IVF

Daniel Eberl daniel-eberl at UIOWA.EDU
Mon Jul 19 10:07:16 EST 1999


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"Robert Butcher" <RDJBUTCHER at biolsci.dundee.ac.uk> wrote:
>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
(truncated)

On a related note, I became interested a number of years ago in developing
a method for freezing sperm as a way of long term maintenance of mutations,
and using artificial insemination (AI) with the thawed sperm to recover the
mutations into live stocks.  It became clear to me that this would require
much more than the "spare time" I had. While I did not succeed in doing AI
in live flies (I didn't try very hard), I followed the report of success in
AI from many years ago [Gottschewski G: K=FCnstliche Befruchtung bei
Drosophila. Naturwissenschaften 1937, 25:650.].

This paper is in German, and as I had translated it crudely at the time, I
thought I would share it here (it's quite short).  Of course those of you
who can read the German are advised to go directly to the source.  I think
this paper is interesting not only for those interested in AI, long-term
storage of Drosophila stocks, or IVF, but for the general biologist or
Drosophila historian as well.....Enjoy:


		Artificial Insemination in Drosophila.

	The development of methods for artifical insemination in Drosophila
offers new perspectives for many problems in Drosophila genetics.  Only two
will be described.  The demonstration of mutations with ultraviolet
irradiation:  The studies of Stadler with ultraviolet irradiation of corn
pollen show new ways to   interpret the mutation event.  In Drosophila such
studies could extend the previous results, if irradiating sperm directly
without surrounding material could be accomplished, and then inseminating
females with this irradiated sperm.  The advantage of Drosophila over corn
lies namely in this: that the ultraviolet rays strike directly on the
nucleus, whereas in the irradiation of pollen the position of the nucleus
in the pollen grain varies, and secondly, that in the corn seed the
radiation-absorbing material which surrounds the nucleus interferes with
the evaluation of apparent results.  Above all, however, a second problem
could come into our grasp through the use of artificial insemination:
namely, the crossing of sterile strains, in which the sterility is based on
sexual or mechanical isolation.  Through implantation of ovaries of one
strain into females of another, and subsequent insemination of the
recipient females with males of the "same" strain, it has been possible in
a few cases to recover offspring; but this method has the disadvantage that
only in a very limited percentage of studies do the implanted ovaries
functionally grow into the reproductive tract.
	The methods of artificial insemination in Drosophila follow:
Virgin females were paired with males, copulation was observed and after
copulation took place the females were dissected and the ejaculate was
sucked out with a fine glass needle.  During dissection, which is done in
Ringer's solution, care should be taken that the vagina remains intact to
beyond the opening of the seminal receptacle.  For injection, the
Standard-Chambers-Microinjection apparatus is used, in the same way as for
transplantation experiments.  The needle is then inserted into the
recipient females, which obviously must also be virgin.  The etherized
females are immobilized with forceps under enough pressure to evert the
vagina.  The dissection and injection are done under a binocular microscope
with 24X magnification.  The movement of the sperm can already be
positively identified under 48X magnification.
	In preliminary experiments the seminal vesicles were initially
removed from males and the sperm obtained.  This method however did not
lead to the desired result, since for a successful fertilization the
secretion from the accessory glands is apparently also required.  One other
preliminary study, in which sperm was obtained by artificial masturbation
of Drosophila males, was actually successful, but this method is not useful
because in a series of 24 males an ejaculate could only be demonstrated in
2 cases, and the time engaged is in no proportion to the result.
	In the following I present the results of studies with Drosophila
melanogaster.  I.  Thirteen 6-14 day old "white" virgin females were
injected with sperm obtained from the seminal vesicles of "wildtype" males.
In only 2 cases were progeny recovered: 2 "wildtype" F1 females and 1
"white" F1 male.  Three females died shortly after injection before they
laid eggs, 8 females had no offspring.  IIa.  In this series of studies
five 8-day old virgin "white" females were injected with sperm taken from
the vagina of other females 0-30 minutes after copulation with "wildtype"
males. Copulation lasts 15-25 minutes.  In one dissection, motile sperm
already existed in the seminal receptacle.  All 5 females were successfully
artificially inseminated: (72 "wildtype" F1 females and 50 "white" F1
males).  In order that obviously functional sperm were injected, the result
of artificial fertilization was proven through the next series of
experiments (IIb), by whether or not the seminal receptacle carries motile
sperm 30 minutes after injection.  Nine females were artificially
inseminated.  The sperm were again taken from the vagina of other females
0-30 minutes after copulation with "wildtype" males as in study IIa.  In
all 9 females the seminal receptacles carried motile sperm 30 minutes after
injection.
		G. Gottschewski, Berlin-Dahlem, Kaiser Wilhelm-Institut fur
			Biologie, at the time (or currently??) at California
			Institute of Technology, Pasadena, Cal. USA,
			in August 1937.
uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu

  Daniel Eberl
  Assistant Professor			Office 	319 335-1323
  Department of Biological Sciences     Lab     319 353-2055
  Rm 238 BB                             Fax     319 335-1069
  University of Iowa
  Iowa City, IA 52242-1324              Email   daniel-eberl at uiowa.edu

	http://www.biology.uiowa.edu/faculty/eberl.htm
nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn
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"Robert Butcher" <<RDJBUTCHER at biolsci.dundee.ac.uk> wrote:

>Hi all,

> i have had a number of requests for info on our crude IVFsystem in=20

>drosophila, and it has been suggested i reply on the newsgroup, so=20

(truncated)


On a related note, I became interested a number of years ago in
developing a method for freezing sperm as a way of long term
maintenance of mutations, and using artificial insemination (AI) with
the thawed sperm to recover the mutations into live stocks.  It became
clear to me that this would require much more than the "spare time" I
had. While I did not succeed in doing AI in live flies (I didn't try
very hard), I followed the report of success in AI from many years ago
[<fontfamily><param>Geneva</param>Gottschewski G: <bold>K=FCnstliche
Befruchtung bei Drosophila. </bold><italic>Naturwissenschaften</italic>
1937, <bold>25</bold>:650.</fontfamily>].


This paper is in German, and as I had translated it crudely at the
time, I thought I would share it here (it's quite short).  Of course
those of you who can read the German are advised to go directly to the
source.  I think this paper is interesting not only for those
interested in AI, long-term storage of Drosophila stocks, or IVF, but
for the general biologist or Drosophila historian as well.....Enjoy:



		Artificial Insemination in Drosophila.


	The development of methods for artifical insemination in Drosophila
offers new perspectives for many problems in Drosophila genetics.  Only
two will be described.  The demonstration of mutations with ultraviolet
irradiation:  The studies of Stadler with ultraviolet irradiation of
corn pollen show new ways to   interpret the mutation event.  In
Drosophila such studies could extend the previous results, if
irradiating sperm directly without surrounding material could be
accomplished, and then inseminating females with this irradiated sperm.
 The advantage of Drosophila over corn lies namely in this: that the
ultraviolet rays strike directly on the nucleus, whereas in the
irradiation of pollen the position of the nucleus in the pollen grain
varies, and secondly, that in the corn seed the radiation-absorbing
material which surrounds the nucleus interferes with the evaluation of
apparent results.  Above all, however, a second problem could come into
our grasp through the use of artificial insemination: namely, the
crossing of sterile strains, in which the sterility is based on sexual
or mechanical isolation.  Through implantation of ovaries of one strain
into females of another, and subsequent insemination of the recipient
females with males of the "same" strain, it has been possible in a few
cases to recover offspring; but this method has the disadvantage that
only in a very limited percentage of studies do the implanted ovaries
functionally grow into the reproductive tract.

	The methods of artificial insemination in Drosophila follow:  Virgin
females were paired with males, copulation was observed and after
copulation took place the females were dissected and the ejaculate was
sucked out with a fine glass needle.  During dissection, which is done
in Ringer's solution, care should be taken that the vagina remains
intact to beyond the opening of the seminal receptacle.  For injection,
the Standard-Chambers-Microinjection apparatus is used, in the same way
as for transplantation experiments.  The needle is then inserted into
the recipient females, which obviously must also be virgin.  The
etherized females are immobilized with forceps under enough pressure to
evert the vagina.  The dissection and injection are done under a
binocular microscope with 24X magnification.  The movement of the sperm
can already be positively identified under 48X magnification.

	In preliminary experiments the seminal vesicles were initially removed
from males and the sperm obtained.  This method however did not lead to
the desired result, since for a successful fertilization the secretion
from the accessory glands is apparently also required.  One other
preliminary study, in which sperm was obtained by artificial
masturbation of Drosophila males, was actually successful, but this
method is not useful because in a series of 24 males an ejaculate could
only be demonstrated in 2 cases, and the time engaged is in no
proportion to the result.

	In the following I present the results of studies with Drosophila
melanogaster.  I.  Thirteen 6-14 day old "white" virgin females were
injected with sperm obtained from the seminal vesicles of "wildtype"
males.  In only 2 cases were progeny recovered: 2 "wildtype" F1 females
and 1 "white" F1 male.  Three females died shortly after injection
before they laid eggs, 8 females had no offspring.  IIa.  In this
series of studies five 8-day old virgin "white" females were injected
with sperm taken from the vagina of other females 0-30 minutes after
copulation with "wildtype" males. Copulation lasts 15-25 minutes.  In
one dissection, motile sperm already existed in the seminal receptacle.
 All 5 females were successfully artificially inseminated: (72
"wildtype" F1 females and 50 "white" F1 males).  In order that
obviously functional sperm were injected, the result of artificial
fertilization was proven through the next series of experiments (IIb),
by whether or not the seminal receptacle carries motile sperm 30
minutes after injection.  Nine females were artificially inseminated.=20
The sperm were again taken from the vagina of other females 0-30
minutes after copulation with "wildtype" males as in study IIa.  In all
9 females the seminal receptacles carried motile sperm 30 minutes after
injection.

		G. Gottschewski, Berlin-Dahlem, Kaiser Wilhelm-Institut fur

			Biologie, at the time (or currently??) at California

			Institute of Technology, Pasadena, Cal. USA,=20

			in August 1937.=20

uuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuuu


  Daniel Eberl

  Assistant Professor			Office 	319 335-1323

  Department of Biological Sciences     Lab     319 353-2055

  Rm 238 BB                             Fax     319 335-1069

  University of Iowa

  Iowa City, IA 52242-1324              Email   daniel-eberl at uiowa.edu


	http://www.biology.uiowa.edu/faculty/eberl.htm

nnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnnn

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