SUPPLEMENTARY INFORMATION for

A Novel Gene Required for Male Fertility and Functional CatSper Channel Formation in Spermatozoa

Jean-Ju Chung, Betsy Navarro, Grigory Krapivinsky, Luba Krapivinsky, and David E. Clapham*

b

Supplementary Figure S1. Molecular cloning of Tmem146-l. (a) Schematic diagram of Tmem146 splice variants and nucleotide sequence alignment of two RT-PCR products shown in Fig. 2b (upper). The additional 118 bp corresponding to Exon 5 (orange lower case) causes a frame-shift and extends the ORF to a new start site (green). (b) Pairwise comparison of protein sequences of human and mouse CATSPERδ encoded by Tmem146-l. Proteinencoded by Tmem146-s starts from methionie underlined in blue.

99

198

298

398

498

598

695

795

798805

795695

595

495

395

295

197

96

mousehuman

Exon 5

CAAGATGCTGGTGTTGATGCTAGCAGCAGCAGTGGCCACCATGGTCAGGGCTCACACACTGTGCCGGGTGCACACAGTGACAAGATGCTGGTGTTGATGCTAGCAGCAGCAGTGGCCACCATGGTCAGGGCTCACACACTGTGCCGGGTGCACACAGTGA

GGACCGGGAAAGTGTTTAAGTCCAATATACAACTTCAAGGGGACCCACTGTTCTACGCCTTCCCCAACACATTTGTGCTCGGACCGGGAAAGTGTTTAAGTCCAATATACAACTTCAAGGGGACCCACTGTTCTACGCCTTCCCCAACACATTTGTGCTC

AAGAACGTGTGCAAGGCTGACATTTCTGTGTATCTAGGGCAGAAAGTTTTCTTGACGATAGACAACTTTGAGTCCAGCCTAAGAACGTGTGCAAGGCTGACATTTCTGTGTATCTAGGGCAGAAAGTTTTCTTGACGATAGACAACTTTGAGTCCAGCCT

CTTGCCGCTCACCGTTCCCAAGTCATTGGCGgtgggggtgccatcaatcacaagcgcccactttgtctccggctcactagCTTGCCGCTCACCGTTCCCAAGTCATTGGCG-------------------------------------------------

tactgtttgtgatcagtgtaaaaggctacagttacgattactatgagaacacctggcgtaaattggaagGTATATCAGAA---------------------------------------------------------------------GTATATCAGAA

CCTGTCTCTCACATTAGTGGTGACGTTTGTTGTTTCAAAGGCTCCTTTTGCCTGGAGTTATCAAATAACCTGTTTGCATACCTGTCTCTCACATTAGTGGTGACGTTTGTTGTTTCAAAGGCTCCTTTTGCCTGGAGTTATCAAATAACCTGTTTGCATA

TCTCCGTGGTGGCCAGATCCCTGGGACCAACATATATTTCTCTGACAATGGAGGATTTTCATTCCAGCTCATGAATACTGTCTCCGTGGTGGCCAGATCCCTGGGACCAACATATATTTCTCTGACAATGGAGGATTTTCATTCCAGCTCATGAATACTG

ATC

ACAAACTGAGTCACCTGACAGGAACACTAGGAGGCATCTTCCACTTACATTCCATGTCTCAGGTGGGGGTACTCATGGTTACAAACTGAGTCACCTGACAGGAACACTAGGAGGCATCTTCCACTTACATTCCATGTCTCAGGTGGGGGTACTCATGGTT

GAAAATAATTTGGGCACATTTCATTACATGGAGTACCCCCTGAACCATAGTATGGGGATAGCCTTTTCATACAAGAATCTGAAAATAATTTGGGCACATTTCATTACATGGAGTACCCCCTGAACCATAGTATGGGGATAGCCTTTTCATACAAGAATCT

CCTTGAAGTCATTATGAAACCATACCAGAGGGGGTTTATGGTCCTCTGGAATCAGAAAAGTATATTGGTGTCCTCCAATTCCTTGAAGTCATTATGAAACCATACCAGAGGGGGTTTATGGTCCTCTGGAATCAGAAAAGTATATTGGTGTCCTCCAATT

CAGGCCAGATTGTTGAACACGTGCGGCTGATAGATCAGAAGATCTTTACAGACTTAGATGTCGAACATGCCAACATCAACCAGGCCAGATTGTTGAACACGTGCGGCTGATAGATCAGAAGATCTTTACAGACTTAGATGTCGAACATGCCAACATCAAC

ATC

8080

160160

240240

320271400282

480362

560442

640522

720602800682

880762

883765

Exon 1(partial) Exon 2

Exon 3 Exon 4

Exon 6 Exon 7

Exon 8

Exon 9

Exon 10(partial)

RT-5’

GSP3

GSP2

mTmem146-lmTmem146-s

aTmem146-l

Tmem146-s

Tmem146-l

Tmem146-s

a

b t=0 s 0.033 s 0.067 s 0.100 s

0 2 4 6

0.133 s 0.167 s 0.200 s 0.233 s

8 10 12 14

One beat cycle equals ~108 ms (9.3 Hz)

116 ms 116 ms 100 ms

Frame no.0 2 4 6 8 10 12 14

t=0 s 0.033 s 0.067 s 0.100 s

0.133 s 0.167 s 0.200 s 0.233 s

0 2 4 6

8 10 12 14

One beat cycle equals ~225 ms ( 4.4 Hz)

Frame no.0 2 4 6 8 10 12 14

233 ms 217 ms 217 ms

δ het 1 δ het 2 δ het 3

δ null 1 δ null 2 δ null 3

Supplementary Figure S2. Spermatozoa 90 min after capacitation. (a) CatSperδ-het sperm cells tethered to a glass coverslip (frame rate = 60 Hz). Average beat cycle = 225 ms. Flagellar capture in each frame is shown in cyan (het 1). Superimposed traces are coloured as shown in the spectrum. Aligned traces from two additional representative sperm cells are shown (het 2 and het 3). (b) CatSperδ-null sperm cells tethered to a glass coverslip. Average beat cycle = 108 ms. Traces from two beat cycles (null 1) are aligned to show the same number of flagellar captures. Aligned traces from two additional representative sperm cells are shown (null 2 and null 3).

Supplementary Figure S3. mRNA expression of CatSper genes over time. (a) Quantitative expressionanalysis of CatSper1, β, and γ mRNAs from CatSperδ-het and null testes using qRT-PCR. Expression ratio (2 ) was calculated by the ddCt method (ref.30). Mean ddCt (null-het) and corresponding expression ratio are from 3 independent experiments. TATA binding protein (TBP) used as an internal control. (b, c) Temporal expression of CatSper1, β, and γ mRNAs during post-natal development of mouse testis. CatSpers mRNAs are measured by real-time RT-PCR. The levels of CatSper1 ( ), δ ( ) (b) and CatSperβ ( ), γ ( ) (c) mRNAs are normalized to TBP and expressed as ratios relative to 80-day-old adult mouse testis. Results are averaged from 3 independent experiments for all stages. The data are presented as mean ± SEM. Significance calculations by t test refer to previous time point (significant increase over previous value. *P<0.05, **P<0.005, ***P<0.001).

0

0.2

0.4

0.6

0.8

1.0

1.2

CatSper1 CatSperβ CatSperγE

xpre

ssio

n ra

tio

a

-ddCt

7 17 20 23 45 80

1.4

1.2

1.0

0.8

0.6

0.4

0.2

0

Days post-partum

Exp

ress

ion

ratio

Exp

ress

ion

ratio

*

**

*

**

***

7 17 20 23 45 80

1.41.2

1.00.8

0.60.40.2

0

1.61.8

*

*

Days post-partum

c

b