Molecular Human Reproduction, Vol. 6, No. 8, 694-698,
August 2000
© 2000 European Society of Human Reproduction and Embryology
Ovary and oogenesis |
The effects of insulin, and insulin-like growth factors I and II on human ovarian follicles in long-term culture
1 Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 Helsinki, Finland, 2 Karolinska Institute, Department of Obstetrics and Gynaecology, Huddinge University Hospital, S-14186 Huddinge, Sweden, and 3 Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, FIN-00290 Helsinki, Finland
Abstract
The aim of this study was to investigate the effects of insulin and insulin-like growth factors I and II (IGF-I and IGF-II) on human ovarian follicles in vitro. Ovarian cortical tissue slices (0.1–0.3 cm) were cultured for 7 or 14 days on an artificial extracellular matrix and with FSH. The ovarian tissue cultures were stimulated by insulin (33 ng/ml), IGF-I (20 or 50 ng/ml) or IGF-II (20 ng/ml). Combined effects of IGF-I (20 ng/ml) or IGF-II (20 ng/ml) and insulin (33 ng/ml) were also studied. Proliferating cell nuclear antigen (PCNA) was selected for immunohistochemical examination activation of the mitotic cell cycle in granulosa cells. After 1 week of culture the number of follicles had decreased in all cases. After 2 weeks of culture the number of healthy follicles had decreased dramatically in control cultures. However, the loss of follicles could be prevented with insulin and IGFs. The number of atretic follicles was significantly lower in insulin cultures compared with control cultures after 2 weeks. The proportion of primary follicles was significantly increased in cultures treated with insulin, IGF-I (50 ng/ml) or IGF-II (20 ng/ml) compared with control cultures after 2 weeks. A similar effect was seen after co-treatment with IGF-II and insulin. There were significantly more PCNA-positive follicles in IGF-I cultures than in control cultures. These results suggest that insulin, IGF-I and IGF-II may act as survival factors for early stage human follicles. IGFs may also be involved in activation of the mitotic cell cycle of granulosa cells.
follicle/follicle culture/IGF/insulin/ovary
Introduction
The early development of mammalian ovarian follicles is a poorly understood process. Although FSH and LH are essential for follicular development from the secondary stage onwards, they are not thought to regulate the onset of growth of primordial follicles. This is supported by the fact that hypogonadal mice, which are deficient in hypothalamic gonadotrophin-releasing hormone (GnRH) and have no detectable levels of gonadotrophins, nevertheless have a pool of small growing follicles (Halpin et al., 1986
). Similarly, women carrying an inactive form of FSH receptor and who, therefore, practically lack FSH bioactivity, produce follicles which can occasionally reach the secondary stage (Aittomäki et al., 1996). Recent studies, however, have indicated that FSH might have effects on the development of early stage follicles (Oktay et al., 1997; Wright et al., 1999).
Factors initiating the transition of quiescent primordial follicles to the pool of growing follicles are still unknown. Studies on animals have indicated that several locally produced growth factors are involved in the multifactorial regulation of the growth of primary and secondary follicles. For example, female growth differentiation factor-9 (GDF-9)-deficient mice are infertile as a result of a block of folliculogenesis at the primary follicle stage (Dong et al., 1996). In addition, stem cell factor (SCF) increases the proportion of developing rat follicles in vitro (Parrot and Skinner, 1999). Blocking the SCF receptor disturbs the onset of mouse primordial follicle development, primary follicle growth and development of later stage follicles (Yoshida et al., 1997). Activin may also be involved in the paracrine or autocrine regulation of early mammalian folliculogenesis. Activin promotes preantral growth of mouse follicles and stimulates rat granulosa cell proliferation. It also induces reaggregation of isolated oocytes and granulosa cells into follicle-like structures (Li et al., 1995, 1998).
There is an increasing body of evidence indicating that insulin-like growth factors I and II (IGF-I and IGF-II) regulate mammalian early follicular development. Female IGF-I knock-out mice are infertile because follicles do not develop beyond the secondary stage and they fail to ovulate even after administration of exogenous gonadotrophins (Baker et al., 1996). IGF gene expression appears to be regulated differently in rodents and humans. In mice and rats, IGF-I gene expression is confined to the granulosa cells of small growing follicles (Zhou et al., 1991; Adashi et al., 1997). In humans, IGF-II mRNA and protein have been localized to theca cells of small antral follicles and granulosa cells of dominant follicles, whereas IGF-I gene expression is confined to the theca cells (El Roeiy et al., 1993; Voutilainen et al., 1996).
Recent studies with human ovarian cortical biopsies have indicated that human primordial and primary follicles may sustain their viability for several weeks in vitro and that initiation of follicular growth is possible (Hovatta et al., 1997; Wright et al., 1999). In work on animals it has been shown that viable mice can be produced from oocytes matured in vitro from primordial stage follicles (Eppig and O'Brien, 1996). Mouse oocytes from mechanically isolated early stage follicles are meiotically competent and fertilizable (Cortvrindt et al., 1996). For human follicles, an ovarian cortical culture system which maintains normal intercellular contacts between follicular and stromal cells, appears to be better than isolation, as isolated follicles can be cultured for short periods only (Roy and Treacy, 1993; Hovatta et al., 1999). Partial isolation also promotes atresia (Hovatta et al., 1999).
The objective of this study was to investigate the effects of insulin, IGF-I and IGF-II on the early development of human ovarian follicles in organ cultures of ovarian cortical tissue.
Materials and methods
Subjects
Ovarian cortical tissue was obtained during gynaecological operations by biopsy or from total oophorectomy specimens. In all, 52 women aged 20–37 years (mean 31 years), donated tissue after informed consent. The study was accepted by the Ethics Committees of the Family Federation of Finland and the Department of Obstetrics and Gynaecology, Helsinki University Central Hospital, Finland.
Tissue culture
Ovarian tissue was cut into slices of 0.5–2 mm, using a scalpel. The slices were transferred to Millicell CM inserts (6 mm diameter, 1.0 µm pore size; Becton Dickinson Labware, Bedford, MA, USA), one to three slices in each well. The inserts were pre-coated with 150 µl extracellular matrix (Matrigel, Becton Dickinson Labware) consisting of laminin, collagen IV and proteoglycans as major components, and placed into 24-well plates (Becton Dickinson Labware).
The culture medium consisted of Earle's balanced salt solution (Gibco, Life Technologies Ltd, Paisley, Scotland, UK) supplemented with 5% inactivated human serum (Finnish Red Cross), 0.47 mmol/l sodium pyruvate (Sigma, St Louis, MO, USA), 0.5 IU/ml recombinant human FSH (Puregon; Organon, Cambridge, UK) and antibiotics: penicillin G (50 IU/ml), streptomycin sulphate (50 µg/ml) and amphotericin B (0.125 µg/ml) (HyClone Laboratories Inc, Utah, USA). Insulin (33 ng/ml; Sigma), IGF-I (20 or 50 ng/ml) or IGF-II (20 ng/ml; R&D Systems Europe Ltd, Abingdon, UK) or insulin and IGF-I/IGF-II (33 and 20 ng/ml respectively) were added to the medium. All the experiments were repeated 5–15 times. Pieces of control tissue were cultured in the absence of these hormones. The medium was changed every second day. The tissue was cultured in a humidified incubator in 5% CO2 in air at 37°C.
On day 0, and on day 7 or 14 of culture, pieces of ovarian tissue were fixed in Bouin's solution or in Histochoice solution (Histochoice tissue fixative; Amresco, Solon, Ohio, USA) for histology. The tissue was embedded in paraffin wax, cut into 4 µm (Bouin-fixed) or 7 µm (Histochoice-fixed) sections and stained with haematoxylin and eosin. Sections (8–10) from each tissue slice were analysed, and the primordial, primary, secondary and tertiary follicles were classified as described previously (Gougeon, 1996). Eosinophilia of the ooplasm, contraction and clumping of the cromatin, wrinkling of the nuclear membrane of the oocyte and pyknotic granulosa cells were regarded as signs of atresia (Gougeon, 1996).
Immunohistochemistry
Some pieces of ovarian cortex fixed for normal histology were cut into sections of 4 µm (Bouin-fixed) or 7 µm (Histochoice-fixed) and placed on Histogrip (Zymed Laboratories Inc, San Francisco, CA, USA) coated slides for immunolocalization of proliferating cell nuclear antigen (PCNA). Sections were placed in an oven (57°C) for 30 min and deparaffinized with xylene for 2x5 min, quickly rehydrated, and microwaved for 3x5 min in 10 mmol/l citrate buffer to enhance antigen retrieval. The first antibody, monoclonal mouse anti-PCNA (Dako, San Francisco, CA, USA) was added (1:100), and the sections were incubated for 30 min at room temperature. The second antibody (goat anti-mouse immunoglobulin) was applied for 15 min at room temperature. Binding of the primary antibody was detected and visualized by using a Dako LSAB kit (Dako) with diaminobenzidine, according to the manufacturer's instructions. The sections were counterstained with haematoxylin. Control sections were stained without the first antibody.
Statistical analyses
Fischer's exact test was used for statistical analysis of the PCNA staining. All the other statistical analyses were performed using the 
2 test. P < 0.05 was considered statistically significant.
Results
Human ovarian cortical tissue slices were cultured for 7 or 14 days on artificial extracellular matrix (Matrigel). The cultures were stimulated by insulin, IGF-I or IGF-II and thereafter analysed histologically.
In fresh tissue, the proportion of primordial follicles was 85%, and the rest (10–15%) were mostly at primary stage. Usually only a few (<5%) atretic follicles were seen. In 7-day cultures, no differences in the developmental stages of the follicles were observed after stimulation, compared with control cultures (data not shown), but the number of atretic follicles was increased significantly in both control (P < 0.01) and insulin-treated cultures (P < 0.01) as compared to the fresh tissue (Figure 1).
|
After 14 days of culture, the proportion of atretic follicles in control cultures had increased extensively as compared to fresh (P < 0.001) or 7-day control cultures (P < 0.05). Insulin had a positive effect on the viability of the follicles; the proportion of atretic follicles was about half of that in the controls (Figure 1
, P < 0.001). A parallel although weaker effect was found with IGF-I and IGF-II (data not shown). In addition to the increase in the proportion of visible atretic follicles, the total number of follicles tended to decrease during the culture.
Both insulin and IGFs increased the proportions of primary follicles during the 14-day culture period. There were significantly more primary follicles in IGF-I (50 ng/ml, P < 0.05) and IGF-II (20 ng/ml, P < 0.01) stimulated cultures than in control cultures (Figure 2). Also insulin (P < 0.001) and insulin + IGF-II co-treatment (P < 0.01) significantly increased the proportion of primary follicles (Figure 3). Similar results were obtained with insulin + IGF-I co-treatment. However, due to the low number of follicles in these cultures, no statistical difference was seen (Figure 3). In contrast to the increase in the proportion of primary follicles, the proportion of primordial follicles decreased significantly in cultures treated with IGF-I (50 ng/ml, P < 0.01), or with insulin + IGF-II (P < 0.05; Figures 2 and 3).
|
|
The detection of PCNA protein expression was used to assess activation of the mitotic cell cycle of the granulosa cells of cultured follicles. No immunostaining was observed in fresh or cultured control tissue. In contrast, granulosa cells in several primary and secondary stage follicles showed positive staining for PCNA in 14-day IGF-I cultures (50 ng/ml; P < 0.01; Table I
). One secondary follicle was stained in a 14-day culture stimulated by IGF-II (20 ng/ml; Figure 4). Primordial follicles did not show positive staining in any of the cultures (Table I).
|
|
Discussion
The present data indicates that insulin and insulin-like growth factors are able to regulate early human ovarian folliculogenesis in vitro. Insulin clearly improved the viability of the cultured follicles in 2 week cultures and increased the proportion of primary follicles. Similar effects were seen with IGF-I and IGF-II. IGF-I was also shown to induce expression of PCNA in the granulosa cells of primary follicles.
Insulin is widely used in different kinds of cell and tissue cultures to increase the viability of the cells. In addition to its well-known effects on glucose homeostasis, insulin also regulates various other intracellular processes such as amino acid transport, lipid metabolism, gene transcription and protein synthesis (Cheatham and Khan, 1995). Insulin receptors are ubiquitously expressed in almost all mammalian tissues, including human ovaries, where its mRNA has been located in both stromal and follicular cells (El-Roeiy et al., 1993). Recent studies have shown that a high concentration (10 µg/ml) of insulin may stimulate follicular growth in vitro (Wright et al. 1999). This is in line with the present results with a substantially lower concentration (33 ng/ml) of insulin and further confirms the possibility that insulin has a positive effect on human ovarian folliculogenesis. Our results also indicate that insulin significantly reduces the proportion of atretic follicles in 2 week cultures hence improving the overall viability of the cultured follicles. Whether the growth supporting effect of insulin is direct, or due to the increased viability of the follicles, remains to be elucidated.
In human ovaries, IGF-II mRNA and protein have been localized in theca cells of small antral follicles and in granulosa cells of dominant follicles (El Roeiy et al., 1993). IGF-I receptor mRNA is expressed in granulosa cells of small antral and dominant follicles and by immunohistochemical methods, the protein has been detected in oocytes and granulosa cells of primordial and pre-antral human follicles (Qu et al., 2000). IGF-I mRNA is localized in theca cells of small antral follicles (El Roeiy et al., 1993) but the expression of IGF-I protein in human follicles is controversial. IGF-1 protein has been localized to the theca cells (Hernandez et al., 1992) but this finding has not been confirmed by others (El Roeiy et al., 1993; Zhou and Bondy, 1993).
IGF-I augments the effects of FSH on steroidogenesis in human granulosa and granulosa–luteal cells (Erickson et al., 1989). Insulin and IGF-I promote the proliferation of cultured human theca cells (Duleba et al., 1998). IGF-II may mediate the steroidogenic and growth promoting actions of FSH on human secondary follicles, as the production of oestradiol in granulosa cells is completely inhibited in the presence of IGF-II antagonist in cultured human preantral follicles (Yuan and Giudice, 1999). In addition, FSH stimulates IGF-II mRNA expression in preantral follicles (Yuan and Giudice, 1999). Insulin may also modulate IGF-II bioactivity. Preincubation with insulin enhances the action of IGF-II on steroidogenesis in human granulosa cells obtained from small and mid-sized antral follicles (Mason et al., 1994).
In our studies, stimulation with IGFs significantly increased the proportion of primary follicles. The increase may have resulted from activation of the growth of primordial follicles. Alternatively, IGFs may have improved survival of the pre-existing primary follicles. The proportion of atretic follicles increased during the culture, and the total number of follicles appeared to decrease as a result of apoptotic cell death. Treatment with IGFs may have supported the survival of activated follicles and hence increased their proportion. This would be in line with previous studies that have shown that IGF-I is able to suppress apoptotic DNA -fragmentation in cultured rat secondary follicles, acting as an antiatretic factor (Chun et al. 1994).
Studies with murine ovaries have shown that IGF-I augments FSH receptor expression in granulosa cells. The lack of IGF-I bioactivity decreases the expression of FSH receptors, which in turn leads to imperfect late folliculogenesis (Zhou et al., 1997). In human ovaries, FSH receptor mRNA is expressed as early as in primary follicles (Oktay et al., 1997). Recently, it was shown that FSH acts as a survival factor for small human follicles (Wright et al., 1999). These results suggest that FSH might have effects on the development of human follicles earlier than has been previously thought. Our results indicate that IGFs may also be survival factors for early stage follicles. The effects of IGFs on the survival of follicles could be direct. It is also possible that IGFs augment the expression of FSH receptors in primary follicles and hence make them more receptive to FSH. The connection between the effects of IGFs and FSH on human early stage follicles was not examined in this study.
A clear PCNA immunostaining was detected in the granulosa cells of primary and secondary follicles in the IGF-I treated cultures. In contrast, no PCNA-positive granulosa cells were found in follicles of the control cultures. Thus, IGF-I appears to affect activation of the mitotic cell cycle of granulosa cells in vitro. Interestingly, only one PCNA-positive secondary follicle but no positive primary follicles was detected in IGF-II-stimulated cultures.
The results obtained with IGF-I and IGF-II were slightly different. The concentration of IGF-I needed for a significant increase in the number of primary follicles was higher than that of IGF-II. PCNA staining showed clear results with IGF-I but not with IGF-II, regardless of comparable effects on follicular development. A similar discrepancy in their bioactivities has been observed in human granulosa cells obtained from pre-ovulatory antral follicles. In these cells, IGF-I was shown to be more effective in stimulating steroidogenesis than IGF-II, although the receptor mediating their effects appeared to be the same (Willis et al., 1998). Recent results indicate that IGF-II, but not IGF-I, is able to bind with high affinity to insulin receptor isoform A (IR-A) and activate its intracellular down-stream signals (Frasca et al., 1999). The IR-A isoform is produced from alternatively spliced IR mRNA and it is expressed at least in human muscle, adipocytes, placenta, lung, breast and colon (Moller et al., 1989; Frasca et al., 1999). Expression of the IR-A isoform in human ovaries has not been studied, but its presence in ovaries could explain the divergence in our results obtained with IGF-I and IGF-II.
Previous studies have shown that IGFs are important mediators of the actions of gonadotrophins in secondary stage follicles and onward. Our results indicate that IGFs and insulin are also able to regulate the early development of human ovarian follicles. Whether they are involved in the actual activation of the primordial follicles or in the survival of activated follicles, remains to be elucidated.
Acknowledgments
The authors wish to thank the clinical staff of the Department of Obstetrics and Gynaecology, Helsinki University Central Hospital. We are also grateful to Ms Marjut Pesonen for help in treating our tissue cultures.
Notes
4 To whom correspondence should be addressed at: Infertility Clinic, The Family Federation of Finland, Kalevankatu 16, FIN-00100 Helsinki, Finland. E-mail: timo.tuuri{at}vaestoliitto.fi 
References
Adashi, E.Y., Resnick, C.E., Payne, D.W. et al. (1997) The mouse intraovarian insulin-like growth factor I system: Departures from the rat paradigm. Endocrinology, 138, 3881–3890.
Aittomäki, K., Herva, R., Stenman, U-H. et al. (1996) Clinical features of primary ovarian failure caused by a point mutation in the follicle-stimulating hormone receptor gene. J. Clin. Endocrinol. Metab., 81, 3722–3726.[Abstract]
Baker, J., Hardy, M.P., Zhou, J. et al. (1996) Effects of an Igf1 gene null mutation on mouse reproduction. Mol. Endocrinol., 10, 903–918.[Abstract]
Cheatham, B. and Kahn, C.R. (1995) Insulin action and the insulin signaling network. Endocr. Rev., 16, 117–142.[ISI][Medline]
Chun, S.-Y., Billig, H., Tilly, J.L. et al. (1994) Gonadotropin suppression of apoptosis in cultured preovulatory follicles: Mediatory role of endogenous insulin-like growth factor I. Endocrinology, 135, 1845–1853.[Abstract]
Cortvrindt, R., Smitz, J. and Van Steirteghem, A.C. (1996) In-vitro maturation, ferilization and embryo development of immature oocytes from early preantral follicles from prepuberal mice in a simplified culture system. Hum. Reprod., 11, 2656–2666.
Dong, J., Albertini, D.F., Nishimori, K. et al. (1996) Growth differentiation factor-9 is required during early ovarian folliculogenesis. Nature, 383, 531–535.[Medline]
Duleba, A.J., Spaczynski, R.Z. and Olive, D.L. (1998) Insulin and insulin-like growth factor I stimulate the proliferation of human ovarian theca-interstitial cells. Fertil. Steril., 69, 335–340.[ISI][Medline]
El Roeiy, A., Chen, X., Roberts, V.J. et al. (1993) Expression of insulin-like growth factor-I (IGF-I) and IGF-II and the IGF-I, IGF-II, and insulin receptor genes and localization of the gene products in the human ovary. J. Clin. Endocrinol. Metab., 77, 1411–1418.[Abstract]
Eppig, J.J. and O'Brien, M.J. (1996) Development in vitro of mouse oocytes from primordial follicles. Biol. Reprod., 54, 197–207.[Abstract]
Erickson, G.F., Garzo, V.G. and Magoffin, D.A. (1989) Insulin-like growth factor-I regulates aromatase activity in human granulosa and granulosa–luteal cells. J. Clin. Endocrinol. Metab., 69, 716–724.[Abstract]
Frasca, F., Pandini, G., Scalia, P. et al. (1999) Insulin receptor isoform A, a newly recognized high-affinity insulin-like growth factor II receptor in fetal and cancer cells. Mol. Cell. Biol., 19, 3278–3288.
Gougeon, A. (1996) Regulation of ovarian follicular development in primates: Facts and hypotheses. Endocr. Rev., 17, 121–155.[ISI][Medline]
Halpin, D.M.G., Jones, A., Fink, G. and Charlton, H.M. (1986) Postnatal ovarian follicle development in hypogonadal (hpg) and normal mice and associated changes in the hypothalamic-pituitary ovarian axis. J. Reprod. Fertil., 77, 287–296.[Abstract]
Hernandez, E.R., Hurwitz, A., Vera, A. et al. (1992) Expression of the genes encoding the insulin-like growth factors and their receptors in the human ovary. J. Clin. Endocrinol. Metab., 74, 419–425.[Abstract]
Hovatta, O., Silye, R., Abir, R. et al. (1997) Extracellular matrix improves survival of both stored and fresh human primordial and primary ovarian follicles in long-term culture. Hum. Reprod., 12, 1032–1036.
Hovatta, O., Wright, C., Krausz, T. et al. (1999) Human primordial, primary and secondary ovarian follicles in long-term culture: effect of partial isolation. Hum. Reprod., 14, 2519–2524.
Li, R., Phillips, D.M. and Mather, J.P. (1995) Activin promotes ovarian follicle development in vitro. Endocrinology, 136, 849–856.[Abstract]
Liu, X., Andoh, K., Yokota, H. et al. (1998) Effects of growth hormone, activin, and follistatin on the development of preantral follicle from immature female mice. Endocrinology, 139, 2342–2347.
Mason, H.D., Willis, D.S., Holly, J.M.P. and Franks, S. (1994) Insulin preincubation enhances insulin-like growth factor-II (IGF-II) action on steroidogenesis in human granulosa cells. J. Clin. Endocrinol. Metab., 78, 1265–1267.[Abstract]
Moller, D.E., Yokota, A., Caro, J.F. and Flier, J.S. (1989) Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man. Mol. Endocrinol., 3, 1263–1269.[Abstract]
Oktay, K., Briggs, D. and Gosden, R.G. (1997) Ontogeny of follicle-stimulating hormone receptor gene expression in isolated human ovarian follicles. J. Clin. Endocrinol. Metab., 82, 3748–3751.
Parrot, J.A. and Skinner, M.K. (1999) Kit-ligand/stem cell factor induces primordial follicle development and initiates folliculogenesis. Endocrinology, 140, 4262–4271.
Qu, J., Godin, P.A., Nisolle, M., Donnez, J. (2000) Expression of receptors for insulin-like growth factor-I and transforming growth factor-ß in human follicles. Mol. Hum. Reprod., 6, 137–145.
Roy, S.K. and Treacy, B.J. (1993) Isolation and long-term culture of human pre-antral follicles. Fertil. Steril., 59, 783–790.[ISI][Medline]
Voutilainen, R., Franks, S., Mason, H.D. and Martikainen, H. (1996) Expression of insulin-like growth factor (IGF), IGF-binding protein, and IGF receptor messenger ribonucleic acids in normal and polycystic ovaries. J. Clin. Endocrinol. Metab., 81, 1003–1008.[Abstract]
Willis, D.S., Mason, H.D., Watson, H. and Franks, S. (1998) Developmentally regulated response of human granulosa cells to insulin-like growth factors (IGFs): IGF-I and IGF-II action mediated via the type-I IGF receptor. J. Clin. Endocrinol. Metab., 83, 1256–1259.
Wright, C.S., Hovatta, O., Margara, R. et al. (1999) Effects of follicle-stimulating hormone and serum substitution on the in-vitro growth of human ovarian follicles. Hum. Reprod., 14, 1555–1562.
Yoshida, H., Takakura, N., Kataoka, H. et al. (1997) Stepwise requirement of c-kit tyrosine kinase in mouse ovarian follicle development. Dev. Biol., 184, 122–137.[ISI][Medline]
Yuan, W. and Giudice, L.C. (1999) Insulin-like growth factor-II mediates the steroidogenic and growth promoting actions of follicle stimulating hormone on human ovarian pre-antral follicles cultured in vitro. J. Clin. Endocrinol. Metab., 84, 1479–1482.
Zhou, J., Chin, E. and Bondy, C. (1991) Cellular pattern of insulin-like growth factor-I (IGF-I) and IGF-I receptor gene expression in the developing and mature ovarian follicle. Endocrinology, 129, 3281–3288.[Abstract]
Zhou, J. and Bondy, C. (1993) Anatomy of the human ovarian insulin-like growth factor system. Biol. Reprod., 48, 467–482.[Abstract]
Zhou, J., Kumar, T.R., Matzuk, M.M. and Bondy, C. (1997) Insulin-like growth factor I regulates gonadotropin responsiveness in the murine ovary. Mol. Endocrinol., 11, 1924–1933.
Submitted on December 16, 1999; accepted on June 5, 2000.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Franks, J. Stark, and K. Hardy Follicle dynamics and anovulation in polycystic ovary syndrome Hum. Reprod. Update, May 22, 2008; (2008) dmn015v1. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. B Carlsson, M. P E Laitinen, J. E Scott, H. Louhio, L. Velentzis, T. Tuuri, J. Aaltonen, O. Ritvos, R. M L Winston, and O. Hovatta Kit ligand and c-Kit are expressed during early human ovarian follicular development and their interaction is required for the survival of follicles in long-term culture. Reproduction, April 1, 2006; 131(4): 641 - 649. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K. A Walters, J. P Binnie, B. K Campbell, D. G Armstrong, and E. E Telfer The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent. Reproduction, March 1, 2006; 131(3): 515 - 523. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
I Demeestere, J Centner, C Gervy, Y Englert, and A Delbaere Impact of various endocrine and paracrine factors on in vitro culture of preantral follicles in rodents Reproduction, August 1, 2005; 130(2): 147 - 156. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 I. Demeestere, C. Gervy, J. Centner, F. Devreker, Y. Englert, and A. Delbaere Effect of Insulin-Like Growth Factor-I During Preantral Follicular Culture on Steroidogenesis, In Vitro Oocyte Maturation, and Embryo Development in Mice Biol Reprod, June 1, 2004; 70(6): 1664 - 1669. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Otala, K. Erkkila, T. Tuuri, J. Sjoberg, L. Suomalainen, A-M. Suikkari, V. Pentikainen, and L. Dunkel Cell death and its suppression in human ovarian tissue culture Mol. Hum. Reprod., March 1, 2002; 8(3): 228 - 236. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||