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Male contraceptiveMale contraceptive

Maturation

in

Epididymis

Mitosis Meiosis ???

Stem cells

Reversible??

Segregation

chromosomes

Aneuploidy??

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TSSK mouse literatureTSSK mouse literature

Tssk-1 : isolated by virtue of homology kinase domain

chr. 16

Testis-specific serine/threonine kinase

Tssk-2 identified via similar approach chr 16 tandem

Late spermatids residual bodies

Via 2-hybrid p65 substrate.

p65 is co-expressed with Tssk-1 and Tssk-2 Tssk-3 Leydig cells

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Tssk human literatureTssk human literature

Tssk-2 found on chr. 22

Tssk-1 on chr. 22 is pseudogene

Location in region linked to DiGeorge syndrome p65 homolog found in human: testis-specific (528 aa)

Tssk-3 also identified.

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Tssk-2 as target male-Tssk-2 as target male-

contraceptioncontraception Tssk-2 expressed in spermatids

Tssk-2 is testis-specifically expressed in both mouseand human

Kinase is drugable family

No Tssk-1 exists.

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Organon workflowOrganon workflow Knockout generation for functional validation:

Tssk-1/Tssk-2 double KO at Lexicon Genetics

Single KO strategy failed

Recombinant human Tssk-2 protein production:

Many attempts: E. coli, Baculo,

Recombinant human p65:

GST-fusion in E.coli

HTS development

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Tssk ISHTssk ISH

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Tssk-protein localizationTssk-protein localization

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Recombinant Tssk2

phosphorylates p65

10

1100

10

1100

10

1100

Tssk2

GST-P65 - GST

GST-P65Tssk2

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Depletion of Tssk activityDepletion of Tssk activity

Tssk2 depletion by tssk2 antibodi

0

20

40

60

80

100

120

inp inp

verdund

prot G

dep1

IgG1

dep1

tssk2

dep1

prot G

dep2

IgG1

dep2

tssk2

dep2

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P65 mutantsP65 mutants

1-216

1-88

1-164

78-216

136-216

216-592

1-592

GST-fusion p65 proteins for phosphorylation site analysis

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GST-p65 mutantsGST-p65 mutants

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HTS assayHTS assay

TSSK2 Activity

0

50

100

150

200250

300

10 100 1000 10000 100000dilution

mP

TSSK2, Ni-NTA purified

TSSK2, SEC purified

Purification of mock transfected Sf21 cells

TSSK2 DM purification

TSSK2, Ni-NTA purified, concentrated, -ATP

TSSK2, SEC purified, -ATP

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TSSK1-TSSK2 KOTSSK1-TSSK2 KO

constructionconstruction1 kb

Screening strategy:

5 external probe : NheI 3 external probe: NheI

wt: 5.2 Kb wt: 7.6Kb

targeted: 2.3 Kb targeted: 9.6Kb

Targeted allele

Neo

Frt Lox PFrtLox P

NheI NheI

5 probe 3 probe

NheI

TSSK1 TSSK2

NheI

NheI

NheI

wt allele

1 kb

Screening strategy:

5 external probe : NheI 3 external probe: NheI

wt: 5.2 Kb wt: 7.6Kb

targeted: 2.3 Kb targeted: 9.6Kb

Screening strategy:

5 external probe : NheI 3 external probe: NheI

wt: 5.2 Kb wt: 7.6Kb

targeted: 2.3 Kb targeted: 9.6Kb

Targeted allele

Neo

Frt Lox PFrtLox P

NheI NheI

5 probe 3 probe

NheI

TSSK1 TSSK2

NheINheI

NheINheI

NheINheI

wt allele

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TSSK1/2 KOTSSK1/2 KO

WT TSSK1/2 KO

Testis

Epididymis

MALE INFERTILITY

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Tssk-1?Tssk-1?

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Tssk-1 human expressionTssk-1 human expression

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Tssk-1Tssk-1

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TSSK genomeTSSK genome

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Tssk-4Tssk-4

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Human Tssk-4Human Tssk-4

TSSK-4

25x

30x

600bp-

600bp-

M Te

Ut

Ki

Th

Li

Br

He

Lu

Sp

Ov

Pa

Be

Ag

Mq

G

TSSK-4

25x

30x

600bp-

600bp-

M Te

Ut

Ki

Th

Li

Br

He

Lu

Sp

Ov

Pa

Be

Ag

Mq

G

+RT

-RT

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Mouse TSSK-4Mouse TSSK-4M A

gBr

Ep

He

Ki

Li

Mu

Te

1

Te

2

Te

3

Te

4

Lu

Sp

Pr

TSSK-4

25x600bp-

600bp-

Mq

G

30x

M Ag

Br

Ep

He

Ki

Li

Mu

Te

1

Te

2

Te

3

Te

4

Lu

Sp

Pr

TSSK-4

25x600bp-

600bp-

Mq

G

30x

+RT

-RT

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TSSK-4 mouse mRNATSSK-4 mouse mRNA

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Stage of expression TSSK-Stage of expression TSSK-

44

M 3 5 7 10

16

20

24

Mq

TSSK-4

25x

30x

600bp-

600bp-

3 5 7 10

16

20

24

Mq

M

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TSSK-4 ISHTSSK-4 ISH

Early + late spermatids and weakly late spermatocytes