Steroidogenesis and androgen/estrogen signaling pathways are altered in in vitro matured testicular tissues of prepubertal mice
- Univ Rouen Normandie, Inserm, Normandie Univ, NorDiC UMR 1239, Adrenal and Gonadal Pathophysiology team, F-76000, France
- Department of General Biochemistry, Rouen University Hospital, France
- Normandie Univ, UNICAEN, OeReCa, France
Figures
Figure 1 with 1 supplement
Leydig cells are partially mature after 30 days of organotypic culture.
(A) Representative images of 3β-hydroxysteroid dehydrogenase (3β-HSD) expression by Leydig cells during mouse postnatal development (6 dpp, 22 dpp, and 36 dpp) and in in vitro cultured tissues after …
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Figure 1—source data 1
Source data of Figure 1.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig1-data1-v1.xlsx
Figure 1—figure supplement 1
Impact of controlled slow freezing on Leydig cells in organotypic cultures.
(A) Representative images of 3β-hydroxysteroid dehydrogenase (3β-HSD) expression in Leydig cells at 6 dpp and in in vitro cultured frozen/thawed (CSF) testicular tissues (D16 and D30). Testicular …
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Figure 1—figure supplement 1—source data 1
Source data of Figure 1—figure supplement 1.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig1-figsupp1-data1-v1.xlsx
Figure 2
The expression of several actors of steroidogenesis is downregulated in 30 day organotypic cultures.
(A–I) Relative mRNA levels of Lhcgr, Star, Cyp11a1, Hsd3b1, Cyp17a1, Hsd17b3, and Hsd17b2 (normalized to Gapdh and Actb or to Hsd3b1) and relative protein levels of 3β-hydroxysteroid dehydrogenase …
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Figure 2—source data 1
Source data of Figure 2.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig2-data1-v1.xlsx
Figure 3 with 1 supplement
An increased production of progesterone and estradiol and a decreased production of androstenedione are observed after 16 and 30 days of culture of prepubertal mouse testicular tissues.
Intratesticular concentrations of (A) progesterone, (B) androstenedione, (C) testosterone, and (D) estradiol during mouse postnatal development (6 dpp, 22 dpp, and 36 dpp) and in in vitro cultured …
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Figure 3—source data 1
Source data of Figure 3.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig3-data1-v1.xlsx
Figure 3—figure supplement 1
Impact of controlled slow freezing on steroids production by Leydig cells in organotypic cultures.
Concentrations of androgens (androstenedione and testosterone) in the culture medium of fresh (FT) or frozen/thawed (CSF) testicular tissues. Data are presented as means ± SEM with n=4 biological …
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Figure 3—figure supplement 1—source data 1
Source data of Figure 3—figure supplement 1.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig3-figsupp1-data1-v1.xlsx
Figure 4
Androgen signaling is altered in 30 days organotypic cultures.
(A) Relative mRNA levels of Ar (normalized to Gapdh and Actb) and (B) relative protein levels of androgen receptor (AR) (normalized to ACTB) during mouse postnatal development (6 dpp, 22 dpp, and 36 …
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Figure 4—source data 1
Source data of Figure 4.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig4-data1-v1.xlsx
Figure 5
The expression of aromatase and estrogen signaling is impaired after 30 days of organotypic culture.
(A) Relative mRNA levels of Cyp19a1 (normalized to Gapdh and Actb) and (B) relative protein levels of CYP19A1 (normalized to ACTB) during mouse postnatal development (6 dpp, 22 dpp, and 36 dpp) and …
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Figure 5—source data 1
Source data of Figure 5.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig5-data1-v1.xlsx
Figure 6 with 2 supplements
Human chorionic gonadotropin (hCG) is insufficient to stimulate Leydig cell differentiation and restore steroidogenesis and steroid hormone signaling.
(A–D) Levels of androgens (androstenedione and testosterone) (A–B) in testicular tissues and (C–D) in culture medium with 1 nM hCG or without supplementation. (E–S) Relative mRNA levels of genes …
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Figure 6—source data 1
Source data of Figure 6.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig6-data1-v1.xlsx
Figure 6—figure supplement 1
Impact of different human chorionic gonadotropin (hCG) concentrations on testosterone production in organotypic cultures.
(A) Concentrations of testosterone in the culture medium after supplementation with different hCG concentrations (5 pM, 50 pM, 1 nM, 5 nM, 50 nM) from D16 to D30. (B) Concentrations of testosterone …
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Figure 6—figure supplement 1—source data 1
Source data of Figure 6—figure supplement 1.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig6-figsupp1-data1-v1.xlsx
Figure 6—figure supplement 2
Impact of human chorionic gonadotropin (hCG) supplementation on mRNA levels of genes involved in steroidogenesis and androgen signaling in organotypic cultures.
Relative mRNA levels of Leydig cell markers, actors of steroidogenesis, and androgen signaling (normalized to Gapdh and Actb or to Hsd3b1) after supplementation with 1 nM hCG. Data are presented as …
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Figure 6—figure supplement 2—source data 1
Source data of figure 6 – figure supplement 2.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig6-figsupp2-data1-v1.xlsx
Figure 7
Altered steroidogenesis and androgen/estrogen signaling pathways in in vitro matured testicular tissues of prepubertal mice.
A similar density of Leydig cells is found after 30 days of organotypic culture (D30) and at 36 days postpartum, the corresponding in vivo time point. However, Leydig cells are partially mature in …
Figure 8 with 1 supplement
Scheme of the study design.
At 6 dpp (D0), the testis contains only spermatogonia and the initiation of meiosis occurs between 7 and 9 dpp. At 22 dpp (D16), meiosis ends and the first round of spermatids appear. At 36 dpp …
Figure 8—figure supplement 1
Morphological analysis of tissue sections after 16 or 30 days of organotypic cultures.
(A) Testicular tissue area (in mm²) measured on tissue sections from mice of different ages (6 dpp, 22 dpp, and 36 dpp) and after 16 days (D16) or 30 days (D30) of organotypic cultures. (B) Necrotic …
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Figure 8—figure supplement 1—source data 1
Source data of Figure 8—figure supplement 1.
- https://cdn.elifesciences.org/articles/85562/elife-85562-fig8-figsupp1-data1-v1.xlsx
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Mus musculus, male) | Testis | CD-1 Mice from Charles River | Freshly isolated from male Mus musculus (CD-1) | |
| Antibody | Anti-3β-HSD (mouse monoclonal) | Santa Cruz Biot. | sc-515120 (AF488) | IF (1:100), WB (1:1000) |
| Antibody | Anti-β-Actin (mouse monoclonal) | Abcam | ab8226 | WB (1:5000) |
| Antibody | Anti-AR (rabbit monoclonal) | Abcam | ab133273 | IF (1:100), WB (1:5000) |
| Antibody | Anti-CC3 (rabbit polyclonal) | Abcam | ab49822 | IF (1:200) |
| Antibody | Anti-CYP17A1 (rabbit polyclonal) | Abcam | ab231794 | IF (1:200), WB (1:10000) |
| Antibody | Anti-CYP19A1 (mouse monoclonal) | BioRad | MCA2077S | IHC (1:50), WB (1:250) |
| Antibody | Anti-FAAH (rabbit polyclonal) | Proteintech | 17909–1-AP | WB (1:1000) |
| Antibody | Anti-Ki67 (rabbit monoclonal) | Abcam | ab16667 | IF (1:100) |
| Antibody | Anti-mouse Alexa 488 (goat) | Abcam | ab150113 | IF (1:200) |
| Antibody | Anti-rabbit Alexa 488 (goat) | Abcam | ab150077 | IF (1:200) |
| Antibody | Anti-rabbit Alexa 594 (goat) | Abcam | ab150080 | IF (1:200) |
| Antibody | Anti-mouse HRP (goat) | Invitrogen | 31430 | WB (1:5000) |
| Antibody | Anti-rabbit HRP (goat) | Invitrogen | A16110 | WB (1:5000) |
| Commercial assay or kit | RNeasy Micro kit | Qiagen | 74004 | |
| Commercial assay or kit | qScript cDNA SuperMix | QuantaBio | 95048 | |
| Commercial assay or kit | Lipid Extraction kit | Abcam | ab211044 | |
| Commercial assay or kit | Cholesterol/Cholesteryl Ester assay kit | Abcam | ab65359 | |
| Commercial assay or kit | Progesterone ELISA kit | Cayman Chemical Company | 582601 | |
| Commercial assay or kit | Estradiol ELISA kit | Cayman Chemical Company | 501890 | |
| Commercial assay or kit | Aromatase (CYP19A) Activity assay kit | Abcam | ab273306 | |
| Commercial assay or kit | INSL3 RIA kit | Phoenix Pharmaceuticals | RK-035–27 | |
| Chemical compound, drug | KSR | Gibco by Life Technologies | 10828010 | (10%) |
| Chemical compound, drug | Retinol | Sigma-Aldrich | R7632 | 1 µM |
| Chemical compound, drug | hCG | MSD France | Ovitrelle | 1 nM |
| Chemical compound, drug | SYBR Green | Thermo Fisher Scientific | 4385616 |
Additional files
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Supplementary file 1
Detailed tables of antibodies, primers and LC-MS/MS's parameters used.
(a) Detailed list of antibodies used in this study HRP: horseradish peroxidase; IF: immunofluorescence; IHC: immunohistochemistry; O/N: overnight; RT: room temperature; WB: western blot. (b) List of PCR primers used in this study. (c) Compound-specific MRM parameters for LC-MS/MS CE: collision energy; DHEA: Dehydroepiandrosterone; MRM: multiple reaction monitoring
- https://cdn.elifesciences.org/articles/85562/elife-85562-supp1-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/85562/elife-85562-mdarchecklist1-v1.pdf
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Source data 1
Western Blot for 3β-HSD, CYP17A1, AR, CYP19A1, and FAAH.
- https://cdn.elifesciences.org/articles/85562/elife-85562-data1-v1.pdf
