THERIOGENOLOGY - Utrecht University Repository

R.M. Moor, Th.A.M. Kruip* and D. Green**
Institute of Animal Physiology
Animal Research Station
307 Huntingdon Road
Cambridge C63 OJQ
* Clinic for Veterinary Obstetrics, A.I.
and Reproduction, State University of
Utrecht, Utrecht, The Netherlands.
** Premier Embryos, Vallum Farm, Military
Road, Stamfordham, Newcastle-upon-Tyne.
The ability to increase the ovulation rate in domestic animals by
gonadotrophin treatment originated from experiments carried out more
than sixty years ago (1).
These seminal discoveries were however,
rapidly followed by reports of excessive variability in the numbers of
eggs shed in response to a standardized amount of injected hormone (2).
Despite numerous attempts to overcome this problem in the intervening
years little substantial progress has been made towards the objective
of developing reliable methods for the hormonal stimulation of adequate
It is the
and predictable numbersof embryos for transplantation.
objective of this paper to identify the hormonal and intraovarian
factors which determine both the number and the quality of eggs shed
In addition, the probable limits to
after gonadotrophin stimulation.
gonadotrophic stimulation of ovarian function will be discussed and
methods for their circumvention outlined.
We shall suggest in the first section of the paper that the mechanisms that control follicle population dynamics dominate all others in
The interdetermining the extent of superovulation in each animal.
relationship between these physiological regulators and superovulation
can best be appreciated by considering the question of exactly how
The factors
exogenous gonadotrophins increase the ovulation rate.
which determine the quality of oocytes shed after hormone injection will
1984 VOL. 21 NO. 1
be considered in the second section of this contribution. Particular
emphasis will be placed on our recent studies,which show how different
gonadotrophins alter to varying degrees the normal development of
New methods of avoiding the limitations
oocytes during maturation.
imposed by superovulation will be outlined in the concluding section of
The opportunity offered by this in vitro method for the
the paper.
production of large numbers of viable eggs frzabattoir
material will
be of the utmost importance in the future improvement of livestock by
genetic manipulation.
It is apparent that exogenous gonadotrophins increase the ovulation
rate by activating responsive follicles.
Of much less certainty,
however, are the characteristics which endow certain classes of follicles
with the capacity to respond to injected hormones.
It is by examining
the normal process of folliculogenesis and the modifications induced by
superovulation that the practical limitations of this process can be
Dynamics of follicular development during the bovine estrous cycle
The available evidence suggests that bovine follicles leave the
non-growing pool and commence development at privileged periods during
the cycle (3).
Thereafter, growth is continuous until the follicle
ruptures at ovulation or else undergoes atresia (4, 5).
Despite the
continuity of growth a number of intrafollicular factors including
estradiol-176 (E2178), inhibin and secreted proteins modulate the rate
of growth in antral follicles (6,7,8). Estradiol output increases
sharply as the bovine follicle reaches 8mm diameter (9); synthesis of
this hormone is critical since it promotes follicular vascularization
and thereby increases the uptake of gonadotrophins and other nutrients
In this way the most active follicle stimulates its own growth,
whilst at the same time inhibiting growth and differentiation of other
follicles by reducing, centrally or locally, the available levels of
FSH and other gonadotrophins. Although circulating levels of FSH in the
luteal phase are sufficient to stimulate follicle growth, they are unable to support the very largest follicles which consequently degenerate, thus enabling smaller follicles to grow.
The above concepts regarding folliculogenic
control can be presented at a practical level by examining the distribution of follicles
in the ovaries of 32 cows slaughtered at random during the estrous
cycle (Fig. 1 and 2).
The analysis shows that each ovary contained
8-10 follicles larger than 2mm diameter and that 85% of these were undergoing atresia (6).
Medium sized non-atretic folliclesweremost abundant
on day 0 - 5 and from day 9 - 13 of the cycle while large non-atretic
follicles (>lOmm diameter)were found on day 4 - 9 and day 13 - 18. The
follicle that finally ovulated generally originated from the medium
sized class and grew rapidly after regression of the corpus luteum.
Occasionally the preovulatory follicle developedfrom the large follicles
found on day 13 - 18.
1984 VOL. 21 NO. 1
Distribution according to size of all follicles
Fig. 1 (Lower diagram)
over 2n diameter in the ovaries of 32 cows slaughtered at random
The total number of follicles
throughout the estrous cycle.
(>21m1diameter) from each cow is shown adjacent to the asterisk*.
Size distribution of the non-atretic follicles
Fig. 2 (Upper diagram)
(>2tmndiameter) from the 32 cows slaughtered during the estrous
*Total number of follicles (>2mm) per cow; t Number of
non-atretic follicles (>2mm) per cow.
1984 VOL. 21 NO. 1
Information on bovine folliculogenesis raises the associated
question about the state of the oocytes at different cyclic stages and
This has been analysed by examining
in different follicle classes.
the nuclear state of oocytes in the entire follicle population (>2mn
diameter) of 11 animals slaughtered at random throughout the estrous
The results presented in Table 1 show that all oocytes in nonatretic follicles, and almost all in follicles undergoing early atresia,
With the progression of
remain in the normal germinal vesicle stage.
atresia the percentage of oocytes showing degenerative changes, generally first observed as a clumping of the chromatin, increases. Resumption
of meiosis occurs in the preovulatory follicle just after the LH surge;
oocytes in smaller non-atretic follicles are unaffected by the elevated
levels of gonadotrophin at estrus.
Percentages of different classes of bovine follicles and the
nuclear stage of their oocytes in normal cycling, untreated
and FSH/PMSG treated COWS (GV= germinal vesicle; PM= pre-metaphase; MI= meiosis I; Deg.= degenerate).
% of oocytes at various
nuclear stages
11 cows
222 follicles
4 cows
99 follicles
Key to classes
1 - Non atretic
3 - Atretic
2 - Slightly atretic
4 - Severely atretic
The most detailed study of the short-term action of gonadotrophins
on the bovine follicle population has been carried out by Monniaux and
colleagues (11).
This study shows that PMSG injection significantly
increases the mitotic index in preantral and very small antral follicles
but is without effect on the larger antral follicle population. Consequently numbers of preantral follicles increase after PMSG but the
number of normal antral follicles (>1.7mm diameter) remains unchanged.
Since PMSG does not increase the number of antral follicles how is
superovulation induced? The answer lies in the capacity of exogenous
gonadotrophins to prevent or reverse the process of atresia.
reduction in atresia within those classes of follicles over 1.7mm diameter directly increases the number of follicles able to respond fully
to gonadotrophin; follicles under this minimum size are incapable of
reaching the preovulatory size in the 4-5 days between gonadotrophin
1984 VOL. 21 NO. 1
injection and the LH surge.
In calculating the number of responsive
follicles it must, however, be stressed that some of the atretic
follicles luteinize rather than ovulate after rescue and are therefore
of no value in the superovulatory procedure.
Our recent findings strongly support the concept that exogenous
gonadotrophin reduces atresia in superovulated animals.
Four animals
were treated with gonadotrophin and 48h thereafter the entire antral
follicle population ( 3.0mm) was dissected out and analysed by the method
of Kruip (6).
The results, summarized in Table 1, showed that 70% of
follicles in the treated animals were either entirely non-atretic or
only lightly atretic as compared with only 35% in untreated animals.
It is perhaps appropriate to examine the extent to which these
observations on follicle population dynamics and on gonadotrophin action
can explain or improve superovulation procedures.
Firstly, the results
demonstrate clearly why gonadotrophin injection on day 8-10 elicits the
highest ovarian response.
It is evident from Fig. 1 and 2 that bovine
ovaries at that stage contain the largest number of medium sized antral
follicles; the hormonal response experiments show that these classes of
follicles respond most favourably to exogenous gonadotrophins. Secondly,
it might be possible to increase the number of responsive follicles by
exploiting the known stimulatory action of exogenous gonadotrophin on
the preantral follicle classes.
A protocol of repeated hormone treatments together with the alteration of steroid balances by immunization
might be productive in this regard.
An additional important question concerns the long term effect of
exogenous gonadotrophin on ovarian function.
The conflicting evidence
on the effect of repeated hormonal treatments has been clearly documented
by Gordon (12).
The majority of reports indicate that repeated hormonal
stimulation is generally accompanied by a reduction in ovarian responsiveness.
These conclusions have received recent support from a large
study in which 1192 beef cows were repeatedly superovulated with FSH-P
It was concluded from the study that embryo production per
collection, analysed on a within group basis, declined with repeated
Moreover, the decline in embryo production could not
be corrected by increasing the amount of FSH-P injected.
We have recently carried out a similar analysis on the effect of
repeated superovulations in 68 normal and problem cows treated with PMSG
rather than FSH-P (14).
Each animal in this study was injected with
2700 iu PMSG at the first treatment; the dose was increased by approximately 200 iu for each treatment thereafter.
The effect of repeated
treatments on ovulation rate, number of viable embryos recovered, proportion of viable embryos (number of viable embryos over ovulation rate) and
proportion of total embryos recovered at each flushing was analysed by
JANUARY 1984VOL. 21 NO. 1
Fig. 3. The effect on ovulation rate of superovulating
68 normal and problem breeder
cows at consecutive intervals
of 40-60 days with PMSG (14).
For details of statistical
differences see Fig. 4 below.
* . 4.
Mean number of viable
%em ryos recovered from normal
and problem breeder cows after
repekted superovulations. The
DIFFERENCE column shows the
means and standard errors of
the differences between the
two groups of cows. Absence
of statistical differences
(denoted by asterisks) between
groups during the fourth and
fifth treatment reflected the
small numbers of cows treated
at these times.
The findings summarised above (Figs 3 and 4) demonstrate that the
variable was maintained at a constant level throughout the experiments.
An analysis of the differences in the variable at different treatments
failed to reveal any effect of repeated superovulations on either the
It will
ovulation rate or on the number of viable embryos recovered.
be observed that the superiority of normal animals over the problem
breeders was evident at the first treatment and maintained thereafter.
When the data for each animal was summed over all treatments it was
found that the proportion of viable embryos was significantly higher for
the normal cows: 87.1% as compared with 75.7%, PcO.01.
It is not possible to ascertain from our results the extent to
which the gradually increasing dose of PMSG contributed to the consistency of response between the first and fifth hormonal treatment. It is,
however, clear that an acceptable response to PMSG can be sustained for
Equally important is the observation that
at least five treatments.
embryo quality does not decline in cows which have been repeatedly
1984 VOL. 21 NO. 1
Superimposed on the problems of inherent ovarian variability are
those associated with the quality of embryos produced by superovulation.
Results summarized by Newcomb (15) indicate that 55% of embryos recovered
from superovulated donors on day 8 show developmental abnormalities.
The cause of these abnormalities has generally been ascribed to an
unfavourable uterine environment induced by the grossly distorted level
of circulating steroids in these animals (16).
However, inappropriate
interactions between exogenous gonadotrophins and partially differentiated thecal or granulosa cells could equally initiate lesions during
maturation which may not be expressed until the morula or blastocyst
A clear example of delayed expression of an early abnormality
has been reported by Moor and Trounson (17).
These workers found that
ovine oocytes matured in the presence of inadequate levels of oestrogen
underwent fertilization and cleavage but later showed aberrant blastulation patterns.
It has been to determine whether similar events occur
during superovulation that recent experiments have been carried out in
our laboratory (18).
The hypothesis under test has been that egg
quality is predetermined by an early series of interactions between the
exogenous gonadotrophin and follicle cells.
The experiments were carried out using ovine follicles obtained
from untreated animals or those treated 36h previously with an equivalent dose of either PMSG or an equine pituitary preparation (FSH-E).
After dissection, the follicles were cultured for 24h in the absence of
Oocytes enclosed by follicle cells were then labelled
for 3h in 35s methionine and the radiolabelled proteins in each oocyte
separated by SDS gel electrophoresis.
synthesized by non-activated
dictyate oocytes (GV) and by
maturing oocytes after breakdown of the germinal vesicle
(GVBD) and formation of the
metaphase plate.
1984 VOL. 21 NO. 1
Oocytes obtained from untreated sheep invariably showed the protein
profile which characterizes the inactivated germinal vesicle stage of
Figure 5 shows the protein profile from these control
oocytes together with the profile of an oocyte at the metaphase II stage
of maturation (GVBD stage).
When follicles were obtained from FSH-E
stimulated animals approximately 94% of oocytes showed the standard
germinal vesicle (GV) pattern of labelled proteins.
These results
suggest that FSH stimulates follicular development without prematurely
activating the associated oocyte.
The effect of PMSG was, however,
entirely different.
A superovulatory dose of 1250 iu PMSG not only
stimulated follicular development but also resulted in the premature
activation of over 33% of the oocytes.
We have recently extended these observations by carrying out a
somewhat similar study using bovine oocytes.
Our purpose has been to
determine the degree to which premature oocyte activation occurs after
gonadotrophin injection in cows; no attempt has been made in these
experiments to compare FSH-E with PMSG.
Nuclear configuration rather
than protein synthesis has been used to assess premature activation in
bovine oocytes.
In the first study four animals were injected with gonadotrophin
and the cumulus and oocytes examined 48h later (see Table 1). Expansion
of the cumulus and resumption of meiosis occurred in 18.4% of the large
non-atretic follicles but in none of the atretic classes of follicles.
Animals in a second study were injected with 20mg of FSH-E and slaughtered 24h later.
Oocytes in the majority of large non-atretic follicles
(77.5%) were prematurely activated (Table 2).
In the medium sized
follicle classes (>6mm diameter)9% of oocytes showed signs of activation
and 19% were degenerate.
It is thus evident that premature activation
is an important source of loss during superovulation.
Some of the
activated oocytes will be retained in luteinized follicles while others
will be ovulated as aged eggs and will subsequently contribute to the
pool of abnormal embryos.
Nuclear maturation of oocytes in large (>bmm) and small to
medium sized bovine follicles collected 24h after FSH-E treatment (20 mg equine pituitary extract).
Size of
No. of
3 - 6mm
* Meiosis
% at each stage of nuclear maturation
These different experiments are of interest for two reasons.
Firstly they show that exogenous gonadotrophins grossly disturb the
nuclear and cytoplasmic function of a significant number of oocytes.
Secondly, the results from the experiments on ovine oocytes suggest that
1984 VOL. 21 NO. 1
PMSG is much more liable than FSH to disrupt the normal physiological
This latter finding accords well with the
function of the gamete.
practice in many laboratories of using FSH rather than PMSG for superovulations (19).
The chemical properties of the two gonadotrophic preparations
provide a probable explanation for the differences in hormonal action on
It is, of course, well established that the gonathe oocyte.
dotrophins share basic similarities. They are composed of structurally
In addition
similar a-subunits and biologically specific B-subunits.
each hormone has a characteristic carbohydrate composition consisting of
neutral sugars, hexosamines and a highly variable sialic acid content
Thus, the very low
which determines the half-life of the molecule.
level of sialic acid in LH and the slightly higher content in FSH are
reflected in half-lives of 30 min and 110 min for LH and FSH,respectively
By contrast, PMSG has an exceptionally high sialic acid con(20).
tent which confers upon it a half-life greatly in excess of that of the
In cattle the clearance of PMSG involves a
pituitary gonadotrophins.
The in
rapid (t - 40-50h) and a slow (t - llB-123h) component (21).
complicated by its dual
vitro ac ivity of this molecule I*f further
@iiZTotrophic activity.
Radioreceptor analyses of PMSG suggest an
FSH:LH ratio of 1.08 whereas both the amino acid sequence data and the
bioassay data (FSH:LH ratio 1:12) are strongly indicative of an LH-like
The slow clearance
preponderence in the PMSG molecule (21, 22, 23).
rate of PMSG coupled with its dominant LH-like activity in vivo have the
combined disadvantage of exposing stimulated follicles tocontinuously
high LH levels during development.
The consequence of this is the
premature partial activation of oocytes in those follicles showing the
greatest degree of differentiation at the time of hormone injection.
In conclusion, we postulate that the numbers of prematurely stimulated follicles will be substantially reduced by manipulating the FSH
and LH levels during superovulation.
A reduction in the high LH
content of pituitary gonadotrophin preparations should decrease premaChemical modificature activation of oocytes during superovulation.
tions to the PMSG molecules or a reduction in its half-life by the
injection of anti-PMSG serum (24), should equally increase the biological effectiveness of this hormone.
Despite these projected improvements availability of viable eggs will still be a major limitation
especially if genetic manipulation or nuclear transfer becomes important
in animal breeding.
These requirements could, however be satisfied by
utilizing oocytes from the large pool of ovaries available daily at
commercial abattoirs.
Oocytes recovered from antral follicles are characterized by their
total inability to support even the earliest phases of embryonic life.
The acquisition of biological competence in these cells necessitates
many structural and biochemical changes which together constitute the
The complexity of this process has
process of oocyte maturation.
JANUARY 1984 VOL. 21 NO. 1
repeatedly been illustrated in the past by the inability to induce the
complete range of maturational changes in vitro.
Oocytes removed from
antral follicles readily resume meiosis-in?ijT&re, but otherwise remain
incompetent and show numerous developmental abnormalities at fertilization or during blastulation (17, 25).
It has been to identify the
processes by which competence is conferred upon the oocyte and to
utilize this information in the development of simple techniques for its
induction -in vitro that much of our recent work has been undertaken.
Fig. 6. Effect of difference in
hormonal support on the percentage of
intrafollicular oocytes that developed into expanded blastocysts. The
number of oocytes/group is shown
above each histogram (17).
Fig. 7. Effect of cumulus cells and
hormones on the percentage of extrafollicular oocytes cultured in a
static system that developed into
expanded blastocysts. The number of
oocyteslgroup is shown above each
Fig. 8. The effect of cumulus cell
numbers on the percentage of extrafollicular oocytes, cultured in a
non-static system, that developed
into expanded blastocysts. Essential
hormones (FSH-LH-E 17B) were
included in all cu ?tures (26).
The initial experiments were designed to develop a reliable system
for the maturation of oocytes within the follicle. The essential features of that study, outlined in Fig.6-8,were that a high proportion of
intrafollicular oocytes acquire full biological competence in vitro
provided only that both gonadotrophins and estradiol-17B areiK?llided in
Additional experiments demonstrated that the intrathe medium.
follicular signals elicited by the added hormones originate in the
follicle cells and are required by the oocyte during the first 6h of
1984 VOL. 21 NO. 1
The practical requirement for large numbers of mature oocytes has
necessitated a re-orientation away from the technically demanding culBasic information gained from the intrature of intact follicles.
follicular system has been used to develop a simple system for the
culture of ovine oocytes outside the follicle.
The results outlined in
Figure 7 show that oocytes denuded of their follicle cells do not
acquire developmental competence irrespective of the hormonal support
By contrast, cumulus-enclosed oocytes cultured in a normal
static system with the appropriate hormone additions develop normally in
Two recent findings have provided the
about 30% of instances (27).
basis for a highly significant increase in the percentage of extraOur first observation was
follicular oocytes that mature in vitro.
that gentle agitation of the cunures prevents attachment and consequent
premature luteinization of the granulosa cells; maintenance of the
correct state of somatic cell differentiation during culture markedly
increases the percentage of oocytes undergoing maturation (Fig. 8). A
further improvement was effected by adding supplementary granulosa cells
to the culture system.
The addition of 5 x 106 cells/ml increased to
37% the number of blastocysts from corona enclosed oocytes and to 55%
The 55% of blastothose formed from cumulus enclosed oocytes (26).
cysts formed from oocytes matured in vitro compares favourably with 65%
formed when oocytes matured normalvinvo
were transferred to host
Our results suggest that this simple procedure will, with
minor modifications, be suitable for the maturation of oocytes from
many species including the cow.
The objective of obtaining a large but predictable number of viable
embryos by superovulation depends both upon the presence of responsive
follicles and the administration of appropriate hormones.
We conclude
that neither of these key determinants have yet been optimized.
An analysis of folliculogenesis in untreated and superovulated cows
demonstrates that exogenous gonadotrophin stimulates mitotic activity in
preantral follicles and reduces atresia in antral follicles.
It is,
however, on the number, size distribution and condition of the antral
follicle classes that the degree of stimulation directly depends. From
present results it seems improbable that the intraovarian control mechanisms can be sufficiently controlled to eliminate inherent differences
in follicle dynamics and thereby overcome this primary source of variability during superovulation.
Significant improvements within these
primary physiological constraints can, nevertheless be expected from
new work on the nature of the administered hormones and their mode of
Results show that both the number and viability of
embryos are directly affected by the hormonal schedules administered.
Future demands for large numbers of eggs for twinning, nuclear
transfer and genetic manipulation will be met by utilizing oocytes from
the large number of ovaries available at commercial abattoirs. Full
physiological maturation is induced by the provision of obligatory
1984 VOL. 21 NO. 1
somatic signals and the maintenance of essential cellular interactions
during the culture of these oocytes in an extrafollicular environment.
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