Growth And Differentiation Factor 9 Research Articles

Aquisição da dependência de gonadotrofinas pelos folículos antrais iniciais e os desafios para promover o seu crescimento in vitro

Abstract This review aims to discuss the main factors involved in the development of early antral follicles until gonadotropin dependence. This follicular phase is characterized by intense proliferation of granulosa cells, formation of a fluid-filled cavity, morphological differentiation of cumulus cells, mural granulosa cells and recruitment of theca cells. The interaction between oocyte, granulosa and theca cells is crucial for follicular growth and hormone production. Growth factors produced by the oocyte, such as growth and differentiation factor-9 (GDF-9) and bone morphogenetic protein-15 (BMP-15), regulate granulosa cell proliferation and differentiation and antral cavity development, as well as stimulate the production of follicle-stimulating hormone (FSH) receptors in granulosa cells. In response to FSH, granulosa cells secrete C-type natriuretic peptide (CNP), which acts through its receptor to increase cyclic guanosine monophosphate (cGMP) production and consequently follicular development. Granulosa cells also produce insulin-like growth factor-1 (IGF-1) and increase aromatase enzyme activity, which results in greater sensitivity to gonadotropins and follicular steroidogenesis. The absence of IGF-1 signaling causes cessation of follicular growth at the early antral stage. Many other local factors are involved in the regulation of follicular development. Therefore, this review brings relevant data for a better understanding of the mechanisms involved in the control of early antral follicle growth, emphasizing the role of endocrine and paracrine factors, the oocyte-granulosa cell interaction and the processes of follicular atresia. The challenges for the establishment of efficient culture systems for in vitro growth of early antral follicles are also discussed.

Gene expression of equine oocytes maintained in meiotic blockade

To improve quality, immature horse oocytes may undergo a pre-maturation (PM) period to promote synchronization of nuclear and cytoplasmic maturation. PM can be performed using meiosis blockers, such as Butyrolactone I, or by keeping oocytes at room temperature (Choi et al. Theriogenology 2006;66(4):955-963; Martino et al. Reproductive Biology and Endocrinology.2014;12(1):99-110). In this experiment the gene expression profile in COCs maintained in a commercial embryo medium (Botuembryo) and in DMEM/F12 supplemented with Butyrolactone was studied. Equine COCs were retrieved by follicular curettage from slaughterhouse ovaries and divided into 3 groups (n = 74): Control - COCs were destined to IVM during 24hs in DMEM/F12 with 10% FCS and antibiotics; Botuembryo - COCs underwent PM for 20 hours at room temperature in Botuembryo (21 to 25°C) and then 24hs of IVM at the same conditions as Control group, and Butyrolactone - COCs stayed in PM in DMEM/F12 with 10% FCS and 50 μM Butyrolactone for 20hs in a controlled atmosphere incubator at 38,5°C, and then were destined to IVM to an extra 24hs. Four replicates were used. Timepoint 1 (M1) in controls corresponds to the moment just after collection and in Botuembryo and Butyrolactone groups, M1 corresponds to the end of PM. Timepoint 2 (M2) corresponds to the end of IVM in all groups. Oocytes were denuded of cumulus cells (CCs) and both were stored in separate vials at -80°C. Analysis of mRNA expression was done by RT-qPCR with β-actin (ACTB) as reference gene. Ampiregulin (AREG), Connexin 43 (Cx43), Hyalurone Synthetase 2 (HAS2), FSH Receptor (FSHR) and LH Receptor (LHR) were studied in COCs whereas Bone Morphogenetic Protein 15(BMP15), Connexin 37 (Cx37), Growth and Differentiation Factor 9 (GDF9) and Matrix Metalloproteinase 2 (MMP2) were analyzed in oocytes. Data for each timepoint were tested by ANOVA followed by group comparison with the Tukey Kramer test (JMP software). Atypical gene expression (outliers) was identified by the generalized ESD test (GraphPad PRISM) and excluded. Data are presented as mean ± SEM, with P<0.05 being significant. The results (Table 1) indicate a failure in blocking molecular maturation in the Butyrolactone group, with an increase in the abundance of AREG and HAS2 in M1 compared to M2. On the other hand, the Botuembryo group was superior in M2 in relation to the abundance of BMP15, and GDF9, indicating better quality oocytes. More studies need to be performed to confirm the superiority of Botuembryo in maintaining meiotic arrest during PM. Acknowledgement: FAPESP (process 2020/01646-5); CNPq; CAPES; Omics Animal Biotechnology; Frigofava.

Fator de crescimento e diferenciação - 9 (GDF-9) aumenta a taxa de crescimento in vitro de folículos antrais iniciais caprinos isolados

Abstract This study aimed to investigate the effect of growth and differentiation factor 9 (GDF-9) during the in vitro culture of isolated caprine early antral follicles. The isolated and selected early antral follicles were individually cultured for 18 days, and the following treatments were tested: α-MEM+ (control treatment) or α-MEM+ supplemented with 200 ng/mL GDF-9. The following endpoints were evaluated: follicular growth and morphology, estradiol production, oocyte nuclear maturation, and relative expression of key genes related to steroidogenesis (CYP19A1, CYP17, and insulin receptor) and basement membrane remodeling (MMP-9 and TIMP-2). In both treatments, a decrease was observed in the percentage of morphologically intact follicles with a concomitant increase in the rates of extruded and degenerated follicles (P &lt; 0.05). The GDF-9 treatment showed higher rates of extruded follicles only on day 6 of culture (P &lt; 0.05). Follicle diameter increased progressively throughout the culture period (P &lt; 0.05) with similar diameters between treatments at all culture times (P &gt; 0.05). Growth and differentiation factor 9 increased the daily growth rate from the first to the second third of culture, with higher values (P &lt; 0.05) than control in the second third. Oocyte maturation rate as well as estradiol levels and relative mRNA expression for CYP19A1, CYP17, MMP-9, TIMP-2, and insulin receptor genes were similar between treatments (P &gt; 0.05). This study shows for the first time that GDF-9 added to a culture medium increased the follicle growth rate of goat early antral follicles cultured in vitro.

N-Acetyl cysteine reduces the levels of reactive oxygen species and improves in vitro maturation of oocytes from medium-sized bovine antral follicles.

This study aims to evaluate the effects of N-acetylcysteine (NAC) on bovine oocyte maturation, mitochondrial activity and transzonal projections (TZP), as well as on the levels of reactive oxygen species (ROS) and messenger RNA (mRNA) for catalase (CAT) superoxide dismutase (SOD), periredoxin-6 (Prdx6), glutathione peroxidase (GPx), growth and differentiation factor-9 (GDF9), histone H1Foo, cyclin B1 (CCNB1) and c-Mos. Bovine cumulus-oocyte complexes (COC) of medium-sized antral follicles (3.0-6.0 mm) were prematured in TCM-199 for 8 h at 38.5°C in 5% CO2. After prematuration in the presence of forskolin and C-type natriuretic peptide, COCs were matured in TCM-199 alone or with 0.1, 0.5 or 2.5 mM NAC. Then, oocytes were classified according to the stage of chromatin. Furthermore, mitochondrial activity and intracellular levels of ROS and TZP were also evaluated. The levels of mRNAs for CAT, SOD, Prdx6, GPx, GDF9, H1Foo, CCNB1 and c-Mos were evaluated using real-time polymerase chain reaction (RT-PCR). The results showed that NAC significantly increased the percentages of oocytes with resumption of meiosis when compared with those oocytes matured in control medium. Oocytes had homogeneous mitochondrial distribution, and those cultured with 0.1 and 0.5 mM NAC had lower levels of ROS when compared with the control. In addition, 0.5 mM NAC reduced TZP and the levels of mRNA for CCNB1. In contrast, NAC did not influence the expression of CAT, GPx, Prdx6, SOD, GDF9, H1Foo, and c-Mos. In conclusion, 0.5 mM NAC reduced the levels of ROS, TZP and mRNA for CCNB1, and improved in vitro resumption of meiosis in oocytes from medium-sized bovine antral follicles.

Long-Term Changes in Ovarian Follicles of Gilts Exposed Neonatally to Methoxychlor: Effects on Oocyte-Derived Factors, Anti-Müllerian Hormone, Follicle-Stimulating Hormone, and Cognate Receptors.

In this paper, we investigated the effects of neonatal exposure to methoxychlor (MXC), a synthetic organochlorine used as an insecticide with estrogenic, antiestrogenic, and antiandrogenic activities on ovarian follicles of adult pigs. Piglets were injected with MXC (20 μg/kg body weight) or corn oil (controls) from postnatal Day 1 to Day 10 (n = 5 per group). Then, mRNA expression, protein abundance and immunolocalization of growth and differentiation factor 9 (GDF9), bone morphogenetic protein 15 (BMP15), anti-Müllerian hormone (AMH) and cognate receptors (ACVR1, BMPR1A, BMPR1B, TGFBR1, BMPR2, and AMHR2), as well as FSH receptor (FSHR) were examined in preantral and small antral ovarian follicles of sexually mature gilts. The plasma AMH and FSH levels were also assessed. In preantral follicles, neonatal exposure to MXC increased GDF9, BMPR1B, TGFBR1, and BMPR2 mRNAs, while the levels of AMH and BMP15 mRNAs decreased. In addition, MXC also decreased BMP15 and BMPR1B protein abundance. Regarding small antral follicles, neonatal exposure to MXC upregulated mRNAs for BMPR1B, BMPR2, and AMHR2 and downregulated mRNAs for AMH, BMPR1A, and FSHR. MXC decreased the protein abundance of AMH, and all examined receptors in small antral follicles. GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles of control and treated ovaries. All analyzed receptors were detected in the oocytes and granulosa cells of preantral follicles, and in the granulosa and theca cells of small antral follicles. The exception, however, was FSHR, which was detected only in the granulosa cells of small antral follicles. In addition, MXC decreased the plasma AMH and FSH concentrations. In conclusion, the present study may indicate long-term effects of neonatal MXC exposure on GDF9, BMP15, AMH, and FSH signaling in ovaries of adult pigs. However, the MXC effects varied at different stages of follicular development. It seems that neonatal MXC exposure may result in accelerated initial recruitment of ovarian follicles and impaired cyclic recruitment of antral follicles.

Local regulation of antral follicle development and ovulation in monovulatory species

The identification of mutations in the genes encoding bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) associated with phenotypes of sterility or increased ovulation rate in sheep aroused interest in the study of the role of local factors in preantral and antral folliculogenesis in different species. An additive mutation in the BMP15 receptor, BMPR1b, which determines an increase in the ovulatory rate, has been introduced in several sheep breeds to increase the number of lambs born. Although these mutations indicate extremely relevant functions of these factors, the literature data on the regulation of the expression and function of these proteins and their receptors are very controversial, possibly due to differences in experimental models. The present review discusses the published data and preliminary results obtained by our group on the participation of local factors in the selection of the dominant follicle, ovulation, and follicular atresia in cattle, focusing on transforming growth factors beta and their receptors. The study of the expression pattern and the functionality of proteins produced by follicular cells and their receptors will allow increasing the knowledge about this local system, known to be involved in ovarian physiopathology and with the potential to promote contraception or increase the ovulation rate in mammals.

Influence of FecGE mutation on the reproductive variables of Pelibuey ewes in the anestrus period.

The growth and differentiation factor 9 (GDF9) intervenes in the fecundity and prolificacy of the ewe, which are important variables that participate in the reproductive efficiency of a flock. The objective of this study was to evaluate the influence of FecGE mutation of the gene GDF9 in the natural response of the manifestation to estrus, return to estrus, ovulation rate, pregnancy, lambing, prolificacy, and fecundity rate in Pelibuey ewes, during the anestrus period. The sequences of the exon 2 of the gene GDF9 were obtained from blood samples collected in Whatman™ FTA™ cards from 42 multiparous Pelibuey ewes with reproductive records. For this purpose, the quality of the sequences was analyzed and the polymorphisms and genotypes were searched for. The ewes were grouped according to their group: (a) homozygous or Embrapa (GG), (b) wild (AA), and (c) group without gene (sG). All the ewes studied manifested estrus behavior, but none showed signs of return to estrus after natural mating (p > 0.05); likewise, the pregnancy and lambing rates (p > 0.05) did not show differences between groups. However, the group GG presented higher ovulation rate, prolificacy, and fecundity rate (p < 0.05), compared to groups AA and sG. Although no differences were found in the manifestation of estrus, return to estrus, and percentage of pregnancy and lambing in females from the genotypes studied, the homozygous ewes GG presented 1.22 and 1.72 more corpus luteum (CL, p < 0.05), prolificacy of 0.7 and 0.7, and fecundity rate of 0.8 and 1.0 more lambs per ewe (p < 0.05) than the ones produced by the wild-type AA and sG groups, respectively.

Effects of Growth and Differentiation Factor 9 and Bone Morphogenetic Protein 15 overexpression on the steroidogenic metabolism in bovine granulosa cells in vitro.

Granulosa cells (GCs) play important roles in the regulation of ovarian functions, and in vitro culture is a relevant model for the study of steroidogenesis in ovarian follicles. Thus, growth factors secreted by the oocyte, like Growth and Differentiation Factor 9 (GDF9) and Bone Morphogenetic Protein 15 (BMP15), play an important part in the luteinization of granulosa cells. The aim of this work was to express GDF9 and BMP15 genes in bovine GCs in vitro and evaluate their effects on the luteinization process. Samples of culture medium and GCs transfected with GDF9 and BMP15 were obtained for 21 consecutive days to analyse the steroidogenic hormones' concentration (progesterone (P4 ) and estradiol (E2 )) and the expression of STAR, GDF9 and BMP15 and their respective receptors. The results demonstrated an inhibitory effect of GDF9 and BMPF15 on P4 secretion in bovine GCs cultured in vitro. Moreover, our study demonstrated the entire expression of their respective receptors (TGFBR1, BMPR1B and BMPR2) and the inhibition of the steroidogenic marker, STAR gene. This work sheds light on a novel biological function of BMP15 and GDF9 in bovine GCs physiology, which could elucidate a non-described biological role for GDF9 and BMP15 in bovine granulosa cells' metabolism.

Involvement of Phosphorylated Akt and FOXO3a in the Effects of Growth and Differentiation Factor-9 (GDF-9) on Inhibition of Follicular Apoptosis and Induction of Granulosa Cell Proliferation After In Vitro Culture of Sheep Ovarian Tissue.

This study aimed to evaluate the effects of growth and differentiation factor-9 (GDF-9) on the morphology, activation, apoptosis, and granulosa cell proliferation of ovine preantral follicles cultured within ovarian tissue slices and to verify whether GDF-9 could influence follicular activation through the phosphatidylinositol 3-kinase/protein kinase B/forkhead box O3a (PI3K/Akt/FOXO3a) pathway. Ovine ovarian fragments were cultured in α-MEM+ or α-MEM+ with GDF-9 (1, 50, 100, 200, or 400ng/ml) for 7days. Apoptosis and cell proliferation were analyzed. Next, the activation of the PI3K was inhibited with LY294002, and immunostaining for p-Akt and p-FOXO3a proteins was assessed. The concentration of 50ng/ml GDF-9 had (P < 0.05) more morphologically normal follicles compared to all treatments, except 1ng/ml GDF-9. Moreover, 50ng/ml GDF-9 increased primordial follicle activation compared to all treatments, except α-MEM+ and 1ng/ml GDF-9. However, the concentration of 50ng/ml GDF-9 showed higher cell proliferation and lower apoptosis than α-MEM+ and 1ng/ml GDF-9 treatments. Culture of the ovarian tissue with LY294002 inhibited the activation of primordial follicles and reduced p-Akt immunostaining in both α-MEM+ and 50ng/ml GDF-9 treatments. In addition, after culture with LY294002, the percentage of oocytes with nuclear p-FOXO3 was higher in 50ng/ml GDF-9 than in the control medium (α-MEM+). In conclusion, after culture of ovine ovarian cortical slices, the addition of 50ng/ml GDF-9 reduces follicular apoptosis and promotes granulosa cell proliferation likely through the involvement of phosphorylated Akt and FOXO3a.

183 Effects of epidermal growth factor and progesterone on invitro oocyte growth, meiotic resumption, and expression of maturation-related transcripts in oocytes from bovine small antral follicles

Oocytes from small antral follicles are not able to undergo nuclear maturation because they still need to accumulate mRNA and proteins. We hypothesised that invitro growth of cumulus-oocyte complexes (COCs) of these follicles in the presence of epidermal growth factor (EGF) and progesterone (P4) increase the levels of transcripts and have a positive impact on maturation. This study aimed to evaluate the effects of EGF and P4 on growth, maturation, and expression of mRNAs for growth and differentiation factor 9 (GDF9), cyclin B1, oocyte-specific linker histone (H1FOO), quinase cMOS, poly(A) ribonuclease (PARN), and initiation factor 4E (eIF4E) after growth and prematuration of COCs. Bovine COCs (n=400) were aspirated from antral follicles (1-3mm) and cultured for 48h in control medium (TCM-199 plus 5.0mgmL−1 LH, 0.5mgmL−1 FSH, and 5% fetal bovine serum) alone or with EGF (10ngmL−1), P4 (100 µM), or both EGF and P4. After growth, the COCs were pre-matured for 20h in TCM-199 containing 5.0mgmL−1 LH, 0.5mgmL−1 FSH, 0.4% bovine serum albumin, and 10μM cilostamide. Then, COCs were matured invitro in the same medium used for prematuration, but without cilostamide. Oocyte diameter and meiotic progression were evaluated after growth, prematuration, and maturation periods. After prematuration, oocytes were stored until RNA extraction. Total RNA was extracted using Trizol according to the manufacturer's instructions (Invitrogen). After reverse transcription, quantification of mRNAs for GDF9, cyclin B1, H1FOO, cMOS, PARN, eIF4E, and GAPDH was performed by real-time PCR. The delta-delta-cycle threshold (ΔΔCT) method was used to normalise the data. The percentages of oocytes in each stage of maturation were compared by Mann-Whitney test. Differences in expression of mRNAs were analysed by Kruskal-Wallis test (P&amp;lt;0.05). The results showed that, compared with time 0, an increase in oocyte diameter was observed after 48h of culture for COCs in all groups (P&amp;lt;0.05), except for those cultured in control medium alone or with only P4. However, when compared with control medium, no effects of EGF, P4, or both were seen after the growth and prematuration periods. After prematuration, a higher percentages of oocytes at the germinal vesicle stage was observed for COCs cultured with P4 compared with those cultured in TCM-199 (P&amp;lt;0.05). After IVM, the rate of meiosis resumption was significantly reduced in oocytes cultured with P4 (P&amp;lt;0.05). The levels of mRNA for cMOS, H1FOO, and cyclin B1 in oocytes cultured with P4 were higher than those cultured in the control medium (P&amp;lt;0.05). The mRNA levels for eIF4E in oocytes cultured with both EGF and P4 were increased (P&amp;lt;0.05) compared with those cultured with EGF. The levels of transcripts for PARN in oocytes cultured with both EGF and P4 were higher than those in COCs cultured with only P4. In contrast, mRNA levels of GDF9 did not show differences between treatments. In conclusion, P4 inhibited oocyte meiotic resumption and increased mRNA levels for cMOS, H1FOO, and cyclin B1 in COCs after culture and prematuration invitro.

Neonatal exposure to agonists and antagonists of sex steroid receptors induces changes in the expression of oocyte-derived growth factors and their receptors in ovarian follicles in gilts

The objective of the present study was to examine the effects of neonatal exposure to either agonists or antagonists of androgen and estrogen receptors on the expression of growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) and their cognate receptors (TGFBR1, BMPR1B, and BMPR2) in ovarian follicles of adult pigs. Piglets were injected subcutaneously with testosterone propionate (TP, an androgen, at 20 mg/kg bw), flutamide (FLU, an antiandrogen, at 50 mg/kg bw), 4-tert-octylphenol (OP, an estrogenic compound, 100 mg/kg bw), ICI 182,780 (ICI, an antiestrogen, 400 μg/kg bw), or corn oil (control) between postnatal Days 1 and 10 (n = 5/group). Ovarian follicles were excised from adult pigs on Days 8–11 of the estrous cycle. The expression of GDF9, BMP15, TGFBR1, BMPR1B and BMPR2 were examined in the population of preantral and small antral ovarian follicles using real-time PCR, Western blot and immunohistochemistry. In preantral follicles, the upregulation of GDF9 mRNA and protein expression was found in pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMP15. TGFBR1 and BMPR2 mRNA and protein expression were upregulated in preantral follicles of adult pigs that were neonatally exposed to TP or FLU, while administration of TP or ICI resulted in upregulation of BMPR1B. In small antral follicles, the mRNA and protein for TGFBR1 and BMPR2 were upregulated, while BMPR1B was downregulated in response to neonatal OP treatment. In addition, treatment with FLU upregulated BMPR1B and BMPR2 mRNA and protein expression, while downregulated the expression of TGFBR1. Moreover, GDF9 and BMP15 were immunolocalized in oocytes and granulosa cells of preantral follicles obtained from both control and treated ovaries. TGFBR1, BMPR1B and BMPR2 receptors were observed in the oocytes and granulosa cells of preantral follicles as well as in granulosa and theca cells of small antral follicles. In conclusion, the present study demonstrated neonatal exposure to either agonists or antagonists of androgen and estrogen receptors affected GDF9 and BMP15 signalling in ovaries of adult pigs. It seems that neonatal androgen excess or deficiency may lead to the acceleration of initial follicle recruitment, while neonatal exposure to compounds with antiandrogenic and estrogenic activity may disturb small antral follicles fate. Therefore, it confirms that neonatal window is critical for programming of ovarian function in pigs.

Growth and Differentiation Factor-9 Supplementation Affects Viability and Morphology of Preantral Follicles in Equine Ovarian Fragments During Short-term in vitro Culture

Abstract This study aimed to evaluate different concentrations of growth and differentiation factor-9 (GDF-9) on the development and maintenance of equine preantral follicle morphology during short-term in vitro culture. Ovaries (n=5) from five mares were collected from a local slaughterhouse and transported to the laboratory, where nine fragments (5x5x1mm) were procured from each ovary. One fragment from each was immediately fixed and submitted for histological analysis (control group; D0). The other eight fragments were cultured in situ for two (D2) or six (D6) days in MEM+ or MEM+ supplemented with GDF-9 at different concentrations (i.e., 50, 100 and 200 ng/mL the GDF-9). After culturing with different concentrations of GDF-9 for 2 or 6 days, the fragments were processed for histological analysis. After two days of cultivation, we observed an increase in the percentage of developing follicles for 0 (MEM+), 50, 100 and 200 ng/mL GDF-9 compared to control (D0; P<0.05). When we evaluated all treatments that preserved follicular integrity, the GDF-9 concentration of 100 ng/mL presented results superior to those of the other cultures (P<0.05). While, at six days of culture, the concentration of 200 ng/mL of GDF-9 appeared to be more efficient in providing development compared to MEM+ (P<0.05). The percentage of morphologically intact follicles in the 6 days culture samples treated with 50 ng/mL of GDF-9 indicated that this concentration was effective in maintaining the integrity of the follicle (P<0.05). We conclude, therefore, that graduated GDF-9 addition to the medium ensure follicular development and is sufficient maintain the architecture.

Implication of SLIT3-ROBO1/ROBO2 in granulosa cell proliferation, differentiation and follicle selection in the prehierarchical follicles of hen ovary.

The SLIT/ROBO pathway has been implicated in prehierarchical follicular development of hen ovary by an intrafollicular autocrine and/or paracrine fashion. SLIT3, one of the key components of the SLIT/ROBO family, serves as a ligand that potentially interacts with the four receptors, ROBO1, ROBO2, ROBO3 and ROBO4. But the exact roles and regulatory mechanism of SLIT3 in chicken ovarian follicle development remain largely unclear. The present study was conducted to investigate the potential roles and molecular regulation of SLIT3 in granulosa cell (GC) proliferation, differentiation and follicle selection within the prehierarchical follicles of hen ovary. We found that SLIT3 interacts physically with the four ROBO receptors, but the expression of the ROBO1 and ROBO2 genes are more susceptible to the regulation of SLIT3 ligand than that of the ROBO3 and ROBO4 genes. Moreover, the siRNA-mediated knockdown of SLIT3 in the follicular GCs leads to a significant increase in cell proliferation. Conversely, overexpression of SLIT3 results in a remarkable reduction in GC proliferation. Furthermore, the overexpressed SLIT3 has notably decreased the mRNA and protein expression levels of follicle-stimulating hormone (FSHR), growth and differentiation factor 9 (GDF9), steroidogenic acute regulatory protein (STAR) and cytochrome P450 11A1 (CYP11A1) in the GCs. These results indicated that SLIT3 may play an inhibitory effect on GC proliferation, differentiation and follicle selection, and these suppressive actions of SLIT3 in the GC proliferation can be prohibited by the siRNA-mediated knockdown of ROBO1 and ROBO2 receptors. The current data provide a basis for further investigation of molecular mechanisms of SLIT3-ROBO1/2 pathway in controlling the prehierarchical follicle development of the hen ovary.

Expression of genes for enzymes synthesizing lysophosphatidic acid, its receptors and follicle developmental factors derived from the cumulus-oocyte complex is dependent on the ovarian follicle type in cows

Cumulus-oocyte complexes (COCs) release factors potentially involved in follicular growth and development, such as growth and differentiation factor 9 (GDF9), bone-morphogenetic protein 15 (BMP15), follistatin (FST) and cathepsins (CTSs). Moreover, the quality of the oocytes and follicles may be related to both the lipid composition of the follicle cells and follicular fluid. One of the lipids, locally regulating the reproductive functions in ovaries of cattle, is lysophosphatidic acid (LPA). In this study, the expression was investigated of the genes for LPA and other factors in COCs of follicles at different stages of development and regression. The relative abundances of mRNA were determined by real-time PCR for receptors for LPA (LPARs), enzymes synthesizing LPA (autotaxin (AX) and phospholipase A2 (PLA2)), BMP15, GDF9, CTSZ, CTSB and FST in COCs isolated from healthy, transitional and atretic follicles. The expression of genes for the LPARs, AX, PLA2 and the factors involved in follicular development in cattle COCs is follicle-type dependent. Greater expression of LPAR1-3 and AX genes were detected in the healthy follicles compared to the atretic and transitional follicles (P < 0.05). The relative abundance of GDF9, BMP15, CTSZ and CTSB was also greater in COCs from healthy follicles than from transitional and atretic follicles (P < 0.05). It is postulated that the greater expression of LPARs and AX genes in healthy follicles compared with atretic follicles indicates an enhanced role of LPA in follicular development. Results of the present study also suggest the regulatory role of factors derived from the COCs in the growth and development of follicles.

Genetic variability in equine GDF9 and BMP15 genes in Arabian and Thoroughbred mares

Abstract In horses, multiple ovulation resulting in implantation of multiple embryos is adverse. However, understanding the mechanisms underlying initiation of multiple ovulation (MO) is advantageous and is related to an increase in efficiency of embryo transfer techniques. It has been postulated that MO may have a genetic background. Two major genes: bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are considered to play a crucial role in folliculogenesis and controlling the ovulation rate. Thus, the aim of the presented study was to identify the variation within equine BMP15 and GDF9 genes to verify their potential role on spontaneous, repetitive multiple ovulations in mares. In addition, variation screening of investigated genes in population of Thoroughbred and Arabian breeds was performed together with establishment of transcript abundance of BMP15 and GDF9 genes in equine ovarian tissue. Sanger sequencing of Arabian and Thoroughbred mares divided according to ovulation rate, revealed occurrence of 3 SNPs in BMP15 and STS in GDF9 genes. The PCR-RLFP and statistical analysis indicated that none of the genotype frequencies were significant in any breeds and none of them were claimed as functional according to ovulation rate. Furthermore, evaluation of transcript abundance by RT -PCR of both genes in ovarian tissues showed that expression of both genes was similar but GDF9 was significantly expressed in growing follicles with 21-30 mm diameter and in ovarian parenchyma, which suggest their potential role in folliculogenesis.

Mechanisms regulating follicle selection in ruminants: lessons learned from multiple ovulation models.

Selection of a single dominant follicle from a cohort of growing follicles is a unique biological process, a key step in female reproductive function in monovular species, and lies at the core of reproductive technologies in cattle. Follicle growth and the number of follicles that ovulate are regulated by precise endocrine, paracrine, and autocrine mechanisms. Most of our current understanding about follicle selection focuses on the role of FSH, LH, and the IGF family in follicle growth and selection of the dominant follicle. However, more recently the role of members of the TGF-ß family has been highlighted, particularly in high fecundity genotypes in sheep. Intercellular signaling between the oocyte and granulosa cells (GC) regulates proliferation and differentiation due to actions of bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) within the follicle. Mutations that either knockout or reduce the activity of BMP15 or GDF9 have been found to increase ovulation rate in heterozygotes and generally cause severe follicle abnormalities in homozygotes. A mutation in the intracellular kinase domain of the BMPR1B receptor (Booroola fecundity gene) increases ovulation rate in heterozygotes with further increases in ovulation in homozygotes. The physiological mechanisms linking these mutations to increased ovulation rates are still not well defined. A recently identified high fecundity bovine genotype, Trio, causes increased expression of SMAD6, an intracellular inhibitor of the BMP15/GDF9 signalling pathways. This bovine model has provided insights into the mechanisms associated with selection of multiple dominant follicles and multiple ovulations in carriers of fecundity alleles. The present review focuses on the mechanisms involved in follicle selection in ruminants with a special emphasis on the contribution made by multiple ovulation models in both cattle and sheep. The evaluation of multiple ovulation models in ruminants has allowed us to construct a new physiological model that relates changes in the BMP15/GDF9 signalling pathways to the physiological changes that result in selection of multiple dominant follicles. This model is characterized by acquisition of dominance at a smaller follicle size but at a similar time in the follicular wave with multiple follicles acquiring dominance in a hierarchal sequence, delaying FSH suppression and, thus allowing additional follicles to continue to grow and acquire dominance.

Molecular Aspects and Clinical Relevance of GDF9 and BMP15 in Ovarian Function.

Growth and differentiation factor 9 (GDF9) and bone morphogenetic protein 15 (BMP15) are oocyte-secreted factors with a leading role in the control of ovarian function in female reproduction, modulating both the cell fate of the somatic granulosa cells and the quality and developmental competence of the egg. This short review aims to consolidate the molecular aspects of GDF9 and BMP15 and their integral actions in female fertility to understand particularly their effects on oocyte quality and fetal growth. The significant consequences of mutations in the GDF9 and BMP15 genes in women with dizygotic twins as well as the clinical relevance of these oocyte factors in the pathogenesis of primary ovarian insufficiency and polycystic ovary syndrome are also addressed.

Improved developmental competence in embryos treated with lycopene during in vitro culture system.

In vitro embryo development remains suboptimal compared to in vivo development due to the challenge from various stressors associated with in vitro culturing of oocytes. When 0.2 μM lycopene was added to oocyte in vitro maturation and embryo culture media, to assess its antioxidant effects on embryo development, we observed a significant (p < 0.05) increase in cleavage and blastocyst development rates compared to the corresponding controls (84.3 ± 0.6% vs. 73.1 ± 1.9% and 41.0 ± 1.4% vs. 33.4 ± 0.7%, respectively). Lycopene also significantly reduced (p < 0.05) intracellular reactive oxygen species concentrations in oocytes and blastocysts, whereas lipid peroxidation and mitochondrial activity increased compared to control conditions. The number of apoptotic nuclei was significantly reduced in the lycopene-treated compared to the control group (1.7 ± 0.1 vs. 4.7 ± 0.3), and the quantity of cells in the trophectoderm (207.1 ± 1.6 vs. 171.3 ± 1.0, respectively) and inner cell mass (41.9 ± 0.4 vs. 36.7 ± 0.4, respectively) was higher following treatment-although the inner cell mass-to-trophectoderm ratio was unchanged (1:3.3 vs. 1:3.4 for lycopene vs. control, respectively). Lycopene supplementation also significantly (p < 0.05) attenuated expression of IKBKB (Inhibitor of nuclear factor kappa B kinase, subunit beta) and reduced Caspase 9 and Caspase 3 protein abundance, while up-regulating GDF9 (Growth and differentiation factor 9), BMP15 (Bone morphogenetic protein 15), SOD2 (Superoxide dismutase 2), NDUFA2 (NADH dehydrogenase), ACADL (Acyl-CoA dehydrogenase, long chain), and ACSL3 (Acyl-CoA synthetase 3, long-chain membrane 3) transcription compared to control. Therefore, co-culturing with lycopene during oocyte maturation improved bovine embryo developmental potential during in vitro culture by improving embryonic resilience to stress.

Dynamics of growth and differentiation factor-9 and inhibin B production in macaque developing follicles during 3-dimensional culture

Growth and differentiation factor 9 (GDF-9) and inhibins (A and B) are members of the transforming growth factor-beta superfamily and local factors essential for normal ovarian function and folliculogenesis in mice. However, their production and roles during follicle growth in primates are less clear. The current study quantitated levels of GDF-9 and inhibin B during in vitro growth of macaque preantral to small antral follicles.

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.