Follicle-stimulating hormone and luteinizing hormone increase Ca2+ in the granulosa cells of mouse ovarian follicles†

Abstract

In mammalian ovarian follicles, follicle stimulating hormone (FSH) and luteinizing hormone (LH) signal primarily through the G-protein Gs to elevate cAMP, but both of these hormones can also elevate Ca2+ under some conditions. Here, we investigate FSH- and LH-induced Ca2+ signaling in intact follicles of mice expressing genetically encoded Ca2+ sensors, Twitch-2B and GCaMP6s. At a physiological concentration (1 nM), FSH elevates Ca2+ within the granulosa cells of preantral and antral follicles. The Ca2+ rise begins several minutes after FSH application, peaks at ∼10 min, remains above baseline for another ∼10 min, and depends on extracellular Ca2+. However, suppression of the FSH-induced Ca2+ increase by reducing extracellular Ca2+ does not inhibit FSH-induced phosphorylation of MAP kinase, estradiol production, or the acquisition of LH responsiveness. Like FSH, LH also increases Ca2+, when applied to preovulatory follicles. At a physiological concentration (10 nM), LH elicits Ca2+ oscillations in a subset of cells in the outer mural granulosa layer. These oscillations continue for at least 6 h and depend on the activity of Gq family G-proteins. Suppression of the oscillations by Gq inhibition does not inhibit meiotic resumption, but does delay the time to 50% ovulation by about 3 h. In summary, both FSH and LH increase Ca2+ in the granulosa cells of intact follicles, but the functions of these Ca2+ rises are only starting to be identified.

The optimized research of the in vitro culture of preantral follicles in mice

“…31,32 The expression of these enzymes converts androstenedione synthesized from follicular theca cells into E2. Although studies have reported that FSH affects the development of preantral follicles, [33][34][35] there is no report regarding the effect of FSH on the growth pattern of preantral follicles in vitro. The reason may be that the PM follicles and ES follicles were cultured together in previous studies.…”

“…In 10 mIU/mL r‐FSH culture medium, ES follicles exhibited a tiled growth pattern, with large adherence area; while in 100 mIU/mL r‐FSH culture medium, they presented a three‐dimensional growth pattern, with small adherence area. Although studies have reported that FSH affects the development of preantral follicles, 33‐35 there is no report regarding the effect of FSH on the growth pattern of preantral follicles in vitro. The reason may be that the PM follicles and ES follicles were cultured together in previous studies.…”

A CRISPR toolbox for generating intersectional genetic mice for functional, molecular, and anatomical circuit mapping

“…Thus, our outcomes here may reflect our core facility capabilities as much as inherent limitations in consistent Cas9 mediated oocyte targeting of large constructs. The successful outcomes here and in a prior publication demonstrate the use of the system to develop lines capable of functional imaging (62).…”

“…10 B). The data demonstrate how the targeting system here can be used to build conditional mouse lines that are useful beyond the nervous system and throughout the body (62).…”

Using Genetically Encoded Calcium Indicators to Study Astrocyte Physiology: A Field Guide

“…This fate mapping effect can be circumvented by induction of GECI expression after neuronal and oligodendroglial maturation in inducible Cre driver mouse lines (tamoxifen or tetracycline/doxycycline). For some GECIs, transgenic mouse lines to allow for glia-specific Ca 2+ imaging have been published, but are not commercially available (Yoshikawa et al, 2016;Egbert et al, 2019). According to our knowledge, we indicate whether transgenic mice, rAAVs, or plasmids ready-to-use for astrocytic expression are available and listed those in Supplementary Table 1.…”