In order to get a
In order to get a better understanding of this synergy, we tested the effect of preliminary incubation of BMS 753 synthesis with non-stimulating concentrations of gonadotropins or FSK before subsequent stimulation by FSK or gonadotropins alone, also at sub-stimulating concentrations. We observed that human LH (hLH) and CG (hCG), but not the non-human LHs and CG, exhibit a permissive effect for subsequent stimulation by 10 µM FSK alone, of cyclic AMP accumulation (Fig. 6). In the reverse protocol where the cells were preincubated with 10 µM FSK before the addition of hLH or hCG in the absence of FSK, no stimulation of cyclic AMP accumulation was observed (Fig. 3). This indicates that hLH or hCG alone (but not the non-human LHs or CG) elicit a long-term change permitting subsequent stimulation by 10 µM FSK. The reverse is not observed since FSK alone, after its removal, does not sensitize the cells to subsequent stimulation by sub-stimulating concentrations of hormones. This sensitizing effect of hLH and hCG compared to non-human LHs and CG can be related to the previously described increasing irreversibility of human gonadotropins to the LH receptor (Combarnous et al., 1986, Huhtaniemi and Catt, 1981, Katikineni et al., 1980). Therefore, the sensitizing effect of hLH and hCG to FSK cannot be attributed simply to a higher Ka for LHR but to a step beyond binding, leading to its irreversibility (Combarnous et al., 1986). Indeed even three washes and replacements with hormone-free medium does not decrease sensitization of MLTC cells to FSK. The possible mechanisms of this binding irreversibility can be either internalization of activated hLH or hCG-bound LHR to very early endosomes (Jean-Alphonse and Hanyaloglu, 2011, Sposini and Hanyaloglu, 2017) or, less likely, to a disulfide interchange between the hormone and receptor leading to a covalent bond due to the PDI-like activity of gonadotropins (Boniface and Reichert, 1990, Chew et al., 1995, Grasso et al., 1991). What in human LH and CG structures make them able to irreversibly bind to the LHR and sensitize MLTC-1 cells to subsequent FSK stimulation, in contrast to non-human LH and CG, is still unknown. It cannot be the βCTP that is present in hCG, but absent in hLH. It is also unlikely that it originates from hLH and hCG carbohydrate structures since natural hLH and hCG are synthesized in different cell types (pituitary gonadotropes and placental syncitiotrophoblasts respectively) and consequently exhibit differing sugar moieties (Bahl et al., 1978, Hard et al., 1992, Nilsson et al., 1986). Similarly, recombinant hLH and hCG exhibit the same sensitizing effect to FSK, in spite of their carbohydrate structures (Butnev et al., 2015, Thakur et al., 2009) differing from those of natural hLH and hCG. It is thus likely that this common outstanding property of hLHs and hCGs comes from their common α-subunit polypeptide sequence. In this respect, a remarkable feature, compared to non-human LHs and CGs α-subunits, is the absence of four amino acids near their N-terminal end (Combarnous, 1992, Pierce and Parsons, 1981). Another particular region in hGPHα-subunits is the one between Thr11 and Pro21, in which the replacement of several amino-acid residues by the corresponding basic residues at the same positions in other mammalian sequences, leads to an increase in binding affinity of hCG to LH receptors, and higher cyclic AMP responses in MA-10 Leydig cell line (Szkudlinski et al., 1996). It is difficult to compare these data with those in the present report, as these authors used recombinant hCGs with point mutations in the hα11-21 sequence, only at stimulating concentrations, and on a different Leydig cell line. Nevertheless, the two studies point out and confirm the importance of the common α-subunit of glycoprotein hormones in the binding to their receptor and in their stimulating activity (Combarnous, 1992). In the absence of FSK, the EC50 calculated from cyclic AMP responses for rat LH and rec hLH are 1100 ± 120 pM and 300 ± 40 pM respectively (Klett et al., 2016). In the presence of FSK, the EC50 for cyclic AMP accumulation by the same hormones are 65 ± 7 pM and 15 ± 3 pM respectively (i.e. 15–20-fold lower). It is interesting to point out that the EC50 for hCG alone on progesterone secretion in MLTC (this work and Evaul and Hammes (2008)) is similar to its EC50 on cyclic AMP accumulation in the presence of FSK (∼20 pM). This indicates that LHs and CGs exert an action on MLTC cells in the 1–100 pM range that can be revealed at the cyclic AMP level only in the presence of FSK. However, it can be observed at the level of progesterone secretion in the absence of FSK (Evaul and Hammes, 2008). This means that steroidogenesis in MLTC cells could be stimulated either without cyclic AMP accumulation or at cyclic AMP concentrations that are non-measurable in both the luminescence and the HTRF techniques.