Mechanisms of Ageing and Development, 4 (1975) 391-398 © Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
391
OVARIAN D E V E L O P M E N T IN A T H Y M I C N U D E MICE II. T H E G R O W T H OF T H E OOCYTE A N D F O L L I C L E
SUE LINTERN-MOORE Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, P.O. Box 4, Canberra City, A.C.T., 2601 (Australia)
E. M. PANTELOURIS Department of Biology, University of Strathclyde, Glasgow G1 1XW ( Gt. Britain)
(Received August 19, 1975)
SUMMARY Congenitally athymic mice homozygous for the Mendelian recessive mutation 'nude' develop well defined morphological and quantitative changes in the ovarian follicle population. A decline in follicle numbers at 2 months of age is preceded by a retardation in follicle growth at 1 month of age. The growth of the oocyte and its nucleus are not affected by the nude mutation. However, the rate of growth and maximum size of the oocyte nucleolus are reduced in nudes. These developmeptal events are discussed in relation to the genetic activity of the oocyte, the role of pituitary gonadotrophins in follicular and oocyte growth and the possible role of the thymus gland in these processes.
INTRODUCTION The discovery that homozygous nude mice lack a thymus gland has led to much research into the pathology of the nude lymphoid system and the cesulting immunological dysfunction 1. However, nude mice have been rarely used to examine the long suspected but as yet undefined role of the thymus gland in sexual maturation and reproductive functionL It is well known that reproductive capacity in nude females is severely reduced. The ovaries are small, the first ovulation is delayed and mature females are invariably sterile or show lowered fertilitya-6. As both fertility and puberty may be restored by the grafting of an intact thymus during the neonatal period 3,7, nude mice appear to be a useful tool for the study of ovarian development and the role of the thymus gland in this event. Our initial study demonstrated a clearly defined reduction in ovarian follicle numbers in nudes 8. This was first evident in the primordial and medium follicle population in mice of 2 months and in all fractions of the follicle populatiop in mice of 4 months. However, from l month of age, all growing non-atretic follicles appeared contracted suggesting that a disruption in the normal biphasic pattern 8 of follicle and oocyte growth may precede any decline in follicle
392 numbers. Accordingly we have compared here the growth of the oocyte, oocyte nucleus, oocyte nucleolus and follicle in nude mice of 1 and 2 months of age with that in phenotypically normal littermates or contemporaries of the same age. MATERIALS AND METHODS The stock of mice segregating for the Mendelian recessive mutation 'nude' and the conditions of husbandry have been described elsewhere 6. Ovaries were obtained from 4 nudes and 4 phenotypically normal littermates or contemporaries of the same age (controls) at both 1 and 2 months of age. All nude mice used in this study were found on dissection to lack a thymus gland. Ovaries from each mouse were fixed in Bouin's aqueous fixative, imbedded in paraffin, serially-sectioned at 5/~m and stained with haematoxylin and eosin. Ovarian follicles were classified into 7 arbitrary growth stages according to the classification as modified from Pedersen and Peters 6,9. The mean diameter of the follicle, its oocyte, oocyte nucleus and oocyte nucleolus were measured by taking the mean of the two diameters at right angles to each other in the section of the follicle which passed through the oocyte nucleolus. Only follicles with an intact follicle envelope and oocyte showing no pyknotic nuclei, macrophages or associated signs of atresia were measured. Approximately 10 representatives of each follicle type were measured in each ovary. This was not always possible with the large preovulatory stages of follicle growth which occurred infrequently and were often atretic. Pooled data from nudes and controls at each age were analysed statistically according to Mandl and Zuckerman 1°. Regression equations in the form o f y -- bx + a were calculated for each group of animals and related oocyte diameter to follicle diameter, oocyte nuclear diameter to oocyte diameter and oocyte nucleolar diameter to oocyte nuclear diameter. In the case of the relationship between oocyte diameter and follicle diameter 2 equations have been derived as, in placeqtal mammals, this conforms to a biphasic pattern 8. During Phase A the follicle and oocyte grow together and during the subsequent phase (Phase B) oocyte growth has ceased and the follicle alone grows. Data were tested in all cases foc statistical significance using Students t-test. RESULTS In both 1 and 2 month old nude and control mice the growth of the oocyte in relation to the follicle conformed to the characteristic biphasic pattern. Initially oocyte diameter was positively and significantly correlated with follicle diameter (Phase A). However, the slope of the regression line describing this relationship was significantly greater in nudes, a difference which was more pronounced at 2 months than at 1 month (Table I). The intercepts were not significantly different. To establish whether the rate of growth of the oocyte had increased or the rate of growth of the follicle had decreased, oocyte and follicle diameters were measured in follicles with approximately equal numbers of granulosa cells in the widest cross section in both groups of nudes and controls. From Table II it can be seen that follicle diameter was
Control Nude
2months
+0.980 +0.992
+0.978 +0.975
0.51 ± 0 . 0 1 0.6l ± 0 . 0 2 /317 -- 5.58 P < 0.001
0.50 -- 0.01 0.56 ± 0.01 t~o7 = 3.38 P < 0.001 4.57 2_0.68 3.73 ± 1.04 t317 -- 0.676 N.S.
5.52 ± 0.86 3.52 ± 0.80 t3ov -- 1.464 N.S. +0.062 +0.088
+0.030 +0.035
r*
a
r§
b
Phase B
Phase A
All values of r are statistically significant (P < 0.001). All values of r are not statistically significant. M a x i m u m diameter of oocyte (/~m). Diameter of follicle (/tm) at completion of oocyte growth.
Control Nude
1 month
§ t * +
Group
Age
0.02±0.01 0.02±0.01 t11~ = 1.40 N.S.
0.01 ± 0.01 0.02 ± 0.02 t13G -- 0.72 N.S.
b
69.66 ± 2.40 69.97 ± 2.80 t115 = 0.084 N.S.
68.68 zk 2.01 68.69 ± 4.02 t136 -- 0.002 N.S.
a*
128.89 108.06
126.32 117.42
F+
C O E F F I C I E N T O F S T R A I G H T L I N E R E G R E S S I O N R E L A T I O N S H I P S ( y - bx + a) B E T W E E N T H E D I A M E T E R O F T H E O O C Y T E (pm) A N D F O L L I C L E (pm) F O R P H A S E S A A N D B O F F O L L I C U L A R G R O W T H I N P H E N O T Y P I C A L L Y N O R M A L M I C E (CONTROL) A N D C O N G E N I T A L L Y A T H Y M I C N U D E LITTERMATES OR C O N T E M P O R A R I E S O F THE SAME AGE. ALL V A L U E S A R E E X P R E S S E D AS T H E M E A N 4- SEM
TABLE I
* f § +
1 2 1 2
month months month months
Age
100 98 95 104
55.31 52.78 112.37 104.44
E± ± ±
0.97 1.22 2.91 3.89
36.48 36.94 67.75 68.11
± d± ±
0.85 0.70 1.22 1.01
97 115 120 110
N*
Oocytef
N*
Folliclet
Control
Nude
N u m b e r of follicles measured. Each value represents the mean ± SEM. Follicle. Oocyte.
140-160
60-80
cross section
Numbers o f granulosa cells~widest
61.46 62.29 124.37 123.88
± ± ± ±
Folliclet
0.88 1.00 3.93 1.02
36.24 37.36 69.99 68.83
± 0.70 _L 0.93 ± 0.78 ± 0.64
Oocytet
4.70 6.03 2.46 4.83
t F§
0.22 0.36 1.55 0.60
0+
< < <
391
OVARIAN D E V E L O P M E N T IN A T H Y M I C N U D E MICE II. T H E G R O W T H OF T H E OOCYTE A N D F O L L I C L E
SUE LINTERN-MOORE Department of Experimental Pathology, John Curtin School of Medical Research, Australian National University, P.O. Box 4, Canberra City, A.C.T., 2601 (Australia)
E. M. PANTELOURIS Department of Biology, University of Strathclyde, Glasgow G1 1XW ( Gt. Britain)
(Received August 19, 1975)
SUMMARY Congenitally athymic mice homozygous for the Mendelian recessive mutation 'nude' develop well defined morphological and quantitative changes in the ovarian follicle population. A decline in follicle numbers at 2 months of age is preceded by a retardation in follicle growth at 1 month of age. The growth of the oocyte and its nucleus are not affected by the nude mutation. However, the rate of growth and maximum size of the oocyte nucleolus are reduced in nudes. These developmeptal events are discussed in relation to the genetic activity of the oocyte, the role of pituitary gonadotrophins in follicular and oocyte growth and the possible role of the thymus gland in these processes.
INTRODUCTION The discovery that homozygous nude mice lack a thymus gland has led to much research into the pathology of the nude lymphoid system and the cesulting immunological dysfunction 1. However, nude mice have been rarely used to examine the long suspected but as yet undefined role of the thymus gland in sexual maturation and reproductive functionL It is well known that reproductive capacity in nude females is severely reduced. The ovaries are small, the first ovulation is delayed and mature females are invariably sterile or show lowered fertilitya-6. As both fertility and puberty may be restored by the grafting of an intact thymus during the neonatal period 3,7, nude mice appear to be a useful tool for the study of ovarian development and the role of the thymus gland in this event. Our initial study demonstrated a clearly defined reduction in ovarian follicle numbers in nudes 8. This was first evident in the primordial and medium follicle population in mice of 2 months and in all fractions of the follicle populatiop in mice of 4 months. However, from l month of age, all growing non-atretic follicles appeared contracted suggesting that a disruption in the normal biphasic pattern 8 of follicle and oocyte growth may precede any decline in follicle
392 numbers. Accordingly we have compared here the growth of the oocyte, oocyte nucleus, oocyte nucleolus and follicle in nude mice of 1 and 2 months of age with that in phenotypically normal littermates or contemporaries of the same age. MATERIALS AND METHODS The stock of mice segregating for the Mendelian recessive mutation 'nude' and the conditions of husbandry have been described elsewhere 6. Ovaries were obtained from 4 nudes and 4 phenotypically normal littermates or contemporaries of the same age (controls) at both 1 and 2 months of age. All nude mice used in this study were found on dissection to lack a thymus gland. Ovaries from each mouse were fixed in Bouin's aqueous fixative, imbedded in paraffin, serially-sectioned at 5/~m and stained with haematoxylin and eosin. Ovarian follicles were classified into 7 arbitrary growth stages according to the classification as modified from Pedersen and Peters 6,9. The mean diameter of the follicle, its oocyte, oocyte nucleus and oocyte nucleolus were measured by taking the mean of the two diameters at right angles to each other in the section of the follicle which passed through the oocyte nucleolus. Only follicles with an intact follicle envelope and oocyte showing no pyknotic nuclei, macrophages or associated signs of atresia were measured. Approximately 10 representatives of each follicle type were measured in each ovary. This was not always possible with the large preovulatory stages of follicle growth which occurred infrequently and were often atretic. Pooled data from nudes and controls at each age were analysed statistically according to Mandl and Zuckerman 1°. Regression equations in the form o f y -- bx + a were calculated for each group of animals and related oocyte diameter to follicle diameter, oocyte nuclear diameter to oocyte diameter and oocyte nucleolar diameter to oocyte nuclear diameter. In the case of the relationship between oocyte diameter and follicle diameter 2 equations have been derived as, in placeqtal mammals, this conforms to a biphasic pattern 8. During Phase A the follicle and oocyte grow together and during the subsequent phase (Phase B) oocyte growth has ceased and the follicle alone grows. Data were tested in all cases foc statistical significance using Students t-test. RESULTS In both 1 and 2 month old nude and control mice the growth of the oocyte in relation to the follicle conformed to the characteristic biphasic pattern. Initially oocyte diameter was positively and significantly correlated with follicle diameter (Phase A). However, the slope of the regression line describing this relationship was significantly greater in nudes, a difference which was more pronounced at 2 months than at 1 month (Table I). The intercepts were not significantly different. To establish whether the rate of growth of the oocyte had increased or the rate of growth of the follicle had decreased, oocyte and follicle diameters were measured in follicles with approximately equal numbers of granulosa cells in the widest cross section in both groups of nudes and controls. From Table II it can be seen that follicle diameter was
Control Nude
2months
+0.980 +0.992
+0.978 +0.975
0.51 ± 0 . 0 1 0.6l ± 0 . 0 2 /317 -- 5.58 P < 0.001
0.50 -- 0.01 0.56 ± 0.01 t~o7 = 3.38 P < 0.001 4.57 2_0.68 3.73 ± 1.04 t317 -- 0.676 N.S.
5.52 ± 0.86 3.52 ± 0.80 t3ov -- 1.464 N.S. +0.062 +0.088
+0.030 +0.035
r*
a
r§
b
Phase B
Phase A
All values of r are statistically significant (P < 0.001). All values of r are not statistically significant. M a x i m u m diameter of oocyte (/~m). Diameter of follicle (/tm) at completion of oocyte growth.
Control Nude
1 month
§ t * +
Group
Age
0.02±0.01 0.02±0.01 t11~ = 1.40 N.S.
0.01 ± 0.01 0.02 ± 0.02 t13G -- 0.72 N.S.
b
69.66 ± 2.40 69.97 ± 2.80 t115 = 0.084 N.S.
68.68 zk 2.01 68.69 ± 4.02 t136 -- 0.002 N.S.
a*
128.89 108.06
126.32 117.42
F+
C O E F F I C I E N T O F S T R A I G H T L I N E R E G R E S S I O N R E L A T I O N S H I P S ( y - bx + a) B E T W E E N T H E D I A M E T E R O F T H E O O C Y T E (pm) A N D F O L L I C L E (pm) F O R P H A S E S A A N D B O F F O L L I C U L A R G R O W T H I N P H E N O T Y P I C A L L Y N O R M A L M I C E (CONTROL) A N D C O N G E N I T A L L Y A T H Y M I C N U D E LITTERMATES OR C O N T E M P O R A R I E S O F THE SAME AGE. ALL V A L U E S A R E E X P R E S S E D AS T H E M E A N 4- SEM
TABLE I
* f § +
1 2 1 2
month months month months
Age
100 98 95 104
55.31 52.78 112.37 104.44
E± ± ±
0.97 1.22 2.91 3.89
36.48 36.94 67.75 68.11
± d± ±
0.85 0.70 1.22 1.01
97 115 120 110
N*
Oocytef
N*
Folliclet
Control
Nude
N u m b e r of follicles measured. Each value represents the mean ± SEM. Follicle. Oocyte.
140-160
60-80
cross section
Numbers o f granulosa cells~widest
61.46 62.29 124.37 123.88
± ± ± ±
Folliclet
0.88 1.00 3.93 1.02
36.24 37.36 69.99 68.83
± 0.70 _L 0.93 ± 0.78 ± 0.64
Oocytet
4.70 6.03 2.46 4.83
t F§
0.22 0.36 1.55 0.60
0+
< < <
