Fshb-iCre mice are efficient and specific Cre deleters for the gonadotrope lineage

Abstract

Genetic analysis of development and function of the gonadotrope cell lineage within mouse anterior pituitary has been greatly facilitated by at least three currently available Cre strains in which Cre was either knocked into the Gnrhr locus or expressed as a transgene from Cga and Lhb promoters. However, in each case there are some limitations including CRE expression in thyrotropes within pituitary or ectopic expression outside of pituitary, for example in some populations of neurons or gonads. Hence, these Cre strains often pose problems with regard to undesirable deletion of alleles in non-gonadotrope cells, fertility and germline transmission of mutant alleles. Here, we describe generation and characterization of a new Fshb-iCre deleter strain using 4.7 kb of ovine Fshb promoter regulatory sequences driving iCre expression exclusively in the gonadotrope lineage within anterior pituitary. Fshb-iCre mice develop normally, display no ectopic CRE expression in gonads and are fertile. When crossed onto a loxP recombination-mediated red to green color switch reporter mouse genetic background, in vivo CRE recombinase activity is detectable in gonadotropes at more than 95 % efficiency and the GFP-tagged gonadotropes readily purified by fluorescence activated cell sorting. We demonstrate the applicability of this Fshb-iCre deleter strain in a mouse model in which Dicer is efficiently and selectively deleted in gonadotropes. We further show that loss of DICER-dependent miRNAs in gonadotropes leads to profound suppression of gonadotropins resulting in male and female infertility. Thus, Fshb-iCre mice serve as a new genetic tool to efficiently manipulate gonadotrope-specific gene expression in vivo.

Evaluation of in vivo bioactivities of recombinant hypo- (FSH 21/18 ) and fully- (FSH 24 ) glycosylated human FSH glycoforms in Fshb null mice

“…Approximately 500 ng-1 μg total RNA was reverse transcribed by the oligo dT method using the SuperScript III kit (ThermoScientific) as described [24-26]. Taqman real time qPCR assays were performed on the cDNA samples in triplicate using custom-made or pre-inventoried primer/combo mixes (ThermoScientific or IDT).…”

“…Taqman real time qPCR assays were performed on the cDNA samples in triplicate using custom-made or pre-inventoried primer/combo mixes (ThermoScientific or IDT). Expression of Ppil1 was used as an internal control and the relative amounts of mRNA expression were calculated as described [24-26]. …”

“…Testes and ovaries were processed, later paraffin-embedded and 6 μm sections were cut and stained with either PAS reagent/ hematoxylin or hematoxylin-eosin for histological analysis as described before [24-26]. Quantification of testis tubule size was done on PAS reagent / hemtoxylin – stained testis section images that were digitally captured as described [21].…”

“…Formalin-fixed and paraffin-embedded tissue sections of approximately 6μm thickness were cut and processed for immunofluorescence as described before [24-26]. The following primary antibodies were used: rabbit polyclonal antibody against Sox9 (gift from Dr. K. Morohashi, 1:1,000), rat monoclonal antibody to GCNA (gift from Dr. George Enders, used undiluted) mouse anti BrdU monoclonal antibody (Roche, 1:100), rabbit anti phospho-CREB (Cell Signaling Systems) or rabbit anti phospho-PKA substrate (Cell Signaling Systems, 1:500) both at a dilution of 1:500.…”

“…The following primary antibodies were used: rabbit polyclonal antibody against Sox9 (gift from Dr. K. Morohashi, 1:1,000), rat monoclonal antibody to GCNA (gift from Dr. George Enders, used undiluted) mouse anti BrdU monoclonal antibody (Roche, 1:100), rabbit anti phospho-CREB (Cell Signaling Systems) or rabbit anti phospho-PKA substrate (Cell Signaling Systems, 1:500) both at a dilution of 1:500. The sections were later incubated with the appropriate secondary antibodies conjugated with Alexa fluors (Invitrogen) and/or a nuclear dye and visualized using an epifluorescence microscope as described [24-26]. GCNA + germ cells were counted in ~ 100 tubules per group and three mice were used per group.…”