HORMONES 2005, 4(3):155-16. 
Research paper
Effects of GH and IGF-I on the in vitro maturatio. 
of mouse oocyte. 
Arasmia Kiapekou1, Dimitris Loutradis2, Peter Drakakis2, Evangelia Zapanti1. 
George Mastorakos3, Aristidis Antsaklis. 
11st Endocrine Section, Alexandra Hospital, Athens, Greece, 21st Department of Obstetrics and Gynaecology. 
Alexandra Hospital, Athens University Medical School, Athens, Greece, 3Endocrine Unit, 2nd Department o. 
Obstetrics and Gynaecology, Aretaion Hospital, Athens University Medical School, Athens, Greec. 
ABSTRACTinvitro maturation of oocytes. Their exact effects on follicular growth and oocyte maturation andthe mechanisms involved are still unclear. In the present study, we have investigated the ef-
fects of Growth Hormone (GH) and Insulin-like Growth Factor 1 (IGF-1) on the in vitro mat-
nuded), were obtained from 4- to 8-week old female mice and were cultured in Ham.s F-10medium. GH and IGF-1 were added separately or in combination in gradually increasing con-
centrations in the culture media, while medium-only containing samples were employed ascontrols. Oocyte development was assessed daily for three days and maturation was consid-
(mean ± SE) of denuded oocytes formed a polar body. The achieved maturation rate after theaddition of either GH or IGF-1 or GH plus IGF-1 was significantly higher than in controls.
The highest maturation rates were achieved after the addition of 0.2 ig/ml GH (76%±5%), 50ng/ml IGF-1 (69%±5%) and a combination of 0.2 ig/ml GH plus 10 ng/ml IGF-1 (75%±5%).
oocyte maturation significantly. The lack of a synergistic effect on oocyte cultures when bothhormones were added indicates that both hormones act through the same signaling pathway.
Key words: Denuded oocytes, Growth hormone, Insulin-like growth factor 1, in vitro maturationOBJECTIVE: A number of hormones and growth factors have been reported to affect the 
uration of mouse oocytes. DESIGN: Immature preovulatory oocytes without cumulus cells (de-
ered to be completed when the first polar body appeared. RESULTS: In control samples, 44±3%
CONCLUSIONS: The data suggest that GH and IGF-1, alone or in combination, affect mouse
INTRODUCTIO. 
In vitro maturation of oocytes is a long and com. 
plex process that has not yet been completely eluci. 
Address correspondence and requests for reprints to. 
Kiapekou Erasmia, 25 Karaiskaki Str., 15772 Athens, Greece. 
Oel.: +30 210 7488926, E-mail: nem10@panafonet.g. 
Received 11-06-05, Revised 10-07-05, Accepted 15-07-0. 
dated. Gonadotropins are considered to be of con. 
siderable importance in the maturation of oocytes. 
In addition, a local regulation of the ovarian func. 
tion by steroid and non-steroid substances has re. 
cently been described. The latter include a variet. 
of growth factors, such as Growth Hormone (GH)1. 
Insulin-like Growth Factors (IGFs)2 and the Epi. 
dermal Growth Factor (EGF)3, as well as other hor. 

15. A. KIAPEKOU, ET A. 
mones such as Prolactin (PRL)4. These growth fac. 
tors are secreted from the granulosa and cumulu. 
cells in response to gonadotropins and subsequent. 
ly act on the oocyte via paracrine and autocrine path. 
ways. Although a growing number of studies hav. 
indicated that there are beneficial effects of growt. 
factors in oocyte maturation, it seems that only de. 
nuded oocytes require the supplementation o. 
growth factors in the culture medium for prope. 
maturation5. Many of those agents are produce. 
from the follicle itself and have either an autocrin. 
or paracrine mechanism of action. 
Recently, GH has been implicated in oocyte mat. 
uration. Izadyar et al6 reported an acceleration of i. 
vitro oocyte maturation, of cumulus-enclosed oo. 
cytes, the induction of cumulus expansion and th. 
promotion of subsequent embryonic developmen. 
of bovine oocytes with the addition of GH in th. 
cultures. The GH mRNA expression and GH local. 
isation have been demonstrated in the cumulus cell. 
and the enclosed oocyte of small to medium size. 
bovine follicles7. Moreover, the addition of GH ap. 
pears to increase the in vitro maturation rate of ear. 
ly-antral follicles in the rat8, while the presence o. 
GH receptor mRNA has been established in rat pre. 
antral follicles9. Furthermore, GH promotes prean. 
tral follicular growth in 11-day old immature femal. 
mice10. Growth hormone can act directly on rat fol. 
licles through its receptors at the granulosa cells. 
and/or indirectly through IGF-18. This is also th. 
case in porcine12 and rabbit13 follicles and denude. 
oocytes (oocytes at the late stages of development. 
not surrounded by layers of cumulus cells and th. 
germinal vesicle clear and distinguished). 
The presence of IGF-1 mRNA and IGF-1 re. 
ceptor mRNA in rat somatic follicular cells and i. 
oocytes has been demonstrated11. Furthermore, th. 
addition of IGF-1 to the culture medium increase. 
the in vitro nuclear maturation rate13. Studies in hu. 
mans have shown that IGF-1 receptors are presen. 
in the follicles throughout all stages of developmen. 
(from the primordial through to the preovulator. 
stage)14. In addition, IGF-1 promotes the in vitr. 
maturation of follicles and denuded oocytes15. In. 
terestingly, Insulin-like Growth Factor 1 enhance. 
the stimulatory effects of GH on the in vitro matu. 
ration of mouse preantral follicles10. 
The aim of the present study was to investigat. 
the effect of GH and IGF-1, given separately or i. 
combination, on the in vitro maturation of mous. 
denuded oocytes using a simple in vitro culture sys. 
METHOD. 
Animal. 
Denuded oocytes were obtained from 4- to 8. 
week old female mice without prior hormonal ova. 
rian stimulation [New Zealand Black (NZB) x Ne. 
Zealand White (NZW); (Pasteur Institute, Athens)]. 
The mice were sacrificed by cervical dislocation an. 
the ovaries were removed. 
Material. 
Dulbecco.s phosphate-buffered saline (DBPS. 
and Ham.s F-10 medium without hypoxanthine wer. 
obtained from Invitrogen Life Technologies, (Pais. 
ley, UK); bovine serum albumin (BSA), from Sig. 
ma-Aldrich, (Dorset, UK); Human growth hormon. 
from Eli Lilly (Windlesham Surrey, U.K.); Huma. 
Insulin-like Growth Factor Type 1 from R&D Sys. 
tems (Minneapolis, USA); culture dishes (Falcon). 
from Becton Dickinson Co., (New Jersey, USA). 
Isolation of mouse denuded oocyte. 
Mouse denuded oocytes were collected mechan. 
ically by puncturing the ovaries in Dulbecco.s phos. 
phate-buffered saline, supplemented with bovin. 
serum albumin (9:1). Approximately 1000 mous. 
denuded oocytes were obtained. Oocytes were thu. 
isolated at the late stages of development, when no. 
surrounded by layers of cumulus cells, and the ger. 
minal vesicle was clearly visible (Figure 1). 
Culture of mouse denuded oocyte. 
Collected oocytes were rinsed twice in a DPB. 
and BSA mixture solution (9:1) and cultured i. 
Ham.s F-10 medium without hypoxanthine supple. 
mented with BSA (9:1). Growth hormone and IGF. 
1 were added as described below, while medium. 
only samples were used as controls. Ten to twent. 
oocytes were placed in the central well of the cul. 
ture dishes with 1 ml of Ham.s F-10 medium. Addi. 
tionally, 4 ml of Ham.s F-10 medium were added t. 
the surrounding area of the dish to maintain humi. 
dity. 

Effects of GH and IGF-I on the in vitro maturation of mouse oocyte. 
Culture conditions were adjusted at 37oC, 5. 
CO2 concentration, 95% humidity and pH 7.3 - 7.4. 
Oocyte development was assessed daily for 72 hour. 
for the appearance of the first polar body. 
Dose-Response Experiment. 
Gradually increasing concentrations of huma. 
GH (0.1, 0.2, 0.4 and 0.6 ig/ml) and human IGF-. 
(10, 50, 100 and 200 ng/ml) were added separatel. 
to different cultures and their effect in mouse oo. 
cyte development was recorded. Furthermore, base. 
on the preliminary results obtained from the dose. 
response experiments, all tested doses of GH wer. 
added to different cultures together with 10 ng/m. 
IGF-1. The concentrations of GH and IGF-1 tha. 
we used have been employed and mentioned in pre. 
vious publications8,11,12. 
Statistic. 
The results were analysed by one-way analysi. 
of variance and the z-test for proportions. A valu. 
of P <0.05 was considered statistically significant. 
Percentages of in vitro oocyte maturation were ex. 
pressed as mean±SE. The Bonferroni correctio. 
was applied. 
RESULT. 
The highest maturation rate was observed with. 
Figure 1. Oocytes at the late stages of development, not sur. 
rounded by layers of cumulus cells and the germinal vesicle clear. 
ly distinguished. The oocytes were collected from the ovaries o. 
2- to 8-week old mice, without hormonal ovarian stimulation. 
The germinal vesicles of the oocytes are visible (black arrow. 
(magnification x30). 
in the first 24 hrs. There was a small number o. 
mature oocytes in the second 24hr period, subse. 
quent to which most oocytes degenerated. We ob. 
served their maturation for an additional 24hr peri. 
od but at that point most oocytes had degenerated16. 
Control culture. 
Mouse denuded oocytes, cultured without G. 
or IGF-1 for 72 hours, matured at a rate of 44±%. 
Effects of GH on the in vitro maturatio. 
of mouse denuded oocyte. 
The maturation rate of cultured mouse denude. 
oocytes significantly increased (p <0.05) in the pres. 
ence of 0.1 ig/ml or 0.2 ig/ml of GH (mean±SE. 
67±1% and 76±5%, respectively, versus 44±3% i. 
control cultures) (Figure 2). Further increase in th. 
GH concentrations added to the cultures (0.4 ig/m. 
and 0.6 ig/ml) did not result in any significant ris. 
in the maturation rate as compared to control sam. 
ples. 
Effects of IGF-1 on the in vitro maturatio. 
of mouse denuded oocyte. 
The maturation rate of cultured mouse denude. 
oocytes significantly increased (p <0.05) in the pres. 
ence of 10 and 50 ng/ml of IGF-1 (mean±SE. 
65±2% and 69±5%, respectively, versus 44±3% i. 
control cultures) (Figure 3). Further increase o. 
IGF-1 concentrations did not result in any signifi. 
Figure 2. Effects of GH on the in vitro maturation of mous. 
denuded oocytes. The percentage of cultured oocytes develop. 
ing a polar body after 72 hours (maturation rate), in the ab. 
sence (control) or the presence of GH (ig/ml) at various con. 
centrations. (* . significantly different from the controls. 
p<0.05). 

15. A. KIAPEKOU, ET A. 
cant rise in the maturation rate as compared to con. 
trol samples. 
Effects of GH plus IGF-1 on the in vitr. 
maturation of mouse denuded oocyte. 
The maturation rate of cultured mouse denude. 
oocytes significantly increased (p <0.05) in the pres. 
ence of 10 ng/ml plus either 0.1 ig/ml or 0.2 ig/m. 
of GH (mean±SE; 70±2% and 75±5%), as com. 
pared to control cultures (mean±SE; 44±3%) (Fi. 
gure 4). Further increase of GH concentrations di. 
not result in any significant changes in the matura. 
tion rate as compared to control samples. 
DISCUSSIO. 
We found that the in vitro maturation rates o. 
mouse denuded oocytes are significantly increase. 
in the presence of GH and/or IGF-1.Increasing th. 
concentrations of GH and IGF-1 beyond a certai. 
level does not further increase oocyte maturation. 
This could be attributed to a down-regulation of thei. 
corresponding receptors when higher concentration. 
are employed. The down-regulation phenomeno. 
is often observed when there are increased concen. 
trations of the ligand in the culture media. It is readi. 
ly seen with protein membrane receptors and rep. 
resents a physiological mechanism of cellular adap. 
tation to increased concentrations of the ligand. 
Figure 3. Effects of IGF-1 on the in vitro maturation of mous. 
denuded oocytes. The percentage of cultured oocytes develop. 
ing a polar body after 72 hours (maturation rate), in the ab. 
sence (control) or the presence of IGF-1 (ng/ml) at various con. 
centrations. (* . significantly different from the controls. 
p<0.05). 
cytes
Growth hormone promotes preantral follicula. 
growth in 11-day old immature female mice10. Th. 
mode of action of GH on oocytes is not well known. 
Two different pathways have been suggested. Th. 
first is a direct one, through GH receptors9, whil. 
the second is indirect, through the action of granu. 
losa-derived IGF-112,13. In bovine oocytes, some stud. 
ies showed that GH acts via IGF-117 while other stud. 
ies showed that GH acts directly via a cAMP-medi. 
ated pathway18,19. Growth hormone receptor is ex. 
pressed in bovine and rat cumulus cells and in oo. 
18,20,21. In the past it had been suggested that th. 
effects of GH on oocyte maturation were exerte. 
via cumulus cells in the two species8,18. In the presen. 
study we employed mouse denuded oocytes an. 
found that GH as well as IGF-1 significantly in. 
creased the in vitro maturation rate of these oocytes. 
The absence of any supporting cumulus cells sug. 
gests the presence of GH receptor on oocytes at thi. 
developmental stage in mice. Furthermore, GH an. 
GH receptor mRNA were demonstrated to be ex. 
pressed in bovine oocytes and granulosa cells re. 
covered from small follicles maturing in vitro7. I. 
granulosa cells, GH helps the differentiation an. 
replication22 in addition to the modulation of ste. 
roidogenic gene expression23. 
Previous studies have demonstrated the presenc. 
of IGF-1 and IGF-1 receptor mRNA at the cumu. 
lus cells and the denuded bovine and porcine oo. 
Figure 4. Effects of different combinations of GH (ig/ml) an. 
IGF-1 (ng/ml) on the in vitro maturation of mouse denuded oo. 
cytes. The percentage of cultured oocytes developing a pola. 
body after 72 hours (maturation rate), in the presence of IGF. 
1 and GH at various concentrations and in controls (* . signif. 
icantly different from the controls; p<0.05). 

Effects of GH and IGF-I on the in vitro maturation of mouse oocyte. 
cytes24,25. In this study IGF-1 appears to act directl. 
on the oocytes of late stage development withou. 
the presence of any cumulus cells. In mice, low IGF. 
1 mRNA levels were detected in primary follicle. 
while transcription increased to maximum during th. 
late preantral and early antral stages2,26. Studies o. 
IGF-1 knockout mice have demonstrated the inabil. 
ity of the follicles to reach the antral stage, inducin. 
infertility due to lack of ovulation27, thus confirmin. 
the essential role of IGF-1 during folliculogenesis. 
Demeestere et al have shown that IGF-1 has a stim. 
ulatory effect on follicular steroidogenesis if adde. 
to culture of mouse preantral follicles in the pres. 
ence of FSH. Subsequently, blastocyst developmen. 
was enhanced after follicular culture with IGF-128. 
The presence of IGF-1 receptor mRNA on rat de. 
nuded oocytes and follicles has been described11,29. 
In the present study, IGF-1 stimulated the in vitr. 
maturation of mouse denuded oocytes without th. 
presence of FSH, suggesting that the action of IGF. 
1 in mouse denuded oocytes is not FSH-dependen. 
and that it might be exerted through IGF-1 recep. 
tors. 
The combination of IGF-1 with various concen. 
trations of GH on mouse cultures did not result in . 
higher maturation rate than that observed by th. 
sole utilisation of either IGF-1 or GH, suggestin. 
that the two hormones do not act synergistically. 
possibly because they act through the same signal. 
ing pathway. 
In conclusion, our data showed that GH and/o. 
IGF-1 increase the rate of the in vitro maturation o. 
mice denuded oocytes. In addition, a possible rela. 
tionship between their pathways of action has bee. 
proposed. 
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