Growth Of Preantral Follicles Research Articles

Development of the “waveless” bovine model

Development of a bovine model without ovarian follicular waves (waveless) and transient increases in gonadotropin secretion during estrous cycles may lead to new methods to more consistently regulate ovulatory follicle growth thereby improving efficiency of embryo transfer. We hypothesized that the GnRH antagonist acyline would inhibit gonadotropin secretion thereby blocking follicular waves, ovarian function and ovulation during estrous cycles of cattle. To test this hypothesis, beef heifers (n = 5 per group) were treated twice daily with vehicle (control) or 25 or 50 μg/kg acyline beginning 12 h after GnRH-induced ovulation and ending 21 days later. Each animal was subjected to ovarian ultrasonography for 25 days to monitor number and growth of follicles ≥3 mm in diameter and growth of the corpus luteum (CL). Blood samples were taken at various intervals to determine circulating concentrations of FSH, LH, anti-Müllerian hormone (AMH, marker of small follicle growth) and progesterone. Results show that acyline treatment decreased or blocked: circulating concentrations of LH, transient FSH spikes associated with emergence of follicular waves, preovulatory FSH/LH surges, follicular waves, dominant follicle development, CL growth, and progesterone production. In contrast, the largest acyline dose increased AMH concentrations. In conclusion, long-term acyline treatment blocks follicular waves but not growth of preantral and small antral follicles (≤ 3 mm). Future studies will determine if the waveless bovine model, which has enhanced development of preantral and small antral follicles, can be used to develop new methods to improve predictability of response of cattle to superovulation.

Effects of Supplementation of Human Endometriotic Fluids on In Vitro Mouse Preantral Follicle Culture.

This study aimed to investigate the potential detrimental effect of human endometriotic fluids (EFs) on in vitro growth of mouse preantral follicles. Preantral follicles (isolated from ovaries of 7- to 8-week-old mice) were cultured in growth medium for 10 days and then in maturation medium for 2 days. During in vitro culture, EF supernatants (0%, 2.5%, 5%, and 10%) were supplemented. Meiotic spindle integrity of metaphase II (Mil) oocytes was analyzed. Hormone (l7(3-estradiol and anti-Mullerian hormone [AMH]) levels in the final spent media were measured by enzyme-linked immunosorbent assay. The survival rates offolliclesatday 10 were significantly lower in 3 EF-supplemented groups (56.1%, 30.6%, and 6.2%; 83.6% in the nonsupplemented group). The production of total oocytes per initiated follicle was also significantly lower in the 3 EF-supplemented groups (34.7%, 18.4%, and 4.1 %; 68.1 % in the nonsupplemented group). Proportions of the oocyte with normal spindles were significantly lower in the 3 EF-supplemented groups (10%, 0% and 0%; 52% in the nonsupplemented group). In the final spent media, the level of 17(3-estradiol was significantly lower only in the 10% EF-supplemented group, and the level of AMH was significantly lower in all 3 EF-supplemented groups, when compared with the nonsupplemented group. During in vitro culture of mouse preantral follicles, the survival rate, oocyte acquisition, spindle integrity of Mil oocytes, and AMH production were greatly affected by EF supplementation. These findings suggest a possibility of detrimental effects of endometriotic cysts on folliculogenesis in adjacent ovarian tissues.

Mouse preantral follicle growth in 3D co-culture system using human menstrual blood mesenchymal stem cell

Follicle culture provides a condition which can help investigators to evaluate various aspects of ovarian follicle growth and development and impact of different components and supplementations as well as presumably application of follicle culture approach in fertility preservation procedures. Mesenchymal Stem Cells (MSCs), particularly those isolated from menstrual blood has the potential to be used as a tool for improvement of fertility. In the current study, a 3D co-culture system with mice preantral follicles and human Menstrual Blood Mesenchymal Stem Cells (MenSCs) using either collagen or alginate beads was designed to investigate whether this system allows better preantral follicles growth and development. Results showed that MenSCs increase the indices of follicular growth including survival rate, diameter, and antrum formation as well as the rate of in vitro maturation (IVM) in both collagen and alginates beads. Although statistically not significant, alginate was found to be superior in terms of supporting survival rate and antrum formation. Hormone assay demonstrated that the amount of secreted 17 β-estradiol and progesterone in both 3D systems increased dramatically after 12 days, with the highest levels in system employing MenSCs. Data also demonstrated that relative expression of studied genes increased for Bmp15 and Gdf9 and decreased for Mater when follicles were cultured in the presence of MenSCs. Collectively, results of the present study showed that MenSCs could improve indices of follicular growth and maturation in vitro. Further studies are needed before a clinical application of MenSCs-induced IVM is considered.

C1QTNF3 in the murine ovary and its function in folliculogenesis.

C1q/tumor necrosis factor-related protein 3 (C1QTNF3) is a novel adipokine with modulating effects on metabolism, inflammation and the cardiovascular system. C1QTNF3 expression levels in the sera and omental adipose tissues of women with PCOS are low compared to control subjects. However, the expression and function of C1QTNF3 in the ovary has not previously been examined. Here, we assessed the expression patterns of C1qtnf3 in the ovary and explored its role in folliculogenesis. The C1qtnf3 transcript abundance was higher in large follicles than in small follicles and was under the influence of gonadotropin. C1QTNF3 was detected mainly in the granulosa cells and oocytes of growing follicles and modestly in the granulosa cells of atretic follicles and in luteal cells. Excess androgen significantly decreased C1QTNF3 expression in the ovaries in vivo and in granulosa cells in vitro Recombinant C1QTNF3 protein accelerated the weight gain of ovarian explants and the growth of preantral follicles induced by follicle stimulating hormone (FSH) in vitro The stimulatory effect of C1QTNF3 on ovarian growth was accompanied by the initiation of AKT, mTOR, p70S6K and 4EBP1 phosphorylation, an increase in CCND2 expression and a reduction in cleaved CASP3 levels. Moreover, the addition of C1QTNF3 accelerated proliferation and reduced activated CASP3/7 activity in granulosa cells. In vivo, the ovarian intrabursal administration of the C1QTNF3 antibody delayed gonadotropin-induced antral follicle development. Taken together, our data demonstrate that C1QTNF3 is an intraovarian factor that promotes follicle growth by accelerating proliferation, decelerating apoptosis and promoting AKT/mTOR phosphorylation.

Regulation by 3,5,3'-tri-iodothyronine and FSH of cytochrome P450 family 19 (CYP19) expression in mouse granulosa cells.

Cytochrome P450 family 19 (CYP19) plays an important role in follicular development, which is regulated by FSH. Although 3,5,3'-tri-iodothyronine (T3) combines with FSH to induce preantral follicle growth and granulosa cell development, the mechanism involved remains unclear. The aim of the present study was to determine the cellular and molecular mechanisms by which thyroid hormone (TH) and FSH regulate CYP19 expression and sterol biosynthesis during preantral follicle growth. Mice were injected subcutaneously (s.c.) with eCG (Equine chorionic gonadotropin). The results showed that eCG increased CYP19 expression in ovarian cells. CYP19 expression in granulosa cells was increased after FSH treatment, and this response was enhanced by T3. Knockdown of CYP19 significantly decreased granulosa cell viability and hormone-stimulated proliferation. In addition, CYP19 knockdown also blocked T3- and FSH-induced oestradiol (E2) synthesis in granulosa cells. Furthermore, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway was required for T3 and FSH regulation of CYP19 expression. In conclusion, the results of the present study indicate that CYP19 is important for T3- and FSH-induced granulosa cell development in the early stages. CYP19 could be a downstream effector of the PI3K/Akt pathway in regulating TH and FSH during follicular development and sterol biosynthesis. The findings suggest that CYP19 is a novel mediator of T3- and FSH-induced follicular development.

Insulin promotes preantral follicle growth and antrum formation through temporal expression of genes regulating steroidogenesis and water transport in the cat.

The aims of the present study were to determine the effects of insulin, invitro, on: (1) the viability and growth of domestic cat ovarian follicles; (2) mRNA expression of genes regulating steroidogenesis (cytochrome P450 family 17 subfamily, A polypeptide 1 (Cyp17a1), cytochrome P450 family 19 subfamily, A polypeptide 1 (Cyp19a1) and steroidogenic acute regulatory protein (Star)) and water transport (aquaporins (AQPs) Aqp1, Aqp3, Aqp7, Aqp9); and (3) steroid production (17β-oestradiol (E2), progesterone (P4), androstenedione (A4)). Cat secondary follicles were isolated from ovarian cortices and cultured in 0 (Control), 1 or 10µgmL-1 insulin for 14 days (Day 0=culture onset). Follicle and oocyte viability (based on neutral red staining), diameter and antrum formation were assessed every 72h and at the end of incubation (Day 14). Expression of steroidogenic and water transport genes was evaluated on Days 0, 6 and 12, and E2, P4 and A4 concentrations in the culture medium were determined on Day 12. By Day 14, 1 and 10µgmL-1 insulin had significantly promoted (P<0.05) both antrum formation in a mean (±s.e.m.) 26.9±9.0% and 78.0±10.0% of follicles respectively, and follicle growth (diameter 151.4±4.5 and 169.9±10.5µm respectively) compared with Control (antrum formation in 3.3±3.3% of follicles and follicle diameter 129.1±6.6µm). High insulin (10µgmL-1) treatment increased follicle viability compared with Control (86.0±9.8% vs 38.1±10.9% respectively; P<0.05). However, insulin had no beneficial effect (P>0.05) on oocyte diameter. Cyp17a1 expression on Days 6 and 12 was higher (P<0.05) in follicles cultured in the low (1µgmL-1) compared with high (10µgmL-1) insulin treatment, with no significant difference between low or high insulin vs Control groups. Star expression was higher (P<0.01) in the low insulin compared with Control group on Day 6, but Star was undetectable in the high insulin group by Day 12. Compared with high insulin, low insulin increased (P<0.05) Aqp1 expression on Day 6, but there were no significant differences between these two groups on Day 12. In contrast, high insulin decreased (P<0.05) Aqp9 transcript levels compared with Control. Only P4 production was affected by insulin, with P4 concentrations in the medium being higher (P<0.05) in the low compared with high insulin and Control groups. In summary, the findings indicate that insulin promotes cat ovarian follicle growth and survival invitro, including enhanced antrum formation, with the likely mechanism involving temporal expression of Cyp17a1, Star and Aqp9 genes.

Anti-Müllerian hormone is a survival factor and promotes the growth of rhesus macaque preantral follicles during matrix-free culture.

Anti-Müllerian hormone (AMH) plays a key role during ovarian follicular development, with local actions associated with a dynamic secretion profile by growing follicles. While results for AMH effects on antral follicle growth and function are consistent among studies in various species, any effects on preantral follicle development remain controversial. Therefore, experiments were conducted to investigate the direct actions and role of AMH during follicle development at the preantral stage. Macaque-specific short-hairpin RNAs (shRNAs) targeting AMH mRNA were incorporated into adenoviral vectors to decrease AMH gene expression in rhesus macaque follicles. Secondary follicles were isolated from adult macaque ovaries and cultured individually in the ultra-low-attachment dish containing defined medium supplemented with follicle-stimulating hormone and insulin for 5 weeks. Follicles were randomly assigned to treatment groups: (a) control, (b) nontargeting control shRNA-vector, (c) AMH shRNA-vector, (d) AMH shRNA-vector+recombinant human AMH, and (e) recombinant human AMH. Follicle survival and growth were assessed. Culture media were analyzed for steroid hormone and paracrine factor concentrations. For in vivo study, the nontargeting control shRNA-vector and AMH shRNA-vector were injected into macaque ovaries. Ovaries were collected 9 days postinjection for morphology and immunohistochemistry assessment. Decreased AMH expression reduced preantral follicle survival and growth in nonhuman primates. Supplemental AMH treatment in the culture media promoted preantral follicle growth to the small antral stage in vitro with increased steroid hormone and paracrine factor production, as well as oocyte maturation. These data demonstrate that AMH is a critical follicular paracrine/autocrine factor positively impacting preantral follicle survival and growth in primates.

Role of CYP51 in the Regulation of T3 and FSH-Induced Steroidogenesis in Female Mice.

Cytochrome P450 lanosterol 14α-demethylase (CYP51) is a key enzyme in sterol and steroid biosynthesis that is involved in folliculogenesis and oocyte maturation, which is regulated by follicle-stimulating hormone (FSH), as a key reproductive hormone during follicular development. Thyroid hormone (TH) is also important for normal reproductive function. Although 3,5,3'-triiodothyronine (T3) enhances FSH-induced preantral follicle growth, whether and how TH combines with FSH to regulate CYP51 expression during the preantral to early antral transition stage is unclear. The objective of this study was to determine the cellular and molecular mechanisms by which T3 and FSH regulate CYP51 expression and steroid biosynthesis during preantral follicle growth. Our results indicated that CYP51 expression was upregulated in granulosa cells by FSH, and this response was enhanced by T3. Moreover, knockdown CYP51 decreased cell viability. Meanwhile, gene knockdown also blocked T3 and FSH-induced estradiol (E2) and progesterone (P4) synthesis. These changes were accompanied by upregulation of phospho-GATA-4 content. Results of small interfering RNA analysis showed that knockdown of GATA-4 significantly diminished CYP51 gene expression as well as E2/P4 levels. Furthermore, thyroid hormone receptor β was necessary to the activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt), which was required for the regulation of CYP51 expression; activated GATA-4 was also involved these processes. Our data demonstrate that T3 and FSH cotreatment potentiates cellular development and steroid biosynthesis via CYP51 upregulation, which is mediated through the activation of the PI3K/Akt pathway. Meanwhile, activated GATA-4 is also involved in this regulatory system. These findings suggest that CYP51 is a mediator of T3 and FSH-induced follicular development.

Effect of metabolic stressors on survival and growth of in vitro cultured ovine preantral follicles and enclosed oocytes

The present study was undertaken to study the effect of metabolic stressors like elevated levels of ammonia, urea, Non-esterified fatty acid (NEFA) and β-hydroxybutyric acid (BHB) on preantral follicle growth, survival, growth rates of oocytes enclosed in preantral follicles (PFs), maturation rates of oocytes recovered from cultured follicles, hormone production (estrogen and progesterone), reactive oxygen species (ROS) as well as superoxide dismutase (SOD) activity. Small pre-antral follicles (SPFs, 100–250 μm) and large pre-antral follicles (LPFs, 250–450 μm) were isolated from slaughterhouse ovaries by a mechanical cum enzymatic method. SPFs and LPFs were cultured in vitro for 14 and 7 days respectively and examined for their growth, survival and growth rates of enclosed oocytes in PFs exposed with different concentration of ammonia (0, 100, 150, 200, 250, 300 and 400 μM), urea (0, 4, 4.5, 5, 5.5,6, 7 and 8 mM), NEFA [Basal NEFA (70 μM): stearic acid, SA (10 μM)+Palmitic acid, PA(20 μM)+oleic acid, OA(40 μM), b) Medium combo (140 μM): SA (20 μM)+ PA(40 μM)+ OA(80 μM), c) High combo (210 μM): SA (30 μM)+PA(60 μM)+OA(120 μM), d) Very high Combo (280 μM): SA(40 μM)+PA(80 μM)+OA(160 μM)] and BHB (0, 0.5, 0.75, and 1 μM). Results indicated that ammonia, urea, NEFA and BHB caused inhibition of survival and growth of in vitro cultured ovine PFs and enclosed oocytes at the levels of 300 μM, 8 mM, high combo level of NEFA and 0.75 μM respectively. Our study may contribute to the identification of the mechanisms involved in decline of fertility due to metabolic and nutritional stress in ruminants.

Understanding follicle growth in vitro: Are we getting closer to obtaining mature oocytes from in vitro-grown follicles in human?

Obtaining and fertilizing mature oocytes from immature follicles that were grown outside the body has conceptually attracted scientists for centuries, with initial attempts first documented in the 19th century. Significant progress has been made since then, due in part to a better understanding of folliculogenesis and improved techniques of in vitro follicle growth. Indeed, in vitro growth is now considered a reasonable approach to preserve or restore fertility when immature follicles and their oocytes need to be grown and matured outside the body. Certain patients would benefit from in vitro follicle growth, particularly those who carry a risk of cancer re-seeding after grafting of frozen-thawed ovarian tissue or who are at the risk of premature ovarian failure due to several intrinsic ovarian defects and genetic mutations that lead to accelerated follicle atresia and early exhaustion of the ovarian reserve. This review provides an update on the current status of in vitro growth of preantral human follicles, from initial efforts to the most recent achievements.

Nitric Oxide-Mediated Regulation of GLUT by T3 and Follicle-Stimulating Hormone in Rat Granulosa Cells.

Thyroid hormones are important for normal reproductive function. Although 3,5,3'-triiodothyronine (T3) enhances follicle-stimulating hormone (FSH)-induced preantral follicle growth and granulosa cells development in vitro, little is known about the molecular mechanisms regulating ovarian development via glucose. In this study, we investigated whether and how T3 combines with FSH to regulate glucose transporter protein (GLUT) expression and glucose uptake in granulosa cells. In this study, we present evidence that T3 and FSH cotreatment significantly increased GLUT-1/GLUT-4 expression, and translocation in cells, as well as glucose uptake. These changes were accompanied by upregulation of nitric oxide (NO) synthase (NOS)3 expression, total NOS and NOS3 activity, and NO content in granulosa cells. Furthermore, we found that activation of the mammalian target of rapamycin (mTOR) and phosphoinositide 3-kinase (PI3K)/Akt pathway is required for the regulation of GLUT expression, translocation, and glucose uptake by hormones. We also found that l-arginine upregulated GLUT-1/GLUT-4 expression and translocation, which were related to increased glucose uptake; however, these responses were significantly blocked by N(G)-nitro-l-arginine methylester. In addition, inhibiting NO production attenuated T3- and FSH-induced GLUT expression, translocation, and glucose uptake in granulosa cells. Our data demonstrate that T3 and FSH cotreatment potentiates cellular glucose uptake via GLUT upregulation and translocation, which are mediated through the activation of the mTOR/PI3K/Akt pathway. Meanwhile, NOS3/NO are also involved in this regulatory system. These findings suggest that GLUT is a mediator of T3- and FSH-induced follicular development.

Stimulation of ovarian follicle growth after AMPK inhibition.

Previous studies showed that the protein kinase B (Akt)-mammalian target of rapamycin (mTOR) and Hippo signaling Yes-associated protein (YAP) pathways play important roles in promoting follicle growth. Additionally, other studies demonstrated that 5' adenosine monophosphate-activated protein kinase (AMPK) is an upstream regulatory element of mTOR and YAP. Here, we used AMPK inhibitor (Compound C) to in vitro cultured ovaries from 10-day-old mice followed by in vivo grafting into adult hosts or to in situ treated ovaries of 3-week-old mice by intrabursal injection followed by gonadotropin stimulation. We found that the phosphorylation of ovarian mTOR and downstream proteins (ribosomal protein S6 (S6) and eukaryotic translation initiation factor 4B (eIF4B)) was upregulated following Compound C administration, whereas tuberous sclerosis complex 2 (TSC2) phosphorylation was downregulated. Additionally, treatment with Compound C increased hypoxia-inducible factor 1-alpha (Hif1a), vascular endothelial growth factor A (Vegfa), VEGF receptor 2 (Vegfr2) and connective tissue growth factor (Ctgf) mRNA levels. Furthermore, treatment of 10-day-old mice with Compound C promoted the growth of preantral and antral follicles accompanied by enhanced angiogenesis. In situ intrabursal injection with Compound C, followed by controlled ovarian hyperstimulation, increased the number of ovulated oocytes in 3-week-old mice, and these oocytes could be successfully fertilized, leading to the delivery of healthy pups. Our results demonstrated that treatment with AMPK inhibitor resulted in the activation of the mTOR signaling pathway, increases in Ctgf expression in mouse ovaries, stimulation of follicle development and promotion of ovarian angiogenesis for ovary growth.

Ring Finger Protein 6 Mediates Androgen-Induced Granulosa Cell Proliferation and Follicle Growth via Modulation of Androgen Receptor Signaling.

The destiny of the ovarian follicle (growth or atresia) is tightly regulated by the actions and interactions of endocrine, paracrine, and autocrine factors. Although androgens are known to be important in the regulation of folliculogenesis, whether they facilitate or suppress follicular growth has been controversial, and the mechanisms involved are not fully understood. Moreover, the role and regulation of androgen receptor (AR) in mediating androgen signaling during follicular development is not clear. Here, we report that the active androgen dihydrotestosterone upregulates the expression of AR and its E3 ligase ring finger protein 6 (RNF6), increasing site-specific AR polyubiquitination and AR transcriptional activity for soluble Kit ligand (sKit-L) expression in preantral follicle growth. RNF6 silencing suppressed dihydrotestosterone-induced AR ubiquitination (lysine residue 63) and proliferation and suppressed apoptosis in preantral granulosa cells, with these responses being overcome by the presence of exogenous sKit-L. Taken together, our findings support the notion that RNF6 plays an important role in androgen-induced, follicle-stage-dependent follicle growth and that it acts by facilitating AR-mediated granulosa cell sKit-L expression and proliferation. Our findings offer insights into the regulatory mechanism of androgen action in ovarian follicular growth.

Androgen Stimulates Growth of Mouse Preantral Follicles In Vitro: Interaction With Follicle-Stimulating Hormone and With Growth Factors of the TGFβ Superfamily

Androgens are essential for the normal function of mature antral follicles but also have a role in the early stages of follicle development. Polycystic ovary syndrome (PCOS), the most common cause of anovulatory infertility, is characterized by androgen excess and aberrant follicle development that includes accelerated early follicle growth. We have examined the effects of testosterone and dihydrotestosterone (DHT) on development of isolated mouse preantral follicles in culture with the specific aim of investigating interaction with follicle-stimulating hormone (FSH), the steroidogenic pathway, and growth factors of the TGFβ superfamily that are known to have a role in early follicle development. Both testosterone and DHT stimulated follicle growth and augmented FSH-induced growth and increased the incidence of antrum formation among the granulosa cell layers of these preantral follicles after 72 hours in culture. Effects of both androgens were reversed by the androgen receptor antagonist flutamide. FSH receptor expression was increased in response to both testosterone and DHT, as was that of Star, whereas Cyp11a1 was down-regulated. The key androgen-induced changes in the TGFβ signaling pathway were down-regulation of Amh, Bmp15, and their receptors. Inhibition of Alk6 (Bmpr1b), a putative partner for Amhr2 and Bmpr2, by dorsomorphin resulted in augmentation of androgen-stimulated growth and modification of androgen-induced gene expression. Our findings point to varied effects of androgen on preantral follicle growth and function, including interaction with FSH-activated growth and steroidogenesis, and, importantly, implicate the intrafollicular TGFβ system as a key mediator of androgen action. These findings provide insight into abnormal early follicle development in PCOS.

Corrigendum to: Hypoxia limits mouse follicle growth in vitro.

Ovarian follicle culture is useful for elucidation of factors involved in the regulation of follicular function. We examined the effects of gas phase oxygen concentration, an oil overlay, serum type and medium supplementation with FSH, insulin-transferrin-selenium (ITS) and I-ascorbic acid on cultured preantral mouse follicle growth in a spherical, non-attached follicle culture system. Follicle growth in 5% oxygen was significantly (P<0.01) inferior to growth in 20% oxygen in terms of follicle diameter. This was likely due to hypoxia, as evidenced by significantly (P<0.05) increased follicle secretion of vascular endothelial growth factor (VEGF), a marker of cell hypoxia. Follicular growth was not (P>0.05) affected by an oil overlay, ITS supplementation or serum type. Culture in medium with 5% mouse serum, 1 IU mL-1 FSH, 25 μgmL-1 l-ascorbic acid and 20% oxygen without an oil overlay supported the growth of follicles to a maximum diameter of 380 μm in 6 days. Compared with mature preovulatory mouse follicles in vivo that often have diameters >500 μm within the same time frame, in vitro-grown follicles clearly exhibit limited growth. Thus, adequate oxygenation is an essential factor in the process of optimising follicle growth.

Onset and Heterogeneity of Responsiveness to FSH in Mouse Preantral Follicles in Culture.

The obligatory role of follicle-stimulating hormone (FSH) in normal development and function of ovarian antral follicles is well recognized, but its function in preantral growth is less clear. The specific objective of this study was to investigate the response, in culture, to FSH of mouse preantral follicles of increasing size, focusing particularly on growth rate and gene expression. Preantral follicles were mechanically isolated from ovaries of C57BL/6 mice, 12 to 16 days postpartum, and single follicles cultured for up to 96 hours in medium alone (n = 511) or with recombinant human FSH 10 ng/mL (n = 546). Data were grouped according to initial follicle diameter in 6 strata ranging from <100 to >140 μm. Follicles of all sizes grew in the absence of FSH (P < 0.01, paired t test). All follicles grew at a faster rate (P < 0.0001) in the presence of 10 ng/mL FSH but larger follicles showed the greatest change in response to FSH. Even the smallest follicles expressed FSH receptor messenger RNA (mRNA). FSH-induced growth was inhibited by KT5720, an inhibitor of protein kinase A (PKA), implicating the PKA pathway in FSH-induced follicle growth. In response to FSH in vitro, FSH receptor mRNA (measured by quantitative polymerase chain reaction) was reduced (P < 0.01), as was Amh (P < 0.01), whereas expression of StAR (P < 0.0001) and the steroidogenic enzymes Cyp11a1 (P < 0.01) and Cyp19 (P < 0.0001) was increased. These results show heterogeneous responses to FSH according to initial follicle size, smaller follicles being less FSH dependent than larger preantral follicles. These findings strongly suggest that FSH has a physiological role in preantral follicle growth and function.

Viability and growth of feline preantral follicles in vitro cultured with insulin growth factor and epidermal growth factor supplemented medium.

In vitro culture of ovarian preantral follicles has emerged as a reproductive technology aimed at obtaining large amount of oocytes for in vitro embryo production. The addition of growth factors (GF) in the in vitro culture of preantral follicles of different species has provided superior results of follicular development, antrum formation and proliferation of granulosa cells. However, there are only few reports regarding the use of these factors on feline preantral follicle in vitro culture. Thus, the aim of this study was to investigate the effect of a combination of IGF-1 and EGF on in vitro viability and growth of preantral follicles and enclosed oocytes collected from domestic cats. A total of 64 follicles characterized by multilayer granulosa cells were isolated and individually cultured for 6days (T6) in minimum essential medium supplemented with IGF-1+EGF (100ng/ml each) or without (control). A higher percentage of follicles were viable after culture with GF than without, and an increase in size when IGF-1+EGF were added to the medium (170±32.4μm (T0) vs. 201±22.3μm (T6); p<.05) was observed. An increase in the diameter was also observed in follicles cultured without GF, but this increase was only 8.3% compared to 15.4% of those cultured with GF (p<.05). No differences were found in the diameter of oocytes contained in follicles cultured in the non-supplemented or supplemented media (107.9±11.8μm (T0) vs. 113.2±15.6μm (T6); p>.05). These data suggest that the addition of IGF-1 and EGF to the culture medium promotes the in vitro development of preantral follicles of cats.

Effect of alpha lipoic acid on in vitro development of bovine secondary preantral follicles

The present study aimed to evaluate the in vitro effect of alpha lipoic acid (ALA) addition to the culture medium on the development of the bovine secondary preantral follicles. Bovine secondary preantral follicles were collected and divided into two groups depending on their size (80–100 μm and 100–110 μm). They were cultured in vitro for 15 days (D) using different media including at three different doses of ALA. The genes expression levels of follicle-stimulating hormone beta-subunit (FSHR), insulin-like growth factor (IGF-1), Activin, luteinizing hormone/choriogonadotropin receptor, bone morphogenetic protein receptor type IA, transforming growth factor beta receptor 1, growth differentiation factor 9, BCL2-associated X protein (BAX), and C-Myc were studied using real-time polymerase chain reaction. The protein expression levels of FSHR, IGF-1, and BAX were measured using Western blot analysis. The results of the present work revealed that in vitro addition of ALA-induced significant increase in the growth and development of secondary preantral follicles throughout the culture period as compared to control. The FSHR, IGF-1, luteinizing hormone/choriogonadotropin receptor, bone morphogenetic protein receptor type IA, transforming growth factor beta receptor 1, growth differentiation factor 9, and Activin A genes were upregulated in ALA-treated follicles as compared to the control. On contrary, preapoptotic genes BAX and C-Myc were downregulated in treated follicles compared to control ones. The protein levels of FSHR and IGF-1 were highly expressed in treated follicles compared to control; however, BAX protein was downregulated in the treated follicles groups. The addition of ALA to the culture medium enhances secondary preantral follicles development and growth.

Androgen and estrogen promote macaque preantral follicle survival and growth in the absence of fsh during 3-dimensional culture

In vivo development of primate follicles is generally considered independent of follicle stimulating hormone (FSH) until the antral stage. However, previous data suggest that FSH promotes the survival of human and macaque preantral follicles in vitro. In this study, we investigated whether local factors, androgen, estrogen and anti-Müllerian hormone (AMH), can replace FSH in supporting the development of macaque preantral follicle during 3-dimensional culture.

Interactions between androgens, FSH, anti-Müllerian hormone and estradiol during folliculogenesis in the human normal and polycystic ovary.

Androgens, FSH, anti-Müllerian hormone (AMH) and estradiol (E2) are essential in human ovarian folliculogenesis. However, the interactions between these four players is not fully understood. The purpose of this review is to highlight the chronological sequence of the appearance and function of androgens, FSH, AMH and E2 and to discuss controversies in the relationship between FSH and AMH. A better understanding of this interaction could supplement our current knowledge about the pathophysiology of the polycystic ovary syndrome (PCOS). A literature review was performed using the following search terms: androgens, FSH, FSH receptor, anti-Mullerian hormone, AMHRII, estradiol, follicle, ovary, PCOS, aromatase, granulosa cell, oocyte. The time period searched was 1980-2015 and the databases interrogated were PubMed and Web of Science. During the pre-antral ('gonadotropin-independent') follicle growth, FSH is already active and promotes follicle growth in synergy with theca cell-derived androgens. Conversely, AMH is inhibitory by counteracting FSH. We challenge the hypothesis that AMH is regulated by androgens and propose rather an indirect effect through an androgen-dependent amplification of FSH action on granulosa cells (GCs) from small growing follicles. This hypothesis implies that FSH stimulates AMH expression. During the antral ('gonadotropin-dependent') follicle growth, E2 production results from FSH-dependent activation of aromatase. Conversely, AMH is inhibitory but the decline of its expression, amplified by E2, allows full expression of aromatase, characteristic of the large antral follicles. We propose a theoretical scheme made up of two triangles that follow each other chronologically. In PCOS, pre-antral follicle growth is excessive (triangle 1) because of intrinsic androgen excess that renders GCs hypersensitive to FSH, with consequently excessive AMH expression. Antral follicle growth and differentiation are disturbed (triangle 2) because of the abnormally persisting inhibition of FSH effects by AMH that blocks aromatase. Beside anovulation, this scenario may also serve to explain the higher receptiveness to gonadotropin therapy and the increased risk of ovarian hyperstimulation syndrome (OHSS) in patients with PCOS. Within GCs, the balance between FSH and AMH effects is pivotal in the shift from androgen- to oestrogen-driven follicles. Our two triangles hypothesis, based on updated data from the literature, offers a pedagogic template for the understanding of folliculogenesis in the normal and polycystic ovary. It opens new avenues for the treatment of anovulation due to PCOS.