In this study, luteinized human granulosa cells (GC) obtained during in vitro fertilization procedures were used as a model system to evaluate the effects of ethanol (EtOH), a well‐known reproductive toxin, on epidermal growth factor (EGF) and gonadotropin‐stimulated steroidogenesis. Our results demonstrate that the basal progesterone (P 4 ) and estradiol (E 2 ) secretion by human GC in vitro was dependent on the ovarian stimulation protocol. EGF significantly enhanced P 4 , but not E 2 , secretion in human GC from clomiphene citrate (CC), human menopausal gonadotropin (hMG), and hMG/gonadotropin‐releasing hormone agonist (GnRH‐a)‐treated patients. The effects of EGF plus luteinizing hormone (LH) were additive in cells from the CC group, but less than additive in hMG and hMG/GnRH‐a groups. EtOH at 20 mM or more inhibited EGF stimulated P 4 secretion in human GC from all three patient groups. EtOH inhibited P 4 secretion stimulated by EGF and LH cotreatment in the CC and hMG/GnRH‐a groups, but not in human GC from the hMG‐treated patients. These results suggest that basal and EGF or LH‐stimulated P 4 secretion by human GC, as well as the effects of EtOH, are profoundly influenced by the follicle's hormonal milieu.
ABSTRACT: Macrophages and their secretory products, cytokines, play an integral role in many reproductive processes. In this study we examined the effect of conditioned media from cultured human peritoneal macrophages on progesterone production by granulosa cells and the role of calcium in this process. Macrophages were pretreated with various concentrations of a calcium channel blocker (verapamil) or a calcium ionophore (A23187). Macrophage‐conditioned media (MCM) or cell‐free media that contained calcium channel modifiers were added at three dose levels to cultured porcine granulosa cells. Progesterone production and LH receptor content were determined. Macrophage‐conditioned media alone elevated basal progesterone production, but significantly attenuated granulosa cell LH receptor content. These effects were neither potentiated nor suppressed by pretreating macrophages with verapamil. However, production of the LH receptor lowering factor(s) appeared to be suppressed by calcium ionophore. We conclude that (1) one or more factors produced by macrophages have a net stimulatory effect on basal progesterone production and these factor(s) may not be calcium‐dependent and (2) macrophage‐derived secretory products reduce granulosa cell LH receptor content. The production of these factor(s) may be calcium‐dependent.
During human pregnancy, the production of 17-beta-estradiol (E2) rises steadily to eighty fold at term, and placenta has been found to specifically bind estrogens. We have recently demonstrated the expression of estrogen receptor alpha (ER-alpha) protein in human placenta and its localization in villous cytotrophoblast (CT), vascular pericytes, and amniotic fibroblasts. In vitro, E2 stimulated development of large syncytiotrophoblast (ST) aggregates. In the present study we utilized ER-beta affinity purified polyclonal (N19:sc6820) and ER-alpha monoclonal (clone h-151) antibodies. Western blot analysis revealed a single ~52 kDa ER-beta band in chorionic villi (CV) protein extracts. In CV, strong cytoplasmic ER-beta immunoreactivity was confined to ST. Dual color immunohistochemistry revealed asymmetric segregation of ER-alpha in dividing villous CT cells. Prior to separation, the cell nuclei more distant from ST exhibited high ER-alpha, while cell nuclei associated with ST showed diminution of ER-alpha and appearance of ER-beta. In trophoblast cultures, development of ST aggregates was associated with diminution of ER-alpha and appearance of ER-beta immunoreactivity. ER-beta was also detected in endothelial cells, amniotic epithelial cells and fibroblasts, extravillous trophoblast (nuclear and cytoplasmic) and decidual cells (cytoplasmic only). In addition, CFK-E12 (E12) and CWK-F12 (F12) monoclonal antibodies, which recognize ~64 kDa ER-beta with hormone binding domain, showed nuclear-specific reactivity with villous ST, extravillous trophoblast, and amniotic epithelium and fibroblasts. Western blot analysis indicated abundant expression of a ~64 kDa ER-beta variant in trophoblast cultures, significantly higher when compared to the chorionic villi and freshly isolated trophoblast cell protein extracts. This is the first report on ER-beta expression in human placenta and cultured trophoblast. Our data indicate that during trophoblast differentiation, the ER-alpha is associated with a less, and ER-beta with the more differentiated state. Enhanced expression of ~64 kDa ER-beta variant in trophoblast cultures suggests a unique role of ER-beta hormone binding domain in the regulation of trophoblast differentiation. Our data also indicate that asymmetric segregation of ER-alpha may play a role in asymmetric division of estrogen-dependent cells.
Placenta growth factor (PlGF), a member of the vascular endothelial growth factor family of angiogenic factors, is prominently expressed by trophoblast. In addition to its role as a paracrine angiogenic factor within the placenta and endometrium, presence of its receptor, Flt-1, on trophoblast suggests that PlGF also may have an autocrine role(s) in regulating trophoblast function. To elucidate its role in trophoblast, we examined the signal transduction and functional responses of primary human trophoblast to PlGF. Exogenous PlGF induced specific activation of the stress-activated protein kinase (SAPK) pathways, c-Jun-N terminal kinase (JNK) and p38 kinase, in primary term trophoblast with little to no induction of the extracellular signal regulated kinase (ERK-1 and -2) pathways. In contrast, PlGF induced significant ERK-1 and -2 activity in human umbilical vein endothelial cells but did not induce JNK or p38 activity. PlGF-induced activation of the SAPK signaling pathways protected trophoblast from growth factor withdrawal-induced apoptosis, but it did not protect trophoblast from apoptosis induced by the pro-inflammatory cytokines, interferon γ and tumor necrosis factor α. These results provide the first direct evidence of a biochemical and functional role for PlGF/Flt-1 in normal trophoblast and suggest that aberrant PlGF expression during pregnancy may impact upon trophoblast function as well as vascularity within the placental bed.
The immune system, besides orchestrating the immune response, plays an important role in the regulation of tissue homeostasis. We refer to this later activity as ‘immune physiology.’ In human ovaries, immune system‐related cells and molecules accompany corpus luteum development and regression and cancer progression. They also accompany the origination of new rat and human germ cells by asymmetric division of ovarian surface epithelium cells, symmetric division and migration of germ cells, and follicular growth. Currently prevailing dogma on the preservation of human oocytes from the fetal period until menopause (‘storage’ doctrine) vs. oocyte renewal in invertebrates and lower vertebrates (‘continued formation’ doctrine) raises question as to the disadvantage from an evolutionary point of view of prolonged oocyte storage in humans. We attempted to reconcile these two opposing views by proposing the prime reproductive period (PRP) doctrine as follows: The ‘storage’ doctrine fits two periods of the life in human females, that between the termination of fetal oogenesis and puberty or pre‐menarcheal period (about 10–12 years), and also that period from the end of PRP (at about 38 years of age) until menopause. On the contrary, the ‘continued formation’ doctrine accounts for oocyte and follicular renewal during the PRP, and insures the availability of fresh oocytes for the development of healthy progeny. Further study on ‘immune physiology’ may help us better understand ovarian physiology and pathology in general, including infertility caused by premature ovarian failure, the pathophysiology of degenerative diseases and mechanisms of malignancy and metastasis.
