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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., (© 2015 by the Society for the Study of Reproduction, Inc.)

Background: The objective of the current study was to determine changes to vascular parameters of nonhuman primate dominant ovarian structures by dynamic contrast-enhanced ultrasound (DCE-US)., Materials and Methods: Dynamic contrast-enhanced ultrasound with intravenous microbubble infusion was performed on the rhesus macaque ovary bearing the pre-ovulatory follicle and corpus luteum (CL) sequentially during the natural luteal phase (n = 8) and GnRH antagonist (antide)-induced luteal regression (n = 6)., Results: Changes in luteal blood volume (BV) and vascular flow (VF) were observed between stages of the luteal phase Luteal BV was highest in early stage CL, before decreasing 2.5-fold in late stage CL (P < 0.06); in contrast, luteal VF peaked at mid luteal stage (P < 0.01). Two females identified with luteal insufficiency trended toward lower peak BV, compared to typical CLs. Another female was identified with a luteal cyst on the contralateral ovary, and a CL that regressed before P levels declined. After 72 hours of antide exposure, BV was reduced 2.3-fold (P = 0.03)., Conclusions: DCE-US provides a sensitive, non-invasive measurement of the dynamics of blood volume and flow in dominant ovarian structures., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

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.

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.

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., (Copyright 2010 American Society for Reproductive Medicine. Published by Elsevier Inc. All rights reserved.)

CRH/urocortin-receptor-binding protein (CRH/UCN-R-BP) mRNAs are dynamically expressed in the primate ovary during the menstrual cycle. Therefore, studies were designed to localize CRH/UCN-R-BP mRNAs to ovarian cell types, quantitate protein expression during the corpus luteum (CL) lifespan, and investigate the role of this system in the macaque ovary at midcycle. Monkey ovaries were removed during the preovulatory phase and through the luteal phase to localize CRH/UCN-R-BP mRNAs by in situ hybridization and determine their protein levels in CL by Western blotting. Also, vehicle or a CRH receptor antagonist (astressin) was injected into the preovulatory follicle; daily serum samples were analyzed for hormone levels, and ovaries were removed on d 9 of the luteal phase for histological analysis. There was minimal ligand mRNA staining, whereas receptor and CRHBP was detected in the granulosa and theca cells of the preovulatory follicle. However, ligand and receptor mRNA staining was appreciable in luteal cells of the CL during the early luteal phase (ECL) and diminished in the late luteal phase (LCL). CRHBP staining was low in the ECL and increased markedly in the LCL. Ligand and receptor protein expression was also highest during ECL, whereas CRHBP expression was highest at the LCL. Although astressin injection did not prevent follicle rupture, progesterone levels were significantly less by the mid-luteal phase, and estradiol levels never increased above baseline during the CL lifespan. Histological indices of cell degeneration were observed in the astressin-treated CL. Thus, CRH/UCN-R-BP components are expressed in an ovarian cell-specific manner. The expression pattern and results from antagonist injection are consistent with the hypothesis that CRH/UCN-R activation promotes luteal development and/or structure-function in monkeys during the menstrual cycle.

This study was designed to evaluate the timecourse of ovarian and pituitary endocrine events throughout the menstrual cycle in the vervet monkey, and whether circulating luteinizing hormone (LH) or the uterus regulates the functional lifespan of the vervet corpus luteum. Daily saphenous blood samples were collected from adult females (1) during spontaneous menstrual cycles (n = 7), and (2) during cycles in which a gonadotropin-releasing hormone antagonist (acyline) was administered for 3 days at midluteal phase (n = 3), and (3) for 30 days following recovery from hysterectomy (n = 4). Estradiol (E) and progesterone (P) levels were assayed using electrochemoluminescent assays. Gonadotropin levels were measured by radioimmunoassay using reagents developed for the assay of follicle-stimulating hormone and LH in macaques. Spontaneous cycles exhibited a midcycle E rise (476+/-49 pg/ml), engendering an LH surge, 12+/-1 days after onset of menses, followed by a luteal phase with peak P levels of 4.7+/-0.9 ng/ml. Histologic evaluation of the ovaries at late follicular phase or early luteal phase revealed the presence of a single, large Graafian follicle or developing corpus luteum, respectively. Acyline treatment caused a significant (P<0.05) decline in P levels (2.9+/-0.5 vs 0.5+/-0.3 ng/ml, 0 vs 48 h post-treatment) and premature menstruation compared with untreated controls (P<0.05). Hysterectomy had no apparent effect on the monthly pattern or levels of circulating E or P. Thus, the characteristics and regulation of the ovarian cycle in vervets appear similar to those in women and macaques, with cyclicity dependent on pituitary gonadotropin hormones and independent of a uterine luteolytic factor.

Context: Vascular endothelial growth factor A (VEGF-A) is a potent cytokine that promotes angiogenesis and vascular permeability. After controlled ovarian stimulation (COS) for in vitro fertilization (IVF), excessive VEGF-A production can occur, particularly in women with polycystic ovarian syndrome (PCOS); however, it is unclear whether the regulation of VEGF-A production is different between PCOS and non-PCOS women., Objective: The aim of this study was to determine whether there were differences in the dose- and time-dependent effects of insulin and IGFs on VEGF-A production by luteinized granulosa cells (LGCs) from women with and without PCOS., Design and Setting: A prospective comparative experimental study was conducted at an institutional practice., Patients: Patients included six PCOS and six non-PCOS women undergoing COS and IVF., Interventions: Interventions included COS for IVF., Main Outcome Measures: VEGF-A levels in culture media were collected daily for 3 d from LGCs after incubation with variable doses of insulin, IGF-I, and IGF-II in the presence and absence of LH., Results: In both study groups, exposure to LH alone did not alter VEGF-A levels. However, insulin or IGF increased VEGF-A levels within 1 d and appeared to synergize with LH at 3 d. VEGF-A production by non-PCOS LGCs was more sensitive to IGF exposure, whereas PCOS cells were more sensitive to insulin. Although an increase in DNA content (P < 0.05) was noted in cultures of PCOS cells, progesterone levels were lower compared with non-PCOS LGCs., Conclusion: Insulin and IGFs promote VEGF-A production in LGCs, but the response patterns are different when cells from PCOS and non-PCOS women are compared.

Circulating levels of Ang-2 and sTie-2 receptor were detectable but invariant in women during COS cycles. During the postimplantation period, the rise in Ang-2 (but not sTie-2) levels probably reflects placental rather than luteal production.

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.

Background: In a prospective study we measured circulating levels of vasoactive factors and their soluble receptors in women undergoing controlled ovarian stimulation (COS) for IVF who were at risk for ovarian hyperstimulation syndrome (OHSS), and compared them to those in a primate model, the rhesus monkey., Methods: A total of 23 women were enrolled in the study and serum vascular endothelial growth factor (VEGF)-A (free and total), soluble (s)VEGF-R1 and -R2, and angiogenin levels were compared in pregnant and non-pregnant women, and in monkeys, during follicular stimulation, the luteal phase and early pregnancy., Results: VEGF levels were similar during the period of follicular stimulation in pregnant and non-pregnant women, but a significant rise in both free and total VEGF occurred in pregnant women during the luteal phase (P < 0.05). The level of sVEGF-R1 (but not -R2) increased (P < 0.05) following implantation, and the rise in sVEGF-R1 corresponded to an abrupt fall in free (but not total) VEGF. In contrast, total VEGF levels remained similar to those observed on the day of hCG injection. Angiogenin levels tended to decline during follicular stimulation, then increased marginally during the luteal phase and were unchanged in early pregnancy. In contrast to women, free VEGF levels were non-detectable and total levels remained constant through the natural menstrual cycle and COS protocols in monkeys., Conclusions: The levels of circulating angiogenic factors and soluble receptors demonstrate significant changes during COS cycles and early pregnancy in women. Thus, the systemic effect of these agents is influenced by ligand-receptor protein-binding interactions, and these relationships may exhibit dynamic changes during COS cycles and early pregnancy, and could contribute to the development of OHSS.

Background: A method was sought to control ovulation of the dominant follicle and to test the importance of LH during the late follicular phase of the menstrual cycle. Menstrual cycles of rhesus monkeys were monitored, and treatment initiated at the late follicular phase (after dominant follicle selection, before ovulation)., Methods: The 2-day treatment consisted of GnRH antagonist plus either r-hFSH and r-hLH (1:1 or 2:1 dose ratio) or r-hFSH alone. In addition, half of the females received an ovulatory bolus of hCG., Results: When treatment was initiated at estradiol levels >120 pg/ml, neither the endogenous LH surge, ovulation nor luteal function were controlled. However, when treatment was initiated at estradiol levels 80-120 pg/ml using either 1:1 or 2:1 dose ratios of FSH:LH, the LH surge was prevented, and ovulation occurred following hCG treatment. FSH-only treatment also prevented the LH surge, but follicle development appeared abnormal, and hCG failed to stimulate ovulation., Conclusions: Control over the naturally dominant follicle is possible during the late follicular phase using an abbreviated GnRH antagonist, FSH+LH protocol. This method offers a model to investigate periovulatory events and their regulation by gonadotrophins/local factors during the natural menstrual cycle in primates.

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.

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 menstrual 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 microg/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.

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.

The role of endothelial cell-specific growth factors in the vascularization of the primate peri-ovulatory follicle was examined. Experiments were designed firstly to detect expression of vascular endothelial growth factor (VEGF), angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) in granulosa cells and secondly, to determine whether gonadotrophins and/or steroids regulate their expression during the peri-ovulatory interval. Granulosa cells and follicular fluid were collected from rhesus macaques undergoing ovarian stimulation before (0 h), 12, or 36 h after a bolus of ovulatory human chorionic gonadotrophin (HCG), with or without steroid ablation and progestin replacement. VEGF, Ang-1 and Ang-2 mRNA were all detected prior to the ovulatory stimulus. Whereas follicular fluid VEGF concentrations increased 6-fold (P < 0.05) between 0 and 12 h, VEGF mRNA values were unchanged and were unaffected by steroid ablation. Ang-1 mRNA decreased from 0 to 12 h (P < 0.05), followed by a 30-fold increase (P < 0.05) at 36 h, while Ang-2 mRNA values were unchanged between 0, 12 and 36 h. Steroid ablation decreased (P < 0.05) Ang-1 mRNA at 36 h, and Ang-2 mRNA at 12 h, while only Ang-1 was restored by progestin replacement. These data suggest a dynamic expression of vascular-specific growth factors in a gonadotrophin-dependent, steroid-independent (VEGF) or steroid-dependent (Ang-1) manner in granulosa cells of peri-ovulatory follicles of primates.

Inhibin, a suppressor of pituitary FSH secretion in nonprimate species, may also act in the ovary to regulate follicular development. To examine whether inhibin has similar actions in primates, female rhesus monkeys (n = 3/treatment), exhibiting regular menstrual cycles, received sc injections of either vehicle or 60 micrograms/kg recombinant human inhibin-A at 0800 and 1600 h for 5 days beginning at menses. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 2.5 days, mean +/- SE) and luteal (16.3 +/- 2.5 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first inhibin injection, levels of immunoreactive inhibin A peaked at 10 ng/mL within 1 h and returned to baseline (< 0.1 ng/mL) before the second injection 8 h later. Although serum E and LH did not change, bioactive FSH decreased (to 66% of pretreatment levels, P < 0.05) within 8 h. Within 1 day, circulating bioactive FSH was less (P < 0.05) in inhibin-treated monkeys, compared with controls. By 2-3 days, serum E levels were also markedly (P < 0.05) reduced in inhibin-treated animals, whereas bioactive LH rose 3-fold (P < 0.05). After inhibin treatment, the midcycle rises in serum E and LH were delayed; hence, the follicular phase was prolonged (15.0 +/- 2.6 days, P < 0.05), compared with controls. Although the patterns and levels of serum LH circulating during the subsequent luteal phase seemed comparable in both groups, mean progesterone levels were suppressed to 2-3 ng/mL (P < 0.05) during the midluteal phase in inhibin-treated monkeys. However, the length of the luteal phase in inhibin-treated cycles (13.0 +/- 2.6 days) was not significantly altered. We conclude that exogenous inhibin rapidly diminishes pituitary FSH secretion in female monkeys during the early follicular phase of the menstrual cycle. This action, and/or other actions directly on the ovary, leads to subsequent effects on follicular steroidogenesis and pituitary LH secretion that culminate in an aberrant ovarian cycle characterized by an insufficient luteal phase. The study identifies, for the first time, possible activities and roles of inhibin during the ovarian cycle in primates.

Previous studies in our laboratory indicated that the midcycle gonadotropin surge stimulates progesterone receptor (PR) expression in granulosa cells of the macaque preovulatory follicle. The current experiments were designed to determine whether gonadotropin or steroids continue to regulate PR in luteinized granulosa cells that contain these receptors after the LH surge. Luteinizing granulosa cells obtained from gonadotropin-treated rhesus macaques were cultured in chemically defined medium in the presence of low density lipoprotein (LDL; 100 micrograms/ml) with or without hCG (100 ng/ml) for up to 4 days. Cells were also cultured with various concentrations (0.25-250 ng/ml) of the 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) inhibitor trilostane to reduce progesterone (P) production in vitro. P and estradiol (E) in the media were assayed by RIA; PR mRNA was assessed by RNase protection assay, and cells expressing PR were identified by immunocytochemistry. Whereas hCG stimulated cellular P production through 4 days of culture, trilostane reduced hCG-stimulated P production in a dose-dependent fashion, with P levels decreasing more than 90% during incubation with 250 mg/ml trilostane (p < 0.05). When trilostane was removed from the media, P production returned to hCG-stimulated levels, indicating that trilostane (250 ng/ml) alone did not alter levels compared to those in controls. Before culture, 68 +/- 11% of luteinizing granulosa cells expressed PR; intense nuclear staining was typically observed. After 2 days of culture, 78 +/- 3% of cells remained PR-positive, but nuclear staining was more heterogeneous. Incubation with hCG did not alter the percentage of luteinized granulosa cells staining positive for PR but increased the intensity of PR staining. Trilostane treatment (25 ng/ml) in combination with hCG significantly reduced the percentage of PR-positive cells (54 +/- 9%) when compared with hCG treatment (83 +/- 2%, p < 0.05). These in vitro data suggest that macaque luteinized granulosa cells retain some PR expression in the absence of luteotropic hormones, but that gonadotropin stimulates PR mRNA levels and enhances PR expression as assessed by intensity of nuclear PR staining. In the presence of gonadotropin, trilostane effectively inhibited P production ad reduced the number of PR-positive cells, suggesting that P or a metabolite modulates PR expression in primate luteinized granulosa cells.

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.

The endocrine or local actions of inhibin-related peptides synthesized by the primate corpus luteum (CL) remain undefined. This in vivo study was designed to determine whether exogenous inhibin or activin modulates pituitary gonadotropin secretion and the functional life span of the CL during the luteal phase of the menstrual cycle. Beginning at midluteal phase of the cycle, either vehicle or 1 microgram/h of recombinant human inhibin A or activin A (n = 3-6 per treatment group) was infused into rhesus monkeys via the jugular vein (i.e., peripheral infusion) or directly into the CL (i.e., intraluteal infusion) by means of an osmotic minipump for 7-14 days. Daily samples of saphenous venous serum were assayed for estradiol (E) and progesterone (P) content by RIA, and for FSH and LH levels by bioassay. Intraluteal infusion of inhibin or activin did not alter circulating P levels or the length of the luteal phase compared to those values in vehicle-infused controls. Likewise, LH levels were not different between the three groups. However, FSH levels declined progressively during inhibin infusion to 26% of pretreatment levels (p < 0.05), whereas FSH levels in vehicle-infused controls were unchanged for several days and then rose (p < 0.05) to peak levels around menses. FSH levels did not change significantly during activin infusion into the CL. Although similar results were obtained in monkeys receiving peripheral or intraluteal infusions of inhibin, events following the peripheral infusion of activin were markedly different from those during intraluteal administration.(ABSTRACT TRUNCATED AT 250 WORDS)

Activin, a stimulator of pituitary FSH secretion in nonprimate species, may also act in the ovary to modulate follicular development. To examine whether activin has similar actions in primates, female rhesus monkeys (n = 3/treatment) exhibiting regular menstrual cycles received sc injections of either vehicle or 60 micrograms/kg recombinant human activin-A at 0800 and 1600 h for 1 (acute) or 7 (chronic) days beginning in the early follicular phase. The vehicle-treated monkeys displayed menstrual cycles of normal length, with the follicular (11.3 +/- 1.3 days, mean +/- SE) and luteal (16.6 +/- 1.8 days) phases demarcated by midcycle peaks in serum estradiol (E) and bioactive LH. After the first activin injection, levels of human activin A peaked at 90 ng/mL within 1 h and returned to baseline before the second injection 8 h later. Although serum E and FSH levels did not change, LH increased (273%, P < 0.05) within 8 h. Acute activin treatment increased (P < 0.05) serum E within 24 h to levels (1290 +/- 330 pmol/L) typically observed at midcycle. With chronic treatment, serum E peaked on day 2 (2580 +/- 338 pmol/L; P < 0.05), then declined and rose to a second peak (1680 +/- 279 pmol/L) on day 5. During chronic activin treatment, LH levels peaked on day 2 (603 +/- 270 ng/mL; P < 0.05 compared to day 0, 15 +/- 7 ng/mL) whereas FSH increased progressively until day 5 (937 +/- 320 ng/mL; P < 0.05 compared to day 0, 169 +/- 59 ng/mL). After acute or chronic activin, the expected midcycle rises in serum E and gonadotropins were delayed to greater than or equal to day 20 (n = 4) or did not occur before menses (n = 2). Although an enlarged ovary with one greater than or equal to 4-mm follicle was observed by laparoscopy during the late follicular phase in vehicle-treated monkeys, medium-to-large follicles were not visible on ovaries during chronic activin treatment or later at the expected midcycle interval in activin-treated monkeys. Similar hormonal and ovarian events were obtained after activin treatment of amenorrheic monkeys having serum FSH, LH, and E levels that were comparable to those at menses in spontaneous menstrual cycles. Thus, exogenous activin stimulates pituitary LH and FSH secretion and ovarian estrogen secretion during the early follicular phase in intact monkeys. However, acute or chronic activin treatment did not promote complete follicular development and disrupted subsequent events in the menstrual cycle.(ABSTRACT TRUNCATED AT 400 WORDS)

Local modulation of follicular and gametogenic functions by ovarian androgens and estrogens in mammalian species has been proposed. This study examined the effects of elevated androgen/estrogen ratios during follicular maturation in vivo by inhibiting aromatase activity in rhesus monkeys. To obviate steroid feedback effects, gonadotropin-treated animals were used. Beginning at menses (day 1), animals received human (h) FSH (60 IU/day, im) on days 1-6, followed by hFSH plus hLH (60 IU/day, im) on days 7-9 to promote the growth of multiple follicles. Ovulatory maturation was induced by hCG (1000 IU, im) on day 10. On days 8-10, four animals received an aromatase inhibitor, 1,4,6-androstatrien-3,17-dione (ATD; 1-1.25 g, orally, twice/day), while five served as controls and received no further treatment. Within 8 h of ATD treatment, a 63% reduction in serum estradiol levels relative to control values was evident, which reached maximal suppression (84%) by day 10. A marked elevation (17-fold) in serum androstenedione and a lesser increase (2.6-fold) in serum testosterone occurred with aromatase inhibition, yielding androstenedione/estradiol (18.0) and testosterone/estradiol (1.9) ratios greater than those in controls (0.6 and 0.3, respectively). ATD treatment did not alter follicular diameters or the total number of follicles per animal (20 +/- 3) relative to control values (16 +/- 3). Of the total cohort classified, the proportion of oocytes collected at prophase I was greater (P < 0.05) after ATD treatment (31%) than in controls (11%). Completion of oocyte meiosis to metaphase II was retarded (P < 0.05) in ATD-treated (4%) compared to control (26%) animals. Furthermore, the in vitro fertilization rate of metaphase II oocytes from ATD-treated animals (9%) was reduced (P < 0.05) relative to that in controls (25%). While basal progesterone production by luteinizing granulosa cells in vitro was similar between groups, the addition of hCG in vitro enhanced progesterone secretion by cells from ATD-treated animals (3.1 +/- 0.3-fold over basal) to a greater extent (P = 0.05) than in controls (1.5 +/- 0.3-fold). Progesterone receptor was detected by immunocytochemistry in nuclei of luteinizing granulosa cells from ATD-treated animals as well as controls. Serum progesterone profiles and the length of the luteal phase were similar between groups. Thus, acute elevation of serum androgen/estrogen ratios in vivo during follicular maturation was detrimental to the gametogenic functions of the primate follicle, but did not alter follicular growth, events of early luteinization, or subsequent luteal function.(ABSTRACT TRUNCATED AT 400 WORDS)

A serum-free medium has been developed which supports in vitro function by ovarian cells derived from rat, monkey, and human tissue. This granulosa cell medium (GCM) consists of Dulbecco's Modified Eagle's Medium: Ham's F-12 medium (1:1, v:v) supplemented with insulin, transferrin, aprotinin, selenium, fibronectin, penicillin, and streptomycin. Ovarian cells from three species were compared: rat, macaque, and human. Four types of ovarian cultures were examined: 1) purified granulosa cell cultures and 2) co-cultures containing granulosa-theca-stroma cells, 3) luteal cells, and 4) granulosa-lutein (harvested from in vitro fertilization cultures) cells. Each cell type was characterized by its response to FSH or hCG when cultured in GCM. Morphologic responses to FSH were observed in GCM in rat granulosa and granulosa-theca-stroma cell cultures, macaque and human granulosa-lutein cells, and human granulosa-theca-stroma cell cultures. The FSH-stimulated cells retracted and became rounded, leaving long intercellular connections. Luteal cells did not retract in response to FSH, and the cells remained firmly attached to the fibronectin matrix. Steroidogenic regulation of the GCM-cultured ovarian cells was monitored following stimulation of the cultures with FSH. The ability of the cells to aromatize testosterone was first examined. Rat granulosa cell cultures and granulosa-theca-stroma cell cultures, macaque granulosa-lutein cell cultures, and human granulosa-theca-stroma cell cultures all accumulated estradiol when given FSH and testosterone for 48 h. Moreover, these cell types as well as human luteal cells were able to metabolize 25-hydroxy [1,2-3H]cholesterol to various steroid metabolites. The data indicate that GCM supports normal granulosa cell morphologic response to FSH.(ABSTRACT TRUNCATED AT 250 WORDS)

Although inhibin (IN) is secreted by granulosa cells (GC) of preovulatory follicles, the major source of immunoreactive IN circulating during the primate ovarian cycle is the corpus luteum. The aims of this study were (1) to investigate culture conditions for optimal IN production by luteinized GC (LGC) from rhesus monkeys and (2) to compare IN and progesterone (P) production by nonluteinized GC (NGC) and LGC in response to putative agonists. Animals were treated for up to 9 days with human menopausal gonadotropins to promote the development of multiple preovulatory follicles. GC were obtained from large follicles before (NGC) or 27 h after (LGC) an ovulatory injection of hCG. For Aim 1, cells were cultured in Hams F-10 medium +/- hCG (100 ng/ml) with or without the addition of insulin/transferrin/selenium, 10% fetal bovine serum, or 10% Serum-Plus (JRH Biosciences, Lenexa, KS). Medium was changed on Days 1, 2, 4, 6, and 8, and IN and P concentrations were determined by RIA. Basal (unstimulated) IN production by LGC was enhanced and maintained for 6-8 days in the presence of serum, but rapidly declined in the absence of serum. In contrast, basal P secretion declined regardless of exposure to serum. Human CG consistently increased (p less than 0.05) IN production only in the presence of serum but stimulated (p less than 0.05) P production under all conditions. For Aim 2, cells were cultured for 4 days in Ham's F-10 medium + 10% macaque serum +/- hCG (100 ng/ml), hFSH (100 ng/ml), prostaglandin E2(PGE2; 14 microns), or dibutyryl(db)-cAMP (5 mM).(ABSTRACT TRUNCATED AT 250 WORDS)

The events in granulosa cells that are initiated by the midcycle LH surge during luteinization of the primate follicle are poorly defined. This study was designed 1) to determine whether an ovulatory dose of hCG can induce progesterone receptors (PR) in macaque granulosa cells, and if so, 2) to begin titrating gonadotropin requirements for PR expression and progesterone production by luteinizing granulosa cells. Rhesus monkeys were treated with human FSH and LH for up to 9 days to stimulate the growth of multiple follicles. The next day, animals (n = 4-5/group) received: 1) no ovulatory stimulus; 2) 1000 IU hCG, im; 3) one injection of 100 micrograms GnRH, sc (GnRH-1); 4) three injections of GnRH (GnRH-3) at 3-h intervals (0800, 1100, and 1400 h); or 5) two injections of 50 micrograms GnRH agonist (GnRHa), sc, 8 h apart (0800 and 1700 h). Granulosa cells obtained by follicle aspiration 27 h after the hCG or initial GnRH/GnRHa injection or on days 8 or 10 from animals receiving no ovulatory stimulus were processed for indirect immunocytochemistry using a monoclonal antibody to human PR (JZB39). Specific staining for PR, determined by comparing cells incubated with PR antibody vs. a nonspecific antibody, was undetectable in granulosa cells from monkeys without an ovulatory stimulus. In contrast, the majority (64 +/- 5%) of cells from hCG-treated animals stained intensely for PR. In the GnRH/GnRHa groups, granulosa cells from only one animal (i.e. one GnRH-3 monkey) showed positive staining for PR. During 24-h culture in Ham's F-10 medium containing 10% monkey serum, basal progesterone production by cells from the hCG-treated group (2163 nmol/L.8 x 10(4) cells) was higher than that by cells from the no ovulatory stimulus/GnRH-1/GnRH-3/GnRHa groups (60, 111, 194, and 332 nmol/L, respectively). However, granulosa cells from the hCG-treated group were less responsive to hCG in vitro in terms of enhanced progesterone production (2 times control levels) than cells from the other four groups (up to 30 times control levels). This study provides direct evidence that an ovulatory dose of hCG induces PR expression in granulosa cells of luteinizing follicles during stimulated cycles in rhesus monkeys. However, repeated injections of GnRH/GnRHa that produced surge levels (greater than 100 ng/mL) of endogenous LH for up to 14 h failed to induce PR expression or progesterone production by granulosa cells. Thus, an extended LH surge more typical of that in the normal menstrual cycle (48-50 h) may be necessary for PR expression and luteinization of granulosa cells in primate follicles.

The dynamics of the steroidogenic response of nonprimate gonadal cells to gonadotropins suggests that the biologic action of pituitary LH differs from that of placental CG. To compare the response to LH and CG in primate species, luteinized granulosa cells (LGCs) obtained from rhesus monkeys following follicle stimulation were cultured in vitro. The pattern and levels of progesterone (P) produced during culture was influenced by the concentration (0-10%) and type (fetal bovine or macaque) of serum in the medium and whether LGCs were plated on plastic or extracellular matrix from bovine corneal endothelial cells. After 2-3 days of culture, LGCs were exposed acutely (15-30 min) or chronically (6 h) to 1 or 100 ng/ml human LH (hLH, NIH 1-2) or hCG (CR123), 50 micrograms/ml ovine LH (oLH, NIH-oLH-25), or incubated in the absence of gonadotropins (controls). After the first 15-30 min, the media were changed at 30-min intervals. Both acute and chronic exposure to hLH, hCG, and oLH increased (p less than 0.05) P concentrations above control levels within 15-30 min. There were no differences in the patterns or levels of P elicited by hLH or hCG over time for each treatment condition. Chronic exposure to 1 and 100 ng/ml hLH or hCG and 50 micrograms/ml oLH sustained P levels above that of controls for the 6-h interval. Acute exposure to 1 ng/ml hLH or hCG failed to maintain elevated P levels throughout the experiment.(ABSTRACT TRUNCATED AT 250 WORDS)

Specific nuclear staining for progesterone receptor (PR) was detected by immunocytochemistry in human granulosa cells (GCs) obtained from in vitro fertilization protocols. The percent of PR-positive cells (60% to 80%) remained unchanged during 7 days of culture in media containing fetal calf serum, in the absence or presence of human chorionic gonadotropin (hCG) or the progesterone antagonist RU486. Progesterone (P) production by GCs cultured on extracellular matrix from bovine corneal endothelial cells was stimulated by hCG and prostaglandin E2 (PGE2). However, addition of RU486 or human relaxin had no effect on control, hCG-, or PGE2-stimulated P production. Thus, the receptor data are consistent with an autocrine action of P in luteinizing GCs, but initial experiments in cell culture did not define a role for P or relaxin in modulating luteal steroidogenesis.

Intraluteal infusion of the prostaglandin (PG) synthesis inhibitor, sodium meclofenamate (Mec) causes premature luteolysis in rhesus monkeys. To evaluate further the actions of PG synthesis inhibitors in primate luteal function, we examined the in vitro effects of Mec and another inhibitor, flurbiprofen (Flur), on PG, cAMP, and progesterone (P) production by macaque luteal tissue obtained at midluteal phase of the menstrual cycle. First, collagenase dispersed luteal cells were incubated with 0-100 microM Mec or Flur, either alone or in the presence of 10 microM arachidonic acid (AA) to assess PGF2 alpha and PGE2 synthesis. Levels of both PGF2 alpha and PGE2 were stimulated (P less than 0.05) by AA (3.3- and 5.8-fold, respectively). Maximal suppression (P less than 0.01) of basal and AA-stimulated PGF2 alpha and PGE2 synthesis was elicited by 1 microM Mec and Flur. Second, adenylate cyclase activity, measured by the conversion of alpha 32P-ATP to alpha 32P-cAMP, was monitored in luteal homogenates exposed to increasing doses of Mec and Flur either alone or with maximal stimulatory doses of hCG, PGE2, or PGI2. Mec elicited a dose-dependent reduction (P less than 0.01) in control activity (incubated with 50 microM GTP), as well as inhibiting hCG- and PG-stimulated activity. The presence of 100 microM Mec suppressed (P less than 0.01) hCG-, PGE2- and PGI2-stimulated activity to control levels, but had no effect on activity stimulated by GMP-P(NH)P or forskolin. In contrast, Flur at any dose did not alter control activity or that stimulated by hormonal or nonhormonal activators. Third, P production by dispersed luteal cells was quantified during exposure to 0, 1, and 100 microM Mec or Flur alone or with maximal stimulatory doses of hCG, PGE2, PGD2, 6 beta PGI1, PGA2, or dibutyryl cAMP (dbcAMP). All hormones and dbcAMP stimulated (P less than 0.01) P synthesis 2-3 fold over basal levels, except PGA2, which had no effect. The presence of 100 microM Mec reduced (P less than 0.01) basal P production by 62% and abolished (P less than 0.05) hCG-, PG-, and dbcAMP-induced stimulation. Conversely, neither 1 microM Mec nor either dose of Flur affected P synthesis in the absence or presence of hormones or dbcAMP. These data indicate that: 1) Mec and Flur are potent inhibitors of PG synthesis in primate luteal cells in vitro and 2) higher doses of Mec suppress PG- and gonadotropin-sensitive adenylate cyclase activity and P production.(ABSTRACT TRUNCATED AT 250 WORDS)

The transient steroidogenic response of the macaque corpus luteum to chronic human CG (hCG) treatment beginning on days 9-10 of the luteal phase (i.e. stimulated early pregnancy) is associated with decreased numbers and affinity of available receptors for gonadotropin and homologous desensitization of adenylate cyclase. This study determined if similar changes in the receptor-adenylate cyclase system accompany the persistent steroidogenic response which occurs when hCG treatment begins earlier in the luteal phase. Female rhesus monkeys received increasing doses of hCG (15 up to 5760 LU) twice daily beginning 5-6 days after the midcycle LH surge. The levels of circulating progesterone increased (P less than 0.05) within 24 h of initial hCG exposure and did not decrease throughout the 10-day regimen. The corpus luteum was removed after 0 (n = 8), 6 (n = 4), or 10 (n = 4) days of hCG treatment. Whereas the numbers of available [125I]hCG binding sites in luteal particulates remained unchanged by 10 days of hCG exposure, the dissociation constant (Kd) for gonadotropin binding was greater than at day 0 (6.17 +/- 1.41 vs. 0.91 +/- 0.06 X 10(-10) M, P less than 0.05). Since the number of binding sites occupied by injected hCG increased with treatment (7.81 +/- 1.55 fmol/mg wet wt at day 10), the total number (available + occupied) of gonadotropin receptors was 3-fold greater (P less than 0.05) at day 10 than at day 0. Adenylate cyclase activity in luteal homogenates, assessed by conversion of [alpha-32P]ATP to [32P]cAMP, was stimulated on day 0 by hCG (2.7 +/- 0.7 X control, at 250 nM hCG), prostaglandin E2 (2.5 + 0.5 X control, at 0.5 mM), and prostaglandin I2 (2.3 +/- 0.5 X control at 0.5 mM) as well as forskolin (100 microM) and 5'-guanylyl-imidodiphosphate (50 microM). In contrast, cAMP production by day 6 of treatment was insensitive to hCG, but remained responsive to prostaglandin E2, prostaglandin I2, and nonhormonal activators. We conclude that CG treatment in the early luteal phase did not prevent the development of gonadotropin receptors to levels typically observed in the functional corpus luteum of the menstrual cycle. Also, many changes in the gonadotropin receptor-adenylate cyclase system in macaque luteal tissue were similar after CG treatment beginning on days 5-6 or days 9-10 of the luteal phase.(ABSTRACT TRUNCATED AT 400 WORDS)

Six leucine auxotrophic strains of the white rot basidiomycete Phanerochaete chrysosporium were characterized genetically and biochemically. Complementation studies involving the use of heterokaryons identified three leucine complementation groups. Since all of the leucine auxotrophs grew on minimal medium supplemented with alpha-ketoisocaproate as well as with leucine, the transaminase catalyzing the last step in the leucine pathway was apparently normal in all strains. Therefore, the wild-type, auxotrophic, and several heterokaryotic strains were assayed for the activities of the other enzymes specific to leucine biosynthesis. Leu2 and Leu4 strains (complementation group I) lacked only alpha-isopropylmalate synthase activity; Leu3 and Leu6 strains (group III) lacked isopropylmalate isomerase activity; and Leu1 and Leu5 strains (group II) lacked beta-isopropylmalate dehydrogenase. Heterokaryons formed from leucine auxotrophs of different complementation groups had levels of activity for all three enzymes similar to those found in the wild-type strain.

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.

A phage-mediated transducing system was used in studying certain physiological characteristics of S. lactis C2 wild type, lactose-negative mutants, and lactose-positive transductants. Lac(-) mutants, obtained by acriflavine treatment of the wild type, were similar to the wild type in all characteristics tested except they lacked beta-D-phosphogalactoside galactohydrolase (beta-Pgal) and could not transport [(14)C]lactose; they also had approximately 10% of the proteolytic ability than wild-type cells. The lactose-fermenting characteristic was transduced from the wild type to Lac(-) mutants. The Lac(+) transductants obtained were similar to the wild-type parent with respect to lactose fermentation and level of beta-Pgal activity (0.186 U of protein per mg). These transductants, however, had not regained full proteolytic ability and were similar to the Lac(-) mutant in this respect. Lactic acid production of the transductants in milk was approximately two-thirds that of the wild type. Data suggest that both the lactose-fermenting and proteolytic characters are carried on extrachromasomal particles (plasmids).

Figure 2 summarizes the changes in endocrine function and the factors which regulate the primate corpus luteum during the fertile menstrual cycle. The classical luteotropic role of LH during the menstrual cycle is superceded by CG at or before the time of implantation. The role of local factors in modulating luteal function is an area of continued research, as some factors are deemed less important (i.e., estrogen, at least prior to luteal rescue) and other possibilities (progesterone, prostaglandins, and relaxin) arise. The role of local factors has not yet been studied in the corpus luteum following its rescue in early pregnancy. Finally, it is apparent that a different type of "shift" precedes the recognized luteal-placental shift in early pregnancy, when the corpus luteum enhances or begins new activities as progesterone secretion declines. These new or augmented activities occur despite apparent desensitization of CG-responsive cAMP-mediated pathways in luteal cells. Although the cellular events promoting these changes are not known, it seems reasonable to propose that the resulting products, including estrogen (as discussed in Dr. Moudgal's chapter) and relaxin are important in early pregnancy. Thus the term "luteal-placental shift" may be a misnomer, as other activities which promote gestation continue within the corpus luteum for a limited time.

Stimulation of the primate corpus luteum by endogenous CG in early pregnancy or by exogenous CG in simulated conditions is transient despite continued exposure to this luteotropic hormone. The transitory response to CG is not due to the down-regulation of gonadotropin receptors. The current studies were designed to determine if the transient response involves a postreceptor lesion at the membrane level, i.e. the loss of CG receptor activation of adenylate cyclase. Nonpregnant female rhesus monkeys received increasing doses of hCG for up to 10 days beginning near the typical time of implantation (9 days post-LH surge) to simulate early pregnancy. Corpora lutea were removed at specific intervals after the onset of hCG treatment, luteal homogenates were prepared, and adenylate cyclase activity was assessed by the conversion of [alpha-32P]ATP to [32P] cAMP. Basal activity of adenylate cyclase was unchanged throughout the in vivo hCG treatment interval. Nonhormonal activators, such as forskolin (100 microM) and 5'-guanylylimidodiphosphate (50 microM) stimulated (P less than 0.05) adenylate cyclase to a similar extent (greater than 10-fold the control level) throughout hCG treatment. On day 0, both gonadotropins (hCG and human LH; 250 nM) and prostaglandins (PGE2 and PGI2; 500 nM) stimulated cAMP production (approximately 3-fold the control level; P less than 0.05). The responses of adenylate cyclase to PGE2 and PGI2 did not diminish throughout the in vivo hCG treatment. In contrast, exposure to hCG for 3 days reduced the sensitivity of adenylate cyclase to gonadotropin. Moreover, adenylate cyclase in luteal tissue after 6-10 days of treatment was insensitive to hCG. The loss of gonadotropin sensitivity of adenylate cyclase by 6 days of hCG treatment correlated with the decline in circulating progesterone levels. These results demonstrate that 1) the gonadotropin-responsive adenylate cyclase of the macaque corpus luteum is also stimulated by paracrine factors, notably PGs of the E and I series; and 2) CG exposure stimulating early pregnancy conditions leads to homologous, not heterologous, desensitization of the adenylate cyclase system. We hypothesize that homologous desensitization of the adenylate cyclase system is an important mechanism leading to the transient response of the primate corpus luteum to CG in early pregnancy.

beta-D-Phosphogalactoside galactohydrolase (beta-Pgal) was examined in a number of lactic streptococci by use of the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside-6-phosphate. Specific activity of beta-Pgal ranged from 0.563 units/mg of protein in Streptococcus lactis UN, to 0.120 in S. diacetilactics 18-16. Essentially no beta-D-galactoside galactohydrolase (beta-gal) was found in these organisms when o-nitrophenyl-beta-D-galactopyranoside served as the chromogenic substrate. S. lactis 7962 was the one exception found. This organism contained rather high levels of beta-gal, and very little beta-Pgal could be detected. beta-Pgal activity was examined in streptococci that differed widely in both their proteolytic ability and rates of lactic acid production during growth in milk. Differences in proteolytic ability did not influence beta-Pgal synthesis; also, the rate of lactic acid production was independent of the level of beta-Pgal present in the cell, since the rate of lactic acid production could be increased approximately fourfold without changing the amount of beta-Pgal present in the cell. Various carbohydrates were tested as potential inducers of the enzyme. Although galactose, either as the free sugar or combined with glucose in lactose, was the only inducer, noninducing sugars such as mannose or glucose showed some ability to cause fluctuations in the basal level of beta-Pgal. Cells growing in mannose or glucose exhibited about 30% of the maximal enzyme levels found in cells growing in lactose or galactose. No gratuitous inducers were found.