1. Department of Zoology, Banaras Hindu University Varanasi 221005, India
A possible role of Oxytocin on spermatogenesis and steroidogenesis in mouse: An approach towards development of precocious puberty
Speaker: Dr. Shabana Anjum
(Ph.D.)
Department of Zoology, Banaras Hindu University Varanasi , India

2. General concept of Hypothalamic–Pituitary-Testicular axisLH
FSH
GnRH
Gonadotropins
Spermatogenesis
Steroid Production
Anterior Pituitary H
GnRH is primary regulator of gonadotropins (LH and FSH) secretion
A hypothalamic decapeptide factor, known as Gonadotropin Releasing Hormone (GnRH) was discovered in brain of mammals
OXT-receptor are present on GnRH neuron and Oxytocin modulate the GnRH release from the hypothalamus (Caligioni et al., 2007) P
Posterior
Pituitary
Oxytocin
Feed back loop T
Testosteroneee
Estradiol

3. OXYTOCIN
Oxytocin is a neuro-peptide, produced by the Paraventricular nucleus (PVN) of the hypothalamus and released from the posterior pituitary
It is nanopeptide with disulphide bridge
Traditionally known as female hormone, where it regulate uterine contraction, milk ejection and parturition
In male, OXT modulate sexual behavior, ejaculation and seminiferous tubule contraction that help in transfer of sperm to epididymis

4. Hypothalamic OXT
Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Lec-Gly-NH2
Extra Hypothalamic OXT
Ovaries
Testes
? ? ? ?
Testis
Molecular mechanism by which OXT affect testicular steroidogenesis is
not yet known

5. How it is regulating Steroidogenesis??
Is there any role of OXT in Spermatogenesis and Steroidogenesis??

6. Objective of my study
Role of OXT on steroidogenesis and spermatogenesis
Regulation of OXT through Luteininzing hormone

7. ELISA for Testosterone Steroidogenic factors Spermatogenic Factors
Experimental design
Syntocinon/OXT
In vivo
Serum
Testis
ELISA for Testosterone
Steroidogenic factors
Spermatogenic Factors
(N=8 per groups)
In vitro
(N=3 per groups)
Testes was cultured for 24 hour in culture medium (DMEM + Ham’s F-12
Culture medium
Testis
Steroidogenic factors
ELISA for
Testosterone
Group (1): Control
Group (2): 0.25 IU/BW (Low) OXT
Group (3): 1.0 IU/BW (High) OXT
For 8 days I.P.
On 28th day of postpartum
Group A: (1) Control;
(2) 0.05 IU/mL OXT;
(3) 10 ng/mL LH
(4) 10 ng LH+0.05 IU OXT

8. Effect of OXT on testosterone level in serum and 3β HSD enzyme activity in testis
(ng/mL)
Dose of OT
(IU/day/BW)
0.25
1.0 - + b a
(A) c
(B)
3 β HSD Enzyme Activity (n mol NADH/hr/mg of protein) b a
Dose of OT
(IU/day/BW)
0.25
1.0 - +
Figure 1

9. 1.0 IU (OT) 0.25 IU (OT) Control (OT)
Effect of OXT on histological study in testis (in vivo)
(A)
(B)
(C)
rSt
rSt
rSt
p Sc
p Sc
pl S
p Sc SC
pl Sc SC
pl Sc
B Sg LC LC LC
1.0 IU (OT)
0.25 IU (OT)
Control (OT)
Figure 2

10. Effect of OXT on Oxytocin receptor mRNA in testis (in vivo)
Control
0.25 IU
1.0 IU
350 bp
1000
900
800
700
600
500
400
300
200
100
mRNA levels
Dose of OT
(IU/day/BW)
0.25
1.0 - + b c a
Oxytocin receptor
Forward F(1C) 5'GGACGTCAATGCGCCCAAAGAAG3'
Reverse (R8) 5'ACTCGAGCTGCAACGACTCA3'
Figure 3

11. Effect of OXT on LH-R and StAR Protein expression in testis (in vivo)
(B)
(A)
LH-R
StAR
β- actin
β- actin
% RIDV
Dose of OT
(IU/day/BW)
0.25
1.0 - + a b c c
% RIDV b
Dose of OT
(IU/day/BW)
0.25
1.0 - + a
Figure 4

12. Effect of OXT on PCNA protein expression in testis (in vivo)
(B)
(C)
p Sc
pl Sc
p Sc
B Sg L
pl Sc L
B Sg
Control
0.25 IU
1.0 IU c b
(D)
PCNA
% RIDV a
β-actin
Dose of OT
(IU/day/BW)
0.25
1.0 - +
Figure 5

13. Effect of OXT on Bcl2 and Androgen receptor protein expression in testis (in vivo)
% RIDV
Dose of OT
(IU/day/BW)
0.25
1.0 - + a b c
(A)
Bcl2
β Actin
% RIDV
Dose of OT
(IU/day/BW)
0.25
1.0 - + a b c
(B) AR
β-actin
Figure 6

14. Effect of OXT on GnRH-R protein expression in testis (in vivo)
Dose of OT
(IU/day/BW)
0.25
1.0 - + c b
GnRH-R
β Actin a
% RIDV
Figure 7

15. Effect of OXT on testosterone level in media and StAR protein expression
in testis (in vitro)
(A)
Testosteronelevel (ng/mL) b c d -
Dose of OT (IU/mL) 10
0.05
Dose of LH (ng/mL) a
(B)
StAR
β-actin b c d
% RIDV a
Dose of OT (IU/mL) - 10
0.05
Dose of LH (ng/mL)
Figure 8

16. Effect of OXT on GnRH-R protein expression in testis (in vitro)
Dose of OT (IU/mL) - 10
0.05
Dose of LH (ng/mL) a b c d
% RIDV
GnRH-R
β-actin
Figure 9

17. Conclusion…
OXT treatment causes significant changes in the spermatogenic and steroidogenic
activity in testis of pre-pubertal mice
OXT showed increaased proliferation of germ cell by increased expression of PCNA,
AR and Bcl2 protein in testis
OXT stimulate testosterone synthesis due to stimulatory effect on 3 β HSD activity and
increased expression of StAR and LH-R protein in testis
OXT stimulate the GnRH-R protein expression in testis
Thus, OXT may act as autocrine/paracrine factor for the testicular activity and locally produced OXT may act throgh autocrine/paracrine mechanism by Leydig cell

18. Histopathological changes
Summary of the study..
Syntocinon/OXT
In vitro study
In vivo study
Pre-pubertal Age ST
Leydig cell
Histopathological changes
LH-R
StAR
3βHSD
Germ cells in stage VII seminiferous tubules
Expression of PCNA protein in spermatogonia
Expression of AR expression
GnRH-R
Detection of anti apoptotic germ cells
Testosterone
Expression of Bcl2
As a autocrine/paracrine factor and enhanced reproductive activity /Precocious puberty

19. Thank you