Redundancy in the central tachykinin systems safeguards puberty onset and fertility

The tachykinin neurokinin B (NKB, Tac2) is critical for GnRH release. NKB signaling deficiency leads to infertility in humans. However, some patients reverse this hypogonadism resembling the fertile phenotype of Tac2KO and Tacr3KO (encoding NKB receptor, NK3R) mice despite the absence of NKB signaling. Here, we demonstrate that in the absence of NKB signaling, other tachykinins (substance P and neurokinin A [NKA], encoded by Tac1) may take over to preserve fertility. The complete absence of tachykinins in Tac1/Tac2KO mice leads to delayed puberty onset in both sexes and infertility in 80% of females (but not males), in contrast to the 100% fertile phenotype of Tac1KO and Tac2KO mice separately. Furthermore, we demonstrate that NKA controls puberty onset and LH release through NKB-independent mechanisms in the presence of sex steroids and NKB-dependent mechanisms in their absence. In summary, tachykinins interact in a coordinated manner to ensure reproductive success in female mice.


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TACs act on different G protein-coupled receptors: NK1R (encoded by Tacr1), the receptor for 5 SP; NK2R (Tacr2), the receptor for NKA; and NK3R (Tacr3), the receptor of NKB. These TAC 6 systems are expressed throughout the central nervous system, where they participate in a variety 7 of physiological functions, e.g. nociception and fear conditioning (1,2).

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Recently, the NKB/NK3R system has emerged as a critical neuroendocrine regulator of 9 reproductive function. A growing body of evidence from our lab and others has documented the 10 stimulatory role of NKB on GnRH release in an estradiol and kisspeptin dependent manner in all 11 studied species, including the human (3).

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Moreover, inactivating mutations in TAC3/TACR3 genes lead to hypogonadotropic hypogonadism 13 (HH) in patients, presenting delayed or absent puberty onset and infertility (4, 5). However, a 14 number of human patients bearing these mutations overcome initial pubertal failure and central 15 hypogonadism, with a later 'awakening' of GnRH secretion and hypogonadism reversal (6); a 16 phenotype that resembles that of Tac2KO and Tacr3KO mice, which are sub-fertile (7,8). The 17 underlying mechanism responsible for the reversal phenotype of these patients remains unknown.

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In addition to NKB, the SP/NK1R system also participates in the central regulation of the 19 gonadotropic axis, likely via kisspeptin-dependent mechanisms (9, 10). Supporting this 20 contention, (a) studies in humans, rabbits and rats showed a central stimulatory role of SP on LH

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Interestingly, because Tac1 encodes both SP and NKA we cannot rule out that the reproductive 27 phenotype observed in Tac1KO mice (delayed puberty onset and sub-fertility) (16, 17) is not, at 28 least in part, due to the absence of NKA signaling, whose participation in the control of puberty 29 onset and fertility remains largely unknown. We and others have documented that NKA also 30 induces LH release in rodents (10,18,19) in a kisspeptin-dependent manner (10). Interestingly, 31 the stimulatory action of NKA on LH release is dependent on the presence of physiological levels 32 of circulating sex steroids, while in their absence, NKA inhibits LH release, similar to NKB (10).

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However, unlike NKB, NKA's receptor (NK2R) is not present on Kiss1 or GnRH neurons (10). We 34 therefore hypothesize that NKA must act upstream of Kiss1 neurons, on an unknown population

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In order to test our hypothesis, that there is redundancy in the tachykinin systems that preserves 48 fertility in the absence of NKA/SP signaling (Tac1KO mice) or NKB signaling (Tac2KO and 49 Tacr3KO mice) individually (7,8,16), we generated double Tac1/Tac2 KO mice and compared 50 their reproductive maturation and fertility to their single mutant littermates. Interestingly, while 51 Tac1KO and Tac2KO mice displayed delayed VO, as reported previously, double Tac1/Tac2 KO 52 females presented only a moderate (not significant) delay in VO compared to WT littermates 53 (Figure 1 A-C), however, they failed to show any signs of first estrous for over 30 days post VO 54 (Figure 1 D). Of note, first estrous is a more accurate marker of puberty onset as it indicates 55 central activation of the gonadotropic axis. Furthermore, in a fertility test in which adult females were mated with proven fertile WT males, only 20% of Tac1/Tac2 KO females were able to deliver 57 pups over a 90-day long period of mating (Figure 1 E). Moreover, the parturition latency of these 58 small portion of Tac1/Tac2 KO females was longer than in WT controls (WT: 21.66 ± 0.66 days; 59 Tac1/Tac2 KO: 29.5 ± 0.5 days; **p<0.01 ) (Figure 1 F) and the litter size was significantly smaller 60 than in controls (WT: 7.33 ± 1.08 pups; Tac1/Tac2 KO: 4 ± 0 pups; **p<0.01) (Figure 1 G). This 61 largely infertile phenotype of Tac1/Tac2 KO females is also supported by the reduced number of 62 corpora lutea in their ovaries (WT: 3.5 ± 0.28 CL/ovary; Tac1/Tac2 KO: 0.75 ± 0.25 CL/ovary; 63 ***p<0.001) (Figure 1 H, I)   A-C). Interestingly, when the response of LH to OVX was assessed, we observed a biphasic 76 response in which LH levels were reduced compared to WT in Tac1/Tac2 KO and Tac2KO 77 females 2 days post OVX; however, there was a quick rebound that led to normal LH levels in 78 both KO models compared to WT females at 7 days post OVX (Figure 2 D). Next, we aimed to 79 assess the ability of Tac1KO, Tac2KO and Tac1/Tac2 KO females to respond to the central 80 administration of kisspeptin (Kp-10) and GnRH. As we previously reported, the lack of Tac1 led 81 to a diminished LH response to Kp-10 (17), which was replicated in Tac1/Tac2 KO females and extended to the GnRH response (Figure 2 E, F), while the expression of the tachykinin receptors 83 (Tacr1, Tacr2 and Tacr3), Kiss1 and Pdyn remained unaffected (Figure 2 G, H)

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Similar to the delay in first estrus in female Tac1/Tac2 KO mice, males displayed delayed puberty 88 onset as shown by the timing of preputial separation (Figure 3 A-C). However, and unlike 89 females, Tac1/Tac2 KO males were fertile, with a 100% success ratio in their ability to impregnate 90 WT females (Figure 3 D-F)

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We previously reported that NKA and NKB action on LH release is largely equivalent, i.e. both 123 increase LH release in the presence of physiological circulating levels of E2 but inhibit LH in the 124 absence of sex steroids (10). It was therefore tentative to speculate that NKA could induce LH 125 release through the stimulation of NKB given the absence of NK2R in Kiss1 and GnRH neurons.

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To test this hypothesis, first we co-administered NK2R and NK3R agonists to OVX + E2 WT mice 127 and observed that the increase in LH was similar in groups injected with an individual dose of 128 each agonist or the combination of both (Figure 6 A), discarding the possibility of an additive 129 effect of NKA and NKB action on LH release and suggesting a possible common pathway. Next, 130 to evaluate if NKA requires NKB signaling to induce LH release, the LH response to NK2R-Ag 131 was tested in the presence of an NK3R antagonist (Figure 6 B) and in NKB deficient (Tac2KO) 132 mice (Figure 6 C). In both cases, NK2R-Ag was able to significantly stimulate LH release 133 indicating that NK2R activation induces LH release independently of the presence of NKB or its 134 receptor NK3R. However, NK2R signaling requires the presence of kisspeptin as Kiss1KO mice 135 replaced with E2 did not display any effect on LH release (WT Basal: 0.27 ± 0.06 ng/mL, WT NK2R-Ag: 0.57 ± 0.09 ng/mL; *p < 0.05; Kiss1KO Basal: 0.21 ± 0.02 ng/mL, Kiss1KO NK2R-Ag:

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In the next set of experiments, we sought to determine whether the inhibitory action of NKA/NK2R 141 on LH release in the absence of sex steroids (i.e. OVX) is mediated by NKB and dynorphin. First,

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we showed that the inhibitory action of NK2R-Ag + senktide was similar to that of senktide alone, inhibitory signal of dynorphin leads to a significant decrease in the ability of the mouse to secrete 154 LH, probably due to disruption of the LH pulse generator mechanism (28). Next, we assessed the 155 action of NK2R-Ag in the absence of NKB (Tac2KO mice) to further confirm the data obtained 156 after NK3R blockade. Unexpectedly, we observed that the absence of NKB leads to a robust 157 induction of LH release, uncovering an action that is not present in WT OVX regardless of whether 158 a functional NK3R is present or antagonized.

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Because we have observed that VMH Tac1 neurons co-express Tacr2 (NK2R) (Figure 4), we 161 hypothesized that NKA could also induce LH release through the stimulation of SP from Tac1 162 neurons that, in turn, would activate Kiss1 neurons (10). To test this hypothesis, we administered the NK2R-Ag in the presence of a NK1R antagonist (proven to efficiently block the action of a

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In the present study, we show that all tachykinins are essential for the normal timing of puberty 173 onset in both sexes and critical for the maintenance of fertility in females. Humans and mice with 174 deficient NKB/NK3R signaling present delayed puberty onset (4, 5, 7, 8). We have also described 175 previously that SP is critical for the proper timing of puberty onset in the mouse (16, 17) and here 176 we extend that finding to include NKA as well.

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The reproductive phenotype of TAC3/TACR3 deficient patients (without NKB/NK3R signaling) 179 has spurred controversy as their infertile phenotype has been shown to reverse spontaneously in 180 some patients leading to successful pregnancies (6). This phenotype resembled the fertile 181 phenotype of both Tac2 and Tacr3 KO mice (7,8). The current model of NKB's action proposes 182 that NKB plays a role before puberty by stimulating the release of kisspeptin (29) and that after 183 puberty it participates in the shaping of kisspeptin-GnRH-LH pulses as part of the GnRH pulse 184 generator within the arcuate/infundibular nucleus (30). Therefore, the explanation for the reversal 185 of the HH phenotype in patients and mice without this seemingly critical factor for GnRH pulsatility 186 remained unknown and demanded further investigation. Because tachykinins have been 187 described to cross-activate all three NK receptors (31, 32), we hypothesized that in the absence 188 of NKB signaling, SP and/or NKA could compensate for the role of NKB and eventually be able to activate the gonadotropic axis. Our present results support this hypothesis, as the complete

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In this study we also addressed the question of whether the analogous action of NKA and NKB 230 in the regulation of LH release (i.e. stimulation in the presence of E2 and inhibition in its absence 231 (10)) is due to a converging mechanism of action of NKA onto NKB signaling. Despite these 232 similarities, our data using NK3R antagonists and Tac2KO mice after OVX and E2 replacement, 233 clearly demonstrate that the stimulatory action of NKA on LH is NKB independent but kisspeptin-234 dependent, suggesting the existence of a yet unknown population of NKA-responsive neurons 235 that in turn activate Kiss1 neurons to induce kisspeptin/GnRH release. On the contrary, NKA had 236 been shown to inhibit LH via dynorphin in the rat (19, 26), in a similar mechanism to that described 237 for NKB in the absence of E2 (38). Here, we further demonstrate that this inhibitory action of NKA 238 is mediated by the activation of the NKB-dynorphin pathway because the blockade of both NK3R 239 and KOR receptors ablates the inhibitory action of NKA. However, we unexpectedly observed 240 that in the absence of NKB, NKA no longer inhibits LH in the absence of E2 but rather significantly 241 stimulates it (our present data in Tac2KO mice) in a process that is also SP-independent. This 242 suggests that when NKB is present, its inhibitory action (after being induced by NKA) is downstream of any stimulation induced by NKA which, as documented by our ISH data, would 244 act upstream of Kiss1 neurons. However, in the absence of NKB (and in the presence of a 245 functional NK3R, i.e. in Tac2KO mice), NKA is able to further stimulate LH release through a 246 mechanism that requires the activation of NK3R (because it is absent when this receptor is 247 blocked). This demonstrates that NKA's action on LH release is inherently stimulatory and 248 requires a functional NK3R, but not NKB.

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The testes' ultra-structure was analyzed in adult (3-4 month old) mice of the two genotypes: WT 330 and Tac1/Tac2 KO (n=4). Testes were collected and processed as described above for ovaries.

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Senktinde or vehicle (0.9% NaCl). Blood samples were collected before SB222200 injection (Basal) and at 25 and 60 minutes after injection. Additionally, we further analyzed the action of NK2R-Ag in the absence of NKB signaling using Tac2 KO OVX+E2 females (n≥5 per group) that 406 were injected with NK2R-Ag (600 pmol) and blood samples were collected before and 25 and 60 407 min after injection. Finally, in order to evaluate the whether the action of NKA requires kisspeptin 408 to stimulate LH release, we used Kiss1 KO OVX+E2 females (n≥5 per group) and LH levels were 409 measured 25 min after icv injection of NK2R-Ag (600 pmol).

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Statistical Analysis of LH pulses: Mice LH concentration time series were analyzed using a 476 MATLAB-bases algorithm. It is a for loop written in the code to determine which LH peaks are 477 considered pulses. This for loop states that any peak whose height is 20% greater than the heights 478 of the 2 previous peaks as well as 10% greater than the height of the following peak is considered 479 a pulse. There is also a condition written into the code that is specific for the second time interval 480 (i=2) that states that the peak at the second-time interval only needs to be 20% greater than the 481 single peak that comes before it to be considered a pulse.        WT + NK2R-Ag Tac2