a better understanding of how different biological processes are altered by organosulfurs has become important because of the 
 increasing number of reports on disease alteration by xenobiotic , food , and complex organosulfur compounds . with that as a goal , 
 how do the results reviewed herein with oocytes / embryos , presumably a model system that is simpler than other biological models , 
 contribute to the following questions .
first , is the biological activity of 2me a consequence of the reduced sulfhydryl form or 
 due to a self- and/or mixed- ( with cysteine ) disulfide ? and second ,
which of the postulated mechanisms outlined in the 
 introduction do the data directly or indirectly support ?
the data are quite indisputable that to enhance ivrp processes , 2me and cysteamine absolutely required the presence of either 
 cysteine or cystine ( the same conclusion was made for enhanced lymphoid replication / functions in vitro ) . a 
 potential exception to this requirement was reported   for 2me supplemented ivc in 
 which development of porcine embryos to the blastocyst stage was enhanced from 23.5  2.1% to 30.5 
2.3% in 
 cysteine / cystine - free medium . was development truly enhanced and/or were conditions completely cysteine / cystine free ?
an answer 
 needs to consider that : statistical significance was borderline ; there was no 2me enhancement of development to the 816 cell 
 stage ; maturation of oocytes was done in ivm 199 medium ( carry - over of cystine / cysteine / gsh ? ) ; enhancement occurred only in 
 ncsu-23 , not in ksom media ( both thiol - free ) ; and both these ivc media were equally effective in supporting parthenogenetic and 
 ivf embryo development from the 1-cell stage to blastocyst stage in the absence of any thiol supplementation .
even with this 
 potential exception , all other investigations demonstrated that 2me / cysteamine alterations required the presence of 
 cystine / cysteine , suggesting that the sulfhydryl and self - disulfide forms of 2me or cysteamine are not the extracellular active 
 forms , at least in the absence of cysteine / cystine , but that a mixed disulfide is the most likely active structure .
direct 
 evidence for a mixed disulfide [ 12 , 38 , 63 , 69 ] is the ( a ) identification of a new compound 
 formed within minutes of mixing cystine / cysteine and 2me ( or cysteamine ) in the absence / presence of cells by both embryo and 
 immune investigators and ( b ) bioactivity of mixed disulfides added directly to cultures in place of cysteine / cystine and 2me or 
 cysteamine .
mixed 2me - cysteine was rapidly taken up by cells and internally converted into 2me and several other compounds  the 
 major one was indistinguishable from gsh .
this enhanced uptake was apparently 
 sufficient to overcome the rate limiting constraint of low cysteine levels for gsh synthesis .
this is in agreement with that 
 found for lymphoid enhancement and tumor cell growth [ 12 , 13 , 21 , 25 ] .
however , questions of what 
 other biological processes were influenced by the internalized disulfide remain to be determined .
studies with s 
 mixed disulfides indicated that the majority of 2me was not altered or incorporated into cellular constituents of tumor cells 
 but was recycled extracellular such that its concentration remained relatively constant for 48 h in the culture media .
moreover , mixed disulfides and cystine were reduced to cysteine when dtt was added to 
 the medium [ 12 , 69 ] and yet , in contrast to dtts 
 protection of oocytes during 
[ 1 , 4 , 6 , 13 ] , it ( as well as melatonin and nac ) , alone or in the presence of cysteine did not enhance 
 ivrp development processes .
whether disulfide functions in vitro have relevance for 2mes alteration of in vivo disease 
 processes is intriguing when considering that cystine or cysteine alone enhanced development of oocytes / lymphocytes in 
 vitro .
this is especially interesting in that even though gsh and development generally paralleled one another , 
 there were circumstances in which development occurred in the absence of gsh increases as well as the reverse , namely 
 development did not occur under conditions in which maintenance / increases in gsh did occur .
this was especially apparent at high 
 doses of 2me and/or cysteine , suggesting that they were affecting processes other than those associated with increased synthesis 
 of gsh . in some cases
, even though gsh increases continued as concentrations of 2me increased , oocyte development became 
 inhibited .
for example , supplementation of ovine oocyte ivm medium with cysteamine or 2me at 200 m compared to non - supplemented 
 medium resulted in ( a ) gsh increases to 6.9 and 6.5 from 4.2 pm / oocyte , ( b ) reduction of peroxide levels by 9.8% and 9.9% and 
 ( c ) enhanced blastocyst development from 26.2% to 42.2% by cysteamine but reduced to 21.1% by 2me .
thus , synthesis of gsh to replenish losses or to increase levels maybe critical for enhanced 
 development , however , in some cases an increase may not be sufficient .
thiols must effect other processes , a conclusion that 
 supports the cautionary suggestion ,  one can not overlook , however , the possibility 
 that 2me has an effect other than making cysteine available for glutathione synthesis  .
what these are remain undefined and may 
 or may not involve structural aspects , i.e. , free sh as well as self- or mixed - disulfides .
a second favorite mechanism for antioxidants such as 2me and cysteamine is that they act as scavengers of ros .
basically the 
 evidence to support this mechanism for ivrp was derived by comparing changes in ros , apoptotic , and dna damage that occur in the 
 presence of thiol and non - thiol antioxidants ( discussed in ) .
for example , 2me and 
 vitamin e supplementation of ivc decreased h2o2 content similarly , reduced dna fragmentation similarly , 
 but enhanced blastocyst development dissimilarly  from a control of 28% to 57% and only 40% .
this enhancement was similar to the 
 36.2% from 22.5% achieved in a thiol - free , lower oxygen atmospheric level of 5% . in 
 contrast , nac or extracellular antioxidants like catalase and sod supplementation of ivc in 7% o2 or supplementation 
 of ivm in 21% o2 failed to improve development whereas supplementation of ivm or ivc with cysteine alone increased 
 development to the blastocyst stage [ 31 , 43 ] . 

another example with relevance for the importance of thiol alteration of ros is the findings that 2me alone ( but not cysteine 
 alone even though it increased gsh ) reduced 21% oxygen - induced ros production to the same level as a combination of gsh+cysteine 
 ( but not quite as well as the combination of 2me+cysteine ) and yet only the combinations enhanced development .
thus , neither 
 reduction of ros by 2me alone nor enhancement of gsh synthesis by cysteine alone was sufficient to enhance development .
further 
 melatonin , nac , and dtt individually or in combination with cysteine did not enhance development   suggesting that a reduction in ros like an increase in synthesis of gsh can be uncoupled from 
 development .
these results are in line with the estimate that only 2545% of 2me s benefits for lymphoid functions in 
 vitro are due to antioxidant activity alone . finding that development can be uncoupled from ros and gsh suggest that the other two mechanisms outlined in the introduction 
 may have a role in ivrp 
simple sulfur compounds , such as hydrogen sulfide   and/or 
 sulfane sulfur [ 21 , 25 ] alter gene expression 
 and/or maintain critical sulfhydryl - disulfide 
allosteric disulphide bonds of cytoplasmic enzymes   and/or cell membrane proteins ( see ref in ) .
unfortunately , 
 both these mechanisms are difficult to monitor and neither were considered or tested in ivrp .
however , studies are beginning to 
 be reported ( cysteamine supplementation ) with evidence for differential gene expression .
for example , mrna for anti - apoptotic 
 genes bcl - xl and mcl-1 were increased when cysteamine was added to ivm and ivc media , whereas the expression of the 
 pro - apoptotic gene bax but not bid was reduced at many stages of development .
it is clear that ( a ) xenobiotic thiols enhance ivrp , be it cysteine , cystine , n - acetylcysteine , n - acetylcysteine - amide , 
 cysteamine , or 2me , ( b ) the latter four required the presence of one of the former two to enhance any of the processes ; ( c ) 
 optimal concentrations of 2me and cysteamine appear to differ by 10100-fold , supporting that found for enhancement of lymphoid 
 responses [ 1 , 4 , 6 , 13 ] , ( d ) interdependence of 2me or cysteamine with cysteine / cystine at 
 optimal concentrations have yet to be defined for ivrp , ( e ) experiments with 2me and cysteamine at equivalent concentrations 
 result in little biologically significant information and should be discontinued ; ( f ) multiple variables determine whether 
 supplementation of an ivrp process with 2me or cysteamine results in enhancement , inhibition or no effect ; and ( g ) mechanisms by 
 which thiols enhance ivrp is almost certainly multifaceted  increase synthesis of gsh ( which is not sufficient ) , reduce ros 
 ( which is also not sufficient ) , and others yet to be defined .
indeed , development of an embryo to a stage worthy of transfer to 
 a recipient may depend upon a combination of multiple thiol benefits that may act synergistically .
one other observation worthy of comment regards media components found to enhance the overall process of in 
 vitro embryo development 
thiols , essential and nonessential amino acids , nucleic acid precursors 
 [ 60 , 64 , 89 ] , vitamins are still evolving and are fast approaching reinventing 
 click s ehaa medium described over 40 years ago that was found to be superior to rpmi 1640 and dmem when used either serum - free 
 or supplemented with autologous serum for supporting lymphoid proliferation   and 
 weak - antigen detection .
moreover , culture of grade 3 and 4 bovine blastocysts for 24 
 h in this medium ( supplemented to contain 50 m 2me and 10% fbs ) resulted in an improvement to grade 1 and 2 for freezing or 
 transfer to surrogate recipients [ r. click , unpublished ] .
similarly , others   found 
 that a 24 h culture in a different medium supplemented with 2me after vitrification and thawing improved bovine blastocyst 
 survival , hatching rates , and their total cell numbers .
   descriptions of organosulfurs altering biologically relevant cellular functions began some 40 years ago when murine 
 in vitro cell mediated and humoral immune responses were shown to be dramatically enhanced by any of four 
 xenobiotic , sulfhydryl compounds2-mercaptoethanol ( 2me ) , dithiothreitol ( dtt ) , glutathione , and l - cysteine ; the most 
 effective were 2me and dtt . 
 these findings triggered a plethora of reports defining 2me benefits for a multitude of 
 immunological processes . 
 this in turn led to investigations on 2me alterations of ( a ) immune functions in other species , ( b ) 
 activities of other cell - types , and ( c ) in vivo diseases . 
 in addition , these early findings preceded the 
 identification of previously undefined anticarcinogenic chemicals in specific foods as organosulfurs . taken all together , 
 
 there is little doubt that organosulfur compounds have enormous benefits for cellular functions and for a multitude of 
 diseases . 
 issues of importance still to be resolved are ( a ) clarification of mechanisms that underlie alteration of 
 in vitro and in vivo processes and perhaps more importantly , ( b ) which if any in 
 vitro alterations are relevant for ( i ) alteration of in vivo diseases and ( ii ) identification of 
 other diseases that might therapeutically benefit from organosulfurs . as one means to address these questions , reviews of 
 different processes impacted by thiols could be informative 
 . therefore , the present review on alterations of in 
 vitro fertilization processes by thiols ( mainly 2me , since cysteamine alterations have been reviewed ) was 
 undertaken . 
 alterations found to occur in medium supplemented with 2me were enhancement , no effect , or inhibition . 
 parameters 
 associated with which are discussed as they relate to postulated thiol mechanisms . 
Possible Thiol Mechanisms
Summary