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

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.

Quantification of dynamic changes to blood volume and vascular flow in the primate corpus luteum during the menstrual cycle.

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.

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.

Expression and role of the corticotropin-releasing hormone/urocortin-receptor-binding protein system in the primate corpus luteum during the menstrual cycle.

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.

Insulin and insulin-like growth factor stimulation of vascular endothelial growth factor production by luteinized granulosa cells: comparison between polycystic ovarian syndrome (PCOS) and non-PCOS women.

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.

Characteristics and regulation of the ovarian cycle in vervet monkeys (Chlorocebus aethiops).

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.

Circulating levels of total angiopoietin-2 and the soluble Tie-2 receptor in women during ovarian stimulation and early gestation.

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.

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.

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.

Controlled ovulation of the dominant follicle: a critical role for LH in the late follicular phase of the menstrual cycle.

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.

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 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.