Assessment of gonadotroph functions in mutant mouse pituitary fragments perifused with different GnRH pulse frequencies
OA Version
Citation
Abstract
BACKGROUND: Vertebrate reproduction is regulated through the hypothalamic-pituitary-gonadal (HPG) axis, a hormonal signaling cascade. The hypothalamus integrates internal and external cues to secrete gonadotropin-releasing hormone (GnRH) in a pulsatile manner, resulting in the synthesis and release of follicle stimulating hormone (FSH) and luteinizing hormone (LH) from gonadotroph cells of the pituitary gland. FSH and LH are heterodimeric proteins that share a common alpha subunit and unique beta subunits that derive from separate genes and confer biological specificity. At the gonads, FSH and LH coordinate gametogenesis and steroidogenesis, resulting in the production of mature oocytes or sperm. GnRH pulse frequency is decoded by GnRH receptors (GnRHR) expressed on pituitary gonadotrophs that activate the Gαs and Gαq signaling pathways to promote the differential expression of Fshb or Lhb, genes encoding the FSH and LH beta subunits respectively, by a mechanism that is not yet fully understood. The Gαq-IP3/DAG signaling pathways are essential for FSH and LH synthesis in vitro and in vivo, while the Gαs cAMP signaling pathway has been shown to differentially regulate Fshb synthesis in vitro. The human and mouse Fshb gene promoters harbor a half cAMP response element (CRE) that binds to phosphorylated CRE binding protein (CREB), promoting transcriptional complex formation. The goal of this study was to identify the direct role of CREB in FSH and LH regulation in vivo.
OBJECTIVES: The objectives of this study were to characterize the pubertal phenotype of gonadotroph-specific CREB knock-out (KO) mice and to develop a perifusion assay to study mouse pituitary fragments ex vivo. We hypothesized that gonadotroph-specific CREB deletion would negatively affect gamete maturation, and therefore fertility, due to diminished FSH synthesis in mice.
METHODS: Gonadotroph-specific CREB KO and littermate control mice were generated by Cre-lox recombination and monitored for body weight and onset of puberty from postnatal day 21. Daily vaginal cytology was used to determine the days of vaginal opening and first estrus, markers of pubertal onset in female mice, as well as estrous cyclicity. Male pubertal onset was determined by preputial separation. Mouse pituitary fragments (with unknown genotype) were perifused ex vivo and stimulated with GnRH pulses at low (every 120 min) or high (every 30 min) frequencies for 20 hours and subsequently analyzed by quantitative PCR to measure changes in Lhb, Fshb, and Gnrhr mRNA levels. The pituitaries of mice harboring a mutation in the promoter of Gnrhr (AP-1 KI mice) were ultimately used to validate a proof-of-concept of pituitary fragment perifusion in this Gnrhr deficient model.
RESULTS: There were no observable differences between the body weights of CREB KO and control mice in either males or females from postnatal day (PND) 21 to 3 months of age. There was also no significant difference in the ages of vaginal opening (p=0.18) or first estrus (p=0.75) between CREB KO and control mice. CREB KO and control mice showed normal estrous cyclicity, with similar amounts of time spent in diestrus/metestrus, proestrus, or estrus. Male CREB KO mice tended to show preputial separation at a younger age compared to control mice (p=0.06). Pituitary perifusion with different GnRH pulse frequencies was successfully established, and increased Gnrhr mRNA levels at both GnRH pulse frequencies was seen in some mice; however, this was not consistent with Gnrhr-deficient AP-1 mutant mouse pituitaries. No changes in Lhb or Fshb mRNA levels were observed in random or in AP-1 mutant mice.
CONCLUSIONS: We were not able to detect differences in Fshb or Lhb mRNA levels in response to low or high GnRH pulse frequencies in mouse pituitaries in an ex vivo perifusion system, so this may not be a viable method to analyze differential Fshb or Lhb synthesis. Gonadotroph-specific CREB KO did not impact the onset of puberty in male or female mice, nor was estrous cyclicity affected in females. Ongoing studies will determine the impact of gonadotroph CREB KO on fertility and folliculogenesis.