O-081 High variability of molecular isoforms of AMH in follicular fluid and granulosa cells from human small antral follicles

Abstract Study question Is the composition of AMH isoforms different in follicular fluids (FF) and granulosa cells (GCs) from human small antral follicles? Summary answer There is a high viability of AMH isoforms in FFs and GCs. Even between same size follicles from the same women, the isoform composition differs. What is known already Anti Müllerian Hormone (AMH) is a member of the TGF-β superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive live. AMH is suggested to inhibit aromatase (i.e. CYP19) expression and thereby decreasing the conversion of androgens to oestrogens in humans, especially in small antral follicles before dominance is achieved and thereby act as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH in human follicles is still not clarified. Study design, size, duration This retrospective study measured AMH isoforms in human FF and GCs from small antral follicles using ELISA, Western blot, and immunofluorescence analysis. A total of 41 female adolescents and women aged 15 to 38 years (mean age: 29.7 years), who underwent ovarian tissue cryopreservation (OTC) at the University Hospital of Copenhagen, Rigshospitalet were included between year 2006 and 2020 included. Participants/materials, setting, methods Donated human ovarian medulla tissue were FFs and GCs were obtained in connection with OTC. The following isoforms were evaluated in FFs using ELISA analysis: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. Antibodies specific for the N-terminal and the C-terminal AMH were used in both Western blot and immunofluorescence analysis of FFs and GCs. Main results and the role of chance A negative correlation between follicle diameter and the mentioned AMH forms were detected. Moreover, Western blot analysis detected various AMH forms in both FFs and GCs, which did not match the above-mentioned consensus forms suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size from the same woman. Limitations, reasons for caution The study group is limited and the significance of the variable AMH isoforms compositions between follicles cannot not be clarified from this data. Wider implications of the findings Collectively, these data suggest that intrafollicular processing of AMH is complex and variable, and thus, it may be difficult to develop an antibody based AMH assay that detect all AMH isoforms. Furthermore, the variability between follicles suggests that designing a proper standard will be difficult. Trial registration number not applicable

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High Variability of Molecular Isoforms of AMH in Follicular Fluid and Granulosa Cells From Human Small Antral Follicles

Frontiers in Endocrinology ◽

10.3389/fendo.2021.617523 ◽

2021 ◽

Vol 12 ◽

Author(s):

Linn Salto Mamsen ◽

Jane Alrø Bøtkjær ◽

Stine Gry Kristensen ◽

Susanne Elisabeth Pors ◽

Janni Vikkelsø Jeppesen ◽

...

Keyword(s):

Western Blot ◽

Granulosa Cells ◽

Follicular Fluid ◽

Proteolytic Processing ◽

Late Gestation ◽

Human Follicular Fluid ◽

Antral Follicles ◽

Follicle Diameter ◽

Exact Function ◽

Reproductive Life

Anti-Müllerian hormone (AMH) is a member of the TGF-β superfamily produced by follicular granulosa cells (GCs) in women from late gestation to the end of reproductive life. AMH is thought to inhibit aromatase (i.e., CYP19) expression and decrease the conversion of androgens to oestrogens, especially in small antral follicles before dominance is achieved. Thus, AMH acts as a gatekeeper of ovarian steroidogenesis. However, the exact function and processing of AMH has not been fully elucidated. The present study measured and determined AMH isoforms in human follicular fluid (FF) from small antral follicles and in human GCs using four ELISAs, western blot, and immunofluorescence analysis. We evaluated the presence of the following isoforms: full-length AMH precursor (proAMH), cleaved associated AMH (AMHN,C), N-terminal pro-region (AMHN), and active C-terminal (AMHC) AMH. A negative correlation between follicle diameter and the AMH forms was detected. Moreover, western blot analysis detected various AMH forms in both FFs and GCs, which did not match our consensus forms, suggesting an unknown proteolytic processing of AMH. The presence of these new molecular weight isoforms of AMH differs between individual follicles of identical size in the same woman. This study detected several AMH forms in FF and GCs obtained from human small antral follicles, which suggests that intrafollicular processing of AMH is complex and variable. Thus, it may be difficult to develop an antibody-based AMH assay that detects all AMH isoforms. Furthermore, the variability between follicles suggests that designing a recombinant AMH standard will be difficult.

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Implication of cortisol and 11β-hydroxysteroid dehydrogenase enzymes in the development of porcine (Sus scrofa domestica) ovarian follicles and cysts

Reproduction ◽

10.1530/rep-07-0003 ◽

2007 ◽

Vol 133 (6) ◽

pp. 1149-1158 ◽

Cited By ~ 14

Author(s):

Neera Sunak ◽

Daphne F Green ◽

Lalantha R Abeydeera ◽

Lisa M Thurston ◽

Anthony E Michael

Keyword(s):

Granulosa Cells ◽

Hydroxysteroid Dehydrogenase ◽

Ovarian Follicles ◽

Cyst Fluid ◽

Ovarian Cysts ◽

Follicle Growth ◽

Antral Follicles ◽

Cortisol Metabolism ◽

Porcine Granulosa Cells ◽

Follicle Diameter

This study investigated cortisol inactivation by 11β-hydroxysteroid dehydrogenase (11β HSD) enzymes in porcine granulosa cells from antral follicles at different developmental stages and in ovarian cysts. In granulosa cells, cortisol oxidation increased threefold with antral follicle diameter (P < 0.001). This trend was paralleled by a threefold increase in NADP+-dependent 11β-dehydrogenase activity in granulosa cell homogenates with follicle diameter. Intact granulosa cells from ovarian cysts exhibited significantly lower enzyme activities than cells from large antral follicles. Neither intact cells norcell homogenates displayed net 11-ketosteroid reductase activities. Since porcine follicular fluid (FF) from large antral follicles and ovarian cysts contains hydrophobic inhibitors of glucocorticoid metabolism by type 1 11β HSD, this studyalso investigated whether levels of 11β HSD inhibitors changed during follicle growth and could affect cortisol metabolism in granulosa cells. The extent of inhibition of 11β HSD1 activity in rat kidney homogenates decreased progressively from 50 ± 8% inhibition by FF from small antral follicles (P < 0.001) to 23 ± 6% by large antral FF (P < 0.05). Cyst fluid inhibited 11β HSD1 activity by 59 ± 4% (P < 0.001). Likewise, net cortisol oxidation in granulosa cells was significantly decreased by large antral FF (35–48% inhibition, P < 0.05) and cyst fluid (45–75% inhibition, P < 0.01). We conclude that inactivation of cortisol by 11β HSD enzymes in porcine granulosa cells increases with follicle development but is significantly decreased in ovarian cysts. Moreover, changes in ovarian cortisol metabolism are accompanied by corresponding changes in the levels of paracrine inhibitors of 11β HSD1 within growing ovarian follicles and cysts, implicating cortisol in follicle growth and cyst development.

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11β-Hydroxysteroid dehydrogenase expression and activities in bovine granulosa cells and corpora lutea implicate corticosteroids in bovine ovarian physiology

Journal of Endocrinology ◽

10.1677/joe.1.07025 ◽

2007 ◽

Vol 193 (2) ◽

pp. 299-310 ◽

Cited By ~ 15

Author(s):

L M Thurston ◽

D R E Abayasekara ◽

A E Michael

Keyword(s):

Molecular Mass ◽

Granulosa Cells ◽

Luteal Phase ◽

Hydroxysteroid Dehydrogenase ◽

Follicular Phase ◽

Corpora Lutea ◽

Luteal Cells ◽

Antral Follicles ◽

Follicle Diameter ◽

Dependent Type

Cortisol–cortisone metabolism is catalysed by the bi-directional NADP(H)-dependent type 1 11β-hydroxysteroid dehydrogenase (11βHSD1) enzyme and the oxidative NAD+-dependent type 2 11βHSD (11βHSD2). This study related the expression of 11βHSD1 and 11βHSD2 enzymes (mRNA and protein) to net 11-ketosteroid reductase and 11β-dehydrogenase (11β-DH) activities in bovine follicular granulosa and luteal cells. Granulosa cells were isolated from follicles of < 4, 4–8, > 8 and > 12 mm in diameter in either the follicular or luteal phase of the ovarian cycle. Luteal cells were obtained from corpora lutea (CL) in the early non-pregnant luteal phase. Enzyme expression was assessed by reverse transcription-PCR and western blotting, while enzyme activities were measured over 1 h in cell homogenates using radiometric conversion assays with 100 nM [3H]cortisone or [3H]cortisol and pyridine dinucleotide cofactors. Irrespective of follicle diameter, the expression of 11βHSD2 and NAD+-dependent oxidation of cortisol predominated in granulosa cells harvested in the follicular phase. In contrast, in granulosa cells obtained from luteal phase follicles and in bovine luteal cells, expression of 11βHSD1 exceeded that of 11βHSD2 and the major enzyme activity was NADP+-dependent cortisol oxidation. Increasing follicular diameter was associated with progressive increases in expression and activities of 11βHSD2 and 11βHSD1 in follicular and luteal phase granulosa cells respectively. In follicular phase granulosa cells from antral follicles < 12 mm, 11βHSD1 migrated with a molecular mass of 34 kDa, whereas in the dominant follicle, CL and all luteal phase granulosa cells, a second protein band of 68 kDa was consistently detected. In all samples, 11βHSD2 had a molecular mass of 48 kDa, but in large antral follicles (> 8 mm), there was an additional immunoreactive band at 50 kDa. We conclude that 11βHSD2 is the predominant functional 11βHSD enzyme expressed in follicular phase granulosa cells from growing bovine antral follicles. In contrast, in bovine granulosa cells from dominant or luteal phase follicles, and in bovine luteal cells from early non-pregnant CL, 11βHSD1 is the major glucocorticoid-metabolising enzyme. The increasing levels of cortisol inactivation by the combined NADP+- and NAD+-dependent 11β-DH activities suggest a need to restrict cortisol access to corticosteroid receptors in the final stages of follicle development.

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Composition and morphology of the follicular basal lamina during atresia of bovine antral follicles

Reproduction ◽

10.1530/rep.0.1230097 ◽

2002 ◽

pp. 97-106 ◽

Cited By ~ 33

Author(s):

HF Irving-Rodgers ◽

ML Mussard ◽

JE Kinder ◽

RJ Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Cell Shape ◽

Type Iv Collagen ◽

Basal Cells ◽

Follicle Growth ◽

Electron Microscopic ◽

Antral Follicles ◽

Type Iv ◽

Follicle Diameter

The fate of the follicular basal lamina during atresia was investigated using bovine follicles, in which different follicle phenotypes have been observed. These phenotypes include: healthy follicles with rounded basal granulosa cells with an aligned basal lamina or follicles with columnar basal granulosa cells with a basal lamina of many loops (loopy), and atretic follicles in which either the antral granulosa cells (antral atresia) or the basal cells (basal atresia) die first. Loopy lamina and basal atresia occur only in small antral follicles < 5 mm in diameter. Follicles were collected from cattle of unknown reproductive history and processed for immunohistochemistry and electron microscopy, and from animals in which follicle growth had been monitored by daily measurements of follicle diameter by ultrasonography. Electron microscopic observations of dominant follicles during the growth phase, plateau and regression showed that the basal lamina was still visible and intact upon atresia. These follicles had a conventional aligned basal lamina, which they retained, except for some degree of folding, as they progressed into antral atresia. In small follicles (2-5 mm in diameter), the basal cell shape (rounded or columnar) and appearance of the basal lamina (aligned or of many loops) did not appear to be related to the type of atresia. On atresia the follicular basal laminae retained immunoreactive laminin alpha1 and beta2, type IV collagen alpha1 and nidogen. Laminin alpha2, which may come from the theca, was present in the follicular basal lamina of only 22% of healthy follicles, but was expressed very commonly in 71% of the atretic follicles. Laminin alpha2 expression was found in both phenotypes of healthy follicles, antral and basal atretic follicles, and follicles with aligned or loopy basal laminae. It is concluded that the basal lamina is not degraded upon atresia, but does undergo a variety of other changes.

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Changes in telomere length and senescence markers during human ovarian tissue cryopreservation

Scientific Reports ◽

10.1038/s41598-021-81973-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Boram Kim ◽

Ki-Jin Ryu ◽

Sanghoon Lee ◽

Tak Kim

Keyword(s):

Western Blot ◽

Telomere Length ◽

Blot Analysis ◽

Western Blot Analysis ◽

Ovarian Tissue ◽

University Hospital ◽

Ovarian Tissue Cryopreservation ◽

Human Ovarian Tissue ◽

Tissue Cryopreservation ◽

Human Ovarian Tissue Cryopreservation

AbstractOvarian tissue cryopreservation is considered as a useful option to preserve fertility for cancer patients. This study purposed to evaluate the change of telomere length and senescence markers during human ovarian tissue cryopreservation. Ovarian tissues were obtained from women who underwent benign ovarian surgery in the gynecology research unit of a university hospital with prior consent and IRB approval. DNA was extracted from the ovarian tissues using a DNeasy tissue kit and telomere lengths in the DNA samples were determined by real time PCR before and after cryopreservation. All tissues were stained with hematoxylin–eosin and subjected to immunohistochemistry and TUNEL assays. Other senescence markers, including p53, p16, p21, and phospho-pRb proteins, were evaluated using western blot analysis. Ovarian tissues were collected from ten patients and prepared for slow freezing with the same size of diameter 4 mm and 1 mm thickness. Mean age of patients was 26.7 ± 6.2 years (range, 16–34 years), and ovarian tissues were cryopreserved and thawed 4 weeks after cryopreservation. The mean telomere length was significantly decreased after cryopreservation (9.57 ± 1.47 bp vs. 8.34 ± 1.83 bp, p = 0.001). Western blot analysis revealed that p53, p16, and p21 proteins increased and phospho-pRb protein expression decreased after ovarian tissue cryopreservation. Ovarian tissue cryopreservation and transplantation is regarded as one of promising options for fertility preservation. However, clinicians and researchers should be aware of possible irreversible DNA changes such as shortening of telomere length and alterations of other senescence markers in human ovarian tissues.

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Early postnatal methoxychlor exposure inhibits folliculogenesis and stimulates anti-Mullerian hormone production in the rat ovary

Journal of Endocrinology ◽

10.1677/joe.1.06592 ◽

2006 ◽

Vol 191 (3) ◽

pp. 549-558 ◽

Cited By ~ 52

Author(s):

Mehmet Uzumcu ◽

Peter E Kuhn ◽

Jason E Marano ◽

AnnMarie E Armenti ◽

Lisa Passantino

Keyword(s):

Western Blot ◽

Granulosa Cells ◽

Blot Analysis ◽

Western Blot Analysis ◽

Endocrine Disruptor ◽

Follicular Development ◽

Immature Rat ◽

Antral Follicles ◽

Ovarian Morphology ◽

Rat Ovary

Methoxychlor [1,1,1-trichloro-2,2-bis(4-methoxyphenyl) ethane; MXC] is a chlorinated hydrocarbon pesticide commonly used in the United States as a replacement for DDT [1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane]. While MXC is a weak estrogenic compound, its more active, major metabolite [2,2-bis(p-hydroxyphenyl)-1,1,1-trichloroethane; HPTE] shows estrogenic, anti-estrogenic, or anti-androgenic properties depending on the receptor subtype with which it interacts. Anti-Mullerian hormone (AMH) is a paracrine factor that suppresses initial follicle recruitment in the ovary. Studies have shown the effects of exposure to MXC on adult ovarian morphology and function. However, the effect of exposure to MXC at an early postnatal stage on pre-pubertal follicular development and ovarian AMH production has not been studied. Around postnatal day (P) 4, most of the primordial follicular assembly in rats is complete, and a large number of primordial follicles transition into the primary follicle stage, a process that is inhibited by estrogen. The objective of this study was to examine the effect of early postnatal (P3–P10) MXC exposure on ovarian morphology and size, follicle number, and AMH production in the pre-pubertal (P20) rat ovary and to investigate the effect of HPTE on AMH production in immature rat granulosa cells in vitro. Female rats were injected (s.c.) daily with vehicle (control) or 1, 10, 50, 100, or 500 mg MXC/kg per day (referred to here as 1MXC, 10MXC, and so forth.) between P3 and P10. On P20, uterine and ovarian weights were determined, ovarian histology was examined, and follicles were counted and classified into primordial, primary, secondary, pre-antral, or antral stages using the two largest serial sections at the center of the ovary. Ovarian AMH production was examined using immunohistochemistry and western blot analysis. The effect of HPTE (0.5–25 μM) on AMH production in cultured immature rat granulosa cells was determined by western blot analysis. Ovarian weight was reduced by 50, 100, and 500MXC (P < 0.01). MXC treatment inhibited folliculogenesis. Both 100 and 500MXC had a reduced number of antral follicles (P < 0.05) with a concomitant increase in pre-antral follicles (P < 0.05). Follicle numbers were not significantly affected by 1, 10, or 50MXC. Total follicle number and the number of primordial, primary, or secondary stage follicles were not significantly different in all treatment groups. Immunohistochemistry showed that MXC-treated ovaries had more AMH-positive follicles with stronger AMH immunostaining. Western blot analysis showed that AMH production was 1.6 ± 0.2, 1.85 ± 0.6, and 2.2 ± 0.5 times higher in the 50, 100, and 500MXC ovaries as compared with the control ovaries respectively (P < 0.05). Granulosa cells treated with 1 or 5 μM HPTE had significantly greater AMH production (P < 0.05). These results demonstrate that MXC inhibits early ovarian development and stimulates AMH production directly in the rat ovary. In addition, HPTE was shown to stimulate AMH production in rat granulosa cells. Endocrine disruptors are widespread in the environment, and MXC represents a model endocrine disruptor due to the multiple actions of its metabolites. This study confirms that the endocrine disruptor MXC inhibits follicular development and demonstrates for the first time that MXC and HPTE directly stimulate AMH productionin the ovary. Thisnovel finding suggests that elevated AMH may play a role in MXC’s inhibitory effect in the ovary.

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Ultrastructure of the basal lamina of bovine ovarian follicles and its relationship to the membrana granulosa

Reproduction ◽

10.1530/reprod/118.2.221 ◽

2000 ◽

pp. 221-228 ◽

Cited By ~ 7

Author(s):

HF Irving-Rodgers ◽

RJ Rodgers

Keyword(s):

Basal Lamina ◽

Granulosa Cells ◽

Light And Electron Microscopy ◽

Single Layer ◽

Antral Follicle ◽

Basal Cells ◽

Preantral Follicles ◽

Antral Follicles ◽

Cellular Processes ◽

Innermost Layer

Different morphological phenotypes of follicular basal lamina and of membrana granulosa have been observed. Ten preantral follicles (< 0. 1 mm), and 17 healthy and six atretic antral follicles (0.5-12 mm in diameter) were processed for light and electron microscopy to investigate the relationship the between follicular basal lamina and membrana granulosa. Within each antral follicle, the shape of the basal cells of the membrana granulosa was uniform, and either rounded or columnar. There were equal proportions of follicles </= 4 mm in diameter with columnar basal cells and with rounded basal cells. Larger follicles had only rounded basal cells. Conventional basal laminae of a single layer adjacent to the basal granulosa cells were observed in healthy follicles at the preantral and antral stages. However, at the preantral stage, the conventional types of basal lamina were enlarged or even partially laminated. A second type of basal lamina, described as 'loopy', occurred in about half the preantral follicles and in half the antral follicles </= 4 mm diameter. 'Loopy' basal laminae were not observed in larger follicles. 'Loopy' basal laminae were composed of basal laminae aligning the basal surface of basal granulosa cells, but with additional layers or loops often branching from the innermost layer. Each loop was usually < 1 microm long and had vesicles (20-30 nm) attached to the inner aspect. Basal cellular processes were also common, and vesicles could be seen budding off from these processes. In antral follicles, conventional basal laminae occurred in follicles with rounded basal granulosa cells. Other follicles with columnar cells, and atretic follicles, had the 'loopy' basal lamina phenotype. Thus, follicles have different basal laminae that relate to the morphology of the membrana granulosa.

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Deleted in malignant brain tumor 1 is secreted in the oviduct and involved in the mechanism of fertilization in equine and porcine species

Reproduction ◽

10.1530/rep-13-0007 ◽

2013 ◽

Vol 146 (2) ◽

pp. 119-133 ◽

Cited By ~ 29

Author(s):

Barbara Ambruosi ◽

Gianluca Accogli ◽

Cécile Douet ◽

Sylvie Canepa ◽

Géraldine Pascal ◽

...

Keyword(s):

Brain Tumor ◽

Western Blot ◽

Immunohistochemical Analysis ◽

Fertilization Rate ◽

Malignant Brain Tumor ◽

Estrus Cycle ◽

Immunofluorescence Analysis ◽

Oviductal Fluid ◽

Porcine Spermatozoa

Oviductal environment affects preparation of gametes for fertilization, fertilization itself, and subsequent embryonic development. The aim of this study was to evaluate the effect of oviductal fluid and the possible involvement of deleted in malignant brain tumor 1 (DMBT1) on IVF in porcine and equine species that represent divergent IVF models. We first performed IVF after pre-incubation of oocytes with or without oviductal fluid supplemented or not with antibodies directed against DMBT1. We showed that oviductal fluid induces an increase in the monospermic fertilization rate and that this effect is canceled by the addition of antibodies, in both porcine and equine species. Moreover, pre-incubation of oocytes with recombinant DMBT1 induces an increase in the monospermic fertilization rate in the pig, confirming an involvement of DMBT1 in the fertilization process. The presence of DMBT1 in the oviduct at different stages of the estrus cycle was shown by western blot and confirmed by immunohistochemical analysis of ampulla and isthmus regions. The presence of DMBT1 in cumulus–oocyte complexes was shown by western blot analysis, and the localization of DMBT1 in the zona pellucida and cytoplasm of equine and porcine oocytes was observed using immunofluorescence analysis and confocal microscopy. Moreover, we showed an interaction between DMBT1 and porcine spermatozoa using surface plasmon resonance studies. Finally, a bioinformatic and phylogenetic analysis allowed us to identify the DMBT1 protein as well as a DMBT1-like protein in several mammals. Our results strongly suggest an important role of DMBT1 in the process of fertilization.

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Ovarian tissue freezing and activation after thawing: an update

Middle East Fertility Society Journal ◽

10.1186/s43043-021-00056-5 ◽

2021 ◽

Vol 26 (1) ◽

Author(s):

Li-fan Peng

Keyword(s):

Granulosa Cells ◽

Clinical Decision Making ◽

Ovarian Function ◽

Ovarian Tissue ◽

Clinical Decision ◽

The Body ◽

Endocrine Function ◽

Ovarian Tissue Cryopreservation ◽

Main Body ◽

Tissue Cryopreservation

Abstract Background With the growth of women’s age, ovarian failure can be caused by various factors. For the women who need chemotherapy because of cancer factors, the preservation of fertility is more urgent. The treatment of cancer is also a process in which all tissues and organs of the body are severely damaged, especially in the reproductive system. Main body As a new fertility preservation technology, autologous ovarian tissue cryopreservation and transplantation is developing rapidly and showing great potentiality in preserving ovarian endocrine function of young cervical cancer patients. Vitrification and slow freezing are two common techniques applied for ovarian tissue cryopreservation. Thus, cryopreserved/thawed ovarian tissue and transplantation act as an important method to preserve ovarian function during radiotherapy and chemotherapy, and ovarian cryopreservation by vitrification is a very effective and extensively used method to cryopreserve ovaries. The morphology of oocytes and granulosa cells and the structure of organelles were observed under the microscope of histology; the hormone content in the stratified culture medium of granulosa cells with the diameter of follicle was used to evaluate the development potential of ovarian tissue, and finally the ovarian tissue stimulation was determined by the technique of ovarian tissue transplantation. Conclusions Although there are some limitations, the team members still carry out this review to provide some references and suggestions for clinical decision-making and further clinical research.

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The effects of IGF-I on bovine follicle development and IGFBP-2 expression are dose and stage dependent

Reproduction ◽

10.1530/rep.1.00682 ◽

2006 ◽

Vol 131 (3) ◽

pp. 515-523 ◽

Cited By ~ 30

Author(s):

Kirsty A Walters ◽

John P Binnie ◽

Bruce K Campbell ◽

David G Armstrong ◽

Evelyn E Telfer

Keyword(s):

Growth Factor ◽

Granulosa Cells ◽

Size Range ◽

Regulatory System ◽

Follicular Development ◽

High Dose ◽

Insulin Like Growth Factor ◽

High Concentration ◽

Antral Follicles ◽

Igf I

This study aimed to determine the effect of insulin-like growth factor-I (IGF-I) on early antral bovine follicular development, and the expression of insulin-like growth factor-binding protein-2 (IGFBP-2). Antral follicles separated into three different size groups were cultured for 6 days in medium supplemented with either a low (10 ng/ml) or high (1 μg/ml) dose of human recombinant IGF-I. Oestradiol production by follicles in all size ranges, cultured in the presence of the high concentration of IGF-I, significantly increased by day 6 (P < 0.05). Follicles in the smallest size range, 165–215 μm, cultured in a high dose of IGF-I, were found to be significantly increased in size (P < 0.01). Oocyte health of the largest follicles (281–380 μm) was significantly improved by the addition of IGF-I to the culture medium. mRNA expression of IGFBP-2 was decreased in the granulosa cells of follicles, size range 216–280 μm, cultured with a high dose of IGF-I (P < 0.05). Granulosa cells (P < 0.05) and oocytes (P < 0.01) of the largest follicles (281–380 μm) showed a decrease in IGFBP-2 expression (protein) when cultured in the control and low-IGF-I treatment groups. Therefore, the response of a bovine follicle to IGF-I is both dose and stage dependent. This work supports a role for IGF-I in modulating somatic and germ-cell maturation and development in early antral follicles. Furthermore, the inverse relationship between the level of IGF-I stimulation and IGFBP-2 expression suggests a local regulatory system modulating IGF-I availability.

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