IVF

Robert Butcher r.d.j.butcher at dundee.ac.uk
Mon Jul 19 07:26:16 EST 1999


Hi all,
 i have had a number of requests for info on our crude IVFsystem in 
drosophila, and it has been suggested i reply on the newsgroup, so 
here goes. Sorry for this delay, but i really have been busy on our 
field course and the inevitable preparing for and then dealing with 
all the data and students afterwards. 
These methods were developed on Ichneumonoidea (Hymenoptera), 
modified with some succes for Pyralid moths (Lepidoptera) but have 
only been roughly tested on Drosophila and thus do NOT represent 
optimised systems. Rather this is a very preliminary system. I am 
hoping to optimise them later this year (I am not working with them 
at present) and will report the results and any new protocols on the 
newsgroup.

Note that these preliminary experiments were carried out using males 
and females from the two lines:-
Cyo P(2,3)/Bc1 Egfr1; y*w*/+ 
 +/+; red1e1/red1/e1
 which although far from ideal, were available at hand at the time 
and allowed the biparental nature of any eclosing imagoes to be 
confirmed. 

1. Virgin females (1-3 days old) were allowed to mate with males 
(more than one copulation was allowed) and  held away from 
oviposition sites (i.e. on yeast paste only) over a 5 hours[1] 
period prior to dissection of the female and removal of the 
sperm or spermataphore [2] into modified drosophila ringers (130mM 
NaCl, 5mM KCl, 1.5mM CaCl2, 2mM Na2HPO4, 5% (w/v) BSA, 10% w/v 
filtered homogenate from the spermatheca [3]). Typically the 
free sperm / spilled spermatophore contents of ten females 
spermatheca were microdissected into 20-30ul of ringers, but it 
appears imperative to reduce dessication so both speed and use of a 
locally saturated atmosphere is important [4].

2.  Microdissect out the mature eggs from virgin females [5] and add 
(bath for up to 1 minute)  to the sperm mixture obtained above. 

3. Load into a microinjector (standard inert oil as per embryo 
microinjections) and "oviposit" via the microinjector [6] into 
media[7]. maintain as per normal.

>From the limited preliminary trials this results in some 20-27% 
(range) of embryos hatching, and 10-14% developing as imagoes, but 
 the system has not yet been optimised.

Notes
[1]. The importance of the number of matings and time in spermatheca 
prior to dissection is not known, and needs optimising, but appears 
that sperm removed from the spermatophore shortly after copulation 
may be less suitable for this IVF sytem.

[2]. I havent tried the less time consuming extraction of sperm by 
microdissection from males. If it worked it would be a mjour tisaver 
and is worthy of checking. This would however avoid the male 
accessory gland secretions (importance in an IVF system, as opposed 
to sperm competition and egg maturation / oviposition stimulants, is 
unknown to me and can be evaluated), and maternal secretions 
into the spermatheca (same points), would also add immature (sterile) 
sperm.

[3]. relavance of female spermatheca homogenate in this system is 
unknown, its left over from Leps.

[4]. keeping the microdissected sperm at cooler temperatures(e.g. 
0-4C) to allow larger bulk procedures has not been evaluated yet. If 
they remained viable this would improve yields per day etc.

[5]. This seems important.  I have tried this procedure on embryos 
laid by older virgin females, or those laid by females mated to XO 
males (derived from the gynF9 (2,3) stock following mating to 
ms(3)K81 males) and they failed to develop. Removal of the chorionic 
and vitelline membranes, prior to IVFfertilissation, and maintaining 
under oil afterwards (standard embryo microinjection procedures) all 
failed in this preliminary system.

[6]. This is left over from parasitic wasps... Passage through an 
orafice (microinjection needle) of a slightly narrower dimension than 
the egg is essential in the Hymenoptera case. Its importance to 
drosophila has not been tested yet.

[7]. not important, the IVF embryo could be hatched on a glass slide 
etc, it is just morconvenient as the first instar can burrow in and 
feed immediately.

Lastly, the ability of this IVF system to work with cryopreserved 
sperm, or embryos, obtained by these methods has not been 
checked yet and i will look at that later on.

Any more queries please ask. Hope this helps and sorry for all the 
space to thiose who have no interest in this
Cheers
Rob

Robert Butcher,
Evolutionary and Ecological Entomology Unit,
Department of Biological Sciences,
Dundee University,
Dundee, DD1 4HN,
Tayside, Scotland,
UK.
Work Phone:- 01382-344291 (Office), 01382-344756 (Lab).
Fax:- 01382-344864
e-mail:- r.d.j.butcher at dundee.ac.uk
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