Fig. 2.

Real time qPCR assay (A) shows nearly identical expression of HFSHB mRNA in pituitaries of Fshb^−/− mice expressing either a HFSHB^WT or HFSHB^dgc transgene. Two different lines #30 and #60, expressing the mutant transgene were developed. Dual label immunofluorescence visualization of pituitary sections (B) from adult mice confirmed gonadotrope-specific expression of HFSHB^dgc transgene similar to that of HFSHB^WT transgene (indicated by white arrowheads in merged images in the right panel in B). The absence of any green fluorescence in pituitary sections from Fshb^−/− mice confirmed the specificity of the FSHβ antibody. The white bar in panel B represents 50 µm. Western blot analysis of proteins under denaturing conditions probed with a goat anti-human FSHβ-polyclonal antibody obtained from Santa Cruz Biotechnology, sc 7757 (C) shows a 15–17 KDa hFSHβ^{Asn7Δ Asn24Δ} non-glycosylated subunit (denoted as hFSHβ*) was abundantly expressed in pituitaries of three different lines of HFSHB^dgc transgenic mice (line # 220, line #30 and line #60) on an Fshb null genetic background. Black arrow indicates a non-specific band. Recombinant human (r–h) FSHβ size-matched with that of HFSHB^WT transgene-encoded hFSHβ subunit and weakly immunoreactive mouse (m) FSHβ subunit detected in normal control mouse (Ctrl.) pituitary. Proteins from Fshb^−/− mouse pituitaries served as a negative control. PNGase F treatment followed by Western blot analysis of either r-hFSH or pituitary proteins from a normal control wild-type (Ctrl.) mouse showed an identical size de-glycosylated FSHβ subunit corresponding to that identified in an Fshb^−/− HSFHB^dgc mouse pituitary (D). Electrophoretic separation of proteins under non-denaturing conditions followed by Western blot analysis showed that in both male (E) and female (F) Fshb^−/− HSFHB^dgc mouse pituitaries, the non-glycosylated FSHβ subunit, despite high level expression (C) was present as an interspecies hybrid (mouse α + human FSHβ*) heterodimer (lanes 5 and 6 in panels E and F) at very low levels compared to the FSH heterodimer (mouse α + hFSHβ) in pituitaries of Fshb^−/− HSFHB^WT mice (lane 4 in panels E and F). Pituitary proteins from control and Fshb^−/− mice (Lanes 1 and 2 in panels E and F) are used as negative controls and r-hFSH (Lane 3) as a positive control. Arrows in panel D denote: a = FSHβ subunit, ns = non-specific band, b = deglycosylated FSHβ subunit. Arrows in panel E and F indicate: a = FSHβ subunit, b = double N-glycosylation site mutant FSHβ subunit, c = uncombined/free FSHβ subunit, d = uncombined double N-glycosylation site mutant FSHβ subunit. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)