A Role of Endogenous Progesterone in Stroke Cerebroprotection Revealed by the Neural-Specific Deletion of Its Intracellular Receptors

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Research Articles: Neurobiology of Disease

A role of endogenous progesterone in stroke cerebroprotection revealed by the neural-specific deletion of its intracellular receptors

Xiaoyan Zhu1, Magalie Fréchou1, Philippe Liere1, Shaodong Zhang1,2, Antoine Pianos1, Fernandez Neïké1, Christian Denier1,3, Claudia Mattern4, Michael Schumacher1 and Rachida Guennoun1

1U1195 Inserm and University Paris-Sud and University Paris-Saclay, 80 rue du Général Leclerc, 94276 Kremlin-Bicêtre, France 2Beijing Neurosurgical Institute, Tiantan Xili 6, Beijing 100050, China 3Department of Neurology and Stroke Center, Bicêtre Hospital, Kremlin-Bicêtre, France 4M et P Pharma AG, Schynweg 7, P.O.Box 138, 6376 Emmetten, Switzerland

DOI: 10.1523/JNEUROSCI.3874-16.2017

Received: 20 December 2016

Revised: 28 August 2017

Accepted: 20 September 2017

Published: 6 October 2017

Author contributions: X.Z., M.S., and R.G. designed research; X.Z., M.F., P.L., S.Z., A.P., and N.F. performed research; X.Z., M.F., P.L., S.Z., A.P., N.F., C.D., C.M., M.S., and R.G. analyzed data; M.S. and R.G. wrote the paper.

Conflict of Interest: The authors declare no competing financial interests.

Xiaoyan Zhu was funded by a Scholarship from the China Scholarship Council (CSC). We thank Pr. Pierre Chambon and the Mouse Clinical Institute (MCI, Illkirch, France) for the generous gift of the PR+/loxP mice. We thank Brigitte Delespierre, Fanny Aprahamian and Annie Cambourg for their excellent technical assistance.

Corresponding authors: Dr. Rachida Guennoun and Dr. Michael Schumacher, U1195 Inserm and University Paris-Sud and University Paris-Saclay, 80 rue du Général Leclerc - 94276 Kremlin-Bicêtre, France, RG: Phone: +33 1 49 59 18 80; E-mail: [email protected], MS: Phone: +33 1 49 59 18 95; E-mail: [email protected]

Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3874-16.2017

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Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process.

2 specific deletion of its intracellular receptors

3 Abbreviated title: Progesterone and stroke cerebroprotection

4 Xiaoyan Zhu1£, Magalie Fréchou1, Philippe Liere1, Shaodong Zhang1,2, Antoine Pianos1, Neïké

5 Fernandez1, Christian Denier1,3, Claudia Mattern4, Michael Schumacher1*, Rachida Guennoun1*

6 1U1195 Inserm and University Paris-Sud and University Paris-Saclay, 80 rue du Général Leclerc,

7 94276 Kremlin-Bicêtre, France

8 2Beijing Neurosurgical Institute, Tiantan Xili 6, Beijing 100050, China

9 3Department of Neurology and Stroke Center, Bicêtre Hospital, Kremlin-Bicêtre, France

10 4M et P Pharma AG, Schynweg 7, P.O.Box 138, 6376 Emmetten, Switzerland

11 £Present address: College of Veterinary Medicine, Northwest A&F University, Yangling 712100,

12 Shaanxi, China.

13 *Corresponding authors: Dr. Rachida Guennoun and Dr. Michael Schumacher

14 U1195 Inserm and University Paris-Sud and University Paris-Saclay, 80 rue du Général Leclerc -

15 94276 Kremlin-Bicêtre, France

16 RG: Phone: +33 1 49 59 18 80; E-mail: [email protected]

17 MS: Phone: +33 1 49 59 18 95; E-mail: [email protected]

18 Number of pages: 66

19 Number of figures: 9

20 Number of tables: 5

21 Number of words in abstract: 239

22 Number of words in significance statement: 116

23 Number of words in introduction: 615

Ϯϰ Number of words in discussion: 1242

Ϯϱ Conflicts of interest: The authors declare no competing financial interests.

Ϯϲ Acknowledgements: Xiaoyan Zhu was funded by a Scholarship from the China Scholarship

Ϯϳ Council (CSC). We thank Pr. 3LHUUH&KDPERQDQGWKH0RXVH&OLQLFDO,QVWLWXWH 0&,,OONLUFK

Ϯϴ )UDQFH IRU WKH JHQHURXV JLIW RI WKH 35OR[3 PLFH We thank Brigitte Delespierre, Fanny

Ϯϵ Aprahamian and Annie Cambourg for their excellent technical assistance.

ϯϬ Abstract

ϯϭ Treatment with progesterone protects the male and female brain against damage after middle

ϯϮ cerebral artery occlusion (MCAO). However, in both sexes, the brain contains significant

ϯϯ amounts of endogenous progesterone. It is not known whether endogenously produced

ϯϰ progesterone enhances the resistance of the brain to ischemic insult. Here, we used steroid

ϯϱ profiling by gas chromatography-tandem mass spectrometry (GC-MS/MS) for exploring

ϯϲ adaptive and sex-specific changes in brain levels of progesterone and its metabolites after

ϯϳ MCAO. We show that in the male mouse brain, progesterone is mainly metabolizeG YLD Į-

ϯϴ UHGXFWLRQ OHDGLQJ WR Į-GLK\GURSURJHVWHURQH Į-DHP), also a progesterone receptor (PR)

ϯϵ DJRQLVWOLJDQGLQQHXUDOFHOOVWKHQWRĮĮ-WHWUDK\GURSURJHVWHURQH ĮĮ-THP). In the female

ϰϬ PRXVHEUDLQOHYHOVRIĮ-'+3DQGĮĮ-7+3DUHORZHUZKLOHOHYHOVRIĮ-DHP are higher

ϰϭ than in males. $IWHU 0&$2 OHYHOV RI SURJHVWHURQH DQG Į-DHP are rapidly upregulated to

ϰϮ pregnancy-like levels in the male but not in the female brain. To assess whether endogenous

ϰϯ SURJHVWHURQH DQG Į-DHP contribute to the resistance of neural cells to ischemic damage, we

ϰϰ selectively inactivated PR in the central nervous system. Deletion of PR in the brain reduced its

ϰϱ resistance to MCAO, resulting in increased infarct volumes and neurological deficits in both

ϰϲ sexes. Importantly, endogenous PR ligands continue to protect the brain of aging mice. These

ϰϳ results uncover the unexpected importance of endogenous progesterone and its metabolites in

ϰϴ cerebroprotection. They also reveal that the female reproductive hormone progesterone is an

ϰϵ endogenous cerebroprotective neurosteroid in both sexes.

ϱϬ Significance statement

ϱϭ The brain responds to injury with protective signaling and has a remarkable capacity to

ϱϮ protect itself. We show here that in response to ischemic stroke, levels of progesterone and its

ϱϯ QHXURDFWLYHPHWDEROLWHĮ-dihydroprogesterone are rapidly upregulated in the male mouse brain

ϱϰ but not in the female brain. An important role of endogenous progesterone in cerebroprotection

ϱϱ was demonstrated by the conditional inactivation of its receptor in neural cells. These results

ϱϲ show the importance of endogenous progesterone, its metabolites and neural progesterone

ϱϳ receptors in acute cerebroprotection after stroke. This new concept could be therapeutically

ϱϴ exploited by taking into account the progesterone status of patients and by supplementing and

ϱϵ reinforcing endogenous progesterone signaling for attaining its full cerebroprotective potential.

ϲϬ Introduction

ϲϭ The very rapid loss of brain tissue after the onset of ischemic stroke represents a major

ϲϮ therapeutic challenge (Saver, 2006). To date, the only acute treatments for ischemic stroke are

ϲϯ thrombolysis with tissue plasminogen activator and removal of the thrombus with a retriever

ϲϰ stent (Vivien et al., 2011; Campbell et al., 2015). These reperfusion therapies would benefit from

ϲϱ the combined use of a cerebroprotective agent, which may prolong the survival of neural cells

ϲϲ and extend the very short 4.5-hour time window to reperfusion. In addition, a cerebroprotective

ϲϳ agent may limit tissue damage resulting from reperfusion (Donnan et al., 2011; Fisher, 2011).

ϲϴ Treatment with exogenous progesterone has been demonstrated to protect the male and

ϲϵ female rodent brain against ischemic damage after middle cerebral artery occlusion (MCAO)

ϳϬ (Sayeed and Stein, 2009; Gibson et al., 2011). Notably, progesterone treatment shows long-

ϳϭ lasting cerebroprotective efficacy with a 6 hr window of opportunity (Gibson et al., 2011; Wali

ϳϮ et al., 2014). However, it is unknown whether endogenous progesterone plays a role in

ϳϯ cerebroprotective mechanisms. In adult males and females, neural cells are indeed exposed to

ϳϰ significant levels of progesterone, either derived from the ovaries in females or from the adrenal

ϳϱ glands in both sexes (Schumacher et al., 2014). Moreover, progesterone is a "neurosteroid"

ϳϲ synthesized de novo within the brain (Baulieu, 1997).

ϳϳ The aim of the present study was to assess whether endogenous progesterone and its

ϳϴ metabolites are involved in acute self-protection of the male and female mouse brain against

ϳϵ ischemic damage. This is an important problem, as there is an ongoing interest in taking

ϴϬ advantage of endogenous cerebroprotective mechanisms for therapeutic purposes (Iadecola and

ϴϭ Anrather, 2011; Mergenthaler and Dirnagl, 2011). However, assessment of the cerebroprotective

ϴϮ actions of progesterone requires to take into account its extensive metabolism in the brain

ϴϯ (Schumacher et al., 2014). 7KXVSURJHVWHURQH LVUHDGLO\FRQYHUWHGWR Į-DHP by neural cells

ϴϰ (Celotti et al., 1992). ,QWKHVHFHOOVERWKSURJHVWHURQHDQGĮ-DHP activate gene transcription

ϴϱ via PR (Rupprecht et al., 1993) (Fig. 1). The PR are ligand-activated transcription factors, but

ϴϲ they also interact with membrane associated kinases outside the nucleus (Lange et al., 2007;

ϴϳ Grimm et al., 2016). We have recently demonstrated that PR play a key role in the

ϴϴ cerebroprotective actions of progesterone after MCAO (Liu et al., 2012). The progesterone

ϴϵ PHWDEROLWH Į-'+3 LV IXUWKHU FRQYHUWHG WR ĮĮ-THP, also well-known as allopregnanolone,

ϵϬ which does not bind to PR but functions as a positive modulator of GABAA receptors, resulting

ϵϭ in the inhibition of neuronal activity (Fig. 1) (Hosie et al., 2006; Lambert et al., 2009). The

ϵϮ FRQYHUVLRQ WR Į-GLK\GURSURJHVWHURQH Į-DHP) is an additional metabolic pathway of

ϵϯ SURJHVWHURQH $V Į-'+3 KDV ORZ DIILQLW\ IRU 35 DQG QR SURJHVWRJHQLF DFWLYLW\ WKH Į-

ϵϰ reduction is generally considered as an inactivation pathway of progesterone (Fig. 1). However,

ϵϱ SURJHVWHURQHDQGĮ-'+3DUHLQWHUFRQYHUWLEOHDQGĮ-DHP may thus serve as a reservoir for

ϵϲ progesterone and its biologically active metabolites (Hillisch et al., 2003; Williams et al., 2012).

ϵϳ We used sensitive and specific gas chromatography-tandem mass spectrometry (GC-

ϵϴ MS/MS) for establishing steroid profiles in the brain and plasma of intact and ischemied male

ϵϵ and female C57BL/6 mice. We found that OHYHOVRIĮ-'+3DQGĮĮ-THP are higher while

ϭϬϬ OHYHOVRIĮ-DHP are lower in the male than in the female brain. During the first 6 hrs after

ϭϬϭ MCAO, there was a rapid and major LQFUHDVHLQWKHOHYHOVRISURJHVWHURQHDQGĮ-DHP in the

ϭϬϮ male but surprisingly not in the female brain. To determine whether endogenous PR-active

ϭϬϯ SURJHVWHURQHDQGĮ-DHP are involved in the acute resistance of the brain to ischemic damage,

ϭϬϰ we selectively inactivated PR in neural cells. Lack of neural PR increased the vulnerability of the

ϭϬϱ brain of both male and female, young and aging mice.

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ϭϯϮ FRORQ\:HDOVRJHQHUDWHGDWUDQVJHQLFFRORQ\RI351HV&UHPLFHWKDWVHOHFWLYHO\ODFN35LQWKH

ϭϯϯ QHXUDOFHOOVXVLQJWKH&UH-OR[VWUDWHJ\0DOHPLFH357J 1HV&UH ZHUHPDWHGZLWKIHPDOH

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ϭϰϬ VSHFLILFSULPHUV Screening for LoxP sites: F1: ¶-AACATGGAAGGATGCACCTGTTCCC-¶

ϭϰϭ R1: ¶-GCCTTGAACTCATGAATTCCCAGCTTC-¶)¶-CCTTCCCTCTCATTGTCATG

ϭϰϮ GAGAC-¶ 5 ¶-AGAGAATCTCTCGCCAGTGTCATGG-¶ Screening for Nes Cre

ϭϰϯ transgene: )¶-GCGGTCTGGCAGTAAAAACTATC-¶; R3: ¶-GTGAAACAGCATTGCT

ϭϰϰ GTCACTT-¶. Internal positive control for PCR amplification: F4: ¶-CTAGGCCACAGAATT

ϭϰϱ GAAAGATCT-¶; R4: ¶-GTAGGTGGAAATTCTAGCATCATCC-¶). 0LFH ZHUH JURXS

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ϭϰϳ RQIURPDPWRSP ZLWKIRRGDQGZDWHUad libitum

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ϭϱϬ Transient cerebral ischemia was performed under ketamine (50 mg/kg) and xylazine

ϭϱϭ hydrochloride (6 mg/kg) anesthesia. %RG\ WHPSHUDWXUH RI WKH PLFH ZDV PRQLWRUHG E\ D UHFWDO

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ϭϱϳ A middle cerebral artery occlusion was performed for 30 min as previously described *LEVRQ

ϭϱϴ DQG0XUSK\ Briefly, the left common carotid artery was isolated. A nylon monofilament

ϭϱϵ 'UHQQDQௗPGLDPHWHU FRDWHGZLWKµWKHUPRPHOWLQJ¶JOXH ௗPPORQJௗPGLDPHWHU ZDV

ϭϲϬ introduced through an arteriotomy performed in the common carotid artery and advanced into

ϭϲϭ the internal carotid artery to occlude the origin of the PLGGOHFHUHEUDODUWHU\. The filament was

ϭϲϮ withdrawn 30 min after occlusion to allow reperfusion.

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ϭϳϱ Experimental groups:

ϭϳϲ Set 1: Designed to evaluate the effect of ischemia on steroid levels:&%/\RXQJDGXOWPDOH

ϭϳϳ DQG IHPDOH PLFH PRQWKV ROG ZHUH SXUFKDVHG IURP -DQYLHU /H *HQHVW )UDQFH DQG ZHUH

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ϭϳϵ GLHVWUXV SKDVH The estrus cycle was monitored between 9 and 10 a.m. by the examination of

ϭϴϬ vaginal smears in a large group of intact young adult females. The adopted nomenclature was

ϭϴϭ proestrus, estrus, metestrus and diestrus according to the percentage of different cell types

ϭϴϮ present: proestrus (a majority of nucleated epithelial cells), estrus (a majority of cornified

ϭϴϯ epithelial cells), metestrus (cornified epithelial cells and leukocytes) and diestrus (a majority of

ϭϴϰ leukocytes) (Byers et al., 2012). The estrus cycle stage was checked in all females and those in

ϭϴϱ diestrus were selected to constitute experimental groups of that day.

ϭϴϲ 7ZHOYHPLFH PDOHVDQGIHPDOHV ZHUHXVHGDVFRQWUROVWRSURYLGHUHIHUHQFHYDOXHVIRUEUDLQ

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ϭϴϴ VXEMHFWHGWR0&$2IRUPLQXWHV6HWVRIPLFH PDOHVDQGIHPDOHV ZHUHVDFULILFHGDW

ϭϴϵ DQGKUVDIWHURFFOXVLRQRIWKHPLGGOHFHUHEUDODUWHU\

ϭϵϬ Set 2: Designed to evaluate the effects of neural PR deletion on steroid levels in both sexes:

ϭϵϭ

ϭϵϮ JQ SHUJURXS DQG\RXQJDGXOWIHPDOH35OR[3OR[3DQG351HV&UHPLFH -PRQWKVROG-

ϭϵϯ JGLHVWUXVSKDVHQ SHUJURXS ZHUHXVHG. The estrus cycle stage was checked in all females

ϭϵϰ and those in diestrus were selected to constitute experimental groups of that day.

ϭϵϱ Set 3: Designed to evaluate the effect of neural PR deletion and MCAO on steroid levels:

ϭϵϲ DGXOWPDOH35OR[3OR[3DQG351HV&UHPLFH -PRQWKVROG-JQ SHUJURXS \RXQJDGXOW

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ϮϬϭ Steroids were extracted from plasma and the left hemisphere (180-240 mg) of brain with

ϮϬϮ methanol, and the following internal standards were added to the extracts for steroid

2 ϮϬϯ quantification: 2 ng of H6-Į-DHP (CDN Isotopes, Sainte Foy la Grande, France) for the

13 ϮϬϰ DQDO\VLV RI Įȕ-GLK\GURSURJHVWHURQH Įȕ-DHP), 2 ng of C3-testosterone (Isoscience, PA,

13 ϮϬϱ USA) for the analysis of testosterone, 2 ng of C3-androstenedione for the analysis of

13 13 ϮϬϲ androstenedione, 2 ng of C3-progesterone for the analysis of progesterone, 2 ng of C3-ȕ-

2 ϮϬϳ HVWUDGLROIRUWKHDQDO\VLVRIȕ-estradiol, H13-deoxycorticosterone (DOC) for analysis of DOC,

ϮϬϴ 2 ng of 19 nor-352*IRUWKHDQDO\VLVRIĮ-'+3DQGĮȕ-dihydrodeoxycorticosterone, 2 ng of

ϮϬϵ HSLHWLRFKRODQRORQH 6WHUDORLGV 1HZSRUW 5KRGH ,VODQG IRU WKH DQDO\VLV RI Į-

ϮϭϬ GLK\GURWHVWRVWHURQH Į-DHT), 3ĮĮ-WHWUDK\GURWHVWRVWHURQH SUHJQHQRORQH Į-

Ϯϭϭ GLK\GURSUHJQHQRORQH ĮȕĮȕ-WHWUDK\GURSURJHVWHURQH ĮĮ-tetrahydroprogesterone,

2 ϮϭϮ ĮȕĮȕ-KH[DK\GURSURJHVWHURQH DQG ĮĮȕ-tetrahydrodeoxycorticosterone, H8-corticosterone

Ϯϭϯ for the analysis of corticosterone. Samples were purified and fractionated by solid-phase

Ϯϭϰ extraction with the recycling procedure (Liere et al., 2004). Briefly, the extracts were dissolved

Ϯϭϱ in 1 ml methanol and applied to the C18 cartridge (500 mg, 6 ml, International Sorbent

Ϯϭϲ Technology, IST), followed by 5 ml of methanol/H2O (85/15). The flow-through, containing the

Ϯϭϳ free steroids, was collected and dried. After a previous re-conditioning of the same cartridge with

Ϯϭϴ 5 ml H2O, the dried samples were dissolved in methanol/H2O (2/8) and re-applied. The cartridge

ϭϭ

Ϯϭϵ was then washed with 5 ml H2O and 5 ml methanol/H2O (1/1) and unconjugated steroids were

ϮϮϬ eluted with 5 ml methanol/H2O (9/1).

ϮϮϭ The unconjugated steroid-containing fraction was then filtered and further purified and

ϮϮϮ fractionated by HPLC. The HPLC system was composed of a WPS-3000SL analytical

ϮϮϯ autosampler and a LPG-3400SD quaternary pump gradient coupled with a SR-3000 fraction

ϮϮϰ collector (Thermoscientific, USA). The HPLC separation was achieved with a Lichrosorb Diol

ϮϮϱ column (25 cm, 4.6 mm, 5 m) and a thermostatic block at 30°C. The column was equilibrated

ϮϮϲ in a solvent system of 90% hexane and 10% of a mixture composed of hexane/isopropanol

ϮϮϳ (85/15). Elution was performed at a flow-rate of 1 ml/min, first 90% hexane and 10% of

ϮϮϴ hexane/isopropanol (85/15) for 8 min, then with a linear gradient to 100% of hexane/isopropanol

ϮϮϵ (85/15) in 2 min. This mobile phase was kept constant for 10 min and a linear gradient to 100%

ϮϯϬ methanol was applied. The column was washed with methanol for 15 min.

Ϯϯϭ ThreHIUDFWLRQVZHUH FROOHFWHGIURPWKH+3/& V\VWHP Įȕ-DHPROG was eluted in the

ϮϯϮ first HPLC fraction (3-13 min) and was silylated with 50 l of a mixture N-methyl-N-

Ϯϯϯ trimethylsilyltrifluoroacetamide/ammonium iodide/dithioerythritol (1000:2:5 vol/wt/wt) for 15

Ϯϯϰ min at 70°C. The second fraction (13-25 min) containing pregnenolone, 20Į-

Ϯϯϱ dihydropregnenolone, progesterone, deoxycorticosterone, testosterone and their reduced

Ϯϯϲ metabolites, and ȕ-estradiol, was derivatized with 25 l heptafluorobutyric anhydride (HFBA)

Ϯϯϳ and 25 l anhydrous acetone for 1 hr 20°C. Corticosterone was eluted in the third fraction (25-33

Ϯϯϴ min) and derivatized with 25 l HFBA and 25 l anhydrous acetone for 1 hr at 80°C. All the

Ϯϯϵ fractions were dried under a stream of N2 and resuspended in hexane for GC-MS/MS analysis.

ϮϰϬ GC-MS/MS analysis of the brain extracts was performed using an AI 1310 autosampler, a

Ϯϰϭ Trace 1310 gas chromatograph (GC), and a TSQ 8000 tandem mass spectrometer (MS/MS)

ϭϮ

ϮϰϮ (Thermo Fisher Scientific San Jose, CA) using Argon as collision gas. Injection was performed

Ϯϰϯ in the splitless mode at 250°C (1 min of splitless time) and the temperature of the gas

Ϯϰϰ chromatograph oven was initially maintained at 50°C for 1 min and ramped between 50 to 200°C

Ϯϰϱ at 20°C/min, then ramped to 300°C at 5°C/min and finally ramped to 350°C at 30°C/min. The

Ϯϰϲ helium carrier gas flow was maintained constant at 1 ml/min during the analysis. The transfer

Ϯϰϳ line and ionization chamber temperatures were 330°C and 180°C, respectively. Electron impact

Ϯϰϴ ionization was used for mass spectrometry with ionization energy of 70 eV. GC-MS/MS signals

Ϯϰϵ were evaluated using a computer workstation by means of the software Excalibur®, release 3.0

ϮϱϬ (Thermoscientific, USA). Identification of steroids was supported by their retention time and

Ϯϱϭ according to two or three transitions. Quantification was performed according to the transition

ϮϱϮ giving the more abundant product ion (Table 1) with a previously established calibration curve.

Ϯϱϯ The analytical protocol has been validated for all the targeted steroids by using extracts of

Ϯϱϰ 200 mg from a pool of male mice brain. The evaluation included the limit of detection, linearity,

Ϯϱϱ accuracy, intra- and inter-assay precisions (Table 2). The limit of detection was determined as

Ϯϱϲ the lowest amount of compounds that can be measured by GC-MS/MS with a signal-to-noise

Ϯϱϳ ratio greater than 3. It ranged from 0.5 pg/g to 20 pg/g. The linearity was assessed by analyzing

Ϯϱϴ increasing amounts of mouse brain extracts (20, 50, 100 and 200 mg) in triplicate. The linearity

Ϯϱϵ was satisfactory for all the steroids with a coefficient of correlation ranging from 0.992 to 0.999.

ϮϲϬ The accuracy of the assay was evaluated by determining the analytical recovery, which was

Ϯϲϭ defined as C/(C0+S)x100(%). C is the concentration of the steroid in the spiked brain extract

ϮϲϮ (100 mg), C0 is the concentration of a steroid in the unspiked brain extract (100 mg) and S is the

Ϯϲϯ spiked concentration. The precision of the intra and inter-assays were evaluated by analyzing 5

Ϯϲϰ replicates of 200 mg of brain extracts on 1 day and over 4 days, respectively.

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ϯϱϲ Statistical analysis was performed using GraphPad Prism 7 (GraphPad Software Inc., USA). The

ϯϱϳ data are expressed as mean ± SEM and n refers to the number of animals per experimental group.

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ϯϱϵ multiple comparisons tests between groups. Kruskal-Wallis analysis and Mann-Whitney tests

ϯϲϬ were used for neurological deficits. The correlation of infarct volume and neurological deficits

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ϭϴ

ϯϲϮ Results

ϯϲϯ Steroid profiling in brain and plasma of young adult male and female mice

ϯϲϰ We first established steroid profiles by GC-MS/MS for brain and plasma of 3 months old

ϯϲϱ male and female C57BL/6 mice to determine reference values (Fig. 2 and Table 3). Females

ϯϲϲ were at their diestrus stage according to vaginal cytology. Results for pregnenolone,

ϯϲϳ progesterone and its major metabolites are shown in Figure 2. Brain levels of pregnenolone were

ϯϲϴ similar in both sexes and about 30 times higher than in plasma, consistent with a de novo

ϯϲϵ synthesis by neural cells (Fig. 2A). Levels of progesterone did not differ between sexes in brain,

ϯϳϬ but tended to be higher in the plasma of females. However, in comparison with pregnenolone,

ϯϳϭ brain levels of progesterone were low (< 1 ng/g) (Fig. 2B). Progesterone is indeed rapidly

ϯϳϮ metabolized in the brain, and its measure thus does not provide sufficient information about the

ϯϳϯ dynamics of pregnanes.

ϯϳϰ The analysis of progesterone metabolites by GC-MS/MS indeed provided a completely

ϯϳϱ different and unexpected picture, with marked differences between brain and plasma and

ϯϳϲ EHWZHHQ VH[HV ,Q WKH PDOH EUDLQ OHYHOV RI Į-DHP were 75 times higher than those of

ϯϳϳ progesterone, reaching concentrations of 12 ng/g (about 40 nM) (Fig. 2C). Importantly, brain

ϯϳϴ OHYHOV RI Į-DHP were about 4 times higher in males than in females, and may thus be

ϯϳϵ considered as a major male brain steroid. +RZHYHUSODVPDOHYHOVRIĮ-DHP were also elevated

ϯϴϬ in males, suggesting an important contribution of the adrenal glands (Fig. 2C).

ϯϴϭ &RQVLVWHQWO\EUDLQOHYHOVRIWKHĮ-'+3PHWDEROLWHĮĮ-THP were higher in males than

ϯϴϮ in females (Fig. 2D ,QWHUHVWLQJO\EUDLQOHYHOVRIĮĮ-WHWUDK\GURGHR[\FRUWLFRVWHURQH ĮĮ-

ϯϴϯ THDOC), another potent GABAA receptor modulator derived from the metabolism of adrenal

ϭϵ

ϯϴϰ deoxycorticosterone (DOC) (Sarkar et al., 2011), were also significantly higher in brain of males

ϯϴϱ when compared to females (Table 3).

ϯϴϲ Thus, steroid profiling by GC-MS/MS revealed the surprising fact that the male mouse

ϯϴϳ brain contains more neuroactive metabolites of progesterone than the female brain. In contrast,

ϯϴϴ EUDLQ DQG SODVPD OHYHOV RI Į-DHP were respectively more than 10 or 20 times higher in

ϯϴϵ females as compared to males (Fig. 2E). The reversible conversion of progesterone to Į-DHP

ϯϵϬ may represent a reservoir of brain pregnanes in females. Additional steroids analyzed in brain

ϯϵϭ and plasma of both sexes are shown in Table 3. Of note, LQ FRPSDULVRQ WR WKH LPSRUWDQW Į-

ϯϵϮ UHGXFWLRQRISURJHVWHURQHLQPDOHVWKHIRUPDWLRQRIĮ-reduced metabolites of testosterone was

ϯϵϯ very low (Table 3).

ϯϵϰ

ϯϵϱ Steroid profiling after MCAO in brain and plasma of male and female mice

ϯϵϲ To gain insight into changes of steroid levels in response to ischemic injury, we performed

ϯϵϳ steroid profiling by GC-MS/MS in the left ischemic hemisphere and in plasma of male and

ϯϵϴ diestrus female C57BL/6 mice at 1, 2, 4, 6 or 24 hrs after transient MCAO. The left middle

ϯϵϵ cerebral artery was occluded during 30 min, followed by reperfusion (Fig. 3 and Table 4).

ϰϬϬ In both males and females, brain levels of pregnenolone were about 5 to 10 times higher in

ϰϬϭ brain when compared to plasma, confirming results of the previous experiment (Fig. 3A). In the

ϰϬϮ ipsilateral hemisphere, levels of pregnenolone were significantly increased at 6 hrs after MCAO

ϰϬϯ and returned to control values after 24 hrs in males. There was no such change with time in

ϰϬϰ females. In both sexes, the low plasma levels of pregnenolone were slightly, but significantly,

ϰϬϱ increased in a transient manner at 4 and 6 hrs (Fig. 3A).

ϮϬ

ϰϬϲ In males, brain levels of progesterone were increased at 4 and 6 hrs after MCAO, paralleled

ϰϬϳ by a similar increase in plasma levels, which probably reflects activation of the adrenal glands

ϰϬϴ (Fig. 3B). A similar trend was observed in females, but without reaching statistical significance

ϰϬϵ for the brain. At 6 hrs, brain and plasma levels of progesterone were lower in females when

ϰϭϬ compared with males.

ϰϭϭ The magnitude of brain-specific changes in endogenous progesterone after MCAO may

ϰϭϮ have been partially masked by its rapid and strong Į-reduction (Poletti et al., 1998). Indeed, the

ϰϭϯ PRVW PDUNHG GLIIHUHQFHV EHWZHHQ WLVVXHV DQG VH[HV ZHUH REVHUYHG IRU Į-DHP. In the male

ϰϭϰ LSVLODWHUDOKHPLVSKHUHOHYHOVRIĮ-DHP continuously raised between 1 and 6 hrs after MCAO

ϰϭϱ (Fig. 3C). Maximal and very elevated EUDLQOHYHOVRIĮ-DHP were reached at 6 hrs. In plasma,

ϰϭϲ OHYHOVRIĮ-DHP remained much lower, with a slight increase at 24 hrs. Of note, leveOVRIĮ-

ϰϭϳ DHP remained elevated in the male brain as late as 24 hrs after MCAO. In contrast, levels of 5Į-

ϰϭϴ DHP remained low in brain ( 5 ng/g) and plasma ( 1 ng/ml) of females (Fig. 3C).

ϰϭϵ The GABAA UHFHSWRUDFWLYHSURJHVWHURQHPHWDEROLWHĮĮ-THP did not show significant

ϰϮϬ changes with time after MCAO in both sexes, neither in brain nor in plasma. However, levels of

ϰϮϭ ĮĮ-THP in brain and plasma were higher in males when compared with females (Fig. 3D). In

ϰϮϮ PDOHVOHYHOVRIĮ-DHP were significantly increased in brain and plasma at 4 and 6 hrs after

ϰϮϯ MCAO. No significant changes over time for the constantly elevated levels RI Į-DHP in

ϰϮϰ females (Fig. 3E).

ϰϮϱ 6XPPLQJ WKH OHYHOV RI SURJHVWHURQH DQG Į-DHP well illustrates the rapid and marked

ϰϮϲ increase in PR-active steroids in male brain after MCAO and the noticeable sex difference. Both

ϰϮϳ pregnanes reached maximal levels of nearly 70 ng/g (about 220 nM) at 6 hrs after MCAO in the

ϰϮϴ male brain (Fig. 4A), which is comparable to their pregnancy plasma levels in mice (Murr et al.,

Ϯϭ

ϰϮϵ 1974). In males, plasma levels of progesterone + Į-DHP were about 6 times lower than brain

ϰϯϬ levels, and only showed a small increase at 6 and 24 hrs (Fig. 4B). In contrast to males, summed

ϰϯϭ levels of progesterone + Į-DHP remained low after MCAO in the female brain and plasma

ϰϯϮ (Fig. 4C,D).

ϰϯϯ Additional steroid metabolomic data for brain and plasma, providing reference data, are

ϰϯϰ SUHVHQWHG LQ 7DEOH ,Q FRQWUDVW WR Į-'+3 OHYHOV RI ȕ-DHP were very low in brain and

ϰϯϱ plasma of both sexes (around 10 SJSHUPORUJ %HFDXVHWKHPROHFXODUVWUXFWXUHRIȕ-reduced

ϰϯϲ steroids is not planar but bent, they are considered as biologically inactive and to be involved in

ϰϯϳ hormone clearance (Chen and Penning, 2014). HoweveU VRPH ȕ-reduced metabolites of

ϰϯϴ progesterone have neuroactive properties. Thus Įȕ-WHWUDK\GURSURJHVWHURQH Įȕ-THP,

ϰϯϵ SUHJQDQRORQH LVOLNHĮĮ-THP (allopregnanolone) a positive allosteric modulator of GABAA

ϰϰϬ receptors, both having the same potency (Belelli et al., 1996). Interestingly, whereas brain and

ϰϰϭ SODVPDOHYHOVRIĮĮ-THP were higher in males (Fig. 3D WKRVHRIĮȕ-THP were higher in

ϰϰϮ IHPDOHVDWKUVDIWHU0&$2 7DEOH /HYHOVRIĮȕĮ-hexahydroprogesterone were also

ϰϰϯ higher in females than in males, but the biological significance of the hexahydroprogesterone

ϰϰϰ isomers remains to be explored.

ϰϰϱ Remarkable were sex differences in the levels of neuroactive steroids derived from the

ϰϰϲ adrenal glands. Plasma levels of corticosterone, the principal glucocorticoid in mice and rats,

ϰϰϳ increased from 10-20 ng/ml in controls to about 200 ng/mg at 6 hrs after MCAO in both sexes

ϰϰϴ (effect of time: F(5,47) = 18.3, p < 0.0001) (Table 4). However, in the brain, a different picture

ϰϰϵ was observed. Although levels of corticosterone significantly increased in the ischemic

ϰϱϬ hemisphere of both sexes (effect of time: F(5,76) = 13.2, p < 0.0001), they remained lower than in

ϰϱϭ plasma until 4 hrs after MCAO. Only at 6 hrs, brain levels of corticosterone reached plasma

ϮϮ

ϰϱϮ levels in males, whereas in females brain levels of corticosterone remained lower (effect of sex:

ϰϱϯ F(1,76) = 10.5, p = 0.002; interaction between time and sex (F(5, 76) = 4.31, p = 0.002). The female

ϰϱϰ brain may thus protect itself against the steadily increasing, very high levels of adrenal

ϰϱϱ glucocorticoids. After 24 hrs, levels of the adrenal steroids returned to lower levels, but remained

ϰϱϲ above control levels.

ϰϱϳ Also of note, in the plasma, coQYHUVLRQ RI WKH FRUWLFRVWHURQH SUHFXUVRU '2& WR LWV ȕ-

ϰϱϴ UHGXFHGPHWDEROLWHVȕ-DHDOC (effect of sex: F(1,59) = 244.7, p < 0.0001) DQGĮȕ-THDOC

ϰϱϵ (effect of sex: F(1,50) = 80.7, p < 0.0001) was much higher in females than in males (Table 4). In

ϰϲϬ WKHFLUFXODWLRQWKHVXPRIERWKȕ-reduced metabolites reached 32 ng/ml in females at 4 hrs,

ϰϲϭ which was about 250 times levels measured in males. In the brain, levels of both metabolites

ϰϲϮ ZHUHDOVRKLJKHULQIHPDOHVWKDQLQPDOHV ȕ-DHDOC: effect of sex: F(1,77) = 83.5, p < 0.0001;

ϰϲϯ Įȕ-THDOC: effect of sex: F(1,67) = 6.69, p < 0.05) (Table 4). Thus, females produce

ϰϲϰ significantly higher amounts of GABAA UHFHSWRUDFWLYHȕ-reduced metabolites of progesterone

ϰϲϱ and DOC, respectively Įȕ-THP and by structural analogy Įȕ-THDOC (Gunn et al., 2015).

ϰϲϲ In contrast to the glucocorticoids, levels of testosterone decreased after the ischemic injury

ϰϲϳ in plasma (effect of time: F(5,59) = 5.38, p < 0.001) and brain (effect of time: F(5,73) = 5.89, p <

ϰϲϴ 0.001) of males (Table 4). Moreover, there was a significant decrease in male EUDLQ Į-DHT

ϰϲϵ levels (effect of time: F(5,67) = 5.41, p < 0.001)7KLVGHFUHDVHLQWKHĮ-reduction of testosterone

ϰϳϬ WKXVGLIIHUVIURPWKHPDUNHGLQFUHDVHLQWKHIRUPDWLRQRIĮ-reduced metabolites of progesterone

ϰϳϭ in males (Fig. 3C,D). As expected (Nilsson et al., 2015), low brain concentrations of estradiol

ϰϳϮ were measured and no significant changes over time were observed after MCAO.

ϰϳϯ

Ϯϯ

ϰϳϰ Selective deletion of intracellular progesterone receptors (PR) in the central nervous

ϰϳϱ system

ϰϳϲ We hypothesized that that the rapid and important increase in brain levels of progesterone

ϰϳϳ DQGĮ-DHP may be part of spontaneous cerebroprotective responses. To gain an insight into the

ϰϳϴ potential role of these endogenous PR ligands in the acute resistance of brain tissues against

ϰϳϵ ischemic damage, we selectively inactivated PR expression in neural cells by using the Cre-LoxP

ϰϴϬ site-specific recombination system to generate a new transgenic mice line PRNesCre.

ϰϴϭ Specificity and efficiency of neural PR gene invalidation via Cre recombination were

ϰϴϮ determined by quantitative RT-PCR (qPCR) and immunofluorescence analyses. QPCR showed a

ϰϴϯ nearly complete deletion of PR mRNA expression in the brain of PRNesCre males and females

ϰϴϰ (96% in hypothalamus and 99% in cortex and subcortical regions) (Fig. 5A). In contrast to the

ϰϴϱ brain, PR mRNA expression was similar in wild-type PR+/+, PRloxP/loxP and PRNesCre mice for

ϰϴϲ aorta, kidney and thymus in males, and for uterus, ovary and mammary glands in females (Fig.

ϰϴϳ 5B). Immunofluorescence detection revealed that neural PR protein was expressed in the

ϰϴϴ hypothalamus of PRloxP/loxP mice, but that it was almost completely absent in the hypothalamus of

ϰϴϵ PRNesCre mice (Fig. 5C). In contrast to the hypothalamus, a similar distribution and density of PR

ϰϵϬ immunoreactivity was observed in the uterus of PRloxP/loxP and PRNesCre female mice (Fig. 5C).

ϰϵϭ Altogether, these results demonstrate that the PR gene was efficiently and selectively invalidated

ϰϵϮ in the brain of both male and female PRNesCre mice. As the Cre recombinase was not expressed in

ϰϵϯ microglia, present results also show that in the mouse brain, PR is not present in this cell type.

ϰϵϰ At the age of 1 month, PRNesCre mice had the same weight as sex-matched PRloxP/loxP mice.

ϰϵϱ At 4 months, about the time when experiments of ischemic injury in young animals were

ϰϵϲ performed, PRNesCre mice were 6-7% lighter than PRloxP/loxP mice of the same sex (males:

Ϯϰ

ϰϵϳ PRNesCre: 26.4g ± 0.2; PRloxP/loxP: 28.1g ± 0.2; females: PRNesCre: 20.9g ± 0.2; PRloxP/loxP: 22.4g ±

ϰϵϴ 0.2, means ± SEM; effect of sex: F(1,29) = 1208.1, p < 0.001; effect of genotype: F(1,29) = 100.3, p

ϰϵϵ < 0.001). At 12 months, the weight differences between PRloxP/loxP and PRNesCre mice became

ϱϬϬ even more pronounced, but did not exceed 20% (males: PRNesCre: 28.3 g ± 0.3; PRloxP/loxP: 35.1 g

NesCre loxP/loxP ϱϬϭ ± 1.1; females: PR : 27.3 g ± 0.8; PR : 31.3 g ± 1.5, means ± SEM; effect of sex: F(1,31)

NesCre ϱϬϮ = 5.55, p < 0.05; effect of genotype: F(1,31) = 28.5, p < 0.001). Thus, during aging, PR mice

ϱϬϯ of both sexes gain less weight.

ϱϬϰ Male PRNesCre mice showed normal fertility when mated with female PRloxP/loxP mice. Of 8

ϱϬϱ pairs, all produced a number of offspring comparable to wild-types. In contrast, PRNesCre females

ϱϬϲ exhibited significantly reduced fertility when mated with PRloxP/loxP males. Over a 3 months

ϱϬϳ observation period, only 4 females out of 24, produced offspring, and the size of their litters did

ϱϬϴ not exceed 2 pups. One reason for this low fertility of PRNesCre females were irregular estrus

ϱϬϵ cycles, as determined by vaginal cytology, and a markedly reduced number of corpus lutea

ϱϭϬ within the ovaries of PRNesCre females. This reproductive phenotype is consistent with the

ϱϭϭ previously reported inability of total PR knockout mice to ovulate (Lydon et al., 1995).

ϱϭϮ

ϱϭϯ Steroid profiling in brain and plasma of young adult PRloxP/loxP and PRNesCre mice of both

ϱϭϰ sexes

ϱϭϱ To assess whether conditional inactivation of PR in neural cells of the CNS had an impact

ϱϭϲ on steroid levels, extended brain and plasma profiles were established by GC-MS/MS for young

ϱϭϳ (3 months old) male and female PRloxP/loxP and PRNesCre mice (Table 5). Both PRloxP/loxP and

ϱϭϴ PRNesCre females were again sampled according to vaginal cytology at the diestrus stage.

Ϯϱ

ϱϭϵ Levels of the major steroids measured in the PRloxP/loxP and PRNesCre mice were comparable

ϱϮϬ to those reported in Figure 2 and Table 3 for wild-type C57BL/6 mice. Levels of pregnenolone

ϱϮϭ were much higher in brain than in plasma, whereas similar low levels of progesterone were

ϱϮϮ measured. Important for our purpose was the confirmation of much higher levels of PR active

ϱϮϯ Į-DHP in the male brain when compared to females and plasma (Table 5). Furthermore, levels

ϱϮϰ RIĮ-DHP in brain and plasma were about 10-20 times higher in females when compared with

ϱϮϱ males. However, DEVROXWHOHYHOVRIĮ-DHP in this experiment were much higher than those

ϱϮϲ shown in figure 2. This difference may not necessarily reflect strain variations, but it may have

ϱϮϳ been caused by higher levels of stress and adrenal activity. Indeed, levels of the stress response

ϱϮϴ hormone corticosterone were also elevated in the present experiment. Results further confirm

ϱϮϵ KLJKHUOHYHOVRIȕ-reduced metabolites of progesterone and DOC in females when compared to

ϱϯϬ males.

ϱϯϭ Importantly, steroid levels were comparable between PRloxP/loxP and PRNesCre mice for PR-

ϱϯϮ DFWLYHSURJHVWHURQHDQGĮ-DHP (Table 5). However, plasma and brain levels of corticosterone

ϱϯϯ were lower in PRNesCre males when compared with PRloxP/loxP males. This may also have

ϱϯϰ contributed to slightly reduced levels of VRPHĮ-reduced metabolites of progesterone and DOC

ϱϯϱ in PRNesCre mice.

ϱϯϲ

ϱϯϳ Changes in brain levels of progesterone and its metabolites after MCAO in male and

ϱϯϴ female PRloxP/loxP and PRNesCre mice

ϱϯϵ The PRNesCre mice were generated to uncover the cerebroprotective effects of endogenous

ϱϰϬ EUDLQ SURJHVWHURQH DQG Į-DHP, both active PR ligands, in males. It was thus important to

ϱϰϭ examine whether levels of progesterone and its active metabolites were rapidly increased after

Ϯϲ

ϱϰϮ MCAO in the transgenic male PRloxP/loxP and PRNesCre mice, as they are in wild-type males (see

ϱϰϯ Fig. 3). Brain levels of progesterone and its major reduced metabolites were analyzed by GC-

ϱϰϰ MS/MS in the left infarcted hemisphere of PRloxP/loxP and PRNesCre male and female mice at 6 hrs

ϱϰϱ after sham surgery or MCAO.

ϱϰϲ Analysis by GC-MS/MS revealed similar steroid profiles for PRloxP/loxP and PRNesCre mice at

ϱϰϳ 6 hrs after sham surgery or MCAO (Fig. 6)+RZHYHUEUDLQOHYHOVRIERWKSURJHVWHURQHDQGĮ-

ϱϰϴ DHP were significantly higher in sham-operated PRNesCre males when compared with sham-

ϱϰϵ operated PRloxP/loxP males, suggesting a greater sensitivity of the former to surgical stress (Fig.

ϱϱϬ 6B,E). After MCAO, brain levels of progesterone were elevated in the male brain, and they were

ϱϱϭ 3 times higher in males than in females (Fig. 6B %UDLQOHYHOVRIĮ-DHP were nearly 10 times

ϱϱϮ higher in males when compared to females, and they were markedly up-regulated at 6 hrs after

ϱϱϯ MCAO in males when compared to sham operation (Fig. 6C). No increases in the levels of

ϱϱϰ SURJHVWHURQHDQGĮ-DHP were found after MCAO in females, consistent with results presented

ϱϱϱ in Figure 3 for C57BL/6 mice. /HYHOVRIĮĮ-THP were also higher in males than in females,

ϱϱϲ but were not upregulated in response to MCAO (Fig. 6D /HYHOVRIĮ-'+3DQGĮĮ-THP

ϱϱϳ were low in both sham and MCAO operated males and females (Fig. 6E,F).

ϱϱϴ Thus, the response of brain steroids to MCAO surgery differs between sexes. Whereas

ϱϱϵ males upregulate the production of PR actiYHSURJHVWHURQHDQGĮ-DHP in response to ischemic

ϱϲϬ injury, females do not show such changes.

ϱϲϭ

ϱϲϮ The resistance of the brain to ischemic damage is reduced in young male and female

ϱϲϯ PRNesCre mice

Ϯϳ

ϱϲϰ For all the outcomes measured after MCAO, we first verified that they were similar for

ϱϲϱ C57BL/6 and PRloxP/loxP mice. Neurological scores, time spent on the rotarod and infarct volumes

ϱϲϲ did not significantly differ between the two strains of mice, and we used PRloxP/loxP mice as

ϱϲϳ controls for the effect of targeted PR inactivation in the CNS.

ϱϲϴ ,I WKH HQGRJHQRXV 35 OLJDQGV SURJHVWHURQH DQG Į-DHP play a role in rapid endogenous

ϱϲϵ cerebroprotective mechanisms, deletion of PR in the brain could be expected to reduce its

ϱϳϬ resistance to ischemic injury. We therefore compared the total infarct volume, neurological

ϱϳϭ outcomes, neuron densities and microglial activation between young male and female PRloxP/loxP

ϱϳϮ and PRNesCre mice at 6 hrs after MCAO (Figs. 7-9; left side). Although sham surgery seemed to

ϱϳϯ have an influence on the brain levels of some progesterone metabolites, it did not cause ischemic

ϱϳϰ damage. Indeed, sham surgery did not affect cerebral blood flow, neurological deficit scores and

ϱϳϱ infarct volume.

ϱϳϲ Infarct volumes were significantly increased in PRNesCre mice of both sexes when compared

ϱϳϳ with PRloxP/loxP mice (Fig. 7A,B). In males, the size of the infarct was nearly doubled after

ϱϳϴ deletion of PR, whereas in PRNesCre females, the infarct was increased by 30%. Infarct volumes

ϱϳϵ did not differ between sexes in PRloxP/loxP mice, but they were significantly larger in male than in

ϱϴϬ female PRNesCre mice (Fig. 7B), pointing to the existence of additional PR-independent protective

ϱϴϭ mechanisms in females.

ϱϴϮ Neurological deficits were assessed on a 6-point scale, with elevated scores reflecting

ϱϴϯ higher functional disability (Gibson et al., 2011). In contrast to the infarct size, they were

ϱϴϰ similarly increased in male and female PRNesCre males when compared to PRloxP/loxP mice (Fig.

ϱϴϱ 7C). Thus, endogenous PR-dependent signaling contributed to the preservation of neural

Ϯϴ

ϱϴϲ functions in both sexes. Highly positive correlations were observed between infarct volumes and

ϱϴϳ neurological deficits in both sexes (Fig. 7D).

ϱϴϴ To go further in the evaluation of the tissue damage at 6 hrs after MCAO, the density of

ϱϴϵ neurons labelled with an antibody against the neuronal nuclear antigen NeuN was measured by

ϱϵϬ immunofluorescence analysis. Neurons were counted in the infarct and in the peri-infarct at the

ϱϵϭ levels of the cerebral cortex and striatum (Fig. 8A,B). In both brain regions, MCAO is known to

ϱϵϮ produce widespread ischemic cell death, with the most rapid damage occurring in the striatum

ϱϵϯ (Carmichael, 2010). DAPI counterstaining of cell nuclei allowed to define the demarcation

ϱϵϰ between the infarct and peri-infarct areas.

ϱϵϱ At the infarct core in both cerebral cortex and striatum, neuron density was significantly

ϱϵϲ lower in PRNesCre mice when compared to PRloxP/loxP mice for both sexes (Fig. 8C,D). At the peri-

ϱϵϳ infarct area, the density of neurons was almost twice the one counted in the infarct core, and no

ϱϵϴ differences were observed between PRNesCre mice and PRloxP/loxP mice (Fig. 8E,F). Similar

ϱϵϵ neuronal densities with no differences between PRNesCre mice and PRloxP/loxP mice were also

ϲϬϬ observed in the contralateral hemisphere. Sex differences in the density of NeuN+ neurons were

ϲϬϭ observed after MCAO in the striatum (Fig. 8D,F) but not in the cortex (Fig. 8C,E).

ϲϬϮ The density of activated microglia was assessed by immunofluorescence analysis of Iba1+

ϲϬϯ cells in cerebral cortex and striatum. As early as 6 hrs after MCAO, microglial activation was

ϲϬϰ only observed in the peri-infarct (Fig. 9B-D). In both cortex and striatum, the density of activated

ϲϬϱ microglial cells was significantly lower in PRNesCre mice when compared to PRloxP/loxP mice for

ϲϬϲ both sexes (Fig. 9C,D). The density of Iba1+ microglial cells was low in the contralateral non-

ϲϬϳ ischemic side and was unaffected by the PR deletion (Fig. 9E,F).

ϲϬϴ

Ϯϵ

ϲϬϵ The resistance of the brain to ischemic damage is also reduced in aging male and female

ϲϭϬ mice after deletion of PR

ϲϭϭ To determine whether the endogenous PR-dependent cerebroprotective mechanisms remain

ϲϭϮ functional during aging, infarct volume, neurological outcomes, neuron densities and microglial

ϲϭϯ activation were examined at 6 hrs after MCAO in aging (12-14 months old) male and female

ϲϭϰ PRloxP/loxP and PRNesCre mice (Figs. 7-9; right side) and were compared with the results obtained

ϲϭϱ for young (3 months old) mice (Figs. 7-9; left side).

ϲϭϲ In aging mice, the invalidation of PR also resulted in larger infarct volumes, and there were

ϲϭϳ no differences between sexes (Fig. 7E,F). As expected, infarct volumes were larger in aging

ϲϭϴ when compared to young mice (Fig. 7B,F). Neurological deficit scores were increased to the

ϲϭϵ same extent in both sexes, reflecting higher functional disability (Fig. 7G). Interestingly,

ϲϮϬ neurological deficits tended to be higher in aging than in young mice, but only for the PRNesCre

ϲϮϭ mice. As for the young mice, positive correlations were observed between infarct volumes and

ϲϮϮ neurological deficits (Fig. 7H). Thus, endogenous PR signaling continues to play an important

ϲϮϯ role in cerebroprotection in aging males and females.

ϲϮϰ Densities of neurons were lower in the aging males and females comparatively to young

ϲϮϱ adults in infarct and peri-infarct areas of cortex and striatum (Fig. 8). Sex differences in the

ϲϮϲ density of NeuN+ neurons were only observed in the infarct at the level of the striatum in aging

ϲϮϳ PRloxP/loxP mice (Fig. 8I). Neuron density was significantly lower in PRNesCre mice when

ϲϮϴ compared to PRloxP/loxP mice not only in the infarct region, but also in the peri-infarct area (Fig.

ϲϮϵ 8H-K), suggesting a more rapid spreading of the ischemic lesion in aging comparatively to

ϲϯϬ young mice. The reduced number of neurons after inactivation of PR in the aging brain

ϯϬ

ϲϯϭ uncovered a role for endogenous PR signaling in neuroprotection in cerebral cortex and dorsal

ϲϯϮ striatum in both infarct and peri-infarct.

ϲϯϯ Activated Iba1+ microglial cells were observed in the peri-infarct of aging mice at 6 hrs

ϲϯϰ after MCAO (Fig. 9G-I). The density of Iba1+ cells was lower in aging mice comparatively to

ϲϯϱ young mice, at the level of the striatum. In both cerebral cortex and striatum, the density of

ϲϯϲ activated microglial cells in the peri-infarct was lower in PRNesCre mice when compared to

ϲϯϳ PRloxP/loxP mice, thus confirming a role of endogenous PR signaling in the activation of microglial

ϲϯϴ responses (Fig. 9H,I). In the contralateral non-ischemic side of the cerebral cortex and striatum,

ϲϯϵ the density of Iba1+ cells was low with no effect of PR deletion (Fig. 9J,K).

ϲϰϬ

ϲϰϭ Discussion

ϲϰϮ In this study we demonstrate the importance of endogenous progesterone, its metabolites

ϲϰϯ and neural PR, in cerebroprotection in young and aging mice of both sexes during the early acute

ϲϰϰ phase after stroke. We choose females in diestrus, a stage lasting more than 2 days of the 4-day

ϲϰϱ reproductive cycle (Byers et al., 2012), as it was considered to be advisable to avoid short peak

ϲϰϲ levels of ovarian progesterone and estradiol (Smith et al., 1975; Nilsson et al., 2015) to uncover a

ϲϰϳ role of endogenous brain progesterone in female cerebroprotection. Only limited information is

ϲϰϴ available concerning the influence of the estrus cycle on responses to cerebral ischemia, but one

ϲϰϵ study reported that hypertensive female rats develop smaller cerebral infarcts during the short

ϲϱϬ proestrus stage (Carswell et al., 2000).

ϲϱϭ The unexpected results of brain steroid profiling by GC-MS/MS lent further support to the

ϲϱϮ notion that progesterone and its metabolites are important brain steroids in males. Whereas levels

ϲϱϯ RI QHXURDFWLYH Į-reduced metabolites of progesterone were particularly elevated in the male

ϯϭ

ϲϱϰ brain, females instead produced larger DPRXQWVRIĮ-DHP. This metabolite of progesterone has

ϲϱϱ been reported to have little affinity for PR and no progestogenic activity (Nadeem et al., 2016).

ϲϱϲ Instead, as the formation of the Į-hydroxy-pregnanes is reversible, they may regulate the

ϲϱϳ availability of biologically active 20-keto-pregnanes (Pelletier, 2010). Even more striking was

ϲϱϴ WKHUDSLGDQGPDUNHGLQFUHDVHLQEUDLQOHYHOVRIWKH35OLJDQGVSURJHVWHURQHDQGĮ-DHP after

ϲϱϵ MCAO in males but not in females.

ϲϲϬ Because of its HOHYDWHGHQGRJHQRXVEUDLQOHYHOVRISURJHVWHURQHDQGĮ-DHP, one may have

ϲϲϭ predicted the male brain to be more resistant to ischemic damage than the female brain.

ϲϲϮ However, this was not the case under the present experimental conditions: infarct volume and

ϲϲϯ neurological deficit scores did not differ between male and female PRloxP/loxP mice at 6 hrs after

ϲϲϰ MCAO. Moreover, in both sexes, infarct volumes and neurological deficits were increased and

ϲϲϱ neuronal densities decreased in PRNesCre mice, demonstrating an important role of PR signaling in

ϲϲϲ the resistance of brain tissue to ischemic damage for both sexes. Somehow, the low brain levels

ϲϲϳ of SURJHVWHURQHDQGĮ-DHP in females were sufficient for efficient cerebroprotection. However,

ϲϲϴ males may UHO\ PRUH WKDQ IHPDOHV RQ FHUHEURSURWHFWLRQ E\ HQGRJHQRXV SURJHVWHURQH DQG Į-

ϲϲϵ DHP, as suggested by the larger infarct volumes in PRNesCre males when compared to PRNesCre

ϲϳϬ females. Taken together, these observations point to additional protective mechanisms against

ϲϳϭ ischemic injury in the female brain.

ϲϳϮ An auxiliary cerebroprotective agent may be estradiol in spite the fact that similar low brain

ϲϳϯ levels of the hormone were measured in both sexes, and that they were not increased in response

ϲϳϰ to MCAO. Females may indeed be more sensitive to the cerebroprotective effects of estradiol

ϲϳϱ WKDQ PDOHV DV HVWURJHQ UHFHSWRU DOSKD (5Į LV UDSLGO\ XSUHJXODWHG LQ WKH LVFKHPLF EUDLQ RI

ϲϳϲ females but not of males (Dubal et al., 2006; Westberry et al., 2008). Estrogens may potentiate

ϯϮ

ϲϳϳ the cerebroprotective effects of progesterone in females by upregulating PR expression (Waters

ϲϳϴ et al., 2008).

ϲϳϵ Steroid profiling revealed other potential cerebroprotective mechanisms in females. Thus,

ϲϴϬ ZKHUHDVSURJHVWHURQHLVPDLQO\FRQYHUWHGWRĮ-reduced metabolites in males, females produce

ϲϴϭ higher amounts of GABAA UHFHSWRU DFWLYH ȕ-reduced metabolites of progesterone and DOC,

ϲϴϮ UHVSHFWLYHO\ Įȕ-7+3 DQG Įȕ-THDOC (Gunn et al., 2015). The positive modulation of

ϲϴϯ GABAA UHFHSWRUV E\ Į-reduced metabolites of progesterone and DOC and the resulting

ϲϴϰ decrease in neuronal excitability is indeed a well recognized mechanism involved in

ϲϴϱ neuroprotective responses (Sayeed et al., 2006; Brinton, 2013; Melcangi and Panzica, 2014).

ϲϴϲ Another sex difference deserving attention was the exposure of the male brain to higher

ϲϴϳ amounts of injury-induced corticosterone when compared with females. Endogenous

ϲϴϴ corticosterone and stress have indeed been shown to exacerbate brain injury resulting from

ϲϴϵ cerebral ischemia and the suppression of glucocorticoid production or actions has been proposed

ϲϵϬ as a therapeutic option for stroke patients (Smith-Swintosky et al., 1996). Of note, progesterone

ϲϵϭ has been recently shown to counter the deleterious effects of stress after brain ischemia

ϲϵϮ (Espinosa-Garcia et al., 2017). ,QPDOHVKLJKHUOHYHOVRISURJHVWHURQHDQGĮ-DHP may protect

ϲϵϯ the brain against the ischemia-induced rise in corticosterone, whereas lower levels of

ϲϵϰ glucocorticoids may improve the resistance of the female brain to ischemic damage.

ϲϵϱ Previous studies have shown that CNS levels of progesterone are increased in response to

ϲϵϲ traumatic brain and spinal cord injury and MCAO (Labombarda et al., 2006; Meffre et al., 2007;

ϲϵϳ Liu et al., 2012; Lopez-Rodriguez et al., 2015, 2016). As treatment with progesterone exerts

ϲϵϴ protective effects in these different injury models, it has been presumed that increased production

ϲϵϵ of progesterone may be part of protective responses of neural cells to injury (De Nicola et al.,

ϯϯ

ϳϬϬ 2009). Here, we provide evidence for this concept by showing that deletion of PR in the brain

ϳϬϭ results in increased ischemic damage and aggravation of neurological deficits after MCAO in the

ϳϬϮ absence of treatment with exogenous progesterone.

ϳϬϯ We have previously shown that the resistance of the brain to ischemic damage is decreased

ϳϬϰ in total PR knockout mice (Liu et al., 2012). However, this study did not allow to distinguish

ϳϬϱ between central and peripheral actions of progesterone. Present results demonstrate a key role of

ϳϬϲ neural PR in the cerebroprotective effects of the hormone. This finding has two major

ϳϬϳ implications. First, synthetic progestins, which have been designed to selectively target PR for

ϳϬϴ contraceptive purposes or for the treatment of endocrine disorders, may be used as

ϳϬϵ cerebroprotective agents (Sitruk-Ware and El-Etr, 2013; Schumacher et al., 2014). However,

ϳϭϬ synthetic progestins cannot mimic the complex and regulated metabolism of natural progesterone

ϳϭϭ in the brain (Kumar et al., 2017). Second, a route of progesterone administration with direct

ϳϭϮ access to the brain, such as intranasal delivery, may be particularly advantageous for acute stroke

ϳϭϯ therapy (Fréchou et al., 2015).

ϳϭϰ In men and women, age is the most important risk factor for stroke and a prognostic marker

ϳϭϱ for poor outcomes (Wong et al., 2013). Concordantly, studies involving aging animals report

ϳϭϲ worse stroke outcomes (Liu et al., 2009). The present study confirms that infarct volumes are

ϳϭϳ larger in aging than in young mice. Moreover, neurological deficits were more severe in aging

ϳϭϴ PRNesCre mice. Treatment with progesterone continues to reduce infarct volumes in aging rodents

ϳϭϵ of both sexes (Wang et al., 2010; Gibson et al., 2011; Wali et al., 2016). Here, we show that

ϳϮϬ endogenous PR-dependent cerebroprotective mechanisms remain operational in aging males and

ϳϮϭ females: infarct volumes and neurological deficits were increased and the density of neurons

ϳϮϮ decreased in PRNesCre mice when compared to PRloxP/loxP mice. The preservation of PR-dependent

ϯϰ

ϳϮϯ cerebroprotective mechanisms at the early acute phase after stroke could explain why the 6-hr

ϳϮϰ time window for treatment by progesterone is preserved in aging males and females (Yousuf et

ϳϮϱ al., 2014).

ϳϮϲ In the PRNesCre mice, PR expression was ablated in neurons, astrocytes and

ϳϮϳ oligodendrocytes, but not in microglial cells. The latter do not express PR, even after

ϳϮϴ lipopolysaccharide challenge (Sierra et al., 2008). Cell-specific inactivation of PR will allow us

ϳϮϵ LGHQWLI\RQZKLFKQHXUDOFHOOVSURJHVWHURQHDQGĮ-DHP act.

ϳϯϬ By using GC-MS/MS technology for steroid profiling and conditional CNS-specific PR

ϳϯϭ knockout mice, the present study demonstrates an important role of endogenous progesterone

ϳϯϮ and its metabolites in the resistance of brain tissue to ischemic damage. Importantly, endogenous

ϳϯϯ PR-dependent cerebroprotective mechanisms are operational in young and aging males and

ϳϯϰ females. Remarkably, summed brain levels of PR-active pregnanes are much higher in males

ϳϯϱ than in females, and they are specifically upregulated in males after MCAO. Thus, progesterone

ϳϯϲ and its neuroactive metabolites may play a particularly important role in the male brain.

ϯϱ

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ϰϯ

ϵϭϱ Figure legends

ϵϭϲ Figure 1. Schematic presentation of the single biosynthetic pathway starting with cholesterol and

ϵϭϳ leading to progesterone, its metabolites and their main signaling mechanisms. Enzymes are

ϵϭϴ shown as numbers in italics next to the arrows indicating metabolic steps. Cholesterol is

ϵϭϵ converted to pregnenolone inside the steroidogenic mitochondria by cytochrome P450scc (1).

ϵϮϬ &DUERQV DUH QXPEHUHG DV VKRZQ 3UHJQHQRORQH LV WKHQ FRQYHUWHG WR SURJHVWHURQH E\ WKH ȕ-

ϵϮϭ hydroxysteroid dehydrogenases (2 DQG SURJHVWHURQH WR Į-GLK\GURSURJHVWHURQH Į-DHP) by

ϵϮϮ WKH Į-reductases (3 %RWK SURJHVWHURQH DQG Į-DHP bind to intracellular progesterone

ϵϮϯ receptors (PR), which regulate gene transcription inside the nucleus, but also interact with

ϵϮϰ membrane associated kinases in extranuclear compartments. The conversion oI Į-DHP to

ϵϮϱ ĮĮ-WHWUDK\GURSURJHVWHURQH ĮĮ-7+3 RU DOORSUHJQDQRORQH E\ WKH Į-hydroxysteroid

ϵϮϲ dehydrogenase (4 LVDUHYHUVLEOHUHDFWLRQ,QFRQWUDVWWRSURJHVWHURQHDQGĮ-'+3ĮĮ-THP

ϵϮϳ does not bind to PR but is a positive modulator of GABAA receptors. The interconversion of 20-

ϵϮϴ keto-SUHJQDQHVWRĮ-hydroxy-pregnanes, considered as biologically inactive, is also reversible

ϵϮϵ and FDWDO\]HGE\DĮ-hydroxysteroid dehydrogenase (5).

ϵϯϬ

ϵϯϭ Figure 2. Levels of pregnenolone, progesterone and its major metabolites were analyzed by GC-

ϵϯϮ MS/MS in the left hemisphere and in plasma of young adult males and diestrus females (for an

ϵϯϯ extended steroid profiling, see Table 3). A, Levels of pregnenolone were higher in the brain of

ϵϯϰ males and females than in plasma (effect of tissue: F(1,29) = 27.5, p < 0.0001). B, Levels of

ϵϯϱ progesterone were similar in brain and plasma of of both sexes (effect of tissue: p = 0.4). In

ϵϯϲ plasma, levels of progesterone were higher in females than in males (effect of sex: F(1,25) = 4.74,

ϵϯϳ p < 0.05). C/HYHOVRIĮ-DHP were higher in males than in females for brain and plasma (effect

ϰϰ

ϵϯϴ of sex: F(1,27) = 20.6, p < 0.001). /HYHOVRIĮ-DHP tended to be higher in brain when compared

ϵϯϵ to plasma (effect of tissue: F(1,27) = 3.51, p = 0.072). D%UDLQDQGSODVPDOHYHOVRIĮĮ-THP

ϵϰϬ were higher in males when compared to females (effect of sex: F(1,27) = 8.71, p < 0.01).

ϵϰϭ 0RUHRYHUOHYHOVRIĮĮ-THP tended to be higher in brain when compared to plasma (effect of

ϵϰϮ tissue: F(1,27) = 3.26, p = 0.082). E,QFRQWUDVWWRWKHĮ-reduced 20-keto-pregnanes, levels of

ϵϰϯ Į-DHP were higher in brain and plasma of females (effect of sex: F(1,25) = 33.1, p < 0.0001).

ϵϰϰ /HYHOV RI Į-DHP did not differ between brain and plasma. F, ,Q WKH EUDLQ Į-DHP was

ϵϰϱ convertHG WR ĮĮ-7+3 ,Q SODVPD OHYHOV RI ĮĮ-THP were very low (40 ± 50 pg/ml)

ϵϰϲ (effect of tissue: F(1,24) = 19.0, p < 0.001). Results are presented as means ± SEM (n = 5-6 per

ϵϰϳ group) and were analyzed by two-way ANOVA (sex x tissue) followed by Newman-Keuls

ϵϰϴ multiple comparisons tests. *** p < 0.001, ** p < 0.01, * p < 0.05 when compared to males or as

ϵϰϵ indicated.

ϵϱϬ

ϵϱϭ Figure 3. Levels of pregnenolone, progesterone and its major metabolites were analyzed by GC-

ϵϱϮ MS/MS in the ipsilateral (left, ischemic) hemisphere and in plasma of young adult male and

ϵϱϯ diestrus female mice at different times (hours, hrs) after MCAO (for an extended steroid

ϵϱϰ profiling, see Table 4). A, In males, levels of pregnenolone were significantly increased in the

ϵϱϱ ipsilateral hemisphere at 6 hrs after MCAO (F(5,46) = 3.0, p < 0.05) and slightly in plasma at 4 and

ϵϱϲ 6 hrs (F(5,37) = 8.3, p < 0.001). As in males, plasma levels of pregnenolone were weakly but

ϵϱϳ significantly increased in females at 4 and 6 hrs (F(5,28) = 10.7, p < 0.001). Analysis by 2-way

ϵϱϴ ANOVA (sex x time) indeed revealed a significant effect of sex (F(5,81) = 11.7, p < 0.001). B,In

ϵϱϵ males, progesterone was significantly increased at 4 and 6 hrs after MCAO both in the ipsilateral

ϵϲϬ hemisphere (F(5,46) = 9.67, p < 0.0001) and in plasma (F(5,35) = 11.4, p < 0.0001). Analysis by 2-

ϰϱ

ϵϲϭ way ANOVA (sex x time) of brain progesterone levels showed a significant effect of time (F(5,80)

ϵϲϮ = 7.94, p < 0.0001) and a significant interaction between time and sex (F(5,80) = 3.20, p < 0.05).

ϵϲϯ Similarly, 2-way ANOVA analysis of plasma progesterone levels revealed a significant effect of

ϵϲϰ time (F(5,60) = 12.4, p < 0.0001), a significant interaction between time and sex (F(5,60) = 4.64, p <

ϵϲϱ 0.001) and also an effect of sex (F(1,60) = 17.8, p < 0.0001). C, In the male ipsilateral hemisphere,

ϵϲϲ OHYHOVRIĮ-DHP continuously increased between 1 and 6 hrs after MCAO, and they were still

ϵϲϳ elevated at 24 hrs (F(5,45) = 7.33 p < 0.0001). Analysis by 2-way ANOVA (sex x time) of brain

ϵϲϴ Į-DHP levels showed a significant effect of time (F(5,79) = 7.05, p < 0.0001), of sex (F(1,79) =

ϵϲϵ 158.7, p < 0.0001) and a significant interaction between time and sex (F(5,79) = 5.80, p < 0.0001).

ϵϳϬ D, /HYHOVRIĮĮ-THP in brain and plasma were higher in males when compared with females.

ϵϳϭ This observation was supported by 2-way ANOVA (sex x time) for brain (effect of sex: F(1,80) =

ϵϳϮ 33.7, p < 0.0001) and plasma (effect of sex: F(1,62) = 29.1, p < 0.0001). E, ,QPDOHVOHYHOVRIĮ-

ϵϳϯ DHP were significantly increased in brain (F(5,42) = 9.0, p < 0.0001) and in plasma (F(5,34) = 9.60,

ϵϳϰ p < 0.0001) at 4 and 6 hrs after MCAO. No significant changes over time were observed in

ϵϳϱ females. A two-way ANOVA (sex x time) revealed a significant effect of sex for plasma levels

ϵϳϲ RIĮ-DHP (F(1,61) = 30.5, p < 0.0001). Results are presented as means ± SEM (n = 5-10 per

ϵϳϳ group) and were analyzed by one-way ANOVA followed by Newman-Keuls multiple

ϵϳϴ comparisons tests. *** p < 0.001, ** p < 0.01, * p < 0.05 when compared to the control (CTL).

ϵϳϵ

ϵϴϬ Figure 4. Rapid and marked increase in the summed levels of the PR-active steroids

ϵϴϭ SURJHVWHURQH DQG Į-DHP after MCAO in the ipsilateral hemisphere of males. A, In the male

ϵϴϮ brain, SURJHVWHURQH Į-DHP progressively increased between 1 and 6 hrs after MCAO and

ϵϴϯ remained elevated at 24 hrs (F(5,45) = 7.87, p < 0.0001). B, In the plasma of males, pooled levels

ϰϲ

ϵϴϰ of progesterone + Į-DHP remained low, but showed a small increase over time (F(5,37) = 6.48, p

ϵϴϱ < 0.001). C, ,QFRQWUDVWWRPDOHVOHYHOVRI3URJHVWHURQHĮ-DHP remained very low in the

ϵϴϲ female brain and were not affected by MCAO. D, There was a slight, albeit significant increase

ϵϴϳ LQIHPDOHSODVPDOHYHOVRISURJHVWHURQHĮ-DHP at 4 and 6 hours (F(5,28) = 13.1, p < 0.0001).

ϵϴϴ

ϵϴϵ Figure 5. Verification of the selective deletion of the progesterone receptors in the brain of

ϵϵϬ PRNesCre mice. A, B, PR mRNA expression was analyzed by q-PCR (n = 4 per group). A,In

ϵϵϭ hypothalamus, cerebral cortex and subcortical regions, PR mRNA expression was similar in

ϵϵϮ wild-type (PR+/+) and male and female PRloxP/loxP mice, but was almost completely abolished in

ϵϵϯ PRNesCre mice. B, In contrast to the brain, PR mRNA expression was not affected in peripheral

ϵϵϰ tissues of PRNesCre mice, including aorta, kidney and thymus for males and uterus, ovary and

ϵϵϱ mammary glands for females. Results are presented as means ± SEM. Group differences were

ϵϵϲ analyzed by one-way ANOVA followed by Newman-Keuls multiple comparisons tests. ***, p <

ϵϵϳ 0.001 when compared to PRloxP/loxP mice. C, The successful deletion of PR was also verified in

ϵϵϴ the hypothalamus at the protein level by immunofluorescence. No specific immunostaining was

ϵϵϵ detected in PRNesCre mice. In contrast to the hypothalamus, PR immunostaining was not

ϭϬϬϬ decreased in the uterus of PRNesCre mice when compared to PRloxP/loxP mice.Scale bar = 50 m.

ϭϬϬϭ

ϭϬϬϮ Figure 6. Levels of pregnenolone, progesterone and its major metabolites were analyzed by GC-

ϭϬϬϯ MS/MS in the left hemisphere of males and females PRloxP/loxP and PRNesCre mice at 6 h after

ϭϬϬϰ sham operation or MCAO. A, Levels of pregnenolone did not significantly differ between sexes

ϭϬϬϱ and genotypes, and they were not affected by MCAO. B, Levels of progesterone were

ϭϬϬϲ significantly upregulated in response to MCAO in PRloxP/loxP males. In contrast to PRloxP/loxP

ϰϳ

ϭϬϬϳ males, brain levels of progesterone were elevated after sham surgery and MCAO in PRNesCre

ϭϬϬϴ males (effect of MCAO: F(1,19) = 15.1, p = 0.001; effect of genotype: F(1,19) = 9.85, p = 0.005;

ϭϬϬϵ interaction between MCAO and genotype: F(1,19) = 5.29, p = 0.033). In females, levels of

ϭϬϭϬ progesterone were lower as compared to males and similar in sham and MCAO mice of both

ϭϬϭϭ genotypes. C, /HYHOV RI Į-DHP were much higher in males than in females and were only

ϭϬϭϮ increased in response to MCAO in males (effect of MCAO in males: F(1,21) =23.1, p < 0.0001).

ϭϬϭϯ 7KHUHZHUHQRGLIIHUHQFHVIRUĮ-DHP levels between PRloxP/loxP and PRNesCre mice. D, $VIRUĮ-

ϭϬϭϰ '+3EUDLQOHYHOVRIĮĮ-THP were much higher in males than in females. Neither in males

ϭϬϭϱ nor in females, there was an effect of MCAO or genotype. E, 6LPLODUORZOHYHOVRIĮ-DHP (<

ϭϬϭϲ 2 ng/g) were measured in the brain of PRloxP/loxP and PRNesCre males and females after sham

ϭϬϭϳ VXUJHU\ RU 0&$2 +RZHYHU LQ VSLWH RI WKH ORZ OHYHOV RI Į-DHP, they were significantly

ϭϬϭϴ upregulated after MCAO in PRloxP/loxP males, and they were higher in sham operated PRNesCre

loxP/loxP ϭϬϭϵ males when compared to sham operated PR males (effect of MCAO in males: F(1,21) =13.9,

ϭϬϮϬ p = 0.001; effect of genotype in males: F(1,21) =11.8, p = 0.003). F, As in the normal brain, levels

ϭϬϮϭ RIĮĮ-7+3ZHUHORZDIWHUVKDPVXUJHU\RU0&$2LQERWKVH[HV,QIHPDOHVĮĮ-THP

ϭϬϮϮ levels were slightly upregulated after MCAO in PRloxP/loxP mice (effect of MCAO in females:

ϭϬϮϯ F(1,19) =8.29, p = 0.010; effect of genotype in females: F(1,19) =3.94, p = 0.062). Results are

ϭϬϮϰ presented as means ± SEM (n = 5-7 per group) and were analyzed by two-way ANOVA (MCAO

ϭϬϮϱ x genotype) for males and females separately followed by Newman-Keuls multiple comparisons

ϭϬϮϲ tests. *** p < 0.001, ** p < 0.01, * p < 0.05 when compared to sham or as indicated.

ϭϬϮϳ

ϭϬϮϴ Figure 7. Infarct volumes and neurological deficits at 6 hrs after MCAO were increased in

ϭϬϮϵ young and aging mice of both sexes after deletion of PR in the brain. Total infarct volumes and

ϰϴ

ϭϬϯϬ neurological deficit scores (higher scores reflect higher disability) were analyzed in young (A-D)

ϭϬϯϭ and aging (E-H) PRloxP/loxP and PRNesCre mice. A, E, Representative 1 mm thick coronal brain

ϭϬϯϮ sections of young and aging male and female PRloxP/loxP and PRNesCre mice stained with 2,3,5±

ϭϬϯϯ triphenyltetrazolium chloride. Infarcted tissue appears pale and normal tissue is colored dark red.

ϭϬϯϰ Scale bar = 5 mm. B, In young males and females, total infarct volumes were significantly larger

ϭϬϯϱ in PRNesCre mice when compared to PRloxP/loxP mice. However, for PRNesCre mice, the infarct

ϭϬϯϲ volume was significantly larger in males than in females. Data analysis by two-way ANOVA

ϭϬϯϳ revealed significant effects of genotype (F(1,23) = 36.7, p < 0.0001) and sex (F(1,23) = 17.4, p =

ϭϬϯϴ 0.0004) as well as significant interactions between both factors (F(1,23) = 5.89, p = 0.024). C,

ϭϬϯϵ Neurological deficits were analyzed by Kruskal-Wallis analysis of variance (p = 0.003). In both

ϭϬϰϬ sexes, neurological deficits were greater in PRNesCre mice when compared to PRloxP/loxP mice. D,

ϭϬϰϭ Spearman's test showed highly positive correlations between neurological deficit scores and

ϭϬϰϮ infarct volumes for males (r = 0.86, p = 0.0003) and females (r = 0.70, p = 0.009). F, In aging

ϭϬϰϯ males and females, total infarct volumes were significantly larger in PRNesCre mice when

ϭϬϰϰ compared to PRloxP/loxP mice. In contrast to young mice, infarct volumes were comparable

ϭϬϰϱ between males and females. Indeed, two-way ANOVA only showed a significant effect of

ϭϬϰϲ genotype (F(1,20) = 18.7, p = 0.0003). However, infarct volumes were larger in aging mice when

ϭϬϰϳ compared to young mice, as revealed by a two-way ANOVA with age and sex as factors (effect

loxP/loxP NesCre ϭϬϰϴ of age: PR mice: F(1,20) = 15.2, p = 0.0009; PR mice: F(1,23) = 12.6, p = 0.002). G, As

ϭϬϰϵ for young mice, neurological deficits were greater in PRNesCre mice when compared to PRloxP/loxP

ϭϬϱϬ mice (p = 0.017 by Kruskal-Wallis analysis). Interestingly, neurological deficits were higher in

ϭϬϱϭ aging than in young PRNesCre mice (p = 0.018 by Mann-Whitney test), but there was no such

ϭϬϱϮ effect of age on functional outcomes in PRloxP/loxP mice. H, Spearman's test showed strong

ϰϵ

ϭϬϱϯ positive correlations between neurological deficit scores and infarct volumes for both aging

ϭϬϱϰ males (r = 0.81, p = 0.002) and females (r = 0.92, p < 0.001). Results are presented as means ±

ϭϬϱϱ SEM (n = 5-7 per group). Two-way ANOVA (sex x genotype) followed by Newman-Keuls

ϭϬϱϲ multiple comparisons tests were used for infarct volume analysis. *** p < 0.001, ** p < 0.01, * p

ϭϬϱϳ < 0.05 when compared to PRloxP/loxP mice or as indicated.

ϭϬϱϴ

ϭϬϱϵ Figure 8. The density of NeuN+ neurons at 6 hrs after MCAO was decreased in the ipsilateral

ϭϬϲϬ ischemic hemisphere in young and aging mice after deletion of PR in the brain. A, Localization

ϭϬϲϭ of the 4 regions where neurons were counted at the level of the cerebral cortex and striatum. The

ϭϬϲϮ regions were located on the infarct and peri-infarct, which were delimited by DAPI staining of

ϭϬϲϯ cell nuclei. The regions are drawn on a coronal brain section taken from the atlas of Franklin and

ϭϬϲϰ Paxinos (2008) at the level of bregma +0.14. The infarct area is schematically presented in grey.

ϭϬϲϱ B, G, Representative immunofluorescence staining of NeuN+ neurons in region 1 (infarct in the

ϭϬϲϲ cerebral cortex) of young and aging PRloxP/loxP and PRNesCre mice of both sexes. Scale bar = 100

ϭϬϲϳ m. B-F, Neuronal density in young mice. C, In region 1 (infarct in the cerebral cortex), there

ϭϬϲϴ were no differences between males and females, but the density of NeuN+ neurons was

ϭϬϲϵ significantly lower in PRNesCre mice when compared to PRloxP/loxP mice (two-way ANOVA with

ϭϬϳϬ sex and genotype as factors, effect of genotype: F(1,20) = 20.1, p = 0.0002). D, In region 2 (infarct

ϭϬϳϭ in the striatum), the density of NeuN+ neurons was also significantly lower in PRNesCre mice when

loxP/loxP ϭϬϳϮ compared to PR mice (effect of genotype: F(1,20) = 48.8, p < 0.0001) for both sexes.

ϭϬϳϯ However, neuron density was slightly but significantly higher in females than in males for both

loxP/loxP NesCre ϭϬϳϰ PR and PR mice (effect of sex: F(1,20) = 18.3, p = 0.0004). E, F, in the peri-infarct in

ϭϬϳϱ the cerebral cortex (E) and striatum (F), neuronal density was about twice as high as at the

ϱϬ

ϭϬϳϲ infarct. There were no differences between PRloxP/loxP and PRNesCre mice, but the density of

ϭϬϳϳ neurons tended to be higher in males (effect of sex in region 3: F(1,20) = 5.84, p = 0.025 and in

ϭϬϳϴ region 4: F(1,20) = 10.2, p = 0.005). G-K, Neuronal density in aging mice. H, As in region 1 of

ϭϬϳϵ young mice, the density of NeuN+ neurons was significantly lower in PRNesCre males and females

loxP/loxP ϭϬϴϬ when compared to PR mice (effect of genotype: F(1,20) = 20.7, p = 0.0002). However, the

ϭϬϴϭ density of neurons was lower in aging mice when compared to young mice as revealed by a two-

loxP/loxP ϭϬϴϮ way ANOVA with age and sex as factors (effect of age: PR mice: F(1,19) = 37.8, p <

NesCre + ϭϬϴϯ 0.0001; PR mice: F(1,21) = 16.7, p = 0.0005). I, In region 2, the density of NeuN neurons

ϭϬϴϰ was significantly lower in PRNesCre males when compared to PRloxP/loxP males (effect of genotype:

ϭϬϴϱ F(1,20) = 11.7, p = 0.003). Again, a two-way ANOVA with age and sex as factors showed that the

ϭϬϴϲ density of neurons was lower in aging mice when compared to young mice (effect of age:

loxP/loxP NesCre ϭϬϴϳ PR mice: F(1,19) = 17.2, p = 0.0006; PR mice: F(1,21) = 3.20, p = 0.088). J, K, In

ϭϬϴϴ contrast to the young mice, neuronal density was lower in the peri-infarct in cerebral cortex (J)

ϭϬϴϵ and striatum (K) of PRNesCre mice when compared to PRloxP/loxP (effect of genotype in region 3:

ϭϬϵϬ F(1,20) = 16.7, p = 0.0006 and in region 4: F(1,20) = 10.6, p = 0.004). Again, the density of neurons

ϭϬϵϭ was lower in the aging mice when compared to the young mice (p < 0.001 for regions 3 and 4 of

ϭϬϵϮ PRNesCre and PRloxP/loxP mice). Results are presented as means ± SEM (n = 5-7 per group) and

ϭϬϵϯ were analyzed by Newman-Keuls multiple comparisons tests after two-way ANOVA (sex x

ϭϬϵϰ genotype). *** p < 0.001, ** p < 0.01, * p < 0.05 when compared to PRloxP/loxP mice or as

ϭϬϵϱ indicated.

ϭϬϵϲ

ϭϬϵϳ Figure 9. The density of activated Iba1+ microglial cells at 6 hrs after MCAO was decreased in

ϭϬϵϴ the peri-infarct in young and aging mice after deletion of PR in the brain. A, Localization of the 4

ϱϭ

ϭϬϵϵ regions where microglial cells were counted at the level of the cerebral cortex and striatum. The

ϭϭϬϬ regions are drawn on a coronal brain section taken from the atlas of Franklin and Paxinos (2008)

ϭϭϬϭ at the level of bregma +0.14. The infarct area is schematically presented in grey. B, G,

ϭϭϬϮ Representative immunofluorescence staining of Iba1+ microglial cells in region 3 (peri-infarct in

ϭϭϬϯ the cerebral cortex) of young and aging PRloxP/loxP and PRNesCre mice of both sexes. Scale bar =

ϭϭϬϰ 100 m. B-F, Microglial density in young mice. C, In region 3 (peri-infarct in the cerebral

ϭϭϬϱ cortex), the density of Iba1+ cells was lower in PRNesCre mice when compared to PRloxP/loxP mice

loxP/loxP ϭϭϬϲ (effect of genotype: F(1,18) = 22.8, p = 0.0002). In PR mice, the density of microglial cells

ϭϭϬϳ was slightly higher in females (effect of sex: F(1,18) = 7.68, p = 0.013). D, In region 4 (peri-infarct

ϭϭϬϴ in the striatum), as in cerebral cortex, the density of Iba1+ cells was lower in PRNesCre mice when

loxP/loxP ϭϭϬϵ compared to PR mice (effect of genotype: F(1,18) = 22.8, p = 0.0002). E, F, In contrast, the

ϭϭϭϬ density of microglial cells was low and not affected by the inactivation of PR or the sex of the

ϭϭϭϭ mice in cerebral cortex (E) and striatum (F) of the contralateral non-ischemic hemisphere (in

ϭϭϭϮ region 5 and region 6). G-K, Microglial density in aging mice. H, I, in the peri-infarct in cerebral

ϭϭϭϯ cortex (H) and striatum (I), the density of Iba1+ microglial cells was lower in PRNesCre mice when

loxP/loxP ϭϭϭϰ compared to PR mice (effect of genotype: cerebral cortex: F(1,20) = 15.6, p = 0.0008;

ϭϭϭϱ striatum: F(1,20) = 6.02, p = 0.024). J, K, As for the young mice, the density of microglial cells

ϭϭϭϲ was low and not affected by the inactivation of PR in cerebral cortex (J) and striatum (K) of the

ϭϭϭϳ contralateral non-ischemic hemisphere (in region 5 and region 6) of both sexes. Results are

ϭϭϭϴ presented as means ± SEM (n = 5-7 per group) and were analyzed by Newman-Keuls multiple

ϭϭϭϵ comparisons tests after two-way ANOVA (sex x genotype). *** p < 0.001, ** p < 0.01, * p <

ϭϭϮϬ 0.05 when compared to PRloxP/loxP mice or as indicated.

ϱϮ

ϭϭϮϭ Table 1. GC-MS/MS parameters for steroid identification and quantification in multiple

ϭϭϮϮ reaction monitoring

Steroids Derivatized steroids Retention Transition Collision (Molecular weight) (molecular weight) time (min.) P]ĺP] energy (eV) Pregnenolone (316) Pregnenolone-3-HFB (512) 21.24 ĺ 8 P (314) P-3-HFB (510) 21.34 ĺ 14

Į-DHP (316) Į-DHP-3,20-TMS2 (460) 18.63 ĺ 12

ȕ-DHP (316) ȕ-DHP-3,20-TMS2 (460) 17.30 ĺ 10 ĮĮ-THP (318) ĮĮ-THP-3-HFB (514) 20.23 ĺ 8 ȕĮ-THP (318) ȕĮ -THP-3-HFB (514) 21.62 ĺ 8 Įȕ-THP (318) Įȕ-THP-3-HFB (514) 20.39 ĺ 12

Į-DHP (316) Į-DHP-3,20-HFB2 (708) 18.48 ĺ 10 ĮĮ-THP (318) ĮĮ-THP-3-HFB (514) 22.93 ĺ 8

ĮĮĮ-HHP (320) ĮĮĮ-HHP-3,20-HFB2 (712) 17.40 ĺ 8

ȕĮĮ-HHP (320) ȕĮĮ-HHP-3,20-HFB2 (712) 18.75 ĺ 10

ĮȕĮ-HHP (320) ĮȕĮ-HHP-3,20-HFB2 (712) 17.59 ĺ 8

Į-DHPREG (318) Į-DHPREG-3,20-HFB2 (710) 18.00 ĺ 10

Testosterone (288) Testosterone-3,17-HFB2 (680) 15.98 ĺ 8 Į-DHT (290) Į-DHT-17-HFB (486) 20.16 ĺ 10

ĮĮ-THT (292) ĮĮ-THT-3,17-HFB2 (684) 15.00 ĺ 8

ȕ-estradiol (272) ȕ-estradiol-3,l7-HFB2 (664) 16.60 ĺ 10

DOC (330) DOC-3,21-HFB2 (722) 21.80 ĺ 10 Į-DHDOC (332) Į-DHDOC-3,21-HFB (528) 25.55 ĺ 8 ȕ-DHDOC (332) ȕ-DHDOC-3,21-HFB (528) 25.23 ĺ 8

ĮĮ-THDOC (334) ĮĮ-THDOC-3,17-HFB2 (726) 20.69 ĺ 8

Įȕ-THDOC (334) Įȕ-THDOC-3,17-HFB2 (726) 20.83 ĺ 10

Corticosterone (346) Corticosterone-3,21-HFB2-H2O (720) 20.31 ĺ 10 ϭϭϮϯ

ϭϭϮϰ 6WHURLGVZHUHLGHQWLILHGDFFRUGLQJWRWKHLUUHWHQWLRQWLPHDQGWKHLUVSHFLILFWUDQVLWLRQ7UDQVLWLRQV

ϭϭϮϱ ZHUHSHUIRUPHGE\VHOHFWLQJDSUHFXUVRULRQRIDWDUJHWHGVWHURLGE\WKHILUVWPDVVVSHFWURPHWHU

ϭϭϮϲ 7KLVSUHFXUVRULRQZDVWKHQIUDJPHQWHGDWDFROOLVLRQHQHUJ\FKRVHQWRJLYHULVHWRDPD[LPDO

ϱϯ

ϭϭϮϳ VLJQDO RI WKH SURGXFW LRQ DQDO\]HG E\ WKH VHFRQG PDVV VSHFWURPHWHU 7KH TXDQWLILFDWLRQ ZDV

ϭϭϮϴ GHWHUPLQHG E\ WKH WUDQVLWLRQ JLYLQJ WKH PRVW DEXQGDQW SURGXFW LRQ 3 SURJHVWHURQH '+3

ϭϭϮϵ GLK\GURSURJHVWHURQH 7+3 WHWUDK\GURSURJHVWHURQH ++3 KH[DK\GURSURJHVWHURQH '+35(*

ϭϭϯϬ GLK\GURSUHJQHQRORQH '+7 GLK\GURWHVWRVWHURQH 7+7 WHWUDK\GURWHVWRVWHURQH '2& -

ϭϭϯϭ GHR[\FRUWLFRVWHURQH '+'2& GLK\GUR--GHR[\FRUWLFRVWHURQH 7+'2& WHWUDK\GUR--

ϭϭϯϮ GHR[\FRUWLFRVWHURQH +)% KHSWDIOXRUREXW\UDWH 706 WULPHWK\OVLO\O

ϱϰ

ϭϭϯϯ Table 2. Validation of the GC-MS/MS analytical procedure in mouse brain

Steroids Detection Accuracy Intra-assay Inter-assay (Molecular weight) Limit (pg/g) (%) variation (%) variation (%) Pregnenolone (316) 2 102.9 5.1 5.5 P (314) 20 105.9 2.2 3.6 Į-DHP (316) 5 101.3 3.2 4.8 ȕ-DHP (316) 2 103.4 3.6 6.8 ĮĮ-THP (318) 10 97.5 5.4 7.4 ȕĮ-THP (318) 0.5 99.6 4.5 5.6 Įȕ-THP (318) 10 105.1 6.2 6.5 Į-DHP (316) 10 103.6 8.2 9.2 ĮĮ-THP (318) 1 106.2 4.3 5.1 ĮĮĮ-HHP (320) 5 98.4 3.2 3.5 ȕĮĮ-HHP (320) 5 99.6 2.9 3.0 ĮȕĮ-HHP (320) 5 95.3 2.6 2.7 Į-DHPREG (318) 1 107.1 1.5 2.5 Testosterone (288) 1 99.8 1.1 1.8 Į-DHT (290) 2 101.4 3.9 4.3 ĮĮ-THT (292) 1 102.6 3.6 4.9 ȕ-estradiol (272) 1 96.4 1.3 1.9 DOC (330) 5 98.2 1.5 1.7 Į-DHDOC (332) 5 102.6 5.4 5.8 ȕ-DHDOC (332) 5 104.5 6.1 6.9 ĮĮ-THDOC (334) 2 10.1.4 4.9 6.1 Įȕ-THDOC (334) 2 98.2 3.4 4.0 Corticosterone (346) 20 97.6 2.5 3.0 ϭϭϯϰ $EEUHYLDWLRQVRIVWHURLGVDVIRU7DEOH7KHDQDO\WLFDOSURWRFROKDVEHHQYDOLGDWHGIRUDOOWKH

ϭϭϯϱ WDUJHWHGVWHURLGVE\XVLQJH[WUDFWVRIPJIURPDSRRORIPDOHPRXVHEUDLQV7KHOLPLWRI

ϭϭϯϲ GHWHFWLRQ ZDV GHWHUPLQHG DV WKH ORZHVW DPRXQW RI FRPSRXQGV WKDW FDQ EH PHDVXUHG E\ *&-

ϭϭϯϳ 0606 ZLWK D VLJQDO-WR-QRLVH UDWLR JUHDWHU WKDQ 7KH OLQHDULW\ ZDV DVVHVVHG E\ DQDO\]LQJ

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ϱϱ

ϭϭϰϬ E\ GHWHUPLQLQJ WKH DQDO\WLFDO UHFRYHU\ ZKLFK ZDV GHILQHG DV & &6 [ & LV WKH

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ϭϭϰϰ GD\DQGRYHUGD\VUHVSHFWLYHO\

ϱϲ

ϭϭϰϱ Table 3. Steroid profiling in brain (left hemisphere) and plasma of young adult C57BL6

ϭϭϰϲ mice of both sexes

Steroids Left hemisphere (ng/g) Plasma (ng/ml) Males Females Males Females E-'+3

3E5D-THP

DE-7+3

3D5D20D-HHP

3E5D20D-HHP

3D5E20D-HHP

D-'+35(*

DOC

5DDHDOC

5EDHDOC

3D5DTHDOC

3D5ETHDOC

Corticosterone

Testosterone

5D-DHT

3D5D-THT 1' 1'

Estradiol

ϭϭϰϳ $EEUHYLDWLRQVRIVWHURLGVDVIRU7DEOH5HVXOWVDUHSUHVHQWHGDVPHDQV6(0 Q -SHU

ϭϭϰϴ JURXS DQG ZHUH DQDO\]HG E\ WZR-ZD\ $129$ WLVVXH [ VH[ IROORZHG E\ 1HZPDQ-.HXOV

ϭϭϰϵ PXOWLSOHFRPSDULVRQVWHVWV p p ZKHQFRPSDUHGWRPDOHV1'QRWGHWHFWHG

ϱϳ

ϭϭϱϬ Table 4. Steroid profiling in brain (ipsilateral hemisphere) and plasma of young adult C57BL/6 mice of both sexes: endpoint

ϭϭϱϭ measures at 1, 2, 4, 6 and 24 hrs after MCAO

Steroids Ipsilateral hemisphere (ng/g) Time post-MCAO (hrs) Sex CTL 1 2 4 6 24 0 0.07 0.09 0.09 0.11 0.06 E-'+3 ) 0 0.36 0.44 0.41 0.33 0.43 0.61 3E5D-THP ) 0.09 0.01 0.09 0.07 0.09 0.14 0 0.09 0.06 0.08 0.10 0.10 0.06 DE-7+3 ) 0.12 0.10 0.19 0.47 0.71 0.47

3D5D20D-HHP 0 0.39 0.51 0.46 0.69 0.66

) 0.68 0.62 0.28 0.21 0.14

0 0.04 0.03 0.05 0.04 3E5D20D-HHP ) 0.04 0.04 0.05 0.03 0.03

0 0.005 1' ND ND ND 3D5E20D-HHP ) 0.07 0.04 0.01 0.02 0.03 0.10

0 0.09 0.11 0.11 0.11 D-'+35(* ) 0.21 0.21 0.11

ϱϴ

0 0.02 2.68 4.56 7.90 7.30 1.34 DOC ) 0.11 4.47 3.27 4.79 5.93 0.81

0 0.23 1.16 0.25 1.20 1.96 1.90 1.48 5DDHDOC ) 0.06 0.49 0.75 0.99 0.90 0.35

0 0.01 0.01 0.07 0.15 0.06 5EDHDOC ) 0.21 2.63 3.46 2.43 3.45

0 0.11 0.37 0.33 0.35 0.39 0.61 3D5DTHDOC ) 0.02 0.03 0.04 0.04 0.13 0.01

0 0.003 0.05 0.08 0.05 0.02 3D5ETHDOC ) 0.15 0.99 1.44 0.75 7.71 0.58

0 3.76 27.6 69.0 75.7 214.6 67.0 Corticosterone ) 1.40 17. 6.85 49.2 76.1 68.6 20.4

0 1.21 0.22 0.15 0.14 0.08 0.06 Testosterone ) 0.02 0.04 0.01 0.03 0.02 0.01

0 0.02 0.02 5D-DHT ) 0.02 0.02 0.02 0.002 0.01 0.002

0 0.02 1' 0.01 3D5D-THT ) 1' 1' 1' 1' 1' 1'

ϱϵ

0 Estradiol ) 0.02 0.02 0.01 0.001 0.02

Plasma (ng/ml) Time post-MCAO (hrs) Sex CTL 1 2 4 6 24 0 1' 1' 1' 1' 1' E-'+3 )

0 0.01 3E5D-THP ) 0.02 0.01 0.02 0.01 0.02 0.02

0 DE-7+3 ) 0.33 0.22 0.57 0.74 1.03 0.25

0 0.18 0.35 0.54 0.52 0.34 3D5D20D-HHP ) 0.32 0.54 0.35 0.27 0.22

0 0.02 0.05 0.05 0.06 3E5D20D-HHP ) 0.04 0.10 0.04 0.03 0.03

0 0.01 0.02 0.01 0.01 3D5E20D-HHP ) 0.06 0.02 0.07 0.15 0.23 0.25

0 0.02 0.03 0.02 0.02 D-'+35(* ) 0.01 0.02 0.02

ϲϬ

0 0.28 1.42 2.27 2.78 4.83 0.84 DOC ) 0.20 1.31 2.07 4.08 3.26 0.81

0 0.20 0.35 0.09 0.34 0.50 0.39 0.43 5DDHDOC ) 0.09 0.04 0.05 0.18 0.18 0.08

0 0.03 0.01 0.02 0.03 0.01 0.06 5EDHDOC ) 0.21 1.64 1.79 2.89 2.54

0 0.11 0.26 0.39 0.51 0.38 0.41 3D5DTHDOC ) 0.02 0.05 0.09 0.24 0.17 0.05

0 0.07 0.10 0.03 0.01 0.05 3D5ETHDOC ) 0.39 6.95 13.9 29.6 21.8 4.58

0 10.2 109.2 139.3 215.6 213.2 67.6 Corticosterone ) 21.5 60.5 6.80 92.0 182.4 191.2 66.4

0 2.16 0.34 0.15 0.14 0.11 0.05 Testosterone ) 0.01 0.02 0.01 0.03 0.02 0.02

0 0.04 0.08 5D-DHT ) 0.01 ND 0.02 0.004 0.01 0.004

0 0.06 ND 3D5D-THT

ϲϭ

) 1' 1' 1' 1' 1' 1'

0 Estradiol ) 0.03 0.02 0.06 0.01 0.04

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ϭϭϱϳ Table 5. Steroid profiling in brain (left hemisphere) and plasma of young adult PRloxP/loxP

ϭϭϱϴ and PRNesCre mice of both sexes

Steroids Left hemisphere (ng/g) PRloxP/loxP PRNesCre Two-way ANOVA Males Females Males Females

3UHJQHQRORQH Sex: F(1,17)=20.4 p=0.0003

Genotype: F(1,17)=0.05 p=0.825

Interaction: F(1,17)=0.43 p=0.521

3URJHVWHURQH Sex: F(1,17)=1.09 p=0.311

Genotype: F(1,17)=2.59 p=0.126

Interaction: F(1,17)=3.10 p=0.096

Į-'+3 Sex: F(1,18)=29.2 p<0.0001

Genotype: F(1,18)=0.19 p=0.669

Interaction: F(1,18)=0.44 p=0.515 ĮĮ-7+3 Sex: F(1,18)=31.6 p<0.0001

Genotype: F(1,18)=10.3 p=0.005

Interaction: F(1,18)=4.96 p=0.039

Į-'+3 Sex: F(1,16)=21.9 p=0.0003

Genotype: F(1,16)=0.43 p=0.522

Interaction: F(1,16)=0.13 p=0.728 ĮĮ-7+3 Sex: F(1,17)=3.60 p=0.075

Genotype: F(1,17)=8.38 p=0.010

Interaction: F(1,17)=7.28 p=0.015 E-'+3 1' 1'

3E5D-THP Sex: F(1,17)=27.4 p<0.0001 Genotype: F(1,17)=9.49 p=0.007

Interaction: F(1,17)=9.27 p=0.007

DE-7+3 Sex: F(1,17)=32.3 p<0.0001

Genotype: F(1,17)=4.55 p=0.048

Interaction: F(1,17)=0.30 p=0.592 3D5D20D-HHP Sex: F(1,15)=1.69 p=0.213 Genotype: F(1,15)=4.61 p=0.049

Interaction: F(1,15)=5.82 p= 0.029 3E5D20D-HHP Sex: F(1,15)=8.63 p=0.010 Genotype: F(1,15)=7.17 p=0.017

Interaction: F(1,15)=5.07 p=0.040 3D5E20D-HHP 1'

D-'+35(* Sex: F(1,17)=5.47 p=0.032

Genotype: F(1,17)=0.25 p=0.625

Interaction: F(1,17)=4.16 p=0.057

DOC Sex: F(1,17)=38.1 p<0.0001

Genotype: F(1,17)=1.28 p=0.273

Interaction: F(1,17)=0.11 p=0.743

ϲϯ

5DDHDOC Sex: F(1,17)=0.08 p=0.786

Genotype: F(1,17)=0.0001 p=0.992

Interaction: F(1,17)=1.38 p=0.257 5EDHDOC 1' 1'

3D5DTHDOC Sex: F(1,18)=102.2 p<0.0001

Genotype: F(1,18)=16.2 p=0.0008

Interaction: F(1,18)=27.5 p<0.0001 3D5ETHDOC 1' 1'

Corticosterone Sex: F(1,18)=0.001 p=0.979

Genotype: F(1,18)=9.86 p=0.006

Interaction: F(1,18)=12.6 p=0.002

Testosterone Sex: F(1,14)=23.7 p=0.0003

Genotype: F(1,14)=0.41 p=0.533

Interaction: F(1,14)=0.52 p=0.482

5D-DHT Sex: F(1,16)=2.41 p=0.141 Genotype: F(1,16)=0.02 p=0.901

Interaction: F(1,16)=0.15 p=0.704

3D5D-THT Sex: F(1,16)=13.3 p=0.002 Genotype: F(1,16)=1.85 p=0.193

Interaction: F(1,16)=1.83 p=0.195

Estradiol Sex: F(1,16)=0.89 p=0.361

Genotype: F(1,16)=2.24 p=0.154

Interaction: F(1,16)=3.89 p=0.066 Plasma (ng/ml) PRloxP/loxP PRNesCre Two-way ANOVA Males Females Males Females

3UHJQHQRORQH Sex: F(1,17)=1.54 p=0.232

Genotype: F(1,17)=1.33 p=0.265

Interaction: F(1,17)=0.51 p=0.486

3URJHVWHURQH Sex: F(1,18)=0.34 p=0.565

Genotype: F(1,18)=2.46 p=0.134

Interaction: F(1,18)=0.29 p=0.598

Į-'+3 Sex: F(1,17)=318.2 p<0.0001

Genotype: F(1,17)=0.54 p=0.471

Interaction: F(1,17)=0.90 p=0.357 ĮĮ-7+3 Sex: F(1,16)=33.3 p<0.0001

Genotype: F(1,16)=8.05 p=0.012

Interaction: F(1,16)=8.30 p=0.011

Į-'+3 Sex: F(1,16)=71.3 p<0.0001

Genotype: F(1,16)=0.22 p=0.645

Interaction: F(1,16)=0.71 p=0.412

ĮĮ-7+3 Sex: F(1,16)=0.33 p=0.575

Genotype: F(1,16)=3.28 p=0.089

Interaction: F(1,16)=3.96 p=0.064 E-'+3 1'

ϲϰ

3E5D-THP Sex: F(1,17)=9.21 p=0.008 Genotype: F(1,17)=6.24 p=0.023

Interaction: F(1,17)=7.49 p=0.014 DE-7+3 Sex: F(1,13)=338.9 p<0.0001

Genotype: F(1,13)=5.62 p=0.034

Interaction: F(1,13)=5.71 p=0.033

3D5D20D-HHP Sex: F(1,16)=0.04 p=0.839 Genotype: F(1,16)=6.31 p=0.023

Interaction: F(1,16)=2.10 p=0.167 3E5D20D-HHP Sex: F(1,15)=4.28 p=0.056 Genotype: F(1,15)=6.74 p=0.020

Interaction: F(1,15)=4.61 p=0.049 3D5E20D-HHP 1' 1'

D-'+35(* Sex: F(1,16)=0.39 p=0.540

Genotype: F(1,16)=2.18 p=0.160

Interaction: F(1,16)=0.14 p=0.715

DOC Sex: F(1,18)=12.1 p=0.003

Genotype: F(1,17)=0.30 p=0.593

Interaction: F(1,17)=0.43 p=0.518

5DDHDOC Sex: F(1,16)=12.7 p=0.003

Genotype: F(1,16)=1.24 p=0.282

Interaction: F(1,16)=0.92 p=0.352 5EDHDOC 1' 1'

3D5DTHDOC Sex: F(1,15)=20.4 p=0.0004

Genotype: F(1,15)=1.24 P=0.283

Interaction: F(1,15)=1.87 P=0.192 3D5ETHDOC

Corticosterone r Sex: F(1,17)=0.55 p=0.469 Genotype: F(1,17)=2.09 p=0.167

Interaction: F(1,17)=7.64 p=0.013

Testosterone Sex: F(1,17)=46.1 p<0.0001

Genotype: F(1,17)=0.42 p=0.523

Interaction: F(1,17)=0.01 p=0.913

5D-DHT Sex: F(1,17)=36.5 p<0.0001 Genotype: F(1,17)=0.84 p=0.373

Interaction: F(1,17)=0.78 p=0.389

3D5D-THT Sex: F(1,17)=17.7 p=0.0006 Genotype: F(1,17)=2.72 p=0.117

Interaction: F(1,17)=0.002 p=0.969

Estradiol Sex: F(1,17)=0.11 p=0.741

Genotype: F(1,17)=0.31 p=0.587

Interaction: F(1,17)=2.30 p=0.148

ϲϱ

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