1. The study examined the role of BMP-2 on buffalo ovarian follicular function through immunohistochemical and in vitro studies. 2. Immunohistochemistry results showed that BMPR-II is expressed in granulosa cells of buffalo ovarian follicles of different stages. Expression was also seen in theca cells, oocytes, corpus luteum and blood vessels. 3. In vitro studies treating buffalo granulosa cells with BMP-2 showed increased estradiol production, indicating BMP-2 may regulate granulosa cell function and follicular development in buffalos.

1. “Immunohistochemical and in vitro
studies on the role of BMP-2 on buffalo
ovarian follicular function”
Chairperson-Dr. R.V.PRASAD
Professor & Head, dept of veterinary anatomy & histology,
Bangalore
Co-chairperson- Dr.S.SELVARAJU
Scientist(SS),NIANP , Bangalore
Speaker,
VINOD KUMAR DIVATAR
Dept. of Veterinary Anatomy and Histology
Colloquium-II

2. Antral follicle development in Bovine Lucy et al., 1992 , Fortune 2001
Many members of BMPs subfamily were having role in regulation of
follicular development and steroidogenesis (Shimasaki et al., 2004)
Introduction

3. Introduction
Knight & Glister 2006

4. OBJECTIVES
1. To study the localization of BMPR-II in ovarian follicles of buffalo
2. To assess the level of BMPR-II expression during different stages of
follicular growth and development
3. To study the effect of BMP-2 on granulosa cell function in vitro

5. II. REVIEW OF LITERATURE
 The name bone morphogenetic protein (BMP) was first
given in 1965 by Urist and colleagues to the active
components in demineralized bone and bone extracts
 The presence of a functional BMP system in the
mammalian ovary was first reported by Shimasaki et al.,
in 1999.

6.  BMP system comprises a critical component of the local
regulatory system by which ovarian processes are
physiologically governed.
 BMP ligands and receptors are powerful regulators of
fundamental granulosa cell functions, including mitosis
and FSH-mediated differentiation and steroidogenesis

7.  BMP-2 is secreted from granulosa cells of ovary and it
acts in both autocrine and in paracrine manner to regulate
the function of ovary
 BMP-2 preferentially binds to ALK-3 and/or ALK-6 type-
I receptors (Nishitoh et al., 1999); and BMPR-II receptor
(Liu et al., 1995)

8. 2.1 Immunolocalization of BMPR-II in ovary
 The strong immunostaining for BMPR-1A, BMPR-1B and
BMPR-II in granulosa cells of ovarian follicles from
primary to late antral stages of follicle development was
observed in sheep ovary
 Expression was also seen in oocyte, corpus luteum,
ovarian surface epithelium and a lesser extent in the theca
layer of antral follicles in sheep
(Souza et al., 2002)

9.  The expression of BMPR-II was seen in chicken in both
GC and TC of ovary, but the expression was greater in GC
when compared to TC
 The expression of BMPR-II was increased in follicles
along with the development of follicles
(Onagbesan et al., 2003)

10.  Immunostaining for BMPR-I and BMPR-II was observed in
4-6mm follicles of bovine ovarian follicles, both GC and
TC were showed positive reaction but reaction was weaker
in TC when compared to GC
 considerable heterogeneity of staining was observed for
receptor localization amongst individual oocytes
(Glister et al., 2004)

11.  A positive BMPR-II immunostaining was found in
primordial, primary and secondary follicles both in their
granulosa cells and oocytes in bovine
 The expression of both BMPR-I and BMPR-II were seen
in granulosa cells of human ovaries from both foetus and
adults
(Fatehi et al., 2005).
(Ronit Abir et al., 2008)

12. 2.2 BMP-2 in Granulosa Cell Function
 The effect of BMP-2 on granulosa cell function of 1-3mm
follicles in SHEEP ovary was investigated by addition of
BMP-2 at doses of 0, 3, 10 and 30ng/ml
 BMP-2 at highest dose increased the estradiol production
which was evident from day 2 of culture and remained
throughout the culture period
(Souza et al., 2002)

13.  Granulosa cells were treated with recombinant human
BMP-2 of different doses of 0, 3, 30 or 100 ng/ml in
porcine
 The doses of 30 and 100 ng/ml of BMP-2 significantly
suppressed progesterone synthesis compared to controls
 The mechanism of action found that BMP-2 decreased
cAMP and 3βHSD production
(Brankin et al., 2005)

14. III. MATERIALS AND METHODS
It includes,
 Immunohistochemistry
 Long-term buffalo granulosa cell culture

15. 3.1 Immunohistochemistry
Collection and fixation of tissues (ovaries)
Sectioning of tissues (4µ thickness)
Depaffinization and rehydration of tissuse sections
Antigen retrieval(sodium citrate buffer, for 20 min
at 600W) followed by cooling at RT for 30min

16.  Blocking with normal serum for 30min
 Endogeneous peroxidase blocking with 3%H2O2 for
15min
 Overnight incubation with primary antibody of BMPR-II
rabbit polyclonal antibody from Santa Cruz
Biotechnological at 1:100 dilution at 4°C
 For negative control instead of primary antibody normal
serum was added

17. Tissue sections were then incubated for 1hr at RT
with goat anti-rabbit IgG secondary antibody at
dilution of 1:400
Tissues were incubated for 30min in Avidin Biotin
Complex(ABC) at RT
The sections were then incubated with DAB
(Diaminobenzidine) at RT until color development
Counter stained with Harris hematoxylin

18. 3.2 Long term Granulosa cell culture
 Collection of ovaries
 Collection of follicular fluid
 Separation of granulosa cells
 Counting viable cells using Trypan Blue exclusion method
Plating the cells(1lack viable cells per well)
The plate was incubated in CO2 incubator at 38°C and
5% CO2 with 95% humidity
Gutierrez et al., 1997

19. The 75% (150μl) of media was replaced with
fresh media after every 48 hours
Granulosa cell treatment (on 6th day, BMP-2 at
50ng/ml in media) and after 24hrs, 75% of
media from each well was recovered and stored
for hormone assay by RIA technique

20.  Treatment groups includes control,BM-2(50ng/ml) and
combination of BMP-2 with FasL (10ng/ml)
 Termination of culture by 0.5% trypsin containing 0.2
mg/ml EDTA
 Neutralization of trypsin was done with 10% foetal bovine
serum(FBS)
 The collected granulosa cells were snap frozen and stored at
-70°C till further expression studies were done.

21. IV.RESULTS and
DISCUSSION
 Immunolocalization of BMPR-II was observed in granulosa cells of different
stages of follicles
 Primordial- oocyte and granulosa cells of different stages of follicles
4.1.1 Immunolocalization of BMPR-II - different stages of follicles
4.1 Immunohistochemistry

22.  Theca cells were showed the expression but the intensity was
weaker when compared to granulosa cells
 Apart from the granulosa and theca cells, expression of BMPR-
II was observed in the oocyte, corpus luteum and blood vessels
 Atretic follicles were showed expression but the intensity was
weaker when compared to healthy follicles

23. 4.1.2 Expression of BMPR-II -different sizes of follicles
 Expression of BMPR-II was observed in the different sizes of
follicles,
1-3mm (n=30),
3-5mm (n=55),
5-8mm (n=30) and
>8mm (n=6) follicle
 The different sizes of follicles were showing the positive
staining in granulosa cells and the expression pattern did not
differ among different sizes of follicles
 Expression was higher in granulosa cells closer to the antrum
and also corona radiata

24. Structure of ovary Intensity of BMPR-II expression
Granulosa cells
Primordial follicles ++
Primary follicles ++
Secondary follicles (preantral) +++
Tertiary follicles (Antral follicle) +++
Atretic follicles (all stages) +/++
Theca cells
Primordial follicles -
Primary follicles -
Secondary follicles +/-
Tertiary follicles +
Atretic follicles +
Corpus luteum +++
Blood vessels +++
Cellular localization and relative intensity of BMPR-II expression

25. Relative intensity of BMPR-II expression in granulosa
cells of different size tertiary follicles
Follicle Size BMPR-II Expression
1-3mm ++
3-5mm +++
5-8mm +++
>8mm +++

26. Immunohistochemical staining of BMPR-II in preantral follicles
(a) and negative control section showing absence of reaction (b).
(a) (b)

27. (a) (b)
Immunohistochemical staining of BMPR-II in antral follicle, staining
was observed in denuded granulosa cells (a) and negative control
section showing absence of reaction (b).

28. Immunohistochemical staining of BMPR-II in antral follicle (a)
and negative control section showing absence of reaction (b).
(a) (b)

29. Immunohistochemical staining of BMPR-II in corpus
luteum(a) and negative control section showing absence of
reaction (b).
(b)(a)

30. (b)(a)
Immunohistochemical staining of BMPR-II in healthy (a) and
late atretic (b) antral follicle

31. Immunohistochemical staining of BMPR-II in atretic antral
follicles (a and b)
(a) (b)

32. Immunohistochemical staining of BMPR-II in oocyte (a) and antral
follicle (b).
(a) (b)

33. Immunofluorescence staining of BMPR-II in atretic antral
follicles
(b)(a)

34. Immunofluorescence staining of BMPR-II in corpus luteu

35. Immunofluorescence staining of BMPR-II in blood vessels
of ovary

36. Immunohistochemical staining of BMPR-II in blood vessels of
ovary

37. BMPR-II
expression
 Buffalo ovarian follicle (mainly in granulosa cells, corona
radia, theca cells of non-atretic follicles and oocyte) express
BMPR-II
 BMPR-II appears to bind exclusively to BMP ligands, including
BMP-2 (Liu et al., 1995),
BMP-4 (Nohno et al., 1995),
BMP-6 (Ebisawa et al., 1999),
BMP-7 (Liu et al., 1995),
BMP-15 (Moore et al.,2003 ) and
GDF-9 (Vittu et al., 2002)
Discussion-immunohistochemistry

38. • These ligands are involved in follicular function
including steroidogenesis of many species
including sheep (Souza et al., 2004), cow (Glister
et al., 2004), goat (Silva et al., 2005, Fatehi et al.,
2005) and pig (Brankin et al., 2005)
• Hence BMPR-II may be of important in regulating
BMP action on granulosa cell function in buffalo.

39. 4.2 Granulosa cell culture
 4.2.1 Dose effect:
Sheep granulosa cell culture model: To study effect of different
doses (0, 30, 50 and 100ng/ml) of BMP-2 on granulosa cell function
(three independent cultures were carried out, each treatment having
four replicates)
 Three different doses of BMP-2 had increased the estradiol
production in treatment groups when compared to control groups
 The lowest dose of BMP-2 tested (30ng/ml) had increased the
estradiol production significantly as compared to control group
 50ng/ml dose had non-significantly increased estradiol production
when compared to 30 and 100ng/ml

40. 0
10
20
30
40
50
60
Control BMP-30 BMP-50 BMP-100
Estradiol17β(pg/ml)
BMP-2 dose (ng/ml)
ab
a
bc
c
Effect of BMP-2 different doses on granulosa cell estradiol production in Sheep

41. 4.2.2 Effect of BMP-2 on buffalo granulosa cell function
• Based on the studies carried out in sheep, the dose of 30ng/ml BMP-
2 was selected to study its effect on buffalo granulosa cell function.
• Addition of BMP-2 at dose 30ng/ml to treatment groups had
significantly increased the estradiol production when compared to
control groups.
0
10
20
30
40
50
60
70
80
control BMP-2
Estradiol17β(pg/ml)
b
a
(P<0.05)

42. Granulosa cells on 7th day of culture, Some cells have formed
elongated fibroblast like structures
Granulosa cell culture

43. 4.2.3 Effect of BMP-2 on different sizes of follicles
 BMP-2 had increased the estradiol production in different sizes of follicles (small,
medium and large follicles)
 Effect of BMP-2 was more pronounced in medium and large follicles when
compared to small size follicles.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
Small Medium Large
Estradiolproductionfoldincrease
(control=1)
control BMP
0
10
20
30
40
50
60
70
80
90
100
Small Medium Large
Estradiol17β(ng/ml)
control BMP(P<0.05)

44. 4.2.4 Effect of BMP-2 in combination with FasL
 The addition of combination of BMP-2 (30ng/ml) and FasL
(10ng/ml) significantly decreased the estradiol production in
treatment groups when compared to control groups in all
different sizes of follicles

45. 0
10
20
30
40
50
60
control BMP-2 FasL+BMP-2
Estradiol17β(pg/ml)
Small follicles (3-5mm)
c
b
a
(P<0.05)
Effect of BMP-2 (30ng/ml) on granulosa cell estradiol production in
small sized follicles. Difference between individual groups were
shown by a different superscript above each bar (P<0.05)

46. 0
10
20
30
40
50
60
70
control BMP-2 FasL+BMP-2
Estradiol17β(pg/ml)
Medium follicles (5-8mm)
a
c
b (P<0.05)
Effect of BMP-2 (30ng/ml) on granulosa cell estradiol production in medium
sized follicles. Difference between individual groups were shown by a
different superscript above each bar (P<0.05)

47. 0
10
20
30
40
50
60
70
80
90
100
control BMP-2 FasL+BMP-2
Estradiol17β(pg/ml) Large follicles (>8mm)
a
b
c
(P<0.05)
Effect of BMP-2 (30ng/ml) on granulosa cell estradiol production in
large sized follicles. Difference between individual groups were shown
by a different superscript above each bar (P<0.05)

48. 0
10
20
30
40
50
60
70
80
90
100
control BMP-2 FasL+BMP-2
Estradiol17β(pg/ml)
Small
Medium
Large
Overall effect of BMP-2 and FasL+BMP-2 on estradiol
production in different sizes of follicles in buffalo

49. Discussion- BMP-2 on granulosa cell function
 BMP-2 at 30ng/ml significantly increased estradiol-17β
production .
 Similar findings have been reported in other species,
sheep (Souza et al., 2004), pig (Brankin et al., 2005)
 BMP-2 may act by increasing FSH receptors and
aromatase enzyme activities, while decreasing cAMP, and
StAR expression (Brankin et al., 2005, Shi et al., 2011)

50. BMP-2 and FasL combination
 BMP-2 did not increase estradiol production from
granulosa cells cultured along with FasL.
 No reports were available to compare the present
study
 BMP-2 may not be involved in improving cell
viability and proliferation as reported in pigs. Hence
in apoptic cells induced by Fas, BMP-2 had no
beneficial effect on estradiol production.

51. Conclusion
 The BMPR-II expression was observed in all different stages and
sizes of follicles in buffalo
 BMP-2 had increased estradiol7-β production from cultured
granulosa cells
 To know the exact mechanism how BMP-2 had increased
estradiol production from granulosa cells needs to studied further
 Addition of FasL and BMP-2 combination had not increased
estradiol production, BMP-2 was not able to overcome the effect
of cell death induced by Fas
 According to my knowledge it is the first study carried out to
study immunolocalization of BMPR-II and effect of BMP-2 on
granulosa cell function in buffalo.

52. one N all
NAIP, C1056 PROJECT, NIANP, Adugodi, Bangalore