Ten to fifteen percent of couples experience infertility [1], which for many can be a life crisis [2,3]. Defects in ovulation and spermatogenesis, as well as unexplained causes, account for the major component of impaired fertile aetiologies [1]. Two main factors contribute to these disorders, the age of the woman and reproductive capacity. The current study aimed to understand how hypothalamic-pituitary-gonadal (HPG) axis regulates the pituitary hormone (luteinizing hormone, LH) and ovarian follicle development during prepubertal, pubertal and adulthood in male and female mice.The onset of puberty and sexual function are mainly triggered by the activation of HPG axis, which produces pulsatile gonadotropin (LH and FSH) release during secondary sexual development, and regulates gonadal steroid hormone (estradiol and testosterone) synthesis [4][5][6][7][8][9][10][11][12][13][14][15]. Hormonal indicators can be used as tools for detecting early pubertal onset in mammals, but there are limited studies on the hormonal profile changes that occur during the pubertal transitions from prepuberty to early-adulthood in males and females [16,17]. The mouse, a powerful model often used to elucidate the genetic basis of human physiology and pathophysiology [18], remains highly challenging to study functionally in the context of hormonal release. This is particularly true for LH release, owing to an inability to accurately detect pulsatile LH release in the ultra-small blood fractions available in mice. Quantitative measures of pulsatile LH in pre-pubertal and post-pubertal female and male mice therefore do not exist. As patterns of LH release are more pulsatile compared to FSH, we aimed to examine the pulsatile LH secretion during the transition of puberty in both male and female mice in this project. Dynamic changes in LH release that occur with age and reproductive cycle will provide vital information that predicts optimal reproductive capacity.The current project described the generation of an ultra sensitive mouse LH enzymelinked immunosorbent assay (ELISA) capable of reliably detecting LH to 0.117 ng/mL in a 2-µL fraction of mouse whole blood. Male C57BL/6J mice were used to quantify the pulsatile LH secretion profile during the transition from prepuberty to adulthood. This method has been validated using terminal male mouse plasma and whole blood 3 samples collected using an established frequent blood procedure [19]. Data demonstrated the peak of episodic LH release rarely exceeded 3 ng/mL in both 4 weeks and 16 weeks of age in wild-type C57BL/6J male mice [20]. Mice with an absence or disruption to the kisspeptin receptor (Gpr54/Kiss1r) [21][22][23][24][25] and Kisspeptin 1 (Kiss1) [26] genes do not undergo normal pubertal development and result in impaired fertility in both sexes. Mutations in GPR54 in humans results in absent LH secretion [25, 27,28]. We validated this LH assay by undertaking highresolution serial blood sampling to determine levels of pulsatile LH secretion in 32-week-old male kisspeptin rece...