Prolactin-mediates a lactation-induced suppression of arcuate kisspeptin neuronal activity necessary for lactational infertility in mice

Reviewer #1 (Public Review):

Summary:

In this paper, Hackwell and colleagues performed technically impressive, long-term, GCaMP fiber photometry recordings from Kiss1 neurons in the arcuate nucleus of mice during multiple reproductive states. The data show an immediate suppression of activity of arc Kiss1 neuronal activity during pregnancy that is maintained during lactation. In the absence of any apparent change in suckling stimulus or milk production, mice lacking prolactin receptors in arcuate Kiss1 neurons regained Kiss1 episodic activity and estrous cyclicity faster than control mice, demonstrating that direct prolactin action on Kiss1 neurons is at least partially responsible for suppressing fertility in this species. The effect of loss of prolactin receptors from CamK2a expressing neurons was even greater, indicating either that prolactin sensitivity in Kiss1 neurons of the RP3V contributes to lactational infertility or that other prolactin-sensitive neurons are involved. These data demonstrate the important role of prolactin in suppressing Kiss1 neuron activity and thereby fertility during the lactational period in the mouse.

Strengths:

This is the first study to monitor the activity of the GnRH pulse-generating system across different reproductive states in the same animal. Another strength in the study design is that it isolated the effects of prolactin by maintaining normal lactation and suckling (assessed indirectly using pup growth curves). The study also offers insight into the phenomenon of postpartum ovulation in mice. The results showed a brief reactivation of arcuate Kiss1 activity immediately prior to parturition, attributed to falling progesterone levels at the end of pregnancy. This hypothesis will be of interest to the field and is likely to inspire testing in future studies. With the exceptions mentioned below, the conclusions of the paper are well supported by the data, and the aims of the study were achieved. This paper is likely to raise the standard for technical expectations in the field and spark new interest in the direct impact of prolactin on Kiss1 neurons during lactation in other species.

Weaknesses:

A weakness in the approach is the use of genetic models that do not offer complete deletion of the prolactin receptor from targeted neuronal populations. A substantial proportion of Kiss1 neurons in both models retain the receptor. As a result, it is not clear whether the partial maintenance of cyclicity during lactation in the genetic models is due to incomplete deletion or to the involvement of other factors. This weakness should be more fully discussed in the text. In addition, results showing no impact of progesterone on LH secretion during lactation are surprising, given the effectiveness of progesterone-containing birth control in lactating women. The progesterone-related experiments were not well justified or discussed in the text. While the authors assert their findings may reflect an important role for prolactin in lactational infertility in other mammalian species, that remains to be seen. Hyperprolactinemia is known to suppress GnRH release, but its importance in the suppression of cyclicity during lactation is controversial. Indeed, in several species, the stimulus of suckling is considered to be the main driver of lactational fertility suppression. Data from rats shows that exogenous prolactin was unable to suppress LH release in dams deprived of their pups shortly after birth; both suckling and prolactin were necessary to suppress a post ovariectomy rise in LH levels. The duration of amenorrhea does not correlate with average prolactin levels in humans, and suckling but not prolactin was required to suppress the postpartum rise in LH in the rhesus monkey. The authors should discuss more thoroughly whether the protocol of this or other studies might result in discordant results and whether mice are likely to be an outlier in their mechanism of cycle suppression.

Reviewer #2 (Public Review):

Summary:

The overall goal of Eleni et al. is to determine if the suppression of LH pulses during lactation is mediated by prolactin signaling at kisspeptin neurons. To address this, the authors used GCaMP fiber photometry and serial blood sampling to reveal that in vivo episodic arcuate kisspeptin neuron activity and LH pulses are suppressed throughout pregnancy and lactation. The authors further utilized knockout models to demonstrate that the loss of prolactin receptor signaling at kisspeptin cells prevents the suppression of kisspeptin function and results in early reestablishment of fertility during lactation. The work demonstrates exemplary design and technique, and the outcomes of these experiments are sophistically discussed.

Strengths:

This manuscript demonstrates exemplary skill with powerful techniques and reveals a key role for arcuate kisspeptin neurons in maintaining lactation-induced infertility in mice. In a difficult feat, the authors used fiber photometry to map the activity of arcuate kisspeptin cells into lactation and weaning without disrupting parturition, lactation, or maternal behavior. The authors used a knockout approach to identify if prolactin inhibition of fertility is mediated by direct signaling at arcuate kisspeptin cells. Although the model does not perfectly eliminate prolactin receptor expression in all kisspeptin neurons, results from the achieved knockdown support the conclusion that prolactin signaling at kisspeptin neurons is required to maintain lactational infertility. The methods were advanced and appropriate for the aims, the studies were rigorously conducted, and the conclusions were thoughtfully discussed. Overall, the aims of this study were achieved.

Reviewer #3 (Public Review):

Summary:

Grattan and colleagues were trying to establish the neural mechanism underlying lactational infertility, in particular trying to establish the relative importance of the neurogenic effects of the suckling stimulus versus prolactin per se. They have shown that in the mouse it is rather prolactin and more specifically its action on the hypothalamic arcuate kisspeptin neuronal system, which is the key neural construct underlying gonadotrophin-releasing hormone (GnRH) pulse generation and central to the neuroendocrine control of reproduction, that mediates lactational infertility. The authors have taken a measured tone to emphasise the data pertaining to the mouse without extravagant extrapolation to humans. Nevertheless, the key findings provide a substantial foundation to facilitate interpretation of studies in other species.

Strengths:

The major strength of this study is the use of a combination of cutting-edge technologies, which of course underlie the majority of scientific advances rather than intellectual prowess favoured by the majority of scientists. Their approach avoided the major confounding effects of using pharmacological strategies to suppress prolactin action that has complicated the vast majority of previous studies. The study also provides an elegant and comprehensive contiguous description of GnRH pulse generator frequency across the ovarian cycle, through pregnancy and lactation, and into weaning in individual animals.

Weaknesses:
There are no significant weaknesses.

Author Response

Reviewer #1 (Public Review):

Response to reviewer 1 comments on “weaknesses”:

“A weakness in the approach is the use of genetic models that do not offer complete deletion of the prolactin receptor from targeted neuronal populations...”

We acknowledge that neither model used provided a complete deletion of the prolactin receptor (Prlr) from the targeted neuronal populations. We suspect that incomplete deletion of targeted genes is not uncommon in these sort of studies, but this remains the best approach to addressing our question, and we believe we have been thorough and transparent in reporting the degree of deletion observed. We thought we had appropriately discussed the implications of the low proportion of Kiss1 cells still expressing Prlr, but will certainly revisit to ensure it is discussed thoroughly. This does not detract, however, from the key conclusion that prolactin action is necessary for full suppression of fertility in lactation in the mouse.

“Results showing no impact of progesterone on LH secretion during lactation are surprising, given the effectiveness of progesterone-containing birth control in lactating women...”

We think that this comment misrepresents what has been done in our study. We did not report a lack of impact of progesterone, as exogenous progesterone was never administered to mice. We did, however, give mifepristone as a progesterone receptor antagonist to determine whether endogenous progesterone contributed to the suppression of kisspeptin neuronal activity. We found that mifepristone, at levels sufficient to terminate pregnancy, had no effect on pulsatile LH secretion in lactating mice. This is consistent with our prior observation that progesterone levels are low in mouse lactation, suggesting that progesterone does not contribute significantly to the suppression of kisspeptin neuronal activity during lactation in the mouse. We agree with the reviewer that if we had given exogenous progesterone, it likely would result in suppression of pulsatile LH secretion (as it does in women). Indeed, in other work, we have found that progesterone administration profoundly suppresses activity of the kisspeptin neurons in mice (https://doi.org/10.1210/en.2019-00193). But this was not the point of the present experiment. We will review how we have described this experiment to ensure that this is absolutely clear.

“While the authors assert their findings may reflect an important role for prolactin in lactational infertility in other mammalian species, that remains to be seen….”

We acknowledge that our study cannot address whether prolactin is necessary for the suppression of lactation in other mammalian species. We hope our data may stimulate a re-examination of this question in other species, however, as some of the prior methodology (such as using pharmacological suppression of prolactin) may have had off target effects that confound interpretation. We thought that this point was discussed appropriately in the manuscript but we will certainly check and make sure this is addressed suitably.