BIOLOGY
OF
REPRODUCTION
21,
891-904
(1979)
Spermatogenic Response to Vitamin A in Vitamin A Deficient Rats H. F. S. HUANG’ Department
W. C. HEMBREE”2
of Obstetrics and
Columbia
and
Department
University
College
New
and
Gynecology’
of Medicine2
York,
of physicians
New
York
and
Surgeons,
10032
ABSTRACT
Male
Holtzman
retinoic contain
rats
were
given
acid after the onset of Sertoli cells, spermatogonia
values are low normal, (followed by a regular testosterone ment (PVA),
A deficient (VAD) diet from 21 days of age and A deficiency. At 130 days of age, seminiferous tubules preleptotene spermatocytes; serum LH and testosterone are high. Oral feeding of a single dose of 1 mg vitamin A
vitamin and
while FSH commercial
remains kinetic
a vitamin
low and FSH characteristics
levels rat pellet does based
diet)
causes
reinitiation
not return to upon histologic
normal
and
de
for
of spermatogenesis, 60
days.
After
although
vitamin
A treat-
criteria of the reinitiated spermatogenesis in VAD rats were normal. Pachytene spermatocytes can be seen by Day 14 PVA and spermatids by Day 24 PVA; elongation of spermatids was completed by Day 31 PVA and spermatozoa were formed by ‘Day 41. Quantitatively, sperm production remained below normal at 150 days PVA, although epididymal sperm counts had continued to increase PVA to 50% of that in mature controls. The qualitative normalcy of spermatogenesis in PVA-VAD rats was demonstrated by fertility with normal litter size. Thus, VAD causes reversible germ cell depletion. Reinitated spermatogenesis in the VAD rat provides a kinetically normal, in vivo system in which functionally normal spermatozoa are produced and in which it may be possible to study the biochemical and morphological events of specific stages of spermatogenesis. INTRODUCTION
Vitamin degeneration characterized
A
ing only
Sertoli
number
of
1964;
rats or
either
retinyl
acetate
et a!., Bieri, this the and
Wald, Vitamin
have Mayer 1964) (Howell
al.,
rats
cells
tubules
spermatogonia
and
a small et
Although
(Howell
et al.,
1963;
of
a!.,
biochemical epithelium 1960). A deficient
requirement of the retina rats
were
also
August December
However, in
in
rats
and that
the for
maintained
and
fed
only with (Dowling
vitamin
A
deficient
1949; Bieri,
requirement retinol
is not
deficiency. of sperm-
on
retinoic
period et al.,
rats
with
and
testosterone regeneration
a prolonged (Thompson
to
rats
Truant,
time course and the extent of the the normalcy of the spermatozoa and the reproductive performance
shares
failure
these
Ahluwalia
epithelium
by the retinol-induced
supplement for been reported
acid
of time 1964),
has the
regeneration, so produced of the retinol
have
not
been
studied. that
to
Recently, maxima!
Krueger et a!. (1974) germ cell loss occurs by
of age if weanling deficient diet, later acid to maintain
small seminal vesicles (Mason, 1933; and Truant, 1949; Thompson et al., and low circulating androgen levels et a!., 1963; Krueger et a!., 1974; Rich
Accepted Received
germinal
atogenesis
Thompson
found
the
mediated Although
normal
et al., 1969; Ahluwalia germinal epithelium
1977).
spermatogenesis
hormone therapy (Meyer Mayer and Goddard, 1951; 1971a) provides evidence
is
contain-
(Thompson 1963).
causes which
maintained by retinoic acid in A deficient diet, maintenance of spermatogenesis in these rats retinol or its aldehyde derivative,
1964; Coward 1971b). The
unique pigment
male
germ
seminiferous
et
reinitiation
requires
in of
cells,
can be a vitamin
on
loss
spermatocytes
Howell
growth
deficiency or by
Kretser,
reinitiate
though levels was axis and
4, 1979. 14, 1978.
luteinizing were low,
hormone and follicle stimulating
a vitamin A by retinoic growth. Altestosterone hormone
normal and an intact pituitary-testicular was demonstrated by the elevation FSH following castration. In addition,
testosterone
891
rats are given supplemented normal body
reported 130 days
levels
observed
should
of
be sufficient
LH the
HUANG
892
to
maintain
10%
spermatogenesis,
of
normal
concentration
was
normal Huckins, Thus,
spermatogenesis 1978). we attempted
normalcy
of
A
treatment
can
be
shown
found
that
and
if
functionally (PVA)
to
an
rats in
germ
might
cell
present logical
serve the
development
herein the characteristics
PVA-VAD
male
as
a
useful
in
vivo
kinetics
of
We
could
be
studied.
morphological of germ
cell
and physiorepletion in
rats.
MATERIALS
AND
METHODS
Chemicals Radioactive materials were purchased from New England Nuclear and used without further purification; Ilford nuclear emulsion (K5) was purchased from Ilford Nuclear Research (Essex, England). Ingredients for preparing vitamin A deficient diet were purchased from I.C.N. Life Science Group, Cleveland, OH. Reagents used for radioimmunoassay of LH and FSH were provided by Rat Pituitary Distribution program NIAMDD, NIH. Other general chemicals, all reagent grade, were purchased from Fisher Scientific Co.
method
of Kopriwa
Spermatids
Weanling
A Deficient
Vitamin
male
Madison, WI), 19-21 an air conditioned,
Sprague-Dawley days light
solely for vitamin A given water ad libitum deficient diet of Muto instead of menadione.
of age, controlled
(VAD) rats were
Reproductive
Co.,
in pairs in room used
deficiency studies. They were and the modified vitamin A et al. (1972), using vitamin K Animals were weighed twice
weekly.
Retinoic acid (5 mg/kg) was added to the when the linear weight gain ceased as a result of complete depletion of vitamin A stores (Thompson et al., 1964; Muto et a!., 1972). At 130 days of age, rats were given 1 mg vitamin A orally and, thereafter, fed a regular commercial rat pellet. VAD
histology
diet
and
Radioautography
rats
determine the genie regeneration. 1 H] -thymidine 0.1 ml Ringer’s
ether
anesthesia
the
Deparaffined
at intervals of 4-6 in an effort to
sequential changes during spermatoIn selected VAD animals, 25 .sCi (sp act 41.6 mCi/mg) were injected in solution intratesticularly under light 24 h prior to sacrifice. About one-
identified
by
the
PAS-positive
Performance
Test
were
sacrificed
to
determine
epididymal
sperm
Counts.
Hormone
Assay
under
animals were sacrificed, blood samples light ether anesthesia through
or from
were
kept
after
at
centrifugation.
the retroorbital
4#{176} C overnight
Radioimmunoassay performed using
sinus.
Blood
serum
was
and
Abraham
et
of
were heart
samples collected
gonadotropins was by NIAMDD, Rat Pituitary Hormone Distribution Program; NIAMDDRat FSH-RP-1, Rat-FSI-I-I-3, Anti-Rat FSH-S-6 and NIAMDD-rat-LH-RP-1, Rat-LH-I-4, Anti-Rat-LI’I-S-3 and -S-4 were used for FSH and LH assay, respectively. Hormones were iodinated with 1251 using the chloramine-T method (Greenwood et al., 1963). lodinated hormones were purified through a Bio-Gel P-60 column. Intraassay coefficient of variation for both FSH and LH assays was
OF
REPRODUCTION
21,
891-904
(1979)
Spermatogenic Response to Vitamin A in Vitamin A Deficient Rats H. F. S. HUANG’ Department
W. C. HEMBREE”2
of Obstetrics and
Columbia
and
Department
University
College
New
and
Gynecology’
of Medicine2
York,
of physicians
New
York
and
Surgeons,
10032
ABSTRACT
Male
Holtzman
retinoic contain
rats
were
given
acid after the onset of Sertoli cells, spermatogonia
values are low normal, (followed by a regular testosterone ment (PVA),
A deficient (VAD) diet from 21 days of age and A deficiency. At 130 days of age, seminiferous tubules preleptotene spermatocytes; serum LH and testosterone are high. Oral feeding of a single dose of 1 mg vitamin A
vitamin and
while FSH commercial
remains kinetic
a vitamin
low and FSH characteristics
levels rat pellet does based
diet)
causes
reinitiation
not return to upon histologic
normal
and
de
for
of spermatogenesis, 60
days.
After
although
vitamin
A treat-
criteria of the reinitiated spermatogenesis in VAD rats were normal. Pachytene spermatocytes can be seen by Day 14 PVA and spermatids by Day 24 PVA; elongation of spermatids was completed by Day 31 PVA and spermatozoa were formed by ‘Day 41. Quantitatively, sperm production remained below normal at 150 days PVA, although epididymal sperm counts had continued to increase PVA to 50% of that in mature controls. The qualitative normalcy of spermatogenesis in PVA-VAD rats was demonstrated by fertility with normal litter size. Thus, VAD causes reversible germ cell depletion. Reinitated spermatogenesis in the VAD rat provides a kinetically normal, in vivo system in which functionally normal spermatozoa are produced and in which it may be possible to study the biochemical and morphological events of specific stages of spermatogenesis. INTRODUCTION
Vitamin degeneration characterized
A
ing only
Sertoli
number
of
1964;
rats or
either
retinyl
acetate
et a!., Bieri, this the and
Wald, Vitamin
have Mayer 1964) (Howell
al.,
rats
cells
tubules
spermatogonia
and
a small et
Although
(Howell
et al.,
1963;
of
a!.,
biochemical epithelium 1960). A deficient
requirement of the retina rats
were
also
August December
However, in
in
rats
and that
the for
maintained
and
fed
only with (Dowling
vitamin
A
deficient
1949; Bieri,
requirement retinol
is not
deficiency. of sperm-
on
retinoic
period et al.,
rats
with
and
testosterone regeneration
a prolonged (Thompson
to
rats
Truant,
time course and the extent of the the normalcy of the spermatozoa and the reproductive performance
shares
failure
these
Ahluwalia
epithelium
by the retinol-induced
supplement for been reported
acid
of time 1964),
has the
regeneration, so produced of the retinol
have
not
been
studied. that
to
Recently, maxima!
Krueger et a!. (1974) germ cell loss occurs by
of age if weanling deficient diet, later acid to maintain
small seminal vesicles (Mason, 1933; and Truant, 1949; Thompson et al., and low circulating androgen levels et a!., 1963; Krueger et a!., 1974; Rich
Accepted Received
germinal
atogenesis
Thompson
found
the
mediated Although
normal
et al., 1969; Ahluwalia germinal epithelium
1977).
spermatogenesis
hormone therapy (Meyer Mayer and Goddard, 1951; 1971a) provides evidence
is
contain-
(Thompson 1963).
causes which
maintained by retinoic acid in A deficient diet, maintenance of spermatogenesis in these rats retinol or its aldehyde derivative,
1964; Coward 1971b). The
unique pigment
male
germ
seminiferous
et
reinitiation
requires
in of
cells,
can be a vitamin
on
loss
spermatocytes
Howell
growth
deficiency or by
Kretser,
reinitiate
though levels was axis and
4, 1979. 14, 1978.
luteinizing were low,
hormone and follicle stimulating
a vitamin A by retinoic growth. Altestosterone hormone
normal and an intact pituitary-testicular was demonstrated by the elevation FSH following castration. In addition,
testosterone
891
rats are given supplemented normal body
reported 130 days
levels
observed
should
of
be sufficient
LH the
HUANG
892
to
maintain
10%
spermatogenesis,
of
normal
concentration
was
normal Huckins, Thus,
spermatogenesis 1978). we attempted
normalcy
of
A
treatment
can
be
shown
found
that
and
if
functionally (PVA)
to
an
rats in
germ
might
cell
present logical
serve the
development
herein the characteristics
PVA-VAD
male
as
a
useful
in
vivo
kinetics
of
We
could
be
studied.
morphological of germ
cell
and physiorepletion in
rats.
MATERIALS
AND
METHODS
Chemicals Radioactive materials were purchased from New England Nuclear and used without further purification; Ilford nuclear emulsion (K5) was purchased from Ilford Nuclear Research (Essex, England). Ingredients for preparing vitamin A deficient diet were purchased from I.C.N. Life Science Group, Cleveland, OH. Reagents used for radioimmunoassay of LH and FSH were provided by Rat Pituitary Distribution program NIAMDD, NIH. Other general chemicals, all reagent grade, were purchased from Fisher Scientific Co.
method
of Kopriwa
Spermatids
Weanling
A Deficient
Vitamin
male
Madison, WI), 19-21 an air conditioned,
Sprague-Dawley days light
solely for vitamin A given water ad libitum deficient diet of Muto instead of menadione.
of age, controlled
(VAD) rats were
Reproductive
Co.,
in pairs in room used
deficiency studies. They were and the modified vitamin A et al. (1972), using vitamin K Animals were weighed twice
weekly.
Retinoic acid (5 mg/kg) was added to the when the linear weight gain ceased as a result of complete depletion of vitamin A stores (Thompson et al., 1964; Muto et a!., 1972). At 130 days of age, rats were given 1 mg vitamin A orally and, thereafter, fed a regular commercial rat pellet. VAD
histology
diet
and
Radioautography
rats
determine the genie regeneration. 1 H] -thymidine 0.1 ml Ringer’s
ether
anesthesia
the
Deparaffined
at intervals of 4-6 in an effort to
sequential changes during spermatoIn selected VAD animals, 25 .sCi (sp act 41.6 mCi/mg) were injected in solution intratesticularly under light 24 h prior to sacrifice. About one-
identified
by
the
PAS-positive
Performance
Test
were
sacrificed
to
determine
epididymal
sperm
Counts.
Hormone
Assay
under
animals were sacrificed, blood samples light ether anesthesia through
or from
were
kept
after
at
centrifugation.
the retroorbital
4#{176} C overnight
Radioimmunoassay performed using
sinus.
Blood
serum
was
and
Abraham
et
of
were heart
samples collected
gonadotropins was by NIAMDD, Rat Pituitary Hormone Distribution Program; NIAMDDRat FSH-RP-1, Rat-FSI-I-I-3, Anti-Rat FSH-S-6 and NIAMDD-rat-LH-RP-1, Rat-LH-I-4, Anti-Rat-LI’I-S-3 and -S-4 were used for FSH and LH assay, respectively. Hormones were iodinated with 1251 using the chloramine-T method (Greenwood et al., 1963). lodinated hormones were purified through a Bio-Gel P-60 column. Intraassay coefficient of variation for both FSH and LH assays was
