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.

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.

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.

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.

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.

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.

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.

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.

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.

Circulating levels of free and total vascular endothelial growth factor (VEGF)-A, soluble VEGF receptors-1 and -2, and angiogenin during ovarian stimulation in non-human primates and women.

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.

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.

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.

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.

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.

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).

Vascular endothelial growth factor (VEGF) and angiopoietin regulation by gonadotrophin and steroids in macaque granulosa cells during the peri-ovulatory interval.

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.

Recombinant human inhibin-A administered early in the menstrual cycle alters concurrent pituitary and follicular, plus subsequent luteal, function in rhesus monkeys.

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.

Progesterone receptor messenger ribonucleic acid and protein in luteinized granulosa cells of rhesus monkeys are regulated in vitro by gonadotropins and steroids.

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.

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.

Systemic and intraluteal infusion of inhibin A or activin A in rhesus monkeys during the luteal phase of the menstrual cycle.

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.

Human recombinant activin-A alters pituitary luteinizing hormone and follicle-stimulating hormone secretion, follicular development, and steroidogenesis, during the menstrual cycle in rhesus monkeys.

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.

Administration of an aromatase inhibitor during the late follicular phase of gonadotropin-treated cycles in rhesus monkeys: effects on follicle development, oocyte maturation, and subsequent luteal function.

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.

Comparison of functional response of rat, macaque, and human ovarian cells in hormonally defined medium.

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.

Inhibin production by macaque granulosa cells from pre- and periovulatory follicles: regulation by gonadotropins and prostaglandin E2.

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.

Titrating luteinizing hormone surge requirements for ovulatory changes in primate follicles. II. Progesterone receptor expression in luteinizing granulosa cells.

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.