Our observations indicate that the 3B5 anti-LHR antibody reacts with rat, porcine and human tissues. In western blots, the 3B5 antibody identifies six distinct bands migrating between ~92 and 48 kDa, and this reactivity with protein extracts from all three species diminishes when the antibody is preabsorbed with porcine ovaries. We also show changes in subcellular LHR distribution during differentiation of various cell types. To our knowledge, LHR expression in fibroblasts, striated muscle cells, and microglial cells (CD68+ resident macrophages) in the central nervous system is presented for the first time. We also report tissue differences in vascular LHR expression.
During cellular differentiation, subcellular LHR distribution changed from granular cytoplasmic to perinuclear, surface and nuclear staining; virtually no staining was detected in stem cells and most differentiated and aged cells. Yet, cell surface staining was observed only on classic LH/CG targets, granulosa and theca cells of preovulatory follicles and mature luteal cells, but also in the vaginal epithelium. This indicates that these cells are prepared for the selective accumulation of LH/CG signals from the circulation.
There was also a striking difference in vascular LHR expression between ovaries and other tissues. In ovarian (and also striated muscle) vessels the LHR expression was virtually absent (Fig. 1A, 2A, 3J), while other tissues investigated, including uterus, brain, skin and vagina, showed distinct vascular LHR immunoreactivity. We also reported previously that in the rat the endothelial LHR expression is absent in testicular vessels, and vessels in other tissues involved in LH/CG transport into (pituitary) and from the blood (kidney) . Saturation of LHR in ovarian targets may be dependent on the precise delivery of small amounts of circulating LH/CG from the blood into the extravascular space. A receptor mediated endothelial transport of hCG has been suggested to represent a model for general involvement of specific receptors in transport of other plasma proteins [57, 58]. Yet, application of this theory on our data will indicate that the LH/CG will be delivered to the uterus, brain, skin and vagina rather than to the ovary. Hence, the opposite mechanism should be considered – endothelial LHR expression may suppress the transport of LH/CG from vessels to the extravascular space. The LH/CG molecule is smaller than that of albumin or IgG, i.e., proteins exhibiting ubiquitous distribution, so there is no need to enhance rather only prevent LH/CG transport to inappropriate sites. Binding of LH/CG to endothelial cells expressing LHR may prevent LH/CG transport from circulation by electrostatic forces, and the lack of endothelial LHR expression may be associated with LH/CG transport mediated by a general mechanism involved in protein exchange .
We report that normal term placentae show virtually no LHR expression in trophoblastic syncytium while syncytium of some abnormal placentae (immature phenotypes) exhibits LHR immunoreactivity. This resembles high LHR expression in first trimester placenta , which is a source of high hCG levels . Yet, hCG levels fall during the second trimester, and elevated maternal midtrimester hCG is associated with higher rates of maternal and neonatal complications (pregnancy-induced hypertension, preeclampsia, gestational diabetes, and perinatal death) [60–63].
Hence, some abnormal term placentae appear to preserve syncytium in a younger state, accompanied by high secretion of hCG. In contrast, vascular LHR expression was detected in sinusoids of chorionic villi from normal term placentae, but was virtually absent in all abnormal placentae. If the vascular LHR expression represents a barrier for hCG transport from chorionic villi to the fetal blood (see above), lack of this barrier may cause high levels of fetal hCG resulting in perinatal morbidity and mortality , regardless of normal or abnormal hCG production.
Western blot analysis of placental villi and trophoblast cultures indicates that cultured trophoblast cells show additional bands, including ~170 kDa species, possibly an LHR homodimer . Although the ~170 kDa species persisted in late cultures, other (additional) LHR variants diminished. This suggests an activation of isolated trophoblast cells and enhanced LHR synthesis with formation of additional glycosylated LHR variants, particularly in time 0 and early cultures. When compared to chorionic villi, trophoblast cultures showed stronger ~59 and 48 kDa species, characteristic for cultured fibroblasts. Indeed, it has been reported, that differentiation of cultured trophoblast is associated with activation of accompanying fibroblasts .
Early differentiation of granulosa, luteal, and other cell types expressing LHR was associated with cytoplasmic LHR immunoreactivity, reflecting receptor synthesis prior its surface expression. In addition, the CL of pregnancy, the function of which is highly dependent on CG, also showed cytoplasmic expression. Hence, it is possible that high levels of circulating LH/CG, e.g., during pregnancy and after menopause, may influence not only cells with surface LH expression, but also cells with cytoplasmic, and perhaps exclusively nuclear LHR expression (pelvic floor compartments, including striated muscle). In other words, cells lacking surface LHR may not be influenced by temporary increase of LH production during the preovulatory period of the ovarian cycle due to the "cell membrane LHR barrier," and the LH effect is probably also prevented by the "vascular LHR barrier." Yet, high CG levels during pregnancy may pass the "cell membrane barrier" and act through the cytoplasmic LHR in the CL of pregnancy and other cells with cytoplasmic expression, and such effect could be enhanced if the "vascular LHR barrier" is absent – abnormal placentae of aged phenotype and similar putative age-related vascular changes in other tissues.
But the LH/CG action through the cytoplasmic/perinuclear and nuclear LHRs opens a possibility of more direct LH/CG action within the cells, which may not require a classic second messenger pathway (cyclic AMP dependent signaling mechanism) involvement, or this system can act through the intracellular LHRs. Evidence is accumulating that a number of other factors modulate the actions of gonadotropins in the ovary and testis via activation of alternative signaling pathways and via LHR protein variants, and alternative second messenger pathways for the transmission of the LHR activation effect exist, which may not include stimulation of cyclic AMP levels [65–69]. A question also arises if the nuclear LHR expression in terminally differentiated cells (e.g., striated muscle fibers) may not indicate a possibility of the direct effect of LH/CG on modulation of protein synthesis, reflecting certain effects of sex steroids on their nuclear receptors.
LH/CG causes a relaxation of smooth muscle cells, which express LHR [36–38] (Fig. 7A). The data presented also indicate that additional principal cell types in the pelvic floor show LHR expression, including stromal and fascial fibroblasts and striated muscle cells. This is associated with relatively high expression of LHR protein in western blots, and a fully glycosylated ~92 kDa species in particular. Interestingly, strong ~59 and 48 kDa proteins expressed in cultured fibroblasts were not detected in pelvic floor lysates, but a distinct ~68 kDa band from mesenchymal cells was apparent. We speculate that a ~92 kDa species in the pelvic floor is a result of interaction of resident macrophages with pelvic floor fibroblasts.
The hCG secreted by trophoblastic syncytium during pregnancy may play an important role in the physiologic adaptation of the body, and preparation of the pelvic floor for labor in particular. However, when compared to hCG, LH has a 10-fold higher LHR binding affinity . Consequently, high LH levels after menopause may cause pathologic relaxation of the pelvic floor resulting in pelvic floor disorders.
Strong LHR expression in microglial cells in the brain cortex is of particular interest. Microglial cells are resident macrophages in the central nervous system, and LHR expression has been described in other types of human resident macrophages (ovary, decidua, endometrium and corpora lutea) [22, 33]. Pathological activation of microglia has been reported in a wide range of conditions such as Alzheimer's disease, cerebral ischemia, prion diseases, multiple sclerosis, AIDS dementia, and other degenerative neurological diseases [70, 71]. Some of these degenerative diseases are associated with advanced age and high levels of circulating LH. High LH levels might pass the presumptive LH barrier of brain vessels expressing LHR, or vascular LHR expression might diminish with age, as in abnormal term placentae. Yet, elevated maternal mid-trimester chorionic gonadotropin is associated with fetal cerebral blood flow redistribution and adverse perinatal outcome . In addition, estrogens, which are known to cause a diminution of high LH levels in postmenopausal women, have been reported to be effective in the prevention and treatment of Alzheimer's disease [72–74]. Microglia belong among cells of the mononuclear phagocyte system . Since CG increases secretion of a variety of cytokines by monocytes, and induces their inflammatory reaction and phagocytic activity [76–79], high LH levels in aging individuals may also activate resident macrophages in the central nervous system and contribute to the development of Alzheimer's disease and other inflammation-mediated neurodegenerative diseases.
Outside of the area occupied by microglial cells in the grey matter of the cerebral cortex, weaker LHR expression was also detected in some nerve cells. This suggests that such nerve cells could be directly influenced by LH/CG proteohormones (with unknown consequences), while the nerve cells among microglial branching processes are protected from such an effect.