Estrogen receptor, calcium mobilization and rat sperm motility
Authors: J Choudhary
G Sethi Saberwal
H S Juneja
N Balasinor
M K Sharma
Journal: Molecular and Cellular Biochemistry (2002)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: tamoxifen citrate; male rat fertility; sperm motility; estrogen receptors; calcium
Affiliations: Institute for Research in Reproduction, Parel, Mumbai, India. 

Abstract:  Oral treatment with 0.4 mg/kg/day of tamoxifen citrate, an antiestrogen, has been reported to reduce the fertility of adult male rat, presumably through estrogen receptors expressed throughout the male reproductive tract. During the course of these studies, tamoxifen was observed to gradually alter the pattern of sperm motility in the cauda epididymides without reducing sperm counts. Studies were carried out to understand the mechanism involved in tamoxifen induced change in the sperm motility pattern. In order to study the direct effects of tamoxifen on motility, biochemical levels/activities of sperm calcium, cAMP, phosphodiesterase and dynein ATPase, normally implicated in sperm motility were studied In view of the fact that tamoxifen is a ligand of estrogen receptor, estrogen receptor alpha protein and transcript were localized on rat sperm membrane and the effect of tamoxifen studied. The present study demonstrated presence of estrogen receptor protein and mRNA in the rat sperm by immunofluorescence, western blotting and in situ hybridization respectively. Specificity of sperm estrogen receptors was confirmed by conventional binding studies using [3H]-estradiol. There was no effect of tamoxifen treatment on estrogen receptors in rat sperms. Biochemical analysis of the sperms from tamoxifen treated cauda epididymides revealed a significant increase in the levels of calcium and cAMP. A significant reduction was also apparent in the activity of dynein ATPase. Tamoxifen treatment did not alter phosphodiesterase activity. Estrogen receptors could be identified both in the control as well as tamoxifen treated rat sperms. It was concluded that tamoxifen treatment mobilized calcium from the intra- or extra-cellular pools with a concomitant increase in cAMP and presumably activation of PKA (protein kinase A). Tamoxifen altered the pattern of sperm motility through a calcium induced block in the activity of dynein ATPase, presumably through the activation of sperm phosphatase. The putative estrogen receptor mediated signal transduction pathway appears to be directly affected in the tamoxifen treated, sub-motile rat sperm. 

Retracted: No

PubPeer comments:
 Table 2 of this narrative review is incorrect in a number of ways. The table attempts to calculate a population attributable fraction (PAF) number of adverse pregnancy events due to caffeine intake in pregnant people. However, there are a number of errors that make the figures in the table wrong. This should be corrected as soon as possible. The first column of the table is titled:  Risk (%) per 100 mg* [intake of caffeine day] (95%u2009CI) . The second,  Risk (%) at 200 mgu2020 (column (i) u00d72) . Both of these are incorrectly attributed. The columns in fact contain the relative risk increase per 100mg of caffeine - i.e. a 1.14 RR or 14% INCREASED risk - and this number doubled. These figures come from Greenwood et al. While this study does not give a baseline risk of miscarriage for pregnant people who do not drink caffeine, it is unlikely in the extreme that this risk is 0%. It is also incorrect to misattribute this relative increase as an absolute figure, as the author has done in this table - given a plausible baseline rate of miscarriage from other literature of 16.1% in people who do not drink caffeine (Gaskins et al, 2018), the relative increase of 14% per 100mg day would translate into an absolute increase of 2.254%, giving a figure of 20.61% in the second column. The error is further compounded by the PAF calculation in the final column. This is reportedly the second column multiplied by the fourth column (the 4th column contains the estimated total number of events such as miscarriages). So, for example, 1,000,000 miscarriages multiplied by 28% gives 280,000 miscarriages attributable to caffeine. This is methodologically flawed and incorrect. The miscarriages are a subset of pregnancies, some of whom drank high amounts of caffeine, many of whom doubtless drank little or none at all. Multiplying in this way ignores this heterogeneity, assuming instead that the majority of people who miscarry drink 200mg of coffee. The final number is simply a reflection of this. To demonstrate how absurd the calculation is, if we were instead to look only at mothers drinking 800mg d of caffeine - who are at an 84% increased relative risk - we would find that they represent 112% of miscarriages in the U.S., a singularly absurd mathematical impossibility. Instead, we should use the commonly-accepted [PAF formula] to ascertain the number of adverse events associated with caffeine. This formula gives Pc(1-(1 RR)) where Pc = proportion of the population exposed and RR = relative risk increase. In this case, a reasonable fraction of the population drinking 200mg d or more caffeine during pregnancy as given by Gaskins et al is ~35%. Thus, the PAF for caffeine intake =200mg d = .35(1-(1 1.28)) = 7.7%, or 77,000 out of 1 million miscarriages. The number of 280,000 given in this table is clearly wrong, and should be corrected. Additionally, the table is incorrect in the assumption that the risk per 100mg of caffeine increases linearly, making column two doubly incorrect. In fact, depending on outcome, the Greenwood meta-analysis found a variety of relationships through their fitted cubic splines. Thus, to calculate the risk of 200mg day, it would be more appropriate to use the numbers given in this meta-analysis. , 
 https: twitter.com GidMK status 1298445444188520448?s=19 , 
 #1 This comment raises a number of questions regarding Table 2, and suggests reasonable grounds for debate about published figures and underlying assumptions. Importantly, as is shown below, those questions do not alter the bottom-line inference derived from current evidence that maternal caffeine consumption is harmful. Unfortunately, the author of the comment has chosen to ignore the fact that the contents of Table 2 represent an extrapolation of estimates reported by Greenwood et al. (2014), wherein the article explicitly acknowledges that the table contents are based on certain stated hypotheticals. In particular, the estimates assume linearity based on extensive published evidence of the relationship between maternal caffeine consumption and negative pregnancy outcomes, with no threshold of consumption below which negative outcomes are absent. Additionally, since there is nothing in the Greenwood et al. article to suggest otherwise, the table assumes zero baseline incidence for women who do not consume caffeine. Accordingly, the main point of Table 2 is to show that maternal caffeine consumption is associated with substantial negative pregnancy outcomes, directly contradicting the claim by Greenwood et al. (2014) that effects are u201cmodestu201d. Specifically, Table 2 proffers an estimate of 280,000 excess miscarriages among 1 million miscarriages, if all pregnant women consumed 200 mg caffeine per day (which the article acknowledges is strictly hypothetical). The author of the comment recalculated that figure, coming up with an estimate of 77,000. Perhaps that re-estimate is closer to the true number. So be it. In either case, the main point is confirmed. Namely, we can agree that the evidence indicates that the bottom line still stands at tens of thousands of excess miscarriages attributable to caffeine. In other words, as Table 2 seeks to demonstrate, maternal caffeine consumption appears to be responsible for substantial avoidable harm. That harm occurs absent of any benefit attributable to caffeine. Hence, the conclusion of the article remains unaltered. Pregnant women and women contemplating pregnancy should avoid caffeine. Finally, the author of the comment claims that it would be u201ca singularly absurd mathematical impossibilityu201d for the incidence of miscarriage to exceed the current level if all pregnant women consumed 800 mg caffeine per day. Not so, if all pregnant women consumed 800 mg of caffeine daily, the evidence is unanimous in indicating that the incidence of miscarriage would indeed be greater than where it presently stands. , 
 #3 This response appears to misunderstand the issue with the table. It is not about whether the numbers support one argument or another - the problem is that they are incorrect. It is wrong to claim that there is no risk of adverse events in pregnancy for those who do not drink caffeine, wrong to misrepresent the relative risk increase as an absolute figure, and the wrong method of determining PAF has been used. Whether or not the final product agrees with a specific viewpoint or not is immaterial: the mathematics and numbers extracted are wrong. As to the Greenwood et al study, I would direct the author to the summary nonlinear dose-response curves published in the study, using best fitting cubic splines and showing a decidedly non-linear relationship between caffeine intake and various outcomes. For many of these outcomes, the risk is actually likely higher than the relative risk represented in the table, as the difference between 0-100mg d is smaller than 100-200mg d. This would act to make the correctly calculated PAF larger, but again it is not about whether the final numbers support one argument over another it is whether they are correct. ,