Leukemia inhibitory factor stimulates the transition of primordial to primary follicle and supports the goat primordial follicle viability in vitro

Article (PDF Available)inZygote 20(1):73-8 · March 2011with21 Reads
DOI: 10.1017/S0967199411000074 · Source: PubMed
Abstract
The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control - FC) and the remaining fragments were cultured in supplemented minimum essential medium (MEM) without (cultured control - CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for 1 or 7 days, at 39°C in air with 5% CO2. Fresh control, CC and treated ovarian fragments were processed for histological and fluorescence analysis. The percentage of histological normal preantral follicles cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than in the CC (32.6%; p < 0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC (p < 0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle viability of goat ovarian tissues cultured for 7 days.

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Zygote 20 (February), pp. 73–78. C!Cambridge University Press 2011
doi:10.1017/S0967199411000074 First Published Online 18 March 2011
Leukemia inhibitory factor stimulates the transition of primordial
to primary follicle and supports the goat primordial follicle
viability in vitro
Janduí Escarião da Nóbrega Jr2,PauloBayardDiasGonçalves
2, Roberta Nogueira Chaves3,Deborahde
Melo Magalhães3, Rafael Rossetto3,IsabelBezerraLima-Verde
3,GabrielRibasPereira
2, Cláudio Cabral
Campello3,JoséRicardoFigueiredo
3and João Francisco Coelho de Oliveira1
Laboratory of Biotechnology and Animal Reproduction – BioRep, Federal University of Santa Maria, Santa Maria; and
Laboratory of Oocytes and Preantral Follicle Manipulation – MOIFOPA, State University of Ceará, Fortaleza, Brazil
Date submitted: 15.09.10. Date accepted: 29.11.10
Summary
The aim of this study was to evaluate the effect of leukemia inhibitory factor (LIF) on the activation and
survival of preantral follicles cultured in vitro enclosed in ovarian fragments (in situ). Goat ovarian cortex
was divided into fragments to be used in this study. One fragment was immediately fixed (fresh control
–FC)andtheremainingfragmentswereculturedinsupplementedminimumessentialmedium(MEM)
without (cultured control – CC) or with different concentrations of LIF (1, 10, 50, 100 or 200 ng/ml) for
1or7days,at39
Cinairwith5%CO
2.Freshcontrol,CCandtreatedovarianfragmentswereprocessed
for histological and fluorescence analysis. The percentage of histological normal preantral follicles
cultured for 7 days with 1 ng/ml (49.3%), 10 ng/ml (58.6%) and 50 ng/ml (58%) of LIF was higher than
in the CC (32.6%; p<0.05). After 7 days of culture, the percentage of primordial follicles in situ cultured
with LIF decreased and primary follicles increased in all LIF concentrations compared with FC and CC
(p<0.05). In conclusion, LIF induced primordial follicle activation and supported preantral follicle
viability of goat ovarian tissues cultured for 7 days.
Keywords: Goat, Leukemia inhibitory factor, Ovary, Preantral follicles, Primordial follicle activation
Introduction
The mammalian ovary at birth consists of thousands of
primordial follicles, which are considered the resting
pool of follicles in the ovary. Throughout the female
reproductive life span, a small number of primordial
follicles are stimulated to grow, in a process referred
to as follicular activation; whereas the vast majority
(99.9%) becomes atretic toward ovulation (Skinner,
2005). Until recently, relatively little was known as
1All correspondence to: João Francisco Coelho de Oliveira.
Laboratory of Biotechnology and Animal Reproduction, Av.
Roraima #1000, CEP 97105–900, Santa Maria, RS, Brazil.
Tel: +55 55 3220 8752. Fax: +55 55 3220 8484. e-mail:
jf@biorep.ufsm.br
2Laboratory of Biotechnology and Animal Reproduction –
BioRep, Federal University of Santa Maria, CEP 97105–900,
Santa Maria, RS, Brazil.
3Laboratory of Oocytes and Preantral Follicle Manipulation
– MOIFOPA, State University of Ceará, CEP 60740–903,
Fortaleza, CE, Brazil.
to how these primordial follicles were activated and
stimulated to develop into a more advanced stage.
Moreover, it is known that in mammals, including
primates, early follicular development goes through a
complex process, in which the oocytes grow and their
surrounding somatic cells proliferate and differentiate
through preantral follicle stages (Gougeon, 1996;
Fortune, 2003). However, few long-term in vitro culture
studies have achieved follicular activation in sheep
(Muruvi et al.,2005),human(Sadeuet al.,2006)and
goat (Rossetto et al.,2009).
Leukemia inhibitory factor (LIF) is a glycoprotein
with pleiotropic activity that exerts an important
role on follicular growth, oocyte maturation and
awidevarietyofcelltypesincludingsomatic
and follicular cells (Demeestere et al.,2005).Shen
&Leder(1992)suggestedaspecicroleforLIF
on mouse preimplantation development. Among its
many activities, LIF can maintain embryonic stem
cell monolayers in a pluripotent undifferentiated state.
Recently, LIF was reported to inhibit myeloid leukemic
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74 Nóbrega Jr et al.
cells and can enhance survival, migration, proliferation
and meiosis resumption of primordial primitive cells
in rodents (van den Hurk & Zhao, 2005).
In rats, the level of LIF in the follicular fluid increases
as ovarian follicles develop. There is evidence that LIF
is responsible for oocyte development and maturation
and preantral follicle viability in vitro (Haidari et al.,
2008). Reports on the development of rat preantral
follicles cultured with LIF showed them to promote
the transition from primordial to primary follicles
(Nilsson et al.,2002;Haidariet al.,2006).Although
LIF was identified as being involved in mammalian
folliculogenesis, the role of LIF on preantral follicle
activation is an issue that remains far from understood.
Therefore, the aim of the present study was to
investigate the influence of different concentrations of
LIF on the activation of primordial follicles and further
follicular survival after in vitro culture of caprine
ovarian tissue.
Materials and methods
Unless mentioned otherwise, all chemicals used in the
present study were purchased from Sigma Chemical
Co.
Ovaries collection
Ovaries (n=10) were collected from five non-
pregnant mixed-breed adult goats (1–3-year-old). All
animals were in the follicular phase of the estrous
cycle with absence of corpora lutea. Immediately
after being slaughter, ovaries were dissected from the
surrounding connective tissue and washed once in
70% alcohol, followed by twice in minimum essential
medium (MEM) supplemented with 100 µg/ml
penicillin and 100 µg/ml streptomycin. Then, ovar-
ies were transported to the laboratory at 4C
within 1 h.
Experimental design
In the laboratory, ovaries were stripped of surrounding
tissues and the medulla and visible growing follicles
were manually removed. Ovarian tissue samples
from each ovarian pair were cut in to slices of
9mm
3(approximate size 3 mm ×3mm×1
mm thickness) using a 26-G needle and a scalpel
blade under sterile conditions. The tissue pieces were
then either directly fixed for histological analysis
(fresh control – FC) or placed in culture for 1 or 7
days. The tissues were transferred to 24-well culture
Petri dishes containing 1 ml of culture medium.
Culture was performed at 39Cinairwith5%
CO2in a humidified incubator and medium were
incubated for 1 h prior to use. The basic culture
medium (cultured control – CC) consisted of αMEM
supplemented with ITS (10 µg/ml insulin, 5.5 µg/ml
transferrin and 5 ng/ml selenium), 0.23 mM pyruvate,
2 mM glutamine, 2 mM hypoxanthine and 1.25
mg/ml bovine serum albumin. For the experimental
conditions, basic medium was supplemented or not
with LIF at different concentrations (1, 10, 50, 100 or
200 ng/ml). Each treatment was repeated five times
and the culture medium was replaced every other day
throughout the 7-day period.
Histological assessment of in vitro follicular growth
For the evaluation of follicular morphology (survival),
all the ovarian pieces were fixed in Carnoy’s
solution for 12 h and then dehydrated in increasing
concentrations of ethanol at 70, 80, 95 and 100%, before
culture (FC) and after 1 or 7 days of culture. After
paraplast embedding, caprine tissues fragments were
sliced into 5 µm sections, mounted on glass slides and
stained by periodic acid Schiff–haematoxylin. Follicle
stage and viability were assessed microscopically
on serial sections. Coded anonymized slides were
examined on a microscopy (Nikon, Japan) under ×400
magnification (Silva et al., 2002). Each follicle was
examined in every section and matched with the same
follicle on adjacent sections to avoid double counting,
thus ensuring that each follicle was only counted once,
regardless of its size.
The developmental stages of follicles have been
defined previously as primordial (one layer of
flattened granulosa cells around the oocyte) or
growing follicles (intermediate: one layer of flattened
to cuboidal granulosa cells; primary: one layer of
cuboidal granulosa cells, and secondary: two or more
layers of cuboidal granulosa cells around the oocyte;
Silva et al., 2004). Follicles were classified individually
as histological normal when an intact oocyte was
present and surrounded by a well organized of
one or more layers of granulosa cells without
the appearance of pyknotic nucleus. Degenerated
follicles were defined as those with a retracted
oocyte or a pyknotic nucleus, and/or surrounded by
disorganized granulosa cells, which were detached
from the basement membrane. Overall, 1950 follicles
were evaluated for each treatment [30 follicles ×
13 groups (1 or 7 days of culture) ×five replicates].
To evaluate follicular activation, the percentages of
healthy primordial follicles were calculated before FC
and after culture in each medium with or without LIF.
Viabil ity assessmen t of pre antral follic les by
fluorescence
Based on the results of histological analysis, the
viability of follicles cultured with LIF that provided the
LIF goat primordial follicle 75
Tab l e 1 Histological evaluation of in situ cultured fragments of goat ovarian cortex for 1 or 7 days using
different concentrations of leukemia inhibitory factor (LIF) (ng/ml)
Follicular stage
Primordial follicle Primary follicle
Groups D0 D1 D7 D0 D1 D7
FC 43.7 ±4.5 15.4 ±5.1 –
CC 60.0 ±2.1 42.5 ±6.1 10.9 ±2.7 22.8 ±9.4
LIF 1 43.0 ±6.4 13.6 ±1.2,–22.4±8.2 38.9 ±7.6,
LIF 10 43.9 ±14.3 15.1 ±4.1,–21.9±8.8 41.5 ±9.5,
LIF 50 33.6 ±10.312.6 ±4.7,–28.1±8.150.6 ±6.1,
LIF 100 49.0 ±11.2 15.2 ±2.7,–26.9±4.842.4 ±3.1,
LIF 200 42.2 ±13.7 9.8 ±5.7,–41.0±5.2,53.4 ±6.1,
Differs significantly from fresh control (FC).
Differs significantly from cultured control (CC) in each day of culture (p<0.05).
Data are shown as mean percentage ±standard error of means (S.E.M.) out of all preantral follicle stages
(primordial and primary follicles). Data were pooled from five replicates. (D) Day of culture.
best outcome (10 and 50 ng/ml) was further analyzed
using a more accurate method of assessment based
on fluorescent probes. Goat preantral follicles were
isolated using a mechanical method described by Lucci
et al. (1999). Ovarian fragments were cultured for
7dayswithCC,10or50ng/mlLIF.Briey,using
atissuechopper(MickleLaboratoryEngineering
Co.) adjusted to a sectioning interval of 75 µm,
samples were sliced into small fragments, placed in
MEM and suspended 40 times using a large Pasteur
pipette (diameter of about 1600 µm) and resuspended
subsequently 40 times with a small Pasteur pipette
(diameter of about 600 µm) to dissociate preantral
follicles from stroma. The obtained material was
passed through 100 µmnylonmeshfilters,resulting
in a suspension containing preantral follicles smaller
than 100 µmindiameter.Thisprocedurewascarried
out within 10 min at room temperature.
Thereafter, the viability of preantral follicles were
analyzed using a two-colour fluorescence cell assay
based on the simultaneous determination of viable
or degenerated cells by calcein-AM and ethidium
homodimer-1 (Molecular Probes, Invitrogen), respect-
ively. While the first probe detected intracellular
esterase activity of viable cells, the second probe
labelled nucleic acids of non-viable cells with plas-
matic membrane disruption. Fluorescence analysis
was performed by adding 4 µMcalcein-AMand2µM
ethidium homodimer-1 (LIVE/DEAD Viability kit: L,
Molecular Probes, Invitrogen) to the suspension of
isolated follicles, followed by incubation at 37Cfor15
min in a dark chamber. After being labelled, follicles
were centrifuged at 100 gfor 5 min, washed once
and resuspended in αMEM. Then, labelled follicles
were mounted on a glass microscope slide using 5 µl
of anti-fading medium (DABCO, Sigma) to prevent
photobleaching and finally examined using a DMLB
fluorescence microscope (Leica). The emitted fluores-
cence signals of calcein-AM, and ethidium homodimer
were collected at 488 and 568 µm, respectively. Oocytes
and granulosa cells were considered viable if the
cytoplasm was stained positively with calcein-AM
(green) and chromatin was not labelled with ethidium
homodimer (red); otherwise, they were classified as
degenerated (Lopes et al.,2009).
Statistical analysis
The mean percentages of primordial, viable (at all
stages) obtained after 1 or 7 days of culture were
subjected to analysis of variance (ANOVA) using
the GLM procedure of SAS/STAT software. Data
were tested for normal distribution using Shapiro–
Wilk test and normalized when necessary. Dunnett’s
test was applied to compare LIF-treated groups with
FC and CC groups. Duncan’s test was used to
compare differences among LIF concentrations and
days of culture. Data from fluorescence analysis
were subjected to chi-squared test. Differences were
considered to be significant when p<0.05 and data
were expressed as mean ±standard error of means
(SEM).
Results
Follicular activation and development
The percentages of primordial and primary follicles
during culture with different concentrations of LIF are
shown in Table 1. After 7 days of culture, the number
of primordial follicles decreased (LIF from 9.8% to
15.2% vs. control groups FC 43.7% and CC 42.5%) and
primary follicles increased (LIF from 38.9% to 53.4%
vs. control groups FC 15.4% and CC 22.8%) when LIF
76 Nóbrega Jr et al.
Figure 1 Viability assessment of caprine preantral follicles
using histological and fluorescent analyses. (A) Histological
section of preantral follicle cultured for 7 days showing
the stroma and follicle integrity. (B) Histological section of
preantral follicle cultured for 7 days showing cytoplasmic
vacuolization, disorganized stroma cells and pycnotic
nucleus. (C) An isolated viable preantral follicle labeled by
calcein-AM (green fluorescence). (D) Degenerated preantral
follicle showing chromatin labelled by ethidium homodimer
(red fluorescence). gc: granulosa cells; n: nucleus; o: ooplasm;
arrow: pycnotic nucleus. Bars =50 µm.
was present in the culture (p<0.05). In the presence
of LIF, formation and differentiation (from flattened to
cuboidal cells) of a new cellular layer of granulosa cells
were observed, giving origin to primary follicle. These
findings are evidence of preantral follicle activation
induced by LIF.
Follicular survival
The preantral follicle survival was histologically
evaluated after 1 or 7 days of culture in the presence
or absence of LIF. An example of viable preantral
follicle is depicted in Fig. 1A.Thedegeneratedfollicles
presented a retracted oocyte, pyknotic nucleus, and/or
cytoplasmic vacuolization (Fig. 1B). The viable prean-
tral follicles were also labelled by calcein-AM green
fluorescence (Fig. 1C)andthedegeneratedpreantral
follicles showed chromatin labelled by ethidium
homodimer red fluorescence (Fig. 1D).
The preantral follicle survival did not differ among
treatment and control groups after 24 h of culture.
However, the survival of preantral follicles was
improved when LIF was used in culture for 7 days
(LIF treatments vs. CC groups; p<0.05; Fig. 2),
which was confirmed by fluorescence method
(Fig. 3). Furthermore, the highest percentages of viable
preantral follicles were obtained at concentrations
between 10 and 50 ng/ml of LIF (from 58.6% to
58.0%), decreasing at 200 ng/ml (46.7%; p<0.05). The
survival of isolated preantral follicles before culture
(FC) was above 90%, which demonstrated follicular
health at the time of follicular isolation.
Discussion
The effect of LIF on the activation of primordial and
survival of preantral follicles was demonstrated in
a7-dayculturesystem,usinggoatasamodel.Our
results revealed that: (1) LIF induced activation of
primordial follicle, formation and differentiation of
new cellular layer of granulosa cells, giving origin
to primary follicle; and (2) LIF supported preantral
follicle viability for 7 days in culture, based on
histological and fluorescence analyses.
Slices of goat ovarian cortex were cultured in the
absence and presence of different concentration of LIF
to verify if LIF promotes primordial to primary follicle
transition in ruminants. The percentages of primordial
follicles decreased and primary follicles increased at
all LIF concentration tested at 7 days of culture. The
optimal LIF concentrations to maintain caprine pre-
antral follicles viable and growing ranged from 10 to
50 ng/ml. In fetal rat ovaries, LIF induced premature
follicular development and meiosis resumption when
used at a concentration of 100 ng/ml (Lyrakou et al.,
2002). Recently, Haidari et al. (2008) reported that LIF
at 50 ng/ml promoted follicular survival and preantral
follicle development in vitro using rat follicles.
In the concentrations used in the present study,
LIF induced increase in granulosa cell number and
change from flattened to cuboidal shape, character-
izing the transition to primary follicle. These results
indicate that LIF can promote activation of primordial
follicle and transition to primary follicle. Similarly,
the dramatic decrease of primordial follicle and
corresponding increase of primary follicle numbers
after LIF treatment were observed in the rat ovary
(Nilsson et al.,2002;Haidariet al., 2008). In the rat,
LIF also promoted the transition from primordial to
primary follicles and supported their viability for
14 days in culture. However, there is little information
on the role of LIF in promoting preantral follicle activ-
ation, development and viability in domestic animals.
LIF is expressed in rat preantral follicle (Nilsson
et al., 2002; Haidari et al., 2008) and in the ovarian
stromal cells in human (Arici et al.,1997).Theselast
authors also demonstrated that LIF concentrations
rise in periovulatory follicular fluid and regulate
ovulation, estrogen production and early embryonic
development. Despite being expressed in preantral
follicles and promoting the development of primordial
follicles, it appears that LIF has a role, but is not
essential for activation and transition to primary
follicles, considering that follicles reach ovulation in
LIF goat primordial follicle 77
Figure 2 Histological analysis of the viability of preantral follicles (%) after 7 days of culture. The results are presented as the
mean ±SEM of five independent cultures. a–dBars with no common letters are significantly different (p<0.05).
Figure 3 Viability of preantral follicles analysed by a two-colour fluorescence cell using calcein-AM and ethidium
homodimer-1. The best results observed in the histological analysis were repeated using probes to detected intracellular ester-
ase activity of viable cells and to label nucleic acids of non-viable cells with plasmatic membrane disruption. The results are pre-
sented as the mean ±SEM of five independent cultures. a–cBars with no common letters are significantly different (p<0.05).
LIF knockout mice (Stewart et al., 1992). Therefore,
there is strong evidence that the regulation of initial
preantral follicle activation and growth is orchestrated
by LIF and other signal factors [such as growth
differentiation factor 9 (GDF9), kit ligand (KL), basic
fibroblast growth factor 2 (bFGF2) and nerve growth
factor (NGF)], having a compensatory action when one
factor is missing in the system.
The expression and function of several transcription
factors and hormones responsible for the regulation of
follicle development is species specific and differences
have been demonstrate in rodents, ruminants and
primates (Shimasaki et al.,2004;Young&McNeilly,
2010). For example, the tissue-type plasminogen activ-
ator (tPA) and urokinase-type plasminogen activator
(uPA) activity is different between rodents and cattle
during the periovulatory period (Dow et al.,2002).LIF
and bone morphogenetic proteins (BMPs) are essential
for mouse embryonic stem (ES) cells to maintain
pluripotency (Smith et al.,1992;Yinget al.,2003);
however, human ES requires activin/nodal (Vallier et
al.,2004,2005;Jameset al.,2005).LIFandotherfactors
[such as bFGF, BMP, GDF9, hepatocyte growth factor
(HGF), insulin-like growth factor (IGF), IGF-binding
protein (IGFBP), interleukin-1 (IL1), keratinocyte
growth factor (KGF), luteinizing hormone (LH), stem
cell factor/kit ligand (SCF), transforming growth
factor beta (TGFβ)andtumournecrosisfactoralpha
(TNFα)] have species-specific patterns of expression
and function (for review see Young & McNeilly, 2010).
Therefore, the investigation of the LIF function in
ovarian preantral follicle development is imperative in
ruminants, which have a high economic importance
and have been used as an experimental model for
humans.
In conclusion, LIF induced activation of primor-
dial follicles, differentiation of granulosa cells from
flattened to cuboidal shape and maintenance of
preantral follicle viability for 7 days in culture. To
our knowledge, this is the first evidence that LIF
78 Nóbrega Jr et al.
is involved in the initial follicle development and
viability in ruminant.
Acknowledgements
This work was supported by CNPq, FINEP and
RENORBIO. We would like to thank contribution of
Dr Maria Fátima Teixeira for help with the fluorescence
analyses.
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  • [Show abstract] [Hide abstract] ABSTRACT: We investigated the effect of the leukaemia inhibitory factor (LIF) alone or in association with FSH on the in vitro culture (IVC) of caprine preantral follicles. Preantral follicles >200 μm in size were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/ml) in the absence or presence of FSH. Every 6 days, follicular survival, growth and antrum formation were evaluated. At the end of the culture period, the oocytes underwent in vitro maturation (IVM), and their viability and chromatin configuration were assessed. Follicles of the control group and those cultured in 10 ng/ml LIF maintained the structural integrity (particularly the preservation of the basement membrane) when compared to the oocytes cultured in 50 ng/ml LIF, regardless the presence of FSH. In the absence of FSH, the percentage of antrum formation after 18 days of culture in the 50 ng/ml LIF group was significantly lower than in either the control group or the 10 ng/ml LIF group. However, this effect was not observed in the presence of FSH. The rate of resumption of meiosis was significantly higher in the 50 ng/ml LIF group in the absence of FSH in comparison with the control and 10 ng/ml LIF groups. Metaphase II was observed only when follicles were cultured in a combination of FSH and 50 ng/ml LIF. In conclusion, LIF alone does not interfere with antral formation and oocyte growth, but at concentration of 50 ng/ml and combined with FSH, it promotes oocyte maturation.
    Article · Aug 2011
  • [Show abstract] [Hide abstract] ABSTRACT: We investigated the effect of the leukemia inhibitory factor (LIF) alone or in association with follicle-stimulating hormone (FSH) on the in vitro growth and antrum formation of sheep preantral follicles. To evaluate oocyte quality, parthenogenetic activation of the oocytes recovered from in vitro grown preantral follicles was performed. Preantral follicles >110 μm in diameter were isolated and cultured for 18 days in basic medium either alone (control) or supplemented with LIF (10 or 50 ng/mL) in the absence or presence of FSH. Every 6 days the follicular survival, growth, and antrum formation were evaluated. When compared to control (P < .05), antrum formation was increased in follicles cultured in the presence of LIF10 and FSH. At the end of the culture, the oocytes underwent in vitro maturation (IVM); their viability and chromatin configuration were assessed. Although IVM was not affect by the treatments (P > .05), the numerically highest maturation rates (29.63%) were obtained when follicles were cultured in 50 ng/mL LIF (LIF50). Therefore, their oocytes were submitted to parthenogenetic activation; from which 58.3% of the mature oocytes resulted in 8-cell stage parthenotes. In conclusion, although LIF10 + FSH increases antrum formation when compared to a nonsupplemented medium (minimum essential medium), oocytes from sheep preantral follicles are capable of growing and maturing in vitro independent of LIF addition to the medium, which resulted in the formation of 8-cell parthenotes.
    Full-text · Article · May 2012
  • [Show abstract] [Hide abstract] ABSTRACT: The purpose of the present study was to investigate the development of follicles and incidence of apoptosis in vitrified neonatal mouse ovaries cultured in vitro in the presence of leukemia inhibitory factor (LIF). The vitrified and non-vitrified ovaries of 1-week-old mouse were cultured in the presence or absence of LIF for 7days. At the beginning and at the end of culture period in each ovary of all groups of study the mean area and the development of ovarian follicles were analyzed; moreover, the incidence of apoptosis was assessed by transmission electron microscopy, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) method, DNA laddering and caspase-3/7 activity technique. The hormonal assay was done on the conditioned media collected during culture period. The proportion of preantral follicles and the levels of hormones increased in all cultured groups and it was significantly higher in LIF treated groups than in their control (P<0.001). The ultrastructural characteristics of cell death, DNA fragmentation and TUNEL positive signals were prominent in vitrified cultured ovaries. The level of caspase-3/7 activity was higher in vitrified cultured ovaries. LIF supplementation during 7days of culture appeared to significantly preserve cells function and increase the follicular development of both vitrified and non-vitrified ovaries.
    Article · Oct 2013
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