Direct actions of androgen, estrogen and anti-Müllerian hormone on primate secondary follicle development in the absence of FSH in vitro.

STUDY QUESTION: What are effects of androgen, estrogen and anti-Müllerian hormone (AMH), independent of FSH action, on the development and function of primate follicles from the preantral to small antral stage in vitro? SUMMARY ANSWER: Androgen and estrogen, but not AMH, promote follicle survival and growth in vitro, in the absence of FSH. However, their growth-promoting effects are limited to the preantral to early antral stage. WHAT IS KNOWN ALREADY: FSH supports primate preantral follicle development in vitro. Androgen and estrogen augment follicle survival and growth in the presence of FSH during culture. STUDY DESIGN SIZE, DURATION: Nonhuman primate model; randomized, control versus treatment groups. Rhesus macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Follicles were encapsulated in 0.25% (w/v) alginate and cultured individually in modified alpha minimum essential media with (i) FSH (1 ng/ml; control), (ii) no FSH, (iii) no FSH + estradiol (E2; 100 pg/ml)/dihydrotestosterone (DHT; 50 ng/ml) and (iv) no FSH + AMH (50 ng/ml). In a second experiment, follicles were cultured with (i) FSH (1 ng/ml), (ii) no FSH, (iii) no FSH + E2 (1 ng/ml), (iv) no FSH + DHT (50 ng/ml) and (v) no FSH + E2/DHT. Follicle survival, antrum formation and growth pattern were evaluated. Progesterone (P4), E2 and AMH concentrations in culture media were measured. MAIN RESULTS AND THE ROLE OF CHANCE: In the first experiment, FSH deprivation significantly decreased (P < 0.05) follicle survival rates in the no FSH group (16 ± 5%), compared to CTRL (66 ± 9%). E2/DHT (49 ± 5%), but not AMH (27 ± 8%), restored follicle survival rate to the CTRL level. Similarly, antrum formation rates were higher (P < 0.05) in CTRL (56 ± 6%) and E2/DHT groups (54 ± 14%), compared to no FSH (0 ± 0%) and AMH (11 ± 11%) groups. However, follicle growth rate after antrum formation and follicle diameter at week 5 was reduced (P < 0.05) in the E2/DHT group (405 ± 25 µm), compared to CTRL (522 ± 29 µm). Indeed, the proportion of fast-grow follicles at week 5 was higher in CTRL (29% ± 5), compared to E2/DHT group (10 ± 3%). No fast-grow follicles were observed in no FSH and AMH groups. AMH levels at week 3 remained similar in all groups. However, media concentrations of P4 and E2 at week 5 were lower (P < 0.05, undetectable) in no FSH, E2/DHT and AMH groups, compared to CTRL (P4 = 93 ± 10 ng/ml; E2 = 4 ± 1 ng/ml). In the second experiment, FSH depletion diminished follicle survival rate (66 ± 8% in control versus 45 ± 9% in no FSH, P = 0.034). E2 plus DHT (31.5 ± 11%) or DHT alone (69 ± 9%) restored follicle survival rate to the control (FSH) level as expected. Also, E2 plus DHT or DHT alone improved antrum formation rate. However, in the absence of FSH, E2 plus DHT or DHT alone did not support growth, in terms of follicle diameter, or steroid (P4 or E2) production after the antral stage. LIMITATIONS REASONS FOR CAUTION: This study is limited to in vitro effects of E2, DHT and AMH during the interval from the secondary to small antral stage of macaque follicular development. In addition, the primate follicle pool is heterogeneous and differs between animals; therefore, even though only secondary follicles were selected, follicle growth and developmental outcomes might differ from one animal to another. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel information on the possible actions of estrogen and androgen during early follicular development in primates. Our results suggest that sequential exposure of preantral follicles to local factors, e.g. E2 and DHT, followed by gonadotropin once the follicle reaches the antral stage, may better mimic primate folliculogenesis in vivo. STUDY FUNDING/COMPETING INTEREST(S): Research reported in this publication was supported by the Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Center for Translational Research on Reproduction and Infertility 5P50HD071836, and the NIH Primate Centers Program 8P510D011092. There are no conflicts of interest.

Differential effects of estrogen and progesterone on development of primate secondary follicles in a steroid-depleted milieu in vitro.

STUDY QUESTION: What are the direct effects of progesterone (P4) and estradiol (E2) on the development and function of primate follicles in vitro from the pre-antral to early antral stage? SUMMARY ANSWER: In a steroid-depleted milieu, E2 improved follicle survival, growth, antrum formation and oocyte health, whereas P4 exerted minimal beneficial effects on follicle survival and reduced oocyte health. WHAT IS KNOWN ALREADY: Effects of P4 and E2 on follicle development have been studied primarily in large antral and pre-ovulatory follicles. Chronic P4 exposure suppresses antral follicle growth, but acute P4 exposure promotes oocyte maturation in pre-ovulatory follicles. Effects of E2 can be stimulatory or inhibitory depending upon species, dose and duration of exposure. STUDY DESIGN, SIZE, DURATION: Non-human primate model, randomized, control versus treatment. Macaque (n = 6) secondary follicles (n = 24 per animal per treatment group) were cultured for 5 weeks. PARTICIPANTS/MATERIALS, SETTING, METHODS: Adult rhesus macaque secondary follicles were encapsulated in 0.25% alginate and cultured individually in media containing follicle stimulating hormone plus (i) vehicle, (ii) a steroid-synthesis inhibitor, trilostane (TRL, 250 ng/ml), (iii) TRL + low E2 (100 pg/ml) or progestin (P, 10 ng/ml R5020) and (iv) TRL + high E2 (1 ng/ml E2) or P (100 ng/ml R5020). Follicles reaching the antral stage (≥750 µm) were treated with human chorionic gonadotrophin for 34 h. End-points included follicle survival, antrum formation, growth pattern, plus oocyte health and maturation status, as well as media concentrations of P4, E2 and anti-Müllerian hormone (AMH). MAIN RESULTS AND THE ROLE OF CHANCE: In a steroid-depleted milieu, low dose, but not high dose, P improved (P < 0.05) follicle survival, but had no effect (P > 0.05) on antrum formation and AMH production. Low-dose P increased (P < 0.05) P4 production in fast-grow follicles, and both doses of P elevated (P < 0.05) E2 production in slow-grow follicles. Additionally, low-dose P increased (P < 0.05) the percentage of no-grow follicles, and high-dose P promoted oocyte degeneration. In contrast, E2, in a steroid-depleted milieu, improved (P < 0.05) follicle survival, growth, antrum formation and oocyte health. E2 had no effect on P4 or E2 production. Follicles exposed to E2 yielded mature oocytes capable of fertilization and early cleavage, at a rate similar to untreated control follicles. LIMITATIONS, REASONS FOR CAUTION: This study is limited to in vitro effects of P and E2 during the interval from the secondary to small antral stage of macaque follicles. WIDER IMPLICATIONS OF THE FINDINGS: This study provides novel information on the direct actions of P4 and E2 on primate pre-antral follicle development. Combined with our previous report on the actions of androgens, our findings suggest that androgens appear to be a survival factor but hinder antral follicle differentiation, E2 appears to be a survival and growth factor at the pre-antral and early antral stage, whereas P4 may not be essential during early folliculogenesis in primates. STUDY FUNDING/COMPETING INTERESTS: NIH P50 HD071836 (NCTRI), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), ONPRC 8P51OD011092. There are no conflicts of interest.

Fibrin promotes development and function of macaque primary follicles during encapsulated three-dimensional culture.

STUDY QUESTION: Does fibrin introduced into the extracellular matrix affect the growth and maturation of individual primate follicles during encapsulated three-dimensional (3D) culture? SUMMARY ANSWER: While not altering follicle survival, fibrin-alginate (FIBRIN) improves macaque primary, but not secondary, follicle development during encapsulated 3D culture in terms of growth, steroidogenesis, anti-Müllerian hormone (AMH)/vascular endothelial growth factor (VEGF) production and oocyte maturation. WHAT IS KNOWN ALREADY: Efforts to grow non-human primate ovarian follicles from the secondary to the antral stage during encapsulated 3D culture have been successful. However, the growth and maturation of primary follicles in vitro has not been reported in primates, especially in chemically defined conditions. STUDY DESIGN, SIZE, DURATION: In vitro follicle maturation was investigated using the rhesus macaque (Macaca mulatta). Ovaries (n = 7 pairs) were obtained during the early follicular phase of the menstrual cycle (cycle day 1-4). Primary (80-120 µm diameter) and secondary (125-225 µm diameter) follicles were isolated mechanically, randomly assigned to experimental groups, encapsulated into alginate (0.25% w/v) or FIBRIN (25 mg/ml fibrinogen-0.25% alginate) and cultured for 13 and 5 weeks, respectively. MATERIALS, SETTING, METHODS: Individual follicles were cultured in alpha minimum essential medium supplemented with FSH. Follicle survival and growth were assessed by microscopy. Follicles that reached the antral stage were treated with recombinant hCG. Metaphase II (MII) oocytes were inseminated via ICSI. Follicle morphology was evaluated by hematoxylin and eosin (H&E) staining. Immunohistochemistry was performed for cytochrome P450 family 17 subfamily A polypeptide 1 (CYP17A1) and 19 subfamily A polypeptide 1 (CYP19A1). Culture medium was analyzed for estradiol (E2) and progesterone by chemiluminescence, androstenedione (A4) by radioimmunoassay, as well as anti-Müllerian hormone (AMH) and vascular endothelial growth factor (VEGF) by enzyme-linked immunosorbent assay. MAIN RESULTS AND THE ROLE OF CHANCE: A total of 105 primary and 133 secondary follicles were collected. The presence of fibrin in the alginate matrix had no effect on either primary or secondary follicle survival. Growing primary and secondary follicles formed an antrum at Weeks 9 and 3, respectively. The percentage of growing follicles was higher (P < 0.05) for primary follicles cultured in FIBRIN than alginate at Week 13. The diameters were larger for the growing secondary follicles cultured in alginate than FIBRIN at Week 5 (P < 0.05). H&E staining revealed the typical morphology for small antral follicles. CPY17A1 immunostaining was detected in theca cells, while CYP19A1 was observed in granulosa cells. E2 increased (P < 0.05) during antrum formation in growing follicles at Week 9 for primary and Week 3 for secondary follicles. AMH levels in medium from growing primary follicles increased (P < 0.05) after Week 4 with peak levels at Weeks 9-11. AMH increased (P < 0.05) in growing secondary follicles at Weeks 3-5. VEGF levels in medium were elevated (P < 0.05) in growing primary follicles at Week 9. VEGF increased (P < 0.05) in medium from growing secondary follicles at Weeks 3-5. E2, AMH and VEGF production was higher (P < 0.05) in primary follicle culture with FIBRIN than alginate alone. One primary follicle cultured in FIBRIN (1 of 5 follicles harvested) and a secondary follicle cultured in alginate alone (1 of 15 follicles harvested) yielded an MII oocyte. The fertilized oocyte from primary follicle culture arrested without cell division after fertilization, while the oocyte from secondary follicle culture cleaved and reached the morula stage. LIMITATIONS, REASONS FOR CAUTION: The study reports on in vitro development and function of individual macaque follicles, that is limited to the interval from the primary and secondary stage to the small antral stage. The findings await translation to human ovarian follicles. WIDER IMPLICATIONS OF THE FINDINGS: The 3D model for primate follicle development offers a unique opportunity to investigate the growth and regulation of primate primary, as well as secondary follicles, and their enclosed oocytes, as they grow to the antral stage by monitoring and manipulating factors or signaling pathways in vitro. Since primate primary follicles, in addition to secondary follicles, can be cultured to the antral stage to provide mature oocytes, they represent an additional source of pre-antral follicles for in vitro follicle maturation with the potential to provide gametes for assisted reproductive technology as an option for fertility preservation in women, including patients with cancer. STUDY FUNDING/COMPETING INTEREST(S): This work was supported by The Oncofertility Consortium (NIH U54 RR024347-HD058294, PL1-EB008542), NIH U54-HD18185 (Eunice Kennedy Shriver Specialized Cooperative Centers Program in Reproduction and Infertility Research), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), Oregon National Primate Research Center 8P51OD011092. There are no conflicts of interest.

Morphological and functional preservation of pre-antral follicles after vitrification of macaque ovarian tissue in a closed system.

STUDY QUESTION: What are the appropriate conditions to vitrify the macaque ovarian cortex in a large-volume, closed system that will preserve functional pre-antral follicles? SUMMARY ANSWER: The combination of glycerol, ethylene glycol (EG) and polymers with cooling in liquid nitrogen (LN2) vapor and a two-step warming procedure was able to preserve tissue and follicle morphology as well as function of a small population of secondary follicles in the macaque ovarian cortex following vitrification in a closed system. WHAT IS KNOWN ALREADY: For prepubertal cancer patients or those who require immediate cancer therapy, ovarian tissue cryopreservation offers the only hope for future fertility. However, the efficacy of live birth from the transplantation of cryopreserved ovarian tissue is still unclear. In addition, live birth from cryopreserved ovarian tissue has only been demonstrated after tissue autotransplantation, which poses the risk of transmitting metastatic cancer cells back to the cancer survivor in certain cancers. STUDY DESIGN, SIZE, DURATION: Non-human primate model, n = 4, randomized, control versus treatment. End-points were collected from tissue histology, tissue culture (48 h) and isolated secondary follicle culture (6 weeks). PARTICIPANTS/MATERIALS, SETTING, METHODS: Two vitrification solutions (VSs) containing EG + glycerol (VEG) and EG + dimethylsulfoxide (VED) were examined for vitrification, devitrification and thermodynamic properties. Once the optimal VS was determined, macaque ovarian cortical pieces (3 × 3 × 0.5 mm(3)) were divided into fresh and two vitrified groups (VEG and VED). For the vitrification groups, tissues were exposed to 1/4, 1/2 and 1× VS for 5 min/step as well as 1× VS + polymers for 1 min at 37°C, loaded into high-security straws with 1 ml of VS + polymers, heat sealed and cooled in LN2 vapor. Samples were warmed in a 40°C water bath and cryoprotective agents were diluted with 1, 0.5, 0.25 and 0 M sucrose. Tissues were fixed for histological analysis and cultured with bromodeoxyuridine (BrdU). Secondary follicles from VEG tissues were encapsulated and cultured (n = 24/treatment/animal). Follicle health, diameter and steroid [progesterone, androstenedione (A4), estradiol (E2)] production were analyzed weekly. MAIN RESULTS AND THE ROLE OF CHANCE: Dense stroma and intact pre-antral follicles were observed using VS containing 27% glycerol, 27% EG and 0.8% polymers with cooling in LN2 vapor and a two-step warming. Higher cooling and warming rates led to fracturing. BrdU uptake was evident in granulosa cells of growing follicles in fresh and vitrified tissues. Secondary follicles from fresh tissues (70 ± 12%) and tissues vitrified with VEG (52 ± 2%) showed similar survival rates (all data: mean ± SEM; P > 0.05). For both groups, the initial follicle diameter was similar and increased (P < 0.05) by Week 3, but diameters in vitrified follicles were smaller (P < 0.05) by Week 6 (566 ± 27 µm) than those of the fresh follicles (757 ± 26 µm). Antrum formation rates were lower (P < 0.05) for vitrified (37 ± 6%) relative to fresh (64 ± 8%) follicles. There was no significant change in levels in culture media of E2, P4 and A4 between fresh and VEG groups at any time point during culture. LIMITATIONS, REASONS FOR CAUTION: Only in vitro studies are reported. Future in vivo tissue transplantation studies will be needed to confirm long-term function and fertility potential of vitrified ovarian tissues. WIDER IMPLICATIONS OF THE FINDINGS: This is the first demonstration of antral follicle development during 3D culture following ovarian tissue vitrification in a closed system using primate ovarian tissue. While diminished antrum formation and slower growth in vitro reflect residual cryodamage, continued development of ovarian tissue vitrification based on cryobiology principles using a non-human primate model will identify safe, practical and efficient protocols for eventual clinical use. Tissue function following heterotopic transplantation is currently being examined. STUDY FUNDING/COMPETING INTEREST(S): National Institutes of Health (NIH) Oncofertility Consortium UL1 RR024926 (1RL1-HD058293, HD058295, PL1 EB008542), the Eunice Kennedy Shriver NICHD/NIH (U54 HD018185) and ONPRC 8P51OD011092-53. G.M.F. works for the company that makes the polymers used in the current study.

Temporal and anatomical sensitivities to the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin leading to premature acyclicity with age in rats.

The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that mediates diverse dioxin toxicities. While the acute effects of activation of the AhR pathway by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have been a focus of past study, the role of this pathway in normal physiology and ageing is unclear. The purpose of this study was to identify the portion of the reproductive axis [ovary vs. hypothalamus and pituitary gland (H-H axis)] and the stages of the reproductive lifespan (foetal and early post-natal life vs. adolescence and adulthood) that are particularly sensitive to the effects of TCDD during female reproductive ageing. Adult pregnant Lewis rat dams were dosed with corn oil vehicle or TCDD (50 ng/kg-week by gavage) on days 14 and 21 of gestation and post-natal days 7 and 14 to provide in utero and lactational (IUL) exposure to pups. Female pups (n = 96) were weaned on post-natal day 21 and dosed with TCDD or vehicle weekly. Half of the pups were used as donors for ovary transplantation while the remainder were recipients. Following ovary transplantation, rats (n = 6-8 per group) received weekly TCDD or vehicle again until sacrifice at 8 months of age. Beginning at vaginal opening, reproductive cycles were monitored by vaginal cytology for 10 days each month. Blood samples were collected at 22.00 h on proestrus to measure concentration of 17beta-oestradiol in serum. Real-time PCR was used to determine differences in Cyp1a1, Cyp19a1, Cyp17a1, LH receptor (LHR), FoxA2 and FoxJ1 genes expression between control and remaining groups. IUL exposure of the H-H axis plus adult exposure of the whole body to TCDD significantly delayed puberty in females rats. Data analysis revealed an accelerated onset of acyclicity by 5 months in all groups involving IUL exposure of the developing ovary to TCDD. 17beta-oestradiol was significantly decreased in animals receiving TCDD during IUL exposure of the H-H axis. CYP1a1 expression was markedly greater in the liver than in ovarian tissue and correlated with ongoing TCDD exposure. Aromatase, 17alpha-hydroxylase and LHR gene expressions were largely unchanged (or occasionally elevated) by TCDD. FoxA2 and FoxJ1 mRNAs were similarly of limited value mechanistically, although FoxJ1 was much higher in TTT females (receiving TCDD as donor, recipient and adult). This study reveals a particular sensitivity of the developing ovary to TCDD leading to early loss of reproductive function with age.

Genetically encoded fluorescent reporters of protein tyrosine kinase activities in living cells.

The complexity and specificity of many forms of signal transduction are widely believed to require spatial compartmentation of protein kinase and phosphatase activities, yet existing methods for measuring kinase activities in cells lack generality or spatial or temporal resolution. We present three genetically encoded fluorescent reporters for the tyrosine kinases Src, Abl, and epidermal growth factor (EGF) receptor. The reporters consist of fusions of cyan fluorescent protein (CFP), a phosphotyrosine binding domain, a consensus substrate for the relevant kinase, and yellow fluorescent protein (YFP). Stimulation of kinase activities in living cells with addition of growth factors causes 20-35% changes in the ratios of yellow to cyan emissions because of phosphorylation-induced changes in fluorescence resonance energy transfer (FRET). Platelet-derived growth factor (PDGF) stimulated Abl activity most strongly in actin-rich membrane ruffles, supporting the importance of this tyrosine kinase in the regulation of cell morphology. These results establish a general strategy for nondestructively imaging dynamic protein tyrosine kinase activities with high spatial and temporal resolution in single living cells.

Probing ion permeation and gating in a K+ channel with backbone mutations in the selectivity filter.

Potassium channels selectively conduct K+ ions across cell membranes, and use diverse mechanisms to control their gating. We studied ion permeation and gating of an inwardly rectifying K+ channel by individually changing the amide carbonyls of two conserved glycines lining the selectivity filter to ester carbonyls using nonsense suppression. Surprisingly, these backbone mutations do not significantly alter ion selectivity. However, they dramatically change the kinetics of single-channel gating and produce distinct subconductance levels. The mutation at the glycine closer to the inner mouth of the pore also abolishes high-affinity binding of Ba2+ to the channel, indicating the importance of this position in ion stabilization in the selectivity filter. Our results demonstrate that K+ ion selectivity can be retained even with significant reduction of electronegativity in the selectivity filter, and that conformational changes of the filter arising from interactions between permeant ions and the backbone carbonyls contribute directly to channel gating.

Phage-display evolution of tyrosine kinases with altered nucleotide specificity.

The problem of identifying downstream targets of kinase phosphorylation remains a challenge despite technological advances in genomics and proteomics. A recent approach involves the generation of kinase mutants that can uniquely use "orthogonal" ATP analogs to phosphorylate substrates in vivo. Using structure-based design, mutants of several protein kinase superfamily members have been found; robust and general methods are needed, however, for altering the nucleotide specificity of the remaining kinases in the genome. Here we demonstrate the application of a new phage display technique for direct functional selection to the identification of a tyrosine kinase mutant with the ability to use N6-benzyl-ATP. Our method produces, in five rounds of selection, a mutant identical to the best orthogonal Src kinase found to date. In addition, we isolate from a larger library of kinase mutants a promiscuous clone capable of using many different ATP analogs. This approach to engineering orthogonal kinases, combined with others, will facilitate the mapping of phosphorylation targets of any kinase in the genome.

Single-molecule fluorescence spectroscopy of enzyme conformational dynamics and cleavage mechanism.

Fluorescence resonance energy transfer and fluorescence polarization anisotropy are used to investigate single molecules of the enzyme staphylococcal nuclease. Intramolecular fluorescence resonance energy transfer and fluorescence polarization anisotropy measurements of fluorescently labeled staphylococcal nuclease molecules reveal distinct patterns of fluctuations that may be attributed to protein conformational dynamics on the millisecond time scale. Intermolecular fluorescence resonance energy transfer measurements provide information about the dynamic interactions of staphylococcal nuclease with single substrate molecules. The experimental methods demonstrated here should prove generally useful in studies of protein folding and enzyme catalysis at single-molecule resolution.

Molecular cloning of a Schizosaccharomyces pombe cDNA encoding lanosterol synthase and investigation of conserved tryptophan residues.

A Schizosaccharomyces pombe cDNA encoding lanosterol synthase was cloned by complementing a Saccharomyces cerevisiae lanosterol synthase mutant. The predicted 83-kDa protein is 54-58% identical to other lanosterol synthases. The previously known lanosterol synthases contain 229 conserved residues, which should encompass the catalytically essential amino acids. This number is decreased dramatically by including the Sc. pombe lanosterol synthase in the analysis; 42 residues are no longer conserved and therefore are catalytically nonessential. We have begun mutagenic studies to identify catalytic residues from the remaining conserved residues. Mutant Sa. cerevisiae lanosterol synthase genes were generated in which phenylalanine was specifically substituted for conserved tryptophan residues. All of the resultant mutant enzymes retained the ability to complement the Sc. cerevisiae lanosterol synthase mutant, suggesting that these conserved tryptophan residues are not catalytically essential.