recently, the IOR has gained attention as a newly described

progenitor for supporting cells of the murine gonad (Mayère et al., 2022). Recent single cell transcriptomics

studies have also identified cells of the RO within human fetal gonads (Lardenois et al., 2023; Taelman et al.,

2022). We reported that the entire RO expresses high levels of PAX8, and used this as a marker to visualize

and characterize cells of the RO. In the present study, we used the Pax

8-rtTa; Tre-H2B-Gfp

RO nuclear

reporter mouse line, advanced imaging techniques, and secretome analysis to characterize the development

of the intact RO in its native context and to investigate the function and heterogeneity of its cells. We found

that the RO arises from a subset of the mesonephric tubules, analogous to the rete testis, and persists into

adulthood. Our studies reveal that the RO is a continuous structure, surrounded by smooth muscle actin, a

dense vascular network, and several macrophage populations. We also show that the RO is directly contacted

by neurons. The enrichment of secretory machinery in RO cells as well as our experimental analysis of

directional flow and luminal contents, together suggest that the RO sends material to the ovary. Based on

these findings, we suggest that the RO plays a role in ovary function and should be investigated as a functional

organ of the female reproductive tract.

Results

Characterization of the IOR, CR, and EOR

We used immunofluorescence (IF) and confocal imaging to investigate the development and sub-regional

structure of the RO from embryonic day (E) 16.5 to postnatal day (P) 7. Three distinct regions of the RO were

originally defined histologically by cell morphology (Byskov and Lintern-Moore, 1973; Lee et al., 2011). Using

IF and confocal imaging, we found that these three regions are maintained throughout development, but their

relative sizes change. While the entirety of the RO is PAX8+, we took different approaches to identify region

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additional RO markers, we performed bulk and single cell RNA sequencing (ScRNA-seq) of cells from the

ovarian complex of mice at E16.5 and 2 months and found that

Krt8

was enriched in the RO (Anbarci et al.,

2023). We validated these findings using IF against KRT8, which revealed that KRT8+ cells were specifically

localized to the EOR (Fig. 1 third row).

To characterize RO development, we next analyzed PAX8 and KRT8 expression from E16.5 to P0. At E16.5,

the CR and IOR were the largest of the three regions. In the EOR, several tubules leading from the region of

the CR converged into a single tube of columnar PAX8+ and KRT8+ epithelial cells that lead to a blind end.

Between E16.5 and E18.5, the EOR underwent rapid expansion, and the blind end of the EOR became

dilated. This structure, which we refer to as the distal dilated tip (DDT) was best visualized from the ventral

side of the ovary (SFig. 1). By E18.5, the EOR was the largest of the three regions. At this stage the CR was

still large, but the IOR began to regress. At P0, the EOR remained the largest region, and the IOR had

regressed to the medullary region of the ovary (McKey et al., 2022). Their distinct structure and protein

expression patterns suggest that each region has a different function.

Integration of the EOR with the extra-ovarian environment

During gonadogenesis, the mesonephros is highly vascularized in both XX and XY embryos (Cool et al.,

2011). In contrast, the ovary and surrounding tissue are more highly innervated than the testis (McKey et al.,

2019). A surprising finding in our bulk transcriptome analysis of the RO was the presence of a high proportion

of immune cells and cells with vascular markers that co-isolated with E16.5 RO cells (Anbarci et al., 2023).

100

To explore the integration of the RO with its environment, we used IF to investigate the expression of

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endothelial marker Endomucin (Fig. 2a), smooth muscle marker alpha smooth muscle actin (aSMA) (Fig. 2b),

102

pan-neuronal marker TUJ1 (Fig. 2c), and macrophage markers F4/80 and LYVE1 (Fig. 2d). We found that at

103

E18.5 the EOR was tightly surrounded by vasculature and ensheathed within a layer of aSMA+ mesenchyme.

104

The EOR, and more specifically the DDT,

was

directly innervated by neurons that contact the PAX8+

105

epithelial cells. We also found that the EOR is specifically associated with F4/80+ macrophages (Fig. 4d).

106

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This multifaceted integration with the environment suggests the RO may respond to or interpret homeostatic

107

cues.

108

109

Flow of luminal material within the EOR

110

Because the EOR lumen is fluid filled, and because the EOR and CR cells are rich in proteoglycans (Byskov,

111

1975) we hypothesized that the EOR produces luminal secretions. To investigate this idea, we first chose to

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determine the direction of flow at P7, when the DDT of the EOR was fully dilated (Fig. 1d). We first injected

113

fluorescently labeled pH-insensitive Dextran into the DDT of the EOR and found that, within just 15 min, the

114

fluorescent fluid had readily traveled from the DDT into the ovary, where it then diffused widely (Fig. 3). By

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contrast, when we injected fluorescently labeled dextran into the P7 ovary near the IOR, the dextran remained

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Figure 2. The EOR is highly integrated with its extra-ovarian 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 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 panel 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

– 100um

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in the ovary and did not travel to the EOR (Fig. 3). These data indicate that, at least at P7, the fluid inside the

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lumen of the EOR travels towards the ovary.

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Potential of the EOR and CR for fluid

120

transfer

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Without the obvious indication of a lumen

122

within the CR at P7 and taking into

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consideration the absence of the epithelial

124

marker KRT8 in those cells, we wondered

125

how fluid could travel through the CR to the

126

ovary. Using an antibody against E-

127

CADHERIN, a marker of cell junctions, we

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found that it was specifically present in the

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EOR at E16.5, but absent from the CR (Fig.

130

4a, second row). However, E-Cadherin

131

expression was gradually gained in the CR

132

and IOR such that, by P7, the entire RO was

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positive for this epithelial marker (Fig. 4a-d,

134

second row). These results suggest that

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tubular epithelial connections between the

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EOR and IOR mature gradually between

137

E16.5-P7.

138

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Next, we investigated mechanisms that could facilitate fluid movement through the RO. Previous reports

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showed that ciliated and non-ciliated cells are present in the RO (Lee et al., 2011). Using an antibody against

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a marker for cilia, ARL13b, we found that primary ciliated cells were abundant in the DDT and throughout the

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tubules of the EOR at E18.5 (Fig. 4g-h). Due to the curvature of the DDT, optical slices often include cilia from

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Figure 3. Fluid moves from the EOR to the ovary.

(a,b) Schematic of dextran injections (syringe) for each group (a-
EOR injections, b- ovary injections) (Oviduct-grey, ovary-light pink,
IOR-yellow, 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

– 100um

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neighboring cells. However, an ultrathin (0.6um) optical section suggested that each cell has a single cilium.

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Since the EOR was covered in a sheath of aSMA+ mesenchyme (Fig. 2b, Fig. 4e-f), we investigated whether

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and when this layer of smooth muscle became contractile. We used an antibody against the contractile

146

smooth muscle protein Calponin (CNN1) and found that CNN1 was absent around the EOR during fetal

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development and in neonates. However, by P7, the mesenchymal sheath around the EOR gained expression

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of CNN1, indicating that it acquired the ability to contract by this stage of development (Fig. 4e-f). These data

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suggest that ciliary mechanosensing and/or muscle contraction may aid the directional movement of the fluid

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from the DDT to the ovary.

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Figure 4. The EOR and CR acquire the potential for fluid transfer

(a,b,c,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

– 100um (h). Airyscan optical section from confocal Z-

stacks of RO complexes at E18.5 immunostained for KRT8 (cyan) and ARL13b (magenta). Scale bar

– 25um

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152

Proteins produced by the EOR

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indicate a role for the SNARE-

154

complex

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Our data showing fluid flow from

156

the EOR to the ovary prompted us

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to investigate the nature and

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identity of the proteins produced

159

and potentially secreted by the

160

EOR. To address this question, we

161

analyzed the protein contents of the

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luminal

material

using

mass

163

spectrometry. Using the

Pax8rtTA; Tre-H2B-GFP

reporter mouse line, we dissected EORs and isolated

164

luminal fluid by gently pressing the tissue with a pestle. Using this method, we anticipated several problems.

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First, we expected that some cells within the EOR and surrounding tissue would be lysed during this

166

procedure, which would release proteins not ordinarily secreted. Second, we expected that some cells closely

167

associated with the EOR would be co-isolated, which could result in contamination with contents from cells

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that are not part of the EOR. To eliminate intracellular proteins arising from lysed cells, we cross-referenced

169

our proteomic dataset with that of the mammalian secretome (Meinken et al., 2015), thereby retaining only

170

known secreted proteins. Next, we compared the resulting list to the E16.5 ScRNA-seq data from cells

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mapping to the RO (Anbarci et al., 2023). This produced a conservative candidate list of 15 proteins (Table

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1). Of the candidate proteins, two were selected for validation,

CLU

and

STXBP2

, due to their roles in protein

173

and vesicle transport (Argraves and Morales, 2004; Saewu et al., 2017; Söllner, 2003).

174

175

Because no validated antibodies were commercially available to visualize protein expression for CLU and

176

STXBP2, validations were performed using Hybridization Chain Reaction (HCR), a method for single -

177

molecule RNA-Fluorescence in situ hybridization (FISH) (https://files.molecularinstruments.com/MI-Protocol-

178

RNAFISH-Mouse-Rev9.pdf). We found that both

Clu

and

Stxbp2

were expressed in the EOR at

E18.5 (Fig.

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Protein

Role

Agt

Pro-peptide for angiotensinogen

Bcam

Glycoprotein

Complement component

Cd200 Glycoprotein
Cfi

Serine proteinase

Clu

Secreted chaperone*

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*

Table 1. Cells of the EOR secrete proteins essential for vesicle transport.

Candidate list of 15 proteins identified by Mass Spectrometry. Bolded proteins were
selected for validation due to role in vesicle and protein transport.

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5a-b) and at P7 (Fig. 5c-d). The presence of

Stxbp2

showed that components of the SNARE-complex were

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actively transcribed in the EOR, suggesting this may serve as a mechanism for secretion. The presence of

181

other components of the SNARE-complex were validated using IF. We found that the T-SNARE complex

182

member STX3was expressed throughout the EOR and was localized to the apical surface, a cellular position

183

that is typically associated with active secretion (Fig. 5e) (Söllner, 2003).

Ras-associated binding (RAB)

184

proteins

are required in the SNARE complex to tether vesicles to the T-SNARE and allow fusion and secretion

185

(Takahashi et al., 2012). Using an antibody against the small GTPase found on the surface of vesicles,

186

RAB11, we found that it was also localized to the sub-apical region of EOR cells (Fig. 5f), which is the typical

187

docking position for vesicles prior to exocytosis (Söllner, 2003). Taken together, these data suggest that the

188

EOR actively secretes proteins into the lumen of the structure possibly encapsulated in extracellular vesicles.

189

190

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) EOR at E18.5 using HCR for 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; Rosa-mTmG (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; Rosa-mTmG (cyan) EOR at E18.5 immunostained for RAB11
(magenta) Outlined higher resolution image acquired with Airyscan Scale bar

– 100um

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Proteins produced by the EOR suggest a role in ovary homeostasis

191

Among proteins captured in our mass spectrometry analysis of the EOR, insulin-like growth factor binding

192

protein 2 (IGFBP2) stood out as a secreted protein with a potential functional role in ovary homeostasis.

193

IGFBP2, found to be secreted by granulosa cells, binds and sequesters IGF1 (Rosenzweig, 2004). The

194

binding of IGFBP2 to IGF1 titrates IGF1 from its receptor IGF1R (Amutha and Rajkumar, 2017). IGF1 has a

195

reported role in ovarian function by

196

amplifying

the

hormonal

action

197

gonadotropins

promote

198

steroidogenesis

and

granulosa

cell

199

proliferation (Talia et al., 2021).

200

201

Because no validated antibodies were

202

commercially available to visualize protein

203

expression for IGFBP2, we again used

204

HCR to determine when and where

Igfbp2

205

was expressed in the epithelial cells of the

206

RO. We found that

Igfbp2

was highly

207

expressed in the EOR at

E18.5 (Fig. 6a).

208

The mRNA was still present, but at lower

209

levels in P7 EOR cells (Fig. 6b).

210

211

Discussion

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The female reproductive tract is often thought to consist of the vulva, vagina, cervix, uterus, oviduct, and

213

ovaries (Rendi et al., 2012). We suggest that the RO be added to this list and investigated as an additional

214

component of female reproductive function. We show that the RO consists of 3 distinct regions that mature

215

during fetal life and persist into adulthood. The cells of the EOR show secretory characteristics consistent

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with the presence of secreted proteins in the lumen identified through mass spectrometry. Importantly, we

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show that contents of the EOR are transported to the ovary by P7, suggesting a role in ovary homeostasis.

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Figure 6. Validation of Igfbp2 expression in the RO.

(a) Maximum intensity projection of confocal Z-stacks of PAX8-rtTa; Tre-
H2B-GFP (cyan) EOR at E18.5 using HCR for 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 detection of
IGFBP2 expression (magenta). Bottom panels are outlined higher resolution
image acquired with Airyscan. Scale bar top panel

– 100um. Scale bar top

panel

– 25um

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219

Historically, the regions of the RO were identified using histological sections, where the EOR was defined as

220

a columnar epithelium, while the CR was defined as pseudo-columnar. Recently we found that the entire RO

221

expresses PAX8+, which is usually considered a marker of urogenital epithelial identity. We also showed that

222

the IOR contained PAX8+/FOXL2+ cells and that the EOR was KRT8+ at E17.5 (McKey et al., 2022). In this

223

study we further characterized KRT8 and E-Cadherin expression throughout RO development as the CR

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acquires epithelial characteristics. We also identified a marker, GFRa1, that is exclusively expressed in the

225

CR. These molecular distinctions predict that the different regions of the RO are functionally distinct.

226

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While PAS-positive staining in the lumen of the EOR of sheep indicated the presence of glycogens,

228

glycoproteins, and proteoglycans (Stein and Anderson, 1979), it remained unclear whether these were

229

secreted by the EOR cells or were accumulated secretions from the ovary. Using injections of a membrane

230

impermeable dye, Dextran, into either the RO or ovary, we found that the fluid within the EOR travels towards

231

the ovary. We also found that the EOR is ensheathed within a layer of aSMA+ mesenchyme that expresses

232

the contractile component Calponin (CNN1) (Gao et al., 2014). Our data suggests that the EOR gains

233

contractility during development, which could facilitate fluid movement within the EOR lumen in conjunction

234

with cilia within the EOR. We found that the ciliary protein ARL13b was expressed in the EOR. While the

235

primary cilium is not motile and does not generate flow, we found in our ScRNA-seq data that cells in the

236

EOR express

Pkd2

. In renal epithelial cells, nonmotile primary cilia expressing PDK2 sense shear stress

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during fluid flow and transduce this sensory information (Nauli et al., 2003). Perhaps fluid movement within

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the EOR is a two-part process, where the primary cilia sense pressure within the lumen inducing contraction

239

of the smooth muscle surrounding the EOR to promote fluid movement towards the ovary. Our E16.5 ScRNA-

240

seq data indicates that the EOR only expresses

Arl13b

and does not express the multicilia marker

Foxj1

241

However, both markers are present in the putative EOR cell clusters in our adult SnRNA-seq data (Anbarci

242

et al., 2023) suggesting that, after maturation, the EOR likely includes

Arl13b+/Foxj1+

multiciliated cells. It is

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well documented that adult oviductal multiciliated cells express both ARL13b and FOXJ1 (Coy et al., 2016).

244

It is possible that the primary cilia give rise to motile multiciliated cells, as shown in the airway epithelium (Jain

245

et al., 2010) and suggested in the oviduct (Shi et al., 2014).

246

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247

We used a “milking” process to extrude the contents of EOR, and mass spectrometry, to identify 4,232

248

proteins. We expected this group to be a mix of proteins from the luminal fluid, and cytoplasmic proteins from

249

lysed EOR cells or from cells closely associated with the EOR. We took a very conservative approach to

250

identify proteins highly likely to be secreted from EOR cells: (1) we compared the proteomics data with the

251

known mammalian secretome to identify only secreted proteins; and (2) we next compared this shortened list

252

with our available E16.5 ScRNA-seq data to find proteins whose transcripts were specific to the EOR at that

253

stage. Although we recognize that the secretome at P7 is likely far more complex, this candidate list of 15

254

proteins reveals the first insight into specific proteins secreted from the RO that may be involved in the

255

secretory process itself or may be transported to the ovary to affect ovary function.

256

257

Within the protein candidate list, we focused on validating clusterin (CLU) and syntaxin binding protein 2

258

(STXBP2) due to their roles in protein and vesicle transport. Consistent with our proteomic results, both

Clu

259

and

Stxbp2

RNAs were detected in the RO. CLU is a chaperone protein that is present in both reproductive

260

and non-reproductive tissues (Argraves and Morales, 2004; Saewu et al., 2017). In the epididymis, CLU acts

261

as a chaperone to direct luminal proteins to the sperm head surface (Saewu et al., 2017). Although the role

262

of CLU in the RO remains unknown, we hypothesize that it is important for post-secretory protein guidance

263

to the ovary. STXBP2 binds t-SNARE protein STX and is essential for the vesicle-apical surface membrane

264

fusion stage of vesicle secretion (Söllner, 2003). The presence of STXBP2 in the candidate list, coupled with

265

validation of the expression of

Stxbp2

RNA in the cells of the EOR, led us to investigate the presence of other

266

components of the SNARE complex in the EOR.

267

268

The SNARE complex is comprised of a vesicle bound v-SNARE (Vamps), membrane bound t-SNARE (STXs),

269

small GTPases (RABs), and syntaxin binding proteins (STXBPs) (Söllner, 2003). Using IF staining, we

270

confirmed the presence of STX3, as well as RAB11. Our ScRNA-seq data indicate that EOR cells also

271

express

Vamp7

and

Vamp

8, which are v-SNAREs typically associated with STX3 (Dingjan et al., 2018). Small

272

GTPases, such as RAB11, are essential in the SNARE complex as they mediate the tethering of vesicle

273

membranes to the apical membrane prior to membrane fusion. Taking into consideration the presence of all

274

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components of the SNARE complex, we hypothesize that the EOR utilizes the SNARE complex to promote

275

apical secretion into the luminal fluid.

276

277

A protein that was prominent on the proteomic candidate list was IGFBP2. We found that

Igfbp2

was highly

278

expressed in both the embryonic and adult RO transcriptome (Anbarci et al., 2023). IGFBP2 is an ideal

279

candidate protein to mediate ovarian function. The IGF1 pathway is essential for regulation of follicular growth

280

and selection (Talia et al., 2021). IGFBP2 can bind and sequester IGF1 (Rosenzweig, 2004), limiting its

281

availability to promote follicle growth through activation of its receptor IGF1R (Amutha and Rajkumar, 2017;

282

Bezerra et al., 2018; Liu et al., 2021). We spatially validated the expression of

Igfbp2

in the EOR using HCR,

283

and noted high expression at E18.5, which was decreased by P7. This decrease correlated with the end of

284

the first-wave of follicle activation immediately after birth (Mork et al., 2012; Zheng et al., 2014). We also found

285

variation in levels of

Igfbp2

in our adult SnRNA-seq data, where

Igfpb2

was enriched specifically in RO cells

286

during the estrus stage compared to the diestrus stage of the estrous cycle. Based on these results, we

287

hypothesize that high levels of estradiol during estrus trigger the EOR to produce IGFBP2 to sequester free

288

IGF1 and decrease follicle growth and production of estradiol, maintaining ovary homeostasis.

289

290

The EOR is highly integrated with the extra-ovarian environment. We found that EOR cells are tightly

291

surrounded in a dense web of vasculature. The RO may send or receive information through the vasculature,

292

similar to other epithelial tissues in the female reproductive tract (Reynolds et al., 2002), raising the possibility

293

that the RO participates in endocrine signaling. We also found that the EOR is directly contacted by TUJ1+

294

neurons, another avenue through which information may be entering or exiting the EOR. It is unclear whether

295

the innervation contacts the epithelial cells specifically, similar to the way in which innervation contacts

296

epithelial cells in the gut (Bohórquez et al., 2015), or whether the innervation contacts the smooth muscle

297

cells that surround the EOR to induce contraction, similar to the innervation-muscle interaction in the uterus

298

(Morizaki et al., 1989). We also found that the EOR is specifically associated with F4/80+ LYVE1-

299

macrophages. LYVE1 macrophages are predicted to have an angiogenic role in the adipose tissue

300

surrounding the epididymis and could possibly be playing a similar role in the EOR (Cho et al., 2007). The

301

integration of the EOR with the extra-ovarian environment, including vasculature, neurons, and macrophages,

302

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places the EOR in an ideal position to act as an antenna to interpret homeostatic signals and send information

303

to the ovary. In

Drosophila

and

C. Elegans

, there is ample evidence that the ovary responds to physiologic

304

levels of glycogen, insulin, amino acid levels, and changes in diet (Hubbard et al., 2013; Laws and Drummond-

305

Barbosa, 2017). The mediator of this response has not been determined in mammals, but we hypothesize

306

that this is the function of the RO. Ongoing work will investigate how the EOR responds to hormones and

307

other physiological signals, and whether proteins secreted by the EOR such as IGFBP2 respond to

308

physiologic stimuli such as diet or immune status, and convey this information to the ovary.

309

310

Methods and Materials

311

Key Resource Table

312

Reagent type

(species) or

resource

Designation

Source or

reference

Identifiers

Additional

information

RRID

Strain, strain

background (

Mus

musculus

)

Crl:CD1(ICR)

Charles River

Strain code: 022

IMSR_CRL:022

Strain, strain

background (

musculus

)

C57BL/6J

Jackson

Laboratory

Stock #:000664

IMSR_JAX:000664

Genetic reagent (

musculus

)

Tre-H2B-Gfp (Tg(tetO-

HIST1H2BJ/GFP)47Efu

/J)

PMID: 14671312

MGI:J:90563

IMSR_JAX:005104

Genetic reagent (

musculus

)

Tre-Cre (B6.Cg-

Tg(tetO-cre)1Jaw/J)

PMID: 12145322

MGI:J:78365

IMSR_JAX:006234

Genetic reagent (

musculus

)

mTmG

Gt(ROSA)26Sortm4(AC

TB-tdTomato,-

EGFP)Luo/J

PMID: 17868096

MGI:J:124702

IMSR_JAX:007576

Genetic reagent (

musculus

)

Pax8-rtTA (B6.Cg-

Tg(Pax8-

rtTA2S*M2)1Koes/J)

PMID: 18724376

MGI:J:140925

IMSR_JAX:007176

Genetic reagent (

musculus

)

Lgr5 (B6.129P2-

Lgr5tm1(cre/ERT2)Cle/

PMID:17934449

MGI:J:127123

IMSR_JAX:008875

Antibody

Smooth muscle alpha

action (aSMA) (Cy3-

conjugated mouse

monoclonal)

Sigma-Aldrich

C6198

1:1000

AB_476856

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available under a

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

The copyright holder for this preprint

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Antibody

E-Cadherin (rat

monoclonal)

Zymed (Thermo

Fisher Scientific)

13-1900

1:500

AB_2533005

Antibody

Endomucin (rat

monoclonal)

Santa Cruz

Biotechnology

sc-65495

1:500

AB_2100037

Antibody

GFP (chicken

polyclonal)

Abcam

ab13970

1:1000

AB_300798

Antibody

GFRa1 (goat

polyclonal)

R&D Systems

AF560

1:150

AB_2110307

Antibody

KRT8 (rat monoclonal)

DSHB

TROMA-I

1:250

AB_531826

Antibody

PAX8 (rabbit polyclonal)

Proteintech

A10336-1-AP

1:500

AB_2918972

Antibody

CNN1 (rabbit

poluclonal)

Proteintech

13938-1-AP

1:200

AB_2082010

Antibody

ARL13b (rabbit

polyclonal)

Proteintech

17711-1-AP

1:1000

AB_2060867

Antibody

RAB11 (rabbit

monoclonal)

Cell Signaling

Technology

5589

1:500

AB_10693925

Antibody

LYVE-1 (goat

polyclonal)

R&D Systems

AF2125

1:500

AB_2297188

Antibody

F4/80 (rat monoclonal)

BioRad

MCA497RT

1:2000

AB_1102558

Antibody

STX3 (rabbit

monoclonal)

Abcam

ab133750

1:200

Antibody

TUJ1 (488-conjugated

mouse monoclonal)

BioLegend

A488-435L

1:1000

AB_10143904

Antibody

AF647 anti-Rabbit

(donkey polyclonal)

Jackson

ImmunoResearch

711-605-152

1:500

AB_2492288

Antibody

AF488 anti-Chicken

(donkey polyclonal)

Jackson

ImmunoResearch

703-545-155

1:500

AB_2340375

Antibody

AF488 anti-Rat (donkey

polyclonal)

Life Technologies

A-21208

1:500

AB_2535794

Antibody

Cy3 anti-Goat (donkey

polyclonal)

Jackson

ImmunoResearch

705-165-147

1:500

AB_2307351

HCR RNA-Fish

probe

Stxbp2 (B3 amplifier)

Molecular

Instruments

Accession #:

XR_001778418.1

HCR RNA-Fish

probe

Clu (B1 amplifier)

Molecular

Instruments

Accession #:

NM_013492.3

HCR RNA-Fish

probe

IGFBP2 (B3 amplifier)

Molecular

Instruments

Accession #:

NM_008342.3

HCR amplifier

F647 (B3 amplifier)

Molecular

Instruments

HCR amplifier

F546 (B1 amplifier)

Molecular

Instruments

CC-BY 4.0 International license

available under a

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made

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Reagent

Dextran, Alexa FluorTM

568; 10,000 MW

ThermoFisher

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)ethylene

diamine

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

313

Mice and Tissue Collection

314

Unless otherwise stated, mice used for experiments were maintained on the CD-1 or mixed CD-1 and

315

C57BL/6J genetic backgrounds. The

Pax8-rtTA

and

Tre-H2BGFP

lines were previously described (Traykova-

316

Brauch et al., 2008; Tumbar et al., 2004) and maintained on a mixed CD-1/C57BL/6J background, and

317

maintained on a mixed CD-1/C57BL/6J background. The

Pax8-rtTa;Tre-Cre;mTmG

line was obtained by

318

crossing the

Pax8-rtTa

line with carriers of the

Tre-Cre

(Perl et al., 2002) and

mTmG

(Muzumdar et al., 2007)

319

alleles, which allowed for visualization of cell membranes of PAX8+ cells. Pregnant and nursing dams were

320

given a doxycycline diet at 625 mg/kg (Teklad Envigo TD.01306) 3 days prior to tissue collection to induce

321

GFP expression in Pax8+ cells. Toe samples were collected from mice for genotyping. Primers used for PCR

322

genotyping are listed in the supplementary table 1. To obtain samples at specific stages of development,

323

males were housed with females for timed matings. Successful mating was determined by the presence of a

324

vaginal plug. Date of plug was considered embryonic day 0.5. Tissue samples were collected in phosphate -

325

buffered saline without calcium or magnesium (PBS -/-), fixed in 4% paraformaldehyde (PFA)/PBS for 30

326

minutes at room temperature and dehydrated stepwise into 100% methanol, followed by storage at -20°C. All

327

mice were housed in accordance with National Institutes of Health guidelines, and experiments were

328

conducted with the approval of the Duke University Medical Center Institutional Animal Care and Use

329

Committee (protocol #: A078-23-03).

330

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331

Immunostaining and Confocal Image Acquisition

Samples were stepwise rehydrated into 100% PBS,

332

followed by a 30-minute permeabilization wash in PBS 0.1% Triton X-100. Samples were then blocked for 1

333

hour with PBS, 1% Triton X-100, 10% horse serum, and 3% BSA. Samples were incubated overnight in

334

primary antibodies diluted in blocking solution at 4°C (key resource table). Samples then underwent three 30-

335

minute washes in permeabilization solution and incubated overnight in secondary antibodies (1:500 dilution)

336

and Hoechst vital dye solution diluted in blocking solution. The next day, samples underwent three 20-minute

337

washes in permeabilization solution before being transferred into 100% PBS and stored at 4°C until ready for

338

confocal imaging. Samples were mounted in DABCO mounting solution and stored at -20°C until imaged.

339

Samples were imaged from both the dorsal and ventral sides using 3D-printed reversible slides that utilize

340

two coverslips that allow for flipping. The 3D model can be downloaded on the NIH 3D Print Exchange website

341

https://3dprint.nih.gov/discover/3DPX-009765

. Samples were imaged

in toto

using laser scanning confocal

342

microscopy captures in the longitudinal plane on Zeiss LSM780 or LSM880 and affiliated Zen software Carl

343

Zeiss, Inc, Germany) using 10x, 20x, and 63x (also used for Airyscan) objectives.

344

345

Hybridization chain reaction

346

The

mouse

embryo

protocol

from

Molecular

Instruments

whole-mount

347

(https://files.molecularinstruments.com/MI-Protocol-RNAFISH-Mouse-Rev9.pdf) was adjusted for P0 and P7

348

ovary samples. Samples were stepwise rehydrated into 0.1% Tween 20 / PBS (PBST). Samples were then

349

subjected to 10 μg/mL proteinase K solution for 10 minutes at room temperature, then washed twice in PBST

350

for 5 minutes. Samples were then post-fixed in 4% PFA for 10 minutes at room temperature, followed by three

351

PBST washes for 5 minutes. Samples were then pre-hybridized for 30 minutes in 500ul of hybridization buffer

352

(Molecular Instruments) at 37°C. Samples were then incubated overnight at 37°C with HCR probes diluted in

353

500ul hybridization buffer (2pmol in 500ul). Samples were then washed for 15 minutes four times in the

354

Molecular Instruments probe wash buffer at 37°C, followed by two 5x 0.1% Tween 20/SSC (SSCT) washes

355

for 5 minutes at room temperature. Fluorescently labeled hairpins were snap cooled and left in a dark drawer

356

at room temperature for 30 minutes. Following hairpin preparation, samples were incubated with hairpins

357

diluted in Molecular Instruments amplification buffer overnight in the dark at room temperature. On the third

358

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day, samples were washed in SSCT four times for 15 minutes each before being transferred to PBS -/- and

359

then mounted in DABCO mounting medium for imaging.

360

361

Image Processing

362

Confocal images were imported into FIJI software for minor image processing (cropping, rotations, maximum

363

intensity projection/ optical slice montage, and color application). The RO was oriented to the left of the ovary

364

with the oviduct at the top of the ovary. Images were then imported into Adobe Photoshop CC (Adobe, Inc,

365

CA) for final processing of channel overlay, brightness, contrast adjustment, and modification of red to

366

“magenta” (hue adjustment to -30). Channel color accessibility was determined via Photoshop colorblind

367

proofing.

368

369

Dextran Injections

370

Postnatal day 7 mice were euthanized, and the entire ovarian complex was dissected in PBS +/+. Samples

371

were then placed on agar blocks (1.5%) soaked in PBS +/+. The distal tip of the EOR was identified and

372

punctured using a tungsten needle (0.001mm tip diameter, Fine Science Tools, 10130-05). A microinjection

373

unit (Picospritzer III microinjector) and fine capillary glass needle (5-10 um) loaded with 2-3 nl dextran solution

374

(1.25mg/ml dextran, PBS, 0.05% phenol red) was used to inject dextran into the opening of the EOR. Samples

375

were then left for 15 minutes before fixing with 4% PFA. During the 15 minutes, samples were monitored to

376

visualize movement. Samples were then fixed, stained, and imaged.

377

378

Luminal Fluid Collection for Proteomics and Analysis

379

Postnatal day 7 EORs were collected from

Pax8-rtTa; Tre-H2B-Gfp

mice (as a guide to only collect EOR) and

380

carefully cleaned up to remove as much non-EOR tissue (40 EOR samples). Samples were then placed in a

381

1.5ml Eppendorf tube and ‘pressed’ for fluid using a disposable pellet pestle. The sample was then spun down

382

and the supernatant was collected and snap frozen in liquid nitrogen. The sample was then submitted to the

383

Duke University Proteomics Core for mass spectrometry. Proteomic analysis uncovered 4,252 proteins

384

present in the pressed fluid. To exclude proteins that may have contributed due to cell lysis, results were

385

cross-referenced against a database of secreted proteins (Meinken et al., 2015). Overlapping proteins were

386

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then cross-referenced to our E16.5 ScRNA-seq data (Anbarci et al., 2023), and candidate proteins were

387

determined by gene expression specific to the EOR cluster.

388

389

LC-MS/MS Proteomics Analysis

390

The sample was subjected to a Bradford (Pierce) protein measurement and 10 ug was removed for

391

downstream processing. The sample was brought to 4% SDS, reduced with 10 mM dithiothreitol for 20 min

392

at 55°C, alkylated with 25mM iodoacetamide for 45 min at room temperature and then subjected to S-trap

393

(Protifi) trypsin digestion using manufacturer recommended protocols. Digested peptides were lyophilized to

394

dryness and resuspended in 50 uL of 0.2% formic acid/2% acetonitrile. The sample was subjected to

395

chromatographic separation on a Waters MClass UPLC equipped with a 1.8 μm Acquity HSS T3 C18 75 μm

396

× 250 mm column (Waters Corp.) with a 90-min linear gradient of 5 to 30% acetonitrile with 0.1% formic acid

397

at a flow rate of 400 nanoliters/minute (nL/min) with a column temperature of 55°C. Data collection on the

398

Fusion Lumos mass spectrometer with a FAIMS Pro device was performed for three difference compensation

399

voltages (-40v, -60v, -80v). Within each CV, a data-dependent acquisition (DDA) mode of acquisition with a

400

r=120,000 (@ m/z 200) full MS scan from m/z 375

– 1500 with a target AGC value of 4e5 ions was performed.

401

MS/MS scans with HCD settings of 30% were acquired in the linear ion trap in “rapid” mode with a target AGC

402

value of 1e4 and max fill time of 35 ms. The total cycle time for each CV was 0.66s, with total cycle times of

403

2 sec between like full MS scans. A 20s dynamic exclusion was employed to increase depth of coverage. The

404

total analysis cycle time for each sample injection was approximately 2 hours.

405

406

Raw LC-MS/MS data files were processed in Proteome Discoverer 3.0 (Thermo Scientific) and then submitted

407

to independent Sequest database searches against a

Mus musculus

protein database containing both

408

forward and reverse entries of each protein. Search tolerances were 2 ppm for precursor ions and 0.8 Da for

409

product ions using trypsin specificity with up to two missed cleavages. Carbamidomethylation (+57.0214 Da

410

on C) was set as a fixed modification, whereas oxidation (+15.9949 Da on M) was considered a dynamic

411

mass modifications. All searched spectra were imported into Scaffold (v5.3, Proteome Software) and scoring

412

thresholds were set to achieve a peptide false discovery rate of 1% using the PeptideProphet algorithm. Data

413

were output as total spectral matches.

414

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415

Acknowledgements

416

The authors would like to thank Benjamin Carlson and Lisa Cameron from the Duke Light Microscopy Core

417

Facility for confocal imaging resources and assistance on high resolution imaging and analysis. The authors

418

would also like to thank Dr. Erik Soderblom from the Duke Center for Genomic and Computational Biology

419

core facility for proteomic analysis and assistance. We are grateful to all members of the Capel and Bagnat

420

laboratories for their constant support, discussion, and suggestions on the work presented in this paper and

421

beyond.

422

423

Funding

424

This project was supported by grants from the National Institutes of Health #1R01HD090050-0 to BC,

425

#1R01DK1321120 to MB, and #K99HD103778 and #R00HD103778 to JM. DNA was supported by

426

#5R01HD039963 to BC and the Duke School of Medicine.

427

428

Author contributions

429

DNA, JM, DSL carried out the experiments; DNA carried out the data curating, validating, and formal

430

analysis; DNA, JM, DSL, and BC designed the experiments; DNA, JM, DSL, MB, and BC drafted, revised,

431

and edited the manuscript; JM, MB, and BC acquired funding for the project.

432

433

Competing interests

434

The authors declare no competing or financial interests.

435

436

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Anbarci, D.N., ORourke, R., Xiang, Y., Peters, D.T., Capel, B., McKey, J., 2023. Transcriptome analysis of

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Bohórquez, D.V., Shahid, R.A., Erdmann, A., Kreger, A.M., Wang, Y., Calakos, N., Wang, F., Liddle, R.A.,

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Byskov, A.G., 1975. The role of the rete ovarii in meiosis and follicle formation in the cat, mink and ferret.

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