Final Report Summary - THYROREPRO (Deciphering the role of thyroid hormones in seasonal reproduction)
Overview & Socio-economic context
Most mammals exhibit annual rhythms in their physiology, which allow them to cope with seasonal variations of their environment such as food availability and harsh weather. One of the most dramatic impact is seen at the level of the reproductive axis, which is turned on and off throughout the course of a year to time birth / lactation when environmental conditions are most favourable. This occurs not only in wild animals but also in domestic animals such as goats and sheep. For domestic animals, seasonal breeding is problematic as it leads to uneven production of dairy products and meat during the year. This in turn impacts farmer’s income and prices on the market.
To counteract this "undesirable feature" and master breeding of domestic animals, several strategies have been developed over the last decades. The most common relies on exogenous hormones such as PMSG (equine chorionic gonadotropin from mare’s blood), Prostaglandins and steroids such as Progesterone. These methods are efficient and allow synchronization of ovulation, hence grouped births, and out-of-season breeding. However, these hormonal treatments come at a high cost for the environment and society, best exemplified by the deleterious impacts of so-called endocrine disruptors. These methods are therefore not sustainable and will have to be replaced by “greener” ones. At the EU level, a call for the development of alternative strategies has been made. However, the development of novel methods requires a better understanding of the central - within the brain - mechanisms which govern seasonal breeding.
Aims of the Project
The overall aim of the ThyroRepro research program was therefore to further our understanding of season perception by the ovine brain. Since daylength is the main driver of seasonal reproduction (metabolic status, social interactions and stress being secondary modulators) we developed approaches to clarify the impact of photoperiod on the medio-basal hypothalamus (MBH), which is the main brain area in decoding and processing photoperiodic cues. More precisely, our aims were to (i) identify in a systematic manner the molecular components within the MBH, which govern the seasonal swings of reproduction in sheep and (ii) better define daylengths which turn on and off the reproductive axis. It was expected that the findings would increase our understanding of the basic biology of time-measurement systems and serve as a starting point for the the development of novel strategies to improve breeding schemes.
Scientific background and Results
Most mammals living at temperate latitudes use the duration of daylength - photoperiod - as the proximal cue to time reproduction. Within the brain, changes in photoperiod translate into changes in the pattern of secretion of a hormone, melatonin, which then acts on specific target sites to trigger the appropriate endocrine changes. Amongst these targets is the MBH. Within the MBH, melatonin acts to yield a local increase in the production of the active thyroid hormone triiodothyronine (T3) during long days. As described initially in birds during the 1940s, this local increase in T3 is crucial to the proper timing of reproduction. While part of the mechanisms linking melatonin to the production of T3 have been worked out over the last decade, the molecular targets and cellular mechanisms through which T3 ultimately impinges on reproduction remain unclear. All experiments were performed in ewes of the Ile-de-France breed and blood sampling was routinely performed twice a week to follow the course of the gonadotropins LH/FSH, which inform us of the reproductive state.
- In a first part, a strategy was devised to characterize the molecular changes which occur within the MBH over the course of the year. Three groups of ewes were culled in May, August and November. We used an unbiased RNAseq approach, which was completed by qRT-PCR and in situ hybridization in other groups of ewes. Taken together, these approaches led to the identification of ~1000 genes which show significant differences in expression from May through to November. The implication of most of these candidates in seasonality was previously unknown. Alternatively, known candidates were also identified, which validates our approach. Our work allowed us to precisely locate, within the MBH, the site(s) of expression of >20 candidate genes. Taking into account what is known about the function of these genes in mouse and human it appears that tissue plasticity, remodeling of the extracellular matrix, epigenetics and cell division are key features of the seasonal program. This solidifies and considerably extends recent findings. This provides us with a shortlist of key genes involved in photoperiod decoding and points to a few select processes as pivotal to the normal unwinding of the seasonal program.
- In a second part we aimed at the identification of the subset of the above-mentioned genes, which relay photoperiod information through T3. To achieve this, we set-up a surgical procedure of thyroidectomy and performed several experiments, in a manner roughly similar to the one described above. This methodology allowed the identification of a small number of genes (5<) which are no longer induced by long daylengths when the thyroid gland - the source of thyroid hormones - is removed. We conclude that these genes are pivotal relays of the photoperiodic information towards the gonadal axis and may represent interesting targets for interefering with seasonal breeding.
- The third part of the project aimed at a better delineation of the limits of entrainment of the seasonal clock in ewes. The entrainment is the process by which light information synchronizes an inner seasonal clock, known as the circannual clock. It is known that long daylengths are much more efficient than short daylengths at entraining the circannual clock. But what exactly is a long daylength? An experiment was therefore performed in winter, when days are at their shortest durations, in which groups of ewes were exposed to daylengths of longer duration in a stepwise manner (i.e. from ~8h of light to 11h, 12h, 13h, 14h). Daylengths interpreted as “long” should turn off the gonadal axis. This procedure is used in breeding schemes, mostly in goats, to advance the breeding cycle of the upcoming year, cycle which then starts ~3 months earlier. Our data show that even daylengths as short as 11h can moderately impact the gonadal axis; daylengths of 13-14h are nevertheless more efficient. We also established molecular correlates of the physiological status in the different groups, looking at the expression of our candidate genes in the MBh of these ewes. Similar experiments were carried out in parallel in rams (in Scotland) and goats (France), under other grant agreements. Taken together, these experiments significantly advance our understanding of important basic features of the photoperiodic read-out system.
Conclusion & Perspectives
The ThyroRepro project provides a much improved picture of the complex molecular mechanisms underlying photoperiodic time-measurement and seasonal breeding. The systematic identification of the actors involved in the MBH processing of information paves the way for future studies, which will clarify the roles of these actors and how they interact. Ultimately, it is expected that these basic findings will lead to the development of novel methods for the control of breeding.
Most mammals exhibit annual rhythms in their physiology, which allow them to cope with seasonal variations of their environment such as food availability and harsh weather. One of the most dramatic impact is seen at the level of the reproductive axis, which is turned on and off throughout the course of a year to time birth / lactation when environmental conditions are most favourable. This occurs not only in wild animals but also in domestic animals such as goats and sheep. For domestic animals, seasonal breeding is problematic as it leads to uneven production of dairy products and meat during the year. This in turn impacts farmer’s income and prices on the market.
To counteract this "undesirable feature" and master breeding of domestic animals, several strategies have been developed over the last decades. The most common relies on exogenous hormones such as PMSG (equine chorionic gonadotropin from mare’s blood), Prostaglandins and steroids such as Progesterone. These methods are efficient and allow synchronization of ovulation, hence grouped births, and out-of-season breeding. However, these hormonal treatments come at a high cost for the environment and society, best exemplified by the deleterious impacts of so-called endocrine disruptors. These methods are therefore not sustainable and will have to be replaced by “greener” ones. At the EU level, a call for the development of alternative strategies has been made. However, the development of novel methods requires a better understanding of the central - within the brain - mechanisms which govern seasonal breeding.
Aims of the Project
The overall aim of the ThyroRepro research program was therefore to further our understanding of season perception by the ovine brain. Since daylength is the main driver of seasonal reproduction (metabolic status, social interactions and stress being secondary modulators) we developed approaches to clarify the impact of photoperiod on the medio-basal hypothalamus (MBH), which is the main brain area in decoding and processing photoperiodic cues. More precisely, our aims were to (i) identify in a systematic manner the molecular components within the MBH, which govern the seasonal swings of reproduction in sheep and (ii) better define daylengths which turn on and off the reproductive axis. It was expected that the findings would increase our understanding of the basic biology of time-measurement systems and serve as a starting point for the the development of novel strategies to improve breeding schemes.
Scientific background and Results
Most mammals living at temperate latitudes use the duration of daylength - photoperiod - as the proximal cue to time reproduction. Within the brain, changes in photoperiod translate into changes in the pattern of secretion of a hormone, melatonin, which then acts on specific target sites to trigger the appropriate endocrine changes. Amongst these targets is the MBH. Within the MBH, melatonin acts to yield a local increase in the production of the active thyroid hormone triiodothyronine (T3) during long days. As described initially in birds during the 1940s, this local increase in T3 is crucial to the proper timing of reproduction. While part of the mechanisms linking melatonin to the production of T3 have been worked out over the last decade, the molecular targets and cellular mechanisms through which T3 ultimately impinges on reproduction remain unclear. All experiments were performed in ewes of the Ile-de-France breed and blood sampling was routinely performed twice a week to follow the course of the gonadotropins LH/FSH, which inform us of the reproductive state.
- In a first part, a strategy was devised to characterize the molecular changes which occur within the MBH over the course of the year. Three groups of ewes were culled in May, August and November. We used an unbiased RNAseq approach, which was completed by qRT-PCR and in situ hybridization in other groups of ewes. Taken together, these approaches led to the identification of ~1000 genes which show significant differences in expression from May through to November. The implication of most of these candidates in seasonality was previously unknown. Alternatively, known candidates were also identified, which validates our approach. Our work allowed us to precisely locate, within the MBH, the site(s) of expression of >20 candidate genes. Taking into account what is known about the function of these genes in mouse and human it appears that tissue plasticity, remodeling of the extracellular matrix, epigenetics and cell division are key features of the seasonal program. This solidifies and considerably extends recent findings. This provides us with a shortlist of key genes involved in photoperiod decoding and points to a few select processes as pivotal to the normal unwinding of the seasonal program.
- In a second part we aimed at the identification of the subset of the above-mentioned genes, which relay photoperiod information through T3. To achieve this, we set-up a surgical procedure of thyroidectomy and performed several experiments, in a manner roughly similar to the one described above. This methodology allowed the identification of a small number of genes (5<) which are no longer induced by long daylengths when the thyroid gland - the source of thyroid hormones - is removed. We conclude that these genes are pivotal relays of the photoperiodic information towards the gonadal axis and may represent interesting targets for interefering with seasonal breeding.
- The third part of the project aimed at a better delineation of the limits of entrainment of the seasonal clock in ewes. The entrainment is the process by which light information synchronizes an inner seasonal clock, known as the circannual clock. It is known that long daylengths are much more efficient than short daylengths at entraining the circannual clock. But what exactly is a long daylength? An experiment was therefore performed in winter, when days are at their shortest durations, in which groups of ewes were exposed to daylengths of longer duration in a stepwise manner (i.e. from ~8h of light to 11h, 12h, 13h, 14h). Daylengths interpreted as “long” should turn off the gonadal axis. This procedure is used in breeding schemes, mostly in goats, to advance the breeding cycle of the upcoming year, cycle which then starts ~3 months earlier. Our data show that even daylengths as short as 11h can moderately impact the gonadal axis; daylengths of 13-14h are nevertheless more efficient. We also established molecular correlates of the physiological status in the different groups, looking at the expression of our candidate genes in the MBh of these ewes. Similar experiments were carried out in parallel in rams (in Scotland) and goats (France), under other grant agreements. Taken together, these experiments significantly advance our understanding of important basic features of the photoperiodic read-out system.
Conclusion & Perspectives
The ThyroRepro project provides a much improved picture of the complex molecular mechanisms underlying photoperiodic time-measurement and seasonal breeding. The systematic identification of the actors involved in the MBH processing of information paves the way for future studies, which will clarify the roles of these actors and how they interact. Ultimately, it is expected that these basic findings will lead to the development of novel methods for the control of breeding.