Oocytes are required for the preantral granulosa cell to cumulus cell transition in mice

Abstract

Preantral granulosa cells (PAGCs) differentiate into cumulus cells following antrum formation. Cumulus cells, but not PAGCs, are competent to undergo expansion. Experiments reported here tested the respective roles of both oocytes and FSH in the transition of preantral granulosa cells to cumulus cells competent to undergo expansion. PAGC-oocyte complexes were cultured with or without a low dose of FSH (0.005 IU/ml) and isolated PAGCs were cultured with or without oocytes. At the end of culture, complexes or isolated PAGCs were tested for their ability to undergo cumulus expansion and upregulate expansion transcripts in response to EGF or FSH (0.5 IU/ml). The ability to undergo expansion in response to EGF required the presence of oocytes but not FSH during the culture period. Likewise, complexes isolated from the ovaries of hypogonadal mice, which lack circulating gonadotropins, underwent expansion in response to EGF, but not FSH. In contrast, the ability to activate MAPK3/1 and MAPK14 and undergo expansion in response to FSH required prior exposure to low doses of FSH. However, these low levels (0.005 or 0.025 IU FSH/ml) suppressed expression of Slc38a3 and Amh, two transcripts highly expressed in cumulus cells, suggesting opposing effects of FSH on cumulus cell differentiation. In conclusion, the ability to undergo expansion in response to FSH requires prior exposure to FSH during development, while oocyte-derived factors alone are sufficient to promote the ability to undergo expansion in response to EGF. These results highlight the crucial role of oocytes in driving the differentiation of PAGCs into cumulus cells during the preantral to antral follicle transition.

Figure 1

Model of follicular development and experimental designs. (A) In-vivo follicular growth beginning with preantral follicles that differentiate into antral follicles with two populations of granulosa cells. The mural cells (green) localize to the follicular wall. Cumulus cells (red) surround the oocyte and express cumulus marker transcripts (Slc38a3 and Amh). Following the LH surge, the cumulus oophorus undergoes expansion with increases in expansion-related transcripts (Has2, Ptgs2, Ptx3 and Tnfaip6). Experimental models used to study acquisition of competence to undergo expansion in vitro. (B) PAGC-oocyte complexes were isolated from ovaries of 12 day old mice and cultured on collagen-coated membranes for 2 to 10 days in medium with or without FSH (0.005 IU/ml). Following culture complexes were removed from the membrane and treated with FSH (0.5 IU/ml) or EGF (10 ng/ml) to stimulate pathways leading to cumulus expansion. (C) To determine whether oocytes play a role in the preantral to antral follicle transition, PAGC-oocyte complexes were oocytectomized and cultured for 6 days on a thin layer of agarose either in medium only or co-cultured with fully-grown oocytes (FGO, 1 oocyte/μl). PAGCs co-cultured with oocytes received freshly isolated oocytes every 2 days. Following the culture period, PAGCs were removed from the agarose and treated with control medium or medium containing EGF (10 ng/ml) during the test period. Freshly isolated FGO (2 oocytes/μl) were also added to all treatment groups during the test period.

Figure 2

Timing of the acquisition of competence to undergo expansion in vitro. Complexes were cultured in control medium or medium supplemented with a low concentration of FSH (0.005 IU/ml) for 2, 4, 6, 8 and 10 days. Following the culture period, complexes were cultured in control medium or medium with EGF (10 ng/ml) for 15 hours during the test period to stimulate cumulus expansion. (+) indicate positive expansion, (−) negative expansion and (+/−) mixed response.

Figure 3

Expression of Has2, Ptgs2, Ptx3 and Tnfaip6 mRNAs in complexes cultured with (A) control medium, or (B) FSH (0.005 IU/ml) for 2, 4, 6, 8 and 10 days followed by treatment with control medium or EGF (10 ng/ml) for 12 hours during the test period. *Denotes value different from control as determined by Student's t-test, P<0.05.

Figure 4

Effect of oocyte co-culture on development of competence to undergo expansion (A) and expression of expansion transcripts (B) in oocytectomized PAGCs cultured in control medium or co-cultured with FGO (1 oocytes/μl) for 6 days (culture period). At the end of the culture period, PAGCs were removed from the agarose and treated with control medium or EGF (10 ng/ml) for 6 hours (mRNA levels) or 15 hours (expansion assay). Freshly isolated oocytes were added to all PAGC treatment groups during the test period only (2 oocytes/μl). Test period treatments, including the oocytes added to provide CEEFs, are shown in the both 4A and 4B. *Denotes value different from control as determined by Student's t-test, P<0.05.

Figure 5

Competence to undergo expansion by COCs collected from hpg^−/− mutant or hpg^+/? littermate controls treated with FSH (0.5 IU/ml) or EGF (10ng/ml) for 15 hours.

Figure 6

Competence to undergo expansion of complexes cultured in control medium or FSH (0.005 IU/ml) for 10 days followed by treatment with control medium (control), 10 ng/ml EGF (middle) or 0.5 IU/ml FSH (bottom) for 15 hours to stimulate expansion.

Figure 7

Steady-state levels of Has2, Ptgs2, Ptx3, and Tnfaip6 mRNA in complexes cultured with control medium or FSH (0.005 IU/ml) for 10 days followed by treatment with EGF (10 ng/ml) (A) or FSH (0.5 IU/ml) (B) for 6 hours. *Denotes value different from control as determined by Student's t-test, P<0.05.

Figure 8

Activation of MAPK14 and MAPK3/1 in complexes cultured with control medium or FSH (0.005 IU/ml) for 10 days followed by treatment with control medium, FSH (0.5 IU/ml), or EGF (10 ng/ml) for 4 hours and immunostaining for MAPK14, MAPK3/1 and ACTB on the same blot. A representative blot of three independent experiments is shown.

Figure 9

Expression of cumulus marker transcripts (Slc38a3 and Amh) in COCs grown in vivo and in vitro (from preantral granulosa cell-oocyte complexes for 10 days with 0, 0.005 or 0.025 IU/ml FSH). ^abcValues with different superscripts are significantly different, p<0.05.