GROWTH OF OOCYTES IN PIG PRIMORDIAL FOLLICLES XENOTRANSPLANTED INTO SCID MICE
Mohammad Moniruzzaman, Shoichiro Senbon and Takashi Miyano*
Graduate School of Science and Technology, and *Faculty of Agriculture, Kobe University
1-1 Rokkodai-cho, Nada-ku, Kobe 657-8501, Japan.
Introduction
Development of primordial follicles of newborn pigs in xenografts
Results
Female mammals have a large pool of small oocytes in primordial follicles
that are the sole source for ovulated eggs over their reproductive life. When
these oocytes are recruited for growth, the surrounding granulosa cells
become cuboidal and start to proliferate. However, the mechanisms for the
growth of small oocytes are not understood well. The technique for
successful growth of oocytes in primordial follicles are still far from
perfected and is not applicable for wide range of species. Ovarian
transplantation are considered to be an alternate method to grow up small
oocytes. Successful xenotransplantation of ovarian tissues to immuno
deficient mice or rats have been achieved in some species. Most of those
reports revealed the growth of oocytes in primordial follicles from fetus and
newborn animals. Recently, Kaneko et al. (Biol. Reprod. 69, 2003) reported
the growth of oocytes in primordial follicles from 20-day-old piglets in nude
mice. On the other hand, Senbon et al. (Zygote. 11, 2003) claimed
Primordial follicles of adult pigs in xenografts
Antral
Fig. 1. Histological examination confirmed that adult pig ovarian tissues contained only primordial follicles (Fig.1-A). After
2 months of transplantation 43 ± 7.8 % follicles survived but none of those were recruited for development (Fig.1-B).
Fig. 3. Ten-day-old pig ovarian tissues
containing only primordial follicles
(Fig. 3-A) were transplanted for 2
months. After transplantation, follicles
developed to the antral stage (Fig. 3-B)
B
Secondary
A
Primary
Primordial
B
A
400 µm
that oocytes in primordial and primary follicles of matured cows did not develop in SCID mice. The present experiment was
conducted to study the growth of oocytes in primordial follicles from adult pigs (6-month-old) in comparison to those of newborn
pigs (10-day-old) in SCID mice. Effect of gender of host mice on the oocyte growth in xenografts was also examined.
40 µm
100 µm
40 µm
Materials and Methods
Before
%
Collection of pig ovarian tissues containing primordial follicles
After
%
100
b
c
Cortical slices containing only primordial follicles were collected under a dissection microscope from newborn (10-day-old; n=4)
and adult (6-month-old; n=12) pigs using fine surgical blades. Size of the slices were about 2×1×0.5 mm. Each slice was cut
into 2 pieces; one part was fixed for immediate histological examination and the other one was used for transplantation.
100
80
Male (n=8,120)
80
Before
After
a
Male (n=5,197)
Female (n=6,617)
Female (n=4,169)
60
Ovariectomized (n=7,373)
60
Ovariectomized (n=4,498)
40
%
100
Pig ovarian tissue containing
only primordial follicles
Divided into two parts
Ovaries were collected from 6-month-old
and 10-day-old (inset) pigs. The scale bar
represents 1cm.
Male (n=549)
80
Ovariectomized (n=484)
40
20
20
0
Primary
Secondary
a
20
20
Male (n=280)
b
a
a
b
0
Ovariectomized (n=209)
Primordial
a
b
ab
Primary
0
Secondary
Primordial
Primary
Secondary
Antral
Fig. 4. Distribution of different types of follicles in newborn pig ovarian tissues after transplantation into male, female and
ovariectomized SCID mice for 2 months. ‘n’ indicates the number of follicles examined.
Primordial
Primary
Secondary
Transplantation
Before
%
100
Fig. 2. Distribution of follicles in adult pig ovarian tissues after transplantation into male, female and ovariectomized
SCID mice for 2 months. ‘n’ indicates the number of follicles examined.
%
100
After
b
a
80
80
a
Male (n=8,120)
Xenotransplantation of ovarian tissues into SCID mice
For transplantation, 6- to 8-week-old male and female SCID (severe combined immune deficiency) mice were anaesthetized, the
left kidneys were exteriorized, and cortical slices were inserted under the kidney capsules with the help of glass pipette. Some
b
Female (n=220)
60
40
Primordial
40
80
Female (n=533)
60
0
Histological
examination
%
100
Female (n=6,617)
60
Male (n=5,197)
Female (n=4,169)
60
Ovariectomized (n=7,373)
Ovariectomized (n=4,498)
Table 1. Oocyte diameter in adult pig ovarian tissues after transplantation
40
40
Transplantation* Types of mouse Diameters of oocytes No. of mice used No. of tissues examined No. of oocytes examined
20
20
female SCID mice were ovariectomized just after the transplantation.
a
c
b
a
Male
Before
SCID mouse
Histological examination of xenografts
After 2 months, the left kidneys of the SCID mice were taken carefully. Then the kidneys together with the grafts were fixed in 3%
paraformaldehyde in phosphate buffered saline and embedded in JB-4 resin. The tissues were cut to make serial sections by 5 µm
and stained with haematoxylin and eosin. All the sections were observed under a light microscope to examine the development of
follicles and to measure the diameter of oocytes as well as the follicles.
After
30.57 ± 0.79
-
9
549
Female
30.71 ± 0.81
-
9
533
Ovariectomized
30.39 ± 0.86
-
9
484
Male
31.62 ± 0.91
4
9
280
Female
31.37 ± 0.90
4
9
220
Ovariectomized
31.57 ± 0.59
4
9
209
0
21- 40
41 <
µm
0
21- 40
41- 60
b
a
61- 80
a
b
a
81- 100
a
b
a
101-120
µm
Fig. 5. Distribution of the oocytes in newborn pig ovarian tissues on the basis of the diameter after transplantation.
‘n’ indicates the number of oocytes examined.
Conclusion
The results showed that the growth of oocytes and follicular development were higher in male SCID mice than those in females.
Oocytes in primordial follicles of adult pigs survived but did not initiate the growth. On the other hand, primordial follicles of
newborn pig ovaries developed to the antral stage in xenografts. The results indicate that the development of primordial follicles in
adult pigs are inhibited by some factors, or they have lost the ability to develop.