Arch. Tierz., Dummerstorf 44 (2001) Special Issue, 9-23
Research Institute for the Biology of Farm Animals, Unit of Reproduetive Biology, Dummerstorf, Germany
'Institute of Animal Physiology and Genetics, Libcchov, Czech Republic
WILHELM KANITZ1, KLAUS-PETER BRÜSSOW1, FRANK BECKER1,
HELMUT TORNER1, Fi
WOLFGANG TOMEK1
Comparative Aspects of Follicular Development, Follicular and
Oocyte Maturation and Ovulation in Cattle and Pigs
Summary
The paper describes comperatively aspects of oogenesis and folliculogenesis, oocyte and follicular growth in
cattle and pigs. Moreover, gonadotropin dependent regulatory processes of the wave like pattern of follicular
growth are discussed in both species as well as functional and morphological changes during oocyte maturation.
Finally, time dependent processes after oecurrence of spontaneous or GnRH induced LH surge are discussed.
Key Words: cattle, pigs, oogenesis, folliculogenesis, oocyte growth, follicular growth, gonadotropins, oocyte
maturation, Ovulation
Zusammenfassung
Titel der Arbeit: Vergleichende Aspekte der Follikelentwicklung, Follikel- und Oozytenreifung sowie
Ovulation bei Rind und Schwein
Der Artikel beschreibt vergleichend Aspekte der Oo- und Follikulogenese sowie des Oozyten- und
FoUikelwachstums ftlr die Spezies Rind und Schwein. Darüber hinaus werden sowohl regulatorische Prozesse
des gonadotropinabhängigen, wellenförmigen FoUikelwachstums als auch funktionelle und morphologische
Veränderungen während der Oozytenreifung ftir beide Spezies besprochen. Abschließend werden zeitabhängige
Ereignisse nach einem endogenen oder GnRH induzierten LH-Gipfels dargelegt.
Schlüsselwörter: Rind, Schwein, Oogenese, Follikulogenese, Oozytenwachstum, Follikelwachstum, GnRH,
Gonadotropine, Oozytenreifung, Ovulation
Oogenesis and Folliculogenesis
The formation of primordial germ cells (PGCs) indicates the commencement of
oogenesis. During early fetal life the primordial germ cells which arise in the yolk sac
endoderm at the caudal aspect ofthe embryo (McGEE et al., 1998) migrate via mesoand entoderm to the presumptive gonads. The migration of the PGCs toward the
presumptive ovaries based initially on passive transport and later on amoeboid-like
movement
in response to chemotactic substances like transforming growth
factor ß (PICTON and GOSDEN, 1998). In cattle the first potential primordial germ
cells were identified in an 18-day-old embryo. In 23- to 25-day-old embryos putative
primordial germ cells (alkaline Phosphatase- and lectin-positive) were situated
predominantly in the axial body region at the level or" the mesonephros. When the
gonadal ridge develops in this region (about day 27) it contains a certain number of
primordial germ cells present from the very beginning (WROBEL and SÜSS, 1998). It
is well known that PGCs play an indispensable role in the induetion of gonadal
development (VAN VOORHIS, 1998). In the pig PGCs display pseudopods and
10
appeared to migrate from the dorsal mesentery of the hindgut (day 18) to the
primordium ofthe gonad (day 23), and enter the genital ridge by day 26.
The number of 3-Fucosyl-N-acetyl-lactosamin positive cells associated with the dorsal
mesentery and genital ridge markedly increased from the 18-day to the 26-day pig
embryo (TAKAGI et al., 1997). During migration ofthe PGCs to the female gonads
cells undergo a species-speciüc number ofmitosis.
PGCs loose their motility after arriving at the developing ovaries. They are called now
oogonia and they show a high frequency of mitotic division. In cattle BECKERS et al.
(1996) found highest mitotic activity of oogonia from day 45 to 150 of fetal
development (approximately 2 million oogonia). Investigation has shown that meiosis
Starts in oogonia from day 80. The highest number of germ cells (oogonia plus
oocytes) was present around day 110 of fetal development. ERICKSON (1966) found
that at this time more than 2.7 million germ cells were present in both ovaries. In fetal
pigs the highest number of germ cells (approximately 1.1 million) was observed on
day fifty (BLACK and ERICKSON, 1968). Similar data (0.9 to 1.4 million germ cells
in dependence on breed) were reported by WISE et al. (2001). Until birth the number
of germ cells decreased by more than 50 % (BLACK and ERICKSON, 1968).
Apoptosis is predominantly involved in this decrease as it was shown in studies on
human oogonia and oocytes (De POL et al., 1998; DRIANCOURT et al., 1998).
Oogonial mitotic proliferation is restricted to prenatal development and shortly after
birth in cattle and pigs (ERICKSON, 1966; BLACK and ERICKSON, 1968). On day
270 of fetal development in cattle 98 % of all germ cells were oocytes and only 2 %
oogonia (ERICKSON, 1966). The process of mitotic division of oogonia leads to the
finite number of oocytes for the reproduetive life. When oogonia stop mitotic divisions
they become arrested in prophase of the first meiotic division. They are called now
primary oocytes.
Coincident with the initiation of meiosis oocytes become enclosed in a single layer of
pregranulosa cells, surrounded by a thin basement membrane. These very early stages
of follicular development are called primordial follicles. Primordial follicles represent
the nongrowing pool of the whole follicular population. The size of the störe of
primordial follicles used for folliculogenesis is the consequence of three processes:
(1) oogonia multiplication, (2) time of meiosis initiation and (3) extent of loss of germ
cells. Apoptosis is causing this loss, but its mechanisms are poorly documented.
Apoptosis also occurs within primordial follicles in adult ovaries and involves oocyte
death (DRIANCOURT et al., 1998; MATSUI, 1998). More than 99 % of ovarian
follicles endowed at early life are destined to undergo apoptosis and the exhaustion of
these follicles serves as a clock for female reproduetive senescence. Once a cohort of
follicles is recruited to grow, it is destined to undergo apoptosis unless rescued by
survival factors (HSUEH et al., 1996). At puberty the pool of primordial follicles will
be around 210.000 and 420.000 in cattle and pigs respectively (GOSDEN and
TELFER, 1987; TELFER, 1996).
Oocyte and follicular growth
Before oocytes reach their competence to resume nuclear maturation for further
fertilization and cleavage they have to grow. During this growth phase several
cytological changes occur. Oocytes acquire a complex cytoplasmic Organisation
dependent on both, new gene products and organelles, and on the modification and
11
redistribution of existing ones. In growing oocytes from cattle and pigs high levels of
RNA synthesis have been observed. The synthesis and uptake of macromolecules into
the oocyte serve two purposes. The first is for growth, development and maturation of
the oocyte itself. The second one is for the storage of RNA and proteins for the
postfertilization development (PICTON and GOSDEN, 1998). During growth phase
oocytes increase their diameter from 20 um to 130 um in cattle and from
approximately 40 pm to 120 pm in pigs. Oocyte and follicular growth are two
processes of one functional system. Granulosa cells are coupled by gap junctions with
the oocyte and influence actively oocyte growth by providing the oocyte with a variety
of biologically active molecules. On the other hand the oocyte actively controls
proliferation, morphogenesis and differentiation of the granulosa cells (PICTON and
GOSDEN, 1998).
Entry of primordial follicles into the growth phase occurs throughout the reproduetive
life. The transition of follicles from the nongrowing into the growing pool is
characterized by conversion of the flattened pregranulosa cells into a single layer of
cuboidal granulosa cells. The follicle is called now a primary follicle. Up to now the
mechanisms responsible for the initiation of follicular growth are poorly understood
although some candidate molecules (gonadotropins, growth factors, c-kit) have been
discussed (WEBB et al., 1999). The number of follicles commencing growth in a
given time is predictable because it is a function of the size of the primordial störe,
which declines exponentially with time (WEBB et al., 1999). In the growing follicle
the zona pellucida is formed around the oocyte. Whether the zona pellucida is a
product of the oocyte, the surrounding granulosa cells or both remains not fully
understood (VAN VOORHIS, 1998) although the mRNA for zona antigens were
found in oocytes and granulosa cells of porcine follicles (KÖLLE et al., 1996) and
bovine follicles (KÖLLE et al., 1998).
Proliferation of granulosa cells accounts for the further development of the primary
follicle. Oocytes of these follicles become surrounded by several layers of granulosa
cells. The follicles of this developmental stage are called secondary follicles.
Secondary follicles are served now by one or two arterioles with capillaries just
outside the basement membrane. This blood supply allows the follicle to be exposed to
factors circulating in the blood. As secondary follicles enlarge, stromal cells near the
basement membrane differentiate and form the theca interna and the theca externa.
Laminin, a major component of the basal lamina, is known to be important in the
differentiation of epithelial cells. The outer granulosa cell layer of ovarian follicles is
attached to a basal lamina surrounding the follicle, and it has been demonstrated that
proteins of the basal lamina can alter the steroidogenic capacity and cytoskeletal
composition of mature granulosa cells (LEE et al., 1996). In the process of theca cell
differentiation. cells become epitheloid and acqutre or^anelles characteristic fot Steroid
secreftng ceils.
In cattle follicles need approximately 180 days for the growth from 0.1 mm to the
ovulatory stage (CAMPBELL et al., 1995). Antrum formation was observed at 0.3 mm
(PÖHLAND, pers. commun.). In pigs almost all follicles > 0.4 mm had an antrum.
Based on estimated growth rates, an activated primordial follicle requires 84 days to
reach the anhal stage aud 14 days in addition to reach a diameter of approximately
3 mm (MORBECK et al., 1992). Table 1 summarizes data about follicular and oocyte
populations in cattle and pigs.
12
Table 1
Follicular and oocyte populations in cattle and pigs (data adapted from LUSSIER et al., 1994 and MORBECK et
Stage of follicular
development
Parameter
Cow
Pig
Primordial
to
early preantral
Diameter of follicles (um)
Diameter of oocytes (pm)
Duration of growth (days)
4 0 - 100
20-45
35-100
44
25
Preantral
stages
Diameter of follicles (pm)
Diameter of oocytes (pm)
Duration of growth (days)
100-150
45-60
10
150-300
68-90
26
Preantral
to
early antral
Diameter of follicles (pm)
Diameter of oocytes (um)
Duration of growth (days)
150-250
6 0 - 100
15
300-400
100
10
Antral
Diameter of follicles (mm)
Diameter of oocytes (pm)
Duration of growth (days)
0.2-3.7
110- 126
19
0.4-1.5
105
11
Diameter of follicles (mm)
Diameter of oocytes (pm)
Duration of growth (days)
3.7 - > 8.5
135
8
1.5-3.5
115-120
3
stages
Antral
preovulatory
In both species the growth of obligatory gonadotropin-dependent follicles occurs in a
wave like pattern (PIERSON and GINTHER, 1988; SA VIO et al., 1988; SIROIS and
FORTUNE, 1988; DRIANCOURT et al., 1991; FORTUNE, 1994; BURKE et al„
2000). In cattle two (GINTHER et al., 1989; KNOPF et al., 1989;
RAJAMAHENDRAN and TAYLOR, 1991; AHMAD et al, 1997, BURKE et al.,
2000; BELLMANN 2001) three (SAVIO et al., 1988; SIROIS and FORTUNE, 1988;
AHMAD et al., 1997, BURKE et al., 2000; BELLMANN 2001) or four waves
(RHODES et al., 1995) have been observed during oestrous cycle. Cycles with three
waves were on average 1.1 day longer and corpora lutea regressed later than in
animals with two waves. Moreover, interval from detection of dominant follicle to
Ovulation and duration of dominance were shorter in animals with three waves
(AHMAD et al., 1997). Normally three to six follicles with a diameter of 4 to 5 mm
occur after recruitment of follicles into in a follicular wave (SAVIO et al., 1988;
SIROIS and FORTUNE, 1988; SUNDERLAND et al., 1994). However, the number of
recruited follicles seems to be higher (BELLMANN, 2001).
In contrast pigs express only one wave of follicular activity during oestrous cycle
(DRIANCOURT, 1991; RATKY and BRÜSSOW, 1998). Results of LOBCHENKO
(1990) and BRÜSSOW et al. (1994) indicate that also in the pig the number of
recruited follicles is higher than the number of selected follicles. QUESNEL et al.
(1998) report that about 50 follicles are simultaneously recruited. Among the cohort of
recruited follicles 15 to 25 follicles are selected to form the ovulatory population.
Gonadotropin dependent regulation of wave like follicular growth
Both in cattle and in pigs, GnRH, FSH and LH are key hormones for the endocrine
regulation of follicular wave oecurrence. In principal, GnRH is released from the
hypothalamus in a pulsatile pattern and binds to the GnRH reeeptor on the
13
gonadotroph cell surface of the anterior pituitary gland to stimulate the synthesis and
release of FSH and LH. The gonadotropins FSH and LH are heterodimeric molecules
comprising a common ct-subunit and a hormone-specific ß-subunit. Transcriptional
regulation of gonadotropin subunits involves basal gene expression and GnRH
upregulated gene expression as well. Moreover, synthesis and secretion of FSH and
LH are both positively and negatively regulated by Steroids and gonadal peptides
(BROWN and McNEILLY, 1999). The gonadotropins FSH and LH bind to their
receptors on follicular cells and induce processes of proliferation and differentiation.
In cattle waves of follicular activity are characterized by processes of recruitment,
selection and dominance (DRIANCOURT, 1991; MIHM et al., 1996). At the
beginning of each wave, from a cohort of growing follicles a single follicle continues
in growth and becomes dominant. The other follicles of the cohort finish their growth
and regress. FSH concentrations increase before follicular wave emergence. During
the parallel growth phase of the recruited follicles FSH concentrations decrease and
reach a nadir at the end of this phase and the beginning of deviation (GINTHER et al
1997, 1999; BERGFELT et al., 2000; GINTHER et al., 1999, 2000). The decline of
FSH concentrations to basal levels is attributed to estradiol secretion of the future
dominant follicle (GINTHER, 2000). Results of XU et al. (1995), MIHM et al (1996
2000), BAO et al. (1997a,b), SCHAMS et al. (1999), and BEG et al. (2001) indicate
that the differentiated expression of the mRNA for steroidogenic enzymes (P450 side
chain cleavage enzyme, 3ß-hydroxysteroid dehydrogenase, P450 aromatase) in theca
and granulosa cells, the expression ofthe mRNA for the LH reeeptor in granulosa cells
of follicles > 8 mm in diameter and the bioavailability of IGF I in association with
IGFBPs play an important role in reaching dominance of one follicle.
As in the rat (RICHARDS et al., 1980), in pigs the increased FSH concentration after
Ovulation is thought to recruit a cohort of follicles to grow. Additional FSH infüsion
during the immediate postovulatory period increased the number of recruited follicles
(RATKY and BRÜSSOW, 1998). However, data from FOXCROFT and Van de VIEL
(1982), SHAW and FOXCROFT (1985) and GUTHRIE and BOLT (1990)
demonstrate that an increase in FSH concentration may not be a prerequisite for
follicular recruitment. The recruitment occurs between day 14 and 16 ofthe oestrous
cycle, eoineident with luteolysis (FOXCROFT and HUNTER, 1985).
Changing the ratio of progesterone and gonadotropin induces further emergence of
ovulatory follicles (GUTHRIE and BOLT, 1990). Follicles undergo the process of
selection until day 17/19 ofthe oestrous cycle (CLARK et al., 1982). During this
follicular phase FSH concentrations decline to basal levels and remain low until the
preovulatory LH surge (GUTHRIE and BOLT, 1990). The growth rate ofthe selected
follicles was approximately 1mm per day (RATKY et al„ 1995). FSH application from
day 4 to day 11 of oestrous cycle (Tab. 2) can prevent the process of selection and
significantly increase the number of follicles on the surface of the ovaries (RATKY
and BRÜSSOW, 1998).
Dominance of follicles seems to be less pronounced in pigs as compared to
monotocous species. However, studies conducted during the follicular phase of the
oestrous cycle by CLARK et al. (1982) and FOXCROFT and HUNTER (1985)
suggested that large follicles create a functional block in the development of nondominant follicles. In addition, GUTHRIE et al. (1993) found abnormal distributions
for estradiol- 17ß and some larger follicles appeared to be less mature. The lack of
14
atresia in largest follicles coupled with extensive atresia in medium-sized follicles
during the follicular phase suggests that the concept of dominance described in
monoovular mammals may be also valid in swine (QUESNEL et al., 1998). Data in
Table 3 summarize knowledge about regulatory aspects of follicular waves in cattle
and pigs.
Table 2
Mean total number of follicles and follicles > 5 mm in diameter in dependence on FSH treatment between day 4
and II of oestrous cycle (RATKY and BRÜSSOW, 1998) (Durchschnittliche Gesamtfollikelzahl und Anzahl
Follikel > 5 mm Durchmesser in Abhängigkeit von einer FSH-Behandlung zwischen den Zyklustagen 4 und 11)
Number of follicles > 5 mm
Number of follicles
Group
Day of
oestrous
(n)
(n)
mean ± SD
cycle
mean ± SD
Control
d4
14.6 + 5.1
FSH
15.0 ±7.6
17.2 ±4.1
d 18
Control
23.4 + 5.0
FSH
25.4 ±16.2
42.4 ±11.8
Table 3
Regulatory and numeric aspects of follicular waves in cattle and pigs (Regulatorische und numerische Aspekte
von Follikelwellen bei Rindern und Schweinen)
Cattle
Pig
Regulated processes
FSH ft?
Recruitment
FSHfl
to50
24 to 28 follicles > 1 mm
Number of follicles (n)
FSH 11/ Progesterone U/ Estradiol ft
Selection
FSH U/ Estradiol ft/ Inhibin ft
20 to 50 follicles 2 to 4 mm
4 to 6 follicles > 4 to 5 mm
Number of follicles (n)
?
IGF / IGF-BP/FSH-R/LH-R
Dominance
15 follicles >6 to 9 mm
1 follicle > 8 to 9 mm
Number of follicles (n)
Our experiments with a long acting GnRH agonist in a depot formulation gave
evidence, that LH and its pulsatile secretion is necessary for maturation of follicles in
cattle and pigs. In heifers, a GnRH agonist given in depot formulation and acting over
28 day long period caused an immediate LH release (data not shown), which was
followed by a significant decrease of LH pulsatility (Fig. 1).
Fig. 1; Characteristics of LH secretion in a control (A) and a GnRH agonist depot treated heifer (B) 15 days
after application of solvent or GnRH agonist. Pulses of LH secretion are signed with asterisk (BELLMANN,
2001) (Charakteristika der LH-Sekretion bei einem mit A bezeichnetem Kontrolltier und einem mit B
bezeichnetem Versuchsrind 15 Tage nach der Applikation eines Lösungsmittels bzw. eines GnRH-Agonisten in
Depotformulierung. LH-Pulse sind durch Sternchen gekennzeichnet)
15
Table 4
Mean number of LH pulses in 6 hours on day 1 to 5, 11 and 15 in control heifers or animals treated with a GnRH
agonist in depot formulation (BELLMANN, 2001) (Durchschnittliche LH-Pulszahl in 6 Stunden an den Tagen 1
bis 5, 11 bzw. 15 bei Kontrolltieren bzw. Versuchstieren, die GnRH-Agonist in Depotformulierung erhielten)
day
Number of pulses
P-value
(LS-Means ± SEM)
Control
GnRH agonist depot
Animals (n)
Pulses
Animals (n)
Pulses
1
13
14
0.02
2.210.5
0.7 ±0.4
2
11
3.0 + 0.5
n = 12
0.01
0.9 ±0.5
3
9
10
0.001
4.6 ±0.5
1.0 ±0.5
4
6
8
0.04
3.8 ±0.8
1.4 ±0.7
5
7
10
4.1 +0.6
0.001
0.9 ±0.5
11
7
10
0.01
3.3 ± 0.4
1.4 ±0.4
15
7
10
3.7 ±0.5
0.001
0.8 1 0.4
Heifers treated with the GnRH agonist had significantly less LH pulses from d 1 to d 5
and on day 11 and 15 after treatment (Tab. 4). As a result of reduced LH pulsatility the
diameter ofthe largest follicle ofthe second follicular wave was significantly lower in
treated (7.2 ± 1.0 mm) than in control animals (14.0 ± 1.6 mm). Similar results were
obtained in our experiments with pigs (BRÜSSOW et al. 1994, 1996). After treatment
with GnRH in depot formulation follicles reached only a diameter < 4 mm. This is an
agreement with results from DRIANCOURT et al. (1995) obtained after application of
a GnRH antagonist.
The pulsatile LH secretion with low frequency but high amplitude Supports the
maintenance of corpora lutea. This is evident in cattle and pigs. However, the luteal
phase is shorter in pigs compared to cattle. Progesterone concentrations start to
decrease after day 13 in pigs but after day 15 in cattle. LH secretion with high
frequency/low amplitude pattern initiates the maturation of follicles and their estradiol
synthesis (QUESNEL et al., 1998).
This is the prerequisite for the priming of the pituitary and the positive estradiol feed
back. In both species, increasing estradiol concentrations present for a longer time
induce the preovulatory LH surge.
Oocyte meiotic maturation
The maturation of oocytes depends on communication between follicle cells and
oocytes, and processes in the follicle cells are under control of gonadotropins.
BALTAR et al. (2000) found an inverse relationship between follicular diameter and
binding capacity for LH/hCG. Oocytes are arrested in prophase I at the so-called
germinal vesicle stage (GV). They overcome this arrest either after hormonal induction
or spontaneously after removal from antral follicles. Both in cattle and pigs cAMP
plays an important role in maintaining meiotic arrest in oocytes (RICE and
McGAUGHEY, 1981; AKTAS et al., 1995a,b). The control of meiotic maturation
involves a complex interplay between somatic and germ cells. In both species meiotic
maturation is triggered by the endogenous preovulatory gonadotropin surge.
Resumption of meiosis appears to be associated with a decrease in cAMP
concentrations within the oocyte caused by elevated LH concentration. However, the
reduction in cAMP concentrations is not the only cause for final maturation of the
oocyte. There is evidence that the production of active maturation-promoting factor
(MPF) in the ooplasm is involved in the processes of nuclear maturation. MPF consist
16
of two components: a 34 kDa protein and cyclin B. Although oocytes from cattle and
pigs do not have the same kinetic of meiotic maturation, MPF activity is low in GVstage, increases during GVBD, and become high at both metaphase I and metaphase II.
The process of meiotic maturation is characterised by nuclear membrane breakdown,
which occurs in cattle oocytes 9 to 12 h (HYTTEL et al., 1989) and in pig oocytes 24 h
(TORNER et al., 1998) after LH peak. Moreover, during maturation the number of
mitochondria decreases and these organelles are located more perinuclear. Cortical
granules, originating in the Golgi move to the periphery of the egg complex
(STRÖMSTEDT and BYSKOV, 1998).
IVM 0 6 8
ERK 1_».
ERK 2—*"
B
18 24
28 h
wnyuyk -itÄ^,
Morphology CV
Histon H1—*•
10 12 14
*
GVBD
* M II
** iiinmmnnii
MBP-»-
IVM 0
6
8
10 12 14
18 24
28 h
120
j j 100
o
< 80
Ai 60
40
^A^AAi.
7y' '
IVM 0 6
0
10
12 14
18 24 28 h
Fig. 2: Activity of cdc2- and MAP Kinase during maturation in vitro of bovine oocytes. A: Analysis of the
phosphorylation of MAP kinase subclasses ERKl and ERK2 during IVM by band shift assay. B: Analysis of
activities of cdc2 kinase and MAP kinase during IVM by in vitro kinase assay. C: Evaluation of cdc2 kinase
(solid line) and MAP kinase (broken line) activities from a gel depicted in Fig 2B (adapted from TORNER et al.,
2001) (Aktivität von cdc2- und MAP-Kinase während der meiotischen Reifung boviner Oozyten in vitro. A:
Analyse der Phosphorylierung von MAP-Kinasesubklassen ERKl und ERK2. B: Analyse der Aktivität von
cdc2-Kinase und MAP-Kinase. C: Quantifizierung der cdc2- und MAP-Kinaseaktivität)
Together with changes in nuclear configuration (e.g. chromatin condensation, GVBD,
spindle formation), meiotic maturation of pig and cattle oocytes is accompanied by
extensive phosphorylation/dephosphorylation of cytoplasmic proteins (LEIBFRIEDRUTLEDGE et al., 1989; KASTROP et al., 1990). At least two major kinases or
kinase cascades are involved in these processes, namely cdc2 kinase (catalytic part of
the maturation promoting factor MPF, with the regulatory subunit cyclin B) and
mitogen-activated protein kinase (MAPK). Analysis of bovine and porcine oocytes
during IVM show that both kinases become activated during the resumption of meiosis
(KUBELKA et al., 1995; TORNER et al., 2001; Fig. 2), however, the physiological
targets of these kinases are known only partially. Apart from other Substrates, MAP
17
kinase can activate transcription factors (JOHNSON and VAILLANCOURT, 1994)
and ribosomal subunit S6 kinase (KALAB et al., 1996), and therefore it seems to be
involved in the regulation of gene expression.
Cdc2 kinase is believed to be responsible for GVBD and chromosome condensation
during the first meiosis, and it was also shown to act as Histone Hl and lamin kinase,
at least in vitro (PETER et al., 1990; VERDE et al, 1990). The essential role of
protein kinases in resumption of meiosis is also documented by the fact that the
inhibition of cdc2 kinase in GV-stage oocytes prevents the onset of meiotic maturation
(KRISCHEK and MEINECKE, 2000; KUBELKA et al., 2000).
Meiotic maturation is also characterised by a strong increase in protein synthesis
around the time of GVBD. This event is tightly correlated with activation of MAP
kinase and with phosphorylation of the translational initiation factor eIF4E (TOMEK
et al, 2001). Since no de novo transcription can be observed at this time point, the
proteins synthesised must originate from a dormant and subsequently activated mRNA
pool. Thus, the regulation of gene expression at this stage of development is regulated
at the level of translation. The cytoplasmic polyadenylation of mRNA is one
mechanism to activate the transcripts. There is some evidence that this occurs in
oocytes and cumulus cells during preovulatory maturation of porcine oocytes
(TORNER et al, 1998b) and also during IVM of bovine oocytes (unpublished results),
but the mRNAs involved in this activation process remain to be analysed.
Table 5
COC morphology in relation to a hCG application after oestrous Synchronisation in pigs (TORNER et al., 1998a)
(COK-Morphologie in Beziehung zu einer hCG-Applikation nach Brunstsynchronisation beim Schwein) '
Time in
Number of
Portion of COC in different classes
relation to
COC
(%\
hCG
(n)
(h)
10
AI
34
J5
47
60
Compact
Slightly
expanded
Corona radiata
Expanded
Denuded
4Z2
25.5
0
30.6'
31.9;
5.0°
18.8
6.4
3.3
~~Ö
25.5°
86.7Jc
ÜT
10 6
S~Ö~
983*'
<T~
59
0
0
L7
Numbers with different superscripts are significantly different in columns p < 0.05
Table 6
Meiotic configuration in oocytes in relation to hCG application after oestrous Synchronisation in pigs (TORNER
et al., 1998a) (Meiosestadien in Oozyten von Schweinen in Beziehung zu einer hCG-Applikation nach
Brunstsynchronisation)
Time in relation to Number of COC
Portion of oocytes
hCG
(%)
(h)
(n)
immature (GV)
resumption of
mature
meiosis (GVBD-A I)
(T I/M II)
-2
81
44.4'
44.4
11.2"
10
42
66.6"
26.2
7.1 a
22
58
17.2C
41.1
41.4"
34
57
0
29.8
70.2C
Although there are some differences between bovine and porcine oocytes in the timing
of specific events occurring during meiotic maturation, and also in the sensitivity to
some kinase inhibitors, the mechanisms described are generally valid in both species.
In cattle and pigs intrafollicular maturation of cumulus-oocyte-complexes (COCs) are
characterized by time dependent changes of COC morphology and oocyte chromatin
configuration. (MEINECKE et al, 1984 ; XIE et al, 1990 ; TORNER et al, 1998a).
TORNER et al. (1998a) observed a close Synchronisation between follicular
maturation after hCG application and the development of intrafollicular COC
morphology and chromatin configuration (Tab. 5 and 6).
Ovulation
In cattle and pigs Ovulation is induced by an increase in LH secretion. The LH surge
triggers a biochemical cascade that leads to the rupture of the preovulatory follicle(s),
the expulsion of the oocyte(s) and the formation of the Corpora lutea. Local acting
factors like Steroids, Prostaglandins and peptides like endothelin 1 are involved in the
process of Ovulation. An increase of Prostaglandins (PGE2 and PGF2a) in follicular
fluid of preovulatory follicles in the cow has been demonstrated by ALGIRE et al.
(1992). More recently ACOSTA et al. (2000) demonstrated an increased local release
of PGF2a and angiotensin II around the time of Ovulation.
In cattle PETERS and BENBOULAID (1997) investigated the oecurrence of Ovulation
after PGF2a/GnRH application in some animals by means of ultrasound. Ovulation
occurred between 24 to 48 h after GnRH injection. More recently, we examined the
time of Ovulation after PGF2a/GnRH application in heifers. Ultrasonographic
examinations of ovaries were done every 6 hours during the periovulatory period. The
mean interval from GnRH to Ovulation was 25 to 33 hours. Our data and results from
PETERS and BENBOULAID (1997) indicate, that ovulatory follicles have a diameter
between 15 and 20 mm. The results of KOT and GINTHER (1999) show, that the
mean time from beginning to completion of evacuation of ovulatory follicles was
4.3 ± 3.3 min (min. 6 s; max. 14.5 min.). The number of ovulations is constant in
cattle.
Duration of Ovulation period in pigs assessed with transrectal ultrasound diagnosis
varied between 1.8 and 4.6 h. Ovulations took place on average 35 ± 8 h after onset of
oestrus (SOEDE et al, 1997). Following GnRH induced Stimulation the
commencement and completion of Ovulation was 35.1 ± 4.1 and 38.3 + 4.3 h,
respectively, and the mean duration of Ovulation was 3.3 ± 1.9 h (BRÜSSOW et al,
1993). SOEDE et al. (1997) observed a mean diameter ofthe ovulatory follicles of 7.1
+ 0.9 mm. This is in agreement with earlier findings of RATKY et al. ( 1995). They
estimated by means of endoscopy a size of preovulatory follicles between 7 to 8 mm.
Interestingly, the number of ovulations was significantly increasing in gilts from first
to third oestrous cycle (RATKY et al, 1995).
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Authors address
PD Dr. WILHELM KANITZ
Research Institute for the Biology of Farm Animals
Department of Reproduetive Biology
Wilhelm-Stahl-Allee 2
D-18196 Dummerstorf
Germany
E-Mail: [email protected]
Arch. Tierz., Dummerstorf 44 (2001) Special Issue, 24-29
Department of Veterinary Preclinical Sludies, University of Glasgow Veterinary School, Glasgow, UK
MONIKA MIHM
Intrafollicular health
development in cattle
markers
during
antral
follicle
wave
(Intrazelluläre Gesundheitsmarker während der Entwicklung von Antralfollikeln beim
Rind)
Summary
Ovarian follicle growth in cattle eulminating in the Ovulation ofa single follicle at the end ofthe oestrous cycle
is strictly regulated and has proven to be very difficult to manipulate. This review will describe in detail antral
follicle waves and in particular the first follicle wave of the cycle, indicating the specific gonadotrophin
dependencies of cohort and dominant follicles, and relating follicle health to steroidogenesis. As intrafollicular
growth factors such as proteins belonging to the inhibin family and the insulin-like growth factor (IGF) system
have important roles in modifying gonadotrophin response of growing antral follicles, characteristics of healthy
or atretic first wave follicles in relation to inhibins, IGFs and IGF-binding proteins will be summarised. Finally,
a model for dominant follicle selection will be proposed including very recent data on proteases possibly
affecting IGF bioavailability.
Key Words: dominant follicle, FSH, inhibin, insulin-like growth factor binding proteins
Introduction
In cattle, follicles from approximately 300 um in diameter form an antrum and
subsequent growth to a size of 3-5 mm is calculated to take more than 30 days (1).
However, growth rates of 0.5 to more than 2 mm per day are seen in larger antral
follicles monitored individually with the aid of transrectal ovarian ultrasound scanning
during their final stages of development. This review will present recent data which
describe the final outcome of antral follicle growth and try to elueidate the regulatory
mechanisms involved in limiting the number of the species specific ovulatory quota to
one in cattle.
Gonadotrophins and growth of an antral follicle wave in cattle
At the beginning of the oestrous cycle and following Ovulation of the last preovulatory
follicle, a transient rise in Follicle Stimulating Hormone (FSH) causes emergence of
up to 24 small antral follicles 3-5 mm in diameter (the cohort) within 24 hours of the
maximum ofthe transient FSH rise (2-4). During declining FSH and over 3 days more
and more follicles from the original cohort become static and regress until only one
single follicle is selected to continue to grow and enhance its steroidogenesis,
specifically its oestradiol secretion when FSH reaches nadir concentrations (3-5). This
is the dominant follicle (DF) and it appears that the DF aids its own selection by
suppressing FSH concentrations below the requirement threshold of the other cohort
members (6,7). The DF attains a much larger size than all other follicles (up to 15-20
mm), is responsible for ovarian oestradiol secretion during its growth phase and
maintains low FSH concentrations to prevent any other cohort growth (6-8). All the
other members of its cohort including the second largest follicle undergo rapid atresia
via apoptosis during the FSH decline due to their acute FSH-dependency (subordinate