Testosterone replacement therapy (TRT) is a critical part of gender affirming care for many transgender men (assigned female at birth). However, TRT, which produces circulating testosterone in the male physiological range, also results in irregular menstrual cycles, diminished reproductive hormone levels, and infertility. This mechanism for the reproductive inhibition by TRT is poorly understood. High doses of dihydrotestosterone, a non-aromatizable androgen, inhibit pulsatile secretion of luteinizing hormone (LH) in females of several animal models including mice; however, it is not well examined if male levels of testosterone, such as those resulting from TRT, will obstruct other aspects of female reproductive hormone secretion such as the preovulatory LH surge which triggers ovulation. To investigate this, we implanted adult female mice with male level testosterone, then ovariectomized them and treated with elevated estradiol known to induce an LH surge. All control mice, without testosterone treatment, showed an LH surge while the testosterone treated mice did not have an LH surge. This indicated that their reproductive axis was inhibited. We next examined the brains of control females or females treated with testosterone and determined that RP3V kisspeptin neurons, known to stimulate the LH surge, had reduced activation in the latter group. Our findings suggest that elevated, male level testosterone in TRT possibly disrupt fertility in females by blocking the LH surge, perhaps by inhibiting kisspeptin neurons. Further research will elucidate the mechanism by which testosterone inhibits the LH surge, which could lead to a therapeutic target to reverse testosterone’s inhibition of female fertility.

Kisspeptin, encoded by the Kiss1 gene, is required for reproductive function. There are several distinct Kiss1 populations in the brain, and how these kisspeptin neurons are regulated is still not fully understood. The two largest hypothalamic Kiss1 populations reside in the ARC and AVPV and respond to sex steroids in opposing manners, one being negatively regulated and the other being positively regulated, respectively. As a result, in research experiments it has been technically difficult to visualize Kiss1 expression of both populations in the same animal. Here, we sought to establish a dose of estradiol (E2) for which both ARC and AVPV kisspeptin populations can be visualized with in situ hybridization. Using this E2 treatment in conjunction with the inducible diphtheria toxin receptor mouse line, we next aimed to exploit the different developmental ontogenies of the Kiss1 populations to selectively remove only the ARC Kiss1 neurons, which have been implicated in the maintenance of the reproductive axis. We discovered that administration of diphtheria toxin (DT) during early juvenile stage to transgenic mice with selective sensitivity to DT led to specific ablation of ARC Kiss1 neurons. Finally, to address lesser understood Kiss1 populations outside the hypothalamus, we used two transgenic mouse models lacking either ERα or ERβ and found that E2 positively regulates Kiss1 cells in the lateral septum (LS) via ERα. Understanding the role of the ARC and LS Kiss1 neurons may lead to a better understanding of how the reproductive axis is regulated and provide potential therapeutics for reproductive disorders.