Growth And Differentiation Factor 9 Research Articles

Using sheep genomes from diverse U.S. breeds to identify missense variants in genes affecting fecundity.

Background: Access to sheep genome sequences significantly improves the chances of identifying genes that may influence the health, welfare, and productivity of these animals. Methods: A public, searchable DNA sequence resource for U.S. sheep was created with whole genome sequence (WGS) of 96 rams. The animals shared minimal pedigree relationships and represent nine popular U.S. breeds and a composite line. The genomes are viewable online with the user-friendly Integrated Genome Viewer environment, and may be used to identify and decode gene variants present in U.S. sheep. Results: The genomes had a combined average read depth of 16, and an average WGS genotype scoring rate and accuracy exceeding 99%. The utility of this resource was illustrated by characterizing three genes with 14 known coding variants affecting litter size in global sheep populations: growth and differentiation factor 9 ( GDF9), bone morphogenetic protein 15 ( BMP15), and bone morphogenetic protein receptor 1B ( BMPR1B). In the 96 U.S. rams, nine missense variants encoding 11 protein variants were identified. However, only one was previously reported to affect litter size ( GDF9 V371M, Finnsheep). Two missense variants in BMP15 were identified that had not previously been reported: R67Q in Dorset, and L252P in Dorper and White Dorper breeds. Also, two novel missense variants were identified in BMPR1B: M64I in Katahdin, and T345N in Romanov and Finnsheep breeds. Based on the strict conservation of amino acid residues across placental mammals, the four variants encoded by BMP15 and BMPR1B are predicted to interfere with their function. However, preliminary analyses of litter sizes in small samples did not reveal a correlation with variants in BMP15 and BMPR1B with daughters of these rams. Conclusions: Collectively, this report describes a new resource for discovering protein variants in silico and identifies alleles for further testing of their effects on litter size in U.S. breeds.

FecXBar a Novel BMP15 mutation responsible for prolificacy and female sterility in Tunisian Barbarine Sheep

BackgroundNaturally occurring mutations in growth and differentiation factor 9 (GDF9) or bone morphogenetic protein 15 (BMP15) genes are associated with increased ovulation rate (OR) and litter size (LS) but also sterility. Observing the Tunisian Barbarine ewes of the “W” flock selected for improved prolificacy, we found prolific and infertile ewes with streaky ovaries. Blood genomic DNA was extracted from a subset of low-ovulating, prolific and infertile ewes of the “W” flock, and the entire coding sequences of GDF9 and BMP15 were sequenced.ResultsWe evidenced a novel polymorphism in the exon 1 of the BMP15 gene associated with increased prolificacy and sterility. This novel mutation called FecXBar is a composite polymorphism associating a single nucleotide substitution (c.301G > T), a 3 bp deletion (c.302_304delCTA) and a C insertion (c.310insC) in the ovine BMP15 cDNA leading to a frame shift at protein position 101. Calculated in the “W” flock, the FecXBar allele increased OR by 0.7 ova and LS by 0.3 lambs (p = 0.08). As for already identified mutations, homozygous females carrying FecXBar exhibited streaky ovaries with a blockade at the primary stage of folliculogenesis as shown by histochemistry.ConclusionsOur investigation demonstrates a new mutation in the BMP15 gene providing a valuable genetic tool to control fecundity in Tunisian Barbarine, usable for diffusion program into conventional flocks looking for prolificacy improvement.

Growth and differentiation factor 9 promotes oocyte growth at the primary but not the early secondary stage in three-dimensional follicle culture.

Factors that differentially regulate oocyte and granulosa cell growth within the early preantral follicle and how these factors differ at each stage of follicle growth remain poorly understood. The aim of this study was to isolate and evaluate the effect of recombinant growth and differentiation factor 9 (GDF9) on oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle growth during in vitro culture. Primary stage follicles (diameters of 50-89μm) and early secondary stage follicles (diameters of 90-120μm) were isolated from immature mice, and individual, intact follicles were cultured in vitro in the presence and absence of recombinant GDF9. The effects of GDF9 on follicle growth were determined by the assessment of changes in the follicle volume during culture. The growth of the granulosa cell and oocyte compartments of the follicles was evaluated separately at each stage. GDF9 significantly increased the growth of isolated follicles at both the primary and early secondary follicle stages. Independent evaluation of the granulosa cell and oocyte compartments revealed that, while GDF9 promoted granulosa cell growth at both stages of folliculogenesis, oocyte growth was stage specific. GDF9 promoted growth of the oocyte at the primary, but not the early secondary, follicle stage. These findings demonstrate a stage-specific role for GDF9 in the regulation of oocyte and granulosa cell growth at the primary and early secondary stages of preantral follicle development.

Expression of growth and differentiation Factor 9 and cognate receptors during final follicular growth in cattle

Mutations in growth and differentiation factor 9 (GDF9) gene are associated to sterility or, paradoxically, increased ovulation rate in ewes. Despite its importance, the exact function of GDF9 in ovarian physiology is still poorly understood. This study aimed to investigate GDF9 function during dominant follicle growth and its regulation in follicular fluid. The regulation of GDF9 receptors in GnRH/LH-stimulated granulosa cells was also investigated. In a first experiment, a new follicular wave was induced and the intrafollicular GDF9 treatment into the largest growing follicle (8.5-9.5 mm) at both 100 (n = 3) and 1000ng/ml (n = 4) had no effect on follicular growth, estrus manifestation and ovulation compared to control (PBSinjected) follicles (n = 3). In a second experiment, follicles were obtained just after follicular deviation (day 4 after follicular emergence) and the abundance of GDF9 in follicular fluid did not differ between healthy dominant (n = 4) and atretic subordinate follicles (n = 4), as assessed by western blot analysis. Finally, mRNA expression of BMPR2 and TGFBR1 receptors was evaluated in granulosa cells obtained from preovulatory follicles (>12 mm diameter) obtained 0, 3, 6, 12 or 24 h after i.m. GnRH administration (n = 4-5 follicles/moment). Both receptors were significantly up regulated 12 h after GnRH treatment. Present results do not confirm the hypothesis that GDF9 inhibits dominant follicle growth and suggests a minor role in determining follicle fate. In the other hand, GDF9 receptors regulation in GnRH/LH-stimulated granulosa cells provides the first in vivo evidence of its involvement in the complex cascade of events that culminates in ovulation and luteinization in cattle.

Identification and characterization of epithelial cells derived from human ovarian follicular fluid.

IntroductionFollicular fluid is important for follicular development and oocyte maturation. Evidence suggests that follicular fluid is not only rich in proteins but cells. Besides oocytes, the follicular fluid contains granulosa, thecal, and ovarian surface epithelial cells, and both granulosa and thecal cells are well-characterized. However, epithelial cells in follicular fluid are poorly studied. This study aims to isolate and characterize in vitro epithelial cells that originate from human ovarian follicular fluid retrieved in the assisted fertilization program.MethodsFollicular fluid samples were collected from 20 women in the assisted reproduction program. Epithelial cells were characterized by flow cytometry assay, immunofluorescence staining, real-time PCR, and time lapse photography.ResultsEpithelial cell cultures were established from 18 samples. A small population of epithelial cells expresses germ-line stem cell markers, such as octamer-binding transcription factor 4 (OCT4), NANOG, and DEAD box polypeptide 4 (DDX4). In the epithelial cell culture system, oocyte-like cells formed spontaneously in vitro and expressed the following transcription markers: deleted in azoospermia-like (DAZL), developmental pluripotency associated protein 3 stella-related protein (STELLA), zona pellucida gene family C (ZPC), Syntaptonemal complex protein (SCP), and growth and differentiation factor 9 (GDF9). Some of the oocyte-like cells developed a zona pellucida-like structure. Both the symmetric and asymmetric division split of epithelial cells and early developing oocytes were observed using time lapse photography. Cell colonies were formed during epithelial culturing, which maintained and proliferated in an undifferentiated way on the feeder layer and expressed some pluripotency markers. These colonies differentiated in vitro into various somatic cell types in all three germ layers, but did not form teratoma when injected into immunodeficient mice. Furthermore, these epithelial cells could be differentiated directly to functional hepatocyte-like cells, which do not exist in ovarian tissues.ConclusionsThe epithelial cells derived from follicular fluid are a potential stem cell source with a pluripotent/multipotent character for safe application in oogenesis and regenerative medicine.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-015-0004-6) contains supplementary material, which is available to authorized users.

Evolutionary Origin of Bone Morphogenetic Protein 15 and Growth and Differentiation Factor 9 and Differential Selective Pressure Between Mono- and Polyovulating Species1

Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are TGFbeta-like oocyte-derived growth factors involved in ovarian folliculogenesis as critical regulators of many granulosa cell processes and ovulation rate. Ovarian phenotypic effect caused by alterations in BMP15 and GDF9 genes appears to differ between species and may be relevant to their mono- or polyovulating status. Through phylogenetic analysis we recently showed that these two paralogous genes are strongly divergent and in rapid evolution as compared to other members of the TGFbeta superfamily. Here, we evaluate the amino acid substitution rates of a set of proteins implicated in the ovarian function, including BMP15 and GDF9, with special attention to the mono- or polyovulating status of the species. Among a panel of mono- and polyovulating mammals, we demonstrate a better conservation of some areas in BMP15 and GDF9 within mono-ovulating species. Homology modeling of BMP15 and GDF9 homodimer and heterodimer 3-D structures was suggestive that these areas may be involved in dimer formation and stability. A phylogenetic study of BMP15/GDF9-related proteins reveals that these two genes diverged from the same ancestral gene along with BMP3 and GDF10, two other paralogous genes. A substitution rate analysis based on this phylogenetic tree leads to the hypothesis of an acquisition of BMP15/GDF9-specific functions in ovarian folliculogenesis in mammals. We propose that high variations observed in specific areas of BMP15 and GDF9 in polyovulating species change the equilibrium between homodimers and heterodimers, modifying the biological activity and thus allowing polyovulation to occur.

The fundamental role of bone morphogenetic protein 15 in ovarian function and its involvement in female fertility disorders.

A large number of studies have contributed to understanding the general mechanisms driving ovarian folliculogenesis in humans and show a complex endocrine dialog between the central nervous system, the pituitary and the ovary, integrated by various intraovarian paracrine messages. The role of intraovarian paracrine regulation has acquired more relevance in the recent years owing to the discovery of previously unknown factors, such as the oocyte-derived bone morphogenetic protein (BMP)15. A thorough literature search was carried out in order to summarize what has been reported so far on the role of BMP15, and the BMP15 paralog, growth and differentiation factor 9 (GDF9), in ovarian function and female fertility. Research articles published in English until March 2014 were included. The biological actions of BMP15 include: (i) the promotion of follicle growth and maturation starting from the primary gonadotrophin-independent phases of folliculogenesis; (ii) the regulation of follicular granulosa cell (GC) sensitivity to FSH action and the determination of ovulation quota; (iii) the prevention of GC apoptosis and (iv) the promotion of oocyte developmental competence. The existence of biologically active heterodimers with GDF9, and/or the synergistic co-operation of BMP15 and GDF9 homodimers are indeed relevant in this context. Experimental disruption of the bmp15 gene in mice resulted in a mild fertility defect limited to females, whereas natural missense mutations in ewes cause variable phenotypes (ranging from hyperprolificacy to complete sterility) depending on a fine gene dosage mechanism also involving GDF9. Strong evidence supports the concept that such a mechanism plays an important role in the regulation of ovulation rate across mammalian and non-mammalian species. Following the discovery of sheep fecundity genes, several research groups have focused on alterations in human BMP15 associated with primary ovarian insufficiency (POI) or polycystic ovary syndrome. Several variants of BMP15 are significantly associated with POI supporting their pathogenic role, but the underlying biological mechanism is still under investigation and of great interest in medicine. BMP15 maps to the Xp locus involved in the determination of the ovarian defect in Turner syndrome and significantly contributes to the determination of ovarian reserve. Pioneering studies in women undergoing controlled ovarian stimulation indicate that BMP15 may represent a marker of ovarian response or oocyte quality. BMP15, an oocyte-derived growth and differentiation factor, is a critical regulator of folliculogenesis and GC activities. Variations in BMP15 gene dosage have a relevant influence on ovarian function and can account for several defects of female fertility. The modulation of BMP15 action may have interesting pharmacological perspectives and the analysis of BMP15 may become a useful marker in IVF procedures. Recent outcomes indicate that the close interactions of BMP15/GDF9 have a critical biological impact that should be taken into account in future studies.

Effects of Vaccination against GDF9 and BMP15 on Fertility and Ovarian Function in the White-tailed Deer

The physiological mechanisms controlling ovarian follicular growth and ovulation involve a complex exchange of systemic signals and a localized exchange of molecules between the oocyte and surrounding somatic cells. It has been demonstrated that the oocyte itself plays an essential role in regulating these processes by secreting two key regulatory proteins: bone morphogenetic protein-15 (BMP15) and growth and differentiation factor-9 (GDF9). Natural mutations in their expression and vaccination against these growth factors have been shown to cause sterility in sheep. The aim of this 3-year study was to determine the effect of vaccination against GDF9 and BMP15 on fertility in female white-tailed deer. Does were randomly assigned to two groups (n = 10/group). Each doe received a primary vaccination followed by a booster 47 days later. After the first year, a subset of animals (n = 4-5/group) received an additional booster vaccination. Blood samples were collected at regular intervals to determine antibody titers and progesterone concentrations. Early pregnancy was assessed by ultrasound, and fawning was subsequently monitored. For the BMP15 group, over the 3 years 80%, 100%, and 75% of does fawned with fawning rates of 2.4, 3.5, and 3.3 fawns/doe, respectively. For the GDF9 group, 80%, 25%, and 25% of does fawned with fawning rates of 2.6, 1, and 2 fawns/doe, respectively. The fawning rate of untreated animals in the herd was 1.8 fawns/doe. Although all animals in the BMP15 group had high antibody titers, they were not made infertile, and they became more fecund. Most animals in the GDF9 group had high antibody titers, but fecundity was not affected the first year; however, they were made infertile in Years 2 and 3. Results from this study demonstrate that vaccination against GDF9 has potential to control fertility in deer. Further research will be required to determine the appropriate timing for administering the vaccine and the longevity of effect.

Effects of culture and transplantation on follicle activation and early follicular growth in neonatal mouse ovaries

Mouse models have been widely utilized to elucidate the basic principles and regulatory mechanisms of primordial follicle activation. Outside their natural environment, the growth of follicles might be affected by unknown factors in vitro and the elimination of regulation in vivo. Currently, in vitro culture and transplantation of ovaries under the kidney capsule are two commonly used incubation methods. However, the limited number of studies that have been published compare various incubation systems and reveal differences between ovaries that are incubated and grown in vivo. We compare the number of primordial, primary and secondary follicles in cultured, transplanted and in-vivo-grown ovaries. We investigate the expression levels of four genes, including zona pellucida 3 (ZP3), growth and differentiation factor-9 (GDF-9), proliferating cell nuclear antigen (PCNA) and anti-Müllerian hormone (AMH). Our results suggest that in vitro culture accelerates follicle activation, delays the transition from primary to secondary follicles and affects the expression patterns of ZP3, GDF-9, PCNA and AMH. A larger number of secondary follicles in ovaries cultured in alpha-minimal essential medium (α-MEM) had intact zona pellucida compared with those grown in Dulbecco’s modified Eagle medium containing Ham’s F-12 nutrient mixture (D/F12), suggesting that α-MEM is a better basal medium. The transplanted ovaries demonstrated the most similar characteristics to the in-vivo-grown ovaries, indicating that transplantation provided an optimal environment for ovarian incubation. This study has thus established the similarities and differences between in-vivo-grown and incubated ovaries, demonstrated that transplantation can mostly mimic the environment of ovarian growth in vivo and determined the optimal basal culture medium between α-MEM and D/F12.Electronic supplementary materialThe online version of this article (doi:10.1007/s00441-013-1678-7) contains supplementary material, which is available to authorized users.

Effects of FSH on the expression of receptors for oocyte-secreted factors and members of the EGF-like family during in vitro maturation in cattle

FSH induces expansion of bovine cumulus-oocyte complexes (COCs) in cattle, which can be enhanced by oocyte-secreted factors (OSFs). In this study it was hypothesised that FSH stimulates COC expansion in part from direct stimulation of the epidermal growth factor (EGF)-like ligands amphiregulin (AREG), epiregulin (EREG) and betacellulin (BTC), but also in part through regulation of OSFs or their receptors in cumulus cells. Bovine COCs were cultured in defined medium with graded doses of FSH. In the absence of FSH, COCs did not expand. FSH caused cumulus expansion, and increased the abundance of AREG and EREG mRNA in a time- and dose-dependent manner, but decreased BTC mRNA levels. FSH had modest stimulatory effects on the levels of mRNA encoding the bone morphogenetic protein 15 (BMP15) receptor, BMPR1B, in cumulus cells, but did not alter mRNA expression of the growth and differentiation factor 9 (GDF9) receptor, TGFBR1. More interestingly, FSH dramatically stimulated levels of mRNA encoding two receptors for fibroblast growth factors (FGF), FGFR2C and FGFR3C, in cumulus cells. FSH also stimulated mRNA expression of FGFR1B, but not of FGFR2B in cumulus cells. Based on dose-response studies, FGFR3C was the receptor most sensitive to the influence of FSH. This study demonstrates that FSH stimulates the expression of EGF-like factors in bovine cumulus cells, and provides evidence that FSH differently regulates the expression of distinct receptors for OSFs in cumulus cells.

Regulation of the ovarian reserve by members of the transforming growth factor beta family

Genetic or environmental factors that affect the endowment of oocytes, their assembly into primordial follicles, or their subsequent entry into the growing follicle pool can disrupt reproductive function and may underlie disorders such as primary ovarian insufficiency. Mouse models have been instrumental in identifying genes important in ovarian development, and a number of genes now associated with ovarian dysfunction in women were first identified as causing reproductive defects in knockout mice. The transforming growth factor beta (TGFB) family consists of developmentally important growth factors that include the TGFBs, anti-Müllerian hormone (AMH), activins, bone morphogenetic proteins (BMPs), and growth and differentiation factor 9 (GDF9). The ovarian primordial follicle pool is the source of oocytes in adults. Development of this pool can be grossly divided into three key processes: (1) establishment of oocytes during embryogenesis followed by (2) assembly and (3) activation of the primordial follicle. Disruptions in any of these processes may cause reproductive dysfunction. Most members of the TGFB family show pivotal roles in each of these areas. Understanding the phenotypes of various mouse models for this protein family will be directly relevant to understanding how disruptions in TGFB family signaling result in reproductive diseases in women and will present new areas for development of tailored diagnostics and interventions for infertility.

Follicle Selection in the Avian Ovary

Follicle development in the highly efficient laying hen is characterized by a well-organized follicular hierarchy. This is not the case in other chickens such as the broiler breeder hen that has excessive follicle development and lower reproductive efficiency. Although management practices can optimize egg production in less productive breeds of chickens, the factors that contribute to this difference are not known. Interactions between the oocyte and surrounding somatic cells are believed to be involved in promoting follicle selection. Anti-Müllerian hormone (AMH) has been shown to have a role in regulating rate of follicle development in mammals. In hens, the expression of AMH is restricted to the growing population of follicles and, similar to mammals, is markedly decreased at around the time of follicle selection. The oocyte factors, growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15), have been identified in the hen, and their expression pattern has been characterized. Anti-Müllerian hormone expression in hens is decreased by a protein factor from the oocyte (not GDF9) and is also decreased by vitamin D. Associated with the decrease in AMH expression by vitamin D, follicle-stimulating hormone receptor mRNA is increased. These data suggest that information about AMH regulation may enhance our understanding of follicle selection, particularly in birds with aberrant follicle development.

Inhibition of Follicular Development Induced by Chronic Unpredictable Stress Is Associated with Growth and Differentiation Factor 9 and Gonadotropin in Mice1

Chronic psychosocial stress negatively affects ovarian function. Ovarian follicular development is regulated by both pituitary-derived gonadotropins and intraovarian regulatory factors. To date, the suppressive effects of chronic stress on the ovary have been observed to be manifested mainly as an inhibition of gonadotropin release. It is not clear whether there are any other intraovarian regulatory mechanisms involved in this process. Growth and differentiation factor 9 (GDF9) is an important, oocyte-specific paracrine regulator required for follicular development. In this study, the chronic unpredictable mild stress model was used to produce psychosocial stress in mice. The number of different developmental stages of follicles was counted on ovarian sections stained with hematoxylin and eosin. Real-time PCR and Western blotting were used to detect the mRNA and protein levels, respectively, of GDF9. The results show that chronic unpredictable stress inhibits follicular development, increases follicular atresia, and suppresses GDF9 expression. Exogenous gonadotropin treatment partly restores the repressed antral follicular development, but has no effect on the repressed secondary follicular development associated with chronic stress. Treatment with recombinant GDF9 restores secondary follicular development. Cotreatments with GDF9 and gonadotropins restore both secondary and antral follicular development in stressed mice. These findings demonstrate that inhibition of follicular development induced by chronic unpredictable stress is associated with GDF9 and gonadotropin.

GDF-9 and bFGF enhance the effect of FSH on the survival, activation, and growth of cattle primordial follicles

This study aims to investigate the effects of follicle stimulating hormone (FSH) in combination with growth and differentiation factor-9 (GDF-9) or basic fibroblast growth factor (bFGF) on the activation, survival and growth of cattle primordial follicles. Ovarian tissues were cultured for 3, 7, 14, 22 days in α minimum essential medium (α-MEM) supplemented with FSH, FSH+GDF-9 or FSH+bFGF. Non-cultured and cultured ovarian fragments were processed for histological and TUNEL analysis. Compared to the FSH medium, the results showed FSH+GDF-9 medium increased the percentage of primary follicles in all culture periods and secondary follicles after 14 days of culture (P<0.05), meanwhile the diameter of primary and secondary follicles were also observed to increase in this medium after 7 days of cultures (P<0.05). FSH+bFGF medium appeared to increase the percentage of primary follicles after 14 days of culture and secondary follicles at day 14 of culture than FSH medium (P<0.05). Furthermore, the FSH+GDF-9 and FSH+bFGF mediums had a greater percentage of normal follicles, and lesser apoptotic cell rates than FSH medium. The results first indicated that FSH in combination with GDF-9 or bFGF can improve the survival, activation, and growth of cattle primordial follicles after the long-term culture of ovarian cortex.

Chronic mastitis is associated with altered ovarian follicle development in dairy cattle

Connection between mastitis and fertility is multifaceted; therefore, several aspects need more elucidation. In particular, the aim was to investigate if naturally occurring chronic mastitis has an effect on ovarian function. At the time of slaughter, a milk sample and both ovaries were collected from 68 cows. The presence and intensity of chronic mastitis was diagnosed by the combined evaluation of bacteriological examination and somatic cell count of the milk of each individual quarter according to the measures of the National Mastitis Council. Animals were divided into 4 groups characterized by a low (n=15), mild (n=14), intense (n=19), or severe (n=16) degree of infection. A count of visible follicles on each ovary was followed by a quantitative analysis of microscopic traits on a selected group of animals (n=16). The latter included the classification and count of the entire preantral follicle population, and the morphometric analysis of the vascular bed extension and connective stroma in the cortical region. Finally, the expression of growth and differentiation factor-9 (GDF-9) was studied. The number of follicles with diameters ranging from 1 to 3mm and 4 to 7mm was not affected by the degree of infection. A significant effect of the degree of udder infection was observed on the number of follicles with a diameter larger than 8mm. Furthermore, the intensity of mastitis had no effect on the number of primordial and primary follicles, but severely affected cows showed a lower number of secondary follicles (0.5±0.1 vs. 0.2±0.03). Quantitative analysis demonstrated a decrease in the density of blood vessels (6.30±1.08 vs. 4.68±0.28) expressed as ratio of vascular bed/total area) and a higher incidence of fibrous stroma (1.60±0.99 vs. 6.04±3.08 expressed as ratio of connective tissue/total area) in the cortical area of the most affected animals. Finally, the level of GDF-9 protein within the oocytes of different follicle size was lower in the animals with the severe form of chronic mastitis (1.34±0.05 vs. 0.78±0.21 expressed as arbitrary units). In conclusion, decreased fertility of cows with chronic mastitis takes place through an effect on the ovary altering the dynamics of folliculogenesis. Within the ovary, this implies a reduction of the vascular bed and an increase in the fibrotic tissue together with a direct effect on oocyte-specific factors as GDF-9, all of which are essential regulatory elements of folliculogenesis.

Expression of growth and differentiation factor 9 (GDF-9) and its effect on the in vitro culture of caprine preantral ovarian follicles

This study examined the expression of growth and differentiation factor 9 (GDF-9) in caprine ovarian follicles, and the effect of GDF-9 with or without FSH on the in vitro culture of preantral follicles. To evaluate the expression of GDF-9 in Experiment 1, follicles were recovered from 32 goat ovaries and the total RNA isolated and transcribed for real-time polymerase chain reaction (PCR). Experiments 2 and 3 each used a further 32 goat ovaries to provide preantral follicles of ≥150μm. These follicles were isolated and cultured individually in 100μL drops. In each experiment at least 45 follicles were used per treatment. Every 6 days, follicles were evaluated for viability, antrum formation and growth rate. At the end of the culture period, oocytes were submitted to in vitro maturation (IVM), viability tests and chromatin evaluation. In Experiment 2, follicles were cultured in a basal medium (control) or this medium supplemented with GDF-9 at a concentration of 100ng/mL (GDF-9 100) or 200ng/mL (GDF-9 200). The same media were used in Experiment 3, supplemented with recombinant FSH at a level of 100ng/mL from day 0, 500ng/mL from day 6 to 12 and 1000ng/mL from day 12 to 18 of culture to form the three treatments: control FSH, GDF-9 (100) plus FSH and GDF-9 (200) plus FSH. Relative GDF-9 expression (Experiment 1) was greater in the secondary (18units) than the primordial (1unit) and the primary (1unit) preantral follicles (P<0.05). In the antral follicles, GDF-9 expression was significantly higher in the cumulus–oocyte complexes COC's<3mm (1.6units) than those of >3mm diameter (1unit; P<0.05), and in COC's<3mm and >3mm (319.2 and 200.1units, respectively), compared to their respective granulosa and theca cells (1unit for each category, P<0.05). In Experiment 2, GDF-9 supplementation significantly improved the survival of the follicles (60.8%, 66.0% and 77.4% for the control, GDF-9 100 and GDF-9 200, respectively; P<0.05), follicular growth rate and antrum formation following 18 days of culture. Oocyte survival was approximately 100% in all treatments. More oocytes were submitted to IVM from GDF-9 100 (78.0%; P<0.05), compared to GDF-9 200 (48.1%), but no suitable oocytes could be retrieved from the control (58.8%). The proportion of oocytes showing a resumption of meiosis, was not significantly different between treatments (41.4%, 35.9% and 36.0% for the control, GDF-9 100 and GDF-9 200, respectively). The addition of GDF-9 to the media supplemented with FSH (Experiment 3) did not significantly affect any of the variables studied. The proportion of oocytes submitted to IVM in Experiment 3 was 53.3%, 56.5% and 63.8% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (no statistical differences). The resumption of meiosis was 75.0%, 60.9% and 60.7% for the control FSH, GDF-9 100 plus FSH and GDF-9 200 plus FSH, respectively (NS). The occurrence of metaphase II was very low in both experiments. It was concluded that the supplementation of a basal medium with GDF-9 had a positive effect on the survival and development of caprine preantral follicles, but had no real effect in the presence of FSH.

A new polymorphism in the Growth and Differentiation Factor 9 (GDF9) gene is associated with increased ovulation rate and prolificacy in homozygous sheep

Brazilian Santa Inês (SI) sheep are very well-adapted to the tropical conditions of Brazil and are an important source of animal protein. A high rate of twin births was reported in some SI flocks. Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15) are the first two genes expressed by the oocyte to be associated with an increased ovulation rate in sheep. All GDF9 and BMP15 variants characterized, until now, present the same phenotype: the heterozygote ewes have an increased ovulation rate and the mutated homozygotes are sterile. In this study, we have found a new allele of GDF9, named FecG(E) (Embrapa), which leads to a substitution of a phenylalanine with a cysteine in a conservative position of the mature peptide. Homozygote ewes presenting the FecG(E) allele have shown an increase in their ovulation rate (82%) and prolificacy (58%). This new phenotype can be very useful in better understanding the genetic control of follicular development; the mechanisms involved in the control of ovulation rate in mammals; and for the improvement of sheep production.

Lower growth factor expression in follicular fluid undergonein-vitrofertilization

ObjectiveThis study was performed to identify whether growth and differentiation factor-9 (GDF-9) and transforming growth factor-β1 (TGF-β1) expressions would be lower in the follicular fluid (FF) of those over age 35 who underwent IVF than under age 35.MethodsA total of 24 IVF cycles (20 patients) were included in this study. All of patients were stimulated for IVF by the GnRH short protocol and divided into two groups for analysis, according to their age: <35 group (14 cycles, 11 patients) vs. ≥35 group (10 cycles, 9 patients). The expression levels of GDF-9 and TGF-β1 were determined by western blotting and quantitative enzyme-linked immunosorbent assay.ResultsThe numbers of retrieved oocytes and metaphase II oocytes were significantly lower in the ≥35 group. Lower expression of GDF-9 and TGF-β1 by western blotting in the ≥35 group were observed as well. The mean GDF-9 and TGF-β1 levels by enzyme-linked immunosorbent assay were lower in the ≥35 group. The values were 6,850.5±928.4 ng/L vs. 3,333.3±1,089.2 ng/L of GDF-9 (p<0.05) and 3,844.1±571.1 ng/L vs. 2,187.7±754.0 ng/L of TGF-β1 (p<0.05). A negative correlation between GDF-9 and age was observed (r=-0.546, p=0.006).ConclusionGDF-9 and TGF-β1 production from stimulated ovaries during IVF appears to decrease with age.

Integral role of GDF‐9 and BMP‐15 in ovarian function

The oocyte plays an important role in regulating and promoting follicle growth, and thereby its own development, by the production of oocyte growth factors that predominantly act on supporting granulosa cells via paracrine signaling. Genetic studies in mice demonstrated critical roles of two key oocyte-derived growth factors belonging to the transforming growth factor-β (TGF-β) superfamily, growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), in ovarian function. The identification of Bmp15 and Gdf9 gene mutations as the causal mechanism underlying the highly prolific or infertile nature of several sheep strains in a dosage-sensitive manner also highlighted the crucial role these two genes play in ovarian function. Similarly, large numbers of mutations in the GDF9 and BMP15 genes have been identified in women with premature ovarian failure and in mothers of dizygotic twins. The purpose of this article is to review the genetic studies of GDF-9 and BMP-15 mutations identified in women and sheep, as well as describing the various knockout and overexpressing mouse models, and to summarize the molecular and biological functions that underlie the crucial role of these two oocyte factors in female fertility.

187 DIFFERENTIAL EXPRESSIONS OF GROWTH AND DIFFERENTIATION FACTOR 9 AND BONE MORPHOGENETIC PROTEIN 15 GENES IN OVARIES OF THE CALF AND COW

It has been reported that prepubertal calf oocytes are less developmentally competent than those obtained from cows. The bone morphogenetic protein (BMP) family of proteins regulate folliculogenesis and the ovulation rate in mammals. Of the members of the BMP family, growth and differentiation factor 9 (GDF9) and BMP15 are oocyte-derived proteins that play critical roles in granulosa cell proliferation and differentiation. In the present study, we characterised the gene expression of bovine GDF9 and BMP15 in calf and adult cow ovaries. The ovaries obtained from 4 calves at 9 to 11 months old and 4 cows at 4 to 6 years old. For quantitative real-time RT-PCR (qPCR), cumulus–oocyte complexes (COC) and mural granulosa cells were collected by aspiration of follicles 2 to 5 mm in diameter from an ovary from each animal. Ovaries from the other side were used for in situ hybridization. The COC and mural granulosa cells were separated and cultured for 22 h according to the protocol for oocyte maturation. Total RNA was isolated from denuded oocytes, cumulus cells, and mural granulosa cells. Bovine glyceraldehyde-3-phosphate dehydrogenase was used to normalise qPCR efficiency. For in situ hybridization, the collected ovaries were immediately fixed with 4% formaldehyde-PBS and embedded in a paraffin block. In situ hybridization was carried out with digoxigenin-labelled RNA probes. We confirmed there was no contamination of oocytes in the collected cumulus and mural granulosa cells by determining the mRNA expression of germ cell–oocyte markers (ZAR1 and VASA). Two, 16, 7, and 10 COC were collected from the ovary on one side of each calf, and 14, 22, 29, and 33 COC were collected from an ovary of each adult cow. Two COC from the calves could not be used for qPCR analysis. Both GDF9 and BMP15 mRNA were detected in oocytes and cumulus cells at the end of maturation culture, whereas only GDF9 mRNA was detected in mural granulosa cells. Quantitative PCR detection revealed that BMP15 and GDF9 mRNA expression of the cumulus cells from adult ovaries was significantly greater than that from calf ovaries. The expression of GDF9 mRNA was significantly greater in calf oocytes than in oocytes from cows. However, BMP15 mRNA expression in the oocytes of calf and adult ovaries was not significantly different. In mural granulosa cells, the intensities of GDF9 mRNA expression were not significantly different between calves and cows. These qPCR results were also ascertained by in situ hybridization. In conclusion, we clarified that the characteristics of bovine GDF9 and BMP15 mRNA expression in oocytes and cumulus cells were different between calves and cows. Our results indicate the possibility that the calf oocytes were less developmentally competent because of excess GDF9 on the oocytes, deficiencies of GDF9 and BMP15 on the cumulus cells, or both.