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Parrish Impact of Heat on Spermatogenesis

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Parrish Impact of Heat on Spermatogenesis Impact of Heat on Spermatogenesis L Enwall, J Schindler, K Gibbs, K Yagoda and J Parrish Come on in Bucky, its great to talk with you about your data. Dr Willenburg shared this with me and it reminds me of some of the research that we do and I might be able to shed some light on what’s going on in these boars. At least help you think about the physiology that is happening So the research we do is on the impact of heat on spermatogenesis which is including a number of my graduate students in these research projects So lets go back and think about the seminiferous tubule where spermatogenesis occurs and on the upper right you can see that the basal membrane bound the outside of each seminiferous tubule. You can then see that there is a basal compartment and then an adluminal compartment as you move closer to the center with finally a lumen present. Now as these germ cells develop, they start as spermatogonia in the basal compartment and go through various changes until they are released into the lumen as a spermatozoa. We can look again at this as a diagram or an animation that one of my students made for me. Here we have the A spermatogonia (A gonia) in the basal compartment, they go through the process of mitosis, dividing to form B spermatogonia (B gonia) and then eventually a primary spermatocyte (1 cyte). All of these cells originate because of mitosis. The primary spermatocyte then moves toward the adluminal compartment, which includes going through the tight junctions that are present between adjacent Sertoli cells and undergoes meiosis once it reaches the adluminal compartment. Here it goes through the different stages of the first division of meiosis to become a secondary spermatocyte (2 cyte). It then divides to form a round spermatid (RS) and now the round spermatid undergoes a process of morphological changes referred to as spermiogenesis in which the nucleus of that round spermatid begins to elongate and eventually as we get development of the tail and other features of the sperm that elongated spermatid which is now a spermatocyte is eventually lost through the process of spermiation into the lumen. Now I’ve shown this sequence here so that I can point out the days it takes for these cells to go through this and eventually be ejaculated. So if we start with the A spermatogonia at the top, once it enters into that process it takes about 45 days, as indicated next to it, before that cell will eventually be ejaculated as a spermatozoa. B spermatogonia is 41 days. The primary spermatocyte, which is going through preleptotene, leptotene, zygotene, pachotene and diplotene [meiosis], has various lengths of time from when these particular cells end up being ejaculated. But notice that it stays for a long period of time as this primary spermatocyte. It then quickly divides to the secondary [spermatocyte] then to the round spermatid. Those first round spermatids take 24 days to complete their morphological changes to sperm and eventually show up in the ejaculate. And even when sperm are released in that process of spermiation it is still 11 days until ejaculation as these sperm must still go through the epididymis and complete maturation there before being ejaculated. Now you can also look at that same information in a diagram such as this where we have the stages of spermatogenesis indicated in the vertical columns and the cycles of spermatogenesis indicated by the horizontal rows. We have the same information with the same cell types, it is just a little bit more broken out here. Below each cell type we have the number of days until ejaculation or before these cells appear in the ejaculate. So this is essentially the same diagram as before, the information is laid out just a little bit differently. We’ve talked about this in the 434 lectures previously. Now our research typically uses scrotal insulation and by insulating the scrotum we can raise the temperature of the testes by about 2°C that’s a little over 3°F. It’s not very much but that slight rise can have a dramatic impact on the process of spermatogenesis. As you’ll see in a minute. So here’s some of the data the we’ve collected where scrotal insulation was applied at day 0 and then we’ve looked at the appearance of primary abnormalities in these ejaculates from these boars. We have [data from] insulated boars in red and we have [data from] control boars in blue at the bottom. And so control boars don’t really change over the course of the experiment in terms of the percentage of primary abnormalities. Now the values that you see here are all the mean ± SD between the boars in each group and the difference from day 0 for those boars. What we are really interested in is where do we have significant differences from the day 0 samples. Those are indicated by the asterisk above the data point for the insulated boars. It turns out that if you look at this carefully, and we draw in some vertical bars, we are actually interested in between days 21 to 37 after the heat insult imposed by scrotal insulation because this is where the significant differences in primary abnormalities occur. Now if we look at that diagram of spermatogenesis and I’ve tried to highlight out those days between day 21 and day 37 shown by the grayed out areas on the graph. These are the cell types impacted by that heat. Eventually they go on to produce those abnormal sperm with abnormal sperm heads (primary abnormalities). We can see here that it is mostly primary spermatocytes from the preleptotene on down to the diplotene stage, secondary spermatocytes and early round spermatids. All of these give rise to the abnormal sperm. Now heres a graph showing the effects of that scrotal insulation on the motility of sperm. Here the data is again the mean ± SEM, and the difference from day 0. We have [data from] insulated boars in red and we have [data from] control boars in blue. What we’ve done here, because we are normalizing to day 0, you can see that in the red line we have a decrease in the percentage of motile sperm that occurs over time. You can see that the control sperm don’t really change, they stay the same as they started out to be. So again if we look for where the significant differences from day 0 are, we can see on the graph, when we put in the bars, that it is between day 30 and day 35 post heat event or post scrotal insulation. We look on our diagram then, you can see that these are primary spermatocytes that range from the zygotene to the pachytene stage [of meiosis]. These are the ones that are impacted by that heat, eventually going on to develop some problems associated with motility of these sperm. Perhaps this is involving problems in centrioles, such that they don’t properly form or act normally when they get to a round spermatid or an elongated spermatid stage and the tails don’t form with the correct axoneme structures present in them. That is yet to be determined. But clearly that impact of that heat is here in a primary spermatocyte that then leads to a decrease in motility once these cells are ejaculated. So the impact of scrotal insulation from our studies are principally on primary spermatocytes and early round spermatid. However, that is not enough to account for some of your data. That explains why you would get increased numbers of abnormal sperm and decreased numbers of motile sperm, but you also looked at total sperm output. That [TSO] is not really accounted for here. In some other data that we have, we have actually quantified how many primary spermatocytes and early round spermatids are present following scrotal insulation. What we find is that the primary spermatocytes actually decrease in number indicating that apoptosis is going on. That can explain at least part of the decrease in total sperm output that occurs. You could use that same figure in terms of spermatogenesis and you could look back to when a potential environmental (heat) event occurred then you could check out what cell type was impacted. We see that it is again the primary spermatocytes. But that can’t explain everything. It’s still not enough to explain why you get a decrease in sperm output. Certainly apoptotic cells won’t be ejaculated. Remember that a lot of the cells are being ejaculated, they just have abnormal form either in terms of morphology or motility. We saw one other thing in our scrotal insulation experiments that is shown here. On the left hand side, there is a seminiferous tubule from a normal boar - a non-insulated boar and you see the seminiferous epithelium present and a large lumen with very little debris present in the lumen. Instead, when you look at the [seminiferous tubule] scrotal insulated boar on the right you see a large amount of debris in that lumen. If we try to identify the cells, they look like they are mostly primary spermatocytes and round spermatids. It’s a little difficult to tell exactly what everything is but those are probably what most of the cells are. Clearly, once they are sloughed off like this they are undergoing apoptosis, but still we can make out some recognizable cells.
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