Meiotic resumption in response to luteinizing hormone is independent of a Gi family G protein or calcium in the mouse oocyte

Abstract

The signaling pathway by which luteinizing hormone (LH) acts on the somatic cells of vertebrate ovarian follicles to stimulate meiotic resumption in the oocyte requires a decrease in cAMP in the oocyte, but how cAMP is decreased is unknown. Activation of Gi family G proteins can lower cAMP by inhibiting adenylate cyclase or stimulating a cyclic nucleotide phosphodiesterase, but we show here that inhibition of this class of G proteins by injection of pertussis toxin into follicle-enclosed mouse oocytes does not prevent meiotic resumption in response to LH. Likewise, elevation of Ca2+ can lower cAMP through its action on Ca2+-sensitive adenylate cyclases or phosphodiesterases, but inhibition of a Ca2+ rise by injection of EGTA into follicle-enclosed mouse oocytes does not inhibit the LH response. Thus, neither of these well-known mechanisms of cAMP regulation can account for LH signaling to the oocyte in the mouse ovary.

Figure One

Concentration dependence of LH stimulation of meiotic resumption in follicle-enclosed oocytes. Antral follicles were exposed to various concentrations of LH for 4–5 hours. The oocytes were then removed from their follicles, and scored for the presence or absence of an intact prophase nucleus. %GVBD = the number of oocytes that had undergone breakdown of the nuclear envelope (germinal vesicle), divided by the number of oocytes that were counted, with oocytes from 4 experiments combined as a single group. The numbers in parentheses indicate the number of oocytes counted for each point. Results for all concentrations of LH were significantly different from the control without LH (p = 0.02 for 0.01 μg/ml LH; p < 0.0001 for 0.1 and 1.0 μg/ml; p = 0.002 for 10 μg/ml; Fisher’s exact test).

Figure Two

A G_i-family G protein does not mediate LH stimulation of meiotic resumption. A. PTX does not inhibit the LH response in follicle-enclosed oocytes. Follicle-enclosed oocytes were injected with PTX (1.8 pg), or left uninjected. 1–3 hours later, they were exposed to 1 μg/ml LH, or control medium, for 3–4 hours. The oocytes were then isolated, to determine %GVBD. The numbers in parentheses indicate the number of oocytes counted for each point, combining the results of 9 experiments. Column 4 is not statistically different from column 3 (p = 0.7), but is different from column 2 (p < 0.0001); column 2 is not different from column 1 (p = 0.09) (Fisher’s exact test). When data for the %GVBD for column 2 were compared to the subset of data for column 1 that were collected on the same days as column 2, the %GVBD in column 1 (12%, n = 33) was very close to that for column 2 (11%, n = 19). B. PTX is an effective inhibitor of G_i-signaling in the oocyte. Isolated oocytes expressing the m2 acetylcholine receptor and the chimeric G protein G_qi were injected with 17 μM calcium green dextran (upper trace), or 17 μM calcium green dextran + 1.8 pg PTX (lower trace). Calcium green fluorescence was recorded before and after addition of 10 μM acetylcholine. The x axis shows time, and the y axis shows the voltage output of the photodiode amplifier, which is proportional to calcium green fluorescence. The initial deflection of the trace during acetylcholine addition is an artifact that is often seen when solution is pipetted into the droplet containing the oocyte (see Mehlmann et al., 1998). C. Immunoblot showing the presence of Gα_z protein in mouse brain (1, 2, or 5 μg) but not in mouse oocytes (6 μg = 230 oocytes). Before the blot was stained for Gα_z, it was stained for total protein, using Ponceau S; this confirmed that the amount of protein transferred to the membrane from the 6 μg oocyte sample was comparable to the amount of protein from the 5 μg brain sample. The brain Gα_z protein migrated with an electrophoretic mobility close to that of a 37 kDa molecular weight standard, close to its predicted molecular weight of 41 kDa; the slight discrepancy in size could be related to the use of a prestained molecular weight standard, which could have migrated more slowly than an unstained protein.

Figure Three

Elevation of oocyte Ca²⁺ does not mediate LH stimulation of meiotic resumption. A. EGTA does not inhibit the LH response in follicle-enclosed oocytes. Follicle-enclosed oocytes were injected with EGTA buffer (10 mM EGTA in the cytoplasm, free Ca²⁺ ~30 nM), and exposed to 1 μg/ml LH for 3–4 hours. The oocytes were then isolated, to determine %GVBD. The numbers in parentheses indicate the number of oocytes counted for each point, combining the results of 3 experiments. Column 2 is statistically different from column 1 (p < 0.0001), but is not different from column 3 of Fig. 2 (p = 0.6) (Fisher’s exact test). In a separate series of experiments, we found that injection of 10 mM of the EGTA buffer did not cause a signficant increase in meiotic resumption (20% GVBD, n = 15). B. EGTA is an effective inhibitor of Ca²⁺ elevation in isolated mouse oocytes. Isolated oocytes expressing the m1 acetylcholine receptor were injected with 20 μM calcium green dextran (upper trace), or 20 μM calcium green dextran + 10 mM of EGTA buffer (lower trace). Calcium green fluorescence was recorded before and after addition of 10 μM acetylcholine. C. EGTA is an effective inhibitor of Ca²⁺ elevation in antral follicle-enclosed mouse oocytes. Antral follicle-enclosed oocytes were injected with 1 μM caged IP₃ and 20 μM calcium green 10 kDa-dextran, with or without 10 mM EGTA buffer. 3–4 hours later, IP₃ was uncaged by exposing the follicle-enclosed oocytes to 330 ± 40 nm light (UV), for a period of 2 sec. In oocytes that had not been injected with EGTA, uncaging of IP₃ resulted in an increase in calcium green fluorescence (n = 6 oocytes). In oocytes that had been injected with 10 mM EGTA buffer, 6/9 oocytes showed no detectable increase in calcium green fluorescence in response to uncaging IP₃, and 3 others showed a small increase (~10%, or in one case ~20%, of that seen without EGTA). Note that the measurement systems used for B and C differed, so the Y axes cannot be directly compared.

Figure Four

Deleterious effect of BAPTA on oocyte structure. Mouse oocytes, which were held in meiotic arrest with 250 μM dbcAMP, were injected with BAPTA (upper panel) or EGTA (lower panel), at a cytoplasmic concentration of 10 mM (~30 nM free Ca²⁺). The oocytes were photographed 3–4 hours after injection.

Figure Five

Some of the possible pathways by which luteinizing hormone (LH) action on the somatic cells of the mammalian ovarian follicle could decrease cAMP in the oocyte, leading to the progression of meiosis. Of the pathways shown, experiments described in this paper argue against those marked with X’s; others remain to be tested. R = receptor; GPR3 = G protein receptor 3; AC3 = adenylate cyclase 3; GC = guanylate cyclase; G_i, G_z, G_s, G_t = G protein isoforms; PDE1, PDE3A, PDE6 = cyclic nucleotide phosphodiesterase isoforms. Green arrows indicate stimuli that increase the activity or amount of the indicated molecule; red lines indicate stimuli that decrease the activity or amount of the molecule. Pathways occuring in the somatic cells of the follicle, including signaling through the LH receptor, production of EGF receptor ligands, and the possible role of a steroid, are not shown (see Introduction and Discussion).