31st British International Congress of Obstetrics and Gynaecology, 4 July 2007

1. July 4th 2007 Mona A. A. Elshafie 1
The Effect Of Leptin On Maturation Of The
Ultra-structural Ovarian Compartments Of
Albino Rats
Dr. Mona Elshafie
Lecturer in Histology
University of Alexandria – Egypt
31st British International Congress of Obstetrics and Gynaecology
July 4th 2007

2. LEPTIN & REPRODUCTION

3. July 4th 2007 Mona A. A. Elshafie 3
 Leptin, an adipocyte secreted hormone, is a
polypeptide of 167 amino acids.
 It acts by binding to a specific receptor, which was
identified in many tissues including hypothalamus,
pituitary gland and ovaries.

4. July 4th 2007 Mona A. A. Elshafie 4
 Leptin could be the permissive factor for puberty by the
activation of hypothalamic-pituitary-ovarian axis,
stimulating the secretion of:
1. GnRH by hypothalamic neurons (Lebrethon et al., 2000)
2. LH & FSH by pituitary cells (Jin et al., 1999)
 Further, it exerts endocrine and/or direct paracrine effects
on the ovaries. (Caprio et al., 1999, Karlsson et al., 1997)

5. July 4th 2007 Mona A. A. Elshafie 5
 In vivo, leptin administration in physiological doses
accelerated the onset of puberty in immature female rats
and stimulated all aspects of their reproductive endocrine
system. (Almog et al., 2001, Barash et al., 1996, Chehab et al., 1996)
 In vitro, leptin administration inhibited folliculogenesis in
immature mice – in a dose related manner (Kikuchi et al, 2001),
and steroidgenesis in rats – in physiological doses. (Zachow et
al, 1999)

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 The rat ovary shows great structural similarity to
the human ovary.
 The main discrepancy between the rat and the
human reproduction is that rats are polyestrus
mammals that repeat the oestrus cycle
throughout the year at intervals of about 4-5
days.
(Buckland et al., 1981)

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WHAT IS NOVEL?

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This is the first study to document the ultra-
structural maturational changes that occur in
the ovarian follicles of immature female albino rats
treated with leptin when compared with controls

9. July 4th 2007 Mona A. A. Elshafie 9
HOW DID WE DO IT?

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50 immature female albino rats aged 22 days (age of weaning),
with an average body weight of 20-25 grams

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 The animals were house kept under the same environmental conditions
& fed a standard rat food.
 No males were housed in same rack.

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Control Group
 26 rats, 2 of them were
sacrificed at the age of 22 days
 The rest (24) received daily
S.C. injection of 5µg of the
solvent
 Rats were sacrificed following
the same regimen as in the
experimental group
Experimental Group
 24 rats
 each received daily S.C.
injection of 5µg leptin
 3 rats were sacrificed every 2
days after injection, i.e. at 24,
26, 28, 30, 32, and 34 days
 Then, on approaching the
expected age of maturity (42-
44 days), they were sacrificed
every 4 days i.e. at 38 and 42
days

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 The rats were sacrificed under light ether anaesthesia 
ovaries were dissected  specimen were prepared for
examination using transmission electron microscope (EM):
 The initial phase of the work was performed in The Histology
Department, Alexandria Medical School, Egypt which is licensed
for animal care and use.
 Preparing and examining the specimens using The EM was
performed in Leicester University, UK.

14. July 4th 2007 Mona A. A. Elshafie 14
WHAT DID WE FIND?

15. 1- Germinal Epithelium & Tunica Albuginea
Control - 22 days
Germinal Epi
Treated - 26 days
Germial Epi

16. 2- Folliculogenesis
Treated - 26 days
Granulosa C
Theca C
PF
SF
Control – 26 days

17. 2- Folliculogenesis
control26
Treated - 26 daysControl - 26 days
Granulosa C

18. 2- Folliculogenesis
Treated - 30 days
L
Granulosa C
Theca C
GC
TC
Control – 30 days
control 30
AF
Control – 30 days
O
ZP
CR
Treated - 30 days

19. 3- Oocyte & Zona Pellucida
Control - 22 days
m
rE
Ovum
Control - 22 days
Zona P
Ovum
ZP
O

20. 3- Oocyte & Zona Pellucida
Treated - 26 days
Zona P
m
Control - 26 days ZP
O
CR
Treated - 26 days

21. 3- Oocyte & Zona Pellucida
ZP
Treated - 30 days
Ovum
Zona P
m
rE
Treated - 30 days
Chromatin
Control – 30 days
ZP

22. 4- Stroma
Control - 22 days
N
g
m
Treated - 26 days
L
L
N
m
g

23. 5- Corpus Luteum
Control - 42 days
Corpus L
L
Treated - 30 days
Corpus L
L
g t

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SUMMARY

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Leptin administration to immature rats led to:
1. Enhanced folliculogenesis:
↓ number of primordial & primary follicles
↑ numbers of growing follicles
Development of antral follicles of different sizes
Formation of corpora lutea
↓ number of atretic follicles

26. July 4th 2007 Mona A. A. Elshafie 26
Leptin administration to immature rats led to:
2. Accelerated maturation of all ovarian components
3. Initiation of steroidogenesis by the granulosa, theca
and stroma cells:
1. Abundant organelles
2. Intracytoplasmic lipid droplets of variable electron-densities
4. Premature ovulation (26-30 days)

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 We documented the ultrastructural evidence that
leptin accelerated the onset of puberty in female
albino rats.
 Nonetheless, the precise role of endogenous
leptin in the physiology of puberty, and the
potential clinical application of exogenous leptin
in cases of impaired reproductive function in
human still need to be elucidated.

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ACKNOWLEDGMENT

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Miss Natalie Allcock, Research Technician,
University of Leicester, UK.
Dr. Ghada Fouad, Assistant Professor in
Histology, University of Alexandria, Egypt.
Prof. Amal Helmy, Professor in Histology,
University of Alexandria, Egypt.
Mr. Ayman Ewies, Consultant Gynaecologist,
The Ipswich Hospital, UK.

30. July 4th 2007 Mona A. A. Elshafie 30
Thank You
Thank You
Thank You
Thank You
Thank You
Thank You

31. July 4th 2007 Mona A. A. Elshafie 31
Any Questions
Alexandria

32. 1- Histological Findings - H&E - Ovary
Control Ovary 22 days
Primordial F
Growing F
Primary F

33. Control Ovary 26 days
Primordial F
Stroma C
Growing F
Atretic F
Treated Ovary 26 days
Stroma C
Stroma C
Graafian F
Graafian F
1- Histological Findings - H&E - Ovary

34. Control Ovary 30 days
Atretic F
Treated Ovary 30 days
Corpus L
Corpus L
Graafian F
Stroma C
1- Histological Findings - H&E - Ovary

35. 1- Histological Findings - Toluidine blue -
Ovary
Control Ovary 32 days
Growing F
Primordial F
Treated Ovary 32 days
Antral F
Antral F
Stroma C
Mitotic figures

36. 1- Histological Findings - H&E - Ovary
Treated Ovary 42 days
Corpus LRuptured F
Stroma C
Control Ovary 42 days
Graafian F
Corpus L
Corpus L
Stroma C

37. 1- Histological Findings - Trichrome - Ovary
Tunica A
TE
Control ovary 24 days
Tunica A
TE
Treated ovary 24 days

38. Control Ovary 30 days
Treated Ovary 30 days
1- Histological Findings - Trichrome - Ovary

39. Control Ovary 42 days
Tunica A
1- Histological Findings - Trichrome - Ovary

40. 2- Histochemical Findings - PAS - Ovary
Control Ovary 24 days
Ovum
Zona P
Control Ovary 30 days
Zona P
Ovum

41. Zona P
Ovum
Treated Ovary 24 days
Cgb
Treated Ovary 30 days
2- Histochemical Findings - PAS - Ovary

42. 3- Histological Findings - Trichrome - Uterus
Control uterus 26 days
Stroma
Treated uterus 26 days

43. 4- Immunohistochemical Findings - Uterus
Control Endometrium 26 days
Stroma
Treated Endometrium 26 days
Stroma

44. 5- Vaginal Smear Findings
Treated 26 days
Epithelial C
Leucocytes
Control 26 days
Leucocytes

45. July 4th 2007 Mona A. A. Elshafie 45
RESULTS
6- Follicular Count
7- Follicular Count
0
0.5
1
1.5
2
2.5
22 24 26 28 30 32 34 38 42
Age (days)
numberoffollicles
leptin-treated Control
Significant increase in the small antral follicle number in leptin-treated ovaries

46. July 4th 2007 Mona A. A. Elshafie 46
RESULTS
6- Follicular Count
7- Follicular Count
0
0.5
1
1.5
2
2.5
22 24 26 28 30 32 34 38 42
Age (days)
numberoffollicles
Leptin-treated Control
Significant increase in the medium antral follicle number in leptin-treated ovaries

47. July 4th 2007 Mona A. A. Elshafie 47
113
The Effect of Leptin on the Ovarian Weight of Rats
0
5
10
15
20
25
30
35
22L 22R 24L 24R 26L 26R 28L 28R 30L 30R 32L 32R 34L 34R 38L 38R 42L 42R
Age (days)
Weight(mg)
Leptin-treated Control
Figure 112: Significant (p<0.01) increase in the weight of the ovaries of rats by daily injection of 5
µg of leptin. n = 24 (treated) and 26 (controls). L = left R = right mg = milligrams
7- Follicular Count
0
0.5
1
1.5
2
2.5
22 24 26 28 30 32 34 38 42
Age (days)
numberoffollicles
Leptin-treated Control
Significant increase in the large antral follicle number in leptin-treated ovaries

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0
5
10
15
20
25
30
35
22L 22R 24L 24R 26L 26R 28L 28R 30L 30R 32L 32R 34L 34R 38L 38R 42L 42R
Age (days)
Weight(mg)
Leptin-treated Control
8- Ovarian Weight
Significant increase in the ovarian weight in leptin-treated rats

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RESULTS
9- Total Body Weight
0
10
20
30
40
50
22 24 26 28 30 32 34 38 42
Age (days)
Weight(g)
Leptin-treated Control
Significant decrease in total body weight in leptin-treated rats