Theodore A. Molskness

Regulation and action of angiogenic factors in the primate ovary.

The ephemerality of the maturing follicle and subsequent corpus luteum as they perform their gametogenic and/or endocrine functions during the ovarian cycle is associated with remarkable changes in local vasculature. Studies on the angiogenic and angiolytic process in the ovary, rare in healthy adult tissues, complement recent efforts to understand vasculogenesis in embryonic tissues and to control angiogenesis in pathologic states such as cancer. Several reports indicate that the newly discovered vascular-specific angiogenic factors are expressed in the ovary, notably members of the vascular endothelial growth factor (VEGF) and angiopoietin (Ang) families plus their receptors (VEGF-Rs, neuropilins, Tie). Unlike in many other tissues, gonadotropic hormones (particularly luteinizing hormone, [LH]) are major stimulators of angiogenesis and VEGF/Ang expression in the ovary. However, local factors such as insulin-like growth factors or oxygen tension likely modulate the angiogenic processes. Recent studies employing systemic or local administration of anti-angiogenic drugs (TNP-470 or fumagillin) or specific VEGF antagonists (VEGF antibody or soluble VEGFR-1) demonstrate a vital role for normal angiogenesis and VEGF action in follicle development, ovulation, or corpus luteum function. Further studies discerning the various angiogenic factors and their roles in controlling the growth, maturation, function, and regression of the vasculature in ovarian compartments during the menstrual cycle could yield novel strategies for manipulating fertility or for alleviating infertility.

Insulin-Like Growth Factors-1 and -2, but not Hypoxia, Synergize with Gonadotropin Hormone to Promote Vascular Endothelial Growth Factor-A Secretion by Monkey Granulosa Cells from Preovulatory Follicles

Abstract The midcycle gonadotropin surge promotes vascular endothelial growth factor-A (VEGF-A) production by granulosa cells in the ovulatory follicle, but it is unclear whether primary regulators of VEGF secretion in other tissues, including hypoxia and growth factors, are also important in the ovary. To address these issues, granulosa cells were collected from rhesus monkeys during controlled ovarian stimulation either before (i.e., nonluteinized granulosa cells, NLGCs) or 27 hours after (i.e., luteinized granulosa cells, LGCs) administration of an ovulatory bolus of hCG, and cultured in fibronectin-coated wells containing a chemically defined media. When NLGCs were transferred to various O2 environments (20%, 5%, or 0% O2) or media containing 100 mM CoCl2, LH (100 ng/ml)-stimulated progesterone (P4) levels were markedly (P < 0.05) suppressed by 0% O2 or CoCl2. VEGF concentrations also declined (P < 0.05) in control, CoCl2, and CoCl2 + LH groups in 0% O2, although CoCl2 modestly increased (75% above control; P < 0.05) VEGF levels in 20% and 5% O2. When NLGCs were cultured in the presence of recombinant human insulin-like growth factor (IGF)-1, IGF-2, or insulin, there was a dose-dependent increase (P < 0.05) in VEGF levels on Day 1 of culture. Whereas optimal doses of IGF-1 or IGF-2 (50 ng/ml), hCG (100 ng/ml), and IGF plus hCG stimulated VEGF levels on Day 1, only the combination of IGF-1 or IGF-2 plus hCG increased VEGF above controls and sustained levels through Day 3 of culture. The synergistic effects of IGF and hCG were also evident in P4 levels, and were not due to changes in DNA content between treatment groups. LGCs produced much higher levels of P4 and VEGF, but the responses to different O2 concentrations and insulin-related factors were qualitatively similar to those of NLGCs. These results suggest that hypoxia is not a primary regulator of VEGF production in primate granulosa cells. However, IGFs may act in concert with the gonadotropin surge to promote VEGF secretion in the ovulatory, luteinizing follicle.

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.

Stimulatory and inhibitory effects of prostaglandins on the gonadotropin-sensitive adenylate cyclase in the monkey corpus luteum

Detailed analysis of the action of prostaglandins (PGs) on the corpus luteum in primate species is very limited. In this study we examined the response of the adenylate cyclase system to PGs in homogenates prepared from the corpus luteum of rhesus monkeys at midluteal phase of the menstrual cycle. The conversion of [alpha 32p] ATP to [32p] cyclic AMP (cAMP) was assessed in the absence (control activity; 50 microM GTP) and presence of various concentrations of seven PGs and arachidonic acid, either alone or in combination with 250 nM hCG. Cyclic AMP production increased up to three-fold in the presence of PGD2, PGE2, PGI2 or PGF2 alpha; however PGA2, PGB2, 13, 14-dihydro-15-keto PGE2 and arachidonic acid alone did not alter cAMP levels. In dose-response studies, adenylate cyclase was 10 and 100-fold more sensitive to PGD2 (Vmax at 1 X 10(-5) M) than to PGE2 or to PGI2 and PGF2 alpha, respectively. Activity in the presence of hCG plus either PGD2, PGE2, PGI2 or PGF2 alpha did not differ from that for hCG (or the PG) alone. In contrast, addition of PGA2 or arachidonate inhibited (p less than 0.05) hCG-stimulated cAMP production by 50 and 100 percent. We conclude that the gonadotropin-sensitive adenylate cyclase of the macaque corpus luteum is also modulated by several PGs. These factors may either mimic (e.g., PGD2, PGE2, PGI2) or suppress (PGA2) gonadotropin-stimulated cAMP production and possibly cAMP-mediated events in luteal cells.

Vascular Endothelial Growth Factor (VEGF) Production by the Monkey Corpus Luteum During the Menstrual Cycle: Isoform-Selective Messenger RNA Expression In Vivo and Hypoxia-Regulated Protein Secretion In Vitro

Abstract Experiments were designed to investigate the expression and regulation of vascular endothelial growth factor (VEGF) in the primate corpus luteum (CL) throughout the luteal life span in the natural menstrual cycle. Corpora lutea were collected during the early (ECL; Days 3–5 post-LH surge), mid (MCL; Day 6– 8 post-LH surge), mid-late (MLCL; Days 10–12 post-LH surge), late (LCL; Days 14–16 post-LH surge), and very late (Days 17– 18 post-LH surge) luteal phase. Specific primers were designed to amplify mRNAs encoding VEGF isoforms 206, 189, 183, 165, 145, and 121. Only two cDNA products were obtained by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends; cloning and sequencing confirmed their 98% homology to the corresponding human VEGF 165 and 121 sequences. Semiquantitative RT-PCR assays indicated that VEGF 165 mRNA levels increased (P < 0.05) from ECL to MLCL but then declined (P < 0.05) by LCL. Although VEGF 121 mRNA levels were limited in ECL, they increased significantly in MCL (P < 0.05). Levels of VEGF protein, as measured by Western blot analysis, were two- to fourfold higher for VEGF 165 versus VEGF 121. Also, VEGF 165 levels were higher (P < 0.05) in ECL and MCL compared to those at later stages. During 2-day culture, preparations of dispersed luteal cells secreted VEGF into the media; the highest levels were observed in ECL and declined (P < 0.05) by LCL. Regardless of luteal stage, hypoxic conditions increased (P < 0.05) VEGF levels, whereas LH exposure increased (P < 0.05) progesterone, but not VEGF, in the media. These results are consistent with a dynamic, local regulation of VEGF production during the life span of the primate CL that is not directly controlled by LH.

Injection of antiangiogenic agents into the macaque preovulatory follicle: disruption of corpus luteum development and function.

Ovulation and conversion of the follicle into the corpus luteum involve remarkable changes in vascular permeability and neovascularization of the luteinizing granulosa layer. To evaluate the importance of these vascular events in follicle rupture and luteal development, sequential experiments were designed in which vehicle or angiogenic inhibitors (TNP-470 or angiostatin) were injected directly into the preovulatory follicle of rhesus monkeys during spontaneous mentstrual cycles. After control injections, 13 of 14 animals exhibited serum levels of progesterone (P) during the subsequent luteal phase that were comparable to untreated animals in our colony. Following low-dose (400 pg/mL) TNP-470, serum P levels increased normally until d 8 of the luteal phase, but then declined prematurely by d 9 (p < 0.05 compared to controls) and remained below controls until menses. Following high-dose (2 µg/mL) TNP-470, serum P levels were diminished in the early luteal phase (d 3–5; p<0.05 compared to controls), but reached typical levels at mid luteal phase, only to decline prematurely by d 9 (p<0.05) and remain low until menses. Control ovaries displayed indices of follicle rupture (protruding stigmata) and luteinization. TNP-470-treated ovaries exhibited signs of distension (torn surface epithelium/tunica albuginea) and luteinization; however, a well-formed stigmata was not observed. A “trapped” oocyte was not observed in serial sections of developing corpora lutea from control or TNP-470-treated animals. However, the early corpus luteum of TNP-470-injected ovaries contained pockets of excessive numbers of blood cells that were absent in controls. Angiostatin did not alter serum P levels or ovarian morphology compared to controls. These data suggest that acute exposure to the antiangiogenic agent TNP-470 impairs the development and functional capacity of the primate corpus luteum in a dose-dependent manner. The results are consistent with a critical role for angiogenesis in cyclic ovarian function in primates.

Vascular endothelial growth factor and angiopoietin production by primate follicles during culture is a function of growth rate, gonadotrophin exposure and oxygen milieu

STUDY QUESTION What is the time course of production of vascular endothelial growth factor-A (VEGF-A), angiopoietin (ANGPT)-1 and ANGPT-2 by primate follicles during encapsulated three-dimensional culture, and what conditions affect their production? SUMMARY ANSWER Primate follicles produce VEGF-A and ANGPT-2 in vitro, particularly after developing to the antral stage, with VEGF production influenced by FSH concentration and O(2) tension. WHAT IS KNOWN ALREADY Folliculogenesis, i.e. the development of primordial follicles into mature, antral follicles, requires the creation of a vascular network in the follicle wall via a process called angiogenesis. Angiogenic factors including VEGFs and ANGPTs have documented roles in angiogenesis. However, direct studies on the production and regulation of angiogenic factors by individual, growing follicles are limited. STUDY DESIGN, SIZE, DURATION Ovaries (n = 9 pairs) were obtained from rhesus macaques during the early follicular phase of the menstrual cycle (cycle days 1-4). Secondary (125-225 µm) follicles were isolated mechanically, encapsulated into alginate (0.25% w/v) and cultured for 40 days. MATERIALS, SETTING, METHODS Individual follicles were cultured in a 5 or 20% O(2) environment in alpha minimum essential medium supplemented with recombinant human (h) FSH. Half of the follicles had recombinant hLH added to the media from Days 30 to 40. Follicle diameters were measured weekly. Follicles were categorized at Week 5 as no-grow (NG; <250 μm in diameter), slow-grow (SG; 251-499 μm) and fast-grow (FG; >500 μm). VEGF-A, ANGPT-1 and -2 concentrations in media were measured by ELISA. MAIN RESULTS AND THE ROLE OF CHANCE VEGF concentrations were low throughout the culture for NG follicles. SG and FG follicles had detectable VEGF concentrations at Week 2, which continued to rise throughout culture. VEGF concentrations were distinct (P < 0.05) among all three follicle categories during Weeks 4 and 5. VEGF concentrations were higher (P < 0.05) in SG follicles in the presence of high/mid-dose FSH at 5% O(2). In contrast, there were no dose-dependent differences in VEGF production for FG follicles based on FSH concentrations or O(2) tension. At Week 5, follicles that produced metaphase II oocytes, following exposure to an ovulatory hCG dose, secreted higher concentrations of VEGF than those containing germinal vesicle-intact oocytes. Media concentrations of ANGPT-1 were low throughout culture for all three follicle categories. ANGPT-2 concentrations were low throughout culture for NG follicles. In contrast, ANGPT-2 concentrations of SG and FG follicles continued to rise from Weeks 1 to 4. During Weeks 2-4, ANGPT-2 concentrations in FG follicles were significantly higher than those of SG and NG follicles (P < 0.05). LIMITATION, REASONS FOR CAUTION This study reports VEGF-A, ANGPT-1 and -2 production by in vitro-developed individual primate (macaque) follicles, that is limited to the interval from the secondary to small antral stage. After VEGF and ANGPT-1 assays, the limited remaining samples did not allow assessment of the independent effects of gonadotrophin and O(2) on the ANGPT-2 production by cultured follicles. Findings await translation to human follicles. WIDER IMPLICATION OF THE FINDINGS The above findings provide novel information on the process of primate follicle maturation. We hypothesize that a symbiotic relationship between elevated concentrations of ANGPT-2 and VEGF allows FG antral follicles to excel in follicle maturation, e.g. by promoting its vascularization. Elevated ANGPT-2 may also offer possible insight into future oocyte quality as early as Week 2, compared with Week 4 for VEGF and follicle size. STUDY FUNDING/COMPETING INTEREST(S) The study was funded by the following grants: NIH U54 RR024347/HD058294/PL1-EB008542 (Oncofertility Consortium), NIH U54-HD018185 (SCCPIR), NIH ORWH/NICHD 2K12HD043488 (BIRCWH), NIH FIC TW/HD-00668, ONPRC 8P51OD011092. There are no conflicts of interest to declare.

Changes in Circulating Levels and Ratios of Angiopoietins during Pregnancy, but not during the Menstrual Cycle and Controlled Ovarian Stimulation

OBJECTIVE To determine whether angiopoietin (ANGPT)-1 and -2 are detectable in the circulation of nonhuman primates and women and whether these levels fluctuate in association with ovarian activity. DESIGN Prospective. SETTING National Primate Research Center, medical center, and infertility clinic. PATIENT(S) Adult female rhesus monkeys; 15 women donating oocytes for infertility treatment. INTERVENTION(S) Controlled ovarian stimulation with gonadotropins, removal of the corpus luteum and ovaries, oocyte retrieval, and ET. MAIN OUTCOME MEASURE(S) Circulating levels of ANGPT-1 and ANGPT-2. RESULT(S) Serum ANGPT-1 and ANGPT-2 levels were detectable and invariant in maintaining an ANGPT-1 to -2 ratio >1 in [1] macaques over the course of the natural menstrual cycle, during a controlled ovulation protocol, and after removal of the corpus luteum or ovaries and [2] women undergoing controlled ovarian simulation. In contrast, the ANGPT-1 to -2 ratio was markedly decreased (<<1) at mid-to-late gestation in macaques and in the follicular fluid of women undergoing controlled ovarian simulation because of increased levels of ANGPT-2. CONCLUSION(S) The ovary and its dominant structures are not major contributors to circulating levels of ANGPT-1 or ANGPT-2. The physiologic importance of the rising levels of ANGPT-2 after the luteal-placental shift in pregnancy is unknown.

Dynamics of Immune Cell Types Within the Macaque Corpus Luteum During the Menstrual Cycle: Role of Progesterone

ABSTRACT The goal of the current study was to characterize the immune cell types within the primate corpus luteum (CL). Luteal tissue was collected from rhesus females at discrete intervals during the luteal phase of the natural menstrual cycle. Dispersed cells were incubated with fluorescently labeled antibodies specific for the immune cell surface proteins CD11b (neutrophils and monocytes/macrophages), CD14 (monocytes/macrophages), CD16 (natural killer [NK] cells), CD20 (B-lymphocytes), and CD3epsilon (T-lymphocytes) for analysis by flow cytometry. Numbers of CD11b-positive (CD11b+) and CD14+ cells increased significantly 3 to 4 days after serum progesterone (P4) concentrations declined below 0.3 ng/ml. CD16+ cells were the most abundant immune cell type in CL during the mid and mid-late luteal phases and were 3-fold increased 3 to 4 days after serum P4 decreased to baseline levels. CD3epsilon+ cells tended to increase 3 to 4 days after P4 decline. To determine whether immune cells were upregulated by the loss of luteotropic (LH) support or through loss of LH-dependent steroid milieu, monkeys were assigned to 4 groups: control (no treatment), the GnRH antagonist Antide, Antide plus synthetic progestin (R5020), or Antide plus the estrogen receptor agonists diarylpropionitrile (DPN)/propyl-pyrazole-triol (PPT) during the mid-late luteal phase. Antide treatment increased the numbers of CD11b+ and CD14+ cells, whereas progestin, but not estrogen, replacement suppressed the numbers of CD11b+, CD14+, and CD16+ cells. Neither Antide nor steroid replacement altered numbers of CD3epsilon+ cells. These data suggest that increased numbers of innate immune cells in primate CL after P4 synthesis declines play a role in onset of structural regression of primate CL.

Anti‐human gonadotropin antibodies generated during in vitro fertilization (IVF)‐related cycles: Effect on fertility of rhesus macaques

Administration of human gonadotropins such as hFSH, hLH, and hCG to rhesus macaques can result in formation of anti‐human gonadotropin antibodies. To determine whether the presence of these antibodies interferes with subsequent fertility, sixteen female rhesus macaques (Macaca mulatta) with known antibody levels were bred with male rhesus macaques. The presence of antibodies did not interfere with conception or maintenance of pregnancy. Furthermore, antibody titers did not increase during gestation or following the resolution of pregnancy.