Ethics authorizations
All human tissues were obtained in accordance with French laws (Good Practice Concerning the Conservation, Transformation, and Transportation of Human Tissue to Be Used Therapeutically, published on December 29, 1998).
Adult COVID-19 patient brains and blood samples and control brains were obtained under authorization for the GonadoCOVID study (French protocol # 2-20-056 id8504) and authorized by the Lille Neurobiobank.
The studies on human fetal tissue were approved by the French agency for biomedical research (Agence de la Biomédecine, Saint-Denis la Plaine, France, protocol no.: PFS16-002). Non-pathological human fetuses were obtained at GW7, GW11 and GW14 from voluntarily terminated pregnancies after written informed consent from the donors (Gynecology Department, Jeanne de Flandre Hospital, Lille, France).
Animal studies were performed with the approval of the Institutional Ethics Committees and the French Ministry of National Education, Higher Education and Research (APAFIS#2617-2015110517317420 v5, APAFIS#25384- 2020041515287655 v6).
COVID-19 ICU patient blood sample and hormonal analysis
Sixty male patients, 35-82 years of age, admitted to the resuscitation unit of the Lille University Hospital with a positive COVID-19 PCR were included in the “GonadoCOVID” study (French protocol # 2-20-056 id8504). Exclusion factors were a previous history of cancer or cirrhosis. Due to the difficulty of following the menstrual cycles of female patients in the ICU, female patients were excluded. For each patient, blood samples yielding 2000 µl of serum were obtained during the first week following admission, at 2 weeks and at 4 weeks if still hospitalized. The sampling week was defined based on the day the samples were obtained, where D1, D3, D5 sampling days were grouped to Week 1; J7, J9, J14 to week 2 and J30 to week 4. Sixteen patients died while in the ICU. Patient profiles are provided in Supplementary Table 1.
The patients were categorized into four groups based on the severity of the decrease in testosterone (T) and LH levels (Group 1: T <= 0.92 ng/ml & LH <=2 IU/L, Group 2: T < 0.92 ng/ml & LH > 2 & LH < 12 IU/L), Group 3: T < 0.92 ng/ml & LH > 12 IU/L, Group 4: T > 0.92 ng/ml). Unpaired t-tests were used to estimate the significance between the groups in terms of testosterone, LH, FSH and TSH levels using GraphPad Prism 8.
Pearson’s product moment correlation of (cor.test() of R stats version 4.0.3) was used to estimate the strength of linear association between leptin vs. testosterone as well as testosterone vs CRP in all patients irrespective of sampling week, while the relation between LH and testosterone levels in patients with normal HPG axis function followed a log-linear model (formula = LH ~ log(T)) determined using lm () of R stats version 4.0.3. The correlation plots as well as the dot plots showing testosterone or CRP levels in alive and deceased patients (n = 16) were generated using ggplot2 version 3.3.5 in R.
COVID-19 patient and control brains
The brains of 4 subjects (3 males and 1 female) who died of COVID-19 infection in the Lille University Hospital and 5 control subjects (4 males and 1 female) who did not test positive for COVID-19, including 2 who died before the pandemic began, were used for this study. COVID-19 and control subjects were matched for age, sex and comorbidities as far as possible. Their clinical characteristics are summarized in Table 1.
In keeping with strict protocols regarding the treatment of SARS-CoV-2-infected human tissues, human brains were immersion-fixed in 10% formalin for 1 week at room temperature. The hypothalamus was then dissected out and immersion-fixed in 4% paraformaldehyde in PBS 0.1M, pH7.4, for an additional 48h at 4°C, cryoprotected in 30% sucrose for an additional week at 4°C, embedded in Tissue-Tek and frozen in liquid nitrogen at the crystallization temperature of isopentane.
Human fetuses
Non-pathological human fetuses (7, 11 and 14 gestational weeks (GW), n = 1 per developmental stage) were obtained from voluntarily terminated pregnancies after written informed consent was obtained from the parents (Gynecology Department, Jeanne de Flandre Hospital, Lille, France). Fetuses were fixed by immersion in 4% PFA at 4°C for 5 days. The tissues were then cryoprotected in PBS containing 30% sucrose at 4°C overnight, embedded in Tissue-Tek OCT compound (Sakura Finetek), frozen on dry ice, and stored at -80°C until sectioning. Frozen samples were cut serially at 20 mm intervals with a Leica CM 3050S cryostat (Leica Biosystems Nussloch GmbH) and immunolabeled, as described below.
K18-hACE2 mice and SARS-CoV-2 infection
Mice expressing human ACE2 (hACE2) under the control of the keratin 18 (K18 or cytokeratin) promoter on a C57BL/6 background (B6.Cg-Tg(K18-hACE2)2Prlmn/J) were purchased from the Jackson Laboratory. Mice were housed under pathogen-free conditions at 21-22°C in a 12h light/dark cycle. Access to food and water was provided ad libitum throughout the experiment.
The BetaCoV/France/IDF0372/2020 strain of SARS-CoV-2 was supplied by the French National Reference Center for Respiratory Viruses hosted by the Institut Pasteur (Paris, France). Viral infection experiments were carried out in a biosafety level 3 (BSL3) facility at the French National Veterinary School in Maisons-Alfort, following a protocol approved by the ANSES/EnvA/UPEC Ethics Committee (CE2A- 16) and authorized by the French ministry of Research under the number APAFIS#25384- 2020041515287655 v6 in accordance with French and European regulations. Male 8- to 10-week-old transgenic mice were anesthetized by i.p. injection of ketamine (100 mg per kg body weight) and xylazine (10 mg per kg body weight) and intranasally infected with 50 µl of DMEM containing 5 × 103 TCID50 of SARS-CoV-2. For brain preparation, animals were euthanized by an i.p. injection of pentobarbital (140 mg/kg body weight) 7 days after infection. Brains were removed and immersion-fixed in 4% PFA in PBS at 4 °C overnight and transferred to a 30% sucrose solution the next day. On the following day, brains were frozen and stored at −80 °C.
FNC-B4 human embryonic GnRH neuronal cell line
FNC-B4 cells 60 were kept in culture in Coon’s modified Ham’s F12 medium complemented with 10% FBS at 37°C and 5% CO2 and medium was changed twice weekly. Cells were used for pseudovirus infection and gene expression analysis when they reached 70% confluency. Gene expression assays for GnRH, ACE2, NRP1 and FPR2 were carried out on uninfected cells by quantitative RT-PCR.
Pseudotyped viral particle infection of cultured FNC-B4 cells for flow cytometry and immunocytofluorescence
Pseudotyped viral particles used for the infection were constructed by Asis Palazon (CIS bioGUNE, Spain) according to the method detailed in Crawford et al., 2020 42 using a five-plasmid system lentiviral backbone 10 (CMV promoter to express ZsGreen Fluorescent Protein), the SARS-CoV-2 spike protein, HDM-Hgpm2, pRC-CMV-Rev1b and HDM-tat1b. Cells infected with the viral particles emit green fluorescence due to the expression of ZsGreen, allowing their detection by flow cytometry. FNC-B4 cells were infected by treatment with 1.1 × 104 viral particles per mL in conditioned medium for 36 hours. Cells were then trypsinized, pelleted at 1000g for 5 minutes and resuspended in PBS for flow cytometry. Flow cytometry was performed using a CytoFLEX LX flow cytometer (Beckman Coulter). The gating strategy was based on measurements of green fluorescence by comparing cell suspensions from green fluorescent positive cells and negative cells. For each replicate, green positive events were counted from a total of 50 000 events..
For immunocytofluorescence, FNC-B4 cells were plated on cover glasses coated with 0.01% Poly-L-lysine at low confluency. Cells were fixed in 4% PFA for 15 minutes and stored at 4°C in PBS containing 0.05% sodium azide. Prior to primary antibody incubation, unspecific binding sites were blocked and cells permeabilized using an incubation solution (0.3%Triton, 0.3% BSA in PBS, pH 7.4) for an hour at room temperature. Cells were then incubated with the primary antibodies (table) in incubation solution overnight at 4°C. After three washes with PBS, cells were incubated with secondary antibodies (table) in incubation solution for an hour at room temperature. After three washes, cells were counterstained with DAPI and mounted in Mowiol.
FACS-based isolation of GnRH neurons from GnRH:GFP mice
FACS isolation of GnRH::GFP neurons has been conducted as elaborated elsewhere54,55. Briefly, the proptic region was microdissected and dissociated using a Papain Dissociation System (Worthington) to obtain single-cell suspension. FACS was performed using an ARIA SORP cell sorter cytometer and FACSDiva 8.0 software (BD Bioscience). Data were analyzed using the Kaluza 2.0 software (Beckman Coulter). The sort decision was based on measurements of EGFP fluorescence (excitation: 488 nm, 50 mW; detection: GFP bandpass 530/30 nm, autofluorescence bandpass 695/40 nm) by comparing cell suspensions from the preoptic region and from the cerebral cortex of Gnrh::Egfp animals.
RNA extraction and quantitative RT-PCR analyses
For cultured human FNC-B4 cells, total RNA extraction was performed with the E.Z.N.A. Total RNA Kit I (cat: R6834-02 ,Omega Bio-tek, Inc.) according to the manufacturer’s instructions. For human brain samples, total RNA was extracted from two fixed unstained hypothalamic slides (18μm each) using the ReliaPrep FFPE Total RNA Miniprep System (cat: Z1002, Promega). For mouse brain samples, the appropriate regions of the whole brain were dissected out and RNA extracted as above. RNA samples were immediately quantified using a Nanodrop apparatus and stored at -80°C until the reverse transcription step. For gene expression analyses, total RNA samples were reverse transcribed using the High-capacity cDNA Reverse Transcription kit (Applied Biosystems ref 4368814). For fixed human brain samples, a linear preamplification step was performed using the TaqMan PreAmp Master Mix Kit protocol (Applied Biosystems ref 4488593). Real-time PCR was then carried out using TaqMan Universal Master Mix II (Applied Biosystems ref 4440049) on the Applied Biosystems 7900HT Fast Real-Time PCR System. The TaqMan probes used in this study are listed below:
Mouse
|
Mm00607939_s1
|
Actb
|
Beta actin
|
Mouse
|
Mm03928990_g1
|
Rn18s
|
18S ribosomal RNA
|
Mouse
|
Mm01159006_m1
|
Ace2
|
Angiotensin I converting enzyme (peptidyl-dipeptidase A) 2
|
Mouse
|
Mm00484464_s1
|
Fpr2
|
Formyl peptide receptor 2
|
Mouse
|
Mm01253208_m1
|
Nrp1
|
Neuropilin 1
|
Mouse
|
Mm00443687_m1
|
Tmprss2
|
Transmembrane protease, serine 2
|
Human
|
Hs00171272_m1
|
GNRH1
|
Gonadotropin-releasing hormone 1
|
Human
|
Hs00826128_m1
|
NRP1
|
Neuropilin 1
|
Human
|
Hs99999901_s1
|
RN18S
|
Eukaryotic 18S RNA
|
The 18S ribosomal RNA was used as the housekeeping transcript for normalization. SARS-CoV-2 N-protein expression was assessed using the CDC 2019-Novel Coronavirus Real-Time RT-PCR Diagnostic Panel, as described elsewhere 61,62. All gene expression data were analyzed using the 2-ΔΔCt method.
RNAscope labeling
Expression of the SARS-Cov2 S-protein in post mortem brains was assessed using a RNAscope® Multiplex Fluorescent Reagent kit v2 Assay and the V-nCov2019-S probe, reference: 848561 (both from Advanced Cell Diagnostics Inc.). Briefly, 20-µm thick hypothalamic sections cut on a cryostat, slides washed twice for 10 min in Gibco® DPBS (ThermoFisher) and dry baked in a HybEZ™ II oven (Advanced Cell Diagnostics Inc.) at 60°C for 30 min. They were then immersion-fixed in 4% paraformaldehyde PBS 0.1M, pH7.4, prepared in DEPC-treated water, for 1h at 4°C and washed again twice for 10 min in Gibco® DPBS. Next, the sections were processed according to manufacturer’s instructions (ethanol dehydration, RNAscope hydrogen peroxide treatment and target retrieval), incubated with RNAscope protease IV for 10 min at room temperature, and the signal revealed using the RNAscope multiplex fluorescent assay.
Immunohistochemistry and quantification for viral and host-cell markers in human and mouse tissues
Dissected, postfixed and cryoprotected blocks of adult human patient brains containing the hypothalamus or olfactory bulb were cut into 20µm sections and mounted. A citrate-buffer antigen retrieval step, 10mM Citrate in TBS-Triton 0.1% pH 6 for 30 min at 70°C, was performed on 20µm sections. After 3 washes of 5 minutes with TBS-Triton 0.1%, sections were blocked in incubation solution (10% normal donkey serum, 1mg/ml BSA in TBS-Triton 0.1% pH 7,4) for 1 hour. Blocking was followed with primary antibody incubation (see Antibody table) in incubation solution for 48h at 4°C. Primary antibodies were then rinsed out, before incubation in fluorophore-coupled secondary antibodies or, in case of amplified immunolabeling, biotinylated secondary antibodies for 1h in TBS-Triton 0.1% at room temperature. For classic immunohistochemistry, secondary antibodies were washed and sections counterstained with DAPI (D9542, Sigma). For amplified immunohistochemistry, after secondary antibodies were rinsed, sections were incubated with VECTASTAIN® Elite ABC-HRP kit (PK-6100, Vector laboratories) following manufacturer’s instructions. Sections were then incubated with biotinyl-tyramide reagent (SAT700001EA, Perkin Elmer) following manufacturer’s recommendations, washed and incubated with fluorophore-coupled streptavidin (1/500 dilution in TBS-Triton 0.1%) before counterstaining with DAPI. Finally, the sections were incubated with Autofluorescence Eliminator Reagent (2160, Millipore) following manufacturer’s instructions and mounted with Fluoromount™ (F4680, Sigma). Immunolabeling in the human brain using the two antibodies to human ACE2 (R&D Systems, with tyramide amplification, and Abcam, without amplification), labeled similar cells.
For immunolabeling of human fetuses, 20 µm-thick sections of entire heads at GW 7, GW 11 and GW 14 were processed as follows. Slides first underwent antigen retrieval for 20 minutes in a 5mM citrate buffer heated to 90°C, then were rinsed in TBS and blocked/permeabilized for 2 hours at room temperature in TBS + 0.3% Triton + 0.25% BSA + 5% Normal Donkey Serum (“Incubation solution”, ICS). Sections were then incubated with primary antibodies (see Antibody table) for two nights at 4°C in ICS. After rinses in TBS, the sections were incubated with secondary antibodies for two hours at RT in ICS, then rinsed again in TBS. Finally, nuclei were stained with DAPI (Sigma D9542, 1:5000 in TBS) for 5 minutes, and sections were rinsed before coverslipping with homemade Mowiol.
For immunolabeling of mouse brain sections, 30 μm-thick floating sections were rinsed 4 times in 0.1 M PBS pH 7.4 and blocked for 1 hour at room temperature in blocking solution (PBS containing 10% normal donkey serum and 0.3% Triton X-100). Sections were incubated overnight at 4°C with a mix of primary antibodies diluted in blocking solution (see Antibody table). The sections were washed three times in 0.1M PBS and incubated at room temperature for 1 hour with Alexa Fluor-conjugated secondary antibodies (1:500 dilution; all purchased from Molecular Probes, Invitrogen, San Diego, CA) in blocking solution. A biotin-streptavidin amplification step was added for TMPRSS2 to verify expression in some tissues. The sections were then rinsed 3 times in 0.1 M PBS. Nuclei were counterstained by incubating the sections for 1 minute in DAPI before mounting and coverslipping as above.
Antibodies for immunohistochemistry
Antibody
|
Manufacturer
|
Reference
|
Dilution
|
Tissue on which used
|
Goat anti human ACE2
|
R&D Systems
|
AF933
|
1/100
|
Human
|
Rabbit anti human ACE2
|
Abcam
|
Ab15348
|
1/100-1/200
|
Human
|
Rabbit anti-TMPRSS2
|
Abcam
|
Ab92323
|
1/100-1/1000
|
Human
|
Mouse anti-FPR2
|
Invitrogen
|
GM1D6
|
1/100-1/400
|
Mouse & Human
|
Chicken anti-vimentin
|
Millipore
|
AB5733
|
1/500
|
Human
|
Goat anti-TAG1
|
R&D Systems
|
AF4439
|
1/500
|
Human
|
Goat anti-OMP
|
Wako
|
544-10001
|
1/200
|
Human
|
Guinea Pig anti GnRH
|
Erik Hrabovszky
|
|
1/3000-1/6000
|
Human, Mouse, Cells
|
Mouse anti-dsRNA
|
SCICONS J2
|
1001050
|
1/500
|
Mouse & Human
|
Mouse anti-SARS-CoV-2 spike protein
|
GeneTex
|
GTX632604
|
1/200
|
Human
|
Rabbit anti-SARS-CoV-2 spike protein
|
Sino Biological
|
40150-T62-Cov2
|
1/200
|
Mouse
|
Rabbit anti-SARS nucleocapsid protein
|
Novus Bio
|
NB100-56576
|
1/100
|
Mouse & Human
|
Goat anti-NRP1
|
R&D Systems
|
AF566
|
1/100
|
Human & Cells
|
Rabbit anti-cleaved caspase 3
|
Cell Signalling
|
#9664
|
1/200
|
Human
|
Statistics
Statistical comparisons were carried out using GraphPad Prism 8. All correlations were performed using R stats version 4.0.3.
Testosterone, LH, FSH and TSH levels were compared between the four COVID-19 patient groups (n=60 patients, 62 samples for Week 1) using an unpaired t-test.
For quantitative RT-PCR experiments (n=5 control patients vs. 4 SARS-CoV-2-infected patients; n=3 mock infected mice vs. 5 SARS-CoV-infected mice), a two-tailed unpaired t-test was used to compare expression levels between infected and control brains. The Friedman multiple comparison test followed by an uncorrected Dunn’s post test was used to compare gene expression levels between the cortex, OB and hypothalamus of infected mice.
For the quantification of the proportion of GnRH neurons showing normal or abnormal morphology in control and COVID-19 patients, a two-way ANOVA was used, followed by Sidak’s post hoc test (n=18 and 20 sections, 85 and 116 GnRH neurons, respectively; N=4 patients per group). To compare the proportion of GnRH neurons with normal or abnormal morphology that expressed cleaved caspase 3 in COVID-19 patients, a two-sided Fisher’s exact test was used (n=66 GnRH neurons, N=4 patients).
Data from flow cytometry experiments were compared using an unpaired t-test.