Abstract
Hypothalamic gonadotropin-releasing hormone (GnRH) neurons are required for fertility in all mammalian species studied to date. In rodents, GnRH neuron cell bodies reside in the rostral hypothalamus, and most extend a single long neuronal process in the caudal direction to terminate at the median eminence (ME), the site of hormone secretion. The molecular cues that GnRH neurites use to grow and navigate to the ME during development, however, remain poorly described. Reverse transcription-PCR (RT-PCR) identified mRNAs encoding Netrin-1, and its receptor, DCC, in the fetal preoptic area (POA) and mediobasal hypothalamus (MBH), respectively, from gestational day 12.5 (GD12.5), a time when the first GnRH neurites extend toward the MBH. Moreover, a subpopulation of GnRH neurons from GD14.5 through GD18.5 express the Netrin-1 receptor, DCC, suggesting a role for Netrin-1/DCC signaling in GnRH neurite growth and/or guidance. In support of this notion, when GD15.5 POA explants, containing GnRH neurons actively extending neurites, were grown in three-dimensional collagen gels and challenged with exogenous Netrin-1 (100 ng/ml or 400 ng/ml) GnRH neurite growth was stimulated. In addition, Netrin-1 provided from a fixed source was able to stimulate outgrowth, although it did not appear to chemoattract GnRH neurites. Finally, the effects of Netrin-1 on the outgrowth of GnRH neurites could be inhibited by blocking either L-type voltage-gated calcium channels (VGCCs) with nifedipine (10 µM), or ryanodine receptors with ryanodine (10 µM). This is consistent with the role of Ca2+ from extra- and intracellular sources in Netrin-1/DCC-dependent growth cone motility in other neurons. These results indicate that Netrin-1 directly stimulates the growth of a subpopulation of GnRH neurites that express DCC, provide further understanding of the mechanisms by which GnRH nerve terminals arrive at their site of hormone secretion, and identify an additional neuronal population whose neurites utilize Netrin-1/DCC signaling for their development.
Highlights
Hypothalamic gonadotropin-releasing hormone (GnRH)-secreting neurons are required for fertility in all mammals studied to date [1,2]
We found that: 1) the Netrin-1 receptor DCC was expressed in a subpopulation of GnRH neurons during the time of their process extension; 2) Netrin-1 was expressed in the preoptic area (POA) and median eminence (ME) across development; 3) Netrin-1 stimulated the growth of, but did not chemoattract, GnRH neurites from fetal POA; 4) The effects of Netrin-1 were attenuated in the presence of either the L-type calcium channel blocker nifedipine, or the intracellular Ca2+ store-release blocker ryanodine
Reverse transcription-PCR (RT-PCR) and immunohistochemistry were used to ascertain the expression patterns of Netrin-1 and DCC mRNAs and DCC protein, respectively, across the developmental window when GnRH neurons extend neurites to the ME. Both Netrin-1 and DCC mRNAs were present in the developing preoptic area (POA) and Netrin-1 was present in the mediobasal hypothalamus (MBH) from GD12.5 through GD18.5 (Fig. 1A)
Summary
Hypothalamic gonadotropin-releasing hormone (GnRH)-secreting neurons are required for fertility in all mammals studied to date [1,2]. GnRH neurons migrate from their site of generation into the brain, once inside the brain, they take a ventro-caudal path to their final site of residence in the rostral hypothalamus [8,9,10,11,12,13]. Much attention has been paid to understanding these early phases of GnRH neuron development, and a clear picture of the underlying molecular and cellular mechanisms is emerging (for reviews see [12,14,15,16,17]). By contrast with the wealth of data on GnRH neuron differentiation and migration, far less is known about the mechanisms underlying the targeting of GnRH neuronal processes to the ME. Several diffusible factors, including fibroblast growth factor 2 (FGF2) [18,19,20], brain-derived neurotrophic factor (BDNF) [21], the Prader-Willi gene candidate necdin [22], and the puberty-driving Kisspeptin peptide [23], have been implicated in GnRH process growth in vitro and/or in vivo
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