It has been known that a preconception paternal alcoholism impacts adversely on the offspring but the mechanism of the effect is uncertain. Several findings suggest that there are signalling systems in testis that are analogous to those known to be altered by alcoholism in brain. We propose that chronic alcohol affects these systems in a manner similar to that in brain. Specifically, we hypothesise that excessive alcohol may disturb glutamatergic-like signalling in testis by increasing expression of the glutamate transporter GLAST (EAAT1). We discuss ways how to test the hypothesis as well as potential significance of some of the tests as tools in the diagnostics of chronic alcoholism

Balcar, Vladimir J.

Kashem, Mohammed Abul

Lee, Aven

Pow, David V.

Sery, Omar

Kashem, M

Lee, A

Pow, D

Sery, O

Balcar, V

RMIT Research Repository

Could ethanol-induced alterations in the expression of glutamate transporters in testes contribute to the effect of paternal drinking on the risk of abnormalities in the offspring?

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Accepted Manuscript
Could ethanol-induced alterations in the expression of glutamate transporters in
testes contribute to the effect of paternal drinking on the risk of abnormalities
in the offspring?
Mohammed Abul Kashem, Aven Lee, David V. Pow, Omar Šerý, Vladimir J.
Balcar
PII: S0306-9877(16)30327-9
DOI: http://dx.doi.org/10.1016/j.mehy.2016.11.015
Reference: YMEHY 8421
To appear in: Medical Hypotheses
Received Date: 4 July 2016
Accepted Date: 23 November 2016
Please cite this article as: M. Abul Kashem, A. Lee, D.V. Pow, O. Šerý, V.J. Balcar, Could ethanol-induced
alterations in the expression of glutamate transporters in testes contribute to the effect of paternal drinking on the
risk of abnormalities in the offspring?, Medical Hypotheses (2016), doi: http://dx.doi.org/10.1016/j.mehy.
2016.11.015
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Could ethanol-induced alterations in the expression of 
glutamate transporters in testes contribute to the effect of 
paternal drinking on the risk of abnormalities in the 
offspring? 
 
 
Mohammed Abul Kashem, MSc1, Aven Lee, PhD2, David V. Pow PhD2,3, Omar 
Šerý PhD4,5 and Vladimir J. Balcar DSc1* 
 
 
 
 
*Correspondence: vibar@anatomy.usyd.edu.au , Ph (+61 2) 9351 2837 Fax (+61 2) 9351 2813 
1 Laboratory of Neurochemistry, Bosch Institute and Discipline of Anatomy and Histology, School of 
Medical Sciences, Sydney Medical school, The University of Sydney, 2006 Sydney, NSW, 
AUSTRALIA. 
2 UQ Centre for Clinical Research, The University of Queensland, Brisbane, QLD 4029, AUSTRALIA 
3 School of Medical Sciences RMIT University, Melbourne, Bundoora, VIC 3083, AUSTRALIA 
4 Laboratory of Neurobiology and Molecular Psychiatry, Department of Biochemistry, Faculty of 
Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic 
5 Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Veveří 97, 
602 00, Brno Czech republic 
 
Email contacts  
MAK: abul.kashem@sydney.edu.au; AL: aven.lee@uq.edu.au; DVP: david.pow@rmit.edu.au; OS: 
omarsery@sci.muni.cz; VJB: vibar@anatomy.usyd.edu.au    
 
Sources of support 
Grant project (no. 16-27243A) of the Agency for Healthcare Research of the Czech Republic (AZV ČR) 
to OS. 
  
2 
 
Abstract 
It has been known that a preconception paternal alcoholism impacts adversely on the offspring but 
the mechanism of the effect is uncertain. Several findings suggest that there are signalling systems in 
testis that are analogous to those known to be altered by alcoholism in brain. We propose that 
chronic alcohol affects these systems in a manner similar to that in brain. Specifically, we 
hypothesise that excessive alcohol may disturb glutamatergic-like signalling in testis by increasing 
expression of the glutamate transporter GLAST (EAAT1). We discuss ways how to test the hypothesis 
as well as potential significance of some of the tests as tools in the diagnostics of chronic alcoholism. 
Background 
Presence of apparent cognitive deficits in children fathered by heavily drinking men has been known 
since antiquity (as noted in [1]). The mechanism(s) by which paternal alcohol-drinking inflicts 
damage on the offspring remain(s), however, obscure. It has been suggested that the DNA in male 
gametes affected by alcohol is altered epigenetically, e.g. by an interference in the methylation of 
DNA cytosine but the evidence, particularly when obtained in humans, is scarce and may be 
contradictory. Some groups have reported a decrease in the expression and activity of a DNA 
methyltransferases [1,2] resulting in a failure of orderly suppression of specific DNA sites in male-
contributed alleles [1]. In contrast, results of other studies, using male mice exposed to ethanol, 
implied that an increased methylation of specific DNA loci, e.g. cytosine-rich sequences (“CpG 
islands”) in the promoter region of the DAT gene encoding a dopamine transporter, could be at fault 
[3]. Disturbed expression of DAT has, indeed, been causally linked to an attention-
deficit/hyperactivity disorder (ADHD)-like behaviour similar to that observed in the offspring of the 
ethanol-exposed male mice [3]; (cf. review of animal models of ADHD [4] and altered expression of 
DAT1/SLC6A3 in human ADHD [5]). Here we propose an alternative hypothesis which, in our view, 
fits better the broad spectrum of deficits encountered in the offspring of alcoholic males. It is based 
on the probable presence in the testis of signalling mechanisms similar to those known to exist in 
the central nervous system. 
The main synaptic transmitters in brain are L-glutamate (excitatory) and GABA (inhibitory); (for a 
historical review see [6]). Both glutamatergic and GABAergic synapses are thought to be strongly 
involved in mediating the effects of alcohol [7], particularly via the NMDA-type of glutamate 
receptor and the alpha4-subunit containing GABA(A) receptor [8,9,10]. Both GABAergic and 
glutamatergic signalling systems, including the two types of receptors mentioned above, are altered 
in alcoholism [11,12,13,14]. Interestingly, it is not only the synaptic receptors which are affected by 
alcohol but also the neurotransmitter-inactivating mechanisms that are changed in alcoholic brains, 
particularly in the case of glutamatergic neurotransmission. 
In the central nervous system (CNS), synaptically released L-glutamate is inactivated by several 
specific transporters located mainly but not exclusively in the plasma membrane of surrounding 
astrocytes. There are five genes coding for the transporters (reviews: [15,16]) but protein products 
of two of them predominate: GLAST transporter encoded by SLC1A3 and GLT1 transporter encoded 
by SLC1A2. GLT1 and GLAST are also referred to as EAAT1 and EAAT2, respectively, particularly when 
discussing human brains. Both transporters require Na+ and K+ transmembrane gradients as the 
driving force  to transport L-glutamate from the extracellular space (reviews: [15,16]). In addition, 
GLAST acts as a chloride-selective ligand- (L-glutamate-) gated channel and thus has a capability to 
  
3 
 
hyperpolarise GLAST-expressing cells in the presence of L-glutamate [review: 16]. Both GLAST and 
GLT are mostly expressed in the central nervous system [15,16]; the only other tissue which 
expresses them in significant quantities is testis [17,18,19,20,21]. 
In the testes, GLT1 (EAAT2) has been detected in the interstitial cells but it is located mainly in the 
seminiferous tubules, both in the Sertoli cells and in the sperm. GLAST is found, apart from the 
Sertoli and interstitial cells, in the sperm, apparently in its anterior part, possibly concentrated in the 
acrosome [19,21]. The other glutamate transporters that have been detected in the testes are 
EAAT5 which acts mostly as an L-glutamate-gated chloride channel [22,23] and EAAT3 [19,21].  
The precise role of glutamate transporters in the testes is not known. However, L-glutamate is the 
most abundant free amino acid in testis [24] and is present inside the seminiferous tubules. 
Moreover, the compartment is separated from the blood stream by a very tight blood-testis barrier 
formed by tight junctions in the Sertoli epithelium. The presence of L-glutamate (together with 
several “synaptic” proteins; cf. [19] and receptors [20,21]) sequestered behind such barrier would 
seem to imply that a signalling apparatus, possibly involving germ cells and sperm, both in their 
mature and immature forms either dormant or active and mediated by L-glutamate, exists and 
functions in testes. Glutamate transporters would then provide a regulating (“inactivating”) 
mechanism analogous to that functioning at brain synapses (reviews: [12,13). Alternatively, L-
glutamate transport, mainly by GLAST (EAAT1) and EAAT5 could trigger chloride influx thus 
hyperpolarizing and activating the sperm [21].  
The Hypothesis 
It has been reported that chronic exposure to large doses of alcohol is associated with significant 
(several-fold) increases in the expression of GLAST (EAAT1), both in mice and men [25,26]. Should a 
similar overexpression of GLAST (EAAT1) occur in testes under similar circumstances (chronic severe 
alcoholism), sperm would become much more susceptible to the activation than the sperm of non-
alcoholic males possibly leading to a formation of active immature sperm. Subsequent fertilization 
by such immature sperm would significantly increase the risk of developmental defects in the 
offspring [27].  
Evaluation 
It should be understood that the developmental defects caused by the paternal alcoholism are 
distinct from the better known Foetal Alcohol Spectrum Disorder (FASD) and would be entirely 
independent of the alcoholic status of the pregnant female. The hypothesis does not negate the role 
of epigenetics as a mediator of the effect of paternal alcohol consumption on the offspring; there is 
ample evidence for such mechanisms from rodent studies (for a review see [28]). The present 
hypothesis adds, however, an extra element to the picture and could help to explain the complexity 
of the phenomenon [28]. 
Testing the hypothesis would present several significant challenges. Firstly, glutamate transporters 
are expressed in a number of splice variants and the splicing pattern in the testes appears very 
different from that in the CNS [23]. Use of single antibodies against GLAST (EAAT1) or GLT1 (EAAT2) 
in the testicular tissue could, therefore, easily miss or underestimate the full extent of the changes in 
GLAST (EAAT1) expression putatively caused by chronic alcoholism. Suitable antibodies against a 
range of splice variants are available [29,30] though not yet on commercial basis. The glutamate 
  
4 
 
transporter immunohistochemistry would have to be complemented by in situ hybridization using 
judiciously selected antisense oligonucleotides to reveal the full extent of the changes and their loci. 
Secondly, not just the presence but also the function of GLAST (EAAT1), GLT1 (EAAT2) and EAAT5 
both in the seminiferous tubules and in the sperm would have to be investigated. Initial approach 
encountered significant methodological hurdles [21] but these can be overcome. Given the 
proposed hypothetical importance of the transporter molecules in the sperm activation, samples of 
sperm should perhaps be used directly in this type of studies. Thirdly, a link between the proposed 
overexpression of GLAST in the testes and/or in the sperm following chronic exposure to alcohol and 
observed deficits in the offspring would have to be established. This experimental component seems 
crucial in testing the validity of the hypothesis but may also be the most difficult, probably requiring 
a large (inter-disciplinary) animal-based approach. In the meantime, GLAST (EAAT1) expression in 
the sperm of alcoholic men could be looked at and perhaps evaluated as a possible diagnostic test 
for the severity of chronic alcoholism. 
Conclusion and Significance of the Hypothesis 
We propose that the mechanism of the effect of paternal alcoholism on inborn deficits in the 
offspring can be at least in part explained by a changed pattern of the expression of glutamate 
transporters in the testes of alcoholic fathers. This would involve, in particular, overexpression of 
GLAST (EAAT1) glutamate transporter. Testing the hypothesis presents significant methodological 
challenges but none of them seem in principle unsurmountable. Additionally, the tests could yield an 
important diagnostic tool potentially useful in assessment of chronic alcoholism in men. Should the 
hyperactivity of glutamate transporter GLAST be an important part of the mechanism, there is 
extensive pharmacological information on structural requirements of glutamate transporters that 
could greatly facilitate design and synthesis of specific inhibitors [31,32]. 
 
Conflict of Interest Statement 
None of the authors has any competing interests  
Funding 
This study was supported by a grant project (no. 16-27243A) of the Agency for Healthcare Research 
of the Czech Republic (AZV ČR) to OS. AHR CR provided research funds to OS and had no further role 
in the design of the current study and collection, analysis, and interpretation of data. MAK, AL, DVP 
and VJB have currently no funding.  
 
List of abbreviations 
ADHD, Attention Deficit Hyperactivity Disorder; CpG, Cytosine and Guanine separated by phosphate 
(the target of DNA methyltransferases); DAT, Dopamine Transporter; EAAT, Excitatory Amino Acid 
Transporter; FASD, Foetal Alcohol Spectrum Disorder; GABA, γ-aminobutyric acid; GLAST, Glutamate 
and Aspartate Transporter; GLT, Glutamate Transporter; SLC, Solute Carrier family of membrane 
transporter proteins.  
  
5 
 
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Could ethanol-induced alterations in the expression of glutamate transporters in testes contribute to the effect of paternal drinking on the risk of abnormalities in the offspring?

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